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Michigan State
University

 

 

 

This is to certify that the
dissertation entitled

Heat—induced Gelation of Myosin with Native or
Heat-Denatured B-Lactoglobulin

presented by

Manee Vittayanont

has been accepted towards fulﬁllment
of the requirements for

Ph.DL degree in Food Science

 

Major professor

Date 5'61 '00

 

MS U is an Afﬁrmative Action/Equal Opportunity Institution 042771

 

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6/01 cJCIFiC/DateDuepss-pJS

HEAT-INDUCED GELATION OF MYOSIN WITH NATIVE OR
HEAT-DENATURED B-LACTOGLOBULIN
By

Manee Vittayanont

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

2000

ABSTRACT

HEAT-INDUCED GELATION OF MYOSIN WITH NATIVE OR
HEAT-DENATURED B-LACTOGLOBULIN

By

Manee Vittayanont

Whey protein is widely used to enhance the textural qualities of processed meat
products, but its effectiveness is often variable as the major protein, B-lactoglobulin ([3-
LG), does not gel at typical processing temperatures (68-71°C). Preheated whey protein
aggregates form gels at lower temperatures than the native protein. The functions of
native or preheated whey protein in meat systems have not been clearly explained due to
the multi-component nature of the proteins. The objective of this research was to study
gelation mechanisms of myosin with native or preheated B-lactoglobulin (B-LG) in a
model system.

There was no interaction between myosin and B-LG during heating as indicated
by the unique denaturation peaks of each protein in the therrnogram of the mixed protein
solution. Use of B-LG in the mixed system increased initial aggregation temperatures
and decreased maximum aggregation rates when compared to myosin. Storage modulus
or stiffness of myosin/B-LG decreased during heating between 55-75°C, then increased
above 80°C and after cooling. B-LG interrupted gel network formation of myosin at 55-

75°C, but enhanced stiffness of the co-gels when heated above its gelling temperatures of

80°C.

Preheating (80°C for 30 min) caused irreversible denaturation and formation of
soluble B-LG aggregates (HDLG). The minimum concentration of HDLG required for
gel formation in 0.6M NaCl, 0.05M sodium phosphate buffer at pH 7.0, was 2%, whereas
B-LG required 4% protein for gel formation. The HDLG gelled at a lower temperature
and had higher stiffness than native B-LG. Decreasing the pH from 7.0 to 6.0 decreased
the gel point of HDLG. The HDLG gel networks were comprised of strands and clumps
of small globular aggregates in contrast to B-LG gels which contained coarse, particulate
networks of compacted globules with large voids.

When heated to 71°C, mixed solutions of 1% myosin/1% HDLG had the same
gel point (48°C) and a higher storage modulus after cooling when compared to 2%
myosin. Myosin/B-LG gelled at 49°C and the G’ was much lower than myosin and
myosin/HDLG gels. Gels of 2% myosin and myosin/HDLG had a similar ﬁne stranded
networks, whereas myosin/B-LG networks were thicker with larger void spaces. HDLG
was incorporated within the myosin networks when heated to 71°C, whereas B-LG
remained soluble.

This work revealed that B-LG interrupted network formation of myosin during
heat-induced gelation. Preheating at 80°C for 30 min modiﬁed the gelling properties of [3-
LG, allowing strong co-gel formation with myosin at lower temperatures than that needed
for unheated B-LG. This work provides direction for the design or modiﬁcation of whey
protein with optimum properties needed by meat processors and may contribute to

enhanced applications of whey products where protein gelation at low temperature is

required.

Dedication

To papa, mama, and other sevens

ACKNOWLEDGMENTS

I would like to thank Dr. Denise M. Smith, my major advisor, for giving me a
chance and leading me the way to an end. I thank very much to the committee, Dr.
Stanley L. Flegler for SEM knowledge and experience, Dr. John A. Partridge for
reviewing the ﬁnal draﬂ, and Dr. James F. Steffe for your help through all the rheology
problems. A lot of thanks to Dr.Mark A. Uebersax for your consideration and helps.

The person I will never forget, Virginia Vega-Warner, thank so much for your
help and being friend. Special thanks to Jay Chick for your friendship and help in
English, Tom Forton for all myosin used throughout the project and also your kindness
and friendship. Many thanks to Dr.Alicia Orta-Ramirez who always there with me no
matter in good or bad time. With great appreciation to Eva Daneielewicz, Geoff William
for all SEM works, Shirley Owens for the last minute rescue.

Thanks to Ivy, Stephanie, Amie, Sarah, Carolyn, Jennifer and J in—shan for making
5 years in lab 110 and 3340 a happy time to remember. To Ubonrat, Kritika, Suriyan,
Nucharin and Korada, I will never forget the hard times we passed through together and
the happiness we share.

To Manut, my beloved brother who never let me feel alone away from home, I
thank you so much. Thanks to all in my family and my husband, Dr.Arnont vittayanont,
for your encouragement. Thank to all Thais for opportunity to fulﬁll my knowledge and

to learn life and people in the world outside Thailand.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. ix
LIST OF FIGURES ................................................................................ xi
CHAPTER 1: INTRODUCTION ................................................................ 1
CHAPTER 2: OBJECTIVES ..................................................................... 4
CHAPTER 3: SIGNIFICANCE AND JUSTIFICATION .................................. 6
CHAPTER 4: LITERATURE REVIEW ....................................................... 9
4.1. Heat-induced gel formation of proteins ................................................. 9
4.1.1. Deﬁnition and types of protein gels ................................................. 9

4.1.2. Mechanism of heat-induced gel formation ........................................ 11

4.1.3. Protein-protein interaction in gels ................................................... 13

4.1.4. Methods to study heat-induced gel formation .................................... 15
4.1.4.1. Thermal denaturation ....................................................... 17

4.1.4.2. Thermal aggregation ........................................................ 17

4.1.4.3. Viscoelastic network formation ........................................... 18

4.1.4.4. Final gel structure ........................................................... 19

4.2. Myosin ...................................................................................... 20
4.2.1. Molecular characteristics ........................................................... 20
4.2.2. Heat-induced gel formation of myosin ........................................... 22

4.2.3. Factors inﬂuencing myosin gelation .............................................. 24
4.2.3.1. Protein concentration ........................................................ 24

4.2.3.2. Environmental conditions ................................................... 25

4.2.3.3. Heat treatment ................................................................ 25

4.3. B-Lactoglobulin ........................................................................... 26
4.3.1. Molecular characteristics ........................................................... 27
4.3.2. Heat-induced gelation ............................................................... 27

4.3.3. Factors inﬂuencing B-lactoglobulin gelation ..................................... 31
4.3.3.1. Protein concentration ........................................................ 31

4.3.3.2. Protein modiﬁcation ......................................................... 32

4.3.3.3. Environmental conditions .................................................... 32

4.4. Mixed protein gel systems ................................................................ 36
4.4.1. Models of two component mixed gel structures ................................. 37

4.4.2. Interaction between meat protein and other proteins ........................... 37

vi

4.5. Pre-heated whey protein and a two-step gelation process ........................... 43

4.6. Cold-set whey protein gels ............................................................... 45
4.6.1. Formation of soluble whey protein aggregates .................................. 46
4.6.2. Network formation of cold-set gels of heat-induced whey protein

aggregates .............................................................................. 48

4.6.2.1. Enzyme-induced cold-set gel formation .................................. 48

4.6.2.2. Salt-induced cold-set gel formation ....................................... 49

4.6.6.3. Acid-induced cold-set gel formation ..................................... 51
CHAPTER 5: HEAT-INDUCED GELATION OF MYOSIN/B-LACTOGLOBULIN
MIXED PROTEINS ............................................................................... 54

5.1. ABSTRACT ................................................................................ 54

5.2. INTRODUCTION ........................................................................ 55

5.3. MATERIALS AND METHODS ........................................................ 56

5.3.1. Protein solution preparation ........................................................ 56
5.3.2. Differential scanning calorimetry ................................................. 57
5.3.3. Thermal aggregation of protein ................................................... 57
5.3.4. Small amplitude dynamic oscillatory testing .................................... 59
5.3.5. Experimental design and statistical analysis ..................................... 61
5.4. RESULTS AND DISCUSSION ......................................................... 61
5.4.1. Thermal denaturation of myosin and B-actoglobulin ........................... 61
5.4.2. Thermal aggregation ................................................................ 64
5.4.3. Rheological properties ............................................................. 73
5.5. CONCLUSIONS .......................................................................... 80
CHAPTER 6: GELLING PROPERTIES OF HEAT-DENATURED B-
LACTOGLOBULIN ............................................................................... 83
6.1. ABSTRACT ............................................................................... 83
6.2. INTRODUCTION ........................................................................ 84
6.3. MATERIALS AND METHODS ....................................................... 86
6.3.1. Preparation of protein solutions .................................................. 86
6.3.2. Electrophoresis ...................................................................... 86
6.3.3. Scanning electron microscopy (SEM) of aggregates .......................... 87
6.3.4. Differential scanning calorimetry ................................................ 88
6.3.5. Rheological measurement ......................................................... 88
6.3.6. Scanning electron microscopy (SEM) of gels ................................... 89
6.3.7. Experimental design and statistical analysis .................................... 90
6.4. RESULTS AND DISCUSSION ....................................................... 90
6.4.1. Electrophoresis ..................................................................... 90
6.4.2. Microstructures of heat-denatured B-lactoglobulin aggregates .............. 92
6.4.3. Thermal denaturation of B-lactoglobulin and heat-denatured
B-lactoglobulin ...................................................................... 94
6.4.4. Rheological properties of heat-denatured B-lactoglobulin gels ............. 96
6.4.5. Microstructures of B-lactoglobulin and heat-denatured B-lactoglobulin
gels .................................................................................. 102

vii

6.5. CONCLUSIONS ...................................................................... 105

CHAPTER 7: INTERACTION OF HEAT-DENATURED B-LACTOGLOBULIN

WITH MYOSIN DURING HEAT-INDUCED GELATION ............................ 107
7.1. ABSTRACT .............................................................................. 107
7.2. INTRODUCTION ..................................................................... 108
7.3. MATERIALS AND METHODS ...................................................... 110

7.3.1. Protein solution preparation ...................................................... 110
7.3.2. Differential scanning calorimetry ............................................... 111
7.3.3. Dynamic rheological measurement .............................................. 111
7.3.4. Scanning electron microscopy (SEM) .......................................... 112
7.3.5. Soluble proteins expressed from co-gels ....................................... 113
7.4. RESULTS AND DISCUSSION ....................................................... 114
7.4.1. Thermal denaturation of mixed proteins ....................................... 114
7.4.2. Rheological properties ............................................................ 116
7.4.3. Microstructure of mixed protein gels ........................................... 123
7.4.4. Soluble proteins expressed from co-gels ....................................... 131
7.5. CONCLUSIONS ........................................................................ 135

CHAPTER 8: CONCLUSIONS ............................................................... 137

CHAPTER 9: FUTURE RESEARCH ....................................................... 139

CHAPTERIO: BIBLIOGRAPHY ............................................................. 141

viii

LIST OF TABLES

Table 4.1. General characteristics of molecular interactions between proteins in aqueous
solution (adapted from Bryant and McClements, 1998) .................................. 14

Table 4.2. Network types and physical properties of whey protein gels (from Foegeding
et al., 1998) ..................................................................................... 35

Table 5.1. Concentrations of myosin and B-lactoglobulin (B-LG) in 0.6M NaCl, 0.05M
sodium phosphate buffer, pH 7.0, used in thermal denaturation (DSC), thermal
aggregation and dynamic oscillatory experiments ......................................... 58

Table 5.2. Initial aggregation temperature (To), maximum aggregation rate (ARmax), and
temperature at maximum aggregation rate (Tm) of myosin, and mixed solutions of
0.25% B-lactoglobulin (B-LG) and 0.1-1.0% myosin in 0.6M NaCl, 0.05M sodium
phosphate buffer, pH 7.0, during heating from 25 to 90°C, holding at 90°C for 30 min
and cooling to 25°C ............................................................................ 66

Table 5.3. Initial aggregation temperature (To), maximum aggregation rate (ARM), and
temperature at maximum aggregation rate (Tm) of B-lactoglobulin (B-LG), and mixed
solutions of myosin and 0.1-1.0% B-LG in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0, during heating from 25 to 90°C, holding at 90°C for 30 min and
cooling to 25°C ................................................................................. 69

Table 5.4. Gel points, storage moduli (G') and tan delta (tan 5) of myosin, and mixed
protein solutions of myosin and B-lactoglobulin (B-LG) during heating in 0.6M NaCl,
0.05M sodium phosphate buffer, pH 7.0, during heating from 25 to 90°C, holding at
90°C for 30 min and after cooling to 25°C .................................................. 74

Table 6.1. Gel point, storage moduli (G’) and tan delta (tan 8) after cooling of 1-3%
heat-denatured B-lactoglobulin (HDLG) heated to 71°C in 0.6M NaCl, 0.05M
sodium phosphate buffer, pH 6.0, 6.5 and 7.0a ............................................ 99

Table 7.1. Gel point, storage moduli (G') and tan delta (tan 6) after cooling of myosin,
1%myosin/ 1% B-lactoglobulin (myosin/B-LG) and 1% myosin/ 1% heat-denatured
B-LG (myosin/HDLG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0,
heated to 71°C for 60 min or 90°C for 30min .............................................. 119

 

Table 7.2. Gel point, storage moduli and tan delta (tan 6) after cooling of myosin and
1% myosin/ 1% heat-denatured soluble aggregates of B-LG (myosin/HDLG)
heated at 71°C for 60 min in 0.6M NaCl, 0.05M sodium phosphate buffer,
pH 6.0, 6.5 and 7.0 ........................................................................... 125

LIST OF FIGURES

Figure 4.1. Network structure of protein gels (a) ﬁne stranded network formed by
connection at points; (b) ﬁne stranded network formed by junction zone connection;
(c) ﬁne stranded string of beads; (d) particulate randomly aggregated gel network
.................................................................................................... 10

Figure 4.2. Schematic model of heat-induced gelation of protein (from Foegeding and
Hamann, 1992) ................................................................................ 12

Figure 4.3. Role of sulﬂrydryl/disulﬁde interchange reactions during protein gelation in
the presence and absence of N-ethylmaleimide (NEM) (from Boye et al., 1997) ..... 16

Figure 4.4. Schematic representation of the myosin molecule: HMM, heavy
meromyosin; LMM, light meromyosin; HMM-Sl, subfragment-l; 82, subfragment-
2; DTNB,5,5-dithiobis-(2-nitrobenzoate) light chain (Adapted from Smith et al.,
1983) ............................................................................................. 23

Figure 4.5. Schematic representation of the two step aggregation of B-lactoglobulin at
pH 7.0 (from Aymard et al., 1996) ........................................................... 30

Figure 4.6. Type I ﬁlled gel network showing network formation by one protein with
soluble polymer entrapped inside (from Browney and Morris, 1988) .................. 38

Figure 4.7. Schematic diagrams of possible structure of type 11 mixed gel (1) coupled
network; (2) phase separated network; (3) interpenetrating network (from Browney
and Morris, 1988) .............................................................................. 38

Figure 4.8. Model for linear polymer formation of ovalbumin (from Doi, 1993) ......... 44

Figure 4.9. Development of a particulate gel structure from heat—induced gelation and

a ﬁlamentous gel structure from cold-set gelation of whey protein (from Bryant
and McClements, 1998) ....................................................................... 50

Figure 5.1a. Heating proﬁle for thermal aggregation and rheological studies ............ 60

Figure 5.1b. Derivative curve of protein aggregation for determination of initial

aggregation temperature (To), maximum aggregation rate (ARmax), and temperature
at ARmax (Tm) ................................................................................... 60

xi

Figure 5.2. Heat capacity proﬁles of (A) 1% myosin and 1% B-lactoglobulin (B-LG)
analyzed separately, and (B) a mixture of 1% myosin/ 1% B-LG in 0.6M NaCl,
0.05Msodium phosphate buffer, pH 7.0 .................................................... 63

Figure 5.3. Aggregation of 0.1-1 .5% myosin in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30
min and cooling to 25°C ...................................................................... 65

Figure 5.4. Aggregation of 0.1-1 .5% B-lactoglobulin in 0.6M NaCl, 0.05M sodium
phosphate buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at
90°C for 30 min and cooling to 25°C ........................................................ 68

Figure 5.5. Aggregation of myosin and mixed solutions of myosin with 0.25% B-
lactoglobulin (B-LG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0,
during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling
to 25°C ........................................................................................... 71

Figure 5.6. Aggregation of B-lactoglobulin (B-LG) and mixed solutions of B-LG with
0.1% myosin in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, during heating
at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C ........ 72

Figure 5.7. Storage moduli of 0.5-2.0% myosin in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30
min and cooling to 25°C ...................................................................... 75

Figure 5.8. Storage moduli of myosin and mixed solutions of myosin and 1.0% B-
lactoglobulin (B-LG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, during
heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C.
.................................................................................................... 77

Figure 5.9. Storage moduli of myosin and mixed solutions of 1.0% myosin and 0.5-3%
B-lactoglobulin (IS-LG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0,
during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling
to 25°C ........................................................................................... 79

Figure 5.10. Frequency dependence of storage (G') and loss (G") moduli of 2% myosin,
4% B-lactoglobulin (B-LG) and 1% myosin/ 1% B-LG gels in 0.6M NaCl, 0.05M
sodium phosphate buffer, pH 7.0, measured at 25°C. Gels were prepared by heating
at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C.... . ...81

Figure 6.1. Electrophoregram of (A) nonreduced and (B) reduced native and heat-

denatured B-lactoglobulin: Lane 1, molecular weight markers; Lane 2, heat-
denatured B-lactoglobulin; and Lane 3, B-lactoglobulin ................................ 91

xii

Figure 6.2. Scanning electron microscopic image of heat-denatured B-lactoglobulin
aggregates at 20,000x magniﬁcation. Bar = 1.0 pm ....................................... 93

Figure 6.3. Heat capacity proﬁles of (A) 1.0% B-lactoglobulin (B-LG) and 1.0% heat-
denatured B-LG (HDLG) in deionized water, pH 7.0, and (B) 1.0% HDLG in 0.6M
NaCl, 0.05M sodium phosphate buffer, pH 6.0, 6.5 and 7.0 ............................. 95

Figure 6.4. Storage moduli of B-lactoglobulin in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30
min and cooling to 25°C ....................................................................... 97

Figure 6.5. Storage moduli of 1-3% heat-denatured B-lactoglobulin in 0.6M NaCl,
0.05M sodium phosphate buffer, pH 7.0, during heating at 1 °C/min from 25 to
71°C, holding at 71°C for 60 min and cooling to 25°C ................................... 98

Figure 6.6. Storage moduli of heat-denatured B-lactoglobulin in 0.6M NaCl, 0.05M
sodium phosphate buffer, pH 6.0, 6.5 and 7.0, during heating at 1 °C/min from 25
to 71°C, holding at 71°C for 60 min and cooling to 25°C ................................ 101

Figure 6.7. Scanning electron microscopic image at 5,000x magniﬁcation of 4% B-
lactoglobulin gels heated to 90°C for 30 min (A) and heat-denatured B-
lactoglobulin gels heated to 71°C for 60 min (B) in 0.6M NaCl, 0.05M sodium
phosphate buffer, (1) pH 6.0; (2) 6.5; and (3) 7.0. Bar = 1.0 pm ....................... 103

Figure 7.1. Heat capacity proﬁles of 1.0% myosin and 1% myosin/ 1% heat-denatured
B-lactoglobulin (HDLG) in 0.6M NaCl, 0.05M sodium phosphate buffer, (A) pH
6.0 and (B) pH 7.0 ............................................................................. 115

Figure 7.2. Storage moduli of myosin, 1%myosin/1% B-lactoglobulin (B-LG) and 1%
myosin/ 1% heat-denatured B-LG (HDLG) in 0.6M NaCl, 0.05M sodimn phosphate
buffer, pH 7.0, during heating at 1°C/min from 25 to 71°C, holding at 71°C for 60
min and cooling to 25°C ..................................................................... 117

Figure 7.3. Frequency dependence of storage (G') and loss (G") moduli of 2% myosin,
1% myosin/ 1% B-lactoglobulin (B-LG) and 1% myosin/ 1% heat-denatured B-LG
(HDLG) gels in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, measured at
25°C. Gels were prepared by heating at1°C/min from 25 to 71°C, holding at 71°C
for 60 min and cooling to 25°C ............................................................. 122

Figure 7.4. Storage modulus of 1%myosin and 1%myosin/1% heat-denatured B-
lactoglobulin (HDLG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 6.0, 6.5

and 7.0, during heating at 1 °C/min from 25 to 71°C, holding for 60 min and cooling
to 25°C .......................................................................................... 124

xiii

Figure 7.5. Scanning electron microscopic images of 2% myosin gels heated to 71°C in
0.6M NaCl, 0.06M sodium phosphate buffer, (1) pH 6.0; (2) 6.5; and (3) 7.0 at
5,000 x magniﬁcation. Bar = 1 pm ........................................................ 126

Figure 7.6. Scanning electron microscopic images of (A) 1% myosin/ 1% B-lactoglobulin
and (B) 1% myosin/ 1% heat-denatured B-lactoglobulin co-gels heated to 71°C in
0.6M NaCl, 0.06M sodium phosphate buffer, (1) pH 6.0; (2) 6.5; and (3) 7.0 at 5,000
x magniﬁcation. Bar = 1 pm ............................................................... 129

Figure 7.7. Electrophoretogram of proteins expressed by centrifugation at 10,000 x g
from myosin, B-lactoglobulin (B-LG), heat-denatured [3-LG (HDLG), myosin/B-LG,
and myosin/HDLG gels heated to 71°C and 90°C. Each lane was loaded with 10 uL
of sample: Lane 1, unheated B-LG; Lane 2, unheated myosin; Lane 3, B-LG heated to
90°C; Lane 4, myosin/B-LG gels heated to 90°C; Lane 5, myosin gels heated to 90°C;
Lane 6, HDLG heated to 71°C; Lane 7, myosin/HDLG heated to 71°C; Lane 8,

myosin/B-LG heated to 71°C; Lane 9, myosin heated to 71°C; Lane 10, broad range
standard molecular weight markers ........................................................ 132

Figure 7.8. Total protein in supernatant expressed from myosin, B-lactoglobulin (B-LG),
heat-denatured B-LG (HDLG), myosin/B-LG and myosin/HDLG gels in 0.6M NaCl,
0.05M sodium phosphate buffer, pH 7.0, heated to 71°C for 60 min or at 90°C for 30
min and cooled to 25°C. Values are means of three observations. Means with
different superscripts are different (p<0.05) ............................................... 134

xiv

CHAPTER 1: INTRODUCTION

Heat induced gelation of salt soluble proteins (SSP) in meat is an important
functional property responsible for texture, water holding, binding and appearance of
meat products (Smith, 1994). Many studies have been performed to understand muscle
protein gelation mechanisms, so that textural qualities and yields of meat products can be
modiﬁed and controlled. Non—meat proteins can be used to substitute for muscle proteins
by maintaining desired texture of meat products and controlling costs.

Whey proteins possess high nutrient and functional properties and can form
emulsions, foams and heat-induced gels. Whey proteins are widely used by the poultry
and meat industries as non-meat binders to enhance yield and textural quality of their
products. However, results from the use of whey protein in meat products are highly
variable due, in part, to differences in source and processing history of whey proteins and
to differences in processing conditions of the meat products in which the whey proteins
are added. Functions of whey proteins in meat systems need to be deﬁned and
characterized to provide direction for designing or modifying whey protein with optimum
properties needed by the meat industry.

Several researchers have attempted to elucidate the heat induced gelling
mechanisms of whey proteins. The studies looked at factors affecting gel properties such
as heating temperature, heating rate, pH, ionic strength, ion type, protein concentration,

other food components i.e. salt and sugar, and solubility of whey protein (Kuhn and

F oegeding, 1991; Beuschel et al., 1992; Xiong et al., 1993; Smith and Rose, 1995; Boye
et al., 1997). Some studies have investigated the interaction and inﬂuence of individual
whey proteins i.e. B-lactoglobulin (B-LG), or- lactalbumin (or-LA) and bovine serum
albumin, on gel properties (Hines and Foegeding, 1993; Matsudomi et al., 1993, 1994).

Other studies have looked at mixed gel systems of salt soluble proteins and whey
protein concentrates (WPC). The solubility of WPC affects meat protein gelation. The
hardness and deformability of a co-gel of 4% SSP and 12% WPC heated at 65°C were
enhanced using WPC of low solubility, whereas mixtures of highly soluble WPC
increased the hardness and deforrnability of gels heated to higher temperatures (Beuschel
et al., 1992). Hung and Smith (1993) reported that WPC shifted the transition
temperatures of storage and loss modulus (G' and G") of SSP to a higher temperature and
increased G' and G" magnitudes of gels heated to 90°C. In isothermal experiments at
65°C, the elasticity of a combination gel of SSP and low solubility WPC was higher than
those contain highly soluble WPC. The microstructure of gels prepared from highly
soluble WPC and SSP was ﬁbrous at 65°C and globular at 90°C. The proportion of each
protein and the concentration of NaCl were found to inﬂuence the gelling properties of
SSP and WPC co-gels (Smith and Rose, 1995).

In the past few years, cold-set gelation of heat-denatured soluble whey protein
aggregates have been intensively investigated (Barbut and Foegeding, 1993; McClements
and Keogh, 1995; Hongsprabhus and Barbut, 1997a; 1997b; Ju and Kilara, 1998b;
1998c). Heat-denatured whey protein aggregates form gels at low temperature upon

addition of salts, acid or proteases (Ju and Kilara, 1998b). Gelling properties of

I .

 

heat-denatured whey protein aggregates are greatly inﬂuenced by heat treatment and
environmental conditions both during preheat treatment and gelation. Cold-set gels were
reported to be stronger and less opaque than heat-set gels (Bryant and McClements,
1998)

Preheated whey protein could be a very useful ingredient in many food products,
especially in low heat processed foods. Recently, the use of heat-denatured whey proteins
in poultry meat batters has shown promising results (Hongsprabhus and Barbut, 1999).
Soluble aggregates of heated-denatured whey protein were produced by heating a solution
of 10% WPC in distilled water, pH 7.0, at 80 °C for 30 min. Two percent substitution of
heat-denatured whey protein in combination with cold-set gelation (16h at 10°C) was
found to improved the texture parameters of raw and cooked poultry meat batters,
especially at S 1.5% salt.

Interactions involved in formation of mixed gels from heat-denatured whey
proteins and SSP remain unclear because of the multi-component nature of the protein
preparations. Studies using pure proteins are needed to simplify the models and allow for
a greater understanding of the mechanisms of co-gel formation. Myosin and B-LG are the
major functional components of meat and whey, respectively. Use of these proteins in
model systems is expected to ease the interpretation of the results. The objective of this
project was to investigate the effects of native B-LG and heat-denatured B-LG on the

heat-induced gelation of myosin in mixed systems.

CHAPTER 2: OBJECTIVES

The overall goal of this project was to investigate the mechanisms involved in the
formation of myosin and B-lactoglobulin (B-LG) co-gels. The effects of B-LG and heat-
denatured B-LG aggregates (HDLG) on the gelation of myosin were also examined. Our
speciﬁc objectives were to:

1. Study interactions between B-LG and myosin during heating by comparing the
denaturation proﬁles (endotherms), aggregation patterns, rheological properties, and
microstructures of mixed protein systems to those of each pure protein;

2. Compare the gelling properties of HDLG to gels formed from native B-LG; and

3. Compare the rheological and microstructural properties of co-gels formed from
myosin/HDLG to those of myosin/B-LG gels.

In Chapter 5, studies of thermally induced gelation of myosin, B-LG and the protein
mixtures were studied. The interactions between myosin and B-LG in the mixed systems
were interpreted by comparing the denaturation, aggregation and rheological properties of
the mixed proteins to those of each pure protein. Results from this experiment led to the
study of heat-denatured B-LG presented in Chapter 6. Thermal denaturation, rheological
properties and microstructures of native and preheated B-LG were compared. The
gelation mechanisms of mixed systems of myosin/native B-LG and myosin/HDLG were

investigated in Chapter 7. Comparison of their thermal denaturation, rheological and

microstructural properties led to an understanding of the inﬂuences of HDLG on myosin
gelation. An understanding of the functional properties of HDLG, mechanisms of heat-
induced gelation of mixed protein systems of myosin with B-LG and HDLG were
integrated to develop suggestions for modifying whey protein to achieve the optimum

functional properties needed by the meat industries.

— 1 \ .H"'..A..

 

CHAPTER 3: SIGNIFICANCE AND JUSTIFICATION

Heat induced gelation is the most signiﬁcant functional property of muscle
proteins and is responsible for the textural quality of many meat products. Protein gels are
characterized as a three-dimensional network of partially unfolded protein, which retains
liquid within the network to form a Viscoelastic solid. Whey proteins may interact with
muscle proteins through a variety of mechanisms to enhance water holding capacity,
reduce cook loss and improve textural qualities of processed meat products. Several
mechanisms may be responsible for these functions:

1) Whey proteins dissolved in the entrapped liquid phase of the muscle protein
gel may result in increased water retention and viscosity. This more viscous
phase enhances strength of the protein network.

2) Whey protein has the ability to form heat induced gels, thus it might form a
second gel network within the muscle protein gel structure (phase separated
network) or form an interpenetrating network. These networks will strengthen
the ﬁnal gel matrix.

3) Whey proteins inﬂuence the formation of the three dimensional muscle
protein networks leading to an alteration in the myosin matrix.

The effects of whey proteins on textural quality and yield of meat products have

been extensively studied. The results were highly variable due to differences in source

and processing history of the whey, which affects protein composition and gelling

properties of the proteins. The processing conditions and formulations (salt and pH) of
meat products in which the whey proteins were added also greatly affected the gelation of
the proteins. Muscle proteins gel at about 55-70°C which are typical processing
temperatures for many meat products. Meanwhile whey proteins need to be heated over
80°C to form a gel. Therefore, gel enhancing effects of whey protein are limited when
used in meat products. The mechanism of interaction between whey proteins and muscle
proteins has never been completely understood.

Heating whey protein in a dilute salt solution at neutral pH denatures whey
proteins and produces soluble aggregates. These soluble whey protein aggregates can
form cold-set gels at ambient or refrigeration temperatures. Preheated or heat-denatured
whey proteins were proposed for use in food products processed at temperatures lower
than the gelling point of native whey. Some examples of these products are comminuted
meat and ﬁsh products, deserts, sauces and dips (Bryant and McClements, 1998).

In this project, we focused on the ability of B-lactoglobulin (B-LG) and heat-
denatured B-LG to form a gel network and/or inﬂuence the gelation of myosin. The
stability of a gel depends on the strength of the protein matrix, which is determined by the
size, shape, concentration and cross-linking of the protein polymers. Hence, it was very
interesting to investigate network formation in a mixed gel system to understand how
whey protein inﬂuences the gelation of meat proteins. This information on gelling
behavior of whey and meat protein could provide signiﬁcant new basic knowledge for

further improving the functional properties of whey proteins for expanded use in meat

products.

Both muscle proteins and whey proteins are multi-component systems. The large
number of proteins in each system may cause confusion when interpreting results. To
minimize these problems, we used myosin and B-LG in a model system. Myosin and B-
LG form strong heat-induced gels and are the major protein components of meat and

whey, respectively.

CHAPTER 4: LITERATURE REVIEW

4.1. Heat induced gel formation of protein

4.1.1. Definition and types of protein gels. Protein gels have been described as
three dimensional matrices or networks in which polymer-polymer and polymer-solvent
interactions occur in an ordered manner resulting in the immobilization of large amount
of water by a small proportion of protein (Flory, 1974; Hermaanson, 1979; Mulvihill and
Kinsella, 1987). Heat induced gel formation is one of the most important functional
properties of many proteins such as egg white proteins, plant proteins, milk proteins and
meat proteins. Functional prOperties contribute to desired textural characteristics and
quality of various kinds of food products containing these proteins.

Protein gel structure may be categorized into 2 main types: ﬁne stranded networks
and particulate aggregates (Figure 4.1). A ﬁne stranded gel is usually formed by long
chain structure or ﬁbrillar protein, such as muscle protein and gelatin, whereas most
globular proteins, such as whey and egg white proteins, can form both gel types
depending on gelling condition. The protein network of the ﬁne strand gels is formed by
cross-linking at points where the interaction occurs between speciﬁc locations of

molecules (Figure 4.1a), or connection at junction zones which occurs via association

(a) (b) (c) (d)

 

 

 

 

 

 

 

 

 

Figure 4.1. Network structure of protein gels (a) ﬁne stranded network formed by
connection at points; (b) ﬁne stranded network formed by junction zone connection; (c)

ﬁne stranded string of beads; (d) particulate randomly aggregated gel network.

between segments of molecules (Figure 4.1b) (Matsumura and Mori, 1996). Tombs
(1974) described ﬁne strand gel formation of globular proteins as a highly oriented string
of beads (Figure 4.1c), whereas random aggregation is responsible for the particulate
structure (Figure 4.1d). The pore size of a protein network was suggested as a way to
distinguish the type of gel. Fine strand gels have pore sizes in the order of nanometers,
whereas pore sizes of particulate gels are in the micrometer range (Langton and

Hermansson, 1992).

4.1.2. Mechanism of heat-induced gel formation. Protein gel formation has
been suggested to be comprised of two main steps. The ﬁrst step involves thermal
unfolding or denaturation of protein molecules. The second step is the aggregation
process of the unfolded proteins resulting in a development of a cross-linked gel network
with Viscoelastic properties (Ferry, 1948). Foegeding and Hamann (1992) proposed a
theoretical model, listing a series of steps within the aggregation process leading to ﬁnal
gel structure (Figure 4.2). In this model, proteins unfold and aggregate to form a
progressively more viscous solution. Once the gel point is reached, a continuous
Viscoelastic gel network is formed. The Viscoelastic properties of the gels may change
with time to form an equilibrium matrix (Smith, 1994).

The gelling process and ﬁnal gel characteristics or properties depend on both
intrinsic and extrinsic factors that signiﬁcantly inﬂuence each step throughout the heat
induced gelation of the protein. The gelling ability of a protein is inﬂuenced by its
concentration, amino acid composition, size, and shape. Meanwhile, the development of

the protein network, governed by the denaturation and aggregation properties, is dictated

ll

Native heat Protein time Protein-protein

 

 

proteins ’ unfolding * interaction
Gel point

Primary time _Equilibrium

matrix 7 matrix

Figure 4.2. Schematic model of heat-induced gelation of protein (from Foegeding and

Hamann, 1992)

12

by the processing conditions, including length and rate of heating and cooling, and
environmental factors, such as ionic strength, pH and buffer composition (Mulvihill and
Kinsella, 1987). The textural properties of gels are largely inﬂuenced by the interactions

between proteins which ultimately cross-link to develop gel network structures.

4.1.3. Protein-protein interaction in gels. A gel matrix is formed via the
assembly of individual molecules into extensive three-dimensional networks. This
assembly requires a balance of attraction and repulsion between protein molecules.
Normally, denaturation or partial unfolding and, in some cases, dissociation of the native
protein subunits, is a prerequisite. The molecular interactions between proteins and their
general characteristics are summarized in Table 4.1.

Heat treatment causes proteins to unfold and expose some hydrophobic residues
which become available for intermolecular bonding and network formation (Oakenfull et
al., 1997). Shimada and Matsushita (1980) showed a correlation between hydrophobic
amino acid content of proteins and a subsequent gel formation. They found that proteins
containing more than 31.5 mole percent of non—polar amino acid residues formed
coagulated type gels, whereas those containing less than this level of non-polar residues
formed transparent gels. However, the hydrophobic amino acid content of a protein is not
the only factor affecting its gelation. The amino acid sequence and local conformation
also inﬂuence protein interactions. Physical/chemical treatments and buffer conditions
can alter the surface hydrophobic areas of proteins and therefore inﬂuence intermolecular

hydrophobic interactions.

Table 4.1. General characteristics of molecular interactions between proteins in aqueous

solution (adapted from Bryant and McClements, 1998)

 

 

Type Force Relative strength Effect Effect of Effect of
direction of pH ionic temperature
strength
Hydrophobic attractive Strong no no Increase
Electrostatic repulsive weak—)stronga yes yes Increase
Hydrogen attractive Weak no no Decrease
bonding
Van der Waals attractive Weak no no -
Steric repulsion repulsive Strong no no -
Disulﬁde bond attractive Weak no no -

 

" Depends on pH and ionic strength

14

It has been suggested that heat treatment results in cleavage of existing disulﬁde
bonds or activation of buried sulfhydryl groups through the unfolding of the protein.
These sulfhydryl groups then can form new intermolecular disulﬁde linkages which are
essential for heat induced gel formation in some globular proteins (Boye et al., 1997).
The role of disulﬁde/sulfhydryl interchange reactions in the cross-linking of protein gels
is illustrated in Figure 4.3.

Electrostatic interactions depend on the exposure of charged groups on the
protein. The surface charge of protein molecules is affected by both the presences of
dissolved salts and solvent pH. Electrostatic repulsion contributes to the dispersion of
protein molecules in solution. The charge repulsion of proteins is enhanced as the
solution pH is adjusted away from the isoelectric point. However, the addition of salts
can alter the net charge of the protein by shielding the excess charges and leading to
inhibition of the exposure of hydrophobic residues buried within the proteins. Divalent
cations may form cross-bridges between adjacent protein molecules contributing to

coagulation and/or gelation.

4.1.4. Methods to study heat-induced gel formation. The gelling mechanisms
of proteins have been investigated by various biochemical and physicochemical
techniques. The choice of appropriate methods is important for obtaining desired results.

The sensitivity of instruments is a determining factor when selecting experimental

conditions.

 

NATIVE PROTEIN

 

 

HEAT
NO ADDITIVE NEM
‘32 6‘6 CYSTEIN E S-NEM
N EM
'8'3- -S-H
«Q .S-S-

 

 

 

Figure 4.3. Role of sulﬂiydryl/disulﬁde interchange reactions during protein gelation in

the presence and absence of N-ethylmaleimide (N EM) (from Boye et al., 1997).

16

4.1.4.1. Thermal denaturation. Differential scanning calorimetry (DSC) is most
commonly used to follow protein unfolding during heating. With this technique,
thermodynamic parameters related to thermally induced conformational changes of
proteins are measured during various rates of heating (Krishnan and Brandts, 1978).
Changes in heat capacity of a protein solution with temperature are recorded as a
thermogram. The temperature at which 50% of the molecule has been unfolded is called
the melting temperature for a single domain protein.

Spectroscopic techniques, such as Fourier transform infrared spectroscopy,
Raman spectroscopy, nuclear magnetic resonance and circular dichroism also have been
reported as tools for studying changes in protein secondary structure (Nonaka et al., 1993;
Li et al., 1994; Matsuura and Manning, 1994). Monoclonal antibodies have been used as

precise probes for speciﬁc conformational changes during unfolding of protein (Wang

and Smith, 1994c).

4.1.4.2. Thermal aggregation. Protein aggregation has been followed using
turbidity measurements, light scattering or other forms of spectroscopy. Electrophoresis
and chromatography are useful tools for investigating bond formation of proteins during
thermal aggregation. The most convenient way to follow thermal aggregation is by
measuring turbidity. With this technique, the differential change in optical density as a
function of temperature is used to determine the initial temperature of aggregation
(thermal transition: To) and transition peak temperature (Tm) of protein-protein

association (temperature at which protein-protein interaction was maximum) (Xiong et

aL,1993)

17

In dynamic light scattering, a laser beam is used to detect molecular size of
particles. The Brownian motion of particles results in a time-dependent ﬂuctuation in the
scattering intensity. The auto-correlation function of ﬂuctuating intensity is derived into
the diffusion coefﬁcient of the scattering particles, providing information of the
hydrodynamic radius of particles. Hoffmann et a1. (1996) studied the effects of
temperatures, salt type and concentration, and initial protein concentration on heat-
induced aggregation of B-lactoglobulin (B-LG) using in situ dynamic light scattering (a
measurement of scattering light from a sample while it is heated in the instrument). The
results showed that aggregate size and aggregation rate of B-LG increased when the
protein concentration was increased. Increasing the heating temperature from 61.5 to
70.0°C resulted in an increase in initial aggregation rate of B-LG. The aggregate size
increased when the concentrations of salts were increased from O to 10 mM. The
aggregate size (30 nm) of B-LG was similar when heated at 685°C for 21h in either 0.5

mM CaCl2 or 10 mM NaCl.

4.1.4.3. Viscoelastic network formation. The formation of a gel network, as
deﬁned by the phase transition of a protein sol to gel upon heating, can be observed by
small deformation dynamic testing. This technique involves continuously recording the
changes in Viscoelastic properties of protein solutions at small deformation, thus avoiding
interruption of network formation during measurement. The complex shear modulus
(G*) is resolved into two components. The storage modulus (G') is a measurement of the

elastic properties, and loss modulus (G") is a measurement of viscous properties of the

18

material. The transition from sol to gel can be monitored by the change in these two
parameters. The phase transition temperature or gel point is usually deﬁned as the
temperature at which the G' and G" crosses over (Stading and Hermansson, 1990). The
ratio of G"/G' or tan delta (tan 6)) is another parameter used to describe Viscoelastic
behavior. Polymer systems show high tan 5 for dilute solutions, 0.2-0.3 for amorphous

polymers and low (near 0.01) for glassy crystalline polymers and gels (steffe, 1996).

4.1.4.4. Final gel structure. The microstructure of the ﬁnal gel matrix is often
studied by light microscopy, transmission electron microscopy, and scanning electron
microscopy. The transmission electron microscopy is relevant for description of a dense
ﬁne-stranded structure, whereas scanning electron microscopy or light microscopy is
better for observing a coarse particulate structure (Hermansson et al., 1993). However,
SEM observation is preferable to TEM because of the easier specimen preparation and
more realistic imaging (three-dimensional imaging) due to the higher depth of ﬁeld.

Concomitantly with electron microscopic observation, ﬁnal gel strength and
character are usually evaluated by large deformation or destructive studies. In large
deformation testing, apparent strain and stress at failure by compression and stress and
strain at failure by torsion analysis are commonly used in model gel systems. Stress at
failure can indicate the ﬁrmness or hardness of the gel and strain at failure is an indication
of gel deformability (Hamann et al., 1987).

Type of gel can be characterized by frequency dependence of the complex shear

modulus (G*) (Clark and Ross-Murphy, 1987). The slope of log-log plots between G' or

19

G* and frequency usually indicate strength of gels. The slope value of a strong gel is less
than that of a weak gel. The G* of a strong cross-linked gel is frequency independent,
whereas G* is slightly dependent on frequency for a physically entanglement gel
network. Stading et a1. (1992) discriminated homogeneous and heterogeneous B-LG gel
structures, formed at different pHs, based on the frequency dependence of G'. An in-
homogeneous network structure containing regions of different density showed a two-
slope frequency dependence (broken) curve, one at low frequency and another at high
frequency. The homogeneous structure was frequency independent and exhibited a

straight line over the frequency sweep range.

4.2. Myosin

There are three fractions of proteins in skeletal muscle: myoﬁbrillar, sarcoplasmic
and stromal. Myosin and actin, deﬁned as contractile proteins, account for about 50%
and 20%, respectively, of the myoﬁbrillar fraction (Obinata et al., 1991; Yates and
Greaser, 1983). Myosin is the most abundant myoﬁbrillar protein in skeletal muscle. It

is also mainly responsible for functional properties of processed meats (Smith, 1988).

4.2.1. Molecular characteristics. Myosin is a highly asymmetric protein
molecule with dimensions of about 150 nm in length and 1.5-2.0 nm diameter in the rod
portion and 8 nm diameter in the globular head (Peason and Young, 1989). Myosin is
composed of two heavy chains (MHC) and two pairs of light chains. Each MHC of

chicken pectoralis myosin is constructed from 1,938 amino acids with a molecular weight

20

of 222.971 KDa (Hayashida et al., 1991). The amino-terminal end of MHC forms the
globular or head region, whereas the remainder of the MHC forms the rod or tail region.
In nature, the rod regions of the two heavy chains form a dimeric, extended coiled-coiled
helical structure.

Each pair of light chains (LC) of myosin is non-covalently associated with the
head of the molecule (Smith et al., 1983). Myosin LC can be divided into 2 types based
on releasing reagent: alkaline LC and 5,5’-dithiobis-(2-nitrobenzoate) or DTNB LC. The
alkaline LC is released from myosin by treatment in an alkaline solution, pH 11. These
LC are non-phosphorylated and are essential for ATPase activity (Pearson and Young,
1989). They are designated as alkaline LCl (A1) or LC-l and alkaline LC2 (A2) or
LC-3. Chicken skeletal muscle myosin LC-l and LC-3 contain 192 and 150 residues
with molecular mass of 20.913 and 16.710 KDa, respectively (Maita et al., 1981;
Umegane,et al., 1982). DTNB LC or LC-2 are dissociated from the myosin molecule by
the sulfhydryl reagent 5,5’-dithiobis-(2-nitrobenzoate) and are not essential for ATPase
activity, but affect the calcium binding ability of myosin. The LC-2 of chicken skeletal
muscle myosin contains 166 amino acid residues with a molecular mass of 18.673 KDa
(Suzuyama et al., 1980).

Myosin can also be divided into sub-fragments by enzymatic digestion. With
limited digestion by trypsin, myosin is cleaved into two subfragments: heavy
meromyosin (HMM) and light meromyosin (LMM) (Lowey et al., 1969). In the same
way, papain hydrolyzes myosin into subfragment-l , which comprises the two globular

heads of myosin, and a rod region, which contains the or-helix (tail) of the molecule.

21

When digested by papain, HMM is cleaved into two subfragments: subfragment—l and
subfragment-Z, which is the rod region of HMM (Figure 4.4) (Smith et al., 1983). The
isoelectric point of myosin is about 5.3 reﬂecting its high content of aspartic and glutamic

acid residues (Harrington, 1979).

4.2.2. Heat induced gel formation of myosin. Myosin has a multi-domain structure
resulting in a very complex denaturation and aggregation process. The mechanisms of
myosin gelation during heating have been extensively studied. During the heating
process, myosin gel network is ﬁrst formed between 30-44°C by the rod region. The
second step of gel formation is attributed to the head portion of myosin between 51-80°C
(Zayas, 1997). Sano et al. (1990) reported that the aggregation and sol-gel transition of
LMM (extracted from the dorsal lateral muscle of live carp) in 0.6M KCl, pH 6.0
occurred at 32-50°C, whereas HMM aggregated above 45°C. The role of six myosin sub-
fragments (LC, S-l, HMM, LMM, rod, and MHC) in gel network formation of chicken
breast muscle myosin in 0.6M NaCl, 0.05M phosphate buffer pH 6.5 were studied by
Smyth et al. (1996). Thermal stability and thermal aggregation proﬁles showed that the
rod was the main fragment involved in myosin gelation. LMM and S-l were responsible
for denaturation and aggregation below 55°C, while S-2 unfolded and aggregated above
55°C. The alkaline LC was less heat stable than the DTNB LC and neither aggregated
during heating, suggesting that LC themselves do not gel. However, the thermal stability

and aggregation of myosin were found to be inﬂuenced by LC when bound to MHC.

22

 

l!

.71—

350,000 HMM "1

Alkali light chain

‘F— 125,000 LMM
115,000

2M1 DTNB light cha\i:@) Wm
T / J® ——1

 

 

 

Trypsin Papain
S2
j.— 40 nm j
Myosin Rod
f 150,000 '1
: 150 um I

 

 

 

 

 

Figure 4.4. Schematic representation of the myosin molecule: HMM, heavy
meromyosin; LMM, light meromyosin; HMM-S], subfragment-l; 82, subfragment- 2;

DTNB, 5,5-dithiobis-(2-nitrobenzoate) light chain (Adapted from Smith et al., 1983).

23

4.2.3. Factors inﬂuencing myosin gelation. The gelling mechanism of myosin
is also inﬂuenced by other factors, including protein concentration, environmental
conditions, and heat treatment. These factors are known to have signiﬁcant effects on

both gel formation and the ﬁnal gel characteristics of myosin.

4.2.3.1. Protein concentration. As a gel network is formed by the
interaction of denatured proteins, the amount of protein needs to be large enough to form
intermolecular bonds. At low concentrations, proteins tend to form intramolecular bonds
resulting in protein precipitation instead of a gel network. The critical or minimum
concentration to form a gel depends on the molecular size and structures. Wang and
Damodaran (1990) demonstrated the importance of polypeptide chain length on network
formation of protein gels. They suggested that increasing the chain length of the protein
increases molecular entanglement and reduces the diffusion coefﬁcient of the gel
structures, therefore increasing the total amount of stable bonds formed between the
peptides per unit cell of the gel. Myosin is a ﬁbrillar protein having a long tail (~130 nm)
that facilitates contact and intermolecular binding, thus promoting interactions. Therefore,
the critical concentration of myosin to form a self supporting gel structure is quite low
when compared to globular proteins, such as B-LG, which has a diameter of 2-3 nm.
Acton et al. (1983) reported that actomyosin required at least 0.6% protein to form a self
supporting gel network in 0.6M KCl, pH 6.0, whereas Paulsson et a1. (1986) suggested
that the minimum concentration needed for B-LG gel formation is about 2% at pH 6.6.

Above the critical concentration, hardness of myosin gels increased with increasing

24

protein concentration as a result of an increase in the number of cross-links within the gel

matrix (Zayas, 1997).

4.2.3.2. Environmental conditions. The pH and ionic strength of the
protein environment have been reported to be the most important factors that inﬂuence
myosin gelation. Charge distribution among amino acid side chains of the myosin
molecule is altered by pH and ionic strength, and hence the attractive and repulsive forces
between molecules are changed. This event subsequently affects the type and stability of
the ﬁnal gel. Hermansson et a1. (1986) reported that bovine semimembranosus myosin
formed two types of gels depending on the ionic strength of the solution. At low ionic
strength (0.1-0.2 M KCl and pH 6.0), myosin formed a thicker ﬁlament and ﬁner gel
matrix than those formed at higher ionic strength (0.6M KCl and pH 6.0). Boye et a1.
(1997) studied thermal gelation of myosin from fast and slow switch rabbit muscles and
found that the direct suspension of protein at a low ionic strength (0.2 M KCl, pH 6.0) led
to very weak gels, whereas a gradual lowering of ionic strength, from 0.6 M KCl to 0.2 M
KCl, by a dialysis technique, produced strong heat induced gels. Maximum gel rigidity

of myosin from chicken breast was found at pH 5.4 to 5.9 in 0.6 M KCl (Asghar et al.,

1984; Morita et al., 1987).

4.2.3.3. Heat treatment. Time, temperature and the rate of heating during
preparation inﬂuence the gelation of myosin. The maximum rigidity of myosin gels
occurs when heated to 65°C in 0.2 and 0.6 M KCl at pH 5.5 and 6.0, respectively (Zayas,

1997). In this temperature range, incubation time is an important factor determining

25

completion of network formation. However, protein concentration will affect the time
period for gel formation. Zayas (1997) suggested that above a critical concentration, the
time necessary to form a gel decreases as protein concentration is increased. Wang and
Smith (1994a) studied the dynamic rheological properties of chicken breast myosin
during isothermal heating at 55, 65 and 75°C in 0.6M NaCl at pH 6.5. They found that
myosin heated at 75°C had higher viscous-like properties as indicated by a higher G",
while myosin heated at 55°C and 65°C formed elastic-like gels. During heating, a three-
dimensional protein network is formed by aggregation of partially unfolded proteins.
Since myosin is a multi-domain molecule, denaturation and aggregation during gel
formation is very complex.

Network formation of strong gels occurs when the proteins are heated at
appropriate heating rates that allow the orientation of unfolded molecules and facilitate
the aggregation process. Foegeding et al. (1986) studied the effect of heating rates on
thermal gelation of myosin and found that myosin gels prepared at a heating rate of 12
°C/h were harder than those prepared at a heating rate of 50 °C/h. Camou et al. (1989)
reported that after cooling to 1°C, a higher force was needed to compress salt soluble

protein gels heated at a rate of 17 °C/h when compared to those prepared by heating at 38

or 85°C/h.
4.3. B—Lactoglobulin

The proteins found in whey include B-LG, a-lactalbumin (or-LA), bovine serum

albumin, immunoglobulins and a proteose-peptone fraction. Among these components,

26

B-LG is the predominant protein, constituting up to 50% of total whey proteins, and plays
the major role in forming heat induced gels (Swaisgood, 1982; Mulvihill and Kinsella,
1987). There are seven known genetic variants of bovine (Bos taurus) B-LG. The most

common in milk are variants A and B (Eigel et al., 1984).

4.3.1. Molecular characteristics. [3- LG is a globular protein with a radius of
about 2-3 nm. It has a single polypeptide chain of 162 residues. The A variant has a
monomeric molecular mass of 18.363 KDa (Oakenfull et al., 1997). The isoelectric point
of B-LG is around 5.2. Under physiological conditions, B-LG exists as a dimer consisting
of 10-15% oc-helix, ~50% B-sheets, 20% turns and the remaining 15% in a random
arrangement (Timasheff et al., 1966; Casal et al., 1988). Below pH 3, B-LG has been
reported to exist as a monomer. It reversibly aggregates to form octamers between pH
3.7-6.5. Above pH 9.0, the protein undergoes time dependent, irreversible denaturation
and aggregation (Boye et al., 1996). Native monomers contain two disulﬁde bridges
between C66/C160 and C106/C119, and one free sulthydryl group (SH-group) at C121
(Creamer et al., 1983; Papiz et al., 1986). The disulﬁde bonds and free SH-group play an

important role in heat-induced gel formation of B-LG.

4.3.2. Heat induced gelation. The heat-induced gelation of B-LG has been
extensively investigated in many laboratories. The mechanism of gelation during heating
involves two main steps; 1) thermal dissociation of dimers into monomers and unfolding

of monomers, and 2) the aggregation of unfolded proteins resulting in network formation.

27

Denaturation of B-LG during heating has been studied under various conditions
and by using different methods, such as DSC, Fourier transform infrared spectroscopy,
nuclear magnetic resonance, circular dichroism and other spectroscopic techniques (Li et
al., 1994; Matsuura and manning, 1994; Qi etal., 1995; Iametti et al., 1996). When
heated above 25°C, native B-LG dissociates into monomers which are further unfolded as
temperature is increased. This step is irreversible at temperatures higher than 65°C
(Iametti et al., 1996).

Sawyer (1968) reported a two step aggregation process for B-LG at neutral pH. It
was suggested that small aggregates were ﬁrst formed by the unfolded proteins which
further associated into bigger aggregates by physical interaction. More details on the
aggregation of B-LG were found using light scattering techniques, size exclusion
chromatography and electrophoresis (Grifﬁn et al., 1993; Roefs and de Kruif, 1994;
Aymard et al., 1996; Hoffmann et al., 1996 Prabakaran and Damodaran, 1997). During
heating, the two step aggregation process began with the formation of small aggregates of
disulﬁde linked B-LG polymers from molten globules. Prabakaran and Damodaran
(1997) suggested that sulfhydryl-disulﬁde interchange reactions between two reactive
monomers initiated further propagation of B-LG polymerization at 60-65°C. In the
second step, the small aggregates formed in the initial stage associated into larger
aggregates by physical interactions. The rate of aggregation depended on heating
temperature, pH and concentration of the monomeric form of B-LG. The size of
aggregates depended on pH, salt type and concentration, and initial concentration of B-

LG (Elofsson et al., 1996; Hoffmann et al., 1996). Roefs and De Kruif (1994) predicted

28

the size of B-LG aggregates to be a proportional to the square root of the initial protein
concentration by using a kinetic model obtained in low salt (<1.0%) conditions.
Aggregates formed in the early stage were suggested to be small rod-like particles
(Grifﬁn and Grifﬁn, 1993). However Aymard et a1. (1996) claimed that the shape of
aggregates was not rod-like, but globular. They found that monomer concentration
decreased by a 2 order kinetic model indicative of the association of monomers into
dimers before formation of the globular aggregates. The globules ﬁnally aggregated to
form fractal structures. The two-step aggregation mechanism of B-LG during heating is
shown in Figure 4.5.

An important chemical reaction during the early stage of B-LG aggregation is the
formation of intermolecular disulﬁde bonds. During thermal denaturation, the cleavage
of disulﬁde bonds and exposure of reactive SH-groups on the surface of the unfolded
proteins facilitate intermolecular disulﬁde bonds and sulfhydryl/disulﬁde interchange
reactions (Shimada and Cheftel, 1988). A sulﬂrydryl-blocking agent such as N-
methylmaleimide (NEM) has been used to demonstrate the effect of disulﬁde bonding on
protein gelation. When NEM was added to a solution of B-LG, heat-induced gel hardness
increased slightly after heating at concentrations up to 5 mM NEM, and then rapidly
decreased when the concentration of NEM exceeded 10 mM (Phillips et al., 1994). Non-
covalent interactions, such as electrostatic, hydrophobic and hydrogen bonding, also play
a major role in the aggregation of B-LG. Aggregation of B-LG leads to gel network
formation. The initial gelling temperature of B-LG is found over a broad range,

depending on protein concentration, pH and salt conditions. Paulsson et a1. (1986)

29

 

Globulc

 

native "denatured"
.. o omer
”“2“" m 'iQ. g. su/s-s
w 'I‘ > 50°C .
native
dimer denatured

dimer

fractal
aggregate

 

 

 

 

Figure 4.5. Schematic representation of the two step aggregation of B-lactoglobulin at

pH 7.0 (from Aymard et al., 1996).

30

reported that 3-9% B-LG in 1% NaCl, pH 6.6 started to gel at 75-80°C.

4.3.3. Factors inﬂuencing B-lactoglobulin gelation. The appearance and
properties of B-LG gels strongly depend on protein concentration, medium conditions,
and heat treatments used in the preparation. The combination of these conditions
inﬂuences the denaturation and aggregation mechanisms during gel formation, which
further alter the ﬁnal gel characteristics. Gels formed under different combinations of pH
and salt concentration may vary from weak to strong, brittle to elastic in texture or

opaque to transparent in appearance.

4.3.3.1. Protein concentration. Increasing the protein concentration
increases the probability of intermolecular interactions, therefore promoting gel
formation. The minimum concentration of B-LG needed to form a gel is highly pH
dependent, with the minimum at the isoelectric point (Stading and Hermansson, 1990).
The critical concentration of B-LG needed to form a gel at temperatures over 80°C was
2.0% in 1.0% NaCl, pH 6.6 (Paulsson et al., 1986), or 5.0% in 0.1M NaCl, 0.02M
imidazole buffer, pH 7.0 (McSwiney et al., 1994) and pH 8.0 (Matsudomi et al., 1991).
The initial gelling temperatures decrease and the gelling rates increase with an increase of
rotein concentration. Above the critical concentration, the gels become more rigid and

opaque as protein concentration was increased.

31

4.3.3.2. Protein modification. B-LG can be modiﬁed to alter the gel point
and rheological properties of the protein. Chen et al. (1994) found that partially
hydrolyzed B-LG gelled more rapidly at the same protein concentration at 80°C. The G'
after cooling of the gels formed from partially hydrolyzed B-LG (7% protein in 20 mM
CaClz, pH 7.0) was twice less than that of the native protein gel heated at 80°C for 3h.
However, when heated at 60°C, the G' of hydrolyzed B-LG gel was 10 time higher than
that of the intact B-LG. Rector et al. (1991) investigated the gelling properties of B-LG
preheated with hot air at 80°C. Preheating decreased the concentration of monomeric [3-
LG and the hardness (force needed for 80% deformation) of the preheated protein gels

(10% protein, 0.1M Tris-HCl buffer, pH 8.0).

4.3.3.3. Environmental conditions. B-LG forms a gel upon heating, and
develops different network structures depending on pH, ionic strength, and type of salt.
The change in pH alters hydrogen bonding and electrostatic interactions, while salts
might neutralize charge and increase non-polar interactions of proteins to further induce
local unfolding or aggregation of B-LG. Xiong et al. (1993) demonstrated the sensitivity
of B-LG aggregation to pH and ionic environment by following turbidity changes during
heating. The aggregation proﬁles of B-LG during heating were displayed by plotting
turbidity changes over time (dA600/dT) against temperature. The maximum aggregation
rate was determined at the transition peak and temperature at the peak was deﬁned as
transition peak temperature. The results showed that an increase in pH from 5.5 to 6.5

decreased the aggregation rate of 0.12% B-LG in distilled water as indicated by a

32

decrease of the transition peaks. Addition of CaCl2 (5-20 mM) decreased transition peak
temperature, indicating a promotion of B-LG aggregation. The addition of 0.02-1.0M
NaCl increased aggregation rate, but increased transition peak temperature of B-LG.
Mulvihill and Kinsella (1988) reported that at neutral pH, salt was required for gel
formation. Maximum gel hardness was obtained in 10 mM CaClZ, pH 7.0 when B-LG
was heated at 80°C. A lower gel point and more rapid gel formation was observed in 20
mM CaCl2 as compared to 100 mM NaCl (Foegeding et al., 1992).

Ca2+ was more effective at a lower concentration than Na+ at inducing B-LG
aggregation. However, the exact role of these cations in the gelation of B-LG is not clear.
Generally, cations modulate thermal gelation by decreasing electrostatic repulsion,
forming salt bridges and facilitating intermolecular disulﬁde bond formation and
hydrophobic interactions. Li et a1. (1994) used nuclear magnetic resonance to look at the
involvement of Na+ and Ca” in the aggregation of B-LG between 10-85°C at pH 7.0.
They found that Ca2+caused conformational changes in B-LG above 40°C. The results
suggested that divalent cations may cross-link anionic carboxyl groups during the
unfolding of polypeptides and stabilize the unfolded form.

J eyarajah et a1. (1994) studied calcium binding to native and preheated B-LG
using an ion selective electrode. Changes in hydrophobic regions and reactive sulﬂrydryl
content of the proteins were also monitored by ﬂuorescence spectroscopy using
8-anilinonaphthalene sulfonic acid and ﬂuorescein mercuric acetate, respectively.
Reactions of mercury with sulfhydryl groups of amino acids quench the ﬂuorescence of

murcurial derivatives of ﬂuoresceins, therefore an increase in the content of reactive

33

sulﬂrydryl groups can be determined by measuring ﬂuorescence quenching of the
ﬂuorescein mercuric acetate. Heat treatment (80°C for 15 min) slightly increased calcium
binding to B-LG. Addition of CaCl2 (1-15 mM) increased 8-anilinonaphthalene sulfonic
acid ﬂuorescence of B-LG. The ﬂuorescence intensity of B-LG preheated at 80°C was
higher than that of B—LG preheated at 60°C and the unheated form. This suggested that
heat treatment unfolded the molecule promoting the exposure of hydrophobic regions
while calcium binding magniﬁed the structural changes of B-LG. A smaller increase in
8-anilinonaphthalene sulfonic acid ﬂuorescence was observed upon the addition of NaCl
compared to addition of CaClZ, indicative of different effects of calcium and sodium on
the structure of B-LG. The reactive sulﬂrydryl group of preheated B-LG was less than
that of unheated B-LG, indicating that sulﬂrydryl/disulﬁde interchanges occurred during
heating. The reactive sulﬂrydryl content of B-LG increased as CaCl2 was increased from
0 to 10 mM. The authors suggested that addition of CaCl2 might cause local unfolding of
B-LG which exposed the reactive sulfhydryl groups buried at the sheet-helix interface.
Addition of NaCl (25-50 mM) decreased the reactive sulfhydryl group content of
preheated B-LG, suggesting that disulﬁde linkages may have been formed.

Foegeding et al. (1998) summarized the network characteristics of whey protein
gels formed at different pHs (Table 4.2). Stranded protein networks, which have a
diameter of one to several molecules, are formed at pHs above and below the p1. The gels
formed at pH > pl are strong and elastic, whereas those formed at pH < pI are weak and
brittle. Particulate gel networks of protein clusters with diameters of 100-1000 times

molecular size are formed at pHs close to the pI (5.1). The network structures of B-LG

34

Table 4.2. Network types and physical properties of whey protein gels (from Foegeding

 

 

 

etaL,1998)
Fracture rheology
Gel type Stress Strain Rigidity Appearance Water
holding
Stranded
i) pH > pI +++ +++ ++ Translucent +++
ii) pH < pI + + ++ Translucent NA
Mixed ++++ ++ ++++ Cloudy/opaque ++
Particulate +++ +++ ++ Opaque +

 

The number of “+” symbols represents the relative magnitude of the property among gel
types, with the greater number indicating a greater magnitude.
“ NA ” indicates no relevant data.

35

gels formed at different pHs follow a similar trend as the whey protein gels. A ﬁne
stranded structure of B-LG was found at pH 4.0 and lower, whereas both ﬁne stranded
and particulate gel structures were formed at pH 6.0 or higher. At pH 4.0-6.0, white
particulate gels were formed (Langton and Hermansson, 1992). Large deformation
measurements indicated that B-LG gels formed at low pH were brittle with low stress and
strain at fracture, whereas those formed at high pH were elastic with high stress and strain

at fracture (Stading and Hermansson, 1991).

4.4. Mixed protein gel systems

Gelling mechanisms of polymers, such as proteins and polysaccharides, are
usually studied in a single component system because it provides a simple ideal model for
the investigation of the gelling process. However, most food gels contain a complex
mixture of different gelling polymers, i.e. different proteins or proteins mixed with
polysaccharides. The gel formation and ﬁnal gel properties of these mixtures are very
complex and are greatly inﬂuenced by the individual gelling characteristics of each
polymer under the processing conditions employed. Researchers are confronted with
difficult interpretations of results obtained from complex gel systems. To begin to
understand the gelation of mixed polymers, experiments have been designed to study

model composite gels of two biopolymers.

4.4.1. Models of two component mixed gel structures. Brownsey and Morris

(1988) proposed two types of two component mixed gels. Type I is called a ﬁlled gel.

36

Only one of the two components forms the gel network. The second polymer remains
soluble or exists as dispersed particles, and is entrapped inside the network as a gel ﬁller
(Figure 4.6). The ﬁller component may inﬂuence gelation of the main network forming
protein.

A type 11 gel is formed when both components independently form a gel network.
The structure of the ﬁnal gel may be divided into three forms (Figure 4.7):

1) Coupled network: two polymers interact to form a gel network, therefore at

least some junction zones involve both polymers.

2) Interpenetrating network: a combination of two separate polymeric networks
is formed. These networks are entangled but there is no common junction
zone.

3) A phase-separate network: one polymer separately forms a network that is
interspersed within another polymeric network.

The properties of two component heat-set gels are the net result of the complex
molecular interactions within each polymer, between each polymer and the solvent, and
between both polymers. Each polymer has a structure that determines its gelling
behavior, thus the gelling mechanism of a mixed system mainly depends on the intrinsic

character of each polymer and the heat treatment employed.

4.4.2. Interactions between meat protein and other proteins. A wide variety

of protein additives from plant and animal origin are used to improve water retention and

textural properties of comminuted meat products. Many studies have been conducted

37

 

Figure 4.6. Type I ﬁlled gel network showing network formation by one protein with

soluble polymer entrapped inside (ﬁ'om Brownsey and Morris, 1988).

(1)

 

Figure 4.7. Schematic diagrams of possible structure of type II mixed gel (1) coupled
network; (2) phase separated network; (3) interpenetrating network (from Brownsey and

Morris, 1988).

38

with these additives in meat systems. Burgarella et a1. (1985) looked at the gel strength
(measured from penetration force) of surimi (minced ﬁsh) in combination with egg white
proteins and whey protein concentrate (WPC). The results showed that the addition of
egg white proteins at a 1:4 ratio with surimi (12% total protein and 2% NaCl w/w)
lowered the rigidity modulus of the gels below 40°C, but strengthened gel rigidity at
temperatures above the gelling temperature of both proteins (70°C). Addition of WPC
increased the rigidity modulus of gels throughout the heat treatment. The authors
suggested egg white protein interfered with network formation of the myoﬁbrillar protein
of surimi, resulting in a decrease in rigidity of the co-gels below 40°C. Egg white protein
gelled above 70°C, leading to enhanced rigidity of the co-gel. Soluble WPC increased the
interstitial ﬂuid viscosity, imparting additional rigidity to the myoﬁbril protein
throughout the heat treatment, and did not interrupt the gelation of surimi protein.
Combinations of surimi and egg white or whey proteins were suggested to form ﬁlled
gels.

The formation of myosin and albumin or ﬁbrinogen co-gels in 0.5M NaCl pH 6.0
was investigated. Foegeding et a1. (1986) found that strength (penetration force) of
myosin-albumin and myosin-ﬁbrinogen gels was affected by heating rate. The myosin-
ﬁbrinogen combination did not form a gel when heated at 70°C for 20 min. When slowly
heated at a rate of 12°C/min to 55°C or 70°C, myosin interacted with ﬁbrinogen resulting
in gels with higher hardness than the sum of the gel hardness of each individual protein.
In myosin-albumin blends, there was no interaction between the proteins until 85°C

where sufficient thermal alteration of the albumin had occurred. The strength of the

39

combination gel formed by isothermal heating at 90 or 95°C was half that of the gel
formed when heated to 90 or 95°C at 12°C/min.

Peng et a1. (1982) studied the interactions between soybean 118 protein and
myosin heated at 34.5, 70, 85, 90, 95 and 100°C for 30 min in 58.3 mM NaPO4, 0.4M
NaCl, pH 6.55, 10 mM 2-mercaptoethanol, 1 mM MgC12, 0.1 mM ATP, and 0.02%
NaN3. This buffer condition was supposed to mimic the pH and ionic environment of a
processed meat system. Gel ﬁltration chromatography and electrophoresis of the
supematants collected after centrifugation (100,000 x g, 30 min) of the proteins after
heating and cooling indicated that 1 1S soy interacted with myosin only in the temperature
range of 85-100°C. Interactions occurred between the myosin heavy chain and partially
dissociated or fully dissociated 1 IS soy protein.

Shiga et a1. (1985) studied interactions of acid precipitated soybean protein (pre-
heated in100°C water bath for 0-15 min) and ground chicken meat. YounG's modulus
and the water holding capacity of the ground chicken meat and preheated soy protein
mixtures heated at 70 or 100°C increased with an increase of preheating time (0-4 min) of
soy protein. These changes in physical properties of the mixed gels were related, in part,
to an increase in the SH-group concentration of soy protein during the ﬁrst 4 min of
preheating. These results indicated that unfolding of soy protein was needed for
interaction with meat protein to enhance the gel strength.

The properties of co-gels of chicken SSP and WPC of various solubilities have
been studied. WPC solubility had different effects on meat protein gelation depending on
heating temperature. Beuschel et a1. (1992) reported that hardness and deformability of

co-gels formed with 4% SSP and 12% WPC in 0.6M NaCl, pH 7.0, heated at 65°C were

40

enhanced by low solubility WPC. Mixtures with highly soluble WPC required a higher
temperature (90°C) to enhance the hardness of the co-gels. Hung and Smith (1993)
studied the rheological properties and microstructure of the same proteins, and found that
WPC shifted the ﬁrst storage and loss moduli (G’ and G") transitions of SSP to higher
temperatures and increased the magnitude of G' and G" of gels heated to 90°C. In
isothermal studies at 65°C, the elasticity of a combination gel of SSP and low solubility
WPC was higher than that of the combination containing highly soluble WPC.
Microstructure of the co-gels formed with highly soluble WPC showed a typical SSP
ﬁbrous structure at 65°C. When heated at 90°C, the globular structure of WPC was
observed in the co-gel networks. The authors suggested that at 65°C, SSP and WPC co-
gels formed a ﬁlled gel network where the WPC was trapped within the SSP network and
competed for available water and increased the viscosity of the liquid phase. When
heated to 90°C, WPC aggregated and probably formed either a ﬁlled gel with low
solubility WPC or a phase separated gel network with highly soluble WPC, leading to an
increase of G' in both co-gels. Addition of WPC, however interrupted the network
formation of SSP as indicated by an increase of G' and G" transition temperatures, and
distortion of the co-gel matrix when compared to SSP gel structures. WPC was added to
SSP at a high concentration (12% WPC + 4% SSP), so the texture enhancement could be
a result of a higher total protein concentration when compared to a control of 4% SSP.

Moreover, the multi-component nature of SSP and WPC caused difﬁculties in the

interpretation of results.

41

Smyth et al. (1998a) investigated the effects of B-LG and or-LA on the heat-
induced gelation of chicken breast SSP in 0.6M NaCl, pH 6.5, using dynamic rheology
and a water holding capacity test. SSP, B-LG, or-LA and the mixtures of SSP with B-LG
or or-LA at ratios of 80:20, 60:40, 40:60 and 20:80 at a total protein content of 2% were
used. SSP had greater G' at 70°C, but lower G’ at 90°C than those of 80:20 and 60:40
SSP/B-LG mixtures. However, the G' of SSP gels were greater than all combinations of
SSP and B-LG after cooling. The water holding capacity of SSP/B-LG co-gels were not
different from SSP gels, whereas water holding capacity of SSP/a-LA co-gels were lower
than those of SSP gels. SSP was primarily responsible for network formation in the
mixed protein systems. Denaturation and aggregation of B-LG above 70°C facilitated a
more rigid gel structure, whereas or-lactalbumin did not participate in the network
formation of the co-gels. The decrease of G' of all mixed protein solutions observed
below 70°C could be due to the dilution of SSP and interruption of SSP network
formation by B-LG or or-LA.

Hongsprabhus and Barbut (1999) reported that addition of 2% preheated (30 min
at 80°C, pH 7.0) WPI followed by cold set gelation (16h at 10°C) increased hardness
(penetration force) and water holding capacity of chicken meat batters after cooking,
especially at low salt concentration (31.5%) when compared to the use of unheated whey
proteins. The function of preheated WPI in gel formation of chicken meat is unclear.
The authors suggested that the texture enhancing effects were probably caused by the

electrostatic interactions of meat and whey proteins during the incubation period.

42

4.5. Pre-heated whey protein and a two-step gelation process

Barbut and Foegeding (1993) reported that 4% preheated WPI solution (at 80°C
for 10-30 min) formed a ﬁne stranded gel by adding 10 mM CaCl2 at ambient
temperature. The gel was more transparent and higher in shear stress at fracture than a
WPI gel formed at 80°C. McClements and Keogh (1995) reported similar results when
200 mM NaCl was added to 9% WPI preheated at 90°C for 30 min. The author also
found that the magnitude of the complex modulus increased with the increasing of
temperature during gelation. It was suggested that preheating whey proteins in absence
of salt caused irreversible denaturation as indicated by a disappearance of the
denaturation peak in a heat capacity proﬁle of the preheated protein. The viscosity of
preheated whey protein solutions suggested the formation of ﬁbrous aggregates or
polymers.

Re-heating the preheated protein solutions (two-step heating method) resulted in
the formation of stronger and more transparent gels over a wider range of pHs and ionic
strengths when compared to gels formed by single step heating of the same protein in
presence of salt. The two-step heating method has been reported to alter the gelling
properties of other globular proteins, such as ovalbumin, bovine serum albumin and egg
lysozyme (Doi, 1993). Mechanisms of aggregate formation and gel network formation of
the aggregates are unclear. Doi (1993) proposed a possible mechanism for ovalbumin
aggregate formation during preheating at 80°C for 6h in 0.02M phosphate buffer, pH 7.0.
Based on the aggregate size observed by TEM and molecular size of the native protein,
the ovalbumin aggregates were formed by partially unfolded globules which ﬁrst bind

together as dimers then arrange into a worm-like cylinder (Figure 4.8). This model

43

 

    

monomer dimer tetramer polymer /<
1k
.*

.I -.
DO L4

56 A/Z molecules
- 28 A/molecule

 

 

 

Figure 4.8. Model for linear polymer formation of ovalbumin (from Doi, 1993).

44

explains the formation of ordered linear polymers which form strong transparent gels.
More evidence is needed to conﬁrm this proposed mechanism.

The pre-aggregation or pre-polymerization alters the gelling properties of globular
proteins, especially whey protein isolate or concentrate. Much research has been
conducted to study various methods to form soluble whey protein aggregates and to

induce gelation of the aggregates.

4.6. Cold-set whey protein gels

Several researchers have indicated that soluble whey protein aggregates can be
formed using acidulants (Ju and Kilara, 1998c), calcium salts (Ju and Kilara, 1998a;
1998d) enzymes (Otte et al., 1996), or heat treatment (Ju and Kilara, 1998b). These
soluble aggregates can gel upon the addition of salts or by changing pH. Since gelation
occurs at temperatures below the gel point of native whey proteins, the gels formed are
called cold-set gels (McClements and Keogh, 1995). As native whey proteins do not
form gels until about 80°C, their inﬂuences on the texture of products that have been
processed at lower temperatures are limited. Therefore, soluble whey protein gels have
potential for applications where protein gelation at low temperature is desirable, such as
in comminuted meat products.

Preparation of cold-set whey protein gels involves two main steps. In the ﬁrst
step, soluble aggregates of whey proteins are formed. The second step involves the
formation of a gel network by the soluble aggregates, which can be initiated by addition

of salt or by changing the pH of the solution.

45

4.6.1. Formation of soluble whey protein aggregates.

Acid-induced aggregates of whey proteins were largest when prepared at pH 5.2,
the isoelectric point of B-LG. The aggregate size decreased when pH was decreased
below the pI, indicating a dissociation of the aggregates (Ju and Kilara, 1998c).
Incubation of WPI in CaCl2 produced stable whey protein aggregates (Ju and Kilara,
1998d). The calcium-induced aggregation rate of WPI increased as the incubation
temperature was increased from 21 to 45°C, while the aggregate size increased with
increased protein and CaCl2 concentrations (Ju and Kilara, 1998a). Acid-induced
aggregation of WPI was more rapid than calcium-induced aggregation.

Otte et a1. (1996) studied the protease-induced aggregation of whey proteins.
Particle sizes of 12% WPI were increased when the protein was hydrolyzed by a protease
as indicated by an increase in turbidity. Size exclusion chromatography of whey proteins
before and after 3h hydrolysis at 40°C showed 35% degradation of B-LG and or-LA.
Average aggregate size, detected by dynamic light scattering, increased from 130-200 nm
to inﬁnity after a lag period of about 50 min, suggesting that the aggregates were formed
from the hydrolyzed proteins.

Soluble whey protein aggregates can also be prepared by heat treatment and does
not require the use of chemicals. This approach is relatively more convenient and less
harsh when compared to other treatments that have been investigated. The treatment
parameters (temperature and time) and the conditions of the initial protein solutions (pH,
ionic strength and protein concentration) need to be controlled to prevent gelation during

heating. Soluble whey protein aggregates were formed when heated above the

46

denaturation temperature of B-LG (70-90°C) and held for 5-60 min. Higher preheating
temperatures and longer times produced larger aggregates than those prepared at the
lower preheating temperatures and shorter times (J u and Kilara, 1998b). An adequate
time/temperature combination allows denaturation of the whey proteins leading to the
exposure of non-polar amino acids and reactive sulfhydryl groups buried in the native
molecules. As a result, hydrophobic attractions and sulfhydryl-disulﬁde interchange
reactions between unfolded protein increase and the protein aggregates are formed.
Prabakaran and Damodaran (1997) reported that sulfhydryl-disulﬁde interchange among
B-LG monomers was initiated at 60-65°C. The pH of the B-LG solution needs to be far
from the isoelectric point (pH 5.2) of the protein and the salt concentration should be very
low so that the aggregation process is limited, resulting in ﬁlamentous aggregates which
remain soluble due to electrostatic repulsion between their surfaces. Most of the
experiments were performed between pH 7.0-8.0. In addition, the concentration of CaCl2
was less than 10 mM or kept within the inherent mineral content of the whey protein
preparations (Hongsprabhus and Barbut, 1998; Ju and Kilara, 1998b).

In and Kilara (1998b) studied the inﬂuence of protein concentration on the
formation of soluble aggregates of whey protein isolate during heating at 80°C at pH 7.0.
Dynamic light scattering revealed formation of soluble aggregates in solutions of protein
concentration higher than 3%. Size of whey protein aggregates increased from 21 nm to
61 run when the protein concentration was increased from 3 to 9%. However, above a
critical concentration, gel formation occurred. Solutions of 12% protein were reported to

form self supporting gels after heating at 80°C for 30 min, pH 7.0, without addition of salt

47

(Ju et al., 1995; Ju and Kilara, 1998c). The viscosity of the heated solution signiﬁcantly
increased when compared to that of the initial protein solution indicative of the formation
of ﬁlamentous aggregates (Barbut and Foegeding, 1993; McClements and Keogh, 1995;
Mleko and Foegeding, 1999b; Vardhanabhuti and Foegeding, 1999b). More research is
needed to understand the mechanisms and factors inﬂuencing the formation of soluble
aggregates, and to understand how the properties of the soluble aggregates inﬂuence the
characteristics of the cold-set gel. This knowledge is important for manipulating the

functional properties of whey protein products.

4.6.2. Network formation of cold-set gels of heat-induced whey protein
aggregates.

A solution of heat-induced soluble whey protein aggregates can form a gel
network upon addition of proteolytic enzyme, salts or changing the pH at ambient
temperature. The cold-set gel network formation and properties are dependent on
aggregate formation and gelling conditions. Heat treatment is a common process used in
whey manufacture and the gel properties of heat-induced aggregates, such as clarity,
hardness and water holding capacity are similar to those needed by food processors.
More research is needed to study the network formation and properties of gels formed

from heat-induced whey protein aggregates.

4.6.2.1. Enzyme-induced gelation. Heat-induced whey protein aggregates
have been reported to form gels upon proteolytic digestion. The proteolytic digestion of

pre-heated soluble aggregates induced network formation via a different mechanism from

48

acid- and salt-induced gelation. Sato et al. (1995) studied gelation of heat-denatured
whey protein by proteolytic digestion at 37°C using several enzymes (pepsin, papain,
pronase, protease and trypsin). Weak gels were obtained when protein solutions were
incubated with all enzymes, except pepsin. Pepsin did not hydrolyze the whey protein
after 8h incubation. Hardness of the gels increased as the degree of hydrolysis was
increased over the incubation time, suggesting gel networks were formed from the protein
hydrolysates. The interactions between the hydrolysates of preheated whey proteins,

which led to a gel network formation, were not known.

4.6.2.2. Salt-induced cold-set gelation. Bryant and McClements (1998)
compared the heat-induced gelation of native whey proteins with cold-set gel formation
of heated whey protein aggregates (Figure 4.9). During heat-induced gelation of native
whey protein in the presence of salts (at a protein concentration higher than the critical
concentration for inducing gelation), the proteins undergo unfolding and aggregation
almost simultaneously when the temperature reaches the gel point. Salts accelerate the
rate of aggregation resulting in large aggregates which cross-link to form particulate gel
structures. In contrast, soluble whey protein polymers with highly negatively charged
surfaces form a ﬁne stranded gel network upon the introduction of salts, at or below
ambient temperature. Salts decrease electrostatic repulsion and form salt bridges between
the soluble aggregates leading to three-dimentional network formation.
Salt type and concentration affected gel properties, suggesting different

mechanisms were involved in network formation. Divalent cations may form intra-

and/or intermolecular salt bridges which affect the texture of the ﬁnal gels (Ju and Kilara,

49

solution viscous solution particulate gel

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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0 O 0 00 O 0"
°o ° N
o° 2 s ° h..._ ”a" is
0 a a 000 o :'
O 0
0 0 0° 0 00 o no salt
9 o a ° 00
o o 000
‘0 oo o
a o 0" O
O O 00 a O
0 0 o O O a
o D o 0 O 0 a
l
unaggregated ﬁlament formation aggregated ﬁlaments

native proteins

Figure 4.9. Development of a particulate gel structure from heat-induced gelation and a
ﬁlamentous gel structure from cold-set gelation of whey protein (from Bryant and

McClements, 1998).

1998c). According to Vardhanabhuti and Foegeding (1999a), the mechanism of cold-set
gel formation is “salt speciﬁc.” A lower concentration of Ca2+ is needed to induce cold
set gelation, when compared to Na“. The most rigid gels were obtained by addition of 20-
30 mM CaCl2 or 200 mM NaCl to 8-10% preheated whey protein. Gel networks became
coarser with decreased water holding capacity as the concentration of salts was increased
above 30 mM CaCl2 or 200 mM NaCl (Hongsprabhus and Barbut, 1997b; 1998; Ju and

Kilara, 1998a; Vardhanabhuti and Foegeding, 1999a).

4.6.2.3. Acid-induced gelation. Lowering the pH of pre-heated whey protein
isolate (soluble aggregates) induced gel formation at or below ambient temperature by
decreasing electrostatic repulsion. Mleko and Foegeding (1999a) reported that weak gels
were formed at 7°C by decreasing the pH of soluble whey protein polymers (preheated at
80°C for 15-53 min) from pH 8.0 to 6.0. Ju and Kilara (1998c) found that addition of
glucono-delta-lactone induced gelation of 8% pre-heated whey protein (preheated at 80 °C
for 30 min) when the pH was decreased from 7.0 to 5.3 at 45 °C. Acid-induced gels had a
maximum hardness at pH 4.7 and were harder than salt-induced gels prepared from the
same protein solutions. No studies have been reported on cold-set gelation induced by
salts at a pH lower than 7.0.

Factors affecting textural properties of cold-set gels induced by salts and acid are
protein concentration and incubation time and temperature. The minimum protein
concentration needed to form a salt or acid induced cold-set gel has been reported to be
3% (diluted from 9% whey protein solution heated at 80°C for 30 min, pH 7.0) (Ju and

Kilara, 1998b). An increase in the concentration of soluble protein aggregates led to the

51

formation of a harder gel (Hongsprabhus and Barbut, 1998; Ju and Kilara, 1998b; l998e).
However, the hardness of the ﬁnal gels was also affected by the concentration of protein
during the preheating step. Increasing the protein concentration from 3% to 9% during
preheating increased the size and content of the soluble aggregates resulting in the
formation of a stronger gel network (Ju and Kilara, 1998b).

Time and temperature have a great effect on the rate of gelation and the ﬁnal
properties of both salt— and acid-induced gels of soluble whey protein aggregates. The
rate of network formation of soluble whey protein aggregates is increased by increasing
temperature (Kitabatake et.al., 1996; Mleko and F oegeding, 1999a). Opacity and
hardness of salt-induced gels increased with an increase in gelling temperature from 20 to
50°C (McClements and Keogh, 1995), suggesting that hydrophobic interactions and
disulﬁde bond formation, may be involved in cold-set gelation at higher temperatures. A
slow addition of calcium ions by dialysis resulted in more translucent gels than direct
addition of calcium to the protein aggregate solution (Roff and Foegeding, 1996). More
research is needed to clarify the factors inﬂuencing gelation of pre-heated whey proteins.

Most research has been focused on cold-set gelation of whey protein aggregates
formed at ambient temperature or lower. However, heat is used during food processing to
destroy pathogenic microorganisms and to produce desired sensory attributes in many
products. Re-heating by the consumer is also a common food preparation practice. This
heating process can affect the properties of gels formed from whey protein aggregates.
Hongsprabhus and Barbut (1998) reported that re-heating at 80°C for 30 min increased
YounG's modulus and shear stress, and reduced shear strain, extensibility and water

holding capacity of a cold-set whey protein gel initially formed by incubation with CaCl2

52

at 24°C for 16h. In some food products, such as sausages, salts, low pH and heat are
used. Therefore, the effects of these factors on the gelation of pre-heated whey protein

polymers need to be studied.

53

CHAPTER 5: HEAT-INDUCED GELATION OF MYOSIN/B-LACTOGLOBULIN

MIXED PROTEINS

5.1. ABSTRACT

The thermal denaturation, aggregation and rheological properties of chicken
breast muscle myosin, B-lactoglobulin (B-LG) and mixed myosin/B-LG systems at
various concentrations in 0.6M NaCl, 0.05 mM sodium phosphate buffer, pH 7.0, were
studied. There was no interaction between myosin and B-LG during heating as indicated
by the unique denaturation peaks of each protein in the therrnograrn of the mixed protein
solution. The maximum aggregation rate (ARmx) of both proteins increased, and the
temperature at the ARmax (Tm) and initial aggregation temperature (To) decreased as the
protein concentration was increased. Above 0.5% protein, concentration had no effect on
To, TIn and ARM of myosin. Aggregation of myosin at <0.5% protein was interrupted by
the presence of 0.25% B-LG in the mixtures. Addition of 0.5-3.0% B-LG decreased
storage moduli of 1% myosin between 55-75°C, but increased storage moduli when
heated to 90°C and after cooling. B-LG had no effect on the gel point of 21 .0% myosin.
Overall, B-LG interrupted the aggregation and gel formation of myosin at 55-75°C, but

enhanced gel strength above 80°C and after cooling. The magnitude of the effects was

concentration dependent.

S4

5.2. INTRODUCTION

Salt soluble proteins form heat-induced gels which are responsible for texture,
water holding, binding and appearance of meat products (Smith, 1994). To improve
yields, reduce costs, or improve quality of meat products, meat processors often use non-
meat proteins to substitute for muscle proteins. Whey proteins possess high nutritional
and functional properties and are used in meat products as binders to enhance yield and
textural quality. However, results from the use of whey proteins in meat products are
highly variable due to differences in the source and processing history of whey protein
concentrates or isolates, and variation in processing conditions used for meat products.
The source and processing history of whey products affects protein composition and
functional properties. While differences in processing condition used for meat products
(time/temperature, salt and pH) greatly affect gelling properties of both proteins.

Many studies have looked at mixed gel systems of salt soluble protein and whey
protein concentrate (Beuschel et. a1, 1992; Hung and Smith, 1993; Smith and Rose, 1995;
McCord et al., 1998). The functions of whey proteins in meat systems have not been
clearly explained due to the multi-component nature of these proteins. Since myosin and
B-lactoglobulin (B-LG) are the major functional proteins of meat and whey products,
respectively, the use of these proteins in a model system might help elucidate the
interactions that occur when these proteins are used.

The mechanism of protein gelation is a multi-step process involving denaturation,
aggregation and network formation of the protein molecules. Bringing together the

information from each step of the process and comparing the data between mixed and

55

pure protein systems will lead to a better understanding of interactions between the
proteins throughout the gelation process. The objective of this experiment was to
elucidate the mechanisms involved in the formation of myosin and B-LG co-gels by:

1) observing the thermal stability of myosin and B-LG during heating of mixed systems,
2) investigating the inﬂuence of B-LG on aggregation of myosin during heat-induced
gelation, and 3) monitoring rheological properties of myosin and B-LG in mixed protein

gel systems.

5.3. MATERIALS AND METHODS
5.3.1. Protein solution preparation

Myosin from breast muscle (M. pectoralis) was extracted immediately after
sacriﬁce from 8-12 week old commercial type broilers (Wang and Smith, 1994b).
Myosin was stored in 48% saturation ammonium sulfate with 30% glycerol (v/w) at
-20°C. Prior to use, myosin was suspended in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0 (PBS), dialyzed against three changes of the same buffer for 48h and
centrifuged at 78,000x g to precipitate denatured protein. Bovine milk B-LG (L0130, lot
#114H0755) containing variants A and B, was purchased from Sigma chemical Co. (St.
Louis, MO). B-LG solutions were prepared by dissolving the protein in PBS overnight at
4°C. The purity of both myosin and B-LG were assessed by sodium dodecyl sulfate
polyacrylarnide gel electrophoresis using 4% and 12% acrylamide for stacking and
resolving gels, respectively (Smyth et al., 1996). Protein concentrations were determined

by absorption using extinction coefﬁcient (Em) of 5.5 at 280 nm for myosin (Smyth et al,

56

1996) and 9.55 at 278 nm for B-LG (Foegeding et al., 1992). Mixed protein solutions
were prepared by mixing an equal volume of each protein prepared at twice the desired
concentration in PBS. The concentrations of myosin, B-LG, and mixed solutions used in

denaturation, aggregation, and dynamic oscillatory experiments are presented in Table

5.1.

5.3.2. Differential scanning calorimetry

The thermal denaturation of myosin, B-LG, and myosin/B-LG in PBS, pH 7.0,
were investigated using an MC-2 differential scanning calorimeter (DSC) (Microcal Inc.,
Amherst, MA). The proteins and blank solutions (PBS) were degassed in a vacuum
chamber before loading 1.24 mL into the DSC. Experiments were conducted at a scan
rate of 1°C/min from 25°C to 90°C. Calorimetric enthalpy (AH,,,) and endothermic peak
or melting temperature (Tm) were determined from heat capacity proﬁles (Cp vs
temperature) using the software (DA-2 Data Acquisition and Analysis system) provided
by the manufacturer. The experiments were conducted using at least three replications

for each sample.

5.3.3. Thermal aggregation of protein
Thermal aggregation of proteins was followed by turbidity measurement at 340
nm using a Lamda 20 UV-visible spectrophotometer connected to a PTP-6 peltier

temperature programmer (Perkin Elmer Inc., Norwalk, CT). Protein solutions of 1.4 mL

57

Table 5.1. Concentrations of myosin and B-lactoglobulin (B-LG) in 0.6M NaCl, 0.05M
sodium phosphate buffer, pH 7.0, used in thermal denaturation (DSC), thermal

aggregation and dynamic oscillatory experiments

 

 

 

Protein type Protein concentration (%)
DSC Thermal aggregation Dynamic oscillatory
Experiment Experiment experiment
Myosin 1.0 0.05, 0.1, 0.25, 0.5, 1.0, 1.5 0.5, 1.0, 1.5, 2.0

B—LG 1.0 0.1, 0.18, 0.25, 0.5, 1.0, 1.5 1.0, 2.0, 3.0
Myosin : B—LG 1.0 : 1.0 0.1 : 0.1 0.1 : 0.25 1.0 : 0.5 0.5 : 1.0
0.1 : 0.25 0.25 : 0.25 1.0 : 1.0 1.0 : 1.0
0.1 :0.5 0.5 : 0.25 1.0 : 2.0 1.5 : 1.0
01:10 1.0 : 0.25 1.0 : 3.0 20:10

 

58

were placed in quartz cuvettes (l-cm path length) and sealed with TeﬂonTM tape to
prevent evaporation. A blank containing the same buffer was used as the control.
Solutions were equilibrated at 25°C for 5 min, then heated to 90°C at 1°C/min, held at
90°C for 30 min, cooled to 25°C within 15 min, followed by a 5 min holding at 25°C
(Figure 5.1a). The change in turbidity of protein solutions was recorded every 0.5°C.
Each study was performed in triplicate. A plot of aggregation rate (Aabsorbance/Atime)
against time or temperature was constructed for determination of the initial aggregation
temperature (To), maximum aggregation rate (ARM), and temperature at ARmax (Tm)
(Figure 5.1b). T0 was deﬁned as the temperature at which the aggregation rate was 2 0.01

min". Peak of the plot determined ARM and temperature at ARmax was determined as Tm,

5.3.4. Small amplitude dynamic oscillatory testing

Dynamic oscillatory tests were performed using a controlled stress rheometer (RS
100, Haake, Karlsruhe, Germany) equipped with a 35 mm diameter stainless steel parallel
plat. Storage (G') and loss (G") moduli were recorded continuously at a ﬁxed frequency
of 0.464 Hz using constant stresses (producing strains from 0.1-0.3%) within the range of
linear Viscoelastic behavior determined from stress sweeps performed for each protein at
90°C and after cooling to 25°C. Protein solutions were loaded between the plate and base
with a gap between 1.0 and 1.2 mm. A few drops of corn oil (Mazola, Best Food, CPC
International, Inc., Englewood Cliffs, NJ) were used to cover the edge of the gap to
prevent evaporation. Solutions were heated using the same temperature proﬁle used in

aggregation experiments (Figure 5. 1 a). The temperature was controlled by a circulating

59

100

 

 

 

 

 

 

 

90 .
,_, 80 -
:3. 70 ~ ‘
93 60 ~
:3
E 50 -
“g. 40 -
g 30 ~
20 —
10 .
0 I u . . .
0 20 40 6O 80 100 120
Time [min]
heating holding cooling
W N H

Figure 5.1a. Heating proﬁle for thermal aggregation and rheological studies.

 

 

 

 

 

 

1 100
0.9 ~ ----- temperature .‘ _ 90
0.8 - » 8O
\‘ H
0-7 : ARM" ~ 70 g
[- 0.6 - 'r.‘ _ 60 '2
.6 ‘. "I
3 0.5 - r 50 5
g 0.4 - ~ 40 a
0.3 ‘ “‘_.I_ 30 £3
0.2 - T - 20
0.1 * T° m _ 10
O r \A - 0
O 20 40 60 80 100 120

Time [min]

Figure 5.1b. Derivative curve of protein aggregation for determination of initial

aggregation temperature (To), maximum aggregation rate (ARM), and temperature at

ARM (Tm)-

60

water bath attached to the rheometer. Frequency sweeps (0.01-100 radian/second) were
performed after cooling using constant stress resulting in strain of 0. 1-0.3%. Protein
solutions used in sweep tests were 2% myosin, 1% myosin/1% B-LG, and 4% B-LG. The
gel point of protein solutions was deﬁned as the temperature at which G' and G" crossed
over in the ﬁxed frequency test (Stading and Hermansson, 1990). Each solution was

tested in triplicate.

5.3.5. Experimental design and statistical analysis

Each experiment was performed in triplicate. Differences between aggregation
and rheological properties (To, Tm, ARM, G' and gel point) affected by protein type or
concentration were statistical analyzed using one way analysis of variance (ANOVA).
Means were compared using the Tukey-Kramer HSD test with the mean square error at

5% probability (JMP, Version 3.2.2, SAS Institute Inc., Cary, NC).

5.4. RESULTS AND DISCUSSION
5.4.1. Thermal denaturation of myosin and B-lactoglobulin

The enthalpy proﬁle of 1% chicken breast myosin in PBS, pH 7.0, contained three
endothermic peaks (Tm) at 48.5, 53.2 and 570°C (Figure 5.2a). The myosin endotherrn
was similar to those reported by other researchers. Chicken breast myosin heated at
1°C/min in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 6.5, exhibited three
endothermic peaks at 47.5, 54.0 and 57.4°C (Smyth et al., 1996). Lui et al (1996)

reported transition temperatures of 48. l , 49.9 and 67°C for chicken breast myosin in 0.6M

61

NaCl at pH 6.0. The denaturation AHcal of myosin (2130 i 80 kcal/mole) was close to the
AH“, of chicken pectoralis myosin reported by Wang and Smith (1994b) (2,140
kcal/mole) and Lui et a1. (1996) (2,216 kcal/mole).

The endotherm of 1% B-LG showed a broad Tm peak at 736°C (Figure 5.2A).
The result was close to a Tm of 734°C reported by Foegeding et a1. (1992) using 10% [3-
LG in 0.1M NaCl, 0.05M TES buffer, pH 7.0 and heating at 10°C/min.

A rapid decrease in heat capacity was observed at 760°C. The intensity of this
peak increased as the concentration of B-LG was decreased from 2% to 0.5% (data not
shown). The sudden drop of heat capacity was previously reported in DSC studies of
myosin at concentrations 5 0.523% (Lui et al., 1996) and was attributed to the
aggregation and precipitation of the unfolded proteins at low concentration. However, a
DSC study using 0.4% B-LG in 0.07M phosphate buffer, pH 6.75, showed no
precipitation when heated at 1.5°C/min (Qi et al., 1995). Moreover, no such phenomenon
occurred when 1% B-LG in distilled water was heated at 1°C/min (data not shown).
Therefore, the rapid decrease of the heat capacity could be a result of the aggregation and
precipitation of B-LG at high ionic strength (0.6M NaCl).

The endotherm of myosin and B-LG mixtures was identical to that expected if the
endotherm of each protein was overlaid on the same axis (Figure 5.2B). Wang and Smith
(1995) studied the inﬂuence of weight ratio of actin to myosin on the gelation of the
reconstituted chicken breast actomyosin. The results revealed interactions between

myosin and actin, which led to changes in the denaturation proﬁle of actomyosin when

62

 

 

 

 

 

 

 

5 -
E)—
E 4 —
3
E 3
Q -
3
3 2’
5
= 1‘
0 4 r T . 1 . 1 . I .
25 30 35 40 45 50 55 60 65 7O 75 80 85 90
Temperature [°C]
6
B
5 _

Heat capacity [mca|/° C]
DJ

 

 

 

25 30 35 40 45 50 55 60 65 70 75 80 85 90
Temperature [°C]
Figure 5.2. Heat capacity proﬁles of (A) 1% myosin and 1% B-lactoglobulin (B-LG)

analyzed separately, and (B) a mixture of 1% myosin/ 1% B-LG mixture in 0.6M NaCl,

0.05M sodium phosphate buffer pH 7.0.

63

compared to pure myosin and pure actin. In this experiment, DSC results indicated that
no interaction occurred between myosin and B-LG during heating as each protein
maintained its unique transition proﬁle. However, B-LG did not precipitate when heated
in the mixed solution with myosin. Since myosin is denatured at a lower temperature
than B-LG, it may form networks which help prevent aggregation and precipitation of [3-

LG during heating in mixed solutions.

5.4.2. Thermal aggregation

UV-visible spectroscopy was employed to investigate the aggregation of pure and
mixed protein solutions at different concentrations. The aggregation patterns of myosin
(Figure 5.3) had a sigmoidal shape similar to those reported by previous researchers (Lui
et al., 1996; Smyth et al., 1996). Myosin rapidly aggregated after the onset temperature.
The turbidity remained constant after reaching a maximum at around 67°C regardless of
concentration, suggesting that myosin aggregation was complete.

The initial aggregation temperature of myosin (To) decreased (p<0.05) from 50.6
to 463°C as the protein concentration was increased from 0.1 to 0.5% (Table 5.2). The
maximum aggregation rate (ARM) and the temperature at maximum aggregation rate
(Tm) also increased with increasing concentrations of myosin from 0.1 to 0.5%. Above
0.5%, concentration had no signiﬁcant effect on either ARmax, Tm or T0 of myosin,
suggesting that myosin aggregation was concentration dependent below 1.0% protein.

The ARM, of 0.1% myosin was 0.44 /min, much lower than 1.96 /min reported by Lui et

64

100

 

+ 0.10%
+7 0.25%
3.5
1A7 0.50%
, m 1.00%
3 : W» 1.50%

Absorbance [340 nm]

 

  
  
  
 

 

 

 

 

 

60
Time [min]

80

100

 

120

[30] aanreaadtual

Figure 5.3. Aggregation of 0.1-1 .5% myosin in 0.6M NaCl, 0.05M sodium phosphate

buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min

and cooling to 25°C.

65

Table 5.2. Initial aggregation temperature (To), maximum aggregation rate (ARM), and
temperature at maximum aggregation rate (Tm) of myosin, and mixed solutions of 0.25%
B-lactoglobulin (B-LG) and 0.1-1.0% myosin in 0.6M NaCl, 0.05M sodium phosphate

buffer, pH 7.0, during heating from 25 to 90°C, holding at 90°C for 30 min and cooling to

 

 

25°C
Protein Myosin To Tm ARmax
type concentration (0C) (CC) (CC)
(%)
Myosin 0.1 50.6 :0.2b 53.2 i0.3b 0.44 3:0.051b
0.25 49.2 i049 51.7 i070 1.07 420.077d
0.5 46.3 :0.2d 50.1 «50.2091 1.36 :01506
1.0 45.8 i0.7d 49.9 :O.6d 1.61 :1:0.136f
1.5 45.3 40.2d 49.3: 0.2d 1.60 i0.167f
0.25% B—LG/ 0.1 77.5105a 901a 003350.002a
myosin
0.25 49.2 $079 54.2 10.39 0.71 i0.180°
0.5 46.3 i0.3d 52.3 450.5bc 1.35:0.1806
1.0 46.1 1:0.2d 50.9 :0.scd 1.79 260.250f

 

' Tm occurred during holding at 90°C.

“‘f Values are means i standard deviations of three observations. Means with different
superscripts in each column are signiﬁcantly different (p<0.05).

66

a1. (1996) using 0.07% chicken pectoralis myosin and heating at 55°C in 0.6M NaCl, pH
6.0. The difference is probably due to differences in pH of the protein solutions.

The aggregation pattern of 0.1-1 5% B-LG (Figure 5.4) was similar to that of
myosin except that B-LG began to aggregate later and had lower ARmax than myosin.
Thermal denaturation of proteins precede thermal aggregation (Sano et al., 1990; Smyth
et al., 1996). DSC results showed that myosin denatured at 48-57°C, whereas B-LG
melted at 736°C. Myosin is a ﬁbrillar protein of 150 nm in length, whereas B-LG is a
globular protein with a radius of about 2-3 nm. Increasing the chain length of protein
increases molecular entanglement and facilitates contact or intermolecular binding,
promoting protein aggregation (Wang and Damodaran, 1990). Therefore, myosin had a
lower To and higher ARmax than B-LG.

T0 of B-LG decreased from 793°C at 0.25% protein to 635°C at 1.0% protein
(Table 5.3). Initial aggregation of 1% B-LG was detected at a temperature (635°C) lower
than its melting point (736°C), indicating that aggregation occurred between partially
unfolded molecules. Prabakaran and Damodaran (1997) reported that small B-LG
particles were ﬁrst formed via the sulﬂrydryl-disulﬁde interchange reaction between two
reactive monomers (molten globules) at 60-65°C. The small particles further associated
into larger aggregates in the propagation stage where the aggregation rate reach its
maximum. The ARmax and Tm of B-LG increased (p<0.05) as the protein concentration
was increased from 0.1 to 1.0%. Many studies have shown that the rate of B-LG
aggregation depended on reactive B-LG monomer concentration (Elofsson et al., 1996;

Hoffmann et al., 1996; Verheul et al., 1998). The aggregation of B-LG at concentrations

67

4.5 100

 

 

 

 

   

 

 

 

470.1%
4 _ —o- 0.25% _ 90
——9— 0.5%
. 6—1.0% *
3.5 -- +— 15% 80
Temperature
_ 70
a 3 -~
5
§ 60 ﬁ
—' 2.5 5
3 1:
g 1. — 50 91
. an
'E 2 + E
8 "A o -1
.2 r: ,1 4 40 g
1 5 , 4 f .9
f - 3O
1 , .
. 20
0.5 _ 10
0 . . . 0
0 20 40 60 80 100 120

Time [min]

Figure 5.4. Aggregation of 0.1-1 5% B-lactoglobulin in 0.6M NaCl, 0.05M sodium
phosphate buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at 90°C for

30 min and cooling to 25°C.

68

Table 5.3. Initial aggregation temperature (To), maximum aggregation rate (ARM), and
temperature at maximum aggregation rate (Tm) of B-lactoglobulin (B-LG), and mixed
solutions of myosin and 0.1-1.0% B-LG in 0.6M NaCl, 0.05M sodium phosphate buffer,

pH 7.0, during heating from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C

 

 

Protein B—LG To Tm ARmax
type concentration (°C) (°C) (min’l)
(%)
B-LG 0.1 845:1:053 901a 0.02 520.0029
0.25 79.3 «:10b 89.3 50.7ab 0.21 1:0.099b
0.5 73.5 i05d 84.0 i1.6b 0.57 360.0469
1.0 63.5 320.5f 78.3 10.29 0.76 50.025d
1.5 63.0 :0.5f 77.35029 0.77 i0.058d
0.1% myosin 0.1 85 i063 89.8 :109 0.02 i0.003a
/B—LG
0.25 77.5 520.59 901a 0.03 50.0024
0.5 70.0 «50.46 88 50.241b 0.1 1 :0006ab
1.0 63.2i0.8f 7951:0le 0.38 450.002bc

 

' Tm occurred during holding at 90°C.
a" Result values are means i standard deviations of three observations. Means with
different superscripts in each column are signiﬁcantly different (p<0.05).

69

higher than 1.5% was not studied due to limitations of the spectophotometric technique.
Both myosin and B-LG formed opaque gels with absorbance values >5.

The effects of B-LG on aggregation of myosin at different concentrations are
illustrated in Figure 5.5. Addition of 0.25% B-LG increased T0 of 0.1% myosin from
50.6 to 775°C and decreased ARmax from 0.44 to 0.03 /min (Table 5.2). When myosin
concentration was increased above 0.25%, the To, ARM, and Tm of the mixed systems
were not (p>0.05) different from those of myosin at the same concentration. The results
indicated that 0.25% B-LG interfered with myosin aggregation in the mixed system
containing _<_0.25% myosin.

The aggregation patterns of mixed protein solutions containing 0.1% myosin and
0.1-1.0% B-LG compared to those of 0.1-1 .0% B-LG are presented in Figure 5.6. Mixed
solutions of 0.1% myosin and 0.1% [3,-LG exhibited two aggregation steps with the ﬁrst
To, Tm and ARmax at 51 .4°C, 55°C and 0.08 /min, respectively. The ﬁrst aggregation step
was attributed to myosin aggregation. However, T0 and Tm were higher and ARM was
lower than those of 0.1% myosin, indicating interruption of myosin aggregation. In the
second step, To, Tm and ARmax occurred at 85°C, 898°C and 0.02 /min, respectively, and
were similar to those of 0.1% B-LG (Table 5.3). The results suggested that both proteins
aggregated independently in mixed systems of low protein concentration. When B-LG
concentration was increased above 0.25%, aggregation of the mixed protein solutions
exhibited a pattern more typical of B-LG aggregation. However, ARmam of all the mixed
proteins were lower than that of B-LG at the same concentration. Results suggested that

both proteins interfered with the aggregation of the other and the proportion of the

70

 

 

 

 

 

~A— 0.1% myosin A 0.25% LG/O. 1% myosin
7+ 0.25% myosin o 0.25% LG/0.25% myosin
, °~ 0.5% myosin 0 0.25% LG/0.5% myosin
4P 1.0% myosin I 0.25% LG/1% myosin
— Temperature
3.5 100
90
3 __ _ '— - "v- --. ..1' '.,... . I
2.5 #5. II |-._._., 4". w; 70
E .’
g — 60
v 2 ‘
Q 1 l
2 . » 50
a , o
f 1.5 . .‘1’1
8 -' 6 L 40
Q 1 1. I 1
< * z,
1 f — 30
g.‘ - 20
0.5 ,'
it 10
- k
.1
0 . . . 0
0 20 40 60 80 100 120

Time [min]

Figure 5.5. Aggregation of myosin and mixed solutions of myosin with 0.25% B-
lactoglobulin (B-LG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, during

heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C.

71

[30] aamnaadural

, * 0.1%LG -k 0.1% myosin/0.1% LG

 

 

 

 

 

7% 0.25% LG *9 0.1% myosin/0.25% LG
°— 0.5%LG ,, 0.1% myosin/0.5% LG
_,.,,1_0% LG 7'— 0.l% myosin/1% LG
Temperature
3.5 100
T . .— ‘ p _ _ 90
3 f . . -.- ,
80
D
, 70
E
c 1 60
v
E
§ . 50
a
.o
I.
3 — 40
n: >
<
- 30
1 20
, 10
1
0
80 100 120

 

Time [min]

[30] aamuadma 1,

Figure 5.6. Aggregation of B-lactoglobulin (B-LG) and mixed solutions of B-LG with

0.1% myosin in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, during heating at

1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C.

72

proteins determined the magnitude of the effects. Since myosin gelled at a lower

temperature, its aggregation was dominant in the mixed system.

5.4.3. Rheological properties

Gel points of B-LG solutions in PBS, pH 7.0, decreased from 79°C at 0.5%
protein to 73°C at 3% protein (Table 5.4). Data could not be obtained during holding at
90°C and cooling at any B-LG concentration due to shrinkage of the gel. The result
suggested that the minimum concentration of B-LG to form a set gel in 0.6M NaCl, pH
7.0, is higher than 3%. McSwiney et a1. (1994) reported that a minimum concentration of
5.0% was required for B-LG gel formation at pH 7.0 in 0.02M imidazole buffer, 0.1M
NaCl.

The gel point of myosin in PBS, pH 7.0, decreased (p<0.05) from 50.3 to 47.8°C
as the concentration was increased from 0.5-2.0% (Table 5.4). Storage modulus (G') of
myosin increased rapidly above its gel point (Figure 5.7). The rheograms of G' contained
two peaks at all myosin concentrations. The G' at each transition peak increased as the
concentrations were increased. The ﬁrst transition peak was observed at about 53-54°C
and a second peak occurred at 73°C. A two-transition temperature pattern was previously
reported in heat-induced gelation of myosin from live carp dorsal lateral muscle (Sano et
al., 1990), chicken breast muscle (Lui, et al., 1996), rabbit Psaos major (Boyer, et al.,
1996), and chicken breast muscle salt soluble proteins (Smyth et al., 1996). Smyth et al.
(1996) suggested that light meromyosin (LMM) and S-l of chicken breast myosin were

responsible for denaturation and aggregation below 55°C, while S-2 unfolded and

73

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74

600 100
70— 0.5°/o myosin

+ 1.0% myosin 90

 

  
 
    

_. 0 '
500 _ ‘— 1.54myosm
,l- 2.0% myosin dial' 80

— Temperature

_. 400

N

a »-a
g S
I— 'U
‘3 300 5
E E
O (9
w _
m 200 _

 

 

 

 

0 20 40 6O 80 100 120

Time [min]

Figure 5.7. Storage moduli of 0.5-2.0% myosin in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min

and cooling to 25°C.

75

aggregated above 55°C. Therefore, the ﬁrst peak of G' possibly reﬂected rigidity of the
network formed by LMM and S-l , while the aggregation of S-2 completed the protein
network and led to the increase of G' at the second peak.

The G’ of myosin decreased above 75°C and during holding at 90°C. In contrast,
Lui et a1. (1996) reported that G' of chicken breast myosin in 0.6M NaCl, pH 6.0
increased during holding at 75°C. Heating at 90°C may extensively unfold protein
molecules, facilitating intramolecular hydrophobic interactions and leading to the
lowering of gel elasticity. Syneresis was also detected during holding at 90°C, suggesting
extensive intramolecular interactions within the protein networks.

The G' of myosin increased sharply during cooling, indicating the formation of
hydrogen bonds which enhanced the ﬁnal rigidity or stiffness of the gels. The G' after
cooling of myosin at each concentration were 1.4-1.6 times greater than (3' at 73 °C (Table
5.4), indicating the important role of network cross-linking by hydrogen bonding.

In mixed protein solutions containing 0.5% myosin, addition of 1% B-LG
increased the gel point and decreased G' of the ﬁrst transition peak when compared to that
of 0.5% myosin (Table 5.4), indicating interruption of myosin network formation. When
myosin in mixed systems was increased 21.0%, 1% B-LG had no effect on the gel point
and the G' at the ﬁrst transition of myosin. The G' at 73°C and after cooling of mixed
solutions increased as myosin concentration was increased, but the G' was still lower than
that of myosin at the same concentration. At 90°C, the G' of all mixed solutions were
greater than those of myosin at the same concentrations (Figure 5.8). The results

suggested that in the mixed system containing 2 1% myosin, B-LG had no effect on gel

76

600
500 -
._ 400 -
a
E.
3
3
o 300 4
E
9
on
2
3
C0 200 «
100 —
O

100

 

 

 

 

 

—+ 0.5% myosin
-—o -].0% myosin * 90
—A-— 1.5% myosin
4 +20% myosin , 80
Temperature ﬂ
. ~ 70
— 6O
- 50
40
r 30

 

 

 

20 4O 6O 80 100 120

Time [min]

Figure 5.8. Storage moduli of myosin and mixed solutions of myosin and 1.0% [3-

lactoglobulin (B-LG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, during

heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C.

77

[30] ”mandala 1,

point but interrupted network formation of myosin during heating at 55-75°C. The gel
enhancing effects were obtained when the mixed protein solutions were heated to the
temperatures over the gel point of B-LG (80°C). Burgarella et a1. (1985) showed that the
addition of egg white proteins at a 1:4 ratio with surimi (12% total protein) lowered the
rigidity modulus of the gels below 40°C, but strengthened gel rigidity at temperatures
above the gelling temperature of both proteins (70°C). Hung and Smith (1993) reported
that addition of 4% WPC increased the ﬁrst G' transition temperature, and increased the
magnitude of G' of 12% chicken salt soluble protein in 0.6M NaCl, pH 7.0 when heated
to 90°C.

A log scale was used in Figure 5.9 to depict the effects of B-LG concentration on
the G' during heating of mixed protein containing 1.0% myosin compared to that of 1.0%
myosin. The G' of mixed protein decreased at 55-75°C and increased above 80°C when [3-
LG concentration was increased from 1% to 3%. The magnitude of the effects increased
with increasing B-LG concentration. The ﬁnal G’ of co-gels from 1% myosin mixed with
0.5%, 1%, 2% and 3% B-LG are 2, 4, 12 and 28 times greater than 6' of 1% myosin. It
was noticed that G' after cooling of 1% myosin/2% B-LG gels was 1447 Pa, almost 2
times greater than 835 Pa of 2% myosin/ 1% B-LG co-gel. This suggested that G' or
stiffness of co-gels could be controlled by an appropriate proportion of myosin and B-LG.
The G' enhancing effects could be a result of B-LG aggregation at temperature higher
than 80°C. These aggregates ﬁlled up the voids within the myosin gel matrix to form a

ﬁlled gel structure, enhancing rigidity of the co-gels.

78

 

 

[30] ummadmal

 

 

 

10000 100
+ 1% myosin/3% LG
—°— 1% myosin/2% LG - 90
0 ‘ 0
1000 _ +1Ameosm/l/0LG
—I— 1%myosin/0.5% LG ~ 80
+ 1.0% myosin
3' 100 Temperature " 70
E;
m . 60
=
3
° 10 - — 50
E
3’»
g - 40
3
m 1 _
- 30
- 20
0.1 i l
H
- 10
0.01 *£_. 5“— . . . . 0
0 20 40 6O 80 100 120
Time [min]

Figure 5.9. Storage moduli of myosin and mixed solutions of 1.0% myosin and 0.5-3%
B-lactoglobulin (B-LG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, during

heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C.

79

Myosin at 1.0% protein or above and myosin/B-LG solution at all proportions
exhibited Viscoelastic gel characteristic as indicated by the low values of tan 5 after
cooling (Table 5.4). Observation of polymer shows very high tan 5 for dilute solutions,
0.2-0.3 for amorphous polymers, and low (near 0.01) for glassy crystalline polymers and
gels (Steffe, 1996).

Gels formed from 2.0% myosin, 4% B-LG and the mixture of 1% myosin/ 1% [3-
LG were strong as G' and G" were independent of frequency with G' 8-10 times greater
than G" (Figure 5.10). A strong cross-linked gel network is characterized by G' and G"
independent of frequency, whereas an entangled weak gel is strongly dependent on
frequency (Stading et al., 1992). Myosin had a stronger gel structure than those of B-LG
and myosin/B-LG gels as indicated by the larger difference between G' and G". The
difference between moduli indicates recovery of the stored energy of the gel. A bigger

difference indicates a stronger gel network (Rao, 1999).

5.5. CONCLUSIONS

Based on our results, we propose the following gelation process for mixed
solutions of myosin and B-LG. During heating, myosin begins to denature and aggregate
below 50°C. The myosin aggregates to form a gel network beginning about 53°C. B-LG
remains soluble until its gel point of 73-79°C is reached. In systems containing less than
0.5% myosin, B-LG interrupted myosin gelation at 50-53°C during the aggregation and

network formation of the rod region. At myosin concentrations above 1.0%, B-LG

80

1 0000

 

I G': 2% myosin D G": 2% myosin
A G': 4% B-LG A G": 4% B-LG
O G': 1% myosin/1% B-LG O G": 1% myosin/1% B-LG

 

 

 

 

 

1000~ .“...
0
I G....“.“:::IIIIII.:
’3
5" A g
ED100 A e 60
E geénégonggnég‘e 6 o A
b D D an all
on DDDDDDDDDUDDDD
10
1 I I
0.01 0.1 1 10 100

Frequency (radian/sec)

Figure 5.10. Frequency dependence of storage (G') and loss (G") moduli of 2% myosin,
4% B-lactoglobulin (B-LG) and 1% myosin/ 1% B-LG gels in 0.6M NaCl, 0.05M sodium
phosphate buffer, pH 7.0, measured at 25°C. Gels were prepared by heating at 1°C/min

from 25 to 90°C, holding at 90°C for 30 min and cooling to 25°C.

81

interfered with network formation of myosin between 55-75°C. At higher temperatures
(>80°C), B-LG began to aggregate which enhanced the G' of the mixed gels. More
research is needed to elucidate the type of interactions between B-LG and myosin during
gel network formation. As B-LG gels at a higher temperature than myosin, the full
synergistic effect expected from addition of B-LG to myosin solutions is not likely to be
achieved until the protein mixture is heated to the gelling point of B-LG (>75°C). This
experiment showed that B-LG did not co-aggregate with myosin, but interrupted the gel
formation of myosin during heat treatment at 55-75°C. This ﬁnding indicates a reduced
potential for use of whey protein in meat products or in other applications where protein
gelation at low temperature is desirable. We hypothesize that rigidity of myosin/B-LG
co-gels could be enhanced if the protein networks can be formed simultaneously. A

modiﬁed B-LG, which has lower gelling temperature than that of the native protein, is

needed.

82

CHAPTER 6: GELLING PROPERTIES OF HEAT-DENATURED

B-LACTOGLOBULIN

6.1. ABSTRACT

The thermal denaturation, rheological and microstructural properties of gels
prepared from B-lactoglobulin (B-LG) and heat-denatured B-LG (HDLG) were studied.
The HDLG was prepared by heating solutions of 4% B-LG in deionized water, pH 7.0, at
80°C for 30 min. This heat treatment caused irreversible denaturation and formation of
soluble B-LG aggregates. In 0.6M NaCl, 0.05M sodium phosphate buffer at pH 7.0,
HDLG formed a set gel at a minimum concentration of 2% when heated to 71°C, whereas
24% protein was needed for B-LG to form set gels at 90°C. The HDLG (3%) gelled at
52°C and had a storage modulus (G') of 2200 Pa after cooling, whereas 3% B-LG had a G'
of 160 Pa upon cooling. When the pH was decreased from 7.0 to 6.0, 3% HDLG gelled
at 42°C and G' was 3100 Pa after cooling. The HDLG gel microstructure was comprised
of strands and clumps of small globular aggregates in contrast to B-LG gels which
contained a coarse, particulate network of compacted globules with large voids. The
HDLG formed a gel at lower concentration and lower temperature than B-LG, suggesting
that thermal denaturation and aggregation during preheating might improve the textural

enhancing effect of whey proteins when used in food products processed to 71°C or less.

83

6.2. INTRODUCTION

Whey protein is highly nutritious and is widely used as a functional ingredient in
comminuted and restructured meat products. Heat-induced gelation is an important
functional property of whey protein. However, commercial whey protein normally gels
above 75°C, which is higher than typical processing temperatures of many food products,
such as comminuted meat products. Therefore, the functional effects expected from
whey protein gelation in these food systems are limited.

The ability to form stong gels at low temperature broadens the potential use of
whey products. Researchers have looked at several ways to lower the gelling temperature
of whey protein. Application of hydrostatic pressure to whey protein concentrate (WPC)
and whey protein isolate (WPI) promoted sulﬂiydryl/disulﬁde interchange reactions,
formation of hydrogen bonds and rupture of hydrophobic interactions to induce gel
formation of the protein at ambient temperature (F untenberger et al., 1997). Partially
hydrolyzed B-LG prepared by limited proteolysis with trypsin had a lower gel point than
native B-LG (Chen et al., 1994). Preheating whey protein produced a product which
formed cold-set gels upon addition of salt or acid at ambient temperature or lower (Barbut
and Foegeding, 1993; McClements and Keogh, 1995; Roff and F oegeding, 1996;
Hongsprabhas and Barbut, 1998; In and Kilara, 1998b). The preheated whey proteins
formed stranded transparent gels with greater strength and water holding ability than a
heat-set whey protein gel prepared with the same concentration of salts.

Cold-set gelation of pre-heated whey protein involves a two-step process. In the

ﬁrst step, heating whey protein at neutral pH under low salt conditions results in the

84

formation of soluble aggregates of denatured protein. In the second step, gel network
formation is induced by increasing ionic strength or changing pH to allow interactions of
the soluble aggregates. Ionic strength, pH, protein concentration, time and temperature
were reported to be factors affecting soluble aggregate formation of whey protein during
preheat treatment (Ju and Kilara, 1998b; Hongsprabhus and Barbut, 1996; Mleko and
Foegeding, 1999b; Vardhanabhuti and Foegeding, 1999b). The mechanisms of soluble
aggregate formation and gelling properties of heat-denatured whey protein are not yet
fully understood.

Doi (1993) reported that a two-step heating method (re—heating the preheated
protein) formed stronger and more transparent gels of some globular proteins, such as
ovalbumin, bovine serum albumin and egg lysozyme when compared to those formed by
single-step heating of the protein in the same NaCl concentration.

Salt-induced cold-set gelation of whey protein aggregates could be beneﬁcial
when used in products containing salt and processed at low temperature, such as
comminuted meat. Since myosin is the major functional component of meat protein,
buffer conditions and heat treatment for myosin gelation (heating to 71°C at 1 °C/min for
60 min in 0.6M NaCl, 0.05M sodium phosphate buffer) was used to study gelling
properties of B-LG and HDLG. The objective of this experiment was to compare the
thermal denaturation, rheological and microstructural properties of B-LG and HDLG gels
prepared by heating to 71°C at 1°C/min for 60 min in 0.6M NaCl, 0.05M sodium

phosphate buffer, pH 6.0, 6.5 and 7.0.

85

6.3. MATERIALS AND METHODS
6.3.1. Preparation of protein solutions

Bovine milk B-LG (L0310 lot # 114H7055) containing variants A and B was
purchased from Sigma Chemical Co. (St. Louis, MO). B-LG was dissolved in 0.6M
NaCl, 0.05M sodium phosphate buffer (PBS) at pH 6.0, 6.5 or 7.0 and kept at 4°C before
use. Protein concentration was determined by spectrometric absorption using an
extinction coefﬁcient (Em) of 9.55 at 278 nm (F oegeding et al., 1992). Heat-denatured [3-
LG was prepared based on In and Kilara (1998b) by dissolving 4% B-LG in deionized
water and adjusting to pH 7.0 with 0.1N NaOH or HCl. The solutions were placed in 16
x 125 mm glass tubes, heated at 80°C for 30 min in a water bath, cooled in an ice bath,
and kept at 4°C. The HDLG was diluted with concentrated buffer to prepare solutions of
1.0, 2.0, and 3.0% protein in PBS. Protein solutions were adjusted to the desired

concentration immediately before use to avoid gel formation during holding at ambient

temperature.

6.3.2. Electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was
used to assess the purity of B-LG and to characterize the molecular weight of B-LG
aggregates. Electrophoresis was run in a mini-Protean 11 Dual Slab Cell (Bio Rad
Laboratories, Hercules, CA) using a Tris (hydroxymethyl) arninomethane glycine
electrode buffer, pH 8.3, and 0.1% SDS as described by Laemmli (1970). The

acrylamide concentration of stacking and resolving gels were 4% and 14%, respectively.

86

 

B-LG and HDLG solutions were diluted to 4 mg/mL with sample buffer (0.0625M
Tris pH 6.8, 2% SDS, 10% glycerol, and 0.2% bromophenol blue), mixed well using a
vortex mixer, heated in boiling water for 5 min, and stored at -20°C until use. Sample
buffer for reducing conditions also contained 5% of 2-mercaptoethanol. About 5 uL of
each protein sample was loaded into a sample well and the gels were run at 85 mA
current and 200 constant voltage for 45 min. Gels were stained for at least 20 min with
Coomassie Brilliant Blue R250 solution (0.25% in 9:45:45 v/v/v of acetic acid: methanol:
water) and were de-stained overnight in acetic acid-methanol-water (6:4:7 v/v/v) solution.
Molecular weight of the proteins was determined from their relative mobilities compared
to those of standard molecular weight markers (Bio-Rad Laboratories, Hercules, CA)

under the same electrophoretic conditions (Weber and Osborn, 1969).

6.3.3. Scanning electron microscopy (SEM) of aggregates

Soluble HDLG aggregates were mixed with an equal volume of 4% glutaldehyde
in 0.1M sodium phosphate buffer, pH 7.0, for 1h at 4°C. After ﬁxation, one drop of the
suspension was placed on a cover slip coated with poly-L-lysine (Sigma, P1399) and
allowed to stand for 5 min. The cover slip was then carefully washed with several drops
of distilled water, follow by dehydration, critical drying and coating as previously
described except that a 15 nm layer of gold was applied. The HDLG aggregates were
examined at an 8 mm working distance using an accelerating voltage of 10 kV and
20,000 x magniﬁcation. The HDLG samples were prepared in duplicate and three

samples from each preparation were examined.

87

6.3.4. Differential scanning calorimetry

The thermal denaturation patterns of 1% B-LG and 1% HDLG in deionized water,
pH 7.0, and in PBS, pH 6.0, 6.5, and 7.0, were investigated using a MC-2 differential
scanning calorimeter (Microcal Inc., Amherst, MA). The protein and blank solutions
(deionized water or PBS) were degassed in a vacuum chamber (N algene, Fisher
Scientiﬁc, Pittsburgh, PA) and 1.24 mL were immediately loaded into the DSC using a
syringe. Each experiment was conducted in triplicate at a heating rate of 1 °C/min from
25°C to 90°C. Heat capacity proﬁles (Cp vs temperature) was used to deﬁne calorimetric
enthalpy (AH,,,) and endothermic peak or melting temperature (Tm) using the software

(DA-2 Data Acquisition and Analysis System) provided by the manufacturer.

6.3.5. Rheological measurement

Dynamic oscillatory tests of 1-3% HDLG and B-LG in PBS at pH 6.0, 6.5 and 7.0
were performed using a controlled stress rheometer (RS 100, Haake, Karlsruhe,
Germany) equipped with a 35 mm diameter stainless steel parallel plate. A circulating
water bath was used to control temperature. Storage (0') and loss (G") moduli were
recorded continuously at a ﬁxed frequency of 0.464 Hz. using constant stresses
(producing strains from 0.1-0.3%) within the range of linear Viscoelastic behavior
determined from stress sweeps performed for each protein at 90°C and after cooling to
25°C. Protein solutions were loaded between the parallel plate and base of 1.0-1.1 mm
gap. A few drops of maize oil (Mazola, Best Food, CPC International, Inc., Englewood

Cliffs, NJ) were used to cover the edge of the plate to prevent evaporation. Solutions

88

were equilibrated at 25°C for 5 min, heated from 25 to 71°C at 1°C/min, held at 71°C for
60 min, cooled to 25°C within 10 min and held at 25°C for 5 min. In addition, the B-LG
solutions were also heated to 90°C, held at 90°C for 30 min and cooled to 25°C within 15
min. Each experiment was performed in triplicate. The gel point was determined as the

temperature at which G' and G" crossed over in the ﬁxed frequency test.

6.3.6. Scanning electron microscopy (SEM) of gels

The B-LG and HDLG gels were prepared at 4% and 2% protein, respectively, in
PBS, pH 6.0, 6.5, and 7.0. One milliliter of each protein solution was transferred into a
12 mm x 75 mm glass tube and sealed with TeﬂonTM tape. The HDLG were heated in a
programmable water bath (Model 9510, PolyScience, Niles, IL) from 25°C to 71°C at
1°C/min, held for 60 min, and cooled in an ice bath. B-LG solutions were heated from 25
to 90°C at 1°C/min and held for 30 min before cooling. Protein gels were cut into 1 x 2 x
2 mm pieces, ﬁxed in 2.0% glutaldehyde in 0.1M sodium phosphate buffer, pH 7.0, for
3h at 4°C and post-ﬁxed in 0.1% osmium tetraoxide overnight at 4°C. Fixed gels were
rinsed with 0.1M sodium phosphate buffer and dehydrated by immersion in a graded
ethanol series of 25, 50, 75, and 95% for 20 min per step, followed by three steps in
100% ethanol. Gels were dried using a Balzers carbon dioxide-critical point dryer
(Balzers CPD, FL-9496, Balzers, Liechtenstein) and coated with a 25-30 nm gold layer in
an Emscope ion-sputter coater (Emscope Laboratories Ltd., Ashford, Kent, UK). Gel
structures were observed with a JEOL scanning microscope (Model J SM-6400V, version

96-2, Tokyo, Japan) at 15 mm working distance using an accelerating voltage of 10-12

89

kV and 5,000x magniﬁcation. Gels were prepared in duplicate and three samples from

each preparation were examined in the SEM.

6.3.7. Experimental design and statistical analysis

All experiments were conducted in triplicate. Differences in gel point and storage
moduli of gels due to treatments (protein concentration and pH) were statistically
analyzed using one way analysis of variance (AN OVA). Means were compared to
determine signiﬁcant differences between treatments using the Tukey-Kramer HSD test
with the mean square error at 5% level of probability (JMP Version 3.2.2, SAS Institute

Inc., Cary, NC).

6.4. RESULTS AND DISCUSSION
6.4.1. Electrophoresis

The electrophoretic patterns of B-LG and HDLG resolved by SDS-PAGE
under non-reducing and reducing condition are shown in Figure 6.1. A band
corresponding to B-LG monomer (18.4 KDa) was observed under both conditions. SDS-
PAGE of HDLG contained a similar protein band under reducing condition, while only a
minor band of B-LG at 18.4 KDa and some proteins too large to pass through the stacking
gel were observed using non-reduced SDS-PAGE. The results suggested that a small
amount of B-LG was present in HDLG. A weak band of about 37 KDa was observed in

both native B-LG and HDLG. It may be a dimeric form of B-LG.

90

 

A 9""! B — uni 211
"'"" W..- 122
H are.» so
”a 51
“3““ 35.9
“TI 28.6
m a ... w gas“? 20.8
ﬂ *3 67

l 2 3 3 2 1

Figure 6.1. Electrophoregram of (A) nonreduced and (B) reduced native and heat-

denature B-lactoglobulin: Lane 1, molecular weight markers; Lane 2, heat-denature B-

lactoglobulin; and Lane 3, B-lactoglobulin.

91

Prabakaran and Damodaran (1997) reported that B-LG dimers were formed via
disulﬁde linkages between monomers during heating at 60-65°C, pH 7.0. A similar
protein band was found in B-LG A, B, and C heated to 72°C for 12.5 min (Manderson et
al., 1998). When heated to higher temperatures, these dimers further formed larger
aggregates or polymers which did not pass through a 4% stacking gel of SDS-PAGE
under non-reducing conditions, suggesting that disulﬁde bonding played a dominant role

in the formation of HDLG aggregates.

6.4.2. Microstructure of heat-denatured B-lactoglobulin aggregates

Heat-denatured B-LG was comprised primarily of small globular aggregates of
30-50 nm, although some of the globules appeared to associate into larger clumps and
long strands (Figure 6.2). Native B-LG has a diameter of 3-5 nm (Elofsson et al., 1996),
suggesting HDLG aggregates were formed by several B-LG molecules. The HDLG
aggregates were similar in size to those of whey protein aggregates (21-61 nm) prepared
by heating 3-9% WPI at 80°C for 30 min, although WPI aggregates were comprised of B-
LG, a-LG, bovine serum albumin and immunoglobulin (Ju and Kilara, 1998b; 1998c).
Since disulﬁde bonds and hydrophobic interactions were found to be important in
polymerization of whey protein and B-LG (Prabakaran and Damodaran, 1997; Mleko and
Foegeding, 1999b; Vardhanabhuti and Foegeding, 1999a), the mechanisms and factors
affecting the formation of soluble aggregates of whey protein and B-LG could be similar.
The size and shape of protein polymers greatly affect network formation of gels (Wang

and Damodaran, 1990). Studies of optimum conditions for the formation of B-LG

92

 

Figure 6.2. Scanning electron microscopic image of heat-denatured B-lactoglobulin

aggregates at 20,000x magniﬁcation. Bar = 1.0 um.

93

polymers could provide information for manipulation of gelling properties of whey

protein polymers.

6.4.3. Thermal denaturation of B-lactoglobulin and heat-denatured B-lactoglobulin

The therrnograms of 1% B-LG and 1% HDLG in deionized water, pH 7.0, were
different (Figure 6.3A). B-LG melted at 79.0°C. Foegeding et a1. (1992) reported a
denaturation temperature (Tm) of 77.3 °C when 10% B-LG was heated in deionized water
at 10°C/min. The difference of Tm is probably due to different protein concentrations
used in the experiments. A more cooperative denaturation peak (narrower peak) and
lower Tm are often observed as the protein concentration is increased (Relkin and Launay,
1990; Qi et al., 1995). No thermal transitions were found for HDLG, suggesting that pre-
heating of B-LG at 80°C for 30 min resulted in irreversible denaturation of the protein
molecules. The irreversible denaturation of B-LG and whey protein above 80°C has been
previously reported (McClements and Keogh, 1995). However, De Wit and Klarenbeek
(1981) and Paulsson et al. (1985) suggested that the complete denaturation of B-LG
probably occurred between 130-140°C as indicated by a second distinct denaturation peak
of B-LG. It is possible that HDLG was a stabilized form of partially denatured or molten
globular form of B-LG occurred during preheating. The molten globule was deﬁned as a
unique molecular state with partially folded conformation, retaining some native
structures, but being distinctive from fully forms (Li-Chan, 1995).

The denaturation proﬁles of HDLG in PBS at pH 6.0, 6.5 and 7.0 showed small

exothermic peaks at about 56, 67 and 71°C, respectively (Figure 6.3B). Since DSC

94

 

 

    

 

 

 

A
6 5 —
9:
3 4
E _
b
'8 3 w
a
a.
8
.. 2
g B-LG
1 l T
HDLG
0 4

25 3O 35 4O 45 50 55 6O 65 7O 75 80 85 90

Temperature [°C]

 

 

 

Heat capacity [meal/°C]
O

 

 

 

25 3O 35 4O 45 50 55 6O 65 70 75 8O 85 90
Temperature [°C]
Figure 6.3. Heat capacity proﬁles of (A) 1.0% B-lactoglobulin (B-LG) and 1.0% heat-

denatured B-LG (HDLG) in deionized water, pH 7.0, and (B) 1.0% HDLG in 0.6M NaCl,

0.05M sodium phosphate buffer, pH 6.0, 6.5 and 7.0.

95

simultaneously detects denaturation and aggregation enthalpy (Gotham et al., 1992), the
exothermic peaks were possibly a reﬂection of HDLG aggregation in the presence of
0.6M NaCl. Additional experiments using a higher concentration of HDLG and/or
different concentrations and types of salts are needed to reveal whether or not these

exothermic peaks indicate HDLG aggregation.

6.4.4. Rheological properties of heat-denatured B-lactoglobulin gels

During heating to 90°C and holding for 30 min, the gel points of B-LG in 0.6M
NaCl, 0.05M sodium phosphate buffer, pH 7.0, decreased from 79°C at 1.0% protein to
73°C at 3.0% protein. The G' of B-LG could not be measured accurately during holding
and cooling due to precipitation and shrinkage of the gels which led to rapid ﬂuctuations
of G' (Figure 6.4). This may be a result of using a lower concentration of B-LG than
optimal to set a gel. McSwiney et a1. (1994) reported that 5% is the critical concentration
of B-LG to form gels at 80°C in 0.1M NaCl at pH 7.0. When heated to 71°C and held at
71°C for 60 min in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, G' of 1-3% [3-
LG were very low (4-9 Pa), indicating lack of network formation when heated at a
temperature lower than its gel point.

Heat-denatured B-LG formed gel networks without precipitation as indicated by a
continuous increase of G' during heating at 71°C and cooling (Figure 6.5). At pH 7.0, the
gel point of HDLG decreased (p<0.05) by 19°C and G' after cooling increased 137 fold as

protein concentration was increased from 1% to 3% (Table 6.1). Gels formed by

96

 

 

 

[30] arniuadmaj,

 

 

 

 

 

700 100
+ 1.0%
+20% ~ 90
600 - +30%
Temperature - 80
500 - ~ 70
'1?
E.
Q ~ 60
3 400 4
3
o ~ 50
E
9 -
3:” 300 _ 40
i-
3
m 0
200 - ’ 3
- 20
100 ~
— 10
O '1 l, l I l O
O 20 4O 6O 80 100 120 140

Time [min]

Figure 6.4. Storage moduli of B-lactoglobulin in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0, during heating at 1°C/min from 25 to 90°C, holding at 90°C for 30 min

and cooling to 25°C.

97

 

2500

 

4w 1.0%
—A- 2.0%
—a— 3.0%

Temperature

2000 -

 

1500 4

1000 «

Storage Modulus [Pa]

500 -

 

 

60 80

Time [min]

100

“Tm“?

 

120

 

140

100

90

#80

-40

DJ
O

~20

~10

Figure 6.5. Storage moduli of 1-3% heat-denatured B-lactoglobulin in 0.6M NaCl,

0.05M sodium phosphate buffer, pH 7.0, during heating at 1 °C/min from 25 to 71°C,

holding at 71°C for 60 min and cooling to 25°C.

98

[30] ”mandala .1.

Table 6.1. Gel point, storage moduli (G') and tan delta (tan 8) after cooling of 1-3% heat-

denatured B-lactoglobulin (HDLG) heated to 71°C in 0.6M NaCl, 0.05M sodium

phosphate buffer, pH 6.0, 6.5 and 7.01

 

 

 

Parameter PH
Concentration 6.0 6.5 7.0
63.0 5. 0.5“ 71°C” 71°96
Gel point (°C) 49.0 i 0.9“ 58.5 i 0.5".y 62.0 i 05‘”
42.0 i 0.5” 50.0 i 1.09y 52.5 i 1.0”
G' (Pa) 100 i143" 40 i- 111'y 16 i 22"y
860 i 50"“ 700 i 108‘” 585 i 58“
3,100 i 57°“ 2,700 i 150°“ 2,200 i 140°“
Tan6 0.12:0.01“ 0.14:0.02“ 0.13:0.01“
0.10 i 0.02bx 0.10 i 0.00“ 0.10 i 0.01“
0.10 i 000'“ 0.09 :t 001‘“ 0.09 i 0.00“

 

' Values are means of three observations i standard deviation.
2" b' ° Means with different superscripts in each column are signiﬁcantly different (p<0.05).
"' y' 7‘ Means with different superscripts in each row are signiﬁcantly different (p<0.05).

99

1% HDLG were very weak (16 Pa at pH 7.0 and 100 Pa at pH 6.0), suggesting that more
than 1% HDLG is needed for the formation of a strong gel network.

The results show HDLG formed a gel at concentrations higher than 1% whereas,
higher than 4% of B-LG was needed to form gel when heated to 71°C in 0.6M NaCl,
0.05M sodium phosphate buffer, pH 7.0. A lower gelling temperature and greater gel
rigidity have been previously reported for a preheated WPI when compared to unheated
whey protein (McClements and Keogh, 1995). In 0.2M NaCl, pH 7.0, 9% preheated WPI
gelled at 48°C, whereas the unheated WPI formed a gel at 77°C or higher. The complex
modulus after cooling of the preheated WPI was much higher than that of untreated WPI.
Other globular proteins, such as ovalbumin, bovine serum albumin and egg lysozyme
have been reported to form more transparent and harder gels by reheating the proteins
which have been preheated without addition of salts (Doi, 1993).

Effects of pH on gelling properties of HDLG were studied (Figure 6.6). When the
pH was decreased from 7.0 to 6.0, the gel point of HDLG at all concentrations decreased
about 10-12°C (Table 6.1). The decrease in gel point at pH 6.0 may be due to an
acceleration of aggregation resulting from the neutralization of the more negatively
charged molecule at pH 7.0. Melko and Foegeding (1999a) reported that a weak gel was
formed from preheated WPI when pH was decreased from 8.0 to 6.0 without addition of
salt. The rigidity of the gels was increased with an increase of temperature from 7 to

25°C, suggesting that both electrostatic and hydrophobic interactions were involved in gel

network formation.

100

 

 

 

 

 

 

3500 100
+pI-I 6.0. _ 90
3000 ‘ —o——pH 6.5
r 80
+pH 7.0
2500 ‘ -— Temperature ' * 70
E .
2‘ - 60 F
.2 2000 r 5
:1 "a
c 91
° — 50 a:
2 '5:
0
500 - a
g 1 - 4O '3‘
3 O
m —
1000 r ' 30
' 20
500 ‘
~ 10
0 . . 0
0 20 40 60 80 100 120 140

Time [min]

Figure 6.6. Storage moduli of heat-denatured B-lactoglobulin in 0.6M NaCl, 0.05M
sodium phosphate buffer, pH 6.0, 6.5 and 7.0, during heating at 1°C/min from 25 to 71°C,

holding at 71°C for 60 min and cooling to 25°C.

101

 

Tan 5 after cooling of 2.0-3.0% HDLG gels were low ($01) at pH 6.0, 6.5 and 7.0
(Table 6.1), indicating a typical Viscoelastic of gel (Steffe, 1996). The G' of HDLG after
cooling were the same (p>0.05) at all pHs (Table 6.1). The results suggested that
electrostatic interactions caused by pH changes affected the aggregation step, while
hydrophobic interactions during heating contributed to the formation and strengthening of
the HDLG gel network. However, the ultimate gel structure and strength also depend on
salt type and concentration. The combined effect of 0.6M NaCl, pH, and temperature

contributed to HDLG gel properties observed.

6.4.5. Microstructure of B-lactoglobulin and heat-denatured B-lactoglobulin gels
Microstructures of 4% B-LG gels prepared at 90°C for 30 min and those of 2%
HDLG gels prepared by heating at 71°C for 60 min were compared (Figure 6.7). B-LG
formed particulate gels comprised of grape-like compacted globular aggregates. Globule
diameter decreased from 1-3 pm at pH 6.0 to 0.2-0.4 pm at pH 7.0. These globule sizes
were similar to those of B-LG gels prepared by heating 0.9% B-LG in 0.5M NaCl, pH 7.0,
at 685°C for 1.5h (Verheul et al., 1998). HDLG formed a protein network comprised of
small globules of 30-70 nm diameter (determined by comparing with bar size in enlarged
images) arranged in clumps and strands. Based on the size and shape of the HDLG
soluble aggregates (Figure 6.7), the HDLG gel networks appeared to form via cross-
linking of the primary soluble aggregates. When compared to B-LG gels, the HDLG gel

structure was much ﬁner with smaller void spaces.

102

Figure 6.7. Scanning electron microscopic images at 5,000 x magniﬁcation of (A) 4%
B-lactglobulin gels heated to 90°C for 30 min and (B) heat-denatured B-lactoglobulin
gels heated to 71°C for 60 min in 0.6M NaCl, 0.05M sodium phosphate buffer, (1) pH

6.0, (2) 6.5, and (3) 7.0. Bar = 1.0 pm

103

 

104

No change in the microstructure of HDLG gels was observed when the pH was
decreased from 7.0 to 6.0. The G' of HDLG gels also did not change with pH (Table 6.1).
The HDLG gels were visually less opaque than B-LG gels corresponding to the
differences in microstructures. B-LG gel networks were comprised of 50 fold bigger
aggregates than those of HDLG gels, resulting in more opacity. Many studies have
shown a relationship between the network microstructure and the opacity of the gels.
Cold-set gels with larger aggregates and bigger pore sizes were more opaque than gels
with ﬁner structure (Hongsprabhus and Barbut, 1998). Bigger aggregates form a gel

network with the higher opacity due to increased light scattering (Barbut and Foegeding,

1993; Foegeding et al., 1998).

6.5. CONCLUSIONS

Pre-heating at 80°C for 30 min in distilled water at pH 7.0 changed the molecular
characteristics of B-LG leading to alteration of its gelling properties. The results from
this experiment indicated that B-LG formed primary aggregates from denatured
molecules via disulﬁde bonding during the preheat treatment. When reheated at 71°C in
0.6M NaCl, networks of HDLG gels were formed through the secondary association of
the primary aggregates. The two-step aggregation process allowed B-LG to form a ﬁner
gel structure at a lower protein concentration and lower temperature when compared to a
one-step aggregation process using untreated B-LG. The results are similar to those
obtained using preheated WPC and WPI (Barbut and Foegeding, 1993; McClements and

Keogh, 1995; Hongsprabhus and Barbut, 1996; Ju and Kilara, 1998b, 1998e). The cold-

105

 

set whey proteins form gels at lower temperatures than traditional WPC or WPI and may
allow increased use of whey proteins in a variety of applications where protein gelation at
low temperature is desirable. Cold-set whey protein could also be used to prepare food
with functional properties different from those of traditional whey ingredients.

This experiment was carried out at temperatures and pH conditions favorable for
myosin gelation, conﬁrming the potential use of whey protein aggregates in meat
systems. More experiments are needed to understand interactions between HDLG and
myosin in mixed systems. An understanding of factors inﬂuencing the characteristics of
the primary aggregates, which ultimately govern the textural properties of the ﬁnal gels,

will provide direction for achieving desirable functional properties of whey products.

106

 

CHAPTER 7: INTERACTIONS OF HEAT-DENATURED ﬂ-LACTOGLOBULIN

WITH MYOSIN DURING HEAT-INDUCED GELATION

7 .1. ABSTRACT

Effectiveness of whey proteins to enhance texture of processed meat products is
often variable as the major component, B-lactoglobulin (B-LG), does not form a gel at
typical processing temperatures (ca. 68-71°C). Pre-heated whey protein isolates (WPI)
gel upon addition of salt at temperatures well below those for the native proteins. To
understand the functions of preheated whey proteins in meat systems, heat-induced
gelation mechanisms of a model system using myosin and heat-denatured B-LG (HDLG),
were studied.

The HDLG was prepared by heating 4% (w/v) B-LG solutions in deionized water,
pH 7.0 at 80°C for 30 min. Thermal denaturation patterns of myosin and myosin/HDLG
at pH 6.0 and 7.0 were similar, except for the appearance of an endothermic peak at 56°C
and 54°C, respectively, in the mixed system. When heated to 71°C at pH 7.0, 2% myosin
began to gel at 48°C and had a storage modulus (G') of 500 Pa after cooling. Mixed
solution of 1% myosin/ 1% HDLG gelled at 48°C and G' was 650 Pa after cooling,
whereas myosin/B-LG gelled at 49°C but the G' was lower (180 Pa). As the pH was
decreased, the gel points of myosin and myosin/HDLG decreased and G' increased.

When compared to 2% myosin gels, myosin/B-LG gel structures were comprised of

107

thicker stranded networks with larger void spaces, whereas myosin/HDLG gels had
similar ﬁne stranded networks with additional clumps of small globules. In the co-gel
systems heated to 71°C, B-LG remained soluble, whereas HDLG was incorporated within
the myosin network. Co-gel formation between preheated whey and meat proteins when
heated at 71°C led to enhanced gel stiffness. Lowering the gelling temperatures of whey

protein may lead to their increased use in meat products.

7.2. INTRODUCTION

Whey protein has been widely used as a functional food ingredient. In meat
products, whey protein is used to improve color, yield and textural quality. Heat induced
gelation of whey protein was reported to be responsible for texture enhancement of some
meat products. However, the effect of whey protein on textural attributes of meat
products is often variable. Commercial whey proteins do not form gels at typical meat
processing temperatures (ca. 68-71°C), which limit their potential for textural attributes.

Many studies have looked at the cold-set gel properties of pre-denatured whey
protein aggregates (Roff and Foegeding, 1996; McClements and Keogh, 1995;
Hongsprabhus and Barbut, 1996; 1998, Ju and Kilara, 1998b). Heating whey protein
solution without addition of salt leads to formation of denatured soluble aggregates or
polymers. Cold-set gels of the aggregates can be induced by increasing the ionic strength
through the addition of salts, lowering the pH or by proteolytic digestion. The gels
formed from pre-heated whey proteins were reported to be clearer and harder than those

from untreated whey protein gels. The preheating of protein solutions before heat-induced

108

gelation (two-step heating process) also enhanced the gel properties (harder and clearer
gels, and able to gel at broader range of NaCl concentrations) of other globular proteins,
such as bovine serum albumin, ovalbumin and egg lysozyme (Doi, 1993).

Recently, Hongsprabhus and Barbut (1999) studied the textural properties and
water-holding capacity of comminuted chicken mixed with preheated whey protein
isolate (WPI). They found that addition of 2% pre-heated WPI aggregates (prepared by
heating at 80°C for 30 min, pH 7.0) in conjunction with cold set gelation (6h at 1°C) l
lowered cook loss and improved hardness of the comminuted chicken aﬁer cooking to
78°C at salt concentrations of 1.5% or below, when compared to unheated WPI. More
work is needed to understand interactions between whey protein aggregates and meat
proteins during heating to optimize the use of whey protein in meat products.

We have previously studied heat-induced gelation of B-LG and myosin, the major
functional components of meat and whey protein. The synergistic effect expected from
addition of B-LG to myosin solutions was not achieved until the protein mixture was
heated to the gelling point of B-LG (over 75°C). At 55-75°C, B-LG interfered with
network formation of myosin. We have also studied gelling properties of B-LG compared
to heat-denatured soluble aggregates of B-LG (HDLG). The HDLG was prepared by
preheating B-LG solutions at 80°C for 30 min at pH 7.0. At pH 6.0-7.0 and 0.6M NaCl
(optimum for myosin gelation), HDLG formed a gel with a ﬁner microstructure and with
much higher gel stifﬁ1ess at lower protein concentration and temperature than B-LG.

The salt-induced gelation of heat-denatured soluble aggregates of B-LG at low

temperature indicates a potential benefit for the use of the whey aggregates in meat

109

products. Salt is commonly added to meat products to extract salt soluble protein and for
ﬂavor. The objective of this experiment was to characterize interactions between myosin

and B-LG or HDLG during heat-induced gelation in 0.6M NaCl at pH 6.0-7.0.

7.3. MATERIALS AND METHODS
7.3.1. Protein solution preparation

Myosin from breast muscle (M. pectoralis) was extracted immediately after
sacriﬁce from 9 week-old commercial type broilers, and stored in 48% saturation of
(NH,,)ZSO4 and 30% glycerol at —20°C (Wang and Smith, 1994b). Immediately prior to
use, myosin was suspended in 0.6M NaCl, 50 mM sodium phosphate buffer (PBS) at the
desired pH, dialyzed against three changes of the same buffer for 48h and centrifuged at
78,000 x g to remove denatured proteins. Solutions of 1% and 2% myosin were prepared
by dilution of this stock solution with PBS at the desired pH.

Bovine milk B-LG, containing valiants A and B, was purchased from Sigma
Chemical Co. (St. Louis, MO). B-LG solutions were prepared by dissolving the protein
in PBS, pH 7.0, overnight at 4°C. Heat-denatured B-LG was prepared by dissolving 4%
(w/v) B-LG in deionized water and pH was adjusted to 7.0 with 0.1M HCl. The solution
was heated in a water bath at 80°C for 30 min, then cooled to 4°C in an ice bath. To
prepare 2.0% HDLG solutions, HDLG was diluted 1:1 with 1.2M NaCl, 0.1M sodium
phosphate buffer.

Mixtures of 1% (w/v) myosin and 1% (w/v) B-LG or HDLG were prepared by

mixing together equal amounts of 2% myosin and 2% B-LG or 2% HDLG. Mixed

110

 

protein solutions were prepared immediately before use. Protein concentrations were
determined by absorption using extinction coefﬁcients (E'%) of 5.5 at 280 nm for myosin

(Smyth et a1, 1996) and 9.55 at 278 nm for B-LG (Foegeding et a1, 1992).

7.3.2. Differential scanning calorimetry

The thermal denaturation patterns of 1% myosin, 1% myosin/ 1% B-LG
(myosin/B-LG) and 1% myosin/ 1% HDLG (myosin/HDLG) in PBS, pH 6.0 and 7.0,
were investigated using a differential scanning calorimeter (DSC) (MC-2, Microcal Inc.,
Amherst, MA). The protein and buffer solutions (PBS) were degassed in a vacuum
chamber before loading into 1.24 mL capacity cells of the DSC. Experiments were
conducted at a scan rate of 1°C/min from 25°C to 90°C. Heat capacity proﬁles
(endotherm; Cp vs temperature), calorimetric enthalpy (AHw) and endothermic peak or
melting temperature (Tm) were obtained by using the software (DA-2 Data Acquisition
and Analysis system) provided by the manufacturer. DSC scans were performed at least

in triplicate using samples from two separate preparations.

7.3.3. Dynamic rheological measurement

A controlled stress rheometer (RS 100, Haake,Karlsruhe, Germany) equipped
with a 35 mm diameter stainless steel parallel plate was used to monitor storage modulus
(G') and loss modulus (G") during heat-induced gelation of 1% and 2% myosin,
myosin/B-LG and myosin/HDLG solutions. The tests were performed at a ﬁxed

frequency of 0.464 Hz. Using constant stresses (producing strains from 0.1-0.3%) within

111

 

the range of linear Viscoelastic behavior determined from stress sweeps performed for
each protein at 71°C and after cooling to 25°C. About 1.0 mL of each protein solution
was loaded between the plate and base with a gap of 1.0-1.1 mm. A few drops of maize
oil (Mazola, Best Food, CPC International, Inc., Englewood Cliffs, NJ) were used to
cover the edge of the plate to prevent evaporation. The solutions were equilibrated at
25°C for 5 min, heated to 71°C or to 90°C at 1°C /min, held for 60 min at 71°C or 30 min
at 90°C, cooled to 25°C within 10 or 15 min, and held at 25°C for 5 min. The frequency
(0.01-100 radian/sec) sweep tests were conducted at the end of the cooling step using
stress which was controlled to produce a strain of 0.1-0.2%. The gel point was deﬁned as

the temperature at which G' and G" crossed over on the rheogram. All experiments were

performed in triplicate.

7.3.4. Scanning electron microscopy (SEM)

To prepare protein gels, 1.0 mL of each protein solution (myosin, myosin/B-LG or
myosin/HDLG) was transferred into 12 mm x 75 mm glass tubes and sealed with
TeﬂonTM tape. Tubes were placed in a water bath (PolyScience, model 9510, Niles, IL)
programmed to heat from 25°C to 71°C or to 90 at 1°C /min, and hold for 60 at 71°C or 30
min at 90°C. The samples were cooled in an ice bath. Protein gels were cut into 1 x 2 x 2
mm pieces. Specimens were preﬁxed for 3h in 2.0% glutaldehyde buffered with 0.1M
sodium phosphate pH 7.0, and postﬁxed ovemight in 0.1% osmium tetraoxide. Fixed gels
were rinsed thoroughly with 0.1M sodium phosphate buffer, pH 7.0, and dehydrated in a

graded series of ethanol (25, 50, 75, and 95%) for 20 min each followed by three 20 min

112

changes in 100% ethanol. Gels were then dried using a Balzers carbon dioxide-critical
point dryer (Balzers CPD, FL-9496, Balzers, Liechtenstein) and coated with a 25-30 nm
gold layer in an Emscope ion-sputter coater (Emscope Laboratories Ltd., Ashford, Kent,
UK). Protein gel microstructures was observed with a JEOL scanning microscope
(Model J SM-6400V, version 96-2, Tokyo, Japan) using an accelerating voltage of 12 kV
at a magniﬁcation of 5000x. Gels were prepared in duplicate and three pieces from each

gel were examined by SEM.

7.3.5. Soluble proteins expressed from co-gels

Proteins remaining in the liquid phase of the protein gel networks were quantiﬁed
by Bradford protein assay and identiﬁed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). A half milliliter of 2% myosin, 4% B-LG, 2% HDLG,
myosin/B-LG, and myosin/HDLG solutions were heated in 1.5 mL micro-centrifuge
tubes using the heating schedule previously described for SEM gel preparation. The caps
of all tubes were punctured with a needle to prevent them from popping up during
heating. After cooling, the protein gels were centrifuged at 10,000 x g for 30 min
(Sorvall RC SB/Plus, Sorvall Products, Newtown, Connecticut). The supematants were
collected and weighed.

Protein concentration in the supematants was determined following the method of
Bradford (1976) using bovine serum albumin as the protein standard. Absorbance was

measured at 595 nm in Minireader II (Thermomax, Molecular Devices Corp, Menlo Park,

113

 

CA), and the protein concentration was estimated by regression analysis (Softmax
program version 231, Molecular Devices).

SDS-PAGE was used to identify soluble protein expressed from gels by
centrifugation. Electrophoresis was run in a Mini-Protein 11 Dual Slab Cell (Bio Rad
Laboratories, Hercules, CA) using a Tris (hydroxymethyl) arninomethane (Tris)-glycine
electrode buffer, pH 8.3, containing 0.1% sodium dodecyl sulfate (SDS) as described by
Laemmli (1970). The acrylamide concentrations of stacking and resolving gels were 4%
and 12%, respectively. Protein solutions were diluted 1:2 with sample buffer (0.0625M
Tris pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.2% bromophenol
blue), mixed well using a vortex mixer and heated in boiling water for 5 min. About 10
pL of each protein sample were loaded into the sample wells and the gels were run at 80
mA current and 200 volts constant voltage for 40 min. Gels were stained for at least 20
min with Coomassie Brilliant Blue R250 solution (0.25%) in acetic acid:methanol:water
(9:45:45 v/v/v) and were destained ovemight in acetic acid-methanol-water (6:4:7, v/v/v)
solution. Molecular weight of the proteins was estimated from their relative mobilities
compared to those of molecular weight standard (Bio-Rad Laboratories) run under the

same electrophoretic conditions (Weber and Osborn, 1969).

7.4. RESULTS AND DISCUSSION
7.4.1. Thermal denaturation of mixed proteins

Denaturation patterns of myosin showed two Tlm at 45°C and 66°C at pH 6.0 and

three Tm at 48.5, 53.2 and 57°C at pH 7.0 (Figure 7.1A and B). The calorimetric

114

 

 

------ myosin/HDLG A

‘ — myosin

Heat capacity [mcal/0C]
U)

 

 

 

25 30 35 40 45 50 55 60 65 70 75 80 85 90

Temperature [°C]

 

------ myosin/HDL B

G g I
-— myos1n ..

Heat capacity [mcal/O C]
M

 

 

 

25 30 35 40 45 50 55 60 65 70 75 80 85 90
Temperature [°C]

Figure 7.1. Heat capacity proﬁles of 1.0% myosin and 1% myosin/ 1% heat-denatured B-
lactoglobulin (HDLG) in 0.6M NaCl, 0.05M sodium phosphate buffer, (A) pH 6.0 and

(B) pH 7.0.

115

enthalpies (AHw) of myosin at pH 6.0 and 7.0 were 2138 i 74 and 2310 :t 80 Kcal/mol,
respectively. Two Tm at 48°C and 67°C with AHcal of 2140 Kcal/mol at pH 6.0 (Lui et al.,
1996), while four Tm at 49.2, 50.2, 57.2 and 668°C with AH“, of 2215.8 Kcal/mol at pH
6.5 (Wang and Smith, 1994b) have been previously reported for chicken breast myosin
(pectoralis). Myosin is a multi-domain protein and has a complex thermal denaturation
pattern (Smyth et al., 1996). Many studies have previously indicated that transition peaks
(Tm) may vary due to differences in ionic strength and pH of the myosin solutions. At pH
6.0 unfolding of chicken breast myosin domains occurred over a broader temperature
range (45-66°C) than at pH 7.0 (48.5-57°C).

The thermograms of myosin/HDLG mixtures were similar to those of pure
myosin at the same pH, except for the increase in transition peak of myosin at 56°C (pH
6.0) and the appearance of an additional endothermic peak at 54°C (pH 7.0) (Figure 7.1A
and B). The source of this signal is unclear. Since HDLG was irreversibly denatured and
had no transition peak (data from section 6.4.3), the new peak might indicate interaction

between the two proteins.

7.4.2. Rheological properties

The G' of 1% and 2% myosin in 0.6M NaCl, pH 7.0, increased during heating and
cooling (Figure 7.2). Myosin at 2% gelled at a lower temperature (47.8°C) than 1%
myosin which reached its gel point at 490°C. The G' began to increase after the gel
point, formed a transition peak at 53°C and then increased rapidly between 58 and 71°C

before reaching a plateau when held at 71°C. The G' decreased during the ﬁrst 5 min of

116

 

700 80

 

 
 
 
   
 

 

[30] ommradmal

 

 

7% 1% myosin
4 2% myosin
600 - & myosin/LG 70
A , myosinzHDLG
-— Temperature 60
500 .
F
E. 50
U2
3 400 -
-5
° 7 40
E
g» 300 -
E 30
m
200 -
L 20
100 - _ 10
0 -1 0

 

 

 

O 10 20 3O 40 50 60 70 80 90 100110120130

Time [min]

Figure 7.2. Storage moduli of myosin, 1% myosin/ 1% B-lactoglobulin (B-LG) and 1%
myosin/ 1% heat-denatured B-LG (HDLG) in 0.6M NaCl, 0.05M sodium phosphate
buffer, pH 7.0, during heating at 1°C/min from 25 to 71°C, holding at 71°C for 60 min

and cooling to 25°C.

117

cooling and then increased rapidly throughout the cooling step. The G' reached a
minimum during cooling at 46-47°C, regardless of the protein concentration (1.0 or
2.0%). A similar G' curve was reported during heating of chicken myosin in 0.6M NaCl,
pH 6.0 (Lui and Foegeding, 1996). The authors noticed that the temperatures
corresponding to the minimum G' during cooling were close to the onset temperatures of
gelation, agreeing with our observation. During heating, hydrogen bonds decrease while
the hydrOphobic interactions of protein increase leading to gel network formation (Lui
and F oegeding, 1996). The decrease of G' at the begimiing of the cooling step may
reﬂect relaxation of the gel networks before reformation of hydrogen bonds which lead to
an increase of gel stiffness during further cooling.
Differences in G' during heating of myosin, myosin/B-LG and myosin/HDLG in 0.6M
NaCl, pH 7.0 can be compared in Figure7.2. When compared to 1% myosin, myosin/[3-
LG had lower G' above 55°C, but G' of the gel was greater during cooling. In contrast,
the G' of myosin/HDLG was greater than myosin throughout the heat treatment. The
addition of B-LG or HDLG had no effect on the gel point, but altered the G' above the gel
point of 1% myosin (Table 7.1). The results suggest that B-LG may interfere with myosin
gel network formation during heating above 55°C and holding at 71°C, while HDLG
enhanced network formation of the mixed protein gels.

In previous experiments, we found that HDLG had a lower gel point and gelled at
a lower concentration than B-LG. However, 1% HDLG did not form a gel at pH 7.0. The

increase in G' of myosin/HDLG co-gels may be due to formation of a “coupled gel

118

Table 7.1. Gel point, storage moduli (G') and tan delta (tan 5) after cooling of myosin,
1% myosin/ 1% B-lactoglobulin (myosin/B-LG) and 1% myosin/ 1% heat-denatured B-LG
(myosin/HDLG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, heated to 71°C

for 60 min or 90°C for 30min3

 

 

Protein Temperature Total protein Gel point G' after Tan 5 after
(°C) concentration (°C) cooling cooling
(%) (Pa)

Myosin 71 1 49.0 i 0.30 120 i 6 0.08 i 0.00

71 2 47.8 :t 0.10 500 i 37 0.08 i 0.00

Myosin/HDLG 71 2 48.0i 0.20 650 i 27 0.08 i 0.00
Myosin/B-LG 71 2 49.0 i 0.30 180 i 16 0.11 i 0.02
Myosin/B-LG 90 2 49.2 i 0.20 500 i 29 0.10 i 0.01

 

a Values are means of three observations i standard deviation.

119

 

network” rather than “phase separated gel network” as described by Brownsey and
Morris (1988). Besides intermolecular hydrophobic interactions, which normally occur
during heating, disulﬁde bonds might be involved in the protein network formation.
Shiga et a1. (1985) suggested that availability of sulﬂrydryl-groups of soy protein
increased with an increase in preheating time (0-4 min) and enhanced the gel strength of a
soy and meat protein system. Chicken breast muscle myosin possess 43 thiol groups
which formed intermolecular disulﬁde bonds, contributing to myosin gel network
development during heating at 45-54°C (Smyth et al., 1998b). More work is needed to
understand the mechanism of network formation of HDLG and myosin co-gels.

The G' did not decrease during cooling when myosin was mixed with either B-LG
or HDLG, suggesting that the gel matrices of mixed protein were formed differently from
myosin gel networks. The G' after cooling of myosin/HDLG gels heated to 71°C was 650
Pa or 3.6 fold greater than the myosin/B-LG gel and 1.3 fold greater than the 2% myosin
gel (Table 7 .1). The G' of myosin/HDLG gels heated to 71°C was also greater than that
of myosin/B-LG gel or 2% myosin (500Pa) heated to 90°C. Results indicated that HDLG
enhanced gel stiffness of myosin at temperatures lower than that needed for B-LG. This
could be attributed to a lower gel point and partially unfolded state of HDLG, allowing
for co-aggregation and network formation of myosin and HDLG during heating. A
similar ﬁnding has been previously reported by Hongsprabhus and Barbut (1999).
Addition of 2% pre-heated WPI aggregates (prepared at 80°C for 30 min, pH 7.0) in
conjunction with cold set gelation (6h at 1°C) improved hardness of comminuted chicken

after cooking to 78°C when compared to the use of unheated WPI. Hardness and

120

 

deformability of co-gels formed with 4% SSP and 12% WPC in 0.6M NaCl heated at
65°C were enhanced by low solubility WPC, whereas a higher temperature (90°C) was
needed for mixtures with highly soluble WPC to enhance the hardness of the co-gels
(Beuschel et al., 1992). Hung and Smith (1993) found that when heated at 65°C, the
elasticity of a combination gel of SSP and low solubility WPC was higher than that of a
combination gel containing highly soluble WPC. The WPC of low solubility was
prepared using higher temperatures and longer holding times than highly soluble WPC.
Therefore, this low solubility WPC may in fact, have similar properties as whey protein
aggregates or HDLG.

Two percent myosin and myosin/HDLG exhibited Viscoelastic gel characteristic
as indicated by their tan delta (tan 5) values after cooling (Table 7.1) and frequency
dependence curves of G' and G" (Figure 7.3). Polymer systems show high tan 5 for dilute
solutions, 0.2-0.3 for amorphous polymers, and low (near 0.01) for glassy crystalline
polymers and gels (Steffe, 1996). The G' and G" were independent of frequency and G'
was 8-10 times greater than G" throughout the frequency range. Frequency independence
(low slope) describes a strong cross-linked gel network, whereas an entangled weak gel is
strongly dependent on frequency (Stading et al., 1992). The G" of myosin/B-LG was
increased at high frequency resulting in a smaller difference between G' and G". A
difference between moduli indicates a lower recovery of stored energy which is a
characteristic of weak gels (Rao, 1999). The results suggested that myosin/B-LG formed

a weaker gel than 2% myosin and myosin/HDLG.

121

 

1 0000

 

 

 

 

 

 

 

I G' myosin/HDLG D G" myosin/HDLG
A G' myosin/LG A G" myosin/LG
O G' 2% myosin O G" 2% myosin
1000- ‘
IIIIII
III-III... .0
::::::oooooooocOO"'
E AAAAAAA‘A“
"T A A AAAAAA““‘
= 100~ '3
CD :1 DDDDDDSEA
ED 0 OOOOOOOOOOOOXZQOOOOoq
A
A AAAAAAAAA A
101
1 . . .
0.01 0.1 1 10 100

Frequency [radian/sec]

Figure 7.3. Frequency dependence of storage (G') and loss (G") moduli of 2% myosin,
1% myosin! 1% B-lactoglobulin (B-LG) and 1% myosin/ 1% heat-denatured B-LG
(HDLG) gels in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 7.0, measured at 25°C.
Gels were prepared by heating at 1°C/min from 25 to 71°C, holding at 71°C for 60 min

and cooling to 25°C.

122

 

The effects of pH on rheological properties of myosin and myosin/HDLG were
examined (Figure 7.4). As the pH was decreased from 7.0 to 6.0, the gel points of both
myosin and myosin/HDLG decreased. The gel point of 1% myosin decreased from 49°C
at pH 7.0 to 44°C at pH 6.0. Meanwhile the gel point of myosin/HDLG decreased from
48°C at pH 7.0 to 435°C at pH 6.0 (Table 7.2). Since the gel points of 1% myosin and
myosin/HDLG were similar at the same pH, it can be concluded that pH mainly affected
the gel point of myosin in the mixed protein system. Conformation of myosin and charge
distribution on its surface is altered by pH leading to changes in protein-protein

interactions (Xiong, 1992). Wang et a1. (1990) reported that chicken salt soluble protein

 

reached the ﬁrst G' transition at a lower temperature at pH 5.5 than at pH 6.5 and 7.5.

The G' after cooling increased for both myosin and myosin/HDLG gels when pH was

decreased. The G' after cooling of 1% myosin at pH 6.0 (510 Pa) was 4.25 times greater
when compared to that at pH 7.0 (120 Pa) (Table 7 .2). Maximum gel rigidity of myosin
from chicken breast was found at pH 5.4 to 5.9 in 0.6M KCl (Asghar etal., 1984; Morita
et al., 1987). The G' after cooling of myosin/HDLG gels at pH 6.0 was 2.3 times greater
than that of the gel at pH 7.0. The effects of pH on G' was less in the mixed system than
in myosin gels. However, the ﬁnal G' of myosin/HDLG gels at pH 6.0-7.0 remained 3-5

times greater than that of 1% myosin gel at the same pH.

7.4.3. Microstructure of mixed protein gels
Microstructures of myosin gel networks were dependent on ionic strength and pH.
When heated at 71°C, 2% myosin formed a ﬁne stranded protein network comprised of

small globular aggregates at pH 6.5 and 7.0 (Figure 7.5). A similar structure has been

123

 

, n * 1%myosin pH 6.0 I myosin/HDLG pH 6.0
,‘_ 1%myosin pH 6.5 J myosin/HDLG pH 6.5
, G— myosin pH 7.0 o 7 myosin/HDLG pH 7.0
—Temperature

1600

 

1400 —

 

1200

1000

800 -

600 ,

Storage Modulus [Pa]

400 ,

200 -

 

 

........

O 10 20 30 4O 50 60 70 80 90 100110120130

Time [min]

 

 

80

70

»60

50

-40

»30

»20

0

Figure 7.4. Storage modulus of 1% myosin and 1% myosin/ 1% heat-denatured B-

[30] arnmndtuol

lactoglobulin (HDLG) in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 6.0, 6.5 and

7.0, during heating at 1 °C/min from 25 to 71°C, holding for 60 min and cooling to 25°C.

124

Table 7.2. Gel point, storage moduli and tan 5 after cooling of myosin and 1%

myosin/ 1% heat-denatured soluble aggregates of B-LG (myosin/HDLG) heated at 71°C

for 60 min in 0.6M NaCl, 0.05M sodium phosphate buffer, pH 6.0, 6.5 and 7.08

 

 

Property pH 1% Myosin 2% Myosin Myosin/HDLG

Gel point 6.0 44.2 i 0.3 40.8 i 0.3 43.5 i 0.1

(°C) 6.5 47.2 i 0.3 46.3 i 0.3 46.8 i 0.3

7.0 49.0 i 0.3 47.8 i 0.1 48.0 i 0.1

Storage moduli 6.0 510 i 23 2,350 i 240 1,482 i 30
after cooling (Pa) 6.5 235 i 4 580 i 30 781 i 29
7.0 120:6 500:37 650i16

Tan 8 after cooling 6.0 0.08 i 0.01 0.08 i 0.01 0.09 i 0.01

6.5 0.1 i 0.02 0.07 i 0.01 0.09 i 0.01

7.0 0.12 i: 0.01 0.08 i 0.01 0.08 i 0.01

 

a Values are means of three observations i standard deviation.

125

Figure 7.5. Scanning electron microscopic images of 2% myosin gels heated to 71°C in
0.6M NaCl, 0.06M sodium phosphate buffer, (1) pH 6.0; (2) 6.5; and (3) 7.0 at 5,000 x

magniﬁcation. Bar = 1 pm.

126

 

127

previously reported in rabbit myosin gels formed in 0.6M KCl at pH 7.0 (Haga and
Ohashi, 1984). The myosin network at pH 6.0 contained thicker, smoother strands than
those formed at a higher pH. Rabbit Psoas major myosin formed at pH 6.0 in 0.6 KCl
had a very similar gel ultrastructure (Boyer et al., 1996).

Myosin/B-LG gels heated to 71°C at all three pHs contained coarser, thicker
protein strands with larger voids when compared to 2% myosin gels at the same pH
(Figure 7.6). The amount of myosin in myosin/B-LG gels was one half of that in 2%
myosin gels. Moreover, some of soluble proteins were washed out during ﬁxing as
indicated by the dark color of the ﬁxing solution, resulting from interactions between
protein and osmiun tetraoxide. Rheological experiments showed a decrease in G' during
heating of myosin/B-LG, indicative of interruption in network formation of the proteins.
Thus, the large void spaces observed in myosin/B-LG co-gels could be a result of a lower
functional protein concentration and/or caused by disruption of the myosin network.

Myosin/HDLG gels had a ﬁner protein networks and smaller voids in the gel
network when compared to myosin/B-LG gels at all pHs (Figure 7.6). The protein
networks in myosin/HDLG gels were denser than myosin/B-LG gel networks, indicating
that more proteins have been incorporated within the co-gel networks. This resulted in a
higher G' of myosin/HDLG gels when compared to myosin/B-LG gels.

The microstructures of myosin/HDLG gel networks contained a homogeneous
granular structure at pH 6.5 and 7.0, similar to that of 2% myosin gels at the same pH
(Figure 7.5 and 7.6). At pH 6.0, myosin/HDLG gels had larger voids and a more uneven

network when compared to 2% myosin gels, suggesting that interaction of myosin and

128

 

Figure 7.6. Scanning electron microscopic images of (A) 1% myosin/ 1% B -lactoglobulin
and (B) 1% myosin/ 1% heat-denatured 0-lactoglobulin co-gels heated to 71°C in 0.6M

NaCl, 0.06M sodium phosphate buffer, (1) pH 6.0; (2) 6.5; and (3) 7.0 at 5,000 x

magniﬁcation. Bar = 1 pm.

 

HDLG at pH 6.0 resulted in a more disordered network structure. There was no distinct
phase separated regions in the gel structure and the gel matrix of myosin/HDLG gels as
dense as that of 2% myosin gels. Heat-denatured B-LG did not form a gel at 1%
concentration, suggesting that HDLG and myosin possibly formed a co-gel in which both
proteins participated in network formation (coupled gel) (Brownsey and Morris, 1988),

especially at pH 6.5 and 7.0.

7.4.4. Soluble proteins expressed from co-gels

Myosin light chains (LCs) were observed in the supematants expressed from
myosin gels heated at 71°C (lane 9) and 90°C (lane 5) (Figure 7.7). The LC-l and LC-3
was detected in supernatant expelled from myosin gels heated to 71°C, while LC-2 was
also found when myosin gels were heated to 90°C. These proteins were present in very
low concentration in unheated myosin (lane 2). The dissociation of LC from myosin by
heat treatment has been previously reported (Dreizen and Richards, 1972; Sharp and
Offer, 1992). The LC fractions were also observed in supernatant of both myosin/HDLG
(lane 7) and myosin/B-LG gels (lane 4 and 8). Since the molecular mass of LC-2 (18.6
KDa) and B-LG (18.4 KDa) are similar, it was difﬁcult to determine which protein was
responsible for the middle band in SDS-PAGE (lane 4) of soluble protein from myosin/B-
LG gel heated to 90°C. Myosin light chains have been identiﬁed in supernatant expressed
from mixed protein gels of myosin and 11S soy protein (Peng et al., 1982), chicken breast
muscle salt soluble protein and WPI, WPC, or soy protein isolate (McCord et al., 1998),

and salt soluble protein and B-LG (Smyth et al., 1998a). Myosin heavy chain was not

131

 

 

 

 

 

‘ ’ , _ _ _ J ,3 KDa
qua-q 210
, ‘ 127
$.79}? 84
49
35
mi? 28
My-thb m :-.-.-.-=:v- W ”
WM ﬂaw mew H A “W“? 20
1 2 3 4 5 6 7 8 9 10

Figure 7.7. Electrophoretogram of proteins expressed by centrifugation at 10,000 x g
from myosin, B-lactoglobulin (B-LG), heat-denatured B-LG (HDLG), myosin/B-LG, and
myosin/HDLG gels heated to 71°C and 90°C. Each lane was loaded with 10 uL of
sample: Lan 1, unheated B-LG; Lane 2, unheated myosin; Lane 3, B-LG heated to 90°C;
Lane 4, myosin/B-LG gels heated to 90°C; Lane 5, myosin gels heated to 90°C; Lane 6,
HDLG heated to 71°C; Lane 7, myosin/HDLG heated to 71°C; Lane 8, myosin/B-LG
heated to 71°C; Lane 9, myosin heated to 71°C; Lane 10, broad range standard molecular

weight markers.

132

detected in the supematants of myosin and mixed protein gels, suggesting that it was a
principal contributor to the network formation of both myosin and mixed protein gels.

B-LG remained soluble in myosin/B-LG gels heated to 71°C as indicated by an
additional band (lane 8) when compared to myosin (lane 9). More B-LG was expressed
from myosin/B-LG gels heated to 71°C (lane 8) when compared to that from the mixed
gels heated to 90°C (lane 4). This agreed with results from total soluble protein analysis
(Figure 7.8). When heated to 90°C, the amount of total protein in the supernatant
expelled from myosin/B-LG gels decreased regardless of the increase of the light chain
fraction of myosin. The results suggested that more B-LG aggregated or was incorporated
into the network of the mixed protein gels when heated to 90°C than at 71°C. Hung and
Smith (1993) studied microstructures of co-gels formed with 4% chicken breast muscle
salt soluble protein and 12% WPC in 0.6M NaCl, pH 7.0. They reported that the co-gels
showed the typical ﬁbrous structure of salt soluble protein at 65°C, whereas the globular
structure of WPC was observed in the co-gel networks heated at 90°C.

A small amount of B-LG was found in the supernatant expressed from both
HDLG and myosin/HDLG gels heated to 71°C (lane 6 and 7), indicating that some B-LG
monomers remained in HDLG. This fraction did not participate in network formation of
either HDLG or myosin/HDLG gels heated to 71°C. The total amount of soluble protein
expressed from myosin/HDLG gel at 71°C was less than that of myosin/B-LG gels heated
to 71 and 90°C, indicating more protein was incorporated within the gel network. This

contributed to a denser protein network (Figure 7 .5) and a greater G' of myosin/HDLG

133

 

0.35

 

0.3 -

0.25 -

0.2 -

0.15 ~

Total protein (mg)

 

 

 

 

Heated at 71C Heated at 90C

Figure 7.8. Total protein in supernatant expressed from myosin, B-lactoglobulin (B-LG),
heat-denatured B-LG (HDLG), myosin/B-LG, and myosin/HDLG gels prepared in 0.6M
NaCl, 0.05M sodium phosphate buffer, pH 7.0, heated at 71°C for 60 min or at 90°C for
30 min and cooled to 25°C. Values are means of three observations. Means with different

superscripts are different (p<0.05).

134

gels heated to 71°C, when compared to myosin/B-LG gels heated to 71 and 90°C (Table

7.1).

7.5. CONCLUSIONS

To begin to understand how whey protein polymers function in meat products, we
used a model system to study thermal denaturation, rheological properties, and
microstructures of the gels of mixtures of heat-denatured polymers of B-LG and myosin,
the major functional whey and meat protein. When heated to 71°C, B-LG did not interact
with myosin and interfered with the network formation in mixed gels, whereas HDLG
interacted with myosin during heating to enhance gel formation. Myosin heavy chains
played a major role in network formation while the LC fractions dissociated and
remained soluble.

Myosin/HDLG co-gels in 0.6M NaCl solution at pH 6.5 and 7.0 had similar
stiffness and microstructure as 2% myosin gels. HDLG/myosin co-gel heated to 71°C
had greater G' than myosin alone and myosin/B-LG gels heated to 90°C, suggesting the
HDLG aggregates could have a texture enhancing effect in meat products processed at
their typical processing temperatures (5 71°C). This work indicates co-gel formation
between preheated whey and meat proteins which enhance texture parameter of the mixed
proteins when heated at 71°C and may contribute to enhanced applications for whey
protein in meat products. The next step would be to optimize the functionality of whey
protein polymers for their use in value-added meat and poultry products. A better

understanding of interactions between whey and meat protein will allow increased use of

135

 

whey protein in meat products, and in a variety of other applications where protein

gelation at low temperature is desirable.

136

here prazsm

CHAPTER 8: CONCLUSIONS

The purpose of this research was to understand gelation mechanisms of whey in
meat products by using myosin and B-lactoglobulin (B-LG), the major functional
components of meat and whey protein, in a model system. B-LG interfered with
aggregation and network formation of myosin when heated between 55 and 75°C.
However, B-LG enhanced stiffness of myosin/B-LG gels when heated above 80°C, which
is the gelling temperature of B-LG. The gelling temperature of whey protein could be one
factor limiting its ability to enhance texture in food products processed at temperatures
below 80°C.

Soluble protein aggregates were formed by heating B-LG solutions in deionized
water, pH 7.0 at 80°C for 30min. Gels were prepared using heating and buffer conditions
used for myosin (71°C for 60 min in 0.6M NaCl, 0.05M sodium phosphate buffer, pH
6.0-7.0) to determine if heat-denatured B-LG aggregates (HDLG) could enhance gel
formation in meat systems. The HDLG formed stiffer gels at lower temperature and lower
concentration when compared to unheated B-LG.

When heated to 71°C, HDLG was incorporated within networks of the co-gels,
whereas B-LG remained soluble. Microstructures of 1% myosin/ 1% HDLG co-gels were
similar to those of 2% myosin gels, while coarser gel networks with bigger voids were

observed in 1% myosin/ 1% B-LG gels. The stiffness of myosin/HDLG co-gels heated to

137

 

71°C was greater than that of myosin gels at the same total concentration. The stiffness
of myosin/HDLG gels was also higher that that of myosin/B-LG gels heated to 90°C. The
results suggested that HDLG and myosin formed a co-gel network with greater stiffness
than those formed from myosin alone or myosin/B-LG.

Results of this research indicate that preheat treatment improved gelling properties
of B-LG by lowering its gelling temperature and allowing the formation of a stronger gel
at lower concentration than the unheated protein. The HDLG might be used to improve
textural qualities of meat products processed at 71°C or lower. Pre-aggregation by
heating could be used as a modifying process to optimize properties of whey products
needed by meat processors and may contribute to broader applications of whey where

protein gelation at low temperature is preferred.

138

 

CHAPTER 9: FUTURE RESEARCH

In this research, we prepared heat-denatured B—lactoglobulin (HDLG) aggregates
by heating at 80°C for 30 min in deionized water, pH 7.0. Protein and salt concentration,
heating time and temperature, and pH were important factors affecting properties of whey
protein polymers (Ju and Kilara, 1998b; Mleko and Foegeding, 1999b). The
characteristics and properties of aggregates or polymers inﬂuence the texture quality of
the gels. Thus, more research is needed to study inﬂuence of these factors on the
characteristics of the aggregates, such as size, shape, viscosity, hydrophobicity and
number of active sulhydryl group.

The size and shape of HDLG aggregates were examined after heating using
scanning electron microscopy. In situ dynamic light scattering allows detection of
sample size during heating, therefore this technique could be very useful for investigating
aggregate formation during heating under different conditions. Understanding the
mechanisms and factors affecting aggregate formation could be greatly beneﬁcial for
manipulating properties and characteristics of the aggregates.

Investigation into the relationship between aggregate characteristics and its
gelling properties is essential in selecting the optimal conditions for the production of the
protein aggregates with the desired functionality. To study the gelling properties of the

protein aggregates, environment conditions, such as pH and salt concentration, should be

139

 

varied to include real conditions found in meat products. Reducing reagents could be
used to elucidate the role of disulﬁde bonds in the co-gel formation.

In this study, pure proteins were used in a model system to avoid confusion in
interpretation of results and allow greater understanding of heat-induced gelation
mechanisms. However, in commercial food systems, whey and meat products contain
many other components which could affect their gelling properties. Therefore, in future
research, whey products should be used to produce the protein aggregates. The
effectiveness of texture enhancing properties of the aggregates in meat products must be
validated in a real meat system. Knowledge gained from studies of B-LG aggregate
formation could be used as directions for the production of whey protein aggregates. The
relationship between aggregate characteristics and the gel properties could be used to
screen protein aggregates to ﬁnd the most promising properties in meat products. Finally
their efﬁcacy in enhancing textural properties should be veriﬁed with meat product in a

pilot scale production.

140

 

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141

 

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