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THESIS

This is to certify that the
dissertation entitled

DEVELOPMENT OF AN INJECTABLE “MODIFIED
MARBLING” SOLUTION FOR WHOLE MUSCLE BEEF CUTS

presented by

CHRISTINE SUZANNE QUINLAN

has been accepted towards fulfillment
of the requirements for the

Doctoral degree in Food Science

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2/05 p1/CIRC/DateDueindd-p1

 

DEVELOPMENT OF AN INJECTABLE “MODIFIED MARBLING” SOLUTION FOR
WHOLE MUSCLE BEEF CUTS

By

Christine Suzanne Quinlan

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

2006

ABSTRACT

DEVELOPMENT OF AN INJECTABLE “MODIFIED MARBLING” SOLUTION
FOR WHOLE MUSCLE BEEF CUTS

By

Christine Suzanne Quinlan

Low quality beef cuts with <3% intramuscular fat are lower in tenderness,
juiciness and flavor. Investigating non-meat ingredients that can mimic the properties of
intramuscular fat and processing technologies that can incorporate this mixture into beef
would be advantageous to the meat industry. The objectives of this research were to
develop and determine the effects of the injection of a “modified marbling” solution
containing sodium alginate (SA), iota carrageenan (1C) whey protein isolate (WPI) and
modified food starch (MFS) on the quality attributes of USDA Select ribeye rolls.

In study 1, twenty-five ingredient combinations (ranging from 0.25 to 0.50%
addition) of the four ingredients were formulated into 500 g solutions using a 24 central
composite design. Solution pH, apparent viscosity and gel (24 h, 4 °C storage) objective
color, water-holding capacity and strength were analyzed to determine the optimal
solution. In study 2, the solution was modified and processing system parameters were
determined on a multi-needle injector to incorporate the solution into whole muscle beef
cuts. The solution was then injected into USDA Select ribeye rolls (5-7% pick-up). In
studies 3 and 4, the properties of the “modified marbling” solution were verified in the
meat by comparing the chemical and sensory attributes to control ribeye rolls (USDA
Select, Low and Average Choice). The injected ribeye rolls were designated to 0, 14, 28,

or 42 days of storage ( 1°C), weighed for ribeye purge and steaks (2.54 cm) were

fabricated on each storage day. A 7-day retail shelf life study (analysis of thiobarbituric
acid reactive substances, color and percent purge) (study 3), Warner-Bratzler shear force
and sensory evaluation (study 4) were conducted on the fabricated steaks.

The data from study 1 resulted in the following recommended levels of non-meat
ingredients for the “modified marbling” solution: 0.4375% SA and IC and 0.375% WPI
and MFS. In study 2, 3% beef tallow and 0.25% beef flavor were added to improve the
hydrophobicity and flavor respectively of the “modified marbling” solution. Parameters
were also set on an automatic, multi-needle injector to acquire the desired percent pick-up
(5-7%) and “modified marbling” pattern. In study 3, the injected Select ribeye rolls had a
significantly higher (P<0.05) ribeye purge than the Average Choice control. For TBARS
values, the injected Select ribeye rolls were significantly higher (P<0.05) than the
controls. There were no significant differences in color scores or steak purge between
treatments in this study. In study 4, the injected ribeye rolls were higher (P<0.05)
compared to the USDA Select control ribeye rolls in beef fat flavor, however a
significant but small off—flavor was found (P<0.05) in the injected ribeye rolls. There
were no differences between the injected and control ribeye rolls for shear force, sensory
tenderness or juiciness.

The results indicate the viability of producing and injecting a “modified
marbling” solution into whole muscle beef cuts. The solution also has the potential to
improve lower quality beef cuts but more research is needed to improve the “modified
marbling” properties. One possibility is that the amount of fat in the solution could be
increased to achieve the benefits of flavor, hydrophobicity and to improve upon the

tenderness, juiciness and marbling appearance of the injected whole muscle beef cuts.

Copyright by
Christine Suzanne Quinlan
2006

This dissertation is dedicated to my husband, Patrick Quinlan. You have been the
motivation and source of support during this degree. First of all, you moved to Michigan
to be with me so that I could complete my education. You were with me throughout the
whole four years, while I was attending classes, conducting research, helping teach
courses, managing the sensory panel and research lab and keeping up with my extra
activities in AMSA and IFT. Amongst all this, we planned a wedding, got married and
had our baby girl. I could not have done this without you and I appreciate everything you
have done and most of all for always loving me!

ACKNOWLEDGMENTS

I would like to thank Dr. Wesley Osburn, my research advisor, for accepting me
into his program and trusting that I could be a Ph.D. student even though I did not have a
strong meat and food science background. You provided me with applied processing
skills, teaching opportunities, leadership experiences and continuous encouragement that
will always help me in the future. I would also like to thank Dr. A1 Booren, my academic
advisor, for helping me finish my degree after Dr. Osburn lefi and for allowing me to be
an independent, free-thinking and responsible graduate student. I appreciate the
opportunity you gave me to help organize and teach your processed meats class and for
always being accommodating, patient and encouraging.

To my guidance committee: Dr. Gale Stasburg, for your food chemistry advice;
Dr. Jim Steffe, for your food engineering involvement; Dr. Bruce Harte for accepting to
be on my committee when I was in a bind; and Dr. Janice Harte for the opportunity to be
the teaching assistant for your sensory evaluation and product development courses and
for providing me with the experience of being the team leader for the 2005 IFT product
development team. The leadership, organization and teaching skills that I gained from
these experiences will continue with me throughout my career and I also thank you for
always being a friend and a mentor.

A special thanks goes to Dr. Matt Doumit. Even though you were not on my
committee, I appreciate the opportunity of being the teaching assistant for your
introductory meat science and advanced muscle biology classes and for your advice and

help with my research project. Thank you for your patience and for allowing me to get in

vi

there and teach lectures and labs and for always asking my opinion throughout the
courses.

Thanks to my past lab mates: Jeff Sindelar, Deanna Hofing, and J in-Shan Shie for
your friendship and help. There are no words that can express my gratitude for you
always being there, helping me through research and life. To the undergraduate
employees: Michelle Grabowski, Courtney Whitfield, Rory McClintock, Rachel Damore,
Mark LaBar, Attalee Brix and Dan Kiesling for all your help. Your contributions are far
more worthy than merely being mentioned here and I appreciate you giving me the
opportunity to be an effective lab technician.

To the sensory panel, Terri Rathsack, Ann Chick, Jerry Allen, Sally Arias, Pete
Schneider and Michelle Grabowski. You were with me from the beginning and I
appreciate your patience as we learned sensory evaluation together. You gave me the
opportunity to start a panel from the beginning and gave me the experiences of recruiting,
training and managing while at the same time having a lot of fun. I truly enjoyed this and
thank each of you for your dedication and commitment to the sensory panel at MSU. I
would also like to thank the meat lab managers, Tom Forton and Jennifer Dominguez, for
the help and patience you gave me in the meat lab. You provided me with the
opportunity to learn and experiment and were always there to answer my many questions.
Thanks also to the color evaluation panel of Chuck Allison, Jennifer Dominguez,
Michelle Grabowski and Courtney Whitfield that was formed for my research study. I
appreciate your time and commitment to the panel especially on the weekends and

holidays.

vii

I would like to give recognition to Dr. Robert Tempelman and Lan Xiao (Shirley)
for your statistical advice and counseling and to Ewa Danielewicz for your help with the
scan electron microscope. Your experience and patience was well appreciated. A special
thanks goes to my fellow graduate students and technicians: Lindsey Keskinen, Kerri
Harris, Teresa Large, Fon Tuntivanich, Emily Helman and Alicia Orta-Ramirez for all
the fun and laughter. You were there during the good times and the bad and pushed me,

encouraged me and stood beside me.

viii

TABLE OF CONTENTS

List of Tables ................................................................................................................... xii
List of Figures ................................................................................................................. xiv
List of Appendices ........................................................................................................... xv
Introduction ........................................................................................................................ 1
Chapter 1 ............................................................................................................................ 4
Review of Literature ............................................................................................................ 4
1.1. Beef carcass quality .................................................................................................... 4
1.1.1. USDA beef carcass quality grades .................................................................. 4
1.1.2. Growth and development of fat depositions ................................................... 6
1.1.3. Types of fat depositions .................................................................................. 7
1.1.4. Factors affecting deposition of intramuscular fat ......................................... 10
1.1.5. Intramuscular fat effects on palatability ....................................................... 13
1.2. Beef carcass quality improvement initiatives ........................................................... 15
1.2.1. National Beef Quality Audits ........................................................................ 15
1.2.2. Beef consumer satisfaction studies ............................................................... 16
1.3. Effects of fat on sensory attributes ............................................................................ 17
1.3.1. Tenderness .................................................................................................... 17
1.3.2. Juiciness ........................................................................................................ 19
1.3.3. Flavor ............................................................................................................ 20
1.4. Fat substitutes in meat products ................................................................................ 21
1.5. Alginate ..................................................................................................................... 22
1.5.1. Background ................................................................................................... 23
1.5.2. Alginate manufacture .................................................................................... 23
1.5.3. Functionality ................................................................................................. 23
1.5.4. Gelation ......................................................................................................... 24
1.5.5. Application of a1 ginate in meat products ...................................................... 24
1.6. Carrageenan .............................................................................................................. 25
1.6.1. Background ................................................................................................... 25
1.6.2. Carrageenan manufacture ............................................................................. 26
1.6.3. Functionality ................................................................................................. 27
1.6.4. Interaction with non-meat ingredients .......................................................... 27

ix

1.6.5. Application of carrageenan in meat products ............................................... 28

1.7. Whey Proteins ........................................................................................................... 29
1.7.1. Background ................................................................................................... 29
1.7.2. Whey protein manufacture ............................................................................ 30
1.7.3. Functionality ................................................................................................. 31
1.7.4. Applications of whey proteins in meat products ........................................... 32

1.8. Modified food starch ................................................................................................. 34
1.8.1. Background ................................................................................................... 34
1.8.2. Modified food starch manufacture ................................................................ 35
1.8.3. Functionality ................................................................................................. 35
1.8.4. Applications of whey proteins in meat products ........................................... 36

1.9. Ingredient combination and interaction .................................................................... 37

1.10. Value-added technologies ......................................................................................... 38
1.10.1. Injection ........................................................................................................ 38
1.10.2. Restructuring ................................................................................................. 40
1.10.3. Mechanical tenderization .............................................................................. 40
1.10.4. Challenges for value-added products ............................................................ 41
1.10.5. Benefits for value-added products ................................................................ 42

1.11. Summary of literature ............................................................................................... 43

References .......................................................................................................................... 45

Chapter 2 .......................................................................................................................... 57

Development of a “modified marbling” solution for whole muscle beef cuts.

1. Abstract ..................................................................................................................... 57
11. Introduction ............................................................................................................... 58
111. Materials and methods .............................................................................................. 60
IV. Results and discussion .............................................................................................. 65
V. Conclusions ............................................................................................................... 75
VI. References ................................................................................................................. 76
Chapter 3 .......................................................................................................................... 78

Modification of a “modified marbling” solution for scale-up to an on-line injection
processing system for whole muscle beef cuts.

1. Abstract ..................................................................................................................... 78
11. Introduction ............................................................................................................... 79
111. Materials and methods .............................................................................................. 81

IV. Results and discussion .............................................................................................. 87
V. Conclusions ............................................................................................................... 92
VI. References ................................................................................................................. 93
Chapter 4 .......................................................................................................................... 94

Comparison of the chemical properties of whole muscle beef cuts injected with the
“modified marbling” solution to non-injected controls.

1. Abstract ..................................................................................................................... 94
11. Introduction ............................................................................................................... 95
111. Materials and methods .............................................................................................. 97
IV. Results and discussion ............................................................................................ 104
V. Conclusions ............................................................................................................. 1 19
VI. References ............................................................................................................... 120
Chapter 5 ........................................................................................................................ 122

Comparison of the sensory properties of whole muscle beef cuts injected with the
“modified marbling” solution to controls.

I. Abstract ................................................................................................................... 122
11. Introduction ............................................................................................................. 123
111. Materials and methods ............................................................................................ 125
IV. Results and discussion ............................................................................................ 129
V. Conclusions ............................................................................................................. 135
VI. References ............................................................................................................... 136
Recommendations for future research ......................................................................... 137
Appendices ...................................................................................................................... 140

xi

LIST OF TABLES

Table 1.1. Types and composition of whey protein products ........................................ 30

Table 3.1. Least square means for the properties of the “modified marbling solution
and gel injected into ribeye rolls ........................................................................................ 89

Table 3.2. Least square means for injection pick-up and tumbling loss of injected
and control ribeye rolls ...................................................................................................... 91

Table 4.1. Least square means for ribeye purge, cooked product yield, steak purge
and TBARS values of injected and control ribeye rolls .................................................. 105

Table 4.2. Least square means for proximate composition and pH of injected and
control ribeye rolls ........................................................................................................... 108

Table 4.3. Least square means for objective and subjective color measurements of
injected and control ribeye rolls ....................................................................................... 112

Table 4.4. Least square means for objective and subjective color measurements and
TBARS values over retail days for storage days 0 and 14 of injected and control ribeye
steaks ................................................................................................................................ 114
Table 4.5. Least square means for objective and subjective color measurements and
TBARS values over retail days for storage days 28 and 42 of injected and control ribeye
steaks ................................................................................................................................ 1 15

Table 5.1. Least square means for Warner-Bratzler shear force and sensory
attribute values of injected and control ribeye steaks ...................................................... 130

Table 5.2. Least square means for sensory attributes of USDA Select ribeye steaks
cooked by different cooking methods to different endpoint temperatures ...................... 133

Appendix 2: Bench top “modified marbling” solution formulation .......................... 142

Appendix 9: Ingredient combinations of non-meat ingredients using central
composite design .............................................................................................................. 150

Appendix 10: Least square means table for viscosity, pH, objective color, water-
holding capacity, water-holding capacity over time and gel strength/hardness of the

“modified marbling” solutions and gels .......................................................................... 151

Appendix 11: Commercial batch “modified marbling” solution formulation ............. 153

xii

Appendix 28: Sensory random order ........................................................................... 182

xiii

LIST OF FIGURES

Figure 1.1. Lower limits of marbling degrees used in beef carcass quality grading ......... 6

Figure 2.1. Response surface curves for significant (P<0.05) total regression
models for viscosity and pH of “modified marbling” solutions ........................................ 66

Figure 2.2. Response surface curves for significant (P<0.05) total regression models
for L*and b* color values of “modified marbling” gels .................................................... 69

Figure 2.3. Response surface curves for significant (P<0.05) total regression models
for water-holding capacity and water-holding capacity over time of “modified marbling”

gels ..................................................................................................................................... 71

Figure 2.4. Response surface curves for significant (P<0.05) total regression models
for gel strength of “modified marbling” gels ..................................................................... 73

Figure 4.1. Endotherrnic peaks of beef ribeye fat and “modified marbling” gels ......... 110

Figure 4.2. Scanning electron microscopy images ........................................................ 118
Appendix 1: Ingredients evaluated for “modified marbling” solution ...................... 141
Appendix 13: Randomization of USDA Select ribeye rolls ........................................ 155
Appendix 14: Randomization of USDA Low Choice and Average Choice ribeye

rolls ................................................................................................................................. 1 56
Appendix 27: Sensory ballot ........................................................................................ 181

xiv

Appendix 3:
Appendix 4:
Appendix 5:
Appendix 6:
Appendix 7:

Appendix 8:

Appendix 12:
procedures .......

Appendix 15:
Appendix 16:
Appendix 17:
Appendix 18:
Appendix 19:
Appendix 20:
Appendix 21:
Appendix 22:
Appendix 23:
Appendix 24:
Appendix 25:

Appendix 26:

LIST OF APPENDICES

Bench top “modified marbling” solution manufacturing procedures 144

Viscosity determination ........................................................................ 145
Objective color measurements (CIE L*, a* and b* values) .................. 146
Water-holding capacity determination .................................................. 147
Water-holding capacity over time determination ................................. 148
TA-HDi-gel strength settings ................................................................ 149
Commercial batch “modified marbling” solution manufacturing
................................................................................................................. 1 54
Determination of ribeye purge .............................................................. 157
Cooked product yield determination ..................................................... 158
Determination of steak purge in retail meat display case ..................... 159
Thiobarbituric acid reactive substances (TBARS) determination ........ 160
Proximate composition determination .................................................. 163
pH determination .................................................................................. 167
Melting point determination ................................................................. 168
Scanning electron microscopy determination ....................................... 170
TA-HDi Warner-Bratzler shear force settings ...................................... 172
TA-HDi texture analyzer calibration and analysis procedures ............. 173
Protocol for use of Taylor Clam Shell Grill .......................................... 176
Protocol for cooking, coring and shearing ............................................ 178

XV

INTRODUCTION

Marbling or intramuscular fat is the fat within the lean cut surface of the
Iongissimus thoracis muscle at the 12th — 13th rib interface of a beef carcass. The amount
or degree of marbling is one of the primary factors for assigning a USDA beef carcass
quality grade (USDA 1997) and has been shown to influence the palatability (tenderness,
juiciness and flavor) of the final beef product. Smith and others (1984) reported minute,
but statistically significant differences in meat palatability as the degree of marbling
decreased from Moderately Abundant (USDA Prime) to Practically Devoid (USDA
Standard). Also, Tatum and others (1980) found that rib steaks from High and Average
Choice carcasses were juicier, more flavorful and more palatable than steaks from Low
Select and High Standard carcasses.

Studies have been conducted to determine quality inconsistencies within the beef
industry chain, from farm to retail. The results from the last National Beef Quality Audit
(McKenna and others 2002) reported that the overall average scores for intramuscular fat
and USDA beef carcass quality grades were Smallo6 (marbling score) and USDA Select79
(USDA Quality Grade) respectively. The fourth challenge in the “top ten quality
challenges” identified from the audit was insufficient marbling since it was found that
45% of carcasses graded USDA Select (Slight degree of marbling), 53% graded USDA
Choice (9% moderate, 26% modest and 65% small degree of marbling) and only 2%
graded USDA Prime. The overall average scores for intramuscular fat and USDA beef
carcass quality grades were below the expectations of the meat industry. These low

scores can influence the consumer’s purchasing decisions. When consumers are not

satisfied with the palatability of beef cuts, their intent to purchase beef may decrease and
along with it the opportunity for the beef industry to generate revenue. Savell and others
(1987) reported that beef packers demand beef carcasses that grade USDA Choice or
higher due to substantial price discounts when carcasses grade less than USDA Choice.

In order to determine the amount of marbling necessary for acceptable
palatability, Savell and Cross (1988) developed a “window of acceptability” for percent
intramuscular fat of retail beef cuts. Broiling cuts (rib, loin, sirloin, etc.) containing 3-7%
intramuscular fat are perceived by consumers to be acceptable in tenderness, juiciness,
flavor and overall palatability but at 3% intramuscular fat there is little room for error in
cookery method or degree of doneness. Three percent intramuscular fat is associated
with the minimum Slight degree of marbling, 5% is related to the midpoint of the Small
amount of marbling and 7% is associated with the lower end of the Moderate amount of
marbling. From the last National Beef Quality Audit, 45% of the carcasses had Slight
degree of marbling or approximately 3% intramuscular fat and are at the lower edge of
the “window of acceptability.” This indicates an opportunity for improvement by
increasing the amount of marbling in whole muscle beef cuts to ensure acceptable
palatability.

Deposition of intramuscular fat is influenced by several pre—harvest factors such
as breed, length of feeding, type of ration fed (concentrate vs. grass) and management but
the palatability of whole muscle cuts fabricated from lower quality (less than USDA
Choice) beef carcasses may be improved through post-harvest, innovative non-meat
ingredient and processing technologies. Several different non-meat ingredients (salt,

phosphate, gums, starches and non-meat proteins) and processing technologies (injection,

restructuring, mechanical tenderization, tumbling and mixing) have all ready been used to
add value to lower quality meat products including whole muscle cuts. Development of a
“modified marbling” solution that can be directly injected into lower quality whole
muscle beef cuts at the level of 5-7% may enhance the overall palatability by mimicking
the organoleptic properties of fat and having an appearance similar to that of marbling.

The hypothesis for this project was that a “modified marbling” solution
manufactured with selected non-meat ingredients injected into less marbled beef ribeye
rolls (USDA Select) would create a steak that is similar to USDA Choice beef steaks in
tenderness, juiciness and flavor. To test this hypothesis, four separate studies were
conducted. In study 1, response surface methodology was utilized to determine the
concentration of each ingredient (sodium alginate, iota carrageenan, whey protein isolate
and modified food starch) to be used in the development of the “modified marbling”
solution. Study 11 was conducted to modify the solution, determine the processing
system parameters and demonstrate that the “modified marbling” solution can be injected
into whole muscle beef cuts. Study 111 and IV were done in order to verify the properties
of the “modified marbling” solution in whole muscle beef cuts and to evaluate the shelf
stability of the injected cuts. Ribeye rolls were injected with the solution, cut into steaks
and the chemical (study III) and sensory (study IV) properties were compared to three
controls (USDA Select, Low Choice and Average Choice).

This dissertation is formatted as five chapters. Chapter 1 is the review of
literature. Chapters 2-5 are manuscript style chapters and Chapter 6 is followed by
recommendations for future research. Finally, appendices are provided with step-by-step

procedures for all protocols used in each study.

CHAPTER 1

LITERATURE REVIEW

1.1. Beef carcass quality

1.1.1. USDA beef carcass quality grades

USDA beef carcass quality grades are based on carcass matmity and the amount
of marbling or intramuscular fat present on the exposed surface of the longissimus
thoracis muscle at the 12m_13m rib interface (USDA 1997). There are eight quality
grades: Prime, Choice, Select, Standard, Commercial, Utility, Cutter and Canner for steer
and heifer carcasses. In order to determine the quality grade, the carcasses are split
equally down the back into two sides and one side is partially separated into hindquarter
and forequarter (ribbed between the 12th and 13‘h rib).

Maturity: Overall carcass maturity is composed of skeletal and lean maturity.
The skeletal maturity is determined by the size, shape, and ossification of the bones and
cartilages, especially of the split chine bones. Ossification occurs at an earlier stage of
maturity in the split chine bones in the sacral vertebrae and at a later stage of maturity in
the lumbar and thoracic vertebrae. The chine bones are also soft and very red in color in
younger carcasses and hard and white in very mature carcasses. The size and shape of
the rib bones are used as well when determining maturity. In younger carcasses, the rib
bones only have a slight tendency toward flatness but in older carcasses, the rib bones are
wide and flat (USDA 1997).

Lean maturity is determined by the color and texture of the lean flesh on the

surface of the exposed ribeye separated between the 12th and 13th ribs. In a younger

carcass, the lean is very fine in texture and light, grayish red in color but as the carcass
maturity increases, the texture of the lean becomes coarser and the color of the lean is a
dzu'ker red. Skeletal and lean maturity is scored in percentages from A0 (youngest) to E100
(oldest). The approximate ages corresponding to each maturity classification are: A: 9 to
30 months, B: 30 to 42 months, C: 42 to 72 months, D: 72 to 96 months and E: more than
96 months. Slightly more emphasis is placed on the skeletal maturity if the skeletal
maturity is different from the lean maturity (USDA 1997).

Marbling: Marbling or the amount of intramuscular fat (fat found between the
muscle bundles) is evaluated on the cut lean surface of the exposed ribeye muscle of beef
carcasses separated between the 12th and 13th rib. The subjective assessment takes into
account the amount, size, number and distribution of intramuscular fat deposits (Dubeski
and others 1997). Marbling is the primary factor affecting quality grade. The degrees of
marbling, in order of descending quantity, are abundant (Ab), moderately abundant
(MAb), slightly abundant (SlAb), moderate (Md), modest (Mt), small (Sm), slight (S1),
traces (Tr) and practically devoid (PD) (Figure l) (USDA 1997). Marbling can be scored
as percentages, for example, if the amount of marbling is higher than the minimum small
but less than the minimum modest, then the marbling level is between Sm0 and Sm‘OO. If
the amount of marbling is 50 percent of the way to modest, then the amount of marbling
is Smso. Percentages should be no smaller than units of 10 (Romans and others 2001).

The relationship between maturity and marbling determines the carcass quality
grade. Beef quality grades are commonly divided into thirds or halves. The most

common divisions are: Prime (thirds), Choice (thirds), Select (halves), Standard (halves),

Commercial (thirds) and Utility (thirds). The symbols most commonly used for the

divisions are: high (+), average (0) and low (-) (AMSA 2001).

     

Very Abundant00 Abundant00 Moderately Abundant00

     

Slightly Abundant00 Moderate00 Modest00

     

Small00 Slight00 Traces00

Figure 1.1. Lower limits of marbling degrees used in beef carcass quality grading.
(Adapted from University of Nebraska Extension fact sheet by D Burson)

1.1.2. Growth and development of fat depositions

The growth and development of adipose tissue begins when it develops into lobes
that are enclosed in a sheath of collagenous fibers. The adipose cells then begin to
accumulate lipid (adipoblasts) and when the cell is filled with lipid it is known as an
adipocyte (Aberle and others 2001). Adipose tissue masses can expand by hyperplasia
(cell proliferation), hypertrophy (cell enlargement) or a combination of the two
(Bjomtorp and Sjostrom 1971; Greenwood and Hirsch 1974; Stern and Greenwood
1974)

Adipoblasts develop at differing rates in different parts of the body. In young

animals, deposits of fat usually develop in the visceral areas first and then deposit

6

beneath the skin (subcutaneous), between the muscles (intermuscular) and between
muscle bundles (intramuscular) (Aberle and others 2001 ). Hood and Allen (1973) and
Garbutt and others (1979) found that hyperplasia is completed in the perirenal and
subcutaneous adipose tissues by the first year of age in cattle and further increases in
adipose mass after one year of age is assumed to be mainly from cell enlargement.
However, since intramuscular fat is the last to develop, these fat cells continue to develop
in growing and adult animals because a smaller proportion of nutrient intake is required
for growth of other tissues and a greater proportion is available for energy storage. Hood
and Allen (1973) found that intramuscular adipose tissue grows by both hyperplasia and
hypertrophy in steers at 14 months of age.

1.1.3. Types of fat depositions

The fat portion represents the greatest source of variation in muscle tissue when
altering the proportion of fat to lean (Allen 1988).

Subcutaneous fat: Subcutaneous fat (external fat) or the fat beneath the skin of
the carcass is useful for predicting total fat content of beef carcasses (Johnson and
Vidyadaran 1981; McIntyre and Ryan 1983). Beef carcass subcutaneous fat depth is
taken at the 12-13th rib site and is more actively mobilized than other fat depots (Butler-
Hogg and others 1985). The amount of subcutaneous fat deposited is influenced by
several factors including: breed (Kempster and others 1976; Truscott and others 1983),
sex (Seideman and others 1982), age, carcass weight and season (Hopkins and others
1993)

The amount of intramuscular fat and quality grade have been shown to affect and

predict beef palatability but still others have found that they provide little assurance that

the beef will be palatable. Therefore, studies have been conducted to determine if the
amount of subcutaneous fat is a predictor of beef palatability. Dolezal and others (1982a)
found progressive increases in palatability of cooked beef as the amount of subcutaneous
fat increased from less than 2.53 m up to 7.61 mm, but subcutaneous fat greater than
7.61 mm did not improve palatability. The mechanism by which the amount of
subcutaneous fat or fattening improved palatability (tenderness) was that the increased
thickness of subcutaneous fat caused carcasses to chill more slowly, which increased
enzyme activity and decreased sacromere shortening thereby improving meat tenderness.
In another study, it was shown that 6-10 mm of subcutaneous fat was sufficient to retard
the postmortem chilling process to assure that beef from young cattle were tender (Smith
and others 1976). Tatum and others (1982), however, reported that subcutaneous fat or fat
thickness alone was not an effective measure of cooked beef palatability compared to
intramuscular fat and would not be a suitable substitute.

It was found though, that the combination of subcutaneous fat and intramuscular
fat was an important factor in the determination of beef palatability. In studies with
young bulls, Riley and others (1983a, b) showed that the combination of subcutaneous fat
thickness and intramuscular fat was important in ensuring that meat from young bulls
would be adequately tender. They found that steaks from Standard and Select bulls and
steers that had less than 7.6 mm fat thickness were significantly less palatable than steaks
from Choice steers or Select bulls with at least 7.6 mm of fat thickness.

Intermuscular fat: Intermuscular fat or seam fat is the fat found between the
muscles and it has been shown that retail beef cuts contain twice as much separable seam

fat as separable subcutaneous fat (Savell and others 1991). Closer trimming of

subcutaneous fat has emphasized intermuscular fat deposits, becoming more evident
during the fabrication of retail cuts. Retail cuts from the beef rib contain the highest
percentage of seam fat compared to cuts from the chuck, loin and round (USDA 1990;
Savell and others 1991) and there is a considerable amount of variation in the amount of
seam fat from anterior to posterior end within the rib (Moore and others 1989). Wulf and
others (1994) found larger amounts of seam fat in the 7th, 8th and 9th rib bone sections.

Since consumers demand leaner beef in the retail case (Cross and others 1986)
and because it is difficult to remove intermuscular fat without destroying the shape and
integrity of the cut, studies have been conducted to identify factors that contribute to
intermuscular fat deposition. Carcass traits connected to the USDA yield grade equation
(hot carcass weight, % kidney, pelvic and heart fat, ribeye area and fat thickness) (USDA
1997) and USDA quality grade (marbling and maturity) have been found to be good
predictors of seam fat. Jones and others (1990) showed that the amount of seam fat
increased as the USDA yield grade and marbling score increased. They determined that
carcasses with high yield grades should be avoided in order to decrease the amount of
seam fat on trimmed retail cuts.

Intramuscular fat: The intramuscular fat (fat found between the muscle bundles)
or marbling is the most variable component of fat deposition. Savell and others (1986)
found that the amount of chemical fat in uncooked longissimus lumborum muscle of 518
beef carcasses varied from 10.42% in Moderately Abundant degree of marbling to 1.77%

in Practically Devoid.

1.1.4. Factors affecting deposition of intramuscular fat

Carcasses vary in composition through genetic, nutritional, hormonal and
management/environmental effects.

Breed: Breed can influence the amount of intramuscular fat since animals of
different breeds grow and develop in a specific manner and produce carcasses with
distinctive characteristics particular to that breed. Dubeski and others (1997) found that
Angus breeds had superior marbling compared to Hereford and Hereford x Angus breeds
and they also found that Holsteins were similar to Hereford x Angus with less
intramuscular fat than Angus but more than Herefords. Dairy breeds including Holsteins,
have less external fat than most beef breeds and the carcasses are superior in USDA yield
grade compared to some beef breeds (Cole and others 1963; Young and others 1978;
Nour and others 1983). Angus cattle have been found to deposit intramuscular fat at an
earlier age and produce more than Herefords or Shorthorns (Kauffman and other 1968;
Cramer and others 1973).

Nutrition: Nutrition can also affect the amount of intramuscular fat deposited
given that nutrition dictates the rate of growth of the animal and the extent of
development. The type of ration fed (concentrate vs. grass) can influence the degree of
intramuscular fat since feeding a high concentrate diet produces a rapid animal grth
rate, which increases the deposition of intramuscular fat. Vestergaard and others (2000)
found that the intramuscular fat content of bulls fed a roughage based diet was 50% lower
in the semitendinosus and longissimus lumborum and 30% lower in the supraspinatus
compared to bulls fed a concentrate based diet. Reduced tenderness of meat from grass-

fed steers has also been reported (Bowling and others 1978; Schroeder and others 1980).

10

This could be partly due to cold shortening because of the thin subcutaneous fat cover
and the rapid growth rate of grain-finished steers, which reduces the effect of connective
tissue on muscle toughness (Allingham and others 1998). Vestergaard and others (2000)
showed that shear force values of the semitendinosus were 33% higher (less tender) for
bulls fed the roughage based diet and sensory panel scores for tenderness, flavor and
juiciness of the longissimus lumborum also were lower. An off-flavor, additionally, was
almost solely detected in meat from roughage fed bulls.

Increasing the amount of time cattle are fed hi gh-concentrate diets can also
enhance the deposition of intramuscular fat in beef carcasses due to increased carcass
maturity and fat deposition, decreased yield grade, and increased percentage of carcasses
grading USDA Choice. Increasing feeding time from 100 to 160 days had a beneficial
effect on flavor but did not affect juiciness, tenderness, or overall palatability (Tatum and
others 1980). Dolezal and others (1982b) found that extending feeding time beyond 90 to
100 days did little to increase beef palatability. It was recommended that the minimum
marbling requirement to grade USDA Choice could be met with no loss in palatability if
cattle were fed a hi gh-concentrate diet for at least 90 days prior to slaughter.

Hormone implants: The use of hormone implants can also affect the deposition
of intramuscular fat. Anabolic implants have been used to improve growth rate and feed
efficiency of cattle. They have also been shown to reduce fat thickness, percentage of
internal fat, USDA yield grade, marbling and USDA quality grade while increasing
carcass weight, carcass conformation and longissimus thoracis muscle area (Galbraith
and others 1981; Rumsey 1982; Trenkle 1987). Estrogens increase protein deposition by

increasing the concentration of somatotropin secreted from the anterior pituitary and

11

insulin secreted from the B-cells of the pancreas (Aberle and others 2001). Androgens
have been shown to increase carcass protein content of cattle by stimulation of muscle
protein synthesis (Muir 1985). Trenbolone acetate (TBA), a synthetic androgen,
however, has been shown to decrease both the rate of protein synthesis and degradation
but the rate of degradation is less than the rate of synthesis, so net muscle protein
deposition is increased (Buttery and others 1978).

Even though the use of implants has been shown to be economically beneficial
(Trenkle 1987), there are concerns regarding the possible harmful effects of implants on
beef quality. Thonney and others (1991) compared the use of implants containing both
an estrogenic and androgenic with a non-implanted control and showed that the implants
reduced tenderness of ribeye steaks. Gerken and others (1995), however, found that the
use of a single estrogenic, androgenic or a combination of an estrogenic and androgenic
had little effect on the deposition of intramuscular fat or on beef tenderness. Apple and
others (1991) found that steers implanted with an estrogenic tended to produce less tender
rib steaks than non-implanted control steers and steers implanted with an androgenic or
an estrogenic plus an androgenic produced rib steaks that were similar in tenderness to
those produced by non-implanted control steers. They also found that 50% of the cattle
implanted with an estrogenic plus an androgenic graded low Choice or higher. It has
been shown that estrogenic and androgenic implants either alone or in combination tend
to reduce marbling scores and quality grades compared to non-implanted controls
(Trenkle 1990; Bartle and others 1992).

Environment: The environmental conditions an animal is raised in may also have

an influence on the grth rate and body composition of the animal, which may

12

influence the deposition of intramuscular fat. Warm-blooded animals need to maintain a
constant body temperature, so heat loss must be equal to heat production in order to
maintain normal physiological processes. In any environmental condition that requires
an animal to generate or dissipate heat, the efficiency of growth is reduced. Changes in
carcass composition can result from changes in energy depending on the growth stage of
the animal and may influence the deposition of intramuscular fat (Aberle and others
2001)

The management or housing system used in raising cattle has also been found to
influence deposition of intramuscular fat and palatability of meat cuts. It has been found
that loose housing compared to tie-stall housing increased shear force value by 25 to 35%
in Iongissimus dorsi (Jensen and Oksarna 1996). Vestergaard and others (2000) found
that the intramuscular fat content of bulls loose housed was 50% lower in the
semitendinosus and longissimus lumborum and 30% lower in the supraspinatus
compared to tie-stall housed bulls. Also, the shear force value of the semitendinosus was
33% higher in loose housed bulls compared to tie-stall housed bulls.

1.1.5. Intramuscular fat effects on palatability

Several studies have been conducted to determine the effects in which degree of
marbling and quality grade have on meat palatability. Tatum and others (1982) showed
that marbling had a low but positive relationship on all beef palatability traits but also
found that 90% of the time steaks with Slight or higher degrees of marbling were more
desirable in tenderness, flavor and overall palatability. Smith and others (1984) reported
minute, but statistically significant differences in meat palatability (juiciness, tenderness,

flavor) as the degree of marbling decreased from Moderately Abundant (USDA Prime) to

13

Practically Devoid (USDA Standard) (Figure 1). Wheeler and others (1994) found that
shear force tenderness ratings and juiciness ratings improved slightly and shear force
variation decreased slightly as marbling increased in meat from Bos taurus and 803
indicus cattle.

A study conducted by Smith and others (1987) determined the relationship
between USDA quality grades and beef palatability. They found that loin steaks from
Prime carcasses were more palatable than steaks from Choice through Canner carcasses
85.7% of the time and more palatable than steaks from Choice through Standard
carcasses in 69.0% of comparisons. Also Longissimus thoracis steaks from USDA High
Choice carcasses tended to have higher tenderness, juiciness and beef flavor intensity
ratings than those from USDA Low Select carcasses (Wheeler and others 1999a). Tatum
and others (1980) found that rib steaks from High and Average Choice carcasses were
juicier, more flavorful and more palatable than steaks from Low Select and High
Standard carcasses.

Savell and Cross (1988) determined that the minimum fat percentage required for
acceptable palatability of broiling cuts is 3% on an uncooked basis (minimum Slight
degree of marbling, USDA Low Select). They came to this conclusion after studying
research conducted over many years and found that steaks with less than 3%
intramuscular fat (Practically Devoid and Traces) were tougher, drier and less flavorful
when evaluated by both trained and consumer sensory panels. However, 3%
intramuscular fat or Slight marbling provides little room for error in cookery method or

degree of doneness to ensure palatability.

14

They also determined two other levels of intramuscular fat related to increased
palatability. Approximately 5% (midpoint of Small degree of marbling) and 7% (low end
of Moderate amount of marbling) were associated with hierarchical degrees in
palatability. From these studies, Savell and Cross (1988) described a “window of
acceptability” for percent intramuscular fat (marbling) of retail beef cuts. Beef cuts
containing 3-7% intramuscular fat (marbling) are perceived by consumers to be
acceptable in tenderness, juiciness, flavor and overall palatability.

Beef palatability is a major concern because when consumers are not satisfied
with the palatability of beef cuts their intent to purchase additional beef products may
decrease. The opportunity for the beef industry to generate revenue also decreases.
Savell and others (1987) reported that beef packers demand beef carcasses that grade
USDA Choice. When carcasses grade less than USDA Choice, a substantial price

discount usually has been paid.

1.2. Beef carcass quality improvement initiatives

1.2.1. National Beef Quality Audits

Studies have been conducted to determine quality inconsistencies within the beef
industry chain, from farm to retail. The National Beef Quality Audit (NBQA)-l991
(Lorenzen and others 1993) established the first major benchmark and showed that the
overall mean marbling score for beef cattle carcasses was a Small24 (USDA Low Choice).
However, the overall mean USDA Quality Grade for carcasses utilized in this study was
Select86 (USDA High Select), indicating that lower quality (less than USDA Choice) beef

carcasses were being produced. The 1995 NBQA (Boleman and others 1998) measured

15

the progress regarding the quality, consistency, and competitiveness of beef since the
initial 1991 NBQA. This study determined that the overall mean marbling score was a
Smallo6 and the mean USDA quality grade was Select”. It was shown that 48.2% of the
carcasses had marbling scores that corresponded to USDA Choice and 46.5% had
marbling scores that corresponded to USDA Select. There was a reduction in marbling
since the 1991 audit and they felt that the industry should be concerned with the observed
decrease in the proportion of carcasses with marbling scores that corresponded to USDA
Prime and Choice quality grades.

The most recent NBQA audit was conducted in 2000 (McKenna and others 2002).
The purpose of this audit was to assess the current status of the quality and consistency of
the US. fed steer and heifer population, to pinpoint inadequacies and shortfalls that the
industry needs to improve upon and to track any progress made since the last audit
(1995). The results from the 2000 audit found that the quality measured by marbling
score and USDA quality grade appeared to be back to the level observed in the early
1990’s.

1.2.2. Beef consumer satisfaction studies

The National Consumer Retail Beef Study (Savell and others 1987) was an
industry-wide program supported by the government, beef producers, packers and
retailers to identify the kind of beef products consumers prefer. The association between
quality grade and taste appeal was looked at. Steaks from carcasses that varied in
marbling were evaluated by 540 households and was the first nationwide study conducted
to determine if consumers, rather than a trained sensory panel could detect differences in

the palatability of beef steaks with different degrees of marbling. From the study, they

16

found that the degree of marbling in top loin steaks impacted palatability (juiciness,
tenderness, flavor). The study also found that tenderness was the single most important
factor affecting consumer perceptions of beef, but Neely and others (1998) found in a
beef consumer satisfaction study that flavor could be as important as tenderness in

determining consumer satisfaction.

1.3. Effects of fat on sensory attributes

Savell and others (1987, 1989) reported that the palatability of beef products
affects consumers’ purchasing decisions and numerous factors have been shown to affect
beef palatability (tenderness, juiciness, flavor and overall acceptability) including the
amount of intramuscular fat. Marbling has been reported to account for 5-10% variation
in tenderness and 16% variation in juiciness (Blumer 1963; Pearson 1966; Parrish 1974;
Jeremiah 1978).

1.3.1. Tenderness

Several theories have been postulated that explain how intramuscular fat
contributes to muscle fiber tenderness. The lubrication theory states that intramuscular
fat present in and around the muscle fibers lubricates the fibers and creates a more tender
and juicy product that stimulates the sensation of tenderness. The bite theory states that
within a bite size piece of meat, marbling decreases the bulk density of the meat by
replacing protein with lipid. Since fat is much less resistant to shear force than protein,
the decrease in bulk density is accompanied by an increase in real or apparent tenderness.

The strain theory states that as marbling is deposited inside the walls of the perimysium

l7

or endomysium, the connective tissue walls on the side of the deposit are thinned,
decreasing their thickness and strength (Savell and Cross 1988).

Intramuscular fat has been shown to have a low to moderate relationship to
tenderness in beef (Smith and Carpenter 1974). Wheeler and others (1994) reported that
Bos taurus carcasses with Slight marbling exhibited higher shear force (less tender)
values compared to beef carcasses with Small through Modest marbling scores.
Carcasses with a Traces marbling score had a higher shear force value than those
carcasses with Slight marbling scores. Davis and others (1979) investigated the
tenderness variations that occurred among beef steaks from carcasses of the same USDA
quality grade. The purpose of the study was to determine why some steaks are less
palatable than others that are from the same USDA quality grade. Steaks from Choice, A
maturity beef loins were used and the most tender steaks were found to have more
intramuscular fat than the steaks found to be less tender. The percentages of
intramuscular fat for steaks from four different tenderness groups of the Choice, A
maturity beef loins were: very tender=7.6%, moderately tender=6. 1%, slightly
tender=5.6% and slightly tough=4.4%.

Moody (1976) concluded that the most important factors that affect meat
tenderness are methods and/or rate of chilling and methods of cooking. Tenderness
decreases as the degree of doneness increases (Cover and others 1962; Parrish and others
1973; Cross and others 1976) and 64% (Branson and others 1986) or 82% (NLSMB
1995) of beef consumers cook meat to a medium to very well degree of doneness. The
negative effects of a higher degree of doneness on tenderness were much greater in less

tender than in more tender longissimus thoracis steaks (Wheeler and others 1999b). It

18

has been hypothesized (Smith and Carpenter 1974; Savell and Cross 1988) that steaks
from carcasses of lower quality grades are more affected by an elevated degree of
doneness than are steaks of higher quality grades.

1.3.2. Juiciness

J uiciness is composed of the combined effects of initial fluid release and the
sustained salivary flow from the stimulating effect of fat (Weir 1960). The initial fluid
release gives the impression of wetness perceived during the first chews, which is
produced by the rapid release of meat fluids. Sustained juiciness is the sensation of
juiciness perceived during continued chewing created by the release of serum within the
meat and partly by the stimulating effect of fat on salivary flow (Bratzler 1971).
Sustained juiciness has been found to be related to intramuscular fat content (Pearson
1966). Intramuscular fat may affect product j uiciness by enhancing the water-holding
capacity of meat, lubricating the muscle fibers during cooking, increasing the tenderness
of meat, simultaneously increasing the sensation of juiciness, and by stimulating salivary
flow during mastication.

It has been shown that intramuscular fat has a low to moderate relationship to
juiciness in beef (Smith and Carpenter 1974). Tatum and others (1980) found that rib
steaks from High and Average Choice carcasses were juicier than steaks from Low Select
and High Standard carcasses. Jones and others (1991) also found that intramuscular fat
influenced juiciness in ribeye steaks. Steaks with Modest, Small and Slight amounts of
marbling had higher mean trained sensory panel juiciness scores than those with Traces

amount of marbling.

l9

1.3.3. Flavor

Fat may affect flavor of meat products in two ways: 1) production of carbonyl
compounds that are potent flavor contributors during fatty acid oxidation and 2) release
of odoriferous compounds stored in fat during heating (Homstein 1971). The species
characteristic flavor tends to come from the lipid fraction of the meat when the volatile
compounds are released from the fat or produced from triglyceride or phospholipid
fractions (Homstein and others 1960). The meaty flavor, however tends to be nonlipid in
origin, but some amount of fat is necessary to give the full, rich beef taste.

Armbruster and others (1983) found that at Slight to Moderately Abundant
degrees of marbling, roasts from Holstein cattle had better flavor which could be
attributed to the higher concentration of water soluble constituents since they have more
active muscle growth (N our and others 1981). Conversely, at higher marbling scores and
increased weight, the accumulation of more fat might have resulted in more flavorful
roasts from Angus than from Holsteins. Branarnan and others (1962) showed that roasts
from beef type steers produced a more intense flavor in the lean than roasts from
Holsteins but the flavor of fat was unaffected by breed.

Smith and Carpenter (1974) reported a low to moderate relationship of
intramuscular fat to beef flavor. Armbruster and others (1983) found that marbling
positively affected the flavor of rib roasts from Angus cattle. Wheeler and others (1999)
also found that longissimus thoracis steaks cooked well done (80 °C) from Top Choice

carcasses had higher beef flavor intensity ratings than those from Low Select carcasses.

20

1.4. Fat substitutes in meat products

In order for an ingredient to successfully replace or substitute fat it must mimic
the taste, texture, and function of the fat it is replacing. The desired function of fat either
flavor, lubrication, or heat transfer determines what properties developers of fat
substitutes seek to achieve (Morrison 1990). Decreasing the fat content in meat products
requires that product palatability — tenderness, juiciness, flavor and mouth-feel or texture
be maintained and/or improved while maintaining economic value (Mandigo 1991).
Functional properties of meat systems are primarily dependent on the interaction of the
protein fraction with the other components. These interactions include: proteinzwater,
proteinzfat and proteinzprotein which determine the textural properties, yield, palatability,
processing behavior and ultimately product value (Shand and Schmidt 1990).

Fat substitutes can be grouped into three general categories, protein-based, fatty
acid-based and carbohydrate-based substitutes. Protein-based substitutes are ingredients
derived from either plant or animal proteins. Plant-based protein additives include soy
flour, soy protein concentrate, soy protein isolate, textured soy protein, corn germ meal,
corn flour, oat flour, wheat flour and vital wheat gluten. Animal-based protein additives
include nonfat dry milk, whey proteins, caseinates, blood plasma, and egg proteins
(Mandigo 1991).

Fatty acid-based substitutes are fatty acids that have been chemically altered to
provide fewer to no calories. Examples of fatty acid based substitutes include olestra
(sucrose polyester), polydextrose, and esterfied propoxylated glycerols (Morrison 1990).

These fat substitutes are used primarily in snack foods rather than in meat products.

21

Carbohydrate-based substitutes include starch derivatives and cellulose based
derivatives. Starch based substitutes are mixtures of starch derivatives and water and are
used to produce a variety of reduced fat foods. The mixtures do not have all the taste and
functional properties of fat so they can only replace part of the fats and oils without a loss
in quality (Morrison 1990). Cellulose based derivatives are gums and hydrocolloids.
They have been used in a variety of food products to stabilize viscosity and emulsions,
suspend particles and form gels.

In developing a fat substitute for intramuscular fat, developers may need to use a
combination of ingredients that mimic the functional and organoleptic properties of fat.
To create a “modified marbling” solution, special consideration must be given to gelation
properties (to create “fat-like” particles resembling marbling), water retention, viscosity
(to allow direct injection into whole muscle), color (lightness or L* values similar to fat)
and melting point temperatures. Non-meat ingredients that may be suitable for
development of a “modified marbling” solution include hydrocolloids, starches and whey

proteins.

1.5. Alginate

Gums, also referred to as hydrocolloids, are long-chain, high-molecular weight
polymers that dissolve or disperse in water. They create a thickening and sometimes a
gelling effect. Used at low levels usually in the range of 0.1-0.5%, they dramatically
increase viscosity and lead to emulsion stability (Glicksman 1982). A gum is not a single
homogenous compound but rather a heterogeneous mixture of several different

polysaccharides. Galactose is the most commonly repeated monomer (Towle 1973).

22

1.5.1. Background

Alginates are monovalent salts derived from brown seaweed. They are
hydrophilic colloids and are widely used as thickeners, stabilizers and gelling agents in
food and can be utilized as a fat replacement with a potential for textural modification
due to gel formation (Mandigo 1991). Alginates are cold soluble and cold-setting and are
heat and freeze thaw stable. Alginate is a linear copolymer composed of two monomeric
units, D-mannuronate and L-guluronate linked together in a flexible chain by glycosidic
linkages of 100-3000 units. The composition of alginate varies depending on the
seaweed species and the part of the plant used.

1.5.2. Alginate manufacture

Alginate occurs in seaweed as a combination of calcium, magnesium, sodium, and
potassium salts. The seaweed goes through more than 20 processing steps in order to
produce alginate. The seaweed is milled and washed with water and acid and then the
alginate is extracted with water and alkali and clarified. It is then filtered with a filter aid
and precipitated with CaClz, which produces calcimn alginate. The calcium alginate is
then washed again with water and acid, which results in the final product (Draget 2000).

1.5.3. Functionality

Dissolving alginate in water causes the molecules to hydrate and the solution will
gain viscosity. The alginate should be added slowly to the water and stirred vigorously to
create a vortex. It should be added slowly into the vortex in order to avoid lumping and
premixing alginate with another powder (sugar) or vegetable oil can help with dispersion.
The dissolved molecules are not completely flexible since rotation around the glycosidic

linkages can be hindered which results in a stiffened chain. Solutions with stiffened

23

molecules are highly viscous (FMC BioPolymer 2005). The viscosity of an alginate
solution depends on both the concentration of the alginate and the number of monomer
units in the chain (the more units in the chain, the higher the viscosity at similar
concentrations). Alginate solutions have shear-thinning characteristics since the viscosity
decreases with increasing shear rate (stirring speed) and the temperature also influences
the effect of alginate to shear force (Draget 2000). As the temperature increases 1 °C, the
viscosity of the solution drops approximately 2.5% (FMC BioPolymer 2005).

1.5.4. Gelation

The gelation of alginate requires polyvalent cations. Polyvalent cations, most
commonly calcium, will react and cross-link with alginate polymers. As the polyvalent
ion content of the solution is increased, thickening and gelation will occur (Pszczola
2003). The alginate needs to contain an adequate amount of guluronate monomers to
react with the polyvalent cation. Regions of guluronate monomers in one alginate
molecule can link to a similar region in another alginate molecule by polyvalent cations
binding the alginate polymers together forming junction zones. This results in gelation of
the solution (FMC BioPolymer 2005). An alginate gel can be considered part solution
and part solid. When a heat stable alginate gel is formed, water is physically entrapped in
the alginate matrix resulting in less released water (Onsoyen 1997) but the water
molecules are flee to migrate.

1.5.5. Applications of alginate in meat products

Alginate has been used as a non-meat ingredient in a variety of meat products. In
a study by Ensor and others (1989), restructured ground turkey and turkey breast meat

patties were formulated with a combination of sodium alginate (0-1.0%), calcium

24

carbonate (0-0.l875%) and lactate (0-0.6%) and compared to a no additive control. All
of the restructured products with the sodium alginate/calcium carbonate binder had
higher cook yields than the no additive control. Raharjo and others (1994) studied the
quality characteristics of restructured steaks with veal trimmings or veal leg meat and
sodium alginate/calcium lactate. The sodium alginate/calcium lactate used as a binder
increased the binding force and sensory bind score, and decreased the cook loss when
used at 0.4%. Berry (1997) found improvements in the acceptability of low fat ground
beef patties by using alginate along with tapioca starch which greatly improved
tenderness and juiciness, and also increased cooking yields. In another study, Devatkal
and Mendiratta (2001) evaluated restructured pork rolls formulated with sodium
alginate/calcium carbonate and found that the raw binding strength was significantly

higher in the restructured pork rolls containing the sodium alginate/calcium carbonate.

1.6. Carrageenan

1.6.1. Background

Carrageenan is a generic term applied to a group of sulfated polygalactoses
extracted from red seaweed. It is made of repeating galactose units and 3,6
anhydrogalactose (sulfated and nonsulfated) and are joined by alternating alpha 1-3 and
beta 1-4 glycosidic linkages. Carrageenan functions as a gelling agent, stabilizer and
thickener and is capable of forming viscous solutions at low concentrations in cold water
with the viscosity dependent upon temperature, pH, concentration, and the type of
carrageenan present (Wallingford and Labuza 1983). The solubility of carrageenan

depends on the number and position of the ester sulfate groups on the repeating galactose

25

units. Higher levels of ester sulfate groups lower the solubility temperature of the
carrageenan and produce a lower strength gel. There are three basic carrageenan types,
kappa, iota and lambda, with each type differing in solubility and gelling properties.

Some but not all carrageenans exhibit cold solubility (Egbert and Huffman 1991).
Kappa carrageenan is soluble in hot water and the addition of potassium ions increases
the formation of a durable gel. The gel is strong, rigid and slightly opaque with normal
usage levels between 0.02-2.0%. Iota carrageenan is soluble in hot water but sodium iota
carrageenan is soluble in cold and hot water. The addition of calcium ions induces the
formation of a durable, clear elastic gel that is freeze thaw stable and used at levels
between 0.2 to 2.0%. Lambda carrageenan is partially soluble in cold water, fully soluble
in hot water but does not form a gel. There is only random distribution of polymer chains
and the solution ranges from low to high viscosity. The addition of cations has little
effect on the viscosity and lambda carrageenan is normally used at levels between 0.1 -
1.0% (FMC BioPolymer 2005).

1.6.2. Carrageenan manufacture

In order to manufacture carrageenan, red seaweed is gathered, dried, baled,
mechanically ground and sieved to eliminate impurities like sand and salt. The seaweed
is then washed and extracted to separate the carrageenan from the extraneous plant fiber.
The cellulosic material is removed by centrifuging the dissolved carrageenan mixture to
eliminate the dense cellulosic particles and then filtered to remove the smaller particles.
The solution is concentrated to accommodate the removal of water and then recovered by
one of two different processing methods. For one method, the concentrated carrageenan

is deposited into a solution of potassium chloride to raise the gelling temperature so the

26

filtrate will gel immediately. The gel is then frozen and compressed during thawing to
remove excess water. In the second method, the concentrated carrageenan is precipitated
in isopropyl alcohol. Since carrageenan is insoluble in alcohol, the filtrate turns into a
coagulum of carrageenan, alcohol and water. The coagulum is compressed to remove the
liquid and vacuum dried to remove the alcohol. Drying is done on a belt drier and the
dried coagulum is ground (Irneson 2000).

1.6.3. Functionality

Carrageenan should be added slowly to the vortex of water produced by a high
speed mixer. Carrageenan can be premixed with a dispersant like sugar or dispersed in
liquid sugar, salt or glycerin to help with dissolving. It should be dispersed in cold water
and then heated above its solubility temperature. The solubility temperature depends on
the level of potassium and calcium ions present with the carrageenan but most
carrageenans are heated to 77-79 °C for solubilization unless it is cold soluble. Cold
soluble carrageenans should be dispersed in cold water by adding the carrageenan slowly
to water with agitation.

The potassium or calcium ions are vital for effective gelation of the carrageenan
solution. Increasing the level of ions improves the dispersion and strength of the gel.
Carrageenan gels are thermally-reversible since the gels become fluid when heated above
the gels’ melting point temperature and resets into a gel when cooled with a minimal loss
of gel strength (FMC BioPolymer 2005).

1.6.4. Interaction with non-meat ingredients

Iota carrageenan has been found to increase the viscosity of starch by as much as

10 times the viscosity of starch alone. Carrageenan can be useful in altering the textural,

27

mouth-feel and processing properties of a starch solution. The increase in viscosity
allows reduction of the overall starch content by as much as 35-40% and improves the
texture and flavor of the finished product.

Carrageenan also has the ability to interact with milk proteins. In milk proteins,
there is a concentration of positive charges at peripheral locations on the casein micelle.
This positive charge attracts the negatively charged sulfate groups on the carrageenan
molecule to form linkages with the dispersed casein micelles. This reaction along with
the normal water gelling capabilities of carrageenan can increase the gel strength by
approximately 10-fold. (Imeson 2000).

1.6.5. Applications of carrageenan in meat products

Carrageenan has been used in a variety of meat product applications. F oegeding
and Ramsey (1986) found that the addition of iota and kappa carrageenan in a low-fat
meat batter resulted in increased water-holding capacity and firmness. In another study,
Huffman and Egbert (1990) added 0.5% carrageenan to low fat beef patties (~9%) and
compared them to all-beef patties with 10 or 20% fat. The carrageenan treatment was
equal in sensory acceptability and beef flavor to the 20% fat beef pattie and more
acceptable than the 10% fat beef pattie. Bater and others (1992) manufactured oven-
roasted turkey breasts with a 70% added brine containing salt, phosphate, nonfat dry milk
and various combinations of kappa carrageenan and starch. The incorporation or 0.5%
kappa carrageenan increased yield, improved the visual appearance, sliceability, rigidity
and decreased the expressible juice compared to the control product. In another study,
Shand and others (1994) studied the effects of adding 0.5-1.0% kappa carrageenan to

structured lean beef rolls. They found that the addition of kappa carrageenan increased

28

cook yield, improved the textural properties (bind, force to fracture, hardness) and
reduced purge of vacuum packaged slices during storage. Rolls with 1.0% kappa
carrageenan had the highest cook yield and highest force to fracture and hardness values.
He and Sebranek (1996) added kappa carrageenan to frankfurters made with lean finely
textured pork and beef. Kappa carrageenan at 0.5% reduced the cooking loss and

increased firmness of the frankfiirters.

1.7. Whey proteins

1.7.1. Background

The application of dairy proteins, especially whey protein concentrates, in the
manufacture of reformed and restructured meat proteins has received much attention in
recent years (Giese 1994). Whey protein, the by-product of cheese or rennet casein and
acid casein manufacture, has been incorporated as water and fat binders and extenders
and has the potential to improve cook yields and modify the textural characteristics of
low-fat comminuted meat products (Comer and others 1986; Ellekj aer and others 1996;
Keeton and others 1997). Whey protein products can be added to processed meat
products at levels up to 3.5% in the finished product (USDA 1999) and are categorized
on the basis of their composition, primarily based on their protein content (Huffman
1996). Products with an increased protein content are sold at higher prices. A variety of

whey protein products are listed in Table 1.1.

29

Table 1.1. Types and composition of whey protein products (From Huffman, 1996).

 

Product name Protein content Fat content Lactose content Ash content
(%) (%) (%) (%)
Whey powder 13 1 76 10
35% WPCa 34-35 4 53 8
50% WPC 53 5 35 7
80% WPC 80 4-7 7 4-7
Whey protein isolate >90 1 1 3

 

a WPC: Whey protein concentrate

1.7.2. Whey protein manufacture

In order to manufacture whey protein, several processing steps are utilized including
clarification, separation, pasteurization, crystallization, ultrafiltration/difiltration, ion
exchange and drying (Mulvihill and Grufferty 1997). First, the liquid whey is recovered
from the cheese or casein manufacturing and clarified to attain low levels of curd and
prevent blocking of the heat exchanger. Clarification is completed by a combination of
settling, screening and centrifugation. Next the fat is separated from the liquid whey by
using a self-discharging separator and then the liquid whey is pasteurized immediately.
Temperature and time for the pasteurization step are 72-75 °C and 15-20 5, respectively.
After pasteurization, the liquid whey is evaporated under vacuum to increase the total
solids to 40-60% and done at low temperatures (below 70 °C) to avoid denaturation of
the proteins. Reverse osmosis can be applied before evaporation to increase the
efficiency of the evaporator, which increases the solid content of the liquid whey to

around 20%.

30

The next step is to crystallize the liquid whey to remove the lactose by using a
crystallization tank seeded with finely ground a-lactose monohydrate or well-crystallized
whey powder. After crystallization, the crystallized lactose can be separated fiom the
liquid whey by centrifugation. The liquid whey is then ultrafiltrated/difiltrated to
increase the protein content and then pasteurized again to reduce the number of microbes.
The minerals in liquid whey can be removed by ion-exchange and after
ultrafitration/difiltration and ion-exchange, whey proteins containing 95% protein can be
produced (Huffman 1996). The final step in the production of whey proteins is drying.
In order to produce non-caking, high solubility and more functional whey powders, a
multi-stage drying process in usually used in the whey industry. Liquid whey is pre-
crystallized before the first stage of drying where the pre-crystallized whey is spray-dried
to achieve 5-8% moisture content. The final drying step and post-crystallization are done
on a fluidized bed to produce whey powders with low bulk densities.

1.7.3. Functionality

The composition of whey protein products affects their functionality. During the
spray drying process, the lactose protects the proteins from denaturation therefore whey
protein products with low lactose contents tend to have a higher degree of denatured
proteins. Also, residual fat from the milk in whey protein products can affect its foaming
properties, so as a result, whey proteins with a lower fat content have superior foaming
properties. The mineral content of the whey protein products is also important for the
functionality. Calcium is the most important mineral and a high concentration of calcium
can cause aggregation and gelation of the whey products during intense heating but at a

neutral pH, calcium phosphate can increase the heat stability. No single whey product

31

has all the functionalities required so often whey products are combined to achieve the
desired functionality (Jost 1993).

One of the key functional properties of whey proteins is their ability to form heat
induced three dimensional gel structures with increased water-holding capacity to
improve cook yield, potential texture modifying properties and improved sliceability
(Morr 1979; Morr and Ha 1993). Whey protein products have also been found to aid in
solubility by creating a smooth texture and reducing the gritty and powdery taste in meat
products. The viscosity is increased with whey protein products by enhancing the body
and texture through thickening and whey proteins also act as emulsifiers by forming
stable fat/oil emulsions, preventing oiling-off and “fat caps” and acting as a meat protein
replacement. Browning is also increased in meat products since whey protein products
enhance the Maillard, non-enzymatic browning reaction and add color and visual appeal.
Whey protein products add to the flavor and aroma by having little or no flavor of their
own, being compatible with cooked meat flavors and with spice/seasoning blends. Whey
protein products also improve the nutrition content since they have a superior amino acid
profile and can serve as a source of calcium enrichment (Keaton 1999).

1.7.4. Applications of whey proteins in meat products

Several studies have been conducted in which the application of whey proteins in
meat products was evaluated. Hemar and others (2002) tested the rheological properties
of whey protein isolate. They added whey protein isolate to kappa-carrageenan mixtures
in aqueous solution and observed no phase separation in the mixture. Whey proteins
have also been shown to reduce cook loss and improve textural parameters. McCord and

others (1998) added whey protein isolate to salt-soluble muscle protein and found that it

32

increased the water-holding capacity of the gels. In another study, Ensor and others
(1987), showed that adding whey protein concentrate to knockwurst proved to be a good
binder compared to soy protein isolate and calcium-reduced non-fat dried milk. Chen
and Trout (1991) reported that adding whey proteins (2.0% whey protein concentrate) in
restructured beef steaks decreased cooking loss. Whey proteins can also be used to
improve emulsion stability in comminuted meat products. Hung and Zayas (1992)
reported that compared to all beef frankfurters (20% fat), beef frankfurters containing
3.5% whey protein concentrate had increased water—holding capacity and decreased
cooking loss.

Due to the ability of whey protein products to bind large amounts of water and fat,
they are good candidates for fat replacers in meat products. El-Magoli and others (1996)
added whey protein concentrate (0 to 4% addition) as a fat-replacer in low fat ground
beef patties formulated to contain 11-22% fat. At the 4% level, whey protein concentrate
served as a fat-replacer without sacrificing product palatability and flavor. Hughes and
others (1998) added whey protein (3.0%) in low fat beef franks to reduce the fat content
from 12 to 5%. Reduced fat (5%) beef franks containing whey protein concentrate had
similar sensory attributes compared to those without whey protein concentrate and had

higher hardness, adhesiveness, gumminess and chewiness values than the 12% fat beef

franks.

33

1.8. Modified food starch

1.8.1. Background

Starches are polysaccharides that consist of repeating glucose units. Starch
molecules have one of two molecular structures, a linear structure, known as amylose;
and a branched structure known as amylopectin (Hegenbart 1996). Amylose and
amylopectin associate through hydrogen bonding and arrange themselves radially in
layers to form granules of starch. Granule size and shape can change greatly due to type
of starch and degree of chemical modification (McCormick 1985). Many varieties of
starches can be isolated from many different sources such as corn, potato, rice, tapioca
and wheat. In addition, each type of starch differs in amylose and amylopectin content as
well as granule size and structure. The amylose generally provides the gel strength and
the amylopectin gives viscosity to solutions (Hegenbart 1996).

Properties of starches can be improved by modification through reactions at the
hydroxyl groups. For modified food starches, only a few of the hydroxyl groups are
modified. Ester or ether groups are attached at very low degrees of substitution (DS) to
the hydroxyl groups. DS values are usually <01 and normally in the range 0.002-0.2, so
on average there is one substituent on every approximately 500 D-glucopyranosyl units.
Modification is completed in order for the starch to withstand various heat, shear and acid
conditions associated with various processing methods to introduce specific
functionalities. Small levels of derivatization can change the properties of starches

dramatically (BeMiller and Whistler 1996).

34

1.8.2. Modified food starch manufacture

Modifications can be done by either a single process or by a combination of
processes. A majority of modified food starches are crosslinked, which alters a starch by
using chemicals that cause intermolecular covalent bonding. Chemicals that can be used
are metaphosphates, phosphorus oxychloride, citric or adipic acid or epichlorohydrin.
Starch chains linked together with crosslinkers reinforce the starch granule and reduce the
rate and degree of granule swelling (McCormick 1985). Another process used to produce
modified food starches is through the use of chemical reactions. Chemical reactions
currently used to produce modified food starches are: esterification, etherification, acid
modification, bleaching and oxidation. The modification process for potato starch cross-
binds phosphorous groups and masks hydroxyl groups with acetyl groups (Skrede 1989),
resulting in changed molecular properties and functionality (Howling 1980).

Pregelatinized or cold-soluble starch can be produced by using starch that has
been cooked, dried and redissolved in cold water. The starch-water slurry flows between
two nearly touching and counter rotating, steam-heated rolls. The starch slurry is
gelatinized and pasted, coats the rolls and dries quickly. The dry film is scraped from the
roll and ground (BeMiller and Whistler 1996). The resulting products are precooked
starches and the process can be completed on both chemically modified and unmodified
starches. Pregelatinized starches contain more ruptured and retro gradated granules in
order to produce the starch paste. They are soluble in cold water and gel without cooking
(McCormick 1985)

1.8.3. Functionality

Starch granules are insoluble in cold water unless they are pregelatinized but

35

when heated in water, they undergo gelatinization. Gelatinization is the disruption of
molecular order within the starch granules. Leaching of amylose occurs during
gelatinization and total gelatinization occurs over a range of temperatures with larger
granules gelatinizing first. Continued heating in excess water results in further granule
swelling, additional leaching of soluble components and eventually total disruption of
granules which results in the formation of a starch paste (BeMiller and Whistler 1996).

A combination of modification processes enhances the functionality of starches.
Changes in functionality include increased solubility, increased or decreased paste
viscosity, increased freeze/thaw stability, enhancement of gel formation and gel strength,
reduction in gel syneresis, improvement of interaction with other substances and
modification of cooking characteristics.

1.8.4. Applications of modified food starch in meat products

Carbohydrates such as starches and flours have been used in the meat industry to
improve cooking yields due to their ability to absorb large amounts of water (Keeton
1991) and have been shown to be effective in reducing purge of low fat/high added-water
bologna (Claus and Hunt 1991; Dexter and others 1993) and other meat products.
Starches also have the ability to improve the texture of meat products. Motzer and others
(1998) studied the addition of modified food starch, kappa-carrageenan and isolated soy
protein in different levels of PSE pork. They found that the modified food starch
decreased bind strength and expressible moisture and increased yields in the 100% PSE
treatment. Modified food starch improved the water retention of PSE pork in restructured
products. In another study, Ioffe and others (2002) used different starches to study the

textural stabilization of extruded beef j erky analogs. They found that a 5% addition of

36

modified starches stabilized the beef j erky analogs after one month of storage to a higher
degree than the treatments containing only potato starch. Beggs and others (1997)
evaluated turkey frankfurters with 2.379 to 6.621% modified cornstarch and 20.93 to
35.07% water. They found that for optimal sensory and physical attributes (internal
color, compression, purge loss and pH) the best levels of modified corn starch and added

water were 2.3 and 33.6% respectively.

1.9. Ingredient combination and interaction

Carbohydrate based fat substitutes or mixtures of gums, starches and/or proteins
appear to offer the most cost effective means of replacing a significant portion of fat in
meat products and duplicating the textural and sensory characteristics of animal fat
(Keeton 1991). It seems that the addition of a single type of a substance is not enough to
achieve this significant replacement but synergistic action between ingredients have been
found to provide the fat replacement desired (Glicksman 1991).

The significance of the addition of more than one type of non-meat ingredient
added to a meat product working in combination was seen in a study by Prabhu and
Sebranek (1997). Eight treatments using kappa carrageenan at 0 or 1.5% and starch at 0,
2, 3.5, or 5% in hams were evaluated for cock yield, purge, color, texture and sensory
attributes. The incorporation of carrageenan at 1.5% increased yield, decreased purge
and had a lower sensory perception of juiciness. Increasing the amount of starch,
however, increased the perception of juiciness. In another study, Suman and Sharma
(2003) investigated the influence of different fat levels (6, 8, 10 or 20%) on the physico-

chemical and sensory characteristics of low-fat ground buffalo meat patties prepared

37

using a combination of carrageenan (0.5%) and sodium alginate (0.1%). The cook yield
and gain in height of the buffalo patties were significantly higher and the shear force
values were significantly lower for patties at all low-fat levels compared to the control
with 20% fat. Due to significantly higher sensory scores, the 10% fat level was chosen as
the optimum for low fat ground buffalo meat patties even though at the 8% fat level the
sensory rating was between good and very good. This was similar to what Lin and
Keeton (1998) found when they formulated low-fat ground beef patties with both alginate
and carrageenan. They were comparable to regular beef patties (20% fat level) in textural

properties.

1.10. Value-added Technologies

Several different processing technologies have been used to improve product
uniformity (color, texture), tenderness, juiciness and flavor in meat products. Swart
(2000) defines value-added as processing steps or technologies that add to the end state of
a product that make the improved product valued by customers. The goal of producing
value-added products is to increase the overall acceptability of meat products by
consumers. Injection, restructuring, mechanical tenderization, tumbling, mixing and use
of ingredients such as salt, phosphate, gums, starches and non-meat proteins can improve
a product’s value. Meat products can be included in this category by implementing
technologies ranging from a slight modification in packaging or creating a new name for
an existing product to producing a restructured or reformed product.

1.10.1. Injection

Injection is used to distribute a brine or marinade into whole muscle meat and

38

poultry through needles that penetrate into the muscle and distribute the brine or
marinade under pressure. Injection is used to improve juiciness, tenderness, and flavor of
a meat product. Research has shown that injection is an ideal method to distribute non-
meat ingredients such as salt, phosphate, nitrite, cure accelerators, sweeteners,
seasonings, non-meat proteins, starches, gums, water, and preservatives in meat products.

The following studies show the effects in which injecting different non-meat
ingredients into lower quality meat products have on tenderness, juiciness, and flavor of
the final product. In a study by McGee and others (2003), USDA Select inside beef
rounds were injected with a solution of sodium lactate, sodium tripolyphosphate and
sodium chloride. Warner-Bratzler shear force, cooking loss and sensory characteristics
were determined. The injected treatments were found to be more tender than the control
products for both Warner-Bratzler shear force and consumer sensory panel scores. The
injected treatments also had a lower cooking and re-heating loss compared to the
controls. Lawrence and others (2003) used semitendinosus and longissimus lumborum
muscles from USDA Select carcasses to study the effects of staged injection of calcium
lactate followed by phosphate and salt on water binding and palatability scores. The
injection of calcium lactate followed by phosphate and salt significantly increased the
pump yield and decreased the expressible moisture values compared to the injection of
calcium lactate only. The staged injection of calcium lactate followed by phosphate and
salt significantly improved the myofibrillar and overall sensory tenderness scores of
longissimus lumborum muscle compared to the non-marinated control.

In another study by Hashim and others (1999), bone-in chicken breast quarters

were marinated with a lemon-pepper marinade by injection or immersion and honey (10,

39

20 and 30%) was substituted for water in the marinades. The injected chicken retained
more marinade, lost less juice during roasting and had a lower shear force value than the
immersed chicken. The addition of honey to the marinade of the injected chicken
increased the honey flavor without affecting the appearance, aroma, and other flavor
attributes or texture.

1.10.2. Restructuring

Restructured products are manufactured from muscle groups that are partially or
completely comminuted and reformed into the same or different form. Restructuring
uses three basic approaches: chunking and forming, flaking and forming, and tearing and
forming (Pearson and Gillett 1996). A number of advantages occur by taking the
muscles apart, physically manipulating them and reforming them into a specific shape.
Restructured products have a texture that closely resembles intact meat cuts but are more
economical to produce. They are produced from less tender muscles and meat trimmings
that are cheaper raw materials compared to boneless intact meat cuts. Restructuring helps
control accurate portion and composition of meat products, easier slicing and serving and
more accurate predictions of yields (Akamittath and others 1990) but problems such as
color instability and fat oxidation occur.

1.10.3. Mechanical Tenderization

Mechanical tenderization is a technology used to improve tenderness of meat
products by destroying connective tissue and muscle fibers (Aberle and others 2001). It
is very effective in improving the tenderness of meat from carcasses with large amounts
of connective tissue (Pearson and Gillet 1996). Huffman (1981) and Booren and others

( 1981) found improvements in tenderness measured by compression and Kramer shear,

40

respectively, by blade tenderizing restructured pork chops, USDA Good (currently called
Select) and Choice beef steaks, and restructured beef steaks respectively. The advantages
of mechanical tenderization are that it improves tenderness of steaks and chops, creates
more uniform tenderness within a product, and improves cost effectiveness and ease of
implementation in a plant setting (Hayward and others 1980).

1.10.4. Challenges for value-added products

Processing technologies can increase the utilization of lower value muscles by
applying processes to increase uniformity of color, texture and tenderness. However, a
number of challenges occur when producing value-added products. Technologies such as
marination by injection, restructuring, blade tenderization and vacuum packaging can be
confusing to the consumer and cause problems with consumer acceptance. The lack of
familiarity with the terminology printed on value-added labels such as “enhanced”
creates confusion as to what has been added or done to the product. Consumers may
wonder whether value-added products compare to traditional products in safety and
wholesomeness.

Another challenge of value-added products is controlling and extending the shelf
life (Sutton and others 1997). Lipid oxidation reduces the shelf life of meat products by
causing rancidity. Rancidity is one of the most serious flavor problems in meat products
(Pearson and Gillett 1996) and is accelerated when mechanical disruption of tissue occurs
during production. Rancidity occurs when fats are oxidized, become free radicals and
react with a number of pre-existing reactants. These products readily decompose into

acids, aldehydes, alcohols, carbonyls, and ketones and some of these compounds can then

41

contribute to strong flavors or odors that contribute to the rancidity of the product
(Schmidt 2000).

Controlling the deve10pment of off-flavors is another challenge associated with
value-added products. “Fresh” flavor or flavors that are recognized as meat-type flavors
by consumers are necessary for acceptance. Off-flavor development is a result of
previously discussed lipid oxidation, warmed-over-flavor (Craig and others 1991), and
the use of non-meat ingredients. Warmed-over-flavor describes the rapid development of
undesirable flavors in cooked meat during refrigerated storage. Oxidation of
phospholipids contributes to the development of this undesirable flavor (Hettiarachchy
and Gnanasambandam 2000).

1.10.5. Benefits of value-added products

Although there appears to be several challenges associated with the development
or manufacture of value-added products, there are several reasons to continue to develop
them. One reason is that value-added products utilize lower value meat, which can be
harder to market. There is little demand for lower value meat but if value-added
technologies can be applied, the demand will increase and therefore raise the value.
Another reason to manufacture value-added products is to improve the uniformity of
existing products. Miller (2000) stated that value-added products allow for
improvements in quality attributes by 1) having a more uniform color of the cut lean
surface and possibly improving the species color or appearance, 2) improving the
tenderness of a product line or improving tenderness uniformity within a product, 3)
improving juiciness of a product line or improving j uiciness uniformity within a product

and 4) extending the shelf life of products.

42

Value-added products increase product variety or choices. Gums, starches and
non-meat proteins can replace expensive animal protein (Keeton and others 1984)
creating the opportunities to produce lower-cost extended products. This can become
increasingly important when developing meat products that are economically competitive

with other protein sources such as beans.

1.11. Summary of literature

The amount of marbling or intramuscular fat has been shown to influence the
palatability (juiciness, tenderness, flavor) of beef cuts. Savell and Cross (1988)
developed a “window of acceptability” for percent intramuscular fat or marbling of retail
beef cuts. Beef cuts containing 3-7% intramuscular fat are perceived by consumers to be
acceptable in tenderness, juiciness, flavor and overall palatability so it is important to
have at least 3% intramuscular fat in whole muscle beef cuts.

The amount of marbling in whole muscle meat cuts has been found to be below
the expectations of the meat industry which can influence the consumer’s purchasing
decisions. When consumers are not satisfied with the palatability of beef cuts, their intent
to purchase beef may decrease and along with it is the opportunity for the beef industry to
generate revenue. The deposition of intramuscular fat or marbling is influenced by many
factors such as breed, length of feeding, type of ration fed and management but it has
been shown that there is plenty of room for improvement in the amount of marbling or
intramuscular fat in whole muscle beef cuts in order to improve the palatability of the
final beef product.

The palatability of whole muscle cuts fabricated from lower quality (less than

43

USDA Choice) beef carcasses may be improved through innovative non-meat ingredient
and processing technologies. Several non-meat ingredients (sodium alginate, iota
carrageenan, whey protein isolate and modified food starch) have been used as fat
substitutes in a variety of processes meat products. Also, several different processing
technologies have been used to add value to lower quality meat products including whole
muscle cuts. The development of a “modified marbling” from selective non-meat
ingredients that can mimic the properties of intramuscular fat and can be directly injected
into lower quality whole muscle beef cuts may enhance its overall palatability by
mimicking the organoleptic properties of fat and having an appearance similar to that of

marbling.

44

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56

CHAPTER 2

DEVELOPMENT OF A “MODIFIED MARBLING” SOLUTION FOR WHOLE
MUSCLE BEEF CUTS

Abstract
A “modified'marbling” solution containing sodium alginate (SA), iota carrageenan (IC),
whey protein isolate (WPI) and modified food starch (MFS) was developed to inject into
low quality beef cuts to mimic the properties of intramuscular fat. Twenty-five
ingredient combinations (ranging from 0.25 to 0.50% addition) of the four ingredients
were formulated into 500 g solutions using a 24 central composite design. Solution pH
and viscosity, and gel (24 h, 4 °C storage) objective color, water-holding capacity, water-
holding capacity over time and gel strength were analyzed to determine the optimal
solution. Higher levels of SA and IC increased (P<0.05) gel viscosity. SA increased
(P<0.05) pH and gel L* (lightness) color value which was comparable to beef rib fat L*
values (77.2 vs. 83.6). SA and IC significantly (P<0.05) affected water-holding capacity
and SA, IC and MFS were significant (P<0.05) factors for water-holding capacity over
time. All four ingredients significantly (P<0.05) affected gel strength. The i
recommended levels of non-meat ingredients for the solution were 0.4375% SA and IC
and 0.375% WPI and MFS. Theses results indicate the feasibility of an inj ectable

“modified marbling” solution.

Keywords: “modified marbling”, solution, intramuscular fat, sodium alginate, iota
carrageenan, whey protein isolate, modified food starch

57

Introduction

The palatability (tenderness, juiciness and flavor) of whole muscle cuts fabricated
from low quality (less than USDA Choice) beef carcasses may be improved through
innovative non-meat ingredient and processing technologies. Several non-meat
ingredients (salt, phosphate, gums, starches and non-meat proteins) and processing
technologies (injection, restructuring, mechanical tenderization, tumbling and mixing)
have already been used to add value to meat products. The development of a “modified
marbling” solution from selective non-meat ingredients that can mimic the properties of
intramuscular fat and can be directly injected into lower quality whole muscle beef cuts
may enhance the overall palatability by mimicking the organoleptic properties of fat and
having an appearance similar to that of marbling.

When decreasing or substituting the fat in meat products, the product palatability:
tenderness, juiciness, flavor and mouth-feel or texture must be maintained or improved
while maintaining economic value (Mandi go 1991). Functional properties of meat
systems are primarily dependent on the interaction of the protein fraction with the other
components. These interactions include: proteinzwater, proteinzfat and protein:protein
which determine the textural properties, yield, palatability, processing behavior and
ultimately product value (Shand and Schmidt 1990).

In developing a fat substitute for intramuscular fat, a combination of ingredients
may be needed in order to mimic the functional and organoleptic properties of
intramuscular fat. Consideration must be given to gelation properties (to create “fat-like”
particles resembling marbling), water retention, viscosity (to allow direct injection into

whole muscle) and color (lightness or L* values similar to fat). Non-meat ingredients

58

chosen for the development of the “modified marbling” solution includes sodium alginate
(SA), iota carrageenan (IC), whey protein isolate (WPI) and modified food starch (MFS).

Alginates have been used in a variety of meat products because they create a
thickening and sometimes gelling effect (Glicksman 1982). Carrageenans function as
gelling agents, stabilizers and thickeners and are capable of forming viscous solutions at
low concentrations in cold water (Wallingford and Labuza 1983) so likewise have been
used in a variety of meat product applications. Whey proteins have been incorporated as
water and/or fat binders and extenders. They have the potential to improve cook yields
and modify the textural characteristics of low-fat comminuted meat products (Comer and
others 1986; Ellekj aer and others 1996; Keeton and others 1997). Modified food starches
have been used in the meat industry to improve cooking yields due to their ability to
absorb large amounts of water (Keeton 1991) and to improve the texture of meat
products.

The objective of this study was to develop a “modified marbling” solution using
these selected non-meat ingredients that mimic the properties of intramuscular fat to
inject into lower quality, less marbled whole muscle beef cuts. To achieve this objective,
response surface methodology was utilized to determine the concentration of each
ingredient (SA, IC, WPI, MFS) to use in the development of the “modified marbling”

solution.

59

Materials and Methods

Ingredient selection

To identify an optimal combination of non-meat ingredients that mimics the
sensory and functional properties of marbling, a variety of non-meat ingredients
(appendix 1) were selected that have specific quality or functional attributes (color, water
binding and retention, gelling properties, viscosity, pH). Each ingredient was dispersed
in water and allowed to gel. After subjectively observing the characteristics of the
dispersions and gels (appendix 1), the non-meat ingredients selected for further

evaluation were SA, IC, WPI and MFS.

Non-meat ingredients

The SA (Protanal RF 6650) and IC (RE 0804-01) were donated by FMC Bio
Polymer (Princeton, NJ). The WPI (Alacen 895) was purchased fiom New Zealand Milk
Products (Lot# 047U45283431314, Lenoyne, PA) and the MFS (PenPlus 47) was
donated by Penford Food Ingredients Co. (Englewood, CO). The calcium sulfate
dihydrate F .C.C. (CAS 10101-41-4) was purchased from Voigt Global Distribution LLC
(Kansas City, MO) and the phosphate was a blend containing sodium tripolyphosphate
and sodium polyphosphate (Brifisol 512) and purchased from BK Giulini (Simi Valley,
CA). Phosphate and calcium sulfate were added to aid in the gelation of the sodium

alginate.

6O

Solution manufacture

The manufacture of the solutions was conducted by weighing and adding the
ingredients to 946.4-ml lidded glass jars with the appropriate amount of water (appendix
2). The ingredients were added in the following order: phosphate, mixture of SA and
vegetable oil (for hydration per recommendations of F MC Bio Polymer), IC, WPI, MF S
and calcium sulfate solution for a mixing time of 2 min per ingredient. The solutions
were mixed using a 4-blade mixing head: 2-blades perpendicular and 2-blades parallel to
the shaft attached to a drill (Model 6220, S-B Power Tool Co., Chicago, IL). Solution pH
and viscosity were determined and after 24 hr of refrigerated storage (4 °C), the gels were
analyzed for color, water-holding capacity, water-holding capacity over time and gel

strength.

Apparent viscosity and pH determination

Apparent viscosity of the solution was determined at 30 °C at speed setting 100
using a Brookfield viscometer (Model HBTD, Brookfield Engineering Laboratories, Inc.,
Stoughton, MA). Measurements were converted to apparent viscosity readings using the

following equation: 11 = M k” and recorded at an average shear rate of 30.5 L
Q s

where

M = % torgue x 57,496 dyne cm N M
100 107 dyne cm

k” = 61,220 @
m3

0: 10.5g(_l
s

61

Solution pH was determined at 22 °C using an Accumet pH Meter (AB 15, Fisher
Scientific, Co., Pittsburgh, PA). The pH meter was calibrated with standard phosphate

buffers pH 4.0 and pH 7.0.

Objective color evaluation

Objective color of the gel was determined by pouring the solution into a Petri
dish, covering with a lid and allowing to gel at 4 °C for 24 hr. Color measurements were
taken using a Minolta Chromarneter CR-310 (Commission International D’Edairerage
(CIE) L*a*b* Ramsey, NJ) with a 5.5 cm reading orifice. Before measruing, the
Chromarneter was calibrated with a standard white tile and the measurements were taken
using the multi-read function. Readings were taken of the exposed surface of each gel

sample for L* (lightness), a* (redness), and b* (yellowness) values.

Determination of water-holding capacity and water-holding capacity over time

The water-holding capacity of the gel was determined by removing the gel from
the jar after the solution was chilled at 4 °C for 24 hr. Approximately 10 g of sample was
placed in a 50 ml polycarbonate tube and centrifuged at 4 °C at 40,000 x g for 30 min
(Honikel and Hamm 1994). Tubes were removed from the centrifuge, supernatant
poured off, and the gel and tube were weighed. Water-holding capacity was measured in
triplicate and determined by the following formula:

weight of gel after centrifugation x 100
weight of gel before centrifugation

The water-holding capacity over time of the gel was determined by removing the

gel from the jar after the solution was chilled at 4 °C for 24 hr. The gel was cut into

62

approximately 2.5 x 2.5 x 1.3 cm samples. A piece of filter paper was laid inside a Petri
dish and both were weighed. The sample cube was placed on the filter paper and the
Petri dish, filter paper, and gel cube were weighed, covered and stored at 22 °C for 2 hr.
The cube was removed from the filter paper and the filter paper and Petri dish were
reweighed. This procedure was used to simulate temperature abuse conditions that may
occur during transportation and storage of meat injected with the solution to determine
how well the gel can retain water and structure under these conditions. Water-holding
capacity over time was measured in triplicate and determined by the following formula:

weight of gel after storage at 22 °C for 2 hr x 100
weight of gel before storage at 22 °C for 2 hr

Gel strength determination

The gel strength of the solution was analyzed on a TA-HDi texture analyzer
utilizing a 5 kg load cell and a 1.3 cm diameter acrylic cylinder attachment 3.5 cm in
height. Gels were analyzed in 50 ml polycarbonate tubes placed in a molded steel pipe
on a heavy-duty platform to eliminate tube movement and variability during analysis.
The acrylic probe penetrated the gel plug in the geometric center of the sample ‘
depressing the gel 1.2 cm before retracting. Peak force was recorded in grams with a

crosshead speed of 1.7 mm/s. All samples were conducted in triplicate.

Experimental design and statistical analysis
Preliminary studies were conducted to determine the selection of ingredients to
use for the “modified marbling” solution and to determine the concentration ranges of the

selected ingredients. Based on the preliminary studies, twenty-five ingredient

63

combinations of the selected non-meat ingredients ranging from 0.25 to 0.5% addition
were formulated into 500 g solutions using a central composite design with one treatment
combination replicated six times to determine error degrees of freedom (appendix 9).
Response surface methodology was used to determine the effect of the four non-meat
ingredients (SA, IC, WPI, and MFS) on solution viscosity, pH, and gel objective color,
water-holding capacity, water-holding capacity over time and gel strength.

The data were analyzed using the Proc GLM procedure of the Statistical Analysis
System (SAS User’s Guide, Version 8.2. Cary, NC: SAS Institute 2002) to determine
which factors were significant (P<0.05) within the total model. Response surface
regression (Proc RSREG) equations were run on those factors that were significant
(P<0.05). Response surface graphs were generated and Proc IML was used to determine

the predicted level of each ingredient.

64

Results and Discussion

Apparent viscosity of the solutions

The apparent viscosity of the solutions was 0.7 Pa 3 at an average shear rate of
30.5 US and Proc GLM showed that the model was significant (P<0.05) so response
surface regression was run. The total model was significant (P<0.05) as well as the linear
(P<0.05) and quadratic (P<0.05) effects. SA and IC were significant (P<0.05) factors
and the following parameters were significant (P<0.05): IC (linear), SA x SA, IC x IC
(quadratic) and MFS x WPI (cross product). SA had the largest influence of all the
ingredients on apparent viscosity (Figure 2.1.a) since apparent viscosity increased as the
percentage of SA increased. The apparent viscosity also increased as the percentage of
IC increased but the shape of the curve was not the same as with SA. This similar pattern
was seen in a study conducted by Marcotte and others (2001) where the apparent
viscosity of several food hydrocolloids was measured at three concentrations and four
temperatures. They found that at higher gum concentrations there was an increase in
apparent viscosity. Also, Rao and Kenny (1975) and Speers and Tung (1986) found that
the effect of concentration on apparent viscosity of hydrocolloids is usually described by
either an exponential or a power relationship. Observations by the researchers in our
study found that the range of apparent viscosities of the solutions would be inj ectable.

Since the WP1 and MFS ingredients of the solution were not significant, they
were held at the center point (0.375%) when creating the graphs and the graphs were
based on the addition of SA and IC. This was consistent with the rest of the attributes

analyzed and the graphs were created in the same manner for all the attributes. For the

65

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apparent viscosity of the solutions, the stationary point was a saddle point and the
recommended use level of each ingredient was 0.305% SA, 0.338% IC and 0.375% WPI

and MFS.

pH of the solutions

The mean pH of the solutions was 6.5 and Proc GLM showed that the model was
significant (P<0.05). Response surface regression found that the total model was
significant (P<0.05) along with the linear (P<0.05) effect and SA was significant as a
factor (P<0.05). This was also seen in a study done by Devatkal and Mendiratta (2001)
where they used calcium lactate with salt-phosphate and alginate-calcium gels in
restructured pork rolls. They found that the pH was significantly (P<0.05) higher in pork
rolls containing alginate and calcium. Means and Schmidt (1986) also saw a significantly
(P<0.05) higher pH when evaluating structured beef steaks containing factorial
treatments of SA and calcium carbonate compared to the control. Figure 2.1 .b shows that
the pH of the solutions increased when the percentage of SA increased and also when the
percentage of IC increased. A pH in the range of fresh meat (5.5-5.9) would be the target
for the solutions and 6.49 is just outside the range. The stationary point was a saddle
point and the recommended use level of each ingredient for pH was 0.494% SA, 0.377%

IC and 0.375% WPI and MFS.

67

Objective color of the gels

The L* color value of the gels had a mean of 77.2 and the model was significant
(P<0.05). Response surface regression showed that the total model was significant
(P<0.05) along with the linear (P<0.05) and cross product (P<0.05) effects. The factor
SA was significant (P<0.05) as well as the parameter MFS x WPI (cross productP
(P<0.05). Figure 223 shows that the L* color value tended to increase as the percentage
of SA increased. The L* color value also increased as the percentage of 1C increased but
only to approximately the center point (0.375%) and then it plateaued. The L* color
values were also comparable to beef rib fat L* values (83.6). The stationary point was a
saddle point and the recommended use levels of each ingredient for L* was 0.702% SA,

0.413% IC and 0.375% WPI and MFS.

The mean a* color value was —4.8 and the model was not significant (P>0.05).
The response surface regression was not run.

The b* color value of the solutions had a mean of 7.2 and the model was significant
(P<0.05). The total model (P<0.05) and linear effect (P<0.05) were significant for response
surface regression. SA was a significant (P<0.05) factor and figure 2.2.b showed that the b*
color value increased as the percentage of SA increased and also increased as the percentage of
IC increased but not to the extent as with the SA. The stationary point was a saddle point and the
recommended level of each ingredient to use in a “modified marbling” solution for b* value was

0.023% SA, 1.081% IC and 0.375% WPI and MFS.

68

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Water-holding capacity and water-holding capacity over time of the gels

The mean for the water-holding capacity of the gels was 98.0% and the model
was significant (P<0.05). The response surface regression showed that the total model
was significant (P<0.05) along with the linear (P<0.05) and quadratic (P<0.05) effects.
The factors SA and IC were significant (P<0.05) along with the following parameters
(P<0.05): IC (linear), IC x IC (quadratic) and IC x SA (cross product). From the
response surface curve (Figure 2.3.3), the water-holding capacity decreased as the
percentage of SA increased and increased as the percentage of IC increased to
approximately the center point (0.375%) and then plateaued. This was also seen by
Foegeding and Ramsey (1986) who found that the addition of iota and kappa carrageenan
in low-fat meat batters resulted in an increased water-holding capacity. Also,
Wallingford and Labuza (1983) found that carrageenan had very good water binding
capacity when evaluating the water biding properties of nine food hydrocolloids in a low
fat meat emulsion. The stationary point was a saddle point and the recommended use
level of each ingredient for water-holding capacity was 0.281% SA, 0.391% [C and
0.375% WPI and MFS.

For water-holding capacity over time of the gels, the mean was 93.0% and the
model was significant (P<0.05). The response surface regression showed that the total
model was significant (P<0.05) as well as the linear (P<0.05) and cross product effects
(P<0.05). The factors SA, IC and MF S were significant (P<0.05) along with the
parameters (P<0.05) IC x SA and MFS x WPI (cross product). From Figure 2.3.b, the
water-holding capacity over time increased proportionally as both the percentage of SA

and IC increased. The water-holding capacity and water-holding capacity over time

70

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71

should be at least in the range of an average cooked product yield (75-85%) and both the
water-holding capacity and water-holding capacity over time were above this range. The
stationary point was a saddle point and the level of each ingredient to use in a “modified

marbling” solution for water-holding capacity over time was 0.434% SA, 0.483% IC and

0.375% WPI and MFS.

Gel strength of the gels

The mean of the strength of the gels was 22.6 g and the model was significant
(P<0.05). From the response surface regression, the total model (P<0.05), linear
(P<0.05), quadratic (P<0.05) and cross product effects (P<0.05) were significant. The
factors SA, IC, WPI and MFS were significant (P<0.05) as well as the following
parameters (P<0.05): IC and WPI (linear), SA x SA and IC x IC (quadratic) and IC x SA
and WPI X 1C (cross product).

From the response surface curve (Figure 2.4), the gel strength increased as the
percentage of SA increased and it also increased as the percentage of IC increased but the
shape of the curve was not the same as with SA. This was also seen by Raharjo and
others (1994) who studied the quality characteristics of restructured steaks with veal
trimmings or veal leg meat and sodium alginate/calcium lactate and found that sodium
alginate/calcium lactate used as a binder increased the binding force. Devatkal and
Mendiratta (2001) found similar results when they evaluated restructured pork rolls
formulated with sodium alginate/calcium carbonate and found that the raw binding
strength was significantly higher in pork rolls containing sodium alginate/calcium

carbonate. In another study, Shand and others (1994) studied the effects of adding

72

Figure 2.4. Response surface curve for significant (P<0.05) total regression models
for gel strength of “modified marbling” gels.

Response surface curve for gel strengthlhardness (P<0.05)
(Whey protein isolate = 0.375% and modified food starch = 0.375%)

0.500

 

  

0.333 Sodium Alginate
(%)

Iota Carrageenan

("l

73

0.5-1.0% kappa carrageenan to structured lean beef rolls. They found that the addition of
kappa carrageenan improved the textural properties (bind, force to fracture, hardness).
The stationary point was at a minimum and the recommended use level of each

ingredient for gel strength was 0.315% SA, 0.300% IC and 0.375% of WPI and MFS.

Development of the solution

After the use levels of each ingredient, for each attribute were made, the overall
levels of ingredients recommended for the final solution were: 0.43 75% SA and IC and
0.3 75% WPI and MFS. The solution was manufactured using these levels and observed
by the researchers to be appropriate in order to give the “modified marbling” solution the
desired functional properties (gelation, water retention, color). The solution was then
scaled up for pilot plant use in a state of the art injector designed to handle solutions of

this type for precise injection at a targeted additive level of 5-7%.

74

Conclusion

A “modified marbling” solution was developed from selected non-meat
ingredients (SA, IC, WPI and MFS) and has the potential to be injected into whole
muscle beef cuts. Higher levels of SA and IC increased gel viscosity. SA increased pH
and gel L* (lightness) color value which was comparable to beef rib fat D“ values. SA
and IC significantly affected water-holding capacity and SA, IC and MFS were
significant factors for water-holding capacity over time. All four ingredients significantly
affected gel strength.

This study was designed to develop a “modified marbling” solution that
mimicked intramuscular fat in appearance and functionality to inject into lower value
whole muscle beef cuts. The results of this study demonstrate that a solution can be
developed which looks like fat and is injectable. The findings of small-scale studies in

the laboratory indicate the feasibility of further development of the solution for injection.

75

References

Comer FW, Chew N, Lovelock L, Allan-Wojtas P. 1986. Comminuted meat products:
Functional and microstructural effects of fillers and meat ingredients. Canadian
Institute of Food Sci and Tech J 19:68-74.

Devatkal S, Mendiratta SK. 2001. Use of calcium lactate with salt-phosphate and
alginate-calcium gels in restructured pork rolls. Meat Sci 58:371-9.

Ellekjaer MR, Naes T, Baardseth P. 1996. Milk proteins affect yield and sensory quality
of cooked sausages. J Food Sci 61:660-6.

Foegeding EA, Ramsey SR. 1986. Effect of gums on low-fat meat batters. J Food Sci
51:33-6, 46.

Glicksman M. 1982. Food applications of gums. In “Food Carbohydrates,” IFT Basic
Symposium Series, an AVI book, p 270. Van Nostrand Reinhold, New York, NY.

Honikel KO, Hamm R. 1994. Advances in meat research, Vol. 9, Pearson AM and
Dutson TR (eds). Chapman and Hall, Glasgow, UK.

Keeton J T. 1991. Fat substitutes and fat modification in processing. Proc Recip Meat
Conf. 44279-91.

Keeton JT. 1997. Non-meat ingredients for low/non fat processed meats. Proc Recip
Meat Conf49223-31.

Mandigo RW. 1991. Problems and solutions for low fat meat products, p100-14. Proc
Meat Ind Res Conf.

Marcotte M, Taherian Hoshahili AR, Ramaswamy HS. 2001. Rheological properties of
selected hydrocolloids as a fimction of concentration and temperature. Food Res

Int 34:695-703.

Means WJ, Schmidt GR. 1986. Algin/calcium gel as a raw and cooked binder in
structured beef steaks. J Food Sci 51 :60-5.

Raharjo S, Dexter DR, Worfel RC, Sofos JN, Solomon MB, Shultz GW, Schmidt GR.
1994. Restructuring veal steaks with salt/phosphate and sodium alginate/calcium
lactate. J Food Sci 59:471—3.

Rao MA, Kenny JF. 1975. Flow properties of selected food gums. Canadian Institute
Food Sci and Tech J 8: 142-8.

SAS Institute, Inc. 2002. SAS User’s Guide, Version 8.2. Cary, NC: SAS Institute.

76

Shand PJ, Schmidt GR. 1990. New technology for low-fat meat products. Proc Recip
Meat Conf 43:37-52.

Shand PJ, Sofos JN, Schmidt GR. 1994. Kappa-carrageenan, sodium chloride and
temperature affect yield and texture of structured beef rolls. J Food Sci 59:282-7.

Speers RA, Tung MA. 1986. Concentration and temperature dependence of flow
behavior of xanthan gum dispersions. J Food Sci 51:96-8, 103.

Wallingford L, Labuza TP. 1983. Evaluation of the water binding properties of food

hydrocolloids by physical/chemical methods and in a low fat meat emulsion. J
Food Sci 4821-5.

77

CHAPTER 3

SCALE-UP OF A “MODIFIED MARBLING” SOLUTION FOR AN ON-LINE
INJECTION PROCESSING SYSTEM FOR WHOLE MUSCLE BEEF CUTS

Abstract
A “modified marbling” solution containing sodium alginate (SA), iota carrageenan (IC),
whey protein isolate (WPI) and modified food starch (MFS) was modified in order to
prevent absorption of muscle myoglobin pigments into the solution and attempt to mimic
the flavor of beef fat. In addition it was scaled to be used in an injection system designed
for high volume processing. Beef tallow was tested at different levels (1-4%) and
different types and levels (0.25-1.0%) of beef flavoring were evaluated. The processing
system and parameters were determined and the solution was manufactured and injected
and tumbled into the whole muscle beef cuts for a pilot plant study. Three percent beef
tallow and 0.25% beef flavor were added to the “modified marbling” solution.
Parameters were set on an automatic, multi-needle injector in order to acquire the desired
percent pick-up (5-7%) and “modified marbling” pattern. The ribeye rolls designated to
different storage days did not significantly differ in injection pick-up and tumbling loss
measured immediately after injection and tumbling, respectively. The injection pick-up
(9.75%) was significantly higher (P<0.05) for the injected Select but there was no

significant difference between the three controls.

Keywords: “modified marbling”, solution, injection, parameters

78

Introduction

Fat substitutes that are protein and carbohydrate-based are hydrophilic ingredients
and since muscle myoglobin pigments are also hydrophilic, the pigments can absorb into
the ingredients or the ingredients can absorb into the pigments. This was an
unanticipated result of preliminary injection observational studies. All the non-meat
ingredients used to manufacture the “modified marbling” solution (sodium alginate (SA),
iota carrageenan (IC), whey protein isolate (WPI) and modified food starch (MFS) are
protein or carbohydrate-based. The muscle myoglobin pigments tended to absorb into
the solution causing color variation among the gelled particles (marbling) when injection
was completed. A possible solution to the problem is to add a hydrophobic ingredient to
the solution in order to prevent or slow down the absorption of the pigments.

Injection has been used to physically distribute a brine or marinade into whole
muscle meat and poultry through needles that penetrate into the muscle and distribute the
brine or marinade under pressure. Injection is used to improve juiciness, tenderness, and
flavor of a meat product. Research has shown that injection is an ideal method to
distribute non-meat ingredients such as salt, phosphate, nitrate, cure accelerators,
sweeteners, seasonings, non-meat proteins, starches, gums, water, and preservatives in
meat products.

In a study by McGee and others (2003), USDA Select inside beef rounds were
injected with a solution of sodium lactate, sodium tripolyphosphate and sodium chloride.
The injected treatments were found to be more tender than the control products for both
Wamer—Bratzler shear force and consumer sensory panel scores. The injected treatments

also had a lower cooking and re-heating loss compared to the controls. In another study

79

by Hashim and others (1999), bone-in chicken breast quarters were marinated with a
lemon-pepper marinade by injection or immersion and honey (10, 20 and 30%) was
substituted for water in the marinades. The injected chicken retained more marinade, loss
less juice during roasting and had a lower shear force value than the immersed chicken.
The addition of honey to the marinade of the injected chicken increased the honey flavor
without affecting the appearance, aroma, and other flavor attributes or texture.

The best processing technique to use in order to get the “modified marbling”
solution into whole muscle beef cuts is injection. Injection should acquire the desired
percent pick-up (5-7%) to achieve an acceptable “modified marbling” pattern.

The objectives of this study were to modify the solution, determine the processing
system and parameters and demonstrate that a “modified marbling” solution can be
injected into whole muscle beef cuts. To achieve this objective, beef tallow and beef
flavor were added to the solution to prevent or slow down the absorption of the muscle
myoglobin pigments into the injected marbling and to mimic the flavor of beef fat
respectively. The processing system used to inject the solution into whole muscle beef
cuts was determined, parameters were set, and the “modified marbling” solution was

injected into the beef cuts in the pilot plant study.

80

Materials and Methods

Solution modification

Results summarized from preliminary injection observation studies using a hand-
held injector indicated that muscle myoglobin pigments absorbed into the “modified
marbling” solution causing color variation among the gelled particles. It was
hypothesized that addition of a hydrophobic ingredient to the “modified marbling”
solution was needed to prevent the absorption of the pigments so beef tallow (B4102,
Proliant Meat Ingredients, Ames, IA) was tested at different levels (1-4%) in the solution.

In addition, informal tasting of the “modified marbling” solution by the
researchers found the need for the addition of a beef flavor. Sensory evaluations were
conducted in a discussion format with a trained sensory panel of six healthy panelists
between ages twenty and sixty-five (four female and two male) using subcutaneous beef
ribeye fat as a standard. They evaluated the beef fat flavor intensity and mouth
coating/texture of the “modified marbling” solution. The “modified marbling” solution
and beef ribeye fat were prepared by cooking the samples in 25-ml covered glass bottles
in a water bath to an internal temperature of 71 °C (the endpoint cooking temperature of a
steak.) The samples were transferred to Souffle’ cups and served to the panelists. From
the evaluations, changes were made to the flavor of the “modified marbling” solution by
experimenting with different beef flavors (powder, solid, liquid) and adjusting the
amounts (0.25-1.0%) in order to try to mimic the flavor of beef fat. Changes were made
to attempt to mimic the mouth coating/texture of the “modified marbling” solution by
adjusting ingredient levels and processing procedures. Modifications were made to the

other ingredients as needed to accommodate the addition of beef tallow and flavoring.

81

Injection parameters

Several tests were conducted to determine the machine parameters to use with the
injector (IMAX 520, Wolf-tee, Inc., Kingston, NY) in order to incorporate the “modified
marbling” solution into whole muscle beef cuts. Ribeye roll sections were injected with
the “modified marbling” solution, weighed to determine percent pick-up of the solution
and the “modified marbling” pattern was observed. Changes were made to the injection
parameters as needed to acquire the desired percent pick-up (5-7%) and “modified
marbling” pattern.

The solution was injected into the ribeye rolls using an IMAX 520 injector (Wolf-
tec, Inc., Kingston, NY) with a 378-L brine tank containing an external, stainless steel
centrifugal pump. The injector contained two hundred 4-mm needles with four 1.5-mm
exit holes. The method of injection was one-way and the pump pressure was 4.5-bar.
The injector had a walking beam to transport the product at a speed of 39-strokes/min and

the solution temperature set point was 35 °C.

Needle injection study

There was a concern that the penetration of the injection needles through the
ribeye rolls could affect the tenderness of the steaks and bias the proposed study, which
studied the effect of the “modified marbling” solution on tenderness. A study was
conducted to compare Warner-Bratzler shear force of needle injected (without the
“modified marbling” solution) and non-injected ribeye rolls. A total of ten ribeye rolls,
112A (2 USDA Average Choice, 2 USDA Low Choice, 3 USDA High Select and 3

USDA Low Select) were purchased at a local meat company (Popoff Quality Food

82

Service, Lansing, MI). The ribeye rolls were cut in half and the anterior end was run
through the injector without solution and the posterior end was not run through the
injector. The injector contained two hundred 4-mm size needles with two 2-mm holes
and the walking beam speed was 30-strokes/min. Two steaks (2.5 cm) were cut fiom the
middle of the ribeye half (opposite from the anterior or posterior end). This resulted in
adjacent steaks being compared for treatment effects in each ribeye

Steaks were cooked on a Taylor clamshell grill (Model QSZ4 Taylor Co, Rockton,
IL). The upper plate was set to 104 °C and the bottom plate at 102 °C with a 2.7-cm gap
between plates. The temperature was monitored using a copper constantan thermocouple
(0.051 cm diameter, 15.2 cm length; Omega Engineering Inc., Stamford, CT) inserted
into the geometric center of the steak and cooked to an endpoint temperature of 71 °C.
Steaks were stored at 4 °C for 24 hr and six 1.27-cm cores were taken parallel to the
longitudinal axis of the fibers using a drill press-mounted corer. Cores were sheared
perpendicular to the fibers using a Warner-Bratzler head on a TA-HDi Texture Analyzer

(Texture Technologies Corp., Scotsdale, NY).

Raw materials and non-meat ingredients .

Ribeye rolls (112A) were selected and purchased from a meat packing plant
(Smithfield Beef Enterprise, Plainwell, MI). Beef carcasses were yield and quality
graded to obtain USDA Select, USDA Low Choice and USDA Average Choice carcasses
for this study. Selected carcasses were tagged for identification and followed through the
fabrication line. The rib sections (6"‘-12th rib) were removed from both sides of the

carcass, boned out to produce a boneless ribeye roll (IMPS 112A), and vacuum packaged.

83

Ribeye rolls were loaded into boxes and transported to the meat laboratory at Michigan
State University. At Michigan State University, replicates were balanced within grade so
that any differences within grades allocated to each treatment were minimized.

The SA, IC, WPI, MF S and calcium sulfate were purchased or donated from the
same sources as in study 1 and the beef tallow (B4102) and the beef stock (B1304) were

donated from Proliant Meat Ingredients (Aimes, IA)

Solution manufacture and analysis

Solutions (SA, IC, WPI and MFS) for the pilot plant study were manufactured at
the Michigan State University meat laboratory. Batches of “modified marbling” solution
(approximately 68 kg) for each of the four replicates were produced using a Rotostat
mixer (Model 80XP63SS, Admix Inc., Londonderry, NH). Ingredients were weighed
(appendix 11) and phosphate was added to half of the water and mixed for 2 min at 1500
rpm. The whey protein isolate and beef tallow were added next followed by the mixture
of SA and vegetable oil (for hydration per recommendation of FMC BioPolymer) and
then the remainder of the water in the solution. The Rotostat speed was increased to
2000 rpm. The beef flavor, IC, MF S and presolubilized calcium sulfate solution were
added and the mixing speed was gradually increased to 3500 rpm. The total mixing time
was 8 min or until the desired thickness was achieved.

The properties of the “modified marbling” solution were determined by
measuring the solution viscosity and pH immediately after the solution was

manufactured. The solution was stored for 24 hr at 4 °C to allow the solution to gel and

84

objective color, water-holding capacity, water-holding capacity over time, and gel

strength/hardness were determined using the same procedures as in study 1.

Processing and injection procedures for subsequent studies

One treatment (injected USDA Select) and three controls (USDA Select control,
USDA Low Choice control, and USDA Average Choice control) were evaluated. The
USDA Select ribeye rolls were processed by taking four ribeye rolls for each replicate
(one ribeye roll for each storage day: 0, 14, 28, and 42) and cutting each in half (anterior
and posterior). For the USDA Low Choice and Average Choice controls, two ribeye rolls
were used for each replicate and were cut in half. The ribeye halves were randomized
across each storage day for each replicate and the Select ribeye halves were also
randomized between the injected and control treatments as a completely randomized,
balanced design (appendix 13 and 14).

The control ribeye rolls (USDA Select control, USDA Low Choice control, and
USDA Average Choice control) were passed through the injector (IMAX 520 using the
set parameters) without solution, weighed, tumbled for 1 min with a Roscherrnatic
tmnbler (Model MM 80, Colrnatic Co., Long Island City, NY) and reweighed. This was
done based on the results of the needle injection study. All control ribeye rolls were
packaged in 30.5 x 61.0 cm vacuum packaged bags (Cryovac Sealed Air Co., Duncan,
SC) and stored in boxes at 1 °C. The injected USDA Select ribeye rolls were weighed,
injected with the “modified marbling” solution, weighed, tumbled for 1 min, and
reweighed. Tumbling was conducted in order to better distribute the “modified

marbling” solution within the ribeye rolls to achieve the desired marbling pattern.

85

Control ribeye rolls were also tumbled in order to keep consistency between the treated
and control ribeye rolls. The ribeye rolls were injected with the needles penetrating into
the non-fat side of the meat at a targeted 5-7 % injection, vacuum packaged and stored in

the same manner as the controls. This process was replicated four times.

Experimental design

Modifications were made to the solution to address encountered problems. An
automatic brine injector was then set-up with the appropriate parameters to inject 5-7% of I
the “modified marbling” solution into whole muscle beef cuts.

The injection verification study was analyzed using the Proc Mixed procedure of
the Statistical Analysis System (SAS User’s Guide, Version 8.2, Cary, NC: SAS Institute,
Inc., 2002) to determine the effect of needle injecting ribeye rolls without the “modified
marbling” solution on Warner-Bratzler shear force. Difference among attribute means
was determined with a predetermined level of significance (P<0.05) using Tukey’s Least
Significant Difference procedure.

The experimental design used for the pilot plant study was a split plot design with
treatment as the whole plot factor and storage day as the split plot factor. The effect of
injecting the “modified marbling” solution into ribeye rolls on quality attributes (injection
pick-up and tumbling loss) of the ribeye rolls was analyzed using the Proc Mixed
procedure of the Statistical Analysis System (SAS User’s Guide, Version 8.2, Cary, NC:
SAS Institute, Inc., 2002). Difference among attribute means was determined with a
predetermined level of significance (P<0.05) using Tukey’s Least Significant Difference

procedure.

86

Results and Discussion

Modification of the solution

Different levels of beef tallow (1-4%) were tested in the “modified marbling”
solution in attempt to decrease the absorption of muscle myoglobin pigments into the
solution. Three percent beef tallow was observed to be the most effective. Also, from
sensory evaluations and follow-up testing in the lab, it was found that a powder beef
flavor at 0.25% be added to the solution in order to attempt to mimic the flavor of beef
fat. Due to the modifications made to the solution, the amount of whey protein isolate
was increased from 0.375 to 1.5% to assist in emulsifying the beef tallow since whey
proteins have been found to improve emulsion stability. Hung and Zayas (1992) reported
that compared to all beef frankfurters (20% fat), beef frankfurters containing 3.5% whey
protein concentrate had increased water-holding capacity and decreased cooking loss.
The amount of sodium alginate was also increased from 0.4375 to 1.0% to strengthen the

gel.

Needle injection study

A study was conducted to compare Wamer—Bratzler shear force values of needle
injected (without the “modified marbling” solution) and non-injected ribeye rolls to
determine if the penetration of the injection needles of the IMAX 520 through the ribeye
rolls would affect the tenderness of the steaks. There was no difference (P>0.05)
between the needle injected (without the “modified marbling” solution) and non-inj ected
ribeye rolls for Warner-Bratzler shear force values. The mean for the ribeye rolls needle

injected was 3.18 kg and for the non-injected ribeye rolls was 3.50 kg. Even though the

87

difference between the needle injected and non-injected ribeye rolls was not significant
(P=0.062), it was close to the predetermined level of significance (P<0.05) so all control
ribeye rolls were needle injected without the “modified marbling” solution during the

subsequent pilot plant study to prevent bias when evaluating tenderness.

Properties of “modified marbling” solution and gel

The properties of the “modified marbling” solution and gel from the pilot plant
study are shown in table 3.1 for each replicate and were consistent across replicates. The
viscosity of the solution was higher than in study 1 due to the need of ingredient addition
based on problems encountered. Beef tallow was added to limit the absorption of the
muscle myoglobin pigments into the “modified marbling” solution. The amount of WPI
and SA was increased to accommodate for the addition of the beef tallow and the
addition of these ingredients increased the viscosity of the solution. The pH of the
solution and the L* value of the gel were similar to values encountered in study 1. The
L* value measured for subcutaneous fat removed from the rib portion of a USDA Choice
carcass was 83.6 which was a little higher but similar to the L* value of the gel.

The water-holding capacity and water-holding capacity over time were similar to
the values optimized from study 1 but the gel strength was higher. The addition of
ingredients to the solution to limit the absorption of the pigments produced a harder gel,
which required a larger amount of force to penetrate it. The moisture, fat and protein

percentages were similar to the ingredient formulation.

88

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Injection pick-up and tumbling loss of ribeye rolls for subsequent study

Table 3.2 shows the percent injection pick-up and tumbling loss for the injected
and control ribeye rolls. The ribeye rolls designated to different storage days did not
significantly differ in injection pick-up and tumbling loss measured immediately afier
injection and tumbling respectively.

The injection pick-up for the injected Select was significantly higher (P<0.05)
than the controls but there was not a significant difference between the three controls.
The controls had a small percentage loss of meat, which was due to small pieces of the
ribeye roll coming off as they were passed through the injector. The average injection
pick-up for the injected Select was a little higher then the targeted injection pick-up of 5-
7%. The tumbling loss for the injected Select ribeye rolls were significantly higher
(P<0.05) than the Select control and the Average Choice control ribeye rolls. The loss
for the injected Select was higher than the Low Choice control but the difference was not

significant.

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91

Conclusion

The “modified marbling” solution was modified to address problems encountered
during plans to scale up for the pilot plant study and the parameters for the injection
processing system were developed. Three percent beef tallow and 0.25% beef flavor
were added to the “modified marbling” solution. Parameters were determined for an
automatic, multi-needle injector in order to acquire the desired percent pick-up (5-7%)
and “modified marbling” pattern for Select ribeye rolls. The ribeye rolls designated to
different storage days did not significantly differ in injection pick-up and tumbling loss
measured immediately after injection and tumbling respectively. The injection pick-up
(9.75%) was significantly higher for the injected Select but there was not a significant
difference between the three controls.

This study was designed to modify the “modified marbling” solution, determine
the parameters for a specific injector and demonstrate that the “modified marbling”
solution can be injected into whole muscle beef cuts. The results of this study indicate
that the “modified marbling” solution could be successfully modified for a specific
equipment piece and the solution could be successfully injected into whole muscle beef
cuts. Further research will be needed to improve upon the “modified marbling” solution

and injection parameters for implementation in an industrial application.

92

References

Hashim IB, McWatters KH, Hung YC. 1999. Marination method and honey level affect
physical and sensory characteristics of roasted chicken. J Food Sci 64:163-6.

Hung SC, Zayas J F . 1992. Functionality of milk proteins and corn germ protein flour in
comminuted meat product. J Food Qual 15:139-52.

McGee MR, Henry KL, Brooks J C, Ray F K, Morgan JB. 2003. Injection of sodium
chloride, sodium tripolyphosphate, and sodium lactate improves Warner-Bratzler
shear and sensory characteristics of pre-cooked inside round roasts. Meat Sci
64:273-7.

SAS Institute, Inc. 2002. SAS User’s Guide, Version 8.2. Cary, NC: SAS Institute.

93

CHAPTER 4
CHEMICAL AND PHYSICAL PROPERTIES OF WHOLE RIBEYE ROLLS
INJECTED WITH THE “MODIFIED MARBLING” SOLUTION COMPARED
TO NON-INJECTED CONTROLS
Abstract
Ribeye rolls (IMPS 112A) injected with the “modified marbling” solution (5-7%

targeted pick-up) were compared to control ribeye rolls in chemical attributes. USDA
Select, Low and Average Choice ribeye rolls were passed through an automatic brine
injector without injecting solution (controls). Ribeye rolls were designated to O, 14, 28,
or 42 days of storage (1°C), weighed for ribeye purge and steaks (2.54 cm) were
fabricated on each storage day. A 7-day retail shelf life study (analysis of TBARS, color
and percent steak purge) was conducted on fabricated steaks from each treatment and
proximate analysis was conducted. The injected Select had a significantly higher
(P<0.05) ribeye purge than the Average Choice control. The injected Select had the
highest percent moisture, lowest percent fat and lowest cooked product yield. For
TBARS values, the injected Select was significantly higher (P<0.05) than all the controls.
There also were no significant differences in color scores between treatments. The
“modified marbling” solution has the potential to improve lower quality beef cuts but
more research is needed to improve the “modified marbling” properties. One possibility
is that the amount of fat in the solution could be increased to achieve the benefits of both
the non-meat ingredients and the fat.

Keywords: intramuscular fat, “modified marbling”, non-meat ingredients, solution,
injectable

94

Introduction

The amount of marbling or intramuscular fat has been shown to influence the
palatability (juiciness, tenderness, flavor) of beef cuts. Savell and Cross (1988)
determined that the minimum fat percentage required for acceptable palatability of
broiling cuts is 3% on an uncooked basis (minimum Slight degree of marbling, USDA
Low Select). They came to this conclusion after studying research conducted over many
years and found that steaks with less than 3% intramuscular fat (Practically Devoid and
Traces) were tougher, drier and less flavorfiil. However, 3% intramuscular fat or Slight
degree of marbling provides little room for error in cookery method or degree of
doneness to ensure palatability. They also determined two other levels of intramuscular
fat related to increased palatability. Approximately 5% (midpoint of Small degree of
marbling) and 7% (low end of Moderate amount of marbling) were associated with
hierarchical degrees in palatability. Beef cuts containing 3-7% intramuscular fat
(marbling) are perceived by consumers to be acceptable in tenderness, juiciness, flavor
and overall palatability.

Studies have been conducted to determine quality inconsistencies within the beef
industry chain, from farm to retail. The results fiom the last National Beef Quality Audit
(McKenna and others 2002) reported that the overall average scores for intramuscular fat
and USDA beef carcass quality grades were Small06 (marbling score) and USDA Select79
(USDA Quality Grade) respectively. The fourth listed challenge in the “top ten quality
challenges” identified from the audit was insufficient marbling since it was found that
45% of carcasses graded USDA Select (Slight degree of marbling), 53% graded USDA

Choice (9% moderate, 26% modest and 65% small degree of marbling) and only 2%

95

graded USDA Prime. F orty-five percent of the carcasses had Slight degree of marbling
or approximately 3% intramuscular fat and are at the lower edge of the “window of
acceptability.” This indicates an opportunity for improvement by increasing the amount
of marbling in whole muscle beef cuts to ensure acceptable palatability.

The palatability of whole muscle cuts fabricated from lower quality (less than
USDA Choice) beef carcasses may be improved through innovative non-meat ingredient
and processing technologies. The development of a “modified marbling” solution from
selective non-meat ingredients (sodium alginate, iota carrageenan, whey protein isolate
and modified food starch) that can mimic the properties of intramuscular fat and can be
directly injected into lower quality whole muscle beef cuts may enhance its overall
palatability by mimicking the organoleptic properties of fat and having an appearance
similar to that of marbling.

The objective of this study was to verify the properties of the “modified
marbling” solution in whole muscle beef cuts. To achieve this objective, USDA Select
ribeye rolls were injected with the solution, cut into steaks and chemical attributes were

compared to non-inj ected USDA Select, Low and Average Choice control ribeye steaks.

96

Materials and Methods
Processing procedures

After the solutions were manufactured and the ribeye rolls were injected as
described in chapter 3, the day 0 ribeye rolls were allowed to equilibrate for 48 hr and
then removed from the package and cut for analysis. Thus the ribeye roll controls for day
0 in this study were actually 48 hr afier injection. Ribeye steaks were cut from the
middle of the ribeye rolls (opposite from the anterior or posterior end) with a 1.3 cm
steak was cut for pH, proximate composition, TBARS analysis and seaming electron
microscopy. A 2.5 cm steak was then cut for the retail meat case shelf-life study and two
more 2.5 cm steaks were cut and randomized for sensory evaluation and Warner-Bratzler
shear force which was done in another study (chapter 5).

The steak designated for the retail meat case shelf-life study was weighed and
placed on 12.7 x 20.3 cm foam trays. The trays were overwrapped with polyvinyl
chloride film (PVC) (RMF-61 HY stretch meat film, Borden Chemical, North Andover,
MA) with a water vapor transmission rate of 26g/254 sq. cm per 24 hr at 38 °C, 90% R.
H. The oxygen transmission rate was 1,400cc/254 sq. cm per 24 hr at 23 °C and the
carbon dioxide transmission rate was 13,400cc/254 sq. cm per 24 hr at 23 °C. The steaks
were placed in a 1 °C retail meat case (Model SC-CMS35-6, Mc Cray Refrigerator Co.,
Inc., Philadelphia, PA). The retail meat case lighting produced a luminance of 122
lumens on the inside shelf of the meat case and 62 lumens on the outside glass surface of
the meat case. The steaks were allowed to equilibrate inside of the meat case for 2 hr and
objective and subjective color was evaluated on the steak through the PVC film. Color

measurements were also taken on day 3, 5, and 7. On day 7 the PVC film was removed.

97

The steaks were reweighed for percent purge determination and a sample was taken for
day 7 TBARS analysis.

On storage day 14, 28, and 42 the appropriate ribeye rolls were removed from the
package, weighed for percent ribeye purge (loss of fluid) and the above steps were

repeated for analysis.

Ribeye purge

Percent ribeye purge was determined on each storage day (14, 28, 42) afler being
stored in boxes at 1 °C. The ribeye rolls were weighed prior to storage and then removed
from the package, blotted dry and reweighed. The percent ribeye purge was determined
by the following equation:

weight before stogge — weight after storage x 100
weight before storage

Cooked product yield

On each storage day (0, 14, 28 and 42), steaks were evaluated for cooked product
yield. Steaks were weighed and then cooked on a Taylor clamshell grill as described in
chapter 4. Steaks were weighed after cooking and allowed to drip for 5 min. Percent
cook yield was calculated as follows:

cooked steakL weight x 100
steak weight before cooking

98

Steak purge

Percent steak purge was determined on day 7 of the retail meat case study for
each storage day (0, 14, 28 and 42) after being stored in a refrigerated (1 °C) retail
display case on foam trays overwrapped with PVC film for 7 days. The steaks were
weighed prior to storage and then removed from the foam trays, blotted dry and
reweighed. The percent steak purge was determined by the following formula:

weight before storage — weight after storage x 100
weight before storage

Thiobarbituric acid reactive substances (TBARS)

On day 0 and 7 of the retail meat case shelf-life study for each storage day (0, 14,
28 and 42), TBARS analysis was conducted as an indicator of oxidative rancidity. Four
replicates were run for each sample according to methods established by Tarladigis and

others (1960) and Zipser and others (1962) as modified by Rhee (1978).

Proximate composition

On each storage day (0, 14, 28 and 42), a 1.3 cm steak was cut from each ribeye
half and one half of the steak was used for proximate composition. Samples were packed
in Whirl-PackTM bags (Fisher Scientific USA, Pittsburg, PA) and frozen at —10 °C for at
least 24 hr before processing. Frozen samples were cut into small pieces and ground with
dry ice into a fine powder using a Tekrnar grinder (Tekmar Co, Cincinnati, OH), packed
in opened Whirl-PackTM bags, placed in the freezer (—1 0 °C) for at least 48 hr (to

evaporate the dry ice), and sealed until further analysis.

99

Moisture, fat and protein contents of samples were determined according to
AOAC (2000) methods 950.46B (oven drying), 991.36 (Soxhlet ether extraction), and
992.15 (combustion method, nitrogen measurement, Model FP-2000, LECO Co., St.

Joseph, MI). Samples were analyzed in triplicate.

pH determination

On each storage day (0, 14, 28 and 42), a 1.3 cm steak was cut from each ribeye
half and one half of the steak was used for pH analysis. Sample (1 :t 0.1 g) was collected
in a 50-ml polycarbonate tube and 10-ml of deionized water was added. Samples were
homogenized using a Polytron mixer (PT-35, Kinematica, AG, Switzerland). The pH of
the homogenized samples was measured at room temperature (22 °C) using an Accumet

pH meter (AB 15, Fisher Scientific, Co., Pittsburgh, PA).

Melting point determination

The melting point of the “modified marbling” gel and beef subcutaneous ribeye
fat was determined using differential scanning calorimetry (DSC) according to ASTM
(1997) methods. Approximately 12 mg of sample was cut with a razor blade and placed
in a DSC pan bottom (T40625, TA Instruments, New Castle, DE) and covered with a
DSC pan lid (T40621, TA Instruments, New Castle, DE). The pan was placed in the
DSC (2010, TA Instruments, New Castle, DE) along with the control pan. The
experiments were conducted in triplicate at a heating rate of 10.1 °C per min from 20 °C

to 80 °C for the beef ribeye fat and 20 °C to 150 °C for the “modified marbling” gel.

100

Objective and subjective color evaluation

On day 0, 3, 5, and 7 of the retail meat case study for each storage day, objective
and subjective color measurements were taken. For objective color, a Minolta
Chromarneter CR-310 (Commission International D’Edairerage (CIE) L*a*b*, Ramsey,
NJ) with a 5.5 cm reading orifice was used to measure L* (lightness), a* (redness), and
b* (yellowness) values of ribeye steaks. Before measuring, the Chromarneter was
calibrated with polyvinyl chloride film on a standard white tile and then one reading was
taken of each steak.

For subjective color analysis, a color panel of four evaluators was used to
determine lean color and marbling score of the ribeye steaks. All color evaluations were
conducted under fluorescent lighting conditions. The lean color was determined on a 7-
point scale adapted from a beef lean maturity scale where 1=extremely bright cherry-red
and 7=extremely dark red (AMSA 2001). The marbling score was evaluated using beef
marbling cards adapted from the official United States standard for grades of carcass beef

(USDA 1997).

Scanning electron microscopy (SEM)

Electron micro graphs were used to study the microstructure and to elucidate the
relationship of the “modified marbling” solution and the muscle proteins. First the
microstructure of the “modified marbling” gel was looked at to determine the structure of
the non-meat ingredients before looking at them in the meat matrix. After the solution
was allowed to gel for 24 hr at 4 °C, small pieces (1.0 x 2.0 x 2.0 mm) of the gel were cut

and prefixed for 2 hr at 22 °C in 4.0% glutaldehyde solution buffered with 0.1 M sodium

101

phosphate pH 7.0. After the prefixation, the gels were postfixed at 4 °C overnight in
0.1% osmium tetraoxide solution. Fixed gels were then rinsed with 0.1 M 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 15 min changes in 100% ethanol. Dehydrated
gels were dried using a carbon dioxide critical point dryer (Balzers CPD, FL-9496,
Balzers, Liechtenstein) and then mounted on stubs and coated with a 25-30 nrn gold layer
in an ion-sputter coater (Emscope laboratories Ltd., Ashford, Kent, UK).

Meat samples injected with the “modified marbling” solution and control samples
were prepared by cutting 2.54 cm cubes and freezing in liquid nitrogen. The samples
were placed between two fiberglass plates and pounded with a rubber mallet in order to
produce sample pieces small enough to analyze. Samples were prefixed for 24 hr in 4%
glutaldehyde buffered with 0.1 M sodium phosphate pH 7.0 at 4 °C. Samples were then
rinsed thoroughly with 0.1 M sodium phosphate buffer (7.0) and dehydrated in a graded
series of ethanol (25, 50, 75, 95%) for 20 min each, followed by three 15 min changes of
100% ethanol. Afier dehydration, samples were dried using a carbon dioxide critical
point dryer, mounted on stubs and coated with a 25-30 nm gold layer in an ion-sputter
coater. The microstructure of both the gels and meat samples was observed using a
scanning electron microscope (JOEL, Model J SM-6400V, version 96-2, Tokyo, Japan) at

a 15 mm working distance using an accelerating voltage of 12 KV.

Experimental design

The experimental design used was a split plot design with treatment as the whole

plot factor and storage day as the split plot factor. The retail meat case shelf-life study

102

was a repeated measurement design. The effect of injecting the “modified marbling”
solution into ribeye rolls on quality attributes (cooked product yield, purge loss, color,
pH, proximate composition, and lipid oxidation) of the ribeye steaks was analyzed using
the Proc Mixed procedure of the Statistical Analysis System (SAS User’s Guide, Version
8.2, Cary, NC: SAS Institute, Inc., 2002). Difference among attribute means was
determined with a predetermined level of significance (P<0.05) using Tukey’s Least

Significant Difference procedure.

103

Results and Discussion

Ribeye purge, cooked product yield, steak purge and TBARS values of ribeye rolls

The ribeye purge, cooked product yield, steak purge and TBARS values are
shown in table 4.1. Afier the ribeye rolls went through the designated time of storage, the
ribeye purge was measured and it significantly (P<0.05) increased (fiom 1.2 to 2.9%) as
the length of storage increased. An increase in purge over storage time is not unusual and
often expected. An increase in purge was also seen in a study by Goddard and others
(1996) where a solution of 2% lactic acid and 2% acetic acid was sprayed on beef strip
loins in order to improve the chemical, physical and microbial attributes. They found that
the amount of purge significantly increased with storage. This was likely caused by the
degradation of muscle proteins, possibly due to the pH nearing the isoelectric point of the
protein allowing the bound water to be released as purge.

The cooked product yield and steak purge did not change across storage days.
The TBARS values, however, significantly increased (P<0.05) as the length of storage
increased from 0.4 to 0.9 mg malonaldehyde/kg of sample. This could be due to the
treatment effect in which the injected ribeye rolls were more prone to lipid oxidation than
the Low or Average Choice ribeye rolls and this effect was probably due to not using
antioxidants in the injected Select treatment. The use of antioxidants to decrease lipid
oxidation was seen in a study by St. Angelo and others (1991). They infused 0.3M
calcium chloride and 1% sodium ascorbate or 0.25% maltol into freshly slaughtered
lambs to look at the differences in tenderization and warmed-over flavor. They found

that with storage, the lamb patties from the lambs infused with either maltol or sodium

104

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105

ascorbate used as antioxidants in addition to the calcium chloride had significantly lower
TBARS than the lamb patties from lambs infused with only the calcium chloride.

For ribeye purge, the injected Select was significantly higher (P<0.05) than the
Average Choice control and higher but not significantly different than the Select control
and the Low Choice control. The significant difference in ribeye purge for the injected
Select was expected since there was an average of 9.75% solution added to the ribeye
rolls. This was also seen in a study by Milligan and others (1997), where a solution of
CaClz was injected into USDA Standard beef inside rounds at 5%. They found that the
purge was significantly greater for the CaClz injected roasts than for the control roasts.

When the steak was cooked, the injected Select had significantly lower (P<0.05)
product yield than the Average Choice control and lower but not significantly different
than the Select control and the Low Choice control. This difference would be expected
since an average of 9.75% solution was added to the ribeye rolls. This was also seen in
the study by Milligan and others (1997). In this study, control roasts had 3.9% less
cooking loss than the roasts injected with the CaClz. There was no difference seen
between the treatments for steak purge (steaks overwrapped with PVC film stored in the
retail meat case for 7 days.) Even though an average of 9.75% “modified marbling”
solution was added to the injected Select, it is speculated that the solution formed such a
strong gel that little liquid came out causing there to be no difference in uncooked steak
purge. Although the injected Select was significantly lower (P<0.05) in cooked product
yield, the differences were not large.

For TBARS values across treatments, the injected Select was significantly higher

(P<0.05) than all the controls. This was probably due to the addition of beef tallow,

106

which did not contain an antioxidant. Beef tallow without antioxidant was used to keep it
consistent with the controls since an antioxidant was not added to any of the controls.
Cannon and others (1995) studied the effect of vitamin E on lipid oxidation.

Longissimus chops from pigs given either 100 mg vitamin E/kg diet or not supplemented
with vitamin B were evaluated for lipid oxidation, microbial growth, sensory
characteristics, cooking/storage losses and reheating losses. The TBARS values were

significantly lower for the vitamin E supplemented chops than for the control chops.

Proximate composition and pH of ribeye rolls

Table 4.2 shows proximate composition and pH values of injected and control
ribeye rolls. The moisture, fat and protein content differed among storage days but there
was not a consistent increase or decrease. This difference could be due to the treatment
effect in which the proximate composition of the injected Select was different from the
Low Choice and Average Choice controls since all ribeye rolls (injected Select, Select,
Low Choice and Average Choice controls) were analyzed together on each storage day.
The pH of the ribeye rolls decreased from 5.6 to 5.1 as the length of storage increased.
This was most likely due to bacteria growth by spoilage organisms since the pH sample
taken was on the outer surface of the ribeye roll half. This was also seen in a study by
Inglis and others (2004) where a meat-based entomophage diet either with or without
antibacterial agents was analyzed for spoilage microorganisms over time. It was found
that the pH of diets not containing antibacterial agents decreased rapidly over time and

was due to an increase in spoilage bacteria.

107

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The moisture content of the injected Select was significantly higher (P<0.05) than
the Low Choice and Average Choice controls and higher but not significantly different
than the Select control. For fat content, the Average Choice control was significantly
higher (P<0.05) than the injected Select and the Low Choice control was higher but not
significantly than the injected Select. Savell and others (1986) also found that the
amount of chemical fat in uncooked longissimus lumborum muscle of beef carcasses
varied across degrees of marbling from 10.42% in Moderately Abundant (USDA Prime
quality grade) to 1.77% in Practically Devoid (USDA Standard quality grade). For
protein content, the injected Select was significantly lower (P<0.05) than the Select
control and the Low Choice control and lower but not significantly than the Average

Choice control. There was no difference in pH across treatments.

Endothermic peaks of beef ribeye fat and “modified marbling” gels

The average melting point of the three subcutaneous beef ribeye fat readings
(Figure 4.1.a) was 35.6 °C, which is a little lower but close to the melting point recorded
for beef tallow (40-48 °C) (Dugan 1987). It was also stated that beef fat melting point
can vary depending on several conditions (breed, age sex and management style of the
animal, type of fat, etc) thus the value of 35.6 °C is reasonable. The melting point
temperature reported by Dugan (1987) was for beef tallow and not subcutaneous fat.
Subcutaneous ribeye fat was used in this study.

The average endothermic peak seen from the three readings for the “modified

marbling” gel was 121.8 °C (Figure 4.1 .b). Since the gel was approximately 90%

109

Figure 4.1. Endothermic peaks of beef ribeye fat and “modified marbling” gels.

4.1.a. Melting point of subcutaneous beef ribeye fat.

 

 

 

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moisture, it was most likely that the water in the solution vaporized at this temperature.
This is a higher vaporization temperature than normally seen for water but the
vaporization temperature increases when it is in a solution (Y an 2000). Since the gel did
not melt and the water did not vaporize until it reached 121.8 °C, at 71 °C, the endpoint
cooking temperature of a steak, the “modified marbling” was still a strong gel and this

may have an effect on the sensory properties.

Objective and subjective color measurements of ribeye rolls

The objective and subjective color measurements are shown in table 4.3. Across
storage days, there was no difference in D“ values. The a* values for steaks became
significantly (P<0.05) less red as the length of storage increased and the b* values
became significantly (P<0.05) less yellow over time. It is reasonable that these changes
could occur during storage. There were no differences in subjective color and marbling
scores across storage days.

Across treatments, for L* value, the injected Select was higher than the Select
control but this difference was not significant. This higher value of reflectance for the
injected Select would probably be due to the amount of solution injected into the ribeye
roll. There was no difference between treatments for a* and b* values. There was also
no difference in subjective color scores across treatments. As far as marbling score, the
Average Choice control and the Low Choice control were significantly higher than the
injected Select and the Select control, which corresponds to the higher proximate fat

content of the Average and Low Choice controls. There was no difference between the

111

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injected Select and Select control for marbling score. The “modified marbling” could not
be seen in the injected Select ribeye steaks during retail display. However, during a
preliminary study, where ribeye rolls were injected with the solution in order for the
injector settings to be modified to acquire the marbling pattern desired, the “modified
marbling” was visible. During the study though, there were problems with the injector
not being designed to handle the solution viscosity so the mixing time of the solutions
were decreased and used for all replicates. This may have altered the properties of the
solution since the ingredients were not as thoroughly mixed as in preliminary studies and
the “modified marbling” was not visible in the injected Select. This problem would
easily be solvable by modifying the solution manufacturing procedures and processing
parameters in order to produce visible “modified marbling”. As the technology is
adopted, there also may be an opportunity for injection equipment to be designed to

handle higher viscosity injection solutions.

Objective and subjective color measurements and TBARS values of ribeye steaks in
the retail meat case shelf-life study

The objective and subjective color measurements and TBARS values for the retail
meat case shelf-life study are shown in tables 4.4 and 4.5. The stande errors of the
mean (SEM) are different across retail days due to the repeated measurement design.

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values significantly decreased or became less red (P<0.05) as the length of storage in the
retail case increased during each storage period except for storage day 0. The b* values
also significantly decreased or became less yellow (P<0.05) as the length of storage in

the retail meat case increased. Jeremiah and Jones (1989) studied the effects of a 10 hr

113

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water spray during chilling on pork carcasses. At 24 hr postmortem, the loins were
removed and cut into 4 equal sized portions and vacuum packaged. One portion from
each loin was randomly assigned to each storage day (0, 14, 28 and 42). Upon removal
from storage, one chop was removed from the center of each portion, wrapped in oxygen
permeable film and placed in a retail display case. They found that the retail display
reduced the redness of all chops and reduced the yellowness of the chops stored for
extended periods. This could be due to the lighting in the retail case, which may discolor
the meat (Kraft and Ayres, 1954) or due to certain species of aerobic bacteria, which have
been shown to discolor meat by reducing the oxygen tension to the meat surface (Robach
and Costilow, 1962).

The subjective color scores significantly increased or became darker brown
(P<0.05) with time in the retail case for each storage day except for storage day 14. The
marbling scores were significantly different (P<0.05) across retail days for storage day 0
but there was no significant difference in the retail case for the other three storage days.
The TBARS values were significantly higher (P<0.05) on retail day 7 than on retail day 0
for each storage day. This could be due to the treatment effect in which the injected
ribeye rolls were more prone to lipid oxidation than the Low or Average Choice ribeye
rolls and this effect was probably due to not using antioxidants as previously described.
There was a significant (P<0.05) interaction between treatment and retail day for both a*
and TBARS values. For 3* value, this was probably a random interaction but for TBARS
values, this interaction is reasonable since there was a significant (P<0.05) difference
between retail days for each storage day and also a significant (P<0.05) difference

between treatments.

116

Scanning electron microscopy (SEM)

The images from the scanning electron microscopy analysis are shown in Figure
4.2. In the “modified marbling” solution (a), the non-meat ingredients tended to interact
with each other (b) in order to form a strong gel with the functional properties needed to
mimic the appearance of intramuscular fat. Once clear images were seen of the
“modified marbling” solution alone, the identification of the solution within the ribeye
roll (injected USDA Select) was attempted and images of the USDA Select control were
used to help identify the “modified marbling” solution injected in the meat. The solution
tended to lie within the meat proteins (c) and did not seem to interact with the meat
proteins ((1). This is in agreement with the differential scanning calorimetry (DSC)
analysis. It was found that the water in the gel did not vaporize until it reached 121.8 °C
so the solution probably formed a strong gel. Since it was such a strong gel, it makes

sense that the gel would lie within the meat proteins and not interact with them.

117

Figure 4.3. Scanning electron microscopy images.

1 11m ,

21) um

 

Figure 4.2. Scanning electron microscopy images. a) Image of the “modified
marbling” solution and b) close-up image of the solution. c) Image of “modified
marbling” solution in the ribeye roll (injected USDA Select) and d) close-up image
of the solution in the ribeye roll.

118

Conclusions

The developed “modified marbling” solution injected into whole muscle beef cuts
has potential for future applications. The injected Select had a significantly higher
(P<0.05) ribeye purge than the Average Choice. This significant difference in ribeye
purge would be expected since there was an average of 9.75% solution added to the
ribeye rolls. The injected Select had the highest percent moisture, lowest percent fat and
lowest cooked product yield. For TBARS values, the injected Select was significantly
higher (P<0.05) than all the controls, which is most likely due to the use of beef tallow
without antioxidants. There also were no significant differences in color scores between
treatments even with the amount of “modified marbling” solution in the injected Select.

This study was designed to verify the “modified marbling” solution in whole
muscle beef cuts by comparing the chemical attributes to controls. The results of this
study indicate that this innovative ingredient and processing technology has the potential
to improve lower quality beef cuts but more research is needed to improve the “modified
marbling” properties. One possibility is that the amount of fat in the solution could be
increased to achieve the benefits of flavor and hydrophobicity and to improve upon the

marbling appearance of the injected whole muscle beef cuts.

119

References

AMSA. 2001. Meat Evaluation Handbook: Lean Maturity Illustrations, p 21. American
Meat Science Association, National Cattlemen’s Beef Association and National
Pork Producers Council, Chicago, IL.

AOAC. 2000. Meat and meat products. In “Official methods of analysis of AOAC
International,” P Cunnifi (ed), p 1-23. AOAC International, Washington, DC.

ASTM. 1997. Standard test method for transition temperatures of polymers by thermal
analysis. In ASTM Designation. American Society of Testing Methods (ASTM)
Committee on Standards, West Conshohocken, PA.

Cannon J E, Morgan JB, Schmidt GR, Dehnore RJ, Sofos JN, Smith GC, Williams SN.
1995. Vacuum-packaged precooked pork from hogs fed supplemental vitamin E:

Chemical, shelf-life and sensory properties. J Food Sci 60:1179-82.

Dugan, Jr. LR. 1987. Meat animal by-products and their utilization. In “The Science of
Meat and Meat Products,” (3rd ed), Price JF, Schweigert BS (eds) ), p 519-20. WH
Freeman, San Francisco, CA.

Goddard BL, Mikel WB, Conner DE, Jones WR. 1996. Use of organic acids to improve

the chemical, physical, and microbial attributes of beef strip loins stored at —1 °C
for 112 days. J Food Prot 59:849-53.

Inglis GD, Cohen AC. 2004. Influence of antimicrobial agents on the spoilage of a meat-
based entomophage diet. J Econ Entomol 97:23 5-50.

Jeremiah LE, Jones SDM. 1989. The effects of spray chilling and vacuum packaged
storage on purge losses and the retail properties of pork. J Food Prot 52:473-6.

Kraft AA, Ayres J C. 1954. Effect of display case lighting on color and bacterial grth
on packaged fresh beef. Food Technol 8:290-5.

McKenna DR, Roebert DL, Bates PK, Schmidt TB, Hale DS, Griffin DB, Savell JW,
Brooks J C, Morgan J B, Montgomery TH, Belk KE, Smith GC. 2002. National
beef quality audit-2000: Survey of targeted cattle and carcass characteristics

related to quality, quantity, and value of fed steers and heifers. J Anim Sci
80:1212-22.

Milligan SD, Miller MF, Oats CN, Ramsey CB. 1997. Calcium chloride injection and

degree of doneness effects on the sensory characteristics of beef inside round
roasts. J Anim Sci 752668-72.

120

Rhee KS. 1878. Minimization of further lipid peroxidation in the distillation 2-
thiobarbutiric acid test of fish and meat. J Food Sci 43:1776-8.

Robach DL, Costilow RN. 1962. Role of bacteria in the oxidation of myoglobin. Appl
Microbiol 92529-33.

SAS Institute, Inc. 2002. SAS User’s Guide, Version 8.2. Cary, NC: SAS Institute.

Savell JW, Cross HR, Smith GC. 1986. Percentage ether extractable fat and moisture

content of beef longissimus muscle as related to USDA marbling score. J Food
Sci 512838, 40.

Savell JW, Cross HR. 1988. The role of fat in the palatability of beef, pork, and lamb. In
“Designing Foods: Animal Product Options in the Marketplace,” p 345-354.
National Academy Press, Washington DC

ST. Angelo AJ, Koohmaraie M, Crippen KL, Crouse J. 1991. Acceleration of
tenderization/inhibition of warmed-over flavor by calcium chloride-antioxidant
infusion into lamb carcasses. J Food Sci 562359-62.

Tarladigis GG, Wats BM, Younthan MT, Dugan L, Jr. 1960. Distillation method for the
quantitative determination of malonaldehyde in rancid foods. J Am Oil Chem Soc
37244-8.

USDA. 1997 . Official United States Standards for Grades of Carcass Beef. Agricultural
Marketing Service, USDA, Washington, DC.

Yan PS. 2000. Chemistry and physics of water. In “Food Chemistry: Principles and
Applications,” Christen GL and Smith J S (eds), p 16. Science Technology
System, West Sacramento, CA.

Zipser MW, Watts BM. 1962. Lipid oxidation (TBA) methods. Food Tech 16:102-4.

121

CHAPTER 5
SENSORY PROPERTIES OF WHOLE RIBEYE ROLLS INJECTED WITH THE
“MODIFIED MARBLING” SOLUTION COMPARED TO NON-INJECTED
CONTROLS
Abstract
Ribeye rolls (IMPS 112A) injected with the developed “modified marbling”
solution (5-7% targeted pick-up) were compared to control ribeye rolls in sensory
attributes. USDA Select, Low and Average Choice ribeye rolls were passed through the
automatic brine injector without injecting solution (controls). Ribeye rolls were
designated to 0, 14, 28, or 42 days of storage (1°C) and steaks (2.54 cm) were fabricated
on each storage day. Wamer—Bratzler shear force and trained sensory evaluation were
conducted on fabricated steaks from each treatment and control. The injected ribeye rolls
were higher (P<0.05) in beef fat flavor compared to the USDA Select control. However
a slight off-flavor was found (P<0.05) in the injected ribeye rolls. There were no
differences between the injected and control ribeye rolls for Warner-Bratzler shear force,
sensory tenderness or juiciness. This innovative ingredient and processing technology
has the potential to improve lower quality beef but more research is needed to improve
the “modified marbling” properties. One possibility is that the amount of fat in the
solution could be increased to achieve the benefits of both the non-meat ingredients and

fat.

Keywords: intramuscular fat, “modified marbling”, non-meat ingredients, solution,
injectable

122

Introduction

Palatability (j uiciness, tenderness, flavor) of beef cuts has been shown to be
influenced by the amount of marbling or intramuscular fat. Tatum and others (1982)
showed that marbling has a low but positive relationship on all beef palatability traits and
also found that 90% of the time steaks with Slight or higher degrees of marbling were
more desirable in tenderness, flavor and overall palatability. Longissimus thoracis steaks
from USDA High Choice carcasses tended to have higher tenderness, juiciness and beef
flavor intensity ratings than those from USDA Low Select carcasses (Wheeler and others
1999a)

Beef palatability is a major concern because when consumers are not satisfied
with the palatability of beef cuts their intent to purchase additional beef products may
decrease. The opportunity for the beef industry to generate revenue also decreases.
Savell and others (1987) reported that beef packers demand beef carcasses that grade
USDA Choice. When carcasses grade less than USDA Choice, a substantial price
discount usually has been paid. Savell and Cross (1988) found that beef cuts containing
3-7% intramuscular fat (marbling) are perceived by consumers to be acceptable in
tenderness, juiciness, flavor and overall palatability.

The deposition of intramuscular fat or marbling is influenced by many factors
such as breed, length of feeding, type of ration fed and management but it has been
shown that there is plenty of room for improvement in the amount of marbling or
intramuscular fat in order to enhance the palatability of the final beef product. The
palatability of whole muscle cuts fabricated from lower quality (less than USDA Choice)
beef carcasses may be improved through innovative non-meat ingredient and processing

technologies. Several different processing technologies have all ready been used to add

123

value to lower quality meat products including whole muscle cuts. The development of a
“modified marbling” solution from selective non-meat ingredients (sodium alginate, iota
carrageenan, whey protein isolate and modified food starch) that can mimic the properties
of intramuscular fat and can be directly injected into lower quality whole muscle beef
cuts may enhance its overall palatability by mimicking the organoleptic properties of fat
and having an appearance similar to that of marbling.

The objective of this study was to verify the properties of the “modified
marbling” solution in whole muscle beef cuts. To achieve this objective, USDA Select
ribeye rolls were injected with the solution, cut into steaks and sensory attributes were

compared to non-injected USDA Select, Low and Average Choice control ribeye steaks.

124

 

Materials and Methods
Processing Procedures
After the “modified marbling” solutions were manufactured and the ribeye rolls

were injected as described in chapter 3, they were processed as described in chapter 4.

Warner-Bratzler shear force

On each storage day, steaks were evaluated for Warner-Bratzler shear force.
Steaks were cooked on a Taylor clamshell grill as described in chapter 4. Steaks were
stored at 4 °C for 24 hr and six 1.3 cm cores were taken parallel to the longitudinal axis
of the fibers using a drill press- mounted corer. Cores were sheared perpendicular to the
fibers using a Wamer—Bratzler head on a TA-HDi Texture Analyzer (Texture

Technologies Corp., Scotsdale, NY).

Sensory evaluation

Sensory attributes of ribeye steaks were determined by a trained sensory panel on
each storage day. Six healthy panelists between twenty and sixty-five (four female and
two male) were trained according to AMSA (1995) and Meilgaard and others (1991). All
panelists had experience in sensory evaluation and were previously trained to evaluate
various meat products. Before product evaluation, three training sessions were held to
familiarize the panelists with the attributes and evaluation procedures. An 8 point
hedonic scale was used to measure 8 sensory attributes: juiciness, muscle fiber
tenderness, connective tissue, overall tenderness, off-flavor intensity, beef broth flavor
intensity, beef fat flavor intensity, and mouth coating. For juiciness, 1=extremely dry and

8=extremely juicy and for muscle fiber tenderness and overall tenderness, 1=extremely

125

tough and 8=extremely tender. For connective tissue, 1=abundant and 8=none and for
beef broth and beef fat flavor intensity, 1=extremely bland and 8=extremely intense. For
off-flavor intensity and mouth coating, 1=none and 8=abundant.

Training for beef broth and beef fat flavor intensity was conducted by using beef
fat and hamburgers made from ground beef with different percentages of fat (80/20,
85/15, and 90/10) and mouth coating was established by using set references (corn
starch=2, ground potato=4 and toothpaste=6, Meilgaard and others, 1991). J uiciness,
muscle fiber tenderness, overall tenderness, connective tissue, and off-flavor intensity
were well established attributes evaluated on a regular basis for whole muscle meat
products using the same trained sensory panel.

Sensory evaluations were conducted in a climate controlled sensory evaluation
room with partitioned booths and incandescent lights. The order of sample preparation
was randomized within each session to minimize positional bias and a 3 digit random
code was used to label the samples. Steaks were cooked on a Taylor clamshell grill as
described in chapter 4 and sample preparation included cutting 1.3 cm cubes fiom the
center portion of each steak and two cubes were placed in 2 oz. Soufflé cups and covered
with a lid. Souffle cups were placed in a 2 quart Pyrex® bowl with a lid and the bowl
was covered with warm towels to insulate the bowl and keep the samples warm. The
insulated bowl was placed in a cooler and transported to the sensory evaluation room.
Each sample was served to the panelists in their booths. Expectorant cups were provided
to prevent taste fatigue and distilled, de-ionized water, unsalted soda crackers and apple
juice were used to clean the palate between samples. The panelists were standardized

each day by evaluating a warm-up sample and discussing the results. Sixteen samples

126

were evaluated on each day and the day was divided into two sessions with a 15 min

break between each session.

Cooking study

A cooking study was conducted on ribeye steaks and attributes were evaluated by
a sensory panel in order to determine whether the lack of differences seen between the
injected and control ribeye steaks for juiciness and tenderness was due to the cookery
method and end-point temperature used. Four USDA Select ribeye rolls (112A) were
purchased from a local meat company (Popoff Quality Food Service, Lansing, MI). The
ribeye rolls were cut in half and two 2.5 cm steaks were cut from the middle (opposite of
the shoulder and loin end) of each half. The four steaks from each ribeye roll were
randomized to four treatments: clamshell grill (71 °C), clamshell grill (77 °C),
F arberware® grill (Model 455ND, Kidde Inc., Bronx, NY) (71 °C), and Farberware®
grill (77 °C). The steaks cooked on the clamshell grill were done using the procedure
described in chapter 4. The steaks cooked on the Farberware® grill (104.7 0C surface
temperature) were laid on the surface of the grill and the temperature of the steak was
monitored using a copper constantan thermocouple (0.051-cm diameter, 15.2 cm length;
Omega Engineering Inc., Stamford, CT) inserted into the geometric center of the steak.
The steaks were cooked to 40 °C and then turned and cooked to the final desired
temperature.

Sample preparation was conducted in the same manner as previously described.
J uiciness, muscle fiber tenderness, overall tenderness, and connective tissue were

evaluated using the same scale and testing procedures. The methods of cookery and

127

Pv-rrr '

endpoint temperatures used were chosen since they resembled consumer’s choice of

cookery and desired endpoint temperatures used in their homes.

Experimental design

The experimental design used was a split plot design with treatment as the whole
plot factor and storage day as the split plot factor. The effect of injecting the “modified
marbling” solution into ribeye rolls on quality attributes (shear force and sensory
attributes) of the ribeye steaks was analyzed using the Proc Mixed procedure of the
Statistical Analysis System (SAS User’s Guide, Version 8.2, Cary, NC: SAS Institute,
Inc., 2002). Difference among attribute means was determined with a predetermined

level of significance (P<0.05) using Tukey’s Least Significant Difference procedure.

128

Results and Discussion
Warner-Bratzler shear force and sensory attribute values of ribeye rolls

The Warner-Bratzler shear force and sensory attribute values for the ribeye rolls
are shown in Table 5.1. The Warner-Bratzler shear force values did not differ across
storage days. The juiciness values decreased from 5.3 to 4.9 and the overall tenderness
values decreased from 5.8 to 5.7 as the length of storage increased but the decreases were
small and not significantly different. The juiciness values were similar to the percent
ribeye purge presented in chapter 4, which also decreased as the length of storage
increased so since there was not as much liquid within the ribeye rolls, the perceived
juiciness was lower. There were not any differences in muscle fiber tenderness and the
amount of connective tissue over storage days.

The off-flavor intensity became significantly (P<0.05) higher from 1.1 to 1.3 as
the length of storage increased (Table 5.1). However, these differences in values were
small. This corresponded to the TBARS values (chapter 4) during storage time, which
could be due to the treatment effect in which the injected ribeye rolls were more prone to
lipid oxidation than the Low or Average Choice ribeye rolls and this effect was probably
due to not using antioxidants in the injected Select treatment. Because the differences
were small, these results may occur during storage at 1 °C in a vacuum package. There
was no difference in mouth coating across storage days. The beef flavor intensity
decreased from 4.3 to 3.9 and beef fat flavor intensity also decreased from 3.8 to 3.5 as
the length of storage increased but the decrease was not significantly different and the
values were again small in scale. The beef flavor added to the solution may have

decreased in intensity over time (42 days).

129

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Across treatments, there were no differences in Warner-Bratzler shear force, or
juiciness, muscle fiber tenderness, amount of connective tissue and overall tenderness
measured by sensory analysis. The “modified marbling” had neither a positive or
negative effect on these attributes. The lack of change across treatments for tenderness
values may have been due to the method of cookery and the end-point temperature used.
The clamshell grill cooking method may be less abrasive on the steaks than open grill
cookery methods, which most consumers use in their home. The end-point temperature
of 71 °C did not show a difference but if a higher end—point temperature (77 °C) would
have been used, perhaps a difference may have been detected. This temperature end-
point may be more what the consumers would choose when cooking steaks in their
homes. Also, the lack of difference for tenderness across treatments could have been
attributed to passing all control ribeye rolls through the injector (single pass without
solution). Even though the controls were passed through the injector to minimize bias
when evaluating tenderness, this could have had a contrary effect and caused the
tenderness values of the injected and control ribeye rolls to be similar. It was reported in
chapter 3 that the injector needles did not affect tenderness. However, the level of
significance for this conclusion was P=0.062.

The lack of difference seen in juiciness values was probably due to the ability of
the solution to form a strong gel so that little liquid was released. The solution did not
actually melt when the steak was cooked. Analysis using differential scanning
calorimetry (DSC) found that the water in the solution vaporized and not until it reached
121.2 °C (chapter 4), which is far beyond the endpoint cooking temperature (71 °C) used

when cooking steaks. When the steaks were cooked, the solution did not melt and release

131

water but probably held together as a strong gel. This is a possibility of why there was no
difference seen in juiciness between the injected and control ribeye steaks.

There also was no difference among treatments for mouth coating. The “modified
marbling” did not affect the mouth coating attribute. For beef flavor intensity, the
injected Select was higher but not significantly different than the Select control and the
Low Choice control. For the beef fat flavor intensity, the injected Select was
significantly higher (P<0.05) than the Select control and higher but not significantly
different than the Low Choice and Average Choice controls. Thus the addition of the
“modified marbling” solution did not have an unfavorable effect on these attributes but
even had a positive effect on the beef fat flavor intensity. This was probably due to the
addition of beef flavor to the solution. The injected Select was also significantly
(P<0.05) higher than the controls in off-flavor intensity, which corresponds to the
TBARS values (chapter 4) and was probably due to not adding antioxidants to the

“modified marbling” solution.

Cooking study of ribeye steaks

To further verify whether the lack of differences seen among treatments for
juiciness and sensory and Warner-Bratzler shear force tenderness values was due to the
cookery method and end-point temperature used, a cooking study was conducted on
USDA Select ribeye rolls. This possibility was stated when discussing the absence of
tenderness change previously observed. There were no differences seen for juiciness,

muscle fiber tenderness, amount of connective tissue or overall tenderness between the

132

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133

two cookery methods (clamshell grill and F arberware® grill) or the two end-point
temperatures (71 °C and 77 °C) (Table 5.2).

The sensory scores for all four attributes were lower but not significantly different
at 77 °C (well done) compared to 71 °C (medium). This observation was also made by
Wulf and others (1996). They studied the effects of animal age, marbling score,
calpastatin activity, subprimal cut, calcium injection and degree of doneness on the
palatability of steaks from Limousin steers. They found that the degree of doneness had a
significant effect (P<0.05) on taste panel tenderness and juiciness scores. The steaks
were less tender and juicy as the degree of doneness increased. It also has been shown
that palatability of meat cooked by dry methods is influenced more by temperature than
by marbling. Cooking losses are increased as end-point temperature increases and the
greater cooking losses decrease meat juiciness. High cooking losses along with protein
hardening and toughening (induced by high cooking temperatured (72-74 °C)) reduces
meat tenderness (Aberle and others 2001).

The sensory scores for all four attributes were lower for the clamshell grill than
the Farberware® grill but not significantly. This showed that there was not a protective
effect with the clamshell grill as previously stated. The clamshell grill may still have
been less abrasive to some extent but the steaks grilled on the Farberware® grill,
probably had higher flavor intensity than the steaks grilled on the clamshell grill. This is
most likely due to the open grilling and the stronger flavor intensity may also lead to the

higher juiciness scores.

134

 

Conclusions

The “modified marbling” solution injected into whole muscle beef cuts has
potential for fixture applications. The injected Select ribeye rolls were higher in beef fat
flavor compared to the USDA Select control, which was probably due to the addition of
beef flavor. The “modified marbling” addition had no effect on other traits. There was
however, a slight off-flavor found in the injected Select ribeyes rolls, which is most likely
due to the use of beef tallow without antioxidants. There was no significant difference
between the injected and control ribeye rolls in Warner-Bratzler shear force, sensory
tenderness or sensory juiciness. The similar tenderness values may be attributed to
passing all the control ribeye rolls through the injector (single pass without solution) to
minimize bias when evaluating tenderness. Similar juiciness values may be the result of
the ability of the solution to form a strong gel so that little liquid was released.

This study was designed to verify the “modified marbling” solution in whole
muscle beef cuts by comparing the sensory attributes to controls. The results of this
study indicate that even though there is potential for the “modified marbling” solution to
improve low quality whole muscle beef cuts additional research is needed. One
possibility is to increase the amount and type of fat used in the solution to achieve the
benefits of flavor, hydrophobicity as well as to improve upon the tenderness and juiciness

of the injected whole muscle beef cuts.

135

 

 

References

Aberle ED, Forrest J C, Gerrard DE, Mills EW. 2001. Growth and development of carcass
tissues; Principles of meat processing. In “Principles of Meat Science,” (4th ed), p
238. Kendal/Hunt Publishing Co, Dubuque, IA.

AMSA. 1995. Research Guidelines for Cookery, Sensory Evaluation and Instrumental
Measurements of Fresh Meat. American Meat Science Association and National
Livestock and Meat Board, Chicago, IL.

Meilgaard M, Civille GV, Carr BT. 1991. Sensory Evaluation Techniques. CRC Press,
Boca Raton, FL.

SAS Institute, Inc. 2002. SAS User’s Guide, Version 8.2. Cary, NC: SAS Institute.

Savell JW, Branson RE, Cross HR, Stiffler DM, Wise J W, Griffin DB, Smith GC. 1987.
National consumer retail beef study: Palatability evaluations of beef loin steaks
that differed in marbling. J Food Sci 52:517-9, 32.

Savell JW, Cross HR. 1988. The role of fat in the palatability of beef, pork, and lamb. In
“Designing Foods: Animal Product Options in the Marketplace,” p 345-54.
National Academy Press, Washington DC.

Tatum JD, Smith GC, Carpenter ZL. 1982. Interrelationships between marbling,
subcutaneous fat thickness and cooked beef palatability. J Anim Sci 54:777-84.

Wheeler TL, Shackelford SD, Koohmaraie M. 1999a. Tenderness classification of beef:
IV. Effect of USDA quality grade on the palatability of “tender” beef longissimus
when cooked well done. J Anim Sci 77:882-8.

Wulf DM, Morgan JB, Tatum JD, Smith GC. 1996. Effects of animal age, marbling
score, calpastatin activity, subprimal cut, calcium injection, and degree of

doneness on the palatability of steaks from Limousin steers. J Anim Sci 74:569-
76.

136

 

Recommendations for future research

The main goal of this study was to develop a “modified marbling” solution from
non-meat ingredients that mimicked the function and appearance of intramuscular fat in
order to provide a fat substitute for whole muscle beef cuts. In study 1, non-meat
ingredients were used for the “modified marbling” solution since it was thought that they
would provide easy mixing and injecting of the solution and in addition give a health
benefit to the consumer. Due to encountered problems of muscle pigments being
absorbed into the hydrophilic “modified marbling” solution, a small amount (3.0%) of fat
(beef tallow) was added in study 2. Future research should optimize the amount of fat
used in the solution to achieve the benefits of flavor and hydrophobicity and to hopefully
improve upon the tenderness, juiciness and marbling appearance of the injected whole
muscle beef cuts.

Continued research is being conducted in this area by focusing on developing the
“modified marbling” solutions from different combinations of lipids. The mixing and
injecting techniques used are similar to those utilized in this study and due to the choice
of lipids, the solution is liquid enough to inject and solidifies once in the meat. This
research is looking at using only lipids to develop the “modified marbling” solution but
there may be possibilities to utilize both lipids and the non-meat ingredients used in this
study to gain the benefits of both. Future research is needed to determine the percentages
of lipids and non-meat ingredients to use to obtain the optimal solution in both function
and appearance.

When developing the solution, all functional properties should be looked at before

incorporating it into the meat. In study 3 it was determined that the solution did not melt

137

but that the water in the solution vaporized. The water did not vaporize until it reached
121.2 °C, which is far beyond the endpoint cooking temperature of steaks (71 °C) and is
one possibility of why there was no difference seen in juiciness between the injected and
control ribeye steaks. At the endpoint cooking temperature of the steaks injected with the
solution, the “modified marbling’ solution was probably still a strong gel and not in
liquid form which should have increased the juiciness.

The results from study 4 indicate that even though the sensory tenderness,
Wamer—Bratzler shear force and sensory juiciness of whole muscle beef cuts injected
with the “modified marbling” solution developed from non-meat ingredients (SA, IC,
WPI and MF S) were not significantly improved from the controls, there is potential for
this solution. The fact that the “modified marbling” solution was significantly higher in
beef fat flavor as measured by a trained sensory panel (probably attributed to the addition
of beef flavoring) gives the solution promise. Also, the similar tenderness and juiciness
values when comparing the injected and non-injected ribeyes may be attributed to passing
all the control ribeye rolls through the injector (single pass without solution) to minimize
bias when evaluating tenderness. The fact that there was just “no difference” found
between the injected ribeye rolls and the controls and not a significant inferior effect
gives potential to the solution.

Another opportunity for future research includes using the “modified marbling”
solution as a carrier for beneficial ingredients. Possibilities include anti-microbials,
antioxidants, and different marinades/flavors. These ingredients could be mixed into the

solution and injected into meat cuts. This study was a new product development project

138

 

and from this project several technologies and applications were discovered, which

provided a strong beginning to this area of research.

 

139

 

 

APPENDICES

140

 

Appendix 1: Ingredients evaluated for “modified marbling” solution

 

Ingredient

Observation

 

Modified food starch (dry blend)

Konj ac

Soy protein isolate

Methylcellulose

Kappa Carrageenan

Iota Carrageenan

Modified [ire-gelatinized food starch
Whey Protein Isolate

Sodium Alginate

Very thin solution, completely separated
out after gelling

Tan color

Tan color, strong aroma

Gels when solution is heated not cooled
Dark tan color, thin gel

Light tan color, thick gel

White color

Translucent color, disperses well in
solution

Light tan color, thick gel

 

141

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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143

 

Appendix 3: Bench top “modified marbling” solution manufacturing procedures

 

1. Add the appropriate amount of water (22 °C) to 946.4-ml lidded glass jars.

2. Add sodium tripolyphosphate.

3. Mix with 4—blade mixing head: 2-blades perpendicular to the shaft and 2-blades
parallel to the shafi attached to a drill (Model 6220, SB Power Tool Co., Chicago,
IL). Mix for two min.

4. Add the sodium alginate mixed with the vegetable oil for hydration and mix for 2
min.

5. Add the iota carrageenan and mix for 2 min.
6. Add whey protein isolate and mix for 2 min.
7. Add modified food starch and mix for 2 min.
8. Mix calcium sulfate with water, add to the mixture and mix for 2 min.

9. Repeat steps for each solution.

144

 

Appendix 4: Viscosity determination

 

Viscometer: Brookfield viscometer (Model HBTD, Brookfield Engineering
Laboratories, Inc., Stoughton, MA)

1.

2.

10.

11.

12.

13.

14.

Turn water bath on and turn knob to desired temperature reading (30 °C).
Turn power button of Brookfield viscometer to on.

Turn speed dial on the side of the viscometer to the desired speed (100).
Fill the metal tube with 18.6-g of solution.

Insert the tube into the viscometer from the bottom and then twist to lock the tube into
place.

Plug the cord of the tube into the temperature recorder.

Insert the desired spindle into viscometer by setting into the tube of solution and then
twisting into place.

Make sure the viscometer reads 0.0. If it does not, then turn the zero knob until it
does.

When the temperature recorder reads the desired temperature, turn the motor button
to on.

Wait for the viscosity reading to become constant and record reading.
Turn off motor button.

Unscrew spindle and take out metal tube.

Clean out metal tube.

Repeat as necessary.

145

Appendix 5: Objective color measurements (CIE L', a. and b. values)

 

Color meter: Minolta Chromarneter CR-310 (Commission D’Edairerage (CIE) L*a*b*,
Ramsey, NJ)

1.

2.

Calibration:

Turn power switch to on.
Press Calibrate.

If the displayed color space in not ny, press Color Space Select repeatedly to change
to ny color space.

Check that indexes are set as desired by pressing Index Set and use the scroll key to
advance through the indexes.

Use the arrow keys and Y/N to change settings is necessary.
Set the “Multi Cal” index to “N”.

Set the calibration channel to 00.

Set the “Multi Measure” to “Y”.

Place the tip of the measuring head flat against the surface of the white calibration
plate.

10. Press the measuring head’s measuring button.

11.After 5 3, “CAL” in the display will be replaced by “End”.

12. Calibration is now completed.

Sample measurement:

1.

2.

3.

4.

Press Color Space Select to set desired color space (L*, a*, b*).
Place tip of measuring head flat against the specimen surface.
Press the measuring head’s measuring button and measured data will be displayed.

Use Kimwipes to clean measuring orifice between measurements.

146

Appendix 6: Water-holding capacity determination

 

8.

9.

. Weigh a 50-ml polycarbonate tube.

. After the solution has gelled for 24 h at 4 oC, remove the gel from the jar by cutting

around the edge of the jar.

Cut the gel into small pieces.

Place approximately IO-g of sample into polycarbonate tube and record weight.
Place tubes in appropriate centrifuge rotor and place in centrifuge.

Centrifuge at 4 °C at 40,000 x g for 30 min.

. Remove tubes from centrifuge.

Pour off supemate.

Weigh tube and gel.

10. Subtract weight of tube in order to determine the weight of the centrifuged gel.

11. Water-holding capacity is determined by the following formula:

Water-holding capacity = weight of gel after centrifugation x 100

weight of gel before centrifugation

147

 

Appendix 7: Water-holding capacity over time determination

 

1. Lay a piece of filter paper inside the bottom of a petri dish and weigh the filter paper
and petri dish together.

2. After the solution has gelled for 24 h at 4 °C, remove the gel from the jar by cutting
around the edge of the jar.

3. Cut the gel into approximately 2.5 x 2.5 x 1.3-cm pieces.

4. Place the sample cube on the filter paper and weigh the petri dish, filter paper and gel
cube.

5. Cover with the petri dish top.

6. Store at 22 0C for 2 h.

7. Remove the cube from the filter paper by scraping away all gel particles.
8. Weigh the filter paper and petri dish.

9. Water-holding capacity over time is determined by the following formula:

Water-holding capacity over time = weigh—tofiel after storage at 22 °C for 2 hr x100
weight of gel before exposure to elevated temp

148

Appendix 8: TA—HDi gel strength settings

 

Texture Analyzer: TA-HDi Texture Analyzer
Texture Technologies Corporation, Scarsdale, NY

Software: Texture Expert Version: 1.22

TA-HDi Settings:
Test Mode:

Option:
Pre-Test Speed:
Test Speed:
Post-Test Speed:

Pre-Travel Distance:

Compression Distance:

Trigger Type:

Data Acquisition Rate:

Attachment/Accessory:

Measure Force in Compression
Return to Start

5.0-mm/s

1.7-mm/s

IO—mm/s

51 .O-mm

12-mm

Return

200 pps

TA-lO; 13-mm AOAC acrylic cylinder, 35-mm tall

S-kg load cell

TA-90; Heavy duty platform

149

Appendix 9: Ingredient combinations of non-meat ingredients using central

composite design

 

 

Treatment

 

NNNNNI—dr—fit—iI—fih—‘r—ip—hu—bv—fip—t
AuNHOomqomhwmwo‘omNO‘M-kaNfi

25

Non-meat Ingredientsa

 

 

Sodium Iota Whey Modified
Alginate Carrageenan Protein Food

Isolate Starch
0.3125‘1 0.3125 0.3125 0.4375
0.3125 0.3125 0.4375 0.3125
0.3125 0.4375 0.3125 0.3125
0.3025 0.4375 0.4375 0.4375
0.4375 0.3125 0.3125 0.3125
0.4375 0.3125 0.4375 0.4375
0.4375 0.4375 0.3125 0.4375
0.4375 0.4375 0.4375 0.3125
0.3125 0.3125 0.3125 0.3125
0.3125 0.3125 0.4375 0.4375
0.3125 0.4375 0.3125 0.4375
0.3125 0.4375 0.4375 0.3125
0.4375 0.3125 0.3125 0.4375
0.4375 0.3125 0.4375 0.3125
0.4375 0.4375 0.3125 0.3125
0.4375 0.4375 0.4375 0.4375
0.2500 0.3750 0.3750 0.3750
0.5000 0.3750 0.3750 0.3750
0.3750 0.2500 0.3750 0.3750
0.3750 0.5000 0.3750 0.3750
0.3750 0.3750 0.2500 0.3750
0.3750 0.3750 0.5000 0.3750
0.3750 0.3750 0.3750 0.2500
0.3750 0.3750 0.3750 0.5000
0.3750 0.3750 0.3750 0.3750

 

a = all ingredient values indicate percentage of ingredient used in formulation.

150

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151

 

 

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152

 

 

Appendix 11: Commercial batch “modified marbling” solution formulation

 

 

Ingredient f/_o g E
Sodium alginate 1.0 681.0 1.5
Sodium Tripolyphosphate 68.1

Vegetable oil 1362 3.0
Calcium Sulfate 1362 3.0
Water 6810 15.0
Iota carrageenan 0.43 75 297.9

Whey Protein Isolate 1.5 1021.5 2.25
Modified Food Starch 0.375 255.3

Beef Tallow 3.0 2043 4.5
Beef Flavor 0.25 170.3

Water 54028.9 119.0/59.5
Total 68100 150

153

 

 

Appendix 12: Commercial batch “modified marbling” solution manufacturing
procedures

 

Temperature of water = approx. 18°C
Temperature of beef tallow = approx. 70°C

1.

Add tripolyphosphate to half the water and mix for 2 min at 1500 rpm using a
Rotostat mixer (Model 80XP63SS, Admix Inc., Londonderry, NH).

Add whey protein isolate and beef tallow and continue mixing at 1500 rpm.
Add sodium alginate hydrated in the vegetable oil.

Add the other half of the water and increase the speed to 2000 rpm.

Add the beef flavor, then the iota carrageenan and the modified food starch.

Mix the calcium sulfate in the water and homogenize to eliminate large particles, add
to the solution and increase speed to 3500 rpm.

Raise mixer head.

Total mixing time - 8 min

154

Appendix 13: Randomization of USDA Select ribeye rolls

 

Replicate 1

 

 

 

 

 

 

 

 

Day 0 Day 14
Anterior Posterior Anterior Posterior
inj Sa cont Sb cont S inj S

Replicate 2
Day 0 Day 14
Anterior Posterior Anterior Posterior
cont S inj S inj S cont S
Replicate 3
Day 0 Day 14
Anterior Posterior Anterior Posterior
inj S cont S cont S inj S
Replicate 4
Day 0 Day 14
Anterior Posterior Anterior Posterior
cont S ini S inj S cont S

 

 

 

 

 

 

 

Day 28 Day 42
Anterior Posterior Anterior Posterior
inj S cont S cont S inj S
Day 28 Day 42
Anterior Posterior Anterior Posterior
cont S inj S inj S cont S
Day 28 Day 42
Anterior Posterior Anterior Posterior
inj 8 cont S cont S inj S
Day 28 Day 42
Anterior Posterior Anterior Posterior
cont S ini S ini S cont S

 

a = injected USDA Select
b = control USDA Select

155

 

Appendix 14: Randomization of USDA Low Choice and Average Choice ribeye rolls

 

Replicate 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

USDA Low Choice Control USDA Average Choice Control
Ribeye 1 Ribeye 2 Ribeye 1 Ribeye 2
Anterior Posterior Anterior Posterior Anterior Posterior Anterior Posterior
Day 14 Day 0 Day 42 Day 28 Day 0 Day 14 Day 28 Day 42

Replicate 2
USDA Low Choice Control USDA Average Choice Control
Ribeye 1 Ribeye 2 Ribeye 1 Ribeye 2
Anterior Posterior Anterior Posterior Anterior Posterior Anterior Posterior
Day 0 Day 14 Day 28 Day 42 Day 14 Day 0 Day 42 Day 28
Replicate 3
QDA Low Choice Coptrol USDA Average Choice Control
Ribeye l Ribeye 2 Ribeye 1 Ribeye 2
Anterior Posterior Anterior Posterior Anterior Posterior Anterior Posterior
Day 14 Day 0 Day 42 Day 28 Day 0 Day 14 Day 28 Day 42
Replicate 4
USDA Low Choice Control USDA Average Choice Control
Ribeye 1 Ribeye 2 Ribeye 1 Ribeye 2
Anterior Posterior Anterior Posterior Anterior Posterior Anterior Posterior
_Day 0 Day 14 Day 28 Day 42 Day 14 Day 0 Day 42 Day 28

 

156

 

Appendix 15: Determination of ribeye purge

 

1. Weigh ribeye rolls and place in vacuum package bag.

2. Vacuum package and store at 1 °C for designated time of storage.

3. Afier designated time of storage, remove ribeye rolls from vacuum package bag.
4. Blot ribeye rolls dry and reweigh.

5. Percent purge loss is determined using the following calculation:

% purge loss = weight before storage — weight after storage x 100
weight before storage

157

 

Appendix 16: Cooked product yield determination

 

p—i

. Weigh steak before cooking.

2. After cooking to 71°C, allow steak to drip for 5 min on a metal rack.

b)

. Weigh cooked steaks.

4. Percent cook yield is determined by the following calculation:

% cook yield = Weight of cooked steak_ x 100
Weight of uncooked steak

158

Appendix 17: Determination of steak purge in retail meat display case

 

1. Weigh steaks on day 0 of each retail meat case study.
2. Place steaks on foam trays and overwrap with PVC film.

3. After 7 days of storage (1 °C) in the retail meat case, remove steaks from the foam
trays.

4. Blot steaks dry and reweigh.

5. Percent purge loss is determined using the following calculation:

% purge loss = weight before storage — weight after storaga x 100
weight before storage

159

Appendix 18: Thiobarbituric acid reactive substances (TBARS) determination

 

Rhee, KS. 1978. Minimization of further lipid peroxidation in the destillation 2-
thiobarbutiric acid test of fish and meat. J Food Sci 43:1776-1778.

Tarladigis, GG, Wats, BM, Younthan, MT, Dugan, L Jr. 1960. J Am Oil Chem 37:44-48.

Zipser, MW, Watts, BM. 1962. Lipid oxidation (TBA) methods. Food Technol
16(7):]02.

Reagents:

1.

TBA Reagent

Prepare the amount of TBA Reagent needed for your samples according to the table
below:

 

 

Thiobarbituric Acid m1 Vol. Water and Acid
1.4416 g 500 m1

0.7208 g 250 ml

0.5766 g 200 m1

0.2883 g 100 ml

0.1442 g 50 ml

Dissolve the Thiobarbituric Acid (Eastman Organic Chemicals) in the distilled water,
about 2/3 the total volume. Place flask in sonic cleaner (several minutes) and shake
occasionally until TBA is dissolved. Allow reagent to come to room temperature
then bring to volume. Store in cooler, may be kept for 2 days.

HCl Solution:
Make volume as needed; 1:2, HCl : H20 (v/v).
Antifoam (Thomas®, Swedeboro, NJ):

The use of antifoam may not be necessary depending on the product. Fish and egg
require antifoam while poultry does not.

Procedures:

1.

Add 10 g of diced sample to 100 m1 plastic bottle containing 50 ml distilled water
plus 10 pl antioxidant solution (Tenox 5 — food grade BHA+BHT).

160

 

10.

11.

12.

13.

14.

15

16.

Homogenize sample using Polytron mixer (PT-35, Kinematica, AG, Switzerland) on
speed setting 4 for 1 minute (Homogenized samples can be held in cooler if needed).

Into 500 ml extraction flasks, add glass beads (Fisher Scientific, Pittsburgh, PA),
homogenized meat sample, 2.5 m1 HCl solution, and if necessary antifoam.

Note: total volume is 50 m1+ 2.5 ml + 47.5 ml = 100 m1

Turn on condenser water and place graduated cylinders under spouts.

Connect extraction flasks to distilling tubes.

Turn heat control knobs to H1.
Distill and collect 50 ml of the distillate.

Transfer distillate to 50 ml centrifuge tubes, cap and hold in refrigerator for TBA
reaction (can be held for 18 hours).

Invert each test tube containing the 50 m1 distillate and pipette 5 ml into each of 2
tubes labeled “A” and “B”. Prepare 2 blanks by pipetting 5 ml distilled water into
both tubes labeled “A” and “B”.

Add 5 m1 of TBA Reagent into each tube containing 5 ml of sample and into both
blanks. Thoroughly mix each tube using Vortex mixer (American Scientific
Products, McGaw Park, IL).

Turn water bath on 100° C.

Place tubes in test tube rack and immerse into boiling water bath (model 9510
PolyScience, Sorvall Co., Niles, IL) for 30 minutes.

Turn on plate reader.

When the tubes are done heating in the water bath cool them in ice for at least 10
minutes.

.Mix each test tube with sample for 10 seconds using Vortex mixer (American

Scientific Products, McGaw Park, IL).

Pipette 200ul into well on plate (done in duplicate).

161

 

17. Place plate in plate reader and set up plate reader. Click on plate reader on computer,
click on experiment 1. Go to “set up” and set the appropriate wavelength and chose
end point analysis. Go to “template” and set up blank and unknowns according to
plate. (Wavelength to 530 for fresh meat) Read samples within 1 hour.

18. Convert % T to optical density and multiply by the constant 7.8 (7.6 for poultry) to
convert to mg malonaldehyde/ 1000 g of sample, i.e. TBA Number.

Absorbance is converted to mg malonaldehyde (MDA) /kg sample (TBARS value) using
the following equation:

TBARS = A532nm x K (mg MDA/kg sample)

Where K = (cone. in moles/5 m1 of distillate x M.W.MDA x 107 x 100) /
(Absorbance x wt. of sample x % recovery)

K is distillation constant and equal to 7.8 in this lab.

162

 

Appendix 19: Proximate composition determination

 

Sample Preparation (modified from section 983.18 Meat and Meat Products)

1.

S”

Section frozen meat into very small (<1 cm squares) pieces. This can be
accomplished by smashing samples with a hammer to decrease size of sample for
ease of grinding.

Add sample to Tekmar grinders (Tekmar Co, Cincinnati, OH) filling grinding
chamber half full.

Then add dry ice to fill up chamber.

Grind 2 to 3 minutes using Tekmar grinder (Tekmar Co, Cincinnati, OH) until sample
is ground into a fine powder. It may be necessary to stop in the middle of grinding
and stir the sample for uniform grinding.

Transfer finely ground powder to labeled whirl pack bags. Loosely close bag so that
dry ice can evaporate and dissipate. This takes about 2 days. Place in freezer
immediately to prevent melting of powder.

Moisture content (oven drying method, AOAC method 950.46B, 2000)

1.

Place a medium weigh boat on scale and zero. This is to keep the scale clean. Add
folded filter paper labeled with sample ID. Record the weight then tare the scale.

Add 2 grams (1 .03 g) of thoroughly mixed sample to the paper. Once desired weight
is reached record weight and fold over top. Place flat on tray. Do all samples in
triplicate. Do not stack samples on tray. This will hinder the drying process.

Once tray is full, place in drying oven set at 100°C for 20 - 24 hours.
After drying, place samples using latex gloves or tongs in desiccators to cool
completely before weighing. Once cool, weigh samples and record. This is your

final weight for moisture and your initial weight for fat analysis. Use the following
formula to determine the percent moisture in your samples:

Moisture (%)= wet sample wt. — dfl sample wt. x 100
wet sample wt.

163

 

Fat content (Soxhlet ether extraction, AOAC method 991.36, 2000)

1.

Take samples from moisture analysis and place in extraction tubes. Make sure that
all the samples are below the level where the ether drains off (curved glass on outside
of tube).

Add petroleum ether to clean boiling flasks until about 3A full. Add 2 to 3 glass beads
as a boiling aid.

Connect the extraction flask to the boiling flask and Soxhlet apparatus. Place
parafilm on the joint. Mount both to the condensing units on top of extraction flasks
using parafilm around joint.

Turn on condensing water so it runs at a steady stream.

Set Rheostats on 4.5 and run for 24 hours.

Place ether soaked samples onto a tray in a hood for 10 min to allow ether to
dissipate.

Place samples in drying oven for 5 to 10 min to remove any possible moisture then
place in desiccators for 1/2 hour to cool.

Weigh and record the weight of the samples. Calculate fat on wet basis with the

following equation:

Fat (%) = drv sam+p1e wt. — extracted sample wt. x 100
wet sample wt.

Protein content (combustion method, AOAC method 992.15, 2000)

1.

Weigh out approximately 1 gram of powdered meat into the tarred ceramic boat with
nickel liner. Write the weight and sample ID on the side of the boat with pencil.

After weighing out samples, dry for 24 hours in the drying oven at 100°C. This

removes moisture that can cause internal malfunctions with the Leco Protein
Analyzer. Do not reweigh samples. Enter wet weight into computer.

164

 

 

1.

Procedures for the LECO FP 2000 Nitrogen Analyzer

Open valves completely on oxygen, helium and compressed air tanks. Make sure
tanks have adequate levels of gas (gauge should read >100psi) and that the pressure
out of the tanks are set at 40 psi.

Press escape on upper left hand corner of touch screen until “front panel” comes up
and then press it. On right hand side of screen a section labeled “analysis gas” can be
found. Push the “on” button to turn gases on to the machine. Check to see that your
furnace temperature is 1050°F (located on left part of screen).

Wait about 5 min for all gases to equilibrate then start your leak tests. Press escape
from the front panel located in upper left corner. A screen with several icons will
appear. Press “maintenance”. This will bring up helium leak test, combustion leak
test and ballast leak test icons. Press the helium leak test, if it passes move onto the
combustion leak test. Run a ballast test only if there is a leak in the combustion
system. Once finished, start running blanks.

Run several air blanks through to purge the system. To do this escape from the
“maintenance” section and push the “analyze” icon. On the bottom of the screen you
will see several commands. Push “select ID code”. Move the highlighted line using
the arrows to "blanks". Then push exit on bottom and push manual weight. This will
bring up a touch screen with 0.2000000 on it. Push the enter button at least 10 times
to bring up 10 rows of 0.20000. Then push analyze. The machine will run through
these ten samples. Numbers should come down to about <0.2000% protein. Wait
until several blanks have approximately the same protein content. Then run EDTA
samples.

. Weigh approx. 1.0 g EDTA samples out in the ceramic boats and write the weight on

the side in pencil (at least four decimal places).

Push “select ID code” on the bottom of the screen. Move the highlighted line using
the arrows to "edta". Select “manual weight” and put your weight into the machine
pressing enter after each entry. Once weights are entered, press analyze. Follow the
directions on the touch screen. Push your first sample into the chamber about one
half inch so the door doesn’t catch the boat. Push okay on the screen when it asks
you to place your sample in the chamber. The next message will tell you to wait
because the system is purging. Then the machine will then tell you to push the boat
into the chamber. The machine will combust and analyze the sample in
approximately 4-5 minutes.

Run 5-6 EDTA samples (approximately 1.0g) to verify machine is operating properly.
EDTA is 59.9% protein. The samples should come out to be 59.9% +/- 0.2%.

If there is more variation in the percentages than 0.2%, DRIFT the samples to
equilibrate the percentages. To drift, press escape until you reach the front panel.

165

 

Press Calibrate, then press Drift Correction. A new screen will pop up; press Carbon
and then OK. Select the closest samples from the list of results by using the up/down
arrows and "Include Result" button at the bottom of the screen. Pressing the "Include
Result" will highlight the result and use it to recalibrate the machine. Once weights
are all selected, choose "Process Results" at the bottom of the screen. Another screen
will pop up asking if you would like to save the new calibration. Choose "Yes."

. After the Drift Correction, escape to the front panel and choose Analyze. Continue
running EDTA samples to ensure that the machine is working properly (59.9% +/-
0.2%). Once it is functioning properly, you may run your test samples. Push “select
ID code” on the bottom of the screen. Move the highlighted line using the arrows to .

166

--_— - ———-;

Appendix 20: pH determination

 

1. Homogenize 1 i 0.1 g diced sample with 50 ml of deionized water in a 50 m1
polycarbonate tube with Polytron mixer (PT-35, Kinematica, AG, Switzerland) set on
speed setting 4 for two 10 5 intervals. Rinse and blot dry Polytron bit between each
sample.

1. Measure pH using an Accumet Scientific pH meter calibrated using buffers 4.0 and
7.0.

3. Rinse pH meter probe with distilled, deionized water between sample readings.

167

 

Appendix 21: Melting point determination

 

Differential Scanning Calorimetry (DSC): DSC (2010, TA Instruments, New Castle,
DE)

Computer set-up

1.

2.

3.

10.

11.

12.

13.

Turn on nitrogen gas to 50 psi.
Click on TA Instrument control.
Click on procedure.

Click on edit.

Double click on segment.

For beef ribeye fat, ramp from 20 °C to 80 °C and for “modified marbling” gel, ramp
from 20 °C to 150 °C.

Click ok.

Click on summary.

Type in sample name (beef fat or solution).

Type in sample weight (approx. 12 mg).

Click on book and scroll arrow to 3% floppy disk.
Insert 3% floppy disk into computer.

Click apply.

Sample preparation

1.

2.

3.

Use a razor blade and out very small pieces of sample.
Place DSC pan bottom (T40625, TA Instruments, New Castle, DE) on scale and tare.
Place approx. 12 mg of sample in pan bottom using a tweezers and record weight.

Place DSC pan lid (T40621, TA Instruments, New Castle, DE) over pan bottom using
a tweezers.

Place pan in crimping die (TA Instruments, New Castle, DE) and press lever down.

168

 

6.

7.

Remove sample pan.

Prepare a control pan by placing a pan lid over an empty pan bottom and denting in
the denting apparatus.

Running the DSC

1.

2.

3.

9.

Take off the glass lid and gold cylinder from the DSC.

Place metal cylinder container on DSC.

Pour liquid nitrogen into cylinder and wait for temperature to drop to approx. 15 °C.
Once temperature has dropped, take off metal cylinder.

Open lid on DSC and place the control pan on the right slot and the sample pan on the
left slot.

Place lid on the DSC along with the gold cylinder and the glass lid.
Press the green run arrow on the top left of the screen.

Once the sample has been run, remove the glass lid, gold cylinder and lid and remove
the sample pan.

The control pan can stay the same for all samples.

10. Repeat as necessary.

169

 

Appendix 22: Scanning electron microscopy determination

 

Operating instructions for J SM 6400V provided by the Center for Electron Optics

Scanning electron microscope: JEOL scanning electron microscope, Model JSM-

6400V, version 96-2, Tokyo, Japan

Caution: 1. Never change the accelerating voltage with the filament saturated.
Change the accelerating voltage only when the filament is desaturated
to the preheat value.

2. Never turn the filament knob faster than the directions describe.

3. At the 8 mm working distance nothing should extend more than 2 mm
above the top of the sample hold.

Analysis procedures:

Start-up

1. Turn up the brightness on CRTs 1 and 2

2. Check the vacuum (10'7 range).

3. Check the heat/preheat light (on). This is located on the box to the right of the SEM.

Sample insertion

1.

2.

Working distance 39, X-25, Y-35, Tilt-0, Rotation-0.

Place samples in the sample holder, adjust height, attach holder to the sample
insertion rod.

Pull the rod back into the spring clip. Place rod on the port, press the red button, and
wait till the light goes out. Do not wait. After the light goes out, the port is no longer
pumped. If you wait, the vacuum will decrease to dangerously low levels.

Turn the flat on the knob to the front and pull it to the right.

Push the rod in, place sample holder on the rail, unscrew the rod, and pull it out.

170

 

6.

7.

Push the knob to the left then turn the flat up.

Push the red button, wait till the vacuum is gone, and remove the rod.

Sample removal

1.

First desaturate filament and turn off the accelerating voltage

2. Working distance 39, X-25, Y-35, Tilt-0, Rotation O.

3. Pull rod back into spring clip. Place the rod on the port, press the red button, and wait
till the light goes out.

4. Turn the flat on the knob to the front and pull it to the right.

5. Push the rod in, screw the rod into the holder, then pull the rod all the way out.

6. Push the knob to the left then turn the flat up.

7. Push the red button, wait till the vacuum is gone, and remove the rod.

Shut-down

1. Desaturate filament, and sample should be removed.

2. Turn down the brightness of CRTs 1 and 2.

3. Turn off the hanging lamp.

171

 

Appendix 23: TA-HDi Warner-Bratzler shear force settings

 

Texture Analyzer: TA-HDi Texture Analyzer
Texture Technologies Corporation, Scarsdale, NY

Software: Texture Expert Version: 1.22

TA—HDi Settings:

Test Mode: Measure Force in Compression
Option: Return to Start
Pre-Test Speed: 5.00 mm/s
Test Speed: 3.30 mm/s
Post-Test Speed: 10 mm/s
Distance: 35.0 mm
Trigger

Type: Return

Distance: 0.5 mm

Stop plot at: Trigger Return

Auto tare: (selected)

Attachment/Accessory: 50 kg load cell

TA-90; Heavy duty platform

172

 

Appendix 24: TA-I-IDi texture analyzer calibration and analysis procedures

 

Calibration Procedure:

Machine Calibration
1. Turn the texture analyzer (TA) on. The power button is located on the bottom right
side toward the front.

N

. Log on to texture analyzer program on computer (Texture Expert Analyzer) found on
computer desktop.

3. Turn TA key to the “run” position.

4. Remove any attachments or platforms that are present on the TA.

Ell

. Attach calibration weight hanger attachment and weight hanger.

6. Turn TA key to machine configuration.

>3

Press “ENT (enter)” until you reach the screen that determines the load cell weight
(Cell).

8. Press " +/-" to acquire appropriate load cell weight. For example: 50 kg load cell will
be indicated by “50” on screen.

9. Turn TA key back to “run” position and then back to machine configuration. This
saves settings in TA.

10. Press the "calibrate" key, then "enter".

11. When TA screen reads the appropriate weight put the actual weight on the TA weight
hanger. For example: 50 kg load cell will utilize a 10 kg weight, 5 kg load cell
utilizes a 2 kg weight.

12. Press "calibrate" and when screen reads "done", switch TA key back the to “run”
position.

13. Remove the actual weight from hanger but do not remove the weight hanger. Do not
put the weight away because you will be using it in the computer calibration.

Computer Calibration
1. Go to heading that reads “TA”.

2. Click on "Calibrate Force".
3. Press "ok". The computer will then ask you to place the actual weight on the hanger.

173

 

9.

Once the weight is placed onto the hanger press “ok”.

The computer will then say “calibration successful”. If this is not indicated, or if the
calibration unsuccessful, re-calibrate the machine.

Remove the weight and the hanger from the TA.

Attach the platform to the TA and the appropriate attachment. For Example: For the
Kramer shear test attach the 5-b1ade attachment to TA and from Gel hardness attach
TA-10 attachment.

When using the WBS attachment an extra set up step is required, if you are not using
the WB attachment you may skip this step. Using the up and down arrows on the TA
control board lower the WB blade into the slit on the platform until you can feel the
blade poke through the platform with the tips of your fingers. Run quick tests and
move the platform to make the force in kg as close to 0.000 as possible. This reduces
the friction during the analyses.

The TA is now ready to analyze samples.

Analysis Procedure and Setting up the Computer Files:

1.

2.

Create a personal file for data collection — go to computer desktop.
Click "My Computer"

Go to Drive "C:\"

Click on "My Documents"

Open the folder in which you wish to save you results. To create a new folder, right
click and scroll to “new” chose the “folder” option.

Name your folder (Example: Set 1). If you choose a file name that is too long the
computer will not read it (Example: Shear Set 1).

In the Texture Expert Program go to your selected project (the minimized window in
the bottom left hand corner of the screen, this is the project window.

Under settings, push dotted button (ellipses) and make sure your correct folder is
selected. Do the same for macro and results. If your desired settings are not entered
you must go to a folder with previous tests in it. Copy the macro file, setting files, as
well as a result file to your folder. This is done so the computer knows which format
to follow.

Push "Restart" on the Texture Expert Analyzer Program. You may receive an error
message because you have not created any results yet.

174

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Under TA go to “Settings” and include the appropriate settings for the test if different
than the settings listed there.

Click on the "TA" heading on the computer screen. Select "run test". Check to where
the results are being sent. The path in black on the middle of the screen indicates this
(Example: C:\mydocu\wbs\johnson\set1 will put the results on the C :\ drive under My
Documents in Johnson's folder under set 1). If the path is incorrect it can be changed
by pressing on the ellipses dots in the large white box. Continually clicking on the
top of this screen will take you back to the C:/ drive. Click on the folder into which
you wish your results to be placed.

Enter the ID of the sample you will analyze under "File ID". Set the "file number" to
one so it can count the samples. A low number is necessary because too many
characters in the file name will cause an error message to appear. Set the file number
to one each time you open a new spreadsheet for results.

Place the sample on the machine to be analyzed.

Enter the ID of the sample. Dates can be entered under "Batch ID".

Press "ok" on the Run Test screen to begin analyzing.

You will be prompted to save the results from the previous test. Click "ok".

Repeat steps 13-16 as necessary.

The analyzer may begin to run slowly after several analyses. If this occurs you
‘should start a new spreadsheet. To start a new spreadsheet click on the results
window to make it active. Select "Save" from the File menu. Then hit the "X" button
on the top right corner of the screen. Do not select "exit", as this will close the
Texture Analyzer Program. You do not need to open a new spreadsheet. One will
start as soon as you run the next test. To get back to the analyzing screen click on the
graph page to make that window active.

If at anytime the "run test" option is not available go back to the Project screen (the

first screen) and click "restart". This should cause the "run test" option to be
available again.

175

 

 

Appendix 25: Protocol for use of Taylor clamshell grill

 

Grill settings:

Gap 2.16 cm (setting on grill between 15 and 16)

Lower grill temp. 102.8 °C

Upper grill temp. 104.4 °C

Cook time — variable (timer set at 900 5, record endpoint time and subtract for final cook
time)

Grill preparation:
1. Turn on power box control on wall (it should be locked out) and both grill controls.

2. Choose STK 4 (setting for item #4) on each grill and allow to warm up until “TOO
COOL” no longer is displayed on LED displays.

3. While the grill is warming up complete the following:
a. Apply the stick-resistant Teflon cloths to both upper grills.
10. Start with left side of grill (top of grill should be in “down position”).
11. Insert left bar through top spring, cloth, and bottom spring in that order.
12. Secure left side.
13. Lift top.
14. Insert right bar through cloth.
15. Pivot bottom of right bar on right back edge of grill and pull the top of the bar

over the top edge.

16. Secure right side.

17. Repeat for right side of grill
4. ake sure display on grill read “STK 4 900”
Cleaning Grill
1. Turn off power to grills and lock-out power box on wall.
2. Let grill cool a minimum of 10 min.
3. Use rubber squeegee to wipe sown stick-resistant Teflon cloths on top burners.

4. Remover cloths, clean in sink, dry and lay flat.

5. Pour a small amount of water on lower grill surface and use metal scraper to release
cooked-on material (repeat as necessary).

6. Use scrub pad dipped in water to rinse both top and bottom grill burners.

7. Apply soap from spray bottle and scrub with pad.

176

8. Rinse out pad and use in clean water to rinse both top and bottom burners.
9. Clean back trough with trough scraper.

10. Remove and clean out fat trap.

11. Let grill dry.

177

 

Appendix 26: Protocol for cooking, coring, and shearing

 

Cutting 2.54 cm steaks

l.

2.

Take fresh, boneless ribeyes and cut 2.54 cm steaks using cutting box and knife.

Make sure each steak is 2.54 cm thick and not more than 2.54 cm thick.

Record steak weight and pre cook (initial) temperature of each steak.

Thermocouples

1.

Insert thin thermocouples into the geometric center of the steak. First, insert probe
(needle) through a small section of meat on the end of the cut, before insertion into
the center of the cut. Insert the needle into the side of the meat (in the center of the
depth of the steak) and push completely through the cut. Remove the probe from the
end of the thermocouple and then pull the thermocouple back into the center of the
meat. The end of the thermocouple should not be touching bone or fat. (See 2f and
figure 2 from The Guidelines for Cooking Procedures — AMSA).

Cooking

1. Open top of grill.

2. Apply thin layer of Crisco® on lower grill surface.

3. Stack grill with steaks from front to back rapidly.

4. Close grill top.

5. Press time button on grill.

6. Cook steaks to 71 °C (remove from grill around 68 °C, temp will rise to 71 °C).

7. Remove steaks from front to back rapidly.

8. Scrape lower grill surface with metal scraper to release buildup.

9. Scrape buildup off upper grill surface (Teflon surface) with rubber squeegee as
needed.

10. Place next rep of steaks on grill and repeat cooking process.

11. Record final cook temperature (highest temperature steak rises to) for each steak.

12. Allow steaks to drain on cooking rack for 5 min.

13. Record weight of cooked steak.

178

 

 

14. Cover steaks with saran wrap and store at 4 °C for 24 h.

Coring steaks with Craftsman 8 in. drill press and 1/z in. corer drill bit.
1. Remove one steak at a time from refiigerated storage to core and shear.

2. Cut cross section off end of steak to determine muscle fiber direction.
3. Position base of drill press to obtain angle parallel to muscle fibers.

4. Slowly and firmly lower the corer through the entire depth of the steak.
5. Remove the corer from the steak.

Warner-Bratzler shear of ‘/2 inch core with Texture Analyzer - HDi
1. Place V2 inch core in the center of the WBS shear plate.

2. Run a test on the TA-HDi.
3. Allow TA-HDi to return to start position.

4. Clean plate and base stand on TA-HDi.

M

. Repeat steps 1-4.

Beef: core and shear 6 samples on each steak.
Standardized Warner-Bratzler Shear Force Procedures for Genetic Evaluation

 

Committee Members:

Jeff Savell, Texas A&M University, Chair; Rhonda Miller, Texas A&M University;
Tommy Wheeler, MARC; Mohammad Koohmaraie, MARC; Steven Shackelford,
MARC; Brad Morgan, Oklahoma State University; Chris Calkins, University of
Nebraska; Mark Miller, Texas Tech University; Michael Dikeman, Kansas State
University; Floyd McKeith, University of Illinois; Glen Dolezal, Oklahoma State
University; Bill Henning, Pennsylvania State University; Jan Busboom, Washington
State University; Roger West, University of Florida; Fred Parrish, Iowa State
University; Scott Williams, University of Georgia.

 

An initiative to standardize the protocol for Warner-Bratzler shear force determinations
was identified at the National Beef Tenderness Plan Conference in April 1994. The
purpose of this protocol is to allow for consistent collection of Warner—Bratzler shear
force determinations across institutions for comparative evaluation. These data then can
be used in progeny testing and in the development of carcass EPDs for meat tenderness.
Any institution abiding by these guidelines can then be certified to collect Wamer-
Bratzler shear force determinations for the beef industry. The objective is to assist the
beef industry ion culling live animals that produce tough meat.

179

Conversion of live animals to carcasses

The process of conversion of the live animal to the carcass can have a significant effect
on meat tenderness. Therefore, the slaughter process and the environmental conditions
during slaughter should be controlled as closely as possible. Conditions that should be
monitored and could affect Warner-Bratzler shear force values include electrical
stimulation and postmortem chilling. Although these factors can affect the ultimate
tenderness of beef, these variables are probably uncontrollable by the researcher.

180

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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181

 

Appendix 28: Sensory random order

 

Storage Day 0
Replicate

hwr—‘Amhwh—‘N—d—‘WNWN

Storage Day 14
Replicate

thww-bNN—‘NP-‘v—‘DJ-h-h—i

Treatment #

MWN-d—‘h—‘NAUJUJHNNh-h

Treatment #

ththWva—‘Nfl—‘Nflh

Treatment

Average Choice Control
Average Choice Control
Injected Select

Injected Select

Control Select

Low Choice Control
Low Choice Control
Injected Select

Average Choice Control
Low Choice Control
Control Select

Control Select

Low Choice Control
Average Choice Control
Control Select

Injected Select

Treatment

Injected Select

Low Choice Control
Average Choice Control
Low Choice Control
Control Select

Average Choice Control
Control Select

Low Choice Control
Injected Select

Low Choice Control
Injected Select

Average Choice Control
Control Select

Injected Select

Average Choice Control
Control Select

182

Random Code
778
699
775
794
475
856
176
797
456
280
861
473
166
883
582
128

Random Code
285
625
426
622
777
180
556
717
109
144
162
809
395
548
707
477

Storage Day 28
Replicate

NW-fi-NhHWWNNv—‘wv—‘Hhh

Storage Day 42
Replicate

W-fiNNH-bAUJHt—‘UJNHUJN-h

Treatment #

hNWWhWWhHNHr-‘ANNH

Treatment #

ANAH-kiA’bLflNUJNN—‘Hwhd

Treatment

Low Choice Control
Average Choice Control
Average Choice Control
Injected Select

Low Choice Control
Control Select

Injected Select

Control Select

Average Choice Control
Control Select

Low Choice Control
Control Select

Low Choice Control
Injected Select

Injected Select

Average Choice Control

Treatment

Low Choice Control
Low Choice Control
Low Choice Control
Control Select

Injected Select

Injected Select

Low Choice Control
Average Choice Control
Control Select

Control Select

Injected Select

Injected Select

Control Select

Average Choice Control
Average Choice Control
Average Choice Control

183

Random Code

566
752
986
706
174
180
627
345
697
555
612
797
385
860
824
1 10

Random Code
511
414
175
434
515
990
735
158
609
755
464
632
598
268
746
226

 

lrgnm‘jgij‘ljjjj:ufljju