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This is to certify that the
dissertation entitled
Oanposition and Functional Properties of

Nbat By-Product Protein Fractions
presented by

Rodney Owen Nuckles

has been accepted towards fulfillment
of the requirements for

Doctoral—_ degree in mm

«9% MW

Major professor

Date 1 1990

MSU is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

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MSU Is An Affirmative Action/Equal Opportunity Institution
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COMPOSITION AND FUNCTIONAL PROPERTIES

OF HEAT BY-PRODUCT PROTEIN FRACTIONS

8}!
Rodney Owen Huckles

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

1990

ABSTRACT

COMPOSITION AND FUNCTIONAL
PROPERTIES OF HEAT BY-PRODUCTS

3?
Rodney Owen Huckles

Composition and functional properties of selected meat
by-products were studied. nechanically deboned chicken meat
(MDCM) and meat by-products (pork lung lobes, pork liver,
beef lung lobes, beef spleen, beef heart) varied in their
proximate composition, amount of the three major protein
fractions [low ionic strength soluble (L18), high ionic
strength soluble (HIS), insoluble] and collagen content.
Least concentration endpoint of the high ionic strength
soluble (HIS) protein gels were beef semitendinosus muscle
(skeletal) 3%, heart 4%, lung 5%, and spleen 6%. The
addition of low ionic strength soluble (LIB) proteins to
high ionic strength protein multicomponent gels initially
decreased expressible moisture (EM) but reduced gel
strength. The addition of insoluble (IN) proteins in
multicomponent gels increased expressible moisture and
reduced gel strength. Variations in microstructure may help
explain the physical properties of the gels.

Beef skeletal meat frankfurter formulations were
modified by heart, lungs and spleen additions. By-product
protein fractions [high ionic strength soluble (HIS), low
ionic strength soluble (L18), insoluble (IN)], percentage
myosin and actin:myosin ratio in formulations varied by

substitution of by-products to investigate functional

Rodney Owen Nuckles

properties. Myosin percentages and actin:myosin ratios
positively correlated to frankfurter water and fat-holding
capacity, reheat yield and strength. Less than or equal to
36% protein as LIB proteins positively correlated to water-
holding capacity and negatively to other tests. Addition of
less than or equal to 38% of IN proteins were not highly
correlated with functional tests. Protein gelation and
frankfurter strength correlated highly. Results suggest
protein gelation can predict processed meats protein

functionality.

This dissertation is dedicated to my parents, Darlene
and Leonard Huckles. There ability to motivate, encourage

and love me has helped me be what I am today.

iv

ACKNOWLEDGMENTS

The author would like to thank Dr. Denise Smith for her
excellent guidance and hard work that has made the PhD.
program a success. He would also like to thank Dr. Stanley
Flegler, Dr. Alden Booren, Dr. Duane Ullrey and Dr. Robert
Merkel for their research insights and for serving on the
guidance committee. Drs. Merkel and Booren were always
available for personal and professional guidance.

Much thanks is owed to Mr. Gordon Jones for his hours
of dissertaion preperation and to Virginia Vega for graphics
assistance.

Thanks are also due to our lab group: Valente Alvarez,
Tom Hearld, Al Antunes, Tan-2i Hung, Bhuefung Wang and nsien
Hsia for their freindships and Bryan Beuschel for his
friendship and research assistance. Bryan and T have changed
the "world of hotdog manufacturing" forever.

A special thanks goes to Tom Porton and the meat lab
crew for allowing me to get in their way and collect the

meat by-products.

TABLE OF CONTENTS

Page
LIST OF TABLES ix
LIST OF FIGURES xii
I. INTRODUCTION 1
A. Introduction to Research 1
8. Dissertation Organization 3
II. Review Of The Literature 5
A. Meat 8y-Products 5
A.1. Definition 5
A.2. Current Pood Uses 5
A.3. Composition and Functionality 7
8. Animal Proteins 9
8.1. Sarcoplasmic Proteins 9
8.2. Myofibrillar Proteins 9
8.3. Stromal Proteins 11
8.4. Functionality 11
8.4.a. Water-binding 12
8.4.b. Emulsion Capacity 17
8.4.c. Gelation 20
C. Gel Evaluation 24
C.1. Textural Properties 24
C.2. Scanning Electron Microscopy of Gels 27
D. Applications 30
D.1. Formed Meat Chunks 30
III. Materials and Methods 32
A. Sources of Meats and Meat by-Products 32
8. Analytical Methods 32
8.1. Compositional Analysis (Studies 1, 2, 3) 32
8.1.a. Protein 32
8.1.b. Moisture 33
8.1.c. Fat 33
C. Electrophoresis of Protein Fractions 33
(Study 1)
D. Timed Emulsification (Study 1) 36
E. Extraction (Studies 1 and 2) and 37
Quantitation (Study 1) of Protein Fractions
P. Collagen Determination (Study 1) 38
P.1. Collagen Solubilization 38
8.2. Color Reaction 39

P.3. Chemicals, Reagents and Calculations 40

vi

vii

G. Heat Induced Protein Gels (Study 2)

6.1.
6.2.

Gel Manufacturing
Gel Evaluation

G.2.a. Least Concentration Endpoint

G.2.b. Expressible Moisture

G.2.c. Apparent Stress and Strain at Failure
G.2.d. Scanning Electron Microscopy

8. Model System Meat Batters (Studies 1 and 3)

3.1.

Formulations

8.1.a. Study 1
8.1.b. Study 3

8.2.
3.3.

Production
Cooked Meat Batter Evaluation

8.3.a. Cook Yield

8.3.b. Severe Reheat Yield

E.3.c. Apparent Stress and Strain at Failure
8.3.d. Apparent Tensile Strength (Study 3)

IV. Study 1: Meat 8y-Product Protein

Composition and Functional
Properties in Model Systems

A. Introduction
8. Material and Methods

B.1.
3.2.

Materials
Methods

8.2.a. Timed Emulsification

8.2.b. Quantitation of Protein Fractions
8.2.c. Collagen Determination

8.2.d. Electrophoresis

8.2.e. Model System Frankfurter

C. Statistics

D. Results and Discussion

V. Study 2: Properties of neat Induced Gels

Prepared from Beef Skeletal, Cardiac
and Smooth Muscle Protein Fractions

A. Introduction
8. Material and Methods

B.1.
B.2.

Materials
Methods

8.2.a. Preparation Of Protein Fractions
8.2.b. Gel Manufacturing

8.2.c. Least Concentration Endpoint
8.2.d. Gel Evaluation

8.2.a. Scanning Electron Microscopy

C. Statistics

D. Results and Discussion

D.1.
0.2.

D03.

SIS Protein Gels Properties

Scanning Electron Microscopy of 818
Protein Gels

High Ionic Strength/Low Ionic Strength
Multicomponent Gels

41
41
42
42
42
43
44
45
45
45
45
45
46
46
46
46
46

48

48
50
50
51
51
52
53
53
54
55
55

66
68
68
68
68
69
70
7O
71
72
72
72
78

D.4.

D.5.

D.6.

D.7.

viii

Scanning Electron Microscopy of High 82
Ionic Strength/Low Ionic Strength Protein
Gels

Sigh Ionic Strength/Insoluble Protein 88
Multicomponent Gels
Scanning Electron Micrographs of High 97

Ionic Strength/Insoluble Multicomponent
Gels

Correlations of Percentages Myosin and 100
Gel Test Parameters

E. Conclusions 100

VI. Study 3: Effect of Meat and Meat 8y-product 102

Protein Fractions on Functional
Properties in Model System Frankfurters

A. Introduction 102
8. Material and Methods 104
8.1. Materials 104
8.2. Methods 105
8.2.a. Meat Batter Formulations 105
8.2.b. Meat Batter Evaluation 106
C. Replications and Statistics 107
D. Results and Discussion 107
D.1. Formulations Containing Different 107

D.2.

D.3.

D04.

D.5.

Percentages of SIS Proteins

Formulations with 35% HIS Proteins and 113
Different Amounts of LIS and IN Proteins
Effect of SIS Proteins on Model System 119
Frankfurters

Effect of LIS Proteins on Model System 119
Frankfurters

Effect of IN Proteins on Model System 121
Frankfurters

D.6. Correlations of Model System Gelation 122
and Frankfurters
E. Conclusions 123
VII. Summary and Conclusions 125
A. Study 1 125
a. Study 2 125
c. Study 3 125
VIII. Recommendations for Future Research 127

Ix. References 128

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

10.

LIST OF TABLES

Page
lorld human food uses of meat
by-products 6
0.8. uses for meat by-products 6
Characteristics of soluble muscle
proteins 12
Proximate composition and collagen
content of meat by-products 55

Proteins fractions as a percentage of
total protein and collagen content of
the low ionic strength soluble (LIS),
high ionic strength (SIS) and insoluble
protein fractions of by-products 57

The percentage of myosin and actin and
the actin/myosin mole ratio of the
by-product high ionic strength soluble
protein fraction 60

Yield and textural characteristics of
model system frankfurters prepared from
mechanically deboned chicken meat and
meat by-products (1:1 based on total
protein content) 62

Correlation coefficients between meat
by-products protein composition and
model system properties 65

Proximate analysis of cooked frank-
furters formulated with different by-
products to alter the percentage

high ionic strength (SIS), low ionic
strength (LIS) and insoluble (IN)

protein fractions 108

Cook yield, severe reheat yield and
textural characteristics of frank-
furters formulated with different by-
products to alter the high ionic

strength (HIS), low ionic strength (LIS)
and insoluble (IN) protein fractions 110

X

Table 11. Proximate analysis of cooked frank-

Table

Table

12.

13.

furters formulated to contain 45% or

35% of high ionic strength soluble

(SIS) proteins by substitution of
by-products 115

Cook yield, severe reheat yield and
textural characteristics of frank-
furters formulated to contain 45% or

35% of high ionic strength soluble

(SIS) proteins by substitution of
by-products 117

Correlation coefficients between meat
by-product low ionic strength (LIS),
insoluble (IN) and high ionic strength
(SIS) protein fraction distribution

and functional tests of model

system frankfurters 120

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

1.

11.

12.

LIST OF FIGURES

Page
Schematic representation of the myosin
molecule 10
Effect of muscle tissue pH on
water-retaining capacity 15

Representative SDS-PAGE electrophoretogram
of by-product high ionic strength soluble
(SIS) proteins (1=beef heart, 2=beef lung,
3=pork lung, 4=molecular weight standards,
5=MDCM, 6=pork liver, 7=beef spleen) 59

Expressible moisture of high ionic
strength plus low ionic strength soluble
protein gels 74

Apparent stress at failure of high ionic
strength plus low ionic strength soluble
protein gels 76

Scanning electron micrographs of high
ionic strength protein gels. A=skeleta1,
8=cardiac , C=lung , D=spleen 7 9

Apparent strain at failure of high ionic
strength plus low ionic strength soluble
protein gels 83

Scanning electron micrographs of high

ionic strength plus low ionic strength
soluble protein gels. A=skeletal,
8=cardiac, C=lung, D=spleen 85

Expressible moisture of high ionic
strength plus insoluble protein gels 89

Apparent stress at failure of high
ionic strength plus insoluble protein
gels 92

Apparent strain at failure of high ionic
strength plus insoluble protein gels 95

Scanning electron micrographs of high
ionic strength soluble plus insoluble
protein gels. A=skeletal, 8=cardiac

C=lung, D=spleen 98

I. INTBQQUQIION

A. Introduction to Research
The food industry must effectively use existing

traditional protein sources and search for new sources of
this valuable nutrient (Gorska et al., 1988). The wastage
of animal by-products which occurs by failure to utilize
them at their highest value plus pollution problems created
by disposal of underutilized by-products are causes for
worldwide concern.

In 1985 the University of Illinois in conjunction with
the National Live Stock and Meat Board conducted a survey to
update information on animal by-product usage. The results
are summarized as follows (Berry, 1986):

1. all by-products saved by packers are being
utilized, but the variety of uses is declining

2. many inedible by-products, especially from pork,
are not being saved or utilized by packers

3. many packers are not saving by-products that are
useful to the pharmaceutical industry, because they are
difficult to collect and store

4. the packers that are saving by-products for the pet
food industry and/or pharmaceutical industry allow these
companies to be the "value adders" to the by-products

5. replacement of by-products by non-meat products
such as bacterial produced pharmaceuticals (insulin, etc.)

and non-meat binders in pet foods is increasing

2
6. exporting of by-products and/or products from by-

products needs to be expanded

7. blood utilization needs to be expanded and

8. in 1984-85 the value of by-products maintained live
steer prices as the wholesale prices declined.

kenney (1986) outlined several areas to improve by-
product utilization, including improved packaging and
presentation of by-products, use of by-products in reformed
meats, texturization of proteins extracted from by-products,
maximizing food uses of by-products especially spleen, lungs
and tripe, and increased use of blood fractions in food
products.

Many processed meat products are currently formulated
using least cost formulation based on bind constants. Least
cost formulation necessitates that a meat product be
formulated using the least expensive meat sources which
exhibit a satisfactory bind value when combined into an
overall formulation (Regenstein, 1988). Saffle (1968)
described the methodology on which current bind values are
based. Emulsifying capacity was measured based on the
formation of a true emulsion in a model system. The bind
values are e emulsifying capacity of the soluble protein
fraction and no measure of emulsion stability is
incorporated into the values. Processed meat product
production failures (greasing out, fat caps and water loss)
can result when bind constants are used because emulsifying

capacity and ingredient functional performance are not

3
highly related (Comer, 1979). Swift et al. (1961)

determined that meat proteins emulsify less fat in meat
products than in emulsion capacity tests. Researchers
recently have suggested the use of a model protein gelation
system rather than emulsion capacity may better predict
protein functionality in a meat product (Comer and Dempster,
1981).

The objectives of this research were:
Study 1. To develop a knowledge base of selected meat by-
product protein fractions composition and functionality to
allow for the practical and effective substitution of by-
products into processed meat products.
Study 2. To test equivalent gelation capabilities of by-
product high ionic strength proteins and determine how the
addition of low ionic strength soluble and insoluble
proteins influence the texture, water-holding capacity and
microstructure of high ionic strength soluble proteins from
skeletal meat and by-products.
Study 3. To determine how the by-product protein fraction
composition (low ionic strength soluble, high ionic strength
soluble and insoluble fractions) influence textural and
water-holding properties when substituted for skeletal meat
in a meat model system. To determine if the data obtained
from protein fraction gels predict the attributes of a model
meat system.

8. Dissertation Organization
The research in this dissertation has been divided into

4
three studies with common sections of text arranged around

them. The first section is a common review of the literature
for the entire dissertation. The next section is a detailed
materials and methods section in which every method used in
the research is described in detail along with the study in
which it was used.

The next three sections are the three studies which
are the dissertation research. It should be noted that the
studies are organized using the format of the gounnnl of
goon Scinnce. Each study contains an introduction,
literature review, materials and methods and results and
discussion sections. The final three sections of the
dissertation are overall conclusions of the research, future

research and references for the entire dissertation.

II EU 0 T TURE
A. Ment ny-Pnoducts
A. . e

Meat by-products are commonly classified as edible or
inedible (Oliveros et al., 1982). Edible meat by-products
are usually referred to as variety meats. As a general
definition, by-products are "everything of economic value,
except for the carcass, that are incidental to the slaughter
of animals" (Eooren and Weiss, 1988). Meat by-products are
underutilized and low priced because they are regarded as an
inferior protein source compared to skeletal muscle meat
(Oliveros et al., 1982). The utilization of meat by-products
has been avoided due to undesirable sensory quality, low
biological value of the proteins and high microbial
contamination (Rosiba, 1983). Mishandling, poor sanitation
and slow chilling decrease quality and increase the
microbial load of meat by-products (8ooren and Weiss, 1988).
The increasing price of meat and processed meat products is
causing the food industry to evaluate the utilization of all

protein sources, including by-products (Gorska et al.,

1988).
A.2. gnnrent Food Uses

In Southeast Asia meat by-products are as prized and as
valuable as carcass meat (Spooncer, 1988). Europe and South
Asia both have high demand for edible by-products. The
export of edible by-products from the 0.8. to Europe and

Southeast Asia is limited because markets in those areas

6
prefer chilled edible by-products, not frozen by-products.

The worldwide uses of by-products as human food are outlined
in Table 1.

- Io ood see of e t b - r ductsA

 

 

Use or

Alternative Cooking
0 an and S ec es nnnn method
Brain All Blanch/Fry
Heart All Roasted/Saute
kidney All Grill
Liver All Lamb's Fry Fry
Lung All Lites Stuffings, Fry
Pancreas Bovine Gutbread Fry
Spleen All Melt Stuffings, Fates
Stomach Porcine Maw (Tripe) Soups, Braise
Rumen ovine, Bovine Blank (Trips) Soup
Reticulum ovine, Bovine Honeycomb (Trips) Soup
Omasum ovine, Bovine Bible Soup
Abomasum ovine, Bovine Soup
Testes ovine, Porcine Fries Fry
Thymus ovine, Porcine Sweetbread Fry
Tongue _All Boil
Udder Bovine Sausages
Uterus Porcine Boil

 

1!adapted from Spooncer, 1988

The most common by-products used for human food in the
0.8. are listed in Table 2. In the U.S. half of poultry by-
products are bought by the pet food industry (Anon., 1988).

Table 2 - U.S. human food uses for meat by-productsa

 

 

By-product Use
Liver Braunschweiger
Tripe Potted meats
Blood Blood sausage
Pork tongues Sulze
Eeart Sausages, luncheon
Intestines Casings, chitterlings
Pork skin Fried pork skins

 

a adapted from Rust and Olson, 1987

. o o t o ‘ ct o t

Information on the functional and physicochemical
properties of by-product proteins is extremely limited. The
protein content and composition of some beef by-products has
been reported (Schaefer and Schierhorn 1974; Oliveros et
al., 1982: Gorska et al., 1988), but the protein fraction
(sarcoplasmic, myofibrillar and stromal) content of by-
products from other species is not known. Gorska et al.
(1988) studied the physiochemical and histological
characteristics of beef gullet meat tissue. Beef gullet meat
tissue contained a low fat and high moisture content.
Compared to lean beef, gullet tissue contained a higher
collagen content and exhibited better water holding capacity
and thus was suitable for use as a raw material in
comminuted meat products.

Oliveros et al. (1982) conducted a study to determine
selected physical and chemical characteristics of beef
tongue, esophagus, tripe, abomasum, small and large
intestines, heart, lungs, spleen, liver and kidneys. Lean
beef was used as control in this study. All meat by-products
had significantly lower protein content than lean beef.
Spleen and liver contained significantly higher protein than
the other by-products. Liver and spleen were similar to lean
in proximate composition. Schaefer and Schierhorn (1974)
reported that the order of protein content of selected beef
by-products is liver > heart > tongue. The majority of the

by-products contained lower amounts of myofibrillar proteins

8
and more sarcoplasmic and stroma proteins than lean meat.

Liver had the highest sarcoplasmic protein content and the
lowest myofibrillar and stromal protein contents. Spleen had
the highest myoglobin content, lowest percent free water
(expressible moisture) and lowest emulsion capacity while
tripe had the highest pH. The stability of emulsions made
from different by-products, except spleen, expressed as
percent cooked yield was similar to the stability of the
emulsion made of lean beef. Spleen had a lower cooked yield
than lean meat.

Eudspeth and May (1969), Maurer et al. (1969), Maurer
and Baker (1966) and Lyon and Thomson (1981) reported on the
physical and functional characteristics of poultry giblets
and concluded that poultry giblets could be utilized in
further-processed meat products. Jimeniz-Colmenero and
Cassens (1987) studied the influence of a water extract of
liver on color and shelf stability of sliced bologna. The
liver extract reduced product color fading and residual
nitrite but did not affect the oxidative rancidity of the
product. Mittal and Laurie (1982, 1985) performed extrusion
studies of soy grits containing 20% or 35% of bovine or
porcine offal. They concluded that the various extruded
products exhibited different textural properties and that
the products could be used as fillers, binders and

texturizers for other food products.

8. in P e n

It is known that meat or meat by-products contain 55-
78% (w/w) water, 15-22% (w/w) protein, 1-15% (w/w) lipid and
less than 4% carbohydrates, minerals and other organic
materials. Studies on meat quality have focused on proteins
which comprise the majority of the solids in meat. Goll et
al. (1977) classified meat proteins on the basis of their
solubility in aqueous solvents into three categories:
sarcoplasmic, myofibrillar and stromal.
B. . arco lasm c P oteins

Sarcoplasmic proteins are soluble at ionic strengths of
0.1 or less and at neutral pH (Goll et al., 1977). They
constitute 30-35% of total proteins in skeletal muscle
(slightly more in cardiac muscle). The sarcoplasmic fraction
is comprised of at least 100-200 different proteins.
Sarcoplasmic proteins are found in the muscle cell cytoplasm
including lysozymal enzymes, mitochondrial enzymes and
myoglobin.
8.2. nyogibrillar Proteins

The myofibrillar proteins are the largest fraction of
proteins in muscle tissue. The myofibrillar proteins are
often defined as insoluble in water and soluble in dilute
salt solutions. This classic definition has been applied to
myofibrillar proteins because high ionic strength (greater
than 0.3 M) is necessary to disrupt the myofibril and
extract myosin, although myosin is water soluble once

extracted from the myofibril (Goll et al., 1977). Ishioroshi

10
et al. (1983) reported that in 0.6 M RC1 myosin monomers are

present, whereas in 0.2 M RC1 myosin is present as

filaments.

Myosin and actin are the major myofibrillar proteins
and constitute approximately 75% of total myofibrillar
proteins. Myosin is a fibrous protein (rod) with two

hydrophobic globular heads and is 1660 A in length (Fig. 1).

Hydrophoblc reglon

 

1660 A

 

Figure 1. Schematic representation of the myosin molecule
(Jones, 1984).

The myosin molecule contains six polypeptide chains
(Smith et al., 1983) consisting of two heavy chains and four
light chains. According to Bechtel (1986) the hydrolysis of
myosin with trypsin yields light meromyosin (LMM) which is a
130 kilodalton fragment of the rod and heavy meromyosin
(HMM) which is the remainder of the rod plus the two
globular heads (290 kilodaltons). Heavy meromyosin contains
one 81 (HMM-Sl) and two 82 (HMM-S2) fragments. HMM-Sl has a
molecular mass of 115 kilodaltons and each HMM-82 fragment

weighs 60 kilodaltons (Goll et al., 1977).

ll
Actin is a monomeric globular shaped molecule (G-actin)

that at monovalent salt concentrations of 50-500 mM or
divalent cations (Ca+2 or Mg+2) aggregates into a double-
stranded, helical filament (F-actin).
. t P 0 e

The stromal proteins are insoluble in water and salt
solutions and are the proteins remaining after exhaustive
extraction of all soluble muscle proteins. Stromal proteins
are a very diverse group of proteins, but collagen and
elastin constitute the majority of this fraction. Collagen
is "a family of molecules" of which at least 11 types have
been identified. Type III is present in muscle perimysium
and plays a role in meat texture. Type III collagen is
thought to be the protein formerly called reticulin. Elastin
is a highly crosslinked, highly insoluble protein derived
from proelastin (72,000-dalton monomer). Elastin is
comparable to collagen in amino acid residues except that it
does not contain hydroxylysine. The proportions of collagen
and elastin vary widely among different muscles in animal
species (Goll et al., 1977).
B. . one t o Anima ote ns

Solubility, emulsifying capacity, gelation and water
binding are the most important protein functional properties
in processed meat products (Rinsella, 1982), although cooked
product stability is currently not thought to rely upon
protein emulsification (Regenstein, 1988). Characteristics

of the soluble muscle proteins are in Table 3.

12
B.4.a. Water-b nd n

The functional water-binding property of foods has
traditionally been called "water holding capacity." Jaurequi
et al. (1981) proposed replacing the term "water holding
capacity" with three more descriptive terms: (1) water-
binding potential, (2) expressible moisture, and (3) free
drip.

Water-binding potential (WBP) and expressible moisture
(EM) are both defined as the ability of a protein system
under the influence of an external force to hold added
water. water-binding potential is the moisture lost per unit
weight of protein in the system, whereas EM is the moisture
lost per unit weight of sample. Free drip is the amount of
liquid lost by a protein system due to gravity.

Table 3 - Characteristics of soluble muscle proteinsa

 

 

Sarcoplasmic Myofibrillar
Property proteins proteins
Conformation Globular Fibrous
Molecular weight Average of Range of 50 -
80 kilodaltons 500 kilodaltons
Primary solubility Water 0.3 M HaCl
Water-binding Very low Very high
capacity
Viscosity Low viscosity High viscosity
Lipid emulsification Slight Extensive
Gel-forming ability None, forms Extensive, forms
coagulum protein matrix

 

3adapted from Acton et al., (1983)
Acton and Dick (1985) defined water-binding capacity as
the "ability of meat to retain its inherent tissue water as

well as any water added during further processing".

13
Water in muscle tissues exists in three forms: bound water,

immobilized water and free water (Acton and Dick, 1985).
Bound water is tightly bound to the protein molecules as a
surface layer about one molecule thick (monolayer). Bound
water accounts for about four to five percent of water in
muscle (Acton and Dick, 1985). Sound water can neither be
added or removed from muscle. Immobilized water is located
adjacent to the bound water and consists of two to five
molecular layers. The amount of immobilized water is
determined by "(1) the amount of ionization or the surface
charge density of the protein's hydrophilic amino acid
groups, (2) the physical forces exerted on the proteins and
(3) the distance of the water location from the protein
molecules" (Acton and Dick, 1985). Free water is held only
by capillary action. Water added during processing is free
water. Expressible moisture (EM) as measured according to
Jauregui et al. (1981), using centrifugation for 15 min at
16,000 rpm, will remove some or all of the free and
immobilized water and may be used to compare EM of different
protein systems.

Regardless of the nomenclature, the ability of foods to
retain moisture is important for several reasons: (1)
economic (reduction of cooking losses), (2) organoleptic
and to some extent (3) fat binding as proteins will unfold
to bind moisture and fat both (Hamm 1981). Protein A
solubilization, as affected by moisture in the food, will

enhance fat binding.

14
The amount of bound, immobilized and free water is

affected by type of protein, salt addition (amount and
type), ionic strength, pH, presence of phosphates, capillary
action and product final internal temperature. Weinberg et
al. (1984) tested nine salts (NaCl, HaI, HaHOa, Hazso‘, RC1,
LiCl, HH4C1, MgClz, CaClz) in comminuted cod muscle and
concluded the EM was similar among the salts, except for
MgClz and CaCl2 which had higher EM (less water binding
ability). Acton et al. (1883) reported that sarcoplasmic
proteins have very low water-binding potential (IBP),
whereas myofibrillar proteins have very high WBP. Venegas et
al. (1988) reported that meat by-products, especially
spleen, had lower EM than skeletal muscle because the by-
products used had a higher pH than the skeletal muscle.
Oliveros et al. (1982) tested beef by-products (tongue,
esophagus, tripe, abomasum, small intestine, large
intestine, heart, lung, spleen, liver, kidney) and lean beef
muscle for EM. They reported that spleen had lower EM than
the other by-products or lean muscle. The order of EM from
most to least was large intestine > tongue > small intestine
> tripe > esphoagus > abomasum > lean muscle > heart >
kidney > lung > liver > spleen.

Figure 2 shows the effect of pH and 2% NaCl on the
water-retaining capacity (water binding capacity) of meat
proteins. Meat has a higher water-retaining capacity at pH
values above and below pH 5.0, the approximate isoelectric

point of meat. The addition of 2% HaCl increased the water-

l5
retaining capacity of meat as the meat pH increased. The

NaCl provided ions that bound to the proteins. Proteins
carrying a negative or positive electric charge will repel
each other and interact with water and retain moisture

(Cheftel et al., 1985).

l l l l
4.5 5.0 5.5 6.0

pH of meat

 

Increasing water-binding capacity

F' e - Ef ect o usc e t sue H on wate -retaining
capaciny Q
a adapted from J. Wismer-Pederson (1987).

Regenstein and Stamm (1979) reported that sodium
pyrophosphate increased water holding capacity (WHC),
whereas divalent cations decreased WHO in chicken breast
muscle. Thomsen and Seuthen (1988) reported that final
internal temperatures of 50-700 C increased WHC while
temperatures greater than 700 C markedly decreased WHO in.
model pork sausages.

Several methods have been developed to determine EM or

WHC in protein products. Hamm (1986) summarized many methods

16
used for evaluating water holding capacity or expressible

moisture of meat and meat products and may be referred to
for further methods. Jaurequi et al. (1981) developed a
centrifugal method to measure moisture loss from protein
gels or meat product samples. The method uses thimble shaped
filter paper, a test tube, sample and high speed
centrifugation at 31,000 x g for 15 min. After
centrifugation, the moisture transferred from the sample to
the filter paper is determined.

Eide et al. (1982) determined EM by placing samples in
test tubes containing a platform of polyester mesh and
centrifuging for 5 min at 1500 x g. After centrifugation,
the moisture that flowed though the mesh to the bottom of
the test tube was measured and EM determined. Hermansson
(1975) used a "net test" to determine WHC. A meat plug was
transferred to a net of stainless steel fastened in a steel
ring. The plug, net and steel ring were placed in a test
tube and centrifuged at 500 x g for 5 min. The moisture lost
from the meat plug was determined and the moisture retained
was calculated.

Jauregui et al. (1981) determined that EM is highly
dependent upon both time and centrifugation speed with the
latter being more important. An advantage to the Jauregui et
al. (1981) method compared to the Side et al. (1982) or
Hermansson methods is that all the moisture is contained in
a removable filter paper that can be analyzed for adhering

fat or proteins. Moisture or meat residues trapped in the

17
polyester mesh or net is more difficult to remove by washing

and may be lost. A disadvantage of high speed centrifugation
is that the product being centrifuged may be torn apart or
deformed and an artificially high EM may be calculated.

Wu et al. (1985) measured EM of cooked starch-fish
protein gels using a Carver Laboratory press. Samples about
2 mm thick were cut from the gels, placed between Plexiglass
sheets and pressed for 1 min at 1000 psi. The wet surface
area and the meat film area on the Plexiglass sheets were

measured and the following equation used:

Percentage = (Total wet snggace aren-neat giln nrea)(61.10)

free water Measured moisture (mg) of gel sample

The 61.10 is a constant derived from relating
centrifugation and Carver Laboratory press methods.
An advantage of low speed centrifugation methods to measure
EM or WHC compared to other physical methods is that it is
easier to apply to weak products such as protein gels
because the gel matrix will be less deformed (crushed) and
the protein and moisture will be easier to separate. Other
physical methods may destroy the protein gel beyond the
point where protein and moisture separation are possible.
8.4.b. Emulsion capacity

Emulsion capacity (EC) is based on the weight of meat
or protein in the model system:
EC (meat basis) = ml of fat (oil) emulsified

per unit weight of meat

18
EC (protein basis) = ml of fat (oil) emulsified

per unit weight of protein

The methodology to measure EC is based on the original
work of Swift et al. (1961). A protein solution or meat
hemogenate of known concentration or amount, and a specific
amount of lard or oil are added to a blender. The blending
process is started and more lard or oil is added until the
viscosity of the mixture in the blender decreases. The
amount of lard or oil added to the blender is measured and
divided by the amount of protein in the blender and called
emulsion capacity. Emulsion capacity is widely reported
(Asghar et al., 1985: Maurer and Baker, 1966: Hudspeth and
May, 1969: Galluzzo and Regenstein, 1978) even though it is
currently not thought to play an important role in cooked
product stability (Regenstein, 1988). Galluzzo and
Regenstein (1978) developed a timed emulsification model
system to define the role of specific muscle proteins in
emulsification and emulsion stability, the amount of oil
remaining in the emulsion after centrifugation.

The EC of a protein depends upon both its amount and
solubility in a model system (Comer and Dempster, 1981).
Protein solubility is one of the major factors causing
differences in EC among ingredients. Gaska and Regenstein.
(1982) showed that myosin, actomyosin and insoluble
myofibrillar proteins all were involved in emulsion

formation. Emulsion capacity is currently one of the

19
parameters used to determine least cost formulation of meat

products. Comer and Dempster (1981) found that EC did not
predict bind values and proposed that "protein quality
factors" be used to predict bind values. Protein quality
factors may include protein gelation tests or other protein
functional property tests that are representative of
ingredient gelation properties. Research is improving EC
data used in least cost formulation and/or will replace BC
with more appropriate methods.

Myofibrillar proteins are capable of emulsifying fats,
while sarcoplasmic proteins only have slight emulsification
properties (Acton et al., 1983). Asghar et al. (1985)
reported the EC of different proteins as: myosin >
actomyosin > sarcoplasmic proteins > actin (in 0.3 M HaCl
isolution). Maurer and Baker (1966) reported that poultry
meats with high levels of collagen had low EC. Skin and
gizzard containing large amounts of connective tissue were
inferior in EC compared to muscle tissue (Hudspeth and May,
1969). Venegas et al. (1988) reported that lean skeletal
meat was better able to emulsify and retain fat compared to
beef by-products. They reported that by-products low in
collagen stabilized fat when cooked at low temperatures (700
C) and by-products high in collagen cooked to high
temperatures (1000 C) stabilized fat. Oliveros et al. (1982)
measured EC (based on Swift's methodology) of lean beef and
beef by-products and reported that by-products high in

myofibrillar proteins and low in stromal proteins and fat,

20
emulsified the most corn oil.

Hydrophilic and lipophilic properties of proteins have
been used to characterize properties of surfactants and
emulsifiers. Based on the work by Griffin (1949),
DeRanterewicz et al. (1987) developed an index to describe
the relative hydrophilic-lipophilic character of proteins
for use in predicting their emulsifying properties of corn
oil. They found that the EC of a protein depends on both its
hydrophilic and lipophilic characteristics rather than each
independently. Myofibrillar proteins exhibit both fat
emulsification (lipophilic interaction) and water-holding
capacity (hydrophilic interactions).

. .c Ge at o

Van Rleef (1986) defined heat induced gelation of
proteins as a denaturation and aggregation process. A change
from the native protein conformation to a more random,
unfolded structure is denaturation, whereas aggregation is
the interaction between proteins that leads to the formation
of very large crosslinked entities. When the protein
concentration is sufficiently high and heat is applied, the
large protein entities eventually precipitate.

Physical and chemical stabilization of fat and water in
comminuted meat products is caused by heat-induced gelation
of muscle proteins (Ziegler and Acton, 1984). Lee (1985).
reasoned that fat is stabilized through physical entrapment
in a protein matrix instead of via emulsification because:

(1) products emulsified, stuffed into casings and cooked

21
(frankfurters) stabilized less fat than coarse ground,

stuffed, cooked products (2) during emulsification in a bowl
chopper a meat batter loses its stability when the fat
reaches its melting temperature, whereas when cooked,
actomyosin and melted fat produced a stable product, (3)
after prolonged emulsification, meat batter instability due
to fat loss is reversed by cooling the batter below the fat
melting temperature, (4) a stable meat emulsion was produced
with fat, but not with oil, (5) some proteins that exhibit
good EC failed to produce a stable meat product matrix, and
(6) synthetic emulsifiers added to meat batters reduced fat
stabilization of the meat batter.

The gelation of muscle proteins chemically or physically
entrap fat and moisture (Foegeding and Lanier, 1987). Gels
can be formed from a single type of polypeptide (Foegeding,
1988) or from complex systems. Tolstoguzov and Braudo (1983)
defined gelation of complex systems such as frankfurters or
other comminuted meats as multicomponent gels. They defined
three types of multicomponent gels: filled, mixed and
complex. Filled gels are formed when one protein forms the
gel matrix and another macromolecule fills the interstitial
spaces. Mixed gels result when at least two macromolecules
gel independently and form three dimensional networks
without interacting. Interactions between different types of
macromolecules lead to the formation of complex gels.

Acton and Ziegler (1983) reported that myofibrillar

proteins have excellent gel forming abilities and form

22
extensive protein matricies, whereas sarcoplasmic proteins

form coagulums and do not posses gel forming abilities.
Foegeding and Lanier (1987) also reported that fish
sarcoplasmic proteins exhibited very poor gelation
capabilities (formed a coagulum) and interfered with the
gelation of myofibrillar proteins.

The myofibrillar protein most responsible for gelation
is myosin (Schmidt et al., 1981). Myosin forms a heat-
induced gel that is irreversible, has high water-binding
capacity and strong elastic properties. Siegel and Schmidt
(1979) studied heat-induced gelation of myosin and found
that gelation was dependent upon an intact myosin heavy
chain (tail). Whiting (1988) described protein gelation as
"disulfide bond formation between myosin heavy chains at 300
to 500 C and followed by hydrophobic interactions and
aggregation at 500 to 700 C of the remaining parts of the
molecule". Yasui et al. (1982) reported that F-actin
enhances the gelation of myosin by forming F-actomyosin
which acts as a crosslinker between the tail portion of
bound and free myosin molecules. Maximum gel strength was in
0.6 M RC1, pH 6.0 and at a myosin to F-actin ratio of 2.7:1
which corresponds to a weight ratio of 15:1. Gelation
between myosin molecules (monomers in 0.6 M RCL) was
dependent upon the tail (rod) section (Ishioroshi et al.,
1983). Myosin filaments in 0.2 M RC1 gelled by head-head A
aggregations. Samejima et al. (1984) reported that myosin

heavy chains (MHC) formed gels almost equal in strength to

23
intact myosin. They also reported that myosin light chains

(MLC) do not contribute to MHC gelling and actin weakened
the MHC gel. The MLC/actin complex as actomyosin may provide
some gel stability at a pH other than 6.0.

Cardiac myosin, as reported by Samejima et al. (1985),
formed a stronger gel than skeletal muscle myosin at pH 5.5,
0.1 M RC1 and 600 C final temperature. Asghar et al. (1984)
reported that under identical situations (pH, ionic
strength, protein concentration, temperature) myosin from
broiler white fiber muscles formed stronger gels than myosin
from red fiber muscles. They related different gelling
abilities of myosin from red and white muscles to myosin
isoforms. Wagner and Anon (1985) studied bovine muscle
depleted of sarcoplasmic proteins and connective tissue
using differential scanning calorimetry (DSC). They reported
that myosin was more sensitive to heat (totally denatured at
57° C) and low pH (5.4 - 5.6) than actin.

Sato and Nakayama (1970) found that myofibrillar
protein content and gelation were related to binding quality
of minced meats. Whiting (1988) stated that successful
gelation requires muscle and fat cell disruption, fat
particle suspension, actin and myosin disassociation and the
prevention of actomyosin formation. Myosin also forms
complex gels with fibrinogen (Foegeding et al., 1987) and
filled gels with starch (Foegeding and Lanier, 1987).

Insoluble proteins have been reported to be both

beneficial and a hinderance to myofibrillar protein gelation

24
in processed meat products. Puolanne and Ruusunen (1981)

studied the effect of cooking temperature on gel water-
holding capacity and strength of meat batters containing 5%
and 15% connective tissue, respectively. They reported that
at 600 C, connective tissue swelled, gelled and bound
water, but cooking to higher temperatures resulted in
greater water loss in the high connective tissue batter.

Schmidt (1987) reported an increase in emulsion
stability when connective tissue proteins were added and the
emulsion was heated to a sufficient temperature to gel the
connect tissue proteins.
C. Gel Evaluation

Observation of gel microstructure can help interpret
gel textural properties (Sone et al., 1983). Gel
microstructure is determined by protein chemical composition
and interactions, therefore understanding the microstructure
will promote understanding of protein functionality.
Microstructural characteristics have been shown to be
related to soybean protein gel hardness (Furukawa, 1978) and
milk gel high stress relaxation or hardness (Ralab and
Harwalker, 1974).
C.l. Textural Pnonerties

Several empirical methods exist for the testing of gel
strength of meat batters or meat products. The methods
include torsion failure tests (Montejano et al., 1983),
texture profile analysis (TPA) (Bourne 1978), 70%

compression (Lee et al., 1987), back extrusion (Hickson et

25
al., 1982), fold test (Rude et al., (1973), tube viscometry

(Toledo et al., 1977) and tensile strength (Trout and
Schmidt, 1984). In this research, protein gel compression
and frankfurter compression and tensile strengths were
measured, thus they will be discussed.

Compression (70%) testing utilizes a compression cell
which is composed of parallel plates between which protein
gels or meat cores can be compressed. Apparent stress at
failure and apparent strain at failure can be calculated
(Diehl et al., 1979). True stress and strain cannot be
calculated because a uniform cylinder is not maintained
during compression. Strain is highly correlated to sensory
and texture profile analysis (TPA) cohesiveness, while
stress to failure (compressive force to failure) is
correlated to TPA hardness and sensory firmness (Montejano
at 81., 1985).

McReith et al. (1988) used compression to evaluate
surimi produced from pork, beef, beef by-products and
commercial fish surimi. They reported that the compressive
force required to crush the surimi produced from pork, beef
and beef heart was similar or stronger than the compressive
force required to crush commercial fish surimi. They
concluded that surimi-like gels could be successfully
produced from pork, beef and beef heart. Van Rleef (1986)
studied the fracture behavior of egg ovalbumin gels and
soybean protein gels using compression. He reported that the

stress at fracture (hardness) of the ovalbumin gels was

26
greater than the soybean protein gels. He explained the

hardness of the ovalbumin gels was greater because of
covalent crosslinkages in the ovalbumin gels and that the
ovalbumin gels formed from dissolved proteins, whereas the
soybean protein gels contained dispersed, not well dissolved
proteins. Morris et al. (1980) tested iota and kappa
carrageenan gels produced with the Hofmeister series of
cations using compression to measure yield stress. They
reported that the use of Rb+ or R+ helped form stronger gels
which they related to the large size of the cations. Randall
and Voisey (1977) used compression to measure firmness of
meat and non-meat (soy protein isolate or whey protein
concentrate) emulsions containing various concentrations of
salt soluble proteins (SSP) and/or connective tissue
proteins (CT). They reported that SSP and CT added firmness
to the emulsions.

Tensile strength measures the force per cross-sectional
area or force per unit weight of sample to tear apart cooked
meat products. Tensile strength was used to measure the
binding strength of chunked and formed products, but
currently is also applied to comminuted meat products (Trout
and Schmidt, 1987). Tensile strength can be measured using
an Instron Universal Testing Machine or apparent tensile
strength can be measured using machines such as the Food
Technology Corporation's Texturerecorder. The advantage of
the texturerecorder is that it is less costly to purchase

than the Instron.

27
Gillett et al. (1978) used the Instron Universal

Testing Machine to test tensile strength of ham slices
containing various concentrations of salt, phosphate and/or
nonfat dry milk. They concluded that tensile strength
successfully measured the increase adhesion of the ham
pieces due to the effect of increased concentrations of
salt, phosphate and/or nonfat dry milk upon the meat
proteins. Bouton et al. (1975) investigated the relationship
between tensile strength and the percentage of myofibrillar
or connective tissue proteins in processed meats. They
reported that the myofibrillar proteins produced the initial
tensile strength reading (increase in force to tear apart
the meat) and that connective tissue proteins produced the
peak force of the tensile strength reading. Trout and
Schmidt (1987) used tensile strength to measure the effects
of ionic strength, pH, pyrophosphate and cooking temperature
on beef muscle homogenates (beef ground through 2.5, 1.5 and
0.5 cm plates). Maximum tensile strength was produced at 660
C for treatments with no cooking loss. Cooking loss from
treatments caused tensile strength to increase linearly with
increased temperature. Overall, tensile strength was maximum
at 66° c, pH 5.0, ionic strength > 0.25 and 0.31%
pyrophosphate.
c o c o o o G

Lee (1985) studied fat stabilization in meat emulsions

via light microscopy (LM), scanning electron microscopy

(SEM) and transmission electron microscopy (TEM). He found

28
that LM was good for evaluation of gross structure and fat

dispersion patterns, whereas SEM and TEM were appropriate
for assessing the interactions between the gel matrix and
fat globules. Sone et al. (1983) reported that in most cases
SEM is used to observe the protein gel microstructure and
that freeze drying, glutaraldehyde fixation followed by
critical point drying, and cryo-SEM are useful tools in food
research. In some cases the use of osmium tetroxide may be
added to help fix the fat in a meat emulsion (Ray et al.,
1979).

Oelker (1988) used SEM to study the structure of dry
sausage. He found that collagen fibril and microfibrils and
myofibrillar and sarcoplasmic proteins are all essential to
form the structure of a dry sausage. Bernal and Stanley
(1986) examined a commercial restructured beef product using
SEM and found that the distinctive elements of intact beef
muscle (muscle fibers surrounded by connective tissue and
grouped into fasciculi) were not apparent, showing that
restructuring had a severe effect on the microstructure of
the product. The surfaces of the restructured particles were
covered by a protein film, due to protein being extracted
during restructuring operations. Hermansson (1986) using SEM
found that cooking of protein, fat and water mixtures above
their gelation point caused random aggregation of proteins
rather than an ordered three dimensional matrix being formed
and increased fat and water loss. Comer and Allan-Wojtas

(1988) studied the role of starch fillers in comminuted meat

29
products using LM and SEM. Light microscopy revealed that

the "all-meat" wieners had a higher degree of fat
agglomeration than did the more stable wieners containing
added starch fillers. Electron microscopy revealed that the
starch granules helped to physically entrap fat globules.
Jones and Mandigo (1982) studied the effects of chopping
temperature on the microstructure of meat emulsions. The use
of SEM revealed that meat emulsion stability is related to
two functions, the interfacial protein film thickness around
the fat globules and the ability of the surrounding emulsion
to maintain the stability of the interfacial protein film
during cooking. Ray et a1. (1979) developed methodology to
identify the protein and fat component in scanning electron
micrographs by fixing two different samples of a cooked meat
emulsion (sample 1 and sample 2). For sample 1 they used
glutaraldehyde to fix the protein and ether to extract the
fat, for sample 2 they fixed the cooked meat emulsion using
osmium tetroxide (fixes both protein and fat). They viewed
and compared the sample to identify the protein and fat
components. Smith and Brekke (1985) used SEM to view
myofibril gels produced from enzymatically modified
mechanically deboned fowl proteins. Gel ultrastructure and
functional properties were related by assessing the scanning
electron micrographs. The control gels were compact and
globular and exhibited high gel strength and gel syneresis.
Gels produced from myofibrils modified to 18% solubility

were fibrous and exhibited high gel strength and water

30
holding ability. Clarke et al. (1988) studied the effect of

algin/calcium, pH and muscle proteins on gelation and
microstructure of gels and restructured beef. They studied
microstructures of sarcoplasmic proteins, myofibrillar
proteins and restructured beef with added algin and calcium.
At the same pH, myofibrillar/algin gels were more fibrous in
structure than sarcoplasmic/algin gels. Low pH (3.9) did not
alter the sarcoplasmic/algin gel structures, whereas the
myofibrillar/algin gels were more globular. The addition of
algin to the restructured beef visibly added globular
structures (algin) to the beef fibrils. Sone et a1. (1983)
reported that meat product microstructures viewed by SEM are
related to gel hardness and firmness.
D. ‘ca 0
D. orm eat C un s

The pet food industry in the 0.8. and both the pet and
human food industry in Canada use a technique to restructure
meat. The meat products are called formed meat chunks (FMC).
Formed meat chunks are manufactured using non-meat binders
(mainly wheat gluten), because the meats do not contain
enough myofibrillar proteins for binding. Formed meat chunks
are cooked by high temperature water bath techniques or high
temperature/pressure cooking.

Myofibrillar proteins, mainly myosin, are responsible
for the binding of restructured meats and FMC (Ring and
MacFarlane, 1987). As the sliced or flaked meats are mixed

with salt and phosphates, myosin is extracted to the meat

31
surface and forms a sticky (tacky) layer. The meat is

pressed together and upon heating the proteins are denatured
and bind the meat pieces together.

Factors that can influence the extraction of
myofibrillar proteins include the following: (1) the state
of rigor development: (2) the ionic environment and the pH
of the system: (3) the temperature history of the meat
during rigor onset: (4) the temperature during extraction:
(5) storage of meat: (6) application of high pressure: and
(7) miscellaneous physical factors such as the duration of
extraction, the degree of agitation of the system (e.g., by
mixing or tumbling), the size of the meat particles, and
application of vacuum (vacuum mixing).

A better understanding of protein functionality will
allow the use of meat by-products and/or meat by-product

proteins in traditional restructured meats or FMC.

. e a et ods

n, Sounces o; nnnts and gent ny-znoductn
Frozen mechanically deboned chicken meat (MDCM)

prepared from the whole carcass (Study 1) was purchased from
Mottawa Gardens Corp., Athens, MI. Beef skeletal muscle used
in Study 2 and 3 was purchased from Monfort of Colorado
Inc., Greeley, CO. By-products [pork lung lobes, pork liver,
beef lung lobes, beef spleen, beef heart (Study 1)] and
[beef lung lobes, beef spleen, beef heart (Studies 2, 3)]
were obtained from market weight hogs and steers slaughtered
at the Michigan State University Meat Laboratory. Additional
beef spleen and beef fat (Study 3) were obtained from Ada
Beef Co., Ada, MI. Pork backfat (Study 1) was purchased from
a local grocery store. All meats, by-products and fats were
stored vacuum packaged (2.5 mil polyethylene) and frozen
until used within three months.
2. Analytical Methods
. . o o t ona Ana s s Studies and 3

B. . P at n

The protein content of the meats, meat by-products,
protein fractions and raw and cooked meat batters was
determined using AOAC Method 24.038 - 24.040 Crude Protein
in Meat: Block Digestion Method (AOAC, 1984). The following
modifications were made in the Block Digestion Method: a
sample weight sufficient to require approximately 8 mL of

HCl acid to titrate the boric acid/nitrogen mixture to its

32

33
original pH, 5 mL instead of 15 mL 8280‘ were used and no

H202 (instead of 3 ml) was added to the sample. All protein
determinations were performed in triplicate and the protein
content was determined on a wet weight basis using a
nitrogen conversion factor of 6.25.

ngl,b2 noisture

Moisture content of the meats, meat by-products and raw
and cooked meat batters was determined using AOAC Method
24.003, Moisture in Meat (AOAC, 1984). Moisture contents
were determined in triplicate.

B.l,n. Fat

Fat content of the meats, meat by-products and raw and
cooked meat batters was determined using AOAC Method 24.005.
Fat contents were determined in triplicate.

C. e tro hores s of Protein Fract on Stud

Sodium dodecyl sulfate polyacrylamide slab gel
electrophoresis (BBS-PAGE) of high ionic strength (HIS)
proteins and insoluble (IN) proteins was performed on 12%
(0.25% bis) acrylamide running gels with a 4% stacking gels
using the system of Laemmli (1970).

The HIS proteins were prepared for electrophoresis by
adding 9 m1 of protein solution, 1 ml 10% sodium dodecyl
sulfate (SDS) and 3 ml of beta mercaptoethanol (BME) to a
screw top test tube. The capped test tube was heated in 95°
C water for 10 min. After heating, the protein mixture was
dialyzed for 18 hr with one change of dialysis buffer. The

dialysis buffer was 25 mM Tris - HCl, 0.2% SDS, 0.2 mM

34
ethylenediamine tetraacetic acid (EDTA) and pH 7.25. After

dialysis, 20% w/w glycerol and one grain (granule) of
bromophenol blue were added to the protein solution. The
insoluble proteins were solubilized for electrophoresis by
adding a 9:1 (w/w) ratio of insoluble proteins and
solubilization buffer to screw top test tubes. The
solubilization buffer was 2.5% SDS, 4M urea, 0.5M Na
phosphate and pH 7.2. Three drops of EMS were added to each
test tube, the tubes were capped and cooked for 2 hr in a
50° C water bath. After 2 hr, the test tubes were boiled in
water for 10 min. The liquid in the test tubes was decanted
into dialysis tubing and dialyzed as discussed for the HIS
proteins.

Electrophoresis was performed with a Hoeffer Vertical
Electrophoresis unit (Model SE 600: Hoeffer Scientific
Instruments, San Francisco, CA) using a constant voltage
power supply (Heathkit Model 1P-17, Benton Harbor, MI). One
hundred micrograms of the HIS protein fraction and 200 ug of
the IN protein solution were put on the stacking gel. A
constant current of 30 mA was applied until the proteins
migrated into the running gel and then the current was
increased to 60 mA until the bromophenol blue tracking dye
reached the bottom of the running gel. The gels were removed
and stained for 6 hr in 0.125% Coomassie Blue in 50/10/40_
(v/v/v) methanol/acetic acid/water. The gels were destained
in 7/5/88 (v/v/v) acetic acid/ methanol/ water until the

protein bands were clearly visible.

35
Subunit molecular weights were estimated using a

mixture of high molecular weight proteins MW-SDS-200 Rit:
Sigma Chemical Corp., St. Louis, MO). The mixture consisted
of the following proteins: carbonic anhydrase (29
kilodalton), egg albumin (45 kilodalton), bovine plasma
albumin (66 kilodalton), phosphorylase 8 subunit (97.4
kilodalton), B-galactosidase subunit (116 kilodalton) and
rabbit muscle myosin subunit (205 kilodalton).

The relative mobility (RM) of the protein standards was

calculated using the formula:

RM = Distance o: Pnotein Migration (cm)

Marker Dye Distance (cm)

and a plot of relative mobility vs log molecular weight was
constructed. The relative mobility of each protein subunit
was calculated and the molecular weight was estimated from
the standard curve. The amount of protein present in each
protein band on the gels was determined using a Shimadzu
Dual Wavelength Thin-Layer Chromato Scanner (Model CS-930,
Ryoto, Japan). The protein bands were identified by their
subunit molecular weights. The percentage of myosin and
actin was calculated by dividing the amount of myosin or
actin by the amount of total protein present on the gel.
Actin:myosin mole ratio was calculated (Goll 1977: Beas et
al., 1988) by dividing the total amount of actin by its

molecular weight (41.785 kilodaltons) and the total myosin

36
by its molecular weight (475 kilodaltons) and then

calculating the ratio of the moles of actin to the moles of
myosin.
D. d 1 at 0 tud

Timed emulsification was developed by Galluzzo and
Regenstein (1978) to measure the ability of proteins, singly
or in mixtures at 5 mg protein/ml in 0.6 M NaCl, to form an
emulsion or cream phase when emulsified with 5 ml of corn
oil. The emulsion or cream phase was formed using a Sorvall
Omni-mixer (Ivan Sorvall, Inc., Norwalk, CT) equipped with
an adapter ring for 50 ml clear polycarbonate tubes. A 3 ml
protein sample was pipeted into the 50 ml clear
polycarbonate tube, overlaid with 6 ml of corn oil and
emulsified for 5 min. The emulsified samples were
centrifuged at 30,000 x g for 15 min to determine stability
(ability to hold the oil) of the emulsion.

In this study, timed emulsification was performed with
modifications because an emulsion was not formed using the
recommended protein concentration of 5 mg/ml. Mechanically
deboned chicken meat and by-products were evaluated in
triplicate at total protein concentrations of 10 to 150
mg/mL. The lowest concentrations of protein required to
produce an emulsion after centrifugation at 30,000 x g for
15 min was recorded and used as an index of protein

functionality.

37
t ac on Studio and 2 a d uantitat on St d 1

f P e ra tio

All extraction procedures were performed in a 4° C cold
room. A.weighed portion of MDCM or by-product was blended
(Waring Blendor, Model 1120, Winsted, CT) for 1 min with 4
volumes of 0.05M Na phosphate buffer, pH 7.4, stirred for 3
hr (Talboy T-Line Laboratory Stirrer, Model 107, Emerson,
NJ) in a cold room and centrifuged at 23,000 x g for 15 min.
The supernatant was saved and the residue re-extracted for 1
hr. Following centrifugation, the supernatants containing
the 0.05M Na phosphate soluble proteins were combined,
designated as low ionic strength soluble (LIS) proteins and
quantitated. The precipitate was mixed with 4 volumes of
0.6M NaCl, 0.05M Na phosphate buffer, pH 7.4 and extracted
twice as described previously. The supernatants, designated
as high ionic strength soluble (HIS) proteins, were combined
and quantitated. The precipitate was designated insoluble
(IN) proteins and weighed. The protein content of each
fraction was determined by Rjeldahl procedures (AOAC, 1984).
Non-protein nitrogen was measured on the low ionic strength
soluble proteins according to Helander (1957) by the
addition of 10% (w/w) of trichloroacetic acid. Non-protein
nitrogen content was subtracted from the low ionic strength
soluble nitrogen before calculating protein (N x 6.25).

The protein fraction percentages were calculated by
dividing the weight of total protein in each fraction by the

weight of total protein in the meat. Percentage protein in a

38
weighed portion of each protein fraction multiplied by the

total fraction weight equaled the total protein in each
fraction.
F e u

Collagen was determined in triplicate on each by-
product, MDCM and the HIS protein and IN protein fractions
(Bergman and Loxley, 1963). Prior to collagen determination,
MDCM, by-products and insoluble proteins were frozen with
dry ice and ground in a IRH - Universal mill M20 (Jankel and
Runkel Co., West Germany).

F.l. Cnllngen Snlnnilization

a. Weigh out, in triplicate, 2 i .030 g of

powdered meat sample (liquid nitrogen ground) into 50
mL screw cap test tubes. Record the net weight of the
sample.

b. Add 20 mL of 6 N HCl and secure the tubes with teflon
lined screw caps.

c. Autoclave the tubes overnight at 121° C, 18-20 psi.

d. Cool tubes to room temperature.

e. Add 1.0 (i .10) g of charcoal to each tube.

f. Shake the tubes (by hand) 20 times and filter the
sample through 15 cm Whatman #2 filter paper into 400
mL beakers.

9. Rinse the tubes and caps 3 times, then rinse the filter
paper 5 times with H20.

h. Adjust the pH of the samples to 6.0 (i 0.1) using 12 N

NaOH. Rinse the pH probe with a squeeze bottle into the

39
sample beaker.

i. Transfer the solutions to a 500 mL volumetric flask or
graduated cylinder, rinsing the beaker 3 times.

j. Dilute to volume with 820 (ie: to 500 mL)

k. Prior to sampling for color reaction, filter a
representative portion through Whatman #1 (or Fisher
no. 09-795) filter paper.
F . o o

a. Prepare the appropriate standards in 50 mL volumetric
flasks using the 50 ug/mL stock solution of hydroxyproline.
b. Prepare Ehrlich's Reagent and oxidant solution.

c. Preheat water bath to 60° C.

d. Pipet 1 mL of 820 into 4 screw cap test tubes. Two are
for zero standards, two are for sample references.

e. Pipet 1 mL of each standard into duplicate screw cap
test tubes.

f. Pipet 1 mL of each sample into duplicate screw cap
test tubes.

9. Add 2.0 mL of isopropanol to each tube, and vortex the
tubes.

h. Start a timer and add 1 mL of oxidant solution to each
tube and vortex.

i. Four minutes after adding the oxidant solution to the
first tube, begin adding 4 mL of Ehrlich's reagent to
each tube, in the same order. Vortex each after
pipeting.

j. Screw the caps on to the tubes and vortex again.

40
k. Set the tubes into the 60° C water bath for 25 min.

1. Cool the tubes for 5 min in chilled water.

m. vortex the tubes and measure the absorbence at 558 nm.
F.3. ca s ea ent Ca c ati ns

a. To produce 6 N HC1, dilute concentrated HC1 1:1 with
n20.

b. Charcoal must be MCB CR 0655-1 or J.T. Baker 8344-7.

c. Prepare stock hydroxyproline standard in .001 N HC1
which is a concentration of 50 ug/mL. Weigh 0.0250 g of L-
hydroxyproline (Sigma H-6002) into a 500 mL volumetric
flask. Add 5 mL of 0.1 N HC1 and dilute to volume with 820.
Solution will be stable for 2-3 weeks when stored under
refrigeration.

d. Prepare acetate citrate buffer by using 0.3 g sodium per
28.5 g H20 (or sodium acetate anhydrous, 17.2 g), 0.29
trisodium citrate into 18.75 g H20 and 0.1 g citric acid
added to 2.75 g 820. Add chemicals to an 800 mL beaker. Add
192.5 mL of isopropanol and approximately 250 mL H20. Adjust
to pH 6.0 with concentrated acetic acid. Transfer to a 500
mL volumetric and dilute to volume with 820. Mixture will be
stable refrigerated for 2-3 weeks.

e. Prepare chloramine T solution by using 1.75 g of
chloramine T diluted to 25 mL with H20. Prepare fresh daily.
f. To prepare oxidant solution, mix 1 volume chloramine T
with 4 volumes acetate citrate buffer. This solution should

be prepared immediately prior to performing the color

reaction.

41
9. To make dimethylaminobenzaldehyde (DABA, Sigma D-2004),

mix 10 g of DABA with 9.4 mL of 70% perchloric acid. Combine
while beaker is surrounded by ice. Place covered beaker on
stir plate to mix contents.

h. Prepare Ehrlich's Reagent by mixing 15 mL of DABA-HClo4
solution with 80 mL of isopropanol in brown-glass jar.
Prepare fresh daily.

i. Use the following equation to convert ug/mL of
hydroxyproline to mg/g of collagen:

mg/g of collagen = (uglnl) (dilution facton) (constant)
(sample weight) (1000)

The dilution factor is 500 and the constant is 7.25.

G, nnnt Inguged Pnotein Gels (Study 2)
. u t

Gels were produced in triplicate from protein solutions
in stoppered glass tubing of 1.0 cm internal diameter and
11.5 cm length. The protein solutions (low ionic strength
soluble, high ionic strength soluble and insoluble proteins
homogenized with buffer) were in 0.05 M Na phosphate, 0.6 M
NaCl and pH 7.0 buffer. For least concentration endpoint,
HIS protein solutions (2% to 6% w/w) were used.
Multicomponent gels (6% protein w/w) were manufactured using
HIS proteins plus LIS proteins or HIS proteins plus IN
proteins. LIS proteins or IN proteins were substituted for
HIS proteins in the composite gels at the following '
percentages (8.33, 16.67, 25.00, 33.33 and 50.00). The HIS

plus LIS protein fractions or HIS plus insoluble protein

42
fractions were combined by blending with a Polytron

homogenizer (PTA 20TS, Brinkman Instruments, Inc., Westbury,
NY.) for 30 sec on speed setting 6. The protein solutions
were pipeted into the stoppered glass tubing. The glass
tubing containing the protein solutions were centrifuged at
750 x g for 5 min (IEC Model R, Damon/IEC Division, Needham
Hts., MA.) to remove air bubbles. After centrifugation, the
protein solutions were cooked for 10 min at 70° C in an
agitating water bath (Magne Whirl, Blue M Electric Co., Blue
Island, IL.) and immediately cooled in an ice bath. Protein

gels were manufactured in triplicate.

G.g, Gel Evaluation

G. .a. east Co cen tio nd o

The least protein concentration or least concentration
endpoint (LCE) was performed on HIS protein gels in 0.05 M
Na phosphate, 0.6 M NaCl, pH 7.0 as described by Trautman
(1966) with modifications. The LCE value was defined as an
apparent stress at failure of greater than or equal to 4 kPa
(a kiloPascal is equal to rather than a subjective analysis
of the gel remaining intact in an inverted tube.
G.g.b. Ennrnssible noisture

Expressible moisture (EM) was measured as modified from
a high speed centrifugation method of Jaurequi et al.
(1981). Three pieces of Whatman #2 filter paper, 9 cm
diameter were folded into a cone shape and placed in a 50 ml

polycarbonate centrifuge tube. A weighed 1.5 +/- 0.3 g

43
sample of protein gel was placed inside the filter paper and

centrifuged at 755 x g for 5 min in a 2° C refrigerated
centrifuge. The weight of the released fluid and weight gain
of the filter papers were recorded. Expressible moisture of
the protein gels was calculated by dividing the weight of
the released fluid plus weight gain of the filter papers by
the weight of the original weighed gel sample. All gel
expressible moistures were determined in triplicate.
G.2. . A a t S a St ess and St a at a

Apparent shear stress and strain at failure of the
heat-induced HIS and multicomponent gels were measured using
an Instron (Model 4202, Canton, MA.) at crosshead speed of
10 mm/min with a 50 N compression cell. The gel cores were
compressed between two lubricated (silicone spray lubricant)
parallel plates. The cores compressed were 1.0 cm in
diameter and 1.0 cm in length which were cut using a razor
blade and template. Cores were equilibrated to 1° C prior to
testing. Six to nine cores were evaluated per replicate.
Strain was measured from the compression peaks measuring the
millimeters (x-direction on recorder paper) the crosshead
travels before the sample fractures divided by the original
sample length. Apparent strain at failure is calculated
using the following equation:

as = '1“ l1 ' dz)
on = apparent strain at failure

ln = natural log

52 strain calculated from recorder paper

44
Stress is measured by determining the height in Newtons

force (N) (y-direction on recorder paper) the crosshead
achieves before the sample fractures. Apparent stress is
calculate using the following equation:

I F

”Pm“ ' W

F = force (Newtons) calculated from recorder paper
B = P1

R2 = radius of sample (meters) squared

O = 0.48 (a constant)

dz = strain calculated from recorder paper

G.2.d. Scanning Electron Micnoscony

Small (5 mm diameter x 2 mm thick) samples of the heat
induced gels (6% HIS only and multicomponent gels with
33.33% substitution of LIS or IN fractions) were cut with a
razor blade and fixed for 6 hr in 4% glutaraldehyde
solution. After fixation, the samples were dehydrated
gradually using an ethanol series (25%, 50%, 75%, 95% and
100%) for 15 min/step. The 100% ethanol step was performed
three times. Following critical point drying with C02
(Balzer FL 9496 Critical Point Dryer, Balzer's Union,
Furstentum, Liechtenstein, West Germany), the fixed and
dried samples were mounted using adhesive tabs (M.E. Taylor
Engineering Inc., Brookeville, MD.) to aluminum mounts
(Electron Microscopy Sciences, Ft. Washington PA.). The

mounted samples were sputter coated with gold (Emscope SC

45
500, Emscope, Ashord, Rent, England) for 3 min at 20 mA.

Examination of the samples at 10,000 x magnification was
done in a JEOL JSM-35 scanning electron microscope (JEOL

Ltd., Tokyo, Japan) using an accelerating voltage of 15 kV.

 

ELl&§&__§§gg!_l

Formulations were prepared using a 50:50 blend (based

on the protein content) of MDCM and meat by-product.
Formulations were standardized to 56% moisture, 30% fat, 12%
protein and 2% salt by the addition of pork backfat or ice.
§.l.b.§tudy 3

Meat batters were formulated to 56% moisture, 30% fat,
12% protein and 2% salt. Meat batters were formulated based
on percentages of protein fractions [low ionic strength
soluble (LIS), high ionic strength soluble (HIS), insoluble
(IN)] contained in the meat and meat by-products based on
Nuckles et al. (1990). During phase 1 the meat batters were
formulated to vary the percentage of HIS proteins while
maintaining the percentage of LIS or IN proteins. During
phase 2 the meat batters were formulated to maintain a
constant percentage of HIS proteins while varying the
percentages of LIS or IN proteins.
H32, gnoduction

Meat batters were prepared in triplicate using a silent
cutter placed in a cold room. Lean meats (MDCM or skeletal

muscle) and by-products, salt and ice were added to the

46
silent cutter bowl and cut for 3 min. The fat (pork backfat

or beef) was added to the mixture and cut for an additional
2 min. The final batter temperatures were not greater than
2° C. The batter was stuffed into 50 mL conical centrifuge
tubes, capped and heated in a 75° C water bath to an
internal temperature of 72° C.
8.3. Cooked Meat Batter Evalu t on
H.§,a, Cook Yield

Cook yield of the model system frankfurters was
calculated by dividing the drained, cooked frankfurter
weight (drained of cook-out fat and moisture) by the
original batter weight.
n.3.b. devene Rehent Yield

Severe reheat yield was calculated after heating a 20 g
sample in 100 mL of water at 95° C for 10 min and cooling at
room temperature for 5 min. Reheat yield equals the
reheated, cooled frankfurter weight divided by the original,
frankfurter sample weight.
n.3.c. Apparent Shear Stress nnd Strain at Failure

Apparent shear stress and strain at failure of the
cooked meat batters were measured and calculated as
described for heat-induced gels (G.2.c.) except that the

cores compressed were 1.5 cm in diameter and 1.5 cm in

length.
H.3.d, npparent Tensile Sinengtn (Study 3)

Apparent tensile strength was measured on the whole

frankfurters using a TR-5 Texturepress with a ST Thin Slice

47
Tensile Test Cell (50 pound cell) attachment and

Texturerecorder (Food Technology Corp., Rockville, MD) at a
speed setting of 4 and x1/10 range setting. Frankfurter
samples (1° C) were weighed prior to apparent tensile
strength testing and loaded on the cell perpendicular to the
cell break-line. The force (N/g) to tear the frankfurters
was calculated by reading the chart peak height and
multiplying the result by 0.01 thus converting the chart
reading to percentage. Multiplying the percentage by 50
(conversion for load cell pounds size) and 0.1 (conversion
for the 81/10 setting) converts percentage to pounds force.
Multiplying the pounds of force by 4.4482 converts pounds to
Newtons (N) of force. Dividing the N of force by the
frankfurter weight determines the N force per gram of
frankfurter (N/q) to tear the frankfurter.

V - - ct P at ' Co o t
W
A. t uct

Meat by-products are commonly classified as edible or
inedible (Oliveros et al., 1982). As a general definition,
by-products are "everything of economic value, except for
the carcass, that are incidental to the slaughter of animals
(8ooren and Weiss, 1988)." Meat by-products are
underutilized and low priced because they are regarded as an
inferior protein source compared to skeletal muscle meat
(Oliveros et al., 1982). The utilization of meat by-
products has been avoided due to undesirable sensory
quality, low biological value of the proteins and high
microbial contamination (Rosiba, 1983). Mishandling, poor
sanitation and slow chilling decrease quality and increase
the microbial load of meat by-products (8ooren and Weiss,
1988). The increasing price of meat and processed meat
products is causing the food industry to evaluate the
utilization of all protein sources, including by-products
(Gorska et al., 1988).

Restructuring techniques, such as formed meat chunk
technology, allows meat by-products to be utilized in value-
added products. Formed meat chunk technology is used to
restructure meats that are difficult to incorporate into
other further-processed products. Meat chunks are formed.
using non-meat binders. The process is used commercially in

Canada for human foods and in the United States for premium

48

49
pet foods. Formed meat chunk technology for human

consumption is limited in the United States due to consumer
perceptions of undesirable sensory quality. Understanding
the composition and function of by-product proteins may
allow for the replacement of non-meat binders with
combinations of by-products to achieve the desired bind,
texture and mouthfeel.

Further-processed meats utilize proteins as the
principal functional and structural components. The proteins
determine the characteristic texture, water-holding and
appearance of these products (Hermansson, 1985). Protein
gelation, fat binding and water-holding are the most
important functional properties in further-processed
products. Cooked product stability is currently not thought
to rely upon protein emulsification (Regenstein, 1988). The
individual product, processing method and stage of
processing will determine the relative importance of each
functional property. As the variety of meat products
increases, the need to understand, modify and control
protein functionality becomes more important (Smith, 1988).

Proteins are denatured and reaggregate into a cross-
linked three dimensional gel matrix during cooking
(Foegeding, 1988). Fat and water are physically or
chemically trapped inside this matrix. The texture and cook
yield of restructured products are directly related to this
matrix formation. The characteristics of the gel formed are

a function of the type and amount of proteins present and

50
are influenced by environmental (e.g., pH, salt) and

processing factors during heating (Whiting, 1988).

Meet by-products exhibit large differences in protein
content and distribution and consequently vary widely in
protein functionality and bind values. Bind values are based
on the emulsion capacity of the soluble protein fraction.
The type and amount of meat by-products in a formulation
influence the texture, fat emulsifying capacity and water-
holding properties of the finished products. Each by-product
contributes different, unique textural attributes to the
finished product. Information on the functional and
physicochemical properties of by-product proteins is
extremely limited. The protein content and composition of
some beef by-products has been reported (Schaefer and
Schierhorn 1974: Oliveros et al., 1982: Gorska et al.,
1988), but the salt soluble protein and stroma protein
content of by-products from other species are not known.

The objective of this study was to develop a knowledge base
of selected meat by-product composition and function for use
in restructured meat products.
8. e a nd Met od
B.1. Ma e a S

Frozen mechanically deboned chicken meat (MDCM)
prepared from the whole carcass was purchased from Nottawa
Gardens Corp., Athens, MI. By-products (pork lung lobes,
pork liver, beef lung lobes, beef spleen, beef heart)

obtained from 15 market weight hogs or 15 market weight

51
steers slaughtered on three different days at the Michigan

State University Meat Laboratory were used in this study.
Five animals were slaughtered each day and like by-products
combined and designated as one replicate. By-products were
removed within 1 hr after exsanguination. By-products were
ground sequentially through the 10, 6, and 3 mm plate of a
Hobart grinder (Model A-200, Troy, OH), vacuum packaged (2.5
mil polyethylene) and frozen until used within three months.
Proximate composition (fat, protein and moisture) were
determined by AOAC (1984) procedures. The pH was determined
after blending 109 of MDCM or by-product with 100g of double
distilled water on setting 3 for 1 min in a Sorvall Omni-
Mixer (Model 17105, Newtown, CT) and was measured using a
Corning 145 pH meter. All experiments were performed in
triplicate.
B. . et ods
n.g.n. Timed Emulsigicatinn

Timed emulsification was performed as described by
Galluzzo and Regenstein (1978) with modifications because a
cream layer was not formed using the recommended protein
concentration of 5 mg/ml. MDCM and by-products were
evaluated in triplicate at total protein concentrations of
10 to 150 mg/ml. The lowest concentration of protein
required to produce a cream layer after centrifugation at

30,000 x g for 15 min was recorded.

52
. . u t of P ote n o

All extraction procedures were performed in a 4° C cold
room. A weighed portion of MDCM or by-product was blended
(Waring Blendor, Model 1120, Winsted, CT) for 1 min with 4
volumes of 0.05M Na phosphate buffer, pH 7.4, stirred for 3
hr (Talboy T-Line Laboratory Stirrer, Model 107, Emerson,
NJ) in a cold room and centrifuged at 23,000 x g for 15 min.
The supernatant was saved and the residue re-extracted for 1
hr. Following centrifugation, the supernatants containing
the 0.05M Na phosphate soluble proteins were combined,
designated as low ionic strength soluble (LIS) proteins and
quantitated. The precipitate was mixed with 4 volumes of
0.6M NaCl, 0.05M Na phosphate buffer, pH 7.4 and extracted
twice as described previously. The supernatants, designated
as high ionic strength soluble (HIS) proteins, were combined
and quantitated. The precipitate was designated insoluble
proteins and weighed. The protein content of each fraction
was determined by Rjeldahl procedures (AOAC, 1984). Non-
protein nitrogen content was measured on the low ionic
strength soluble proteins according to Helander (1957).
Non-protein nitrogen content was subtracted from the low
ionic strength soluble nitrogen before calculating protein
(N x 6.25).

The protein fraction percentages were calculated by
dividing the weight of total protein in each fraction by the
weight of total protein in the meat. Percentage protein in a

weighed portion of each protein fraction multiplied by the

53
total fraction weight equaled the total protein in each

fraction.
. o e at o

Collagen was determined in triplicate on each by-
product, MDCM, the HIS protein fraction and the insoluble
protein fraction according to Bergman and Loxley (1963).
Prior to collagen determination, MDCM, by-products and
insoluble proteins were frozen with dry ice and ground in a
IRE-Universal mill M20 (Jankel and Runkel Co., West
Germany).
B. . ect o hores

Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) of the HIS and insoluble protein
fractions was performed using 12% polyacrylamide gels
according to Smith and Brekke (1985). The insoluble protein
fractions were solubilized prior to electrophoresis
according to Wu et al. (1982). One hundred micrograms of the
HIS protein fraction and 200 ug of the insoluble protein
extract were loaded on separate gels. Protein bands were
stained with Coomassie Blue (Sigma Chemical Co., St. Louis,
MO). Myosin, actin and other protein bands were identified
by comparing relative retention times to molecular weight
standards (MW-SDS-200, Sigma Chemical Co., St. Louis, MO).
Myosin:actin molar ratios of MDCM and by-product protein
fractions were calculated according to Beas et al. (1988).
The protein bands were quantitated by densitometric scans at

580 nm (Schimadzu 08-930, Ryoto, Japan).

54
B O O O Od‘ 8 t

Batters were prepared in triplicate using a silent
cutter as described by Smith and Brekke (1984), except the
batter was stuffed into 50 mL conical centrifuge tubes,
capped and heated in a 75° C water bath to an internal
temperature of 72° C. Formulations were prepared using a
50:50 blend (based on the protein content) of MDCM and meat
by-product. Formulations were standardized to 56% moisture,
30% fat, 12% protein and 2% salt by the addition of pork
backfat or ice.

Cook yield of the model system frankfurters was
calculated by dividing the drained, cooked frankfurter
weight (drained of cook-out fat and moisture) by the
original batter weight. Reheat yield was calculated after
heating a 20g sample in 100 mL of water at 95° C for 10 min
and cooling at room temperature for 5 min. Reheat yield
equals the reheated, cooled frankfurter weight divided by
the original, cooked frankfurter weight.

Apparent shear stress and strain at failure (Diehl et
al., 1978) of model system frankfurters cores were measured
using an Instron (Model 4202, Canton, MA) at a crosshead
speed of 10 mm/min with a 50 N compression cell. The cores
were compressed between two parallel plates. A 1.5 cm
diameter central core was removed along the longitudinal_
axis of the model system frankfurters using a cork borer and

cores were cut to 1.5 cm lengths using a razor blade and

55
template. Cores were equilibrated to 1°C prior to testing.

Six cores were evaluated per replicate.
C. fitntinnign

The statistical design was completely randomized.
Basic statistics, analysis of variance (ANOVA) and
correlations were performed using M-STAT (1988). Two way
ANOVA was performed to test significance within replications
and between treatments. Mean separations (significance) were.
tested using Tukey's test.
D. Results and Discussion

The proximate composition of MDCM and by-products is
shown in Table 4.

Table 4. Proximate composition and collagen content of meat
by-products.

Collagen Collagen
Moisture Fat Protein (% of total (mg/g

 

Sample (%) (95) (%) protein) sample)
Mechanically 65.6cd 14.2” 17.4” 3.9° 6.s°
Deboned

Chicken Meat

Beef Heart 65.5Cd 17.5“ 15.4c 5.5” 8.5”
(cap on)

Beef Lung 79.7“” 1.9d 17.7” 5.5” 9.7“
(lobes only)

Beef spleen 79.9“” 3.s° 15.3° 2.3d 3.5d
Pork Liver 71.7” 1.8“ 22.1“ 1.5a 3.4‘1
Pork Lung 82.5“ 2.0d 15.5° 6.1“ 9.4“<

(lobes only)

sag 1.; iio 0.4 0.3 0.4
I I I I

Means within columns followed by the same letter
do not differ significantly (P< 0.05).

 

56

MDCM was used in this study because it has been
extensively studied, only a small amount of mechanically
separated red meats are produced in the U.S. and much of the
production is used in pet foods (Field, 1988). Pork and beef
lungs, beef spleen and pork liver generally contained higher
moisture and lower fat than beef heart and MDCM. Pork liver
contained significantly more (P< 0.05) total protein than
the other by-products. Beef heart, beef spleen and pork
lungs contained the lowest (P< 0.05) protein content. The
proximate composition of beef heart (with cap) and pork
liver are consistent with values published by Porteous
(1979) and Wiley et al. (1979). The percentages of fat and
moisture in beef heart published by Oliveros et al. (1982)
are significantly lower because our analysis included the
heart cap. Non-protein nitrogen (NPN) content of MDCM and
by-products were not significantly different and contained
an average of 2.38% NPN. Collagen content (mg/g meat) was
significantly higher (P< 0.05) in beef heart and pork lung
compared to MDCM and other by-products. The beef heart and
liver collagen contents were comparable to the published
values of Porteous (1981). Rhalili and Zarkadas (1988)
reported 1.96 to 3.08% collagen in chicken breast and 5.63
to 6.87% in leg muscles.

The protein distribution varied among the by-products

examined (Table 5). Mechanically deboned chicken meat

57

 

 

Table 5. Protein fractions as a percentage of total protein
and collagen content of the low ionic strength
soluble (LIS), high ionic strength soluble (HIS)
and insoluble protein fractions of by-products.
Collagen Collagen

in in
LIS HIS HIS Insoluble
Proteins Proteins Fraction Insoluble Fraction

Sample (%) (%) (%) Proteins (%)

Mechanic- 23.6“ 40.4“ 1.9“ 31.0“ 12.0”

ally Deboned

Chicken Meat

Beef Heart 30.1“ 21.2” 5.1“ 43.7” 10.1“

(cap on)

Beef Lung 36.3“ 9.6“ 3.3“ 54.1“ 10.1“

(lobes only)

Beef 53.6” 21.7” 3.1“ 24.7“ 3.0“

Spleen

Pork Liver 76.1“ 14.3“ 3.3“ 9.1“ 13.2“

Pork Lung 51.6” 10.2“ 4.3” 33.2“ 13.3“

(lobes only)

SEN 2.7 1.6 0.2 2.4 0.3

a-b—c—d—e ‘

' ' ' ' " Means within columns followed by the same letter

do not differ significantly (P< 0.05).

58
contained nearly twice as much HIS protein as meat by-

products. The HIS percentages for beef heart, beef lung and
beef spleen are lower than percentages reported by Oliveros
et al. (1982). The differences may be explained by different
extraction times (7 days versus 4 hr). Beef heart and beef
lung contained the highest percentage of insoluble proteins,
while pork liver contained the highest quantity of LIS
proteins. Beef heart contained the highest percentage of
collagen in the HIS fraction. The sum of the collagen in the
HIS fraction and insoluble protein fractions is consistent
with the amount of total collagen in the samples. Soluble
collagen was measured on the HIS proteins because large
amounts of collagen within this fraction may adversely
affect functionality.

A representative SDS-PAGE eletrophoretogram of the by-
product HIS proteins is shown in Fig. 3. Myosin or actin
were not present on electrophoretograms of the insoluble
proteins. MDCM and beef heart had significantly higher (P<
0.05) percentages of myosin and actin and a lower percentage
of other proteins compared to the other by-products (Table

6).

.283... 88 n s .55: canon u m .69 u m .mcumccfim 3.383
38830.. u n. .83 ton u m .93 when u m 3.82 .88 n o 288.8
6338 Baum €93 mo gaggogo mflmmrmom gflfifimonmmm .m 0.563

‘
3.2.0
29.
53>:

 

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. .. c=o<
ooo me-
ooodo. .

v. . Euro
33.3 3....
Sodom. l . . . Eng:

5925 £221

.a.:oflos.

60

Table 6. The percentage of myosin and actin and the myosin/
actin ratio of the by-product high ionic strength
soluble protein fraction.

 

 

Myosin Actin Actin:myosin
£529.43 (’5) (’61 521.213.9122
Mechanically 50.3“ 22.3“ 5.0
Deboned Chicken Meat
Beef Heart 47.9” 20.6” 5.0
(cap on)
Beef Lung 37.6“ 10.5“ 3.2
(lobes only)
Beef Spleen 22.6“ 10.1“ 4.5
Pork Liver 24.4“ 9.2“ 4.3
Pork Lung 33.6“ 10.6“ 3.0
(lobes only)
SEN 2.7 1.3

 

“'”'°'° Means within columns followed by the same letter do

not differ significantly (P< 0.05).

MDCM and beef heart contained twice as much myosin and
actin compared to beef spleen and pork liver. The
significant differences in the percentages of myosin, actin
and other proteins may be related to differences between
skeletal, cardiac and smooth muscle types. Myosin and actin
are present in cardiac and smooth muscle and are extractable
at high ionic strength (Garrels and Gibson, 1976: Cross at
al., 1988). MDCM and beef heart had lower myosin/actin molar
ratios (1.0:s.0, Table 3) when compared to the other by-
products (1.0:3.0 to l.0:4.5). Potter (1974) and Murakami.
and Uchida (1985) reported that the molar ratio of myosin to

actin in skeletal muscle was 1.0:6.0. The myosin:actin ratio

61
in rabbit skeletal muscle was 1:6.6 based on amino acid

analysis rather than densitometry (Yates and Greaser, 1983).
Murakami and Uchida (1985) also reported a molar ratio of
myosin to actin of 1.0:4.1 for cardiac muscle and 1.0:16.5
for smooth muscle (chicken gizzard). Yasui and Ishioroshi
(1980) found that a weight ratio myosin:actin of 15 formed
more rigid gels. Asghar et al. (1985) stated that the
myosin:actin ratio rather than the actomyosin:actin ratio
may be related to gel strength.

The timed emulsification procedure of Galluzzo and
Regenstein (1978), developed to study the emulsifying
properties of skeletal meat proteins was modified to
evaluate meat by-products. The minimum protein concentration
necessary to produce a cream layer after centrifugation was
used as an index of protein functionality among the by-
products. MCDM, beef spleen and pork liver formed cream
layers (10, 20 and 40 mg protein/ml, respectively) but beef
heart, beef lung and pork lung did not form cream layers at
protein concentrations up to 150 mg/ml. Oliveros et al.
(1982) reported that beef spleen emulsified more oil than
beef meat or beef lung.

Cooked yield, reheat yield, apparent shear stress and
apparent strain at failure of model system frankfurters (pH
6.3) were influenced by meat by-product protein composition

(Table 7).

62

 

 

Table 7. Yield and textural characteristics of model system
frankfurters prepared from mechanically deboned
chicken meat and meat by-products (1:1 based on
total protein content)

Apparent
Cook Reheat stress at Apparent
Yield Yield failure strain at

Sample (%) (%) (RPa) failure

Mechanically 91.3“ 32.6” 42.30” 0.76”

Deboned Chicken

Meat

Beef Heart 92.5“ 30.1“ 42.22“ 0.76”

(cap on)

Beef Lung 93.0” 73.6“ 20.35“ 0.69“

(lobes only)

Beef Spleen 93.7” 73.6“ 20.35“ 0.69“

Pork Liver 91.2“ 74.4“ 10.549 0.56“

Pork Lung 92.3“ 69.3” 17.37“ 0.69“

(lobes only)

0.3 0.7 O.§9 0.01

 

 

fig!

atter formulation:

b,c,d,e,f

2% salt.

12% protein, 30% fat, 56% moisture and

'9 Means within columns followed by the same

letter do not differ significantly (P< 0.05).

63

Apparent strain is highly correlated to sensory and texture
profile analysis (TPA) cohesiveness while apparent stress at
failure is correlated to TPA hardness and sensory firmness
(Montejano et al., 1985). Model system frankfurter
percentage cooked yield ranged from 91.2 to 93.7% while
reheat yield ranged from 69.8 to 82.6%. The model system
frankfurters produced with by-products had significantly
lower (P< 0.05) percentage reheat yield and compressive
force at failure compared to MDCM frankfurters. Among the
by-product frankfurters, beef heart had the highest reheat
yield and pork lung exhibited the lowest reheat yield. The
pH values of the frankfurter batters were not significantly
different.

Changes in the modified timed emulsification test were
positively correlated with the quantity of by-product
insoluble proteins and the percentage collagen contained
within the HIS proteins and negatively correlated with the
quantity of HIS proteins (as HIS proteins increased, less
protein was needed to form a stable cream layer, Table 8).
The quantity of protein required to form a stable layer in
the modified timed emulsification test was not highly
correlated with the percentage of by-products LIS proteins,
myosin or actin within the HIS proteins, collagen in the
insoluble proteins or any parameters measured in the model

system frankfurter test.

64
Reheat yield was positively correlated with the

quantity of HIS proteins and the percentage of myosin and
actin within the HIS proteins fractions (Table 5). Reheat
yield was inversely correlated with LIS proteins. Apparent
shear stress and strain at failure of the model system
frankfurters were negatively correlated with LIS proteins
and positively correlated with HIS proteins and the
percentage of myosin and actin within the HIS fractions.

The HIS proteins are generally considered to impart the
most functionality to processed meats. In this study, larger
quantities of HIS proteins were related to larger reheat
yields and better textural properties in the model system
frankfurters. The quantity of insoluble proteins in the meat
by-products was not strongly correlated with the parameters
measured in the frankfurters. Timed emulsification was not
an adequate tool to evaluate by-products.

These results indicate that the protein distribution of
meat by-products was related to the yield and texture
imparted to comminuted meat products. Results suggest it is
possible that a certain percentage of HIS proteins or ratio
of protein fractions are necessary to produce the desired

bind in frankfurter formulations.

65

Table 8. Correlation coefficients between meat by-products
protein composition and model system properties.

 

 

Modified Apparent Apparent
Timed Reheat stress at strain at
MW
1018 -e31 -e71 -e85 -e82
Proteins
HIS -e71 -e78 -e80 -e61
Proteins
Myosin .36 .64 .93 .98
in HIS
fraction
HIS fraction
HIS fraction
Insoluble .79 .16 .14 .30
Proteins
Proteins
% Collagen
Apparent -.09 .63 .83
strain at
failure
Apparent -.35 .84
stress at
failure

Reheat Yield -.41

V. ud : 0 es o ea induced e s e a ed om

beef skeleta card ac and smooth muscl

212W

A. ENTRODUCIlON

Protein gelation is one of the most important
functional properties in processed meat products. Gelation
of muscle proteins, a transformation from viscous sols to
elastic gels, contributes to desirable texture and the
stabilization of fat and water in processed meat products.
Ferry (1948) defined heat-induced protein gelation as a two
step process where partial unfolding of protein domains is
followed by reaggregation into a crosslinked, three
dimensional network of protein fibers. The simplest type of
protein gel is one formed from a single type of polypeptide
(Foegeding, 1988).

Salt soluble proteins (myofibrillar) contribute the most
functionality to gels (Rnight, 1988). The contribution of
water soluble proteins (sarcoplasmic) and/or insoluble
proteins (stroma and some myofibrillar) to processed meat
products is not well understood (Rnight, 1988). The gel
properties of myosin and fibrinogen or albumin have been
studied (Foegeding, 1983). Myosin and fibrinogen interacted
to form a gel at 55° C and 70° C that was stronger than gels
made from the individual proteins. Myosin and albumin did
not form a gel until 80° C and formed a weaker gel than the

myosin-fibrinogen gel. Mixtures of salt soluble proteins

66

67
plus sarcoplasmic or insoluble proteins have not been

studied. Information from such studies should help explain
the effects of protein interactions on product textures, fat
and water-holding and help meat processors select meats to
achieve desired product attributes. Rnight (1988) reported
that three protein fractions (salt soluble, insoluble
myofibrillar and connective tissues) from pork shoulder meat
had different functional properties. He suggested that
manufacturers could use information on individual protein
fractions to improve or create new products.

Venegas et al. (1988) studied whole offal homogenates
(cattle lungs, spleen, kidneys, testicles and pig stomach)
and beef skeletal muscle for water holding capacity (WHC),
emulsion stability and gel strength. Their results showed
that skeletal muscle exhibited significantly higher gel
strength, but the offal showed better WHC probably due to
its higher pH. Among the offal, lungs showed the best
overall functionality, while spleen had good WHC but low gel
strength.

The gel-forming abilities of proteins from various
sources can be compared. Tests include 1) least
concentration endpoint (LCE) (Trautman, 1966) to compare the
minimum protein concentration required to form a gel that
remains intact within an inverted tube, 2) expressible
moisture (EM) (Jaurequi et al.,1981) to measure the water-
holding capacity of the gels, 3) apparent shear stress and

strain (Diehl et al., 1979) to ascertain gel strength and 4)

68
scanning electron microscopy (SEM) to visually compare gel

attributes. The objectives of this research were to evaluate
gels produced from high ionic strength soluble (HIS)
proteins and multicomponent gels produced from
combinations of H18 and low ionic strength soluble (LIS) or
insoluble (IN) proteins from beef skeletal, cardiac and
smooth muscles.
. METRO S

Bala__§AI£BIAL§

Beef semitendinosus muscle (beef skeletal muscle) and
beef by-products (lung lobes, spleen and heart) were
obtained from 9 market weight steers slaughtered on three
different days at the Michigan State University Meat
Laboratory. Three steers were slaughtered each day and like
by-products combined and designated as one replicate. By-
products were removed within 1 hr after exsanguination,
vacuum packaged in 500 g aliquots (2.5 ml polyethylene) and
frozen until used within three months.
B ODS
B a at o o ro act'o s

Three protein fractions were extracted in triplicate as
described by Nuckles et al. (1990). The fractions were low
ionic strength soluble (LIS) proteins extracted with 0.05 M
Na phosphate buffer, pH 7.4, high ionic strength soluble 4
(HIS) proteins extracted with 0.6 M NaCl, 0.05 M Na
phosphate buffer, pH 7.0 and insoluble (IN) proteins. The

protein extraction procedures were modified so the first

69
extraction used only 2 volumes (rather than 4 volumes) of

buffer. The modification eliminated additional protein
concentration steps when producing the gels. Prior to gel
production, the LIS proteins were dialyzed for 24 hr (two
buffer changes) with 0.05 M Na phosphate, 0.6 M NaCl, pH 7.0
buffer. The nitrogen content of the LIS and IN proteins was
measured and percentage protein calculated (AOAC, 1984).
Non-protein nitrogen (NPN) was determined according to
Helander (1957) and subtracted from the LIS nitrogen before
calculating protein. The LIS, HIS and IN proteins were
adjusted to 6% (w/w) protein with 0.05M Na phosphate, 0.6 M
NaCl, pH 7.0 buffer.
8. Ge actu n

Gels were produced in triplicate from protein solutions
in 0.05M Na phosphate, 0.6M NaCl, pH 7.0 buffer by pipeting
into stoppered glass tubing of 1.0 on internal diameter and
11.5 cm length. For least concentration endpoint, HIS
protein solutions ranging from 2% to 6% (w/w) were prepared.
Multicomponent gels (6% protein w/w) were manufactured using
HIS proteins plus LIS proteins or IN proteins. Low ionic
strength soluble proteins or IN proteins were substituted
for HIS proteins in the multicomponent gels at the following
percentages (8.33, 16.67, 25.00, 33.33, 41.67 and 50.00).
Fractions were combined by blending with a Polytron
homogenizer (PTA 20TS, Brinkman Instruments, Inc., Westbury,’
NY.) for 30 sec on speed setting 6. Protein solutions were

centrifuged at 750 x g for 5 min (IEC Model R, Damon/IEC

70
Division, Needham Hts., MA.) to remove air bubbles. After

centrifugation, the protein solutions were heated for 10 min
at 70° C in an agitating water bath (Magne Whirl, Blue M
Electric Company, Blue Island, IL.) and immediately cooled
in an ice bath. Three separate beatings of each treatment
were performed.
8.2.c. Lea t o cent at d o

The least protein concentration or least concentration
endpoint (LCE) was performed on HIS protein gels in 0.05 M
Na phosphate, 0.6 M NaCl, pH 7.0 as described by Trautman
(1966) with modifications. The LCE value was defined in this
study as an apparent stress at failure of greater than or
equal to 4 kPa rather than a subjective analysis of the gel
remaining intact in an inverted tube.

. . . Ge a uat

Expressible moisture (EM) was measured as modified from
a high speed centrifugation method of Jauregui et al.
(1981). Three pieces of Whatman #2 filter paper, 9 cm
diameter were folded into a cone shape and placed in a 50 ml
polycarbonate centrifuge tube. A weighed 1.5 +/- 0.3 g
sample of protein gel was placed inside the filter paper and
centrifuged at 755 x g for 5 min at 2° C. The weight of the
released fluid and weight gain of the filter papers were
recorded. Expressible moisture of the protein gels was
calculated by dividing the weight of the released fluid plus
weight gain of the filter papers by the weight of the

original weighed gel sample. Expressible moistures were

71
determined in triplicate.

Apparent shear stress and strain at failure (Hamann,
1983) of the heat-induced HIS and multicomponent gels were
measured using an Instron (Model 4202, Canton, MA.) at
crosshead speed of 10 mm/min with a 50 N compression cell.
The gel cores were compressed between two lubricated
(silicone spray lubricant) parallel plates. The cores were
1.0 cm diameter and 1.0 cm length cut using a razor blade
and template. Cores were equilibrated to 1° C prior to
testing. Six to nine cores were evaluated per replicate.
Gels with an apparent stress at failure of < 4 kPa are not
presented because they were too weak for compression
testing.

8. .e. Sc n ect o M o o

Small (5 mm diameter x 2 mm thick) samples of the heat-
induced gels (6% HIS only and multicomponent gels with
33.33% substitution of LIS or IN fractions) were fixed for 6
hr in 4% glutaraldehyde solution. After fixation, the
samples were dehydrated in a graded ethanol series (25%,
50%, 75%, 95%, and 100%) for 15 min/step. The 100% ethanol
dehydration step was performed three times on the fixed
samples. Following critical point drying with C02 (Balzer FL
9496 Critical Point Dryer, Balzer's Union, Furstentum,
Liechtenstein, Germany), the samples were mounted using
adhesive tabs (M.E. Taylor Engineering, Inc., Brookeville,
MD.) to aluminum stubs (Electron Microscopy Sciences, Ft.

Washington, PA.). The mounted samples were sputter coated

72

with gold (Emscope SC 500, Emscope, Ashord, Rent, England)

for 3 min at 20 mA. Samples were

magnification using a JEOL JSM-35

examined at 10,000 x

scanning electron

microscope (JEOL Ltd., Tokyo, Japan) at an accelerating

voltage of 15 kV.
C. C

The statistical design was a

2 x 2 factorial to study

the influence of protein source and type on functional

tests. Basic statistics and analysis of variance (ANOVA)

were performed to test significance within replications and

between treatments. Mean separations (significance) were

tested using Tukey's test (MSTATC, 1989).

D. U US 0

D . H otein G ro t s
The minimum concentrations of HIS
heat-induced gel with an apparent
greater than or equal to 4 kPa in
heart, lung and spleen were 3, 4,
Twice the beef spleen HIS protein

to form a stable (apparent stress

protein required to form a
stress at failure of

beef skeletal muscle,

5 and 6%, respectively.
concentration was required

at failure greater than or

equal to 4 kPa) gel compared to beef semitendinosus muscle

(skeletal) HIS proteins. Spleen contains a higher ratio of

actin to myosin compared to skeletal muscle (Nuckles et al.,

1990) and may explain the lower gel strength of spleen HIS

proteins. Schmidt et al. (1981) reported that myosin is the

protein mainly responsible for gelation. Yasui et al. (1980,

1982) reported that F-actin enhanced myosin gelation, but F-

73
actin alone has poor gelation capabilities. Venegas et al.

(1988) reported that cooked homogenates of cattle spleen or
cattle lungs both exhibited high water-holding capacity, but
the gel strength of the spleen homogenate was low. The HIS
proteins from skeletal, cardiac and smooth muscles do not
exhibit equivalent functional properties. Further studies
relating the HIS protein fraction composition to
functionality are required.

The HIS protein gels produced from beef skeletal muscle
expressed approximately 15% less moisture than beef spleen
HIS gels (Fig. 4). The order of expressed moisture of the
HIS gels from most to least was spleen > lung > heart >
skeletal muscle. Less expressed moisture may be related to a
higher amount of myosin in the HIS protein fraction
(Nuckles, 1990), as higher amounts of myosin may enhance the
production of a gel network that is better able to
chemically and/or physically entrap moisture (Schmidt et
al., 1981).

Apparent stress at failure of the HIS protein gels
showed that skeletal muscle proteins produced significantly
harder gels than the by-product proteins (Fig 5). Apparent
stress at failure has been correlated to texture profile
analysis (TPA) hardness and sensory firmness (Montejano et

al., 1985). The HIS gels produced from skeletal muscle

74

Figure 4. Expressible moisture of high ionic plus low ionic

strength soluble protein gels.

g 8:53:38

00n00 N0“ 3 nnhnn 00t.wm honor find 00.0

 

 

75

\. coo-am comm I

0:3 comm Ill
toe: comm ele
0.3:: .36-8% comm ale

00.0

1000..

 

00.0w

00.0n

100.3.

 

100.00

(%) emisgow elqgsseudxgg

76

Figure 5. Apparent stress at failure of high ionic strength

plus low ionic strength soluble protein gels.

E 8:35.83
oonom hob; nnhnn comma homo. nnd oo.o

 

 

 

 

 

 

 

77

00.0

 

Iced.
cooam comm I

95... .Sam I

too... teem I

0.00:: .3295 .Sam ale

I00.NF

 

00.... P

(Odd) 9Jn|!D_-j 10 scans iueloddv

78
exhibited nearly twice the apparent stress at failure

compared to HIS gels from spleen and lung. The weaker gels
from spleen and lung may be related to a lower percentage of
myosin in those HIS protein fractions (Nuckles et al.,
1990). Heart does not contain a significantly lower
percentage of myosin in the HIS protein fraction (Nuckles et
al., 1990), therefore further studies need to be performed
to explain the lower apparent stress at failure for the
heart HIS protein gels.

Beef skeletal muscle HIS protein gels had significantly
greater apparent strain at failure (0.78) compared to the
spleen HIS proteins gels (0.57) (Fig.4). Apparent strain at
failure has been highly correlated to sensory and TPA
cohesiveness and deformability (Montejano et al., 1985).
Skeletal muscle HIS protein gels were the most cohesive
followed by heart > lung > spleen.

D.2. Scanning Elecnnon Micrographs og HIS Protein Gels

The HIS protein gels of beef semitendinosus muscle
(Fig 6a) and heart (Fig 6b) appeared fibrous (thick fibers)
and honeycomb-like, whereas the lung HIS protein gel was
globular (Fig 6c) and the spleen HIS protein gel (Fig 6d)
appeared filamentous (thin fibers). The fibrous gels of the
semitendinosus muscle and heart HIS proteins may explain the
increased strength and decreased expressible moisture as.
compared to the lung and spleen HIS protein gel. The fibrous
honeycomb-like network may help entrap or chemically

stabilize the moisture, whereas the globular lung HIS

79

Figure 6. Scanning electron micrographs of high ionic
strength soluble protein gels.
A = skeletal
B = cardiac
C = lung
D

= spleen

 

81
proteins may not provide the honeycomb-like protein network.

The filamentous strands of the spleen HIS protein network

may not provide the needed structural strength to entrap or

chemically retain fat and water.

D o c t t w o St 3 t Mu tico one t
dnln

Multicomponent gels (HIS proteins plus LIS proteins or
HIS proteins plus IN proteins) were prepared with 6% protein
because this was the minimum concentration required for
gelation of spleen proteins.

Data presented in Fig. 4 shows the percentage of
moisture expressed from the HIS plus LIS protein
multicomponent gels. The addition of 8.33% LIS proteins to
HIS proteins reduced EM of the gels. At higher LIS
substitutions the EM was equivalent or significantly higher -
than the HIS gels. Acton and Dick (1976) reported that
sarcoplasmic (LIS) proteins do not gel but rather form a
coagulum that may have deposited on the HIS protein gel
matrix, increasing surface area for moisture/protein
interaction and decreasing expressible moisture. Venegas et
al. (1988) reported low expressible moisture for gels
produced from spleen homogenates due to sarcoplasmic (LIS)
proteins and high pH.

Addition of 8.33% LIS proteins to all HIS/LIS
multicomponent gel treatments caused a significant reduction
in apparent stress at failure of approximately 50% (Fig. 5).

The 8.33% substitution of LIS proteins to the HIS protein

82
gels had an immediate, weakening effect while 16.67 to

50.00% LIS substitution caused more gradual gel weakening.
Generally, as the percentage of LIS proteins
substituted into the HIS protein gels increased, the
apparent strain at failure decreased (Fig. 7). A 16.67% LIS
protein substitution caused a 12.5% reduction of apparent
strain at failure. The higher substituted gels (>8.33%
substitution) became less deformable possibly because the
LIS proteins do not form a gel, but rather a coagulum (Acton
and Dick, 1976) that may interrupt or modify the gelation of

the HIS proteins.

D.1. Spanning Electron Micnograpns of High lonic

StnengthLLow lonic dtnength Protein dels
In the scanning electron micrographs of the HIS/LIS

multicomponent gels (Fig. 8a-d) it appeared that the HIS
proteins formed a gel network (as seen in Fig 6a-d) and the
LIS proteins formed a coagulum that deposited on the surface
and in the interstitial spaces of the HIS protein gel
network. The deposited LIS proteins may have increased the
surface area for hydrophilic interactions between the
proteins and moisture, thus the addition of the 8.33% LIS
proteins decreased expressible moisture. Once the sites for
HIS/LIS protein and LIS protein/moisture interactions were
filled the excess moisture was not held by the gel and
expressible moisture (EM) increased.

The addition of LIS proteins to the HIS proteins may

have created a filled gel. Three types of multicomponent

83

Figure 7. Apparent strain at failure of high ionic strength

plus low ionic strength soluble protein gels.

as 8:35.58

 

 

 

 

 

 

84

 

oodm No. I. nnfin OO.mN 506— find 00.0
r — b _ _ _ .
0v 0
./.ll/’ l/
.000
Iomd

ceoEm teem I

95.. team all

too... .Sam I

0.3:: .3285 comm I

 

100...

aiming lo ugons iueloddv

85

Figure 8. Scanning electron micrographs of high ionic
strength soluble plus low ionic strength soluble
protein gels.

A = skeletal

B = cardiac
C = lung
D

= spleen

 

87
gels have been defined by Tolstoguzov and Braudo (1983):

filled, mixed and complex. Filled gels are formed when the
protein gel network is formed by one macromolecule and other
components of the system fill the interstitial spaces. Mixed
gels are formed when two or more different macromolecules
gel independently and do not interact. Complex gels are
produced when the components of the system interact to form
the gel.

The HIS/LIS proteins appeared not to have formed
mixed or complex gels because the LIS proteins did not gel,
singly or in conjunction with the HIS proteins, but rather
formed a coagulum as described by Acton and Dick (1985).
Morris (1985) described a filled gel as a gel matrix with
particulate inclusions such as gas bubbles, liquid droplets,
ice or fat crystals and/or starch granules. Ma et al.,
(1990) reported on co-gels of egg white and oat globulin.
They found that heating the proteins resulted in the egg
white forming a continuous network and the oat globulin was
distributed randomly throughout the matrix. Foegeding and
Ramsey (1987) increased the lipid content in a gelled meat
batter and claimed that a filled gel was formed because of
an increase in shear stress to failure and no significant
change in strain to failure. Lee and Abdollahi (1981)
reported that the firmer the fat used in gelled meat batter,
the larger the increase in force to fracture. Morris (1985)

reported that "inactive" polysaccharides, as defined as

88
polysaccharides that do not form a gel network, enhance

osmotic swelling of gels and reduce water loss. The LIS/HIS
gels are probably filled gels with LIS proteins as inactive
fillers as no increase apparent stress at failure was
measured, rather added LIS proteins formed a soft coagulum
that reduced apparent stress at failure.
o c S t so ubl ote u t com onent
921;

The substitution of 16.67% or more of IN proteins into
HIS protein gels significantly (P<0.05) increased EM (Fig
9). The addition of 16.67% insoluble proteins increased
expressible moisture approximately 5%, higher amounts of
substituted IN proteins increased EM. The substitution of
<16.67% IN proteins may have not been enough to affect the
HIS protein network and therefore the IN proteins acted as
"inactive" proteins in the HIS gel network and did not
increase expressed moisture.

Poulanne and Ruusunen (1981) added isolated thick
epimysial tissue (IN protein) from young bull skeletal
muscle to comminuted meat products and found that at 60° C
the tissue swelled and bound water but increasing cooking
temperatures decreased the amount of water bound in the
protein matrix. Sadowska et al. (1980) increased the amount
of connective tissue added to an emulsified product by 100%
and 200% (from 5% of total protein to 10% and 15%) and '
reported an increase in cookout loss. Rnight (1988) reported

that the role of connective tissue proteins (CTP) in

89

Figure 9. Expressible moisture of high ionic strength plus

insoluble protein gels.

90

so 8:383...
oopom no“; nfinn 00me Soho. nmd
6.00:: .3286 .60m ele
toe: teem ele

9.3 teem ele
52% .Sam I

00.0
00.0

100.0F

 

00.0w

 

 

plus

00.0n

100.9.

 

[00.0m

(%) emisgow olqgssoldxg

91
processed meat products is unclear, but that CT? would bind

water if gelatin was produced upon cooking. Marshall and
Petrie (1980) using differential scanning calorimetry (DSC)
reported that the gelation temperature (T9) of collagen (IN)
was 82.50 c - 87.50 c. since processed meat products are
currently cooked at temperatures less than T9, the role of
collagen and other CT? for binding water are probably
minimal. The substitution of In proteins in multicomponent
gels may have formed filled gels. The IR soluble proteins
had not gelled at the final internal cooking temperature of
72° c, thus mixed or composite gels could not be formed.
The substitution of IN proteins in multicomponent gels may
have formed filled gels.

The addition of 16.67% of IN proteins significantly
reduced the apparent stress at failure of the skeletal,
heart and spleen gels, whereas a 25% substitution was
required to significantly reduce the hardness of the lung
gels (Fig. 10). The 318 proteins from the various meats do
not function equivalently in least concentration endpoint
gelation, therefore the structure of the lung n18 protein
gels may be less disrupted by IR proteins compared to the
other file protein gels, although further investigations are
required. Apparent stress at failure was reduced
approximately 30% by the substitution of IN proteins at_the

25% level to the HIS/IN multicomponent gels. Greater

92

Figure 10. Apparent stress at failure of high ionic strength

plus insoluble protein gels.

 

 

 

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94
substitution of 18 proteins into the BIB/IN protein gels

resulted in weaker gels. The addition of the In proteins
within the HIS/IN gel matrix were less detrimental to gel
apparent stress at failure compared to the addition of LIB
proteins to BIB/L18 multicomponent gels. The addition of IN
proteins may have filled the interstitial spaces and were
"harder" than the LIS proteins and thus decreased apparent
stress at failure less. Lee and Abdollahi (1981) reported
that the addition of harder fats acting as fillers in
processed meat products increased their force to fracture.
Schmidt (1987) reported an increase in firmness when
connective tissue was added to emulsion products, but
Sadowska et al. (1980) reported that added connective tissue
at greater than 5% of the total protein reduced firmness by
lO-23%. Addition of stroma proteins will increase hardness
and decrease rubbery texture common in low fat matrices such
as frankfurters and bologna (Jones, 1984).

In contrast to the apparent stress at failure, the
apparent strain at failure (Fig. 11) was significantly lower
than the control for the lung multicomponent gel at 8.33%
substitution and significantly lower for the skeletal, heart
and spleen multicomponent gels at 16.67% substitution.
Apparent strain at failure (cohesiveness and/or
deformability) was lower for the 8.33% substituted lung gel
while the apparent stress at failure (hardness) was not
significantly reduced. Therefore it appears that IN proteins

have a more negative effect on gel cohesiveness than gel

95

Figure 11. Apparent strain at failure of high ionic strength

plus insoluble protein gels.

 

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97
hardness. Insoluble proteins may disrupt the HIS heat-

induced protein matrix thus reducing gel cohesiveness.

D o a s of h o

 

The addition of 1! proteins (Fig 12a - d) visibly added
more coarse strands or were deposited as globular masses to
the HIS protein gel network. The coarse strands may
interrupt the heat-induced BIB protein gel network and
increase the moisture expressed and decrease the gel
strength. As the coarse insoluble protein strands
interrupted the HIS protein gel structure, at the HIS/IN
protein junctions, moisture trapped during cooking could
have leaked during centrifugation. The IN proteins
disrupting the BIB protein matrix could have created a non-
continuous 318 protein matrix thus reducing the gels!
strength. Smith and Brekke (1985) related structurally
compact and globular chicken myofibrillar protein gels to

high gel strength and increased gel syneresis.

98

Figure 12. Scanning electron micrographs of high ionic
strength plus insoluble protein gels.
A = skeletal
B = cardiac
c = lung
D

= spleen

 

 

 

 

 

100
7 O 0 e

RSISEQEQEQ

Results indicated that HIS protein fractions from
various sources do not function equivalently. Nyosin has
been reported to be the protein most responsible for protein
gelation (Schmidt et al., 1981). The percentage myosin in
the HIS protein fraction for semitendinosus muscle was 50.7%
(Lowey and Risby, 1971) and 47.9, 37.6 and 22.6% for beef
heart, lung and spleen, respectively (Nuckles et al., 1990).
Therefore correlations between gel functional tests and
percentage myosin in the 818 protein fraction were
calculated.

The quantity of myosin in the HIS protein fraction was
strongly correlated to gel apparent stress at failure
(r=o.86), apparent strain at failure (r=o.80) and
expressible moisture (r=-.82). Therefore the percentage
myosin in the HIS protein fraction may help explain gel
functionality. Further work in model gel and meat systems
needs to be performed.

3. CON Us 0 8

Results indicate that the gel forming abilities of HIS
protein fractions from skeletal, cardiac and smooth muscles
are not equivalent and thus functionality of muscle tissues
cannot be compared solely on the basis of the size of the
HIS protein fraction. The addition of LIS and IN proteins
affected HIS protein gel functionality. It may be possible

to predict the gel strength and other functionalities of

 

101
muscle tissues based on the amount of the LIS, HIS and IN

protein fractions and the amount of myosin in the SIS
protein fraction. These results suggest a new method is
needed for measuring bind constants or relative
functionalities of meat ingredients used in least cost

formulation calculations by meat processors.

VI. 8 u 3: ect o eat d eat 8 - roduct

grotein Fractions on Functional
P o e t e ode S s em ankfurte s

A t uct o

Reformulation or designing of meat products to obtain
high cooking yields and desired textures is an empirical
process that often leads to many product failures (Knight,
1988). Product failures are caused in part by the different
types and amounts of protein and fat in the raw meat
ingredients and the lack of understanding of meat protein
functionality. Neat by-product protein functionality is not
well understood. Venegas et al. (1988) and Oliveros et al.
(1982) reported on emulsion and water-holding capacities of
by-products in model systems. They suggested each by-product
would individually contribute different properties to a meat
product, therefore utilisation of by-product mixtures could
be beneficial to water and fat-holding properties and
textures of meat products.

Randall and Voisey (1977) studied the effects of meat
protein fractions on textural characteristics of meat
emulsions. Their results suggested that salt soluble
proteins formed the cooked emulsion matrix and connective
tissue proteins added hardness. Knight (1988) studied three
protein fractions from minced pork shoulder and found that
the salt soluble protein (SSP) fraction formed gels, the
insoluble myofibrillar protein fraction acted as a filler in

the SSP gel. The role of the connective tissue proteins was

102

103
unclear, but appeared to contribute to gelation. Mcxeith et

al. (1988) reported on characteristics of beef, pork and
beef by-product surimi. Results showed that beef, pork and
beef heart SSP formed gels that were comparable to a
commercial fish surimi. The effects of sarcoplasmic proteins
on protein gelation or meat product texture are not well
understood.

Nuckles et al. (1990) quantitated meat by-product
protein, percentage of three protein fractions [low ionic
strength soluble (LIS) high ionic strength soluble (HIS) and
insoluble (IN)], percentage myosin and the actin:myosin
ratio of several by-products (pork liver, pork lung lobes,
beef heart, beef lung lobes, beef spleen). By-products were
generally higher in moisture and lower in fat than skeletal
muscle, varied in their percentage of the three protein
fractions and contained less myosin than skeletal muscle.
Substitution of LIS and IN protein fractions from meat by-
products caused negative effects on functional properties
(increased expressible moisture, lower apparent stress and
strain at failure) of heat-induced HIS gels (Nuckles et al.,
1990b). The effects of substituting by-product proteins for
skeletal HIS proteins in processed meats are not understood.

The objectives of this study were to determine the
effects of beef by-product (heart, lung, spleen) protein,
fractions [low ionic strength soluble (LIS), high ionic
strength soluble (HIS), insoluble (IN)], the percentage of

myosin in the formulation and the myosin:actin ratio of the

104
HIS protein fraction on beef skeletal muscle frankfurters.

The frankfurters were evaluated using functional tests (cook
yield, severe reheat yield, apparent stress and strain at
failure, apparent tensile strength).

In the first experiment, the percentages of high ionic
strength (HIS) proteins were varied in the frankfurter
formulations by addition of by-products to determine a
minimum percentage of HIS proteins required so that the
results of a functional test (severe reheat yield) would not
be significantly lower when the formulation contained a
different percentage HIS proteins. Experiment 2 formulations
contained a constant percentage of HIS proteins to determine
the effect of substituting LIS and IN proteins for HIS
protein on model system frankfurter functionality.

Nuckles et al., 1990b tested beef skeletal muscle and by-
products protein fractions (LIS, HIS, IN) in a model
gelation system. Correlations were performed to test if
results of the gelation model system predicted results in

the frankfurters.

MW
3,;. Materials

Beef semimembranosus muscle (beef skeletal muscle),
beef by-products (lung lobes, spleen and heart) and beef fat
were used in this study. The beef lung lobes and hearts were
obtained from 9 market weight steers slaughtered at the
Michigan State University Neat Laboratory. Beef spleen and

fat were purchased from Ada Beef Co., Ada, MI and the beef

105
semimembranosus muscle was purchased frozen from Monfort of

Colorado, Greeley, co. By-products were removed within 1 hr
after exsanguination, vacuum packaged (2.5 mil polyethylene)
in 500 g aliquots and frozen until used within 1 month.
8. . eth
B. .a Ne t at e o u t on

Neat batters were prepared using a silent cutter as
described by Smith and Brekke (1984), except that the batter
was stuffed into 50 ml conical centrifuge tubes, capped and
heated in a 750 c water bath to an internal temperature of
72° c.

Neat batters were formulated to 56% moisture, 30% fat,
12% protein and 2% salt. Neat batters were formulated to
contain different percentages of protein fractions [low
ionic strength soluble (LIS), high ionic strength soluble
(BIS) and insoluble (IN)]. calculation of percentage protein
fractions, myosin and actin:myosin ratio in the formulations
was based on results reported by Nuckles et al. (1990a) and
Lowey and Risby (1971). In the first experiment, meat
batters were formulated to contain HIS proteins at 25 - 45%
of the total protein by substituting spleen or lung for beef
skeletal muscle. In the second experiment, meat batters were
formulated to a constant percentage of HIS proteins while
the percentages of LIS or IN proteins were varied (27 - 38%)
by substituting beef heart, spleen or lungs singly or in

combination for beef skeletal muscle.

106
.8. O

Proximate composition (fat, protein, and moisture) of
the raw batter and frankfurters were determined in
triplicate by AOAC (1984) procedures. Raw batter and
frankfurter pH was determined in triplicate by blending 10 g
of batter with 100 g of double distilled water on setting 6
for 30 sec using a Polytron homogenizer (Brinkman
Instruments Co., lestbury, N.Y.). The pH was measured using
a Corning 145 pH meter.

Cook yield and severe reheat yield were determined as
described by Nuckles et al. (1990a). Apparent shear stress
and strain at failure (Diehl et al., 1979) of the model
system frankfurters were measured at 1° C on 1.5 cm x 1.5 cm
central cores of the frankfurter using an Instron (Model
4202, Canton, MA) at a crosshead speed of lo mm/min with a
50 N compression cell as described by Nuckles et al. (1990).

Apparent tensile strength was measured on whole
frankfurters using a TR-s Texturepress with a ST Thin Slice
Tensile Test Cell attachment and Texturerecorder (Food
Technology Corp., Rockville, MD) at a speed of 4 and range
x 1/10. Frankfurter (1° C) weights were recorded prior to
tensile strength testing and loaded on the cell
perpendicular to the cell break-line. The force (N79) to
break the frankfurters was calculated. Twelve frankfurters
per formulation (4 from each replicated formula) were

tested.

107
W

The statistical design was completely randcmised. All
data represent a mean value of at least triplicate analyses.
Basic statistics, 2-way analysis of variance (ANOVA),
treatments 1 replications, and correlations were performed
using M-STAT (1988). Correlations were determined using
amount of LIS, HIS or IN proteins, myosin percentage and
actin:myosin ratio (Nuckles et al., 1990a), gel apparent
stress and strain at failure (Nuckles et al., l990b) and the
frankfurter results. Mean separations were performed using
Tukey's test.

D. u t a c
D. :- nu ;t_0's co; - 2 2° -- — e; -e 3.! .oe- - a“;
ote

The proximate composition (fat, moisture and protein)
of the raw meat batters formulated with different amounts of
HIS proteins from beef skeletal muscle and by-products were
not significantly different. The pH of the raw meat batters
and the frankfurters were significantly different from each
other (5.8 and 6.2, respectivelY). but the pH within the
treatments was not significantly different (P < 0.05).

As the protein fraction composition of the formulation
was varied, frankfurter proximate composition (Table 9)
changed due to differences in fat and moisture loss during
cooking (Table 10). As the percentage of low ionic strength
soluble (LIS) protein increased and the percentage myosin

decreased due to substitution of spleen for skeletal meat in

108

Table 9 - Cooked proximate analysis of frankfurters
formulated with different by-products to alter the
percentage high ionic strength (HIS), low ionic strength
(LIS) and insoluble (IN) protein fractions.

 

 

Protein1 Myosin1 Cooked Proximate
Fraction in Analysis
(%) Formula Protein Fat Moisture

Treatment LIS HIS IN (%) (%) (%) (%)

Control, 100% 26 45 29 50.7“ 12.3“ 30.6“ 55.1“
skeletal

Skeletal 31%, 31 41 23 45.4b 12.6a 23.2“ 62.2b
spleen 19%

Skeletal 56%, 33 35 27 33.3“ 12.5“ 22.9“ 62.4b
spleen 44%

Skeletal 35%, 44 30 26 32.4“ 12.3“ 13.3“ 66.5“
spleen 65%

Skeletal 14% 50 25 25 26.5f 12.0“ 13.7“ 66.7“
spleen 86%

Skeletal 35% 27 40 33 43.7“ 12.5a 31.3“ 53.3“
lung 15%

Skeletal 70% 29 35 37 46.3b 12.3“ 31.9“ 53.6“
lung 30%

Skeletal 57% 30 30 40 45.1b 12.5“ 30.9“ 54.4“
lung 43%

Skeletal 43% 32 25 43 43.2“ 12.2“ 23.9b 56.7“
lung 57%

sex 1.1 0.3 1.0 2.1

 

1calculated from Nuckles et al. (1990)

alblcldlel

do not differ significantly (P < 0.05).

fMeans within columns followed by the same letter

109
the formulation, the percentage fat retained in the

frankfurter decreased (fat cook-out increased), and the
percentage moisture in the frankfurter increased compared to
the raw batter. Venegas et al. (1988) and Oliveros et al.
(1982) reported that meat by-products, especially spleen,
containing large amounts of LIS proteins had good water
holding capacity.

Cook yield was significantly lower (P < 0.05) as spleen
was substituted for beef skeletal muscle at percentages
greater than or equal to 44% of the formula. The
frankfurters contained significantly less fat than the meat
batters (Table 9), thus as the percentage of LIS proteins
increased and myosin decreased, fat cooked out of the
frankfurters and reduced cook yield.

Cook yield decreased as the concentration of IN
proteins increased and the percentage of total protein as
myosin decreased (Table 10). A 15% substitution of beef lung
(an increase of 4% insoluble proteins) caused approximately
a 20% decrease in cooked yield. Higher amounts of
substitution of skeletal muscle with lung did not
significantly reduce cooked yield. When the percentages of
HIS proteins were equivalent, the formulas containing 44%
spleen (higher LIS protein concentration) had lower cook
yields and myosin contents than the lung (high IN protein
concentration) formulas.

Severe reheat yield (Table 10) was adversely affected

by the addition of by-products. As the percentage of HIS

Table 10 - Cook yield, severe reheat yield and textural
characteristics of frankfurters formulated with different
by-products to alter the high ionic strength (HIS), low
ionic strength (LIS) and insoluble (IN) protein fractions.

 

 

Apparent
Severe Stress Apparent
Cook Reheat at Strain Tensile
Yield Yield Failure at Strength

Treatment (%) (%) (kPa) Failure (N/g)

Control, 100% 93.15“ 30.39“ 104.3“ 0.93“ 0.32“
skeletal

Skeletal 31%, 95.77“ 59.33b 42.3“ 0.77“ 0.19“
spleen 19%

Skeletal 56%, 36.53b 43.73“ 19.3“ 0.61“ 0.13“
spleen 44%

Skeletal 35%, 76.35“ 43.65“ 15.3“ 0.60“ <0.10“
spleen 65%

Skeletal 14% 70.07“ 43.44“ 10.41 0.55“ <0.10“
spleen 86%

Skeletal 35% 73.65“ 59.331’ 79.3b 0.96“‘ 0.34“
lung 15%

Skeletal 70% 77.77“ 59.731’ 43.4“ 0.33b 0.23b
lung 30%

Skeletal 57% 77.64“ 59.77b 25.7“ 0.77“ 0.25b
lung 43%

Skeletal 43% 76.93“ 52.33“ 23.4“ 0.77“ 0.24b
lung 57%

83M 1.02 2.31 0.2 0.07 0.04

 

a,b,c,d,e,f Means within columns followed by the same letter
do not differ significantly (P< 0.05).

111
proteins and percentage myosin decreased and the percentage

of LIS protein increased due to the addition of spleen the
severe reheat yield was significantly reduced approximately
30% compared to the control frankfurters. HIS protein
concentration of less than 35% in the skeletal meat/spleen
formulas did not cause a significant reduction in severe
reheat yield compared to formulas with 35% HIS proteins.

The substitution of 30% or greater lung into the beef
skeletal muscle formulas which resulted in an increase in IN
proteins and a decrease in HIS proteins and myosin, caused a
reduction in severe reheat yield compared to the control. As
with the spleen formulas, 35% HIS proteins in the
formulation did not give a significantly different severe
reheat yield compared to 25 or 40% HIS proteins.

An increase in LIS proteins in the formulation had a
more severe effect on reheat than an increase in IN protein
concentration. LIS proteins at 38% of the total 12% protein
had significantly lower cook yields than IN proteins at 43%
of the total protein.

The addition of 19% spleen (increase of 4% LIS proteins
and a 5% reduction of percentage myosin in formulation) or
addition of 15% lung (increase of 4% IN proteins but no
significant change in percentage myosin in the formulation)
reduced apparent stress at failure of the frankfurters by
more than 50% compared to the 100% skeletal meat
frankfurter (Table 10). The substitution of higher amounts

of spleen or lung, thus increasing the LIS or IN protein

 

112
fraction and reducing percentage myosin of the total protein

in the meat batter, significantly reduced (P < 0.05)
apparent stress at failure. The addition of lung to the
formulations was less detrimental to the apparent stress at
failure compared to the addition of spleen. Substitution of
35% lung or 19% spleen resulted in comparable apparent
stress at failure of the frankfurters.

Apparent strain at failure or deformability of the
frankfurters (Table 10) was significantly reduced by the
addition of spleen (increase in amount of LIS protein
fraction) to the formulation. The addition of lung to the
frankfurters had a less pronounced effect upon apparent
strain at failure compared to the addition of spleen.
Substitution of 19% spleen into the formulation had the same
effect as a 43% lung substitution. Greater than 15% lung had
to be substituted into the skeletal meat formulas before
apparent strain at failure was significantly (P<0.05)
reduced.

Apparent tensile strength of the cooked meat batters
was reduced by both the addition of spleen or greater than
15% lung to the formulation (Table 10). A 19% substitution
of spleen into the beef skeletal muscle formulation (a 4%
increase of the LIS protein fraction and a 5% decrease in
percentage myosin) caused approximately a 40% reduction in
tensile strength. The substitution of 30% lung (an 8%
increase in the IN protein fraction and 4% decrease in

percentage myosin) compared to the control caused

113
approximately a 30% reduction in tensile strength. The

tensile strength for the skeletal/lung formula containing
35% HIS proteins was approximately 75% stronger than the
skeletal/spleen formula containing the same amount of HIS
proteins. Substitution of 65% or greater of spleen into the
formulation resulted in frankfurter tensile strength < 0.10
N/g.

Nuckles et al. (1990b) reported that HIS proteins from
various meat sources do not function equivalently in
gelation tests and that the addition of LIS or IN proteins
to HIS protein gels increased gel expressible moisture and
reduced gel strength. They proposed that gel functionality
was related to HIS protein fraction size and composition (%
myosin or actin:myosin ratios) and the influence of LIS and
IN proteins. The present research further suggests that
substitution of LIS and IN proteins for HIS proteins by the
addition of by-products modify frankfurter functionality by
decreasing severe reheat yield and frankfurter strength as
measured by apparent stress and strain at failure and
apparent tensile strength.

D.2. Formulations with 35% HIS proteins and different
ou t o S and I ote s

Formulations substituted with spleen or lung for
skeletal meat containing 35% HIS proteins of the total 12%
protein did not have significantly different severe reheat
yields when compared to formulating to a lower percentage

(25 or 30%) HIS proteins. Therefore formulations with 35%

114
HIS proteins were tested to determine the effects of LIS and

IN proteins on frankfurter functionality.

The proximate composition of the raw meat batters
formulated with 35% of the total protein as HIS proteins
from by-products were not significantly different. The
frankfurter proximate compositions (Table 11) and cook yield
(Table 12) varied with the amounts of LIS and IN proteins
and the percentage myosin in the meat batters. The
substitution of 44% beef spleen increased LIS proteins by
10% and increased fat loss as indicated by lower cook yield
and higher moisture in the frankfurters compared to the raw
meat batters. A 30% lung substitution, an increase in IN
proteins by 8%, increased moisture loss as indicated by
lower cook yield and higher fat in frankfurters compared to
raw meat batters. A substitution of 44% heart, which was a
12% increase in LIS proteins, lowered cook yield but did not
cause a significant change in frankfurter proximate
composition compared to the control. This result suggests
that both fat and moisture were lost during cooking.

The skeletal/heart formula contained a higher
percentage LIS proteins but myosin percentage did not
significantly differ from the control. These results
suggested that not only percentage of the three protein
fractions influenced the frankfurters, but that the
percentage myosin and myosin:actin ratios also affected cook
yield. Both beef heart and beef semimembranosus muscle

contain an actin:myosin ratio of 5.0 (Nuckles et al., 1990:

115

Table 11 - Cooked proximate analysis of frankfurters
formulated to contain 45% or 35% of high ionic strength
soluble (HIS) proteins by substitution of by-products.

 

 

Protein1 Myosin1 Cooked Proximate
Fraction in Analysis
(%) Formula Protein Fat Moisture
Treatment LIS HIS IN (%) (%) (%) (%)
Control, 100% 26 45 29 50.7“ 12.3“ 30.6b 55:1“
skeletal
Skeletal 56%, 33 35 27 49.5“ 12.2“ 30.2b 55.2“
heart 44%
Skeletal 70%, 23 3s 37 46.3b 12.3“ 31.9“ 53.61"
lung 30%
Skeletal 56%, 36 35 29 33.3“ 12.3“ 23.9“ 61.4“
spleen 44%
Skeletal 60, 27 35 33 43.6“ 12.2“ 30.9b 54.7“
heart 30%,
lung 10%
Skeletal 55%, 31 35 34 44.4“ 12.5“ 30.3b 54.4“
heart 25%,
spleen 20%
Skeletal 60%, 36 35 29 41.0“ 12.5“ 25.3“ 59.4b
spleen 30%,
lung 10%
Skeletal 60%, 30 35 35 46.0b 12.3“ 31.3“ 53.6f
heart 20%,
spleen 10%,
lung 10%
Skeletal 60%, 32 35 33 43.5“ 12.3“ 27.3“ 57.5“
spleen 20%,
heart 10%,
lung 10%
SEM 1.1 0.1 1.3 0.7
1calculations based on Nuckles et al. 1990

a,b,c,d,

do not differ significantly (P<0.05).

e'fMeans within columns followed by the same letter

116
Risby and Dewey, 1971, respectivelY). whereas spleen and

lung contain a actin:myosin ratio of 4.5 and 3.2,
respectively (Nuckles et al., 1990). The percentage myosin
in the spleen and lung formulations was significantly
(P<0.05) lower than the control, skeletal/heart and
skeletal/heart/lung formulas (Table 11). The frankfurters
produced from skeletal meat plus 2 or 3 by-products showed
that increasing amounts of LIS proteins and decreased
percentage myosin in the formulation caused fat loss.
Increasing amounts of IN proteins and decreasing myosin
percentage in the formulations favored moisture loss.

Severe reheat yield (Table 12) was reduced by the
addition of spleen or lung, whereas the addition of heart
did not significantly reduce severe reheat yield. Severe
reheat yield appeared to be influenced by percentage myosin,
actin:myosin ratio and percentage of LIS or IN proteins in a
formulation. Low ionic strength proteins exerted a more
negative effect upon severe reheat yield than IN proteins.
Skeletal 60/spleen 44% and skeletal 60/spleen 30/lung 10%
formulas contain 10% more LIS proteins but no more IN
proteins compared to the control and exhibited approximately
30% lower severe reheat yields. Formulas containing 6 -9%
more IN proteins had 5 - 14% lower severe reheat yields.

Apparent stress at failure (Table 12) decreased upon
the addition of by-products to the formulations.
Substitution of heart affected frankfurter strength the

least compared to substitution of Spleen and lungs. Heart

117

Table 12 - Cook yield, severe reheat yield and textural
characteristics of frankfurters formulated to contain 45% or
35% of high ionic strength soluble (HIS) proteins by
substitution of by-products.

 

 

 

Apparent
Severe Stress Apparent
Cooked Reheat at Strain Tensile
Yield Yield Failure at Strength
Treatment (%) (%) (kPa) Failure (N/g)
Control, 100% 93.15“ 32.66“ 103.6“ 0.94“ 0.37“
skeletal
Skeletal 56%, 35.33“ 30.39“ 30.2“ 0.92“ 0.41“
heart 44%
Skeletal 70% 37.13“ 73.22“ 50.3“ 0.33“ 0.29“
lung 30%
Skeletal 56% 36.22“ 50.32“ 20.0“ 0.61“ 0.139
spleen 44%
Skeletal 60, 36.55“ 73.33“ 32.0“ 0.93“ 0.42“
heart 30%,
lung 10%
Skeletal 55%, 35.97“ 75.37“ 44.3“ 0.62“ 0.32“
heart 25%,
spleen 20%
Skeletal 60% 35.12“ 55.34“ 42.6“ 0.64“ 0.191'
spleen 30%,
lung 10%
Skeletal 60% 35.67“ 63.93“ 55.1“ 0.70“ 0.32“
heart 20%,
spleen 10%,
lung 10%
Skeletal 60% 34.33“ 53.22“ 41.7“ 0.62“ 0.24“
spleen 20%,
heart 10%,
lung 10%
SSM 0.33 1.23 1.1 0.04 0.02
a'1"°""°Means within columns followed by the same letter do

not differ significantly (P< 0.05).

118
increased the strength of formulas containing 2 or more by-

products compared to the comparable formulas without heart.
Frankfurter apparent strain at failure did not significantly
differ from the control when 44% heart was substituted into
the formula even though the percentage LIS proteins
increased. The percentage myosin in the formula did not
change significantly. The addition of lung or spleen reduced
frankfurter cohesiveness (apparent strain at failure) by 6%
and 35%, respectively (Table 12). Spleen increased the.
percentage LIS proteins and lung increased the percentage IN
proteins in the formula. Both by-products contain less
myosin and a lower myosin:actin ratio in the HIS protein
fraction compared to heart and beef skeletal muscle. The
addition of heart to the frankfurters increased tensile
strength compared to the control. The negative effect, lower
apparent tensile strength, from the addition of spleen
and/or lung to the formulation was less when heart was also
added (Table 12). The skeletal/heart/spleen formula which
contains 5% less LIS proteins and 6% more myosin than the
skeletal/spleen formula exhibited 2.5 times the tensile
strength of the skeletal/spleen formula. The
skeletal/heart/lung formula which contains 11% less LIS and
11% more IN proteins and 2% more myosin than the
skeletal/lung formula, exhibited a tensile strength 1.4.
times stronger than the skeletal/lung formula. Addition of
lung was less detrimental to frankfurter tensile strength

than spleen.

119
D.3 o ot o ode s e s

Frankfurter water- and fat holding capacity, severe
reheat yield, apparent stress and strain at failure and
apparent tensile strength were all correlated 0.70 or
greater to the amount of high ionic strength (HIS) proteins,
percentage myosin in the HIS proteins and actin:myosin
ratios in the HIS protein (Table 13). Schmidt et al. (1981)
reported that myosin is the myofibrillar protein (HIS) most
responsible for gelation. Myosin forms a heat-induced gel
that is irreversible, has high water-binding capacity and
strong elastic properties. Yasui and Ishioroshi (1980)
reported that F-actin enhanced the gelation of myosin.

Cook yield was not highly correlated to HIS protein
concentration, myosin percentage or the actin:myosin ratio.
HIS proteins from various sources do not function
equivalently (Nuckles et al., 1990b) therefore equal
percentages of HIS proteins in the frankfurter formulations
are not capable of equivalent water- and fat holding. Also
the centrifuge tubes used to cook the frankfurters are
impervious to air and moisture, unlike frankfurter casings,
therefore moisture and fat are more readily retained as part
of the frankfurter.

D 4. ect of otei on odel s st r f to s

Cook yield of the frankfurters was not highly
correlated (-0.60) to amount of LIS proteins in the meat
batters. Cook yields were not significantly different even

though the percentage of LIS proteins in experiment 2

120

Table 13 - Correlation coefficients between meat by-product
(LIS), insoluble (IN) and high ionic strength (HIS) protein fractio
and functionality tests of model system frankfurters.

 

 

 

Frank- Frank-
furter furter
apparent apparent
Water- Fat Severe stress strain
holding hold3 Cook reheat at at Tensile
cap. cap' yield yield failure failure strength
Ant. 0.86 -0.92 -0.60 -0.96 -0.93 -0.77 -0.96
of
L18
proteins4
Ant.“ -0.40 -0.45 -0.31 -0.40 0.64 0.30 0.30
of
IN
proteins
Ant.“ 0.92 0.93 0.24 0.79 0.30 0.73 0.90
of
818
proteins
Myosins 0.90 0.88 0.22 0.80 0.70 0.82 0.84
g
Actinz‘ 0.76 0.74 0.31 0.71 0.71 0.71 0.73
myosin
ratio
“Results from Nuckles et al. (199:)
2Water-holding capacity
3Fat holding capacity
4Amount of proteins
sFrom Lowey and Risby (1971); Nuckles et al. (1990)

6Calculated from Nuckles et al. (1990)

121
formulas varied by 10%. This result may be attributed to the

moisture retention (but fat loss) of the frankfurters during
cooking, as shown by high moisture and lower fat percentages
of experiment 1 spleen containing formulas. This result may
also explain why LIS proteins were positively correlated to
water-holding capacity (0.86) and negatively correlated to
fat holding capacity (-0.92). Severe reheat yield was highly
negatively correlated (-0.96), which indicated that as
amount of LIS proteins in the frankfurters increased, severe
reheat yield decreased. Frankfurter apparent stress and
strain at failure and tensile strength were highly
negatively correlated with the quantity of LIS proteins in
the formulation. The strength of the frankfurters decreased
as the amount of LIS proteins in the meat batter increased.
These results agree with results reported by Nuckles et al.
(1990).
D.5. Effect of IN prgteing on model system frankfurters

The amount of insoluble (IN) proteins was not highly
correlated with the parameters measured on the frankfurters
(Table 13) . The amount of IN proteins in the formulations!
which was not highly correlated to any parameter measured!
was most highly correlated (0.64) to frankfurter apparent
strain at failure. The low correlations suggest that IN
protein amounts may act as fillers (rather than binders) in
the meat batters and do not stabilize or destabilize the r34
batters. Nuckles et al. (1990b) reported that IN proteins

act as fillers in HIS protein gels. It may be necessary for

 

 

 

.a.-1

 

122
connective tissue proteins (IN) to gel to influence the

functional properties to frankfurters. Currently processed
meat products are not cooked to temperatures sufficient to
gel the connective tissue proteins (Knight, 1988).

D 6. or at o s of o e t e t o d u ters

The apparent stress and strain at failure of heat-
induced multicomponent gel prepared with HIS and LIS
proteins (Nuckles et al., 1990b) were highly correlated to
frankfurter apparent stress and strain at failure (r=0.90
and 0.91, respectively). Results indicate that the LIS
multicomponent gelation system used by Nuckles et al. (1990)
predicted the functionality of LIS proteins in model system
frankfurters.

The apparent stress and strain at failures of heat-
induced gels prepared with HIS and IN proteins measured by
Nuckles et al. (1990b) were highly positively correlated to
the apparent stress (0.88) and strain (0.91) at failure for
the frankfurters. The HIS gel apparent stress and strain at
failure (Nuckles et al., 199!) were highly correlated to the
measured apparent stress (0.95) and strain (0.93) of the
frankfurters. Expressible moisture (HM) of the protein gels
(Nuckles et al, 1990b) and frankfurter reheat yields were
negatively correlated (-0.73). Gel HM increased and
frankfurter reheat yield decreased by losing moisture or fat
during reheating. The results suggest that the model gel

system adequately predicted the frankfurters results.

123
mm

The results indicate that the proportions of the protein
fractions (LIS:HIS:IN) and the percentage myosin and/or
myosin:actin ratio in the meat batters were related to the
cook and reheat yields, fat and water-holding capacity,
apparent stress and strain at failure and tensile strength
of the frankfurters.

The quantity of the HIS proteins was the most important
factor to provide the most functionality to the model system
frankfurters. The DIS and IN proteins were detrimental to
the model system frankfurters. Low ionic strength (LIS) and
IN proteins acted like fillers in cooked frankfurters. The
LIS proteins decreased fat-holding of the raw meat batters
during cooking, as exhibited by the cooked frankfurters
being higher in moisture and lower in fat than the raw
batters. Both the LIS and IN proteins were detrimental to
severe reheat yield, with IN proteins being less detrimental
to frankfurter production.

The protein gelation system described by Nuckles et al.
(199b) predicted the protein functionality in the by-product
substituted model system frankfurters. These results suggest
that protein gelation in model systems can be used by
processors to predict protein functionality in meat
products. Results also suggest that bind values currently
used by meat processors to formulate meat products may be
improved by incorporating additional information about raw

materials such as the percentage LIS, HIS and IN protein

124
fractions of the total protein, percentage myosin in the HIS

protein fraction and the HIS protein fraction actin:myosin

ratio.

VI . and C c u o s
A. Study I.

Mechanically deboned chicken meat (MDCM), and meat by-
products (pork lung lobes, pork liver, beef lung lobes, beef
spleen, beef heart) varied in their proximate composition,
amount of three major protein fractions [low ionic strength
(LIS), high ionic strength soluble (HIS), insoluble (IN)]
and collagen content. Frankfurters produced with by-products
exhibited lower severe reheat yield and apparent stress and
strain at failure compared to frankfurters manufactured from
mechanically deboned chicken meat.

8. Stag! II.

Results indicate that the gel forming abilities of HIS
protein fractions from skeletal, cardiac and smooth muscles
were not equivalent and thus functionality of muscle tissues
cannot be compared solely on the basis of the size of the
HIS protein fraction. It may be possible to predict the gel
strength and other functionalities of all muscle tissues
based on the size of salt soluble protein fraction and the
amount of myosin in the salt soluble protein fraction.
Results suggested a new method is needed for measuring bind
constants or relative functionalities of meat ingredients

used in least cost formulation calculations of meat

processors.
C, Study III.

The gelation of the HIS proteins provide the most

functionality to the model system frankfurters. The amount

125

126
of HIS, LIS and IN protein fractions in a formulation, the

percentage of myosin in the HIS protein fraction and of the
total protein and the actin:myosin ratio in the HIS protein
fraction were all important factors in HIS protein fraction
functionality.

The DIS proteins were detrimental to production of the
model system frankfurters. Insoluble (IN) protein were less
detrimental that LIS proteins and tended to act like fillers
in cooked model system meat products.

The protein gelation system and results reported by
Nuckles et al. (1990b) predicted the protein functionality
in model system frankfurters. These results suggest that
protein gelation in model systems can be used by processors
to predict protein functionality in meat products. Results
also suggest that bind values currently used by meat
processors to formulate meat products could be improved by
incorporating additional information about raw materials
such as the percentage LIS, HIS and IN protein fractions of
the total protein, percentage myosin in the HIS protein
fraction, % myosin in the formulation and the HIS protein

fraction actin:myosin in ratio.

A.

ec O 0

Test isolated protein fractions in current or novel
food products.

1. Low ionic strength (LIS) proteins to replace added
water in meat products. Various amounts of LIS
proteins substituted for water in meat products
should be tested to determine substitution effects
on color, oxidative rancidity, sensory and other
parameters.

2. High ionic strength (HIS) proteins singly or in
combinations with insoluble proteins (IN) to replace
binders in formed meat chunks (FMC) or restructured
meats (traditional or extruded).

3. Conduct further tests of the use of high ionic
strength soluble (HIS) proteins in surimi-type
products such as human and cat foods.

4. Determine the gelation temperatures of high ionic
strength soluble (HIS) proteins to determine the
optimal temperature to utilize the protein fraction
in processed meats.

Test model meat and/or by-product formulations based on
% myosin and/or myosin to actin ratios. Determine if
the % myosin or myosin/actin ratio within the high
ionic strength soluble proteins determines the
functionality of the meat or by-products in the
formulations.

Work in conjunction with engineers to develop and test
methodology to extract protein fractions on a
commercially feasible basis. Engineers could develop
the extraction machinery and protein scientists could
test the functionality of the extracted proteins.

Extract red meat muscles (or meat cuts) into the
protein fractions (HIS, LIS, and IN) and determine the
% myosin and myosin to actin ratio in the HIS protein
fraction. The information could be used to develop a
computer knowledge base to replace the current bind
constants used in least cost formulation.

Expand the extraction of proteins fractions to include
all red meat by-products and poultry meats and by-‘
products to develop a complete knowledge base to use
for computer program least cost formulation.

127

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