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This is to certify that the
thesis entitled
Verification of Indicator Proteins and Color

Measurements as Methods to Determine Adequate
Thermal Processing of Ground Beef Patties

presented by

Arnie Sair

has been accepted towards fulfillment
of the requirements for

Master degree in Food Science

 

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Date August 12, 1997

 

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VERIFICATION OF INDICATOR PROTEINS AND COLOR MEASUREMENTS AS
METHODS TO DETERMINE ADEQUATE THERMAL PROCESSING OF GROUND
BEEF PATTIES
By

Amie Sair

A THESIS

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

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1997

ABSTRACT
VERIFICATION OF INDICATOR PROTEINS AND COLOR MEASUREMENTS AS
METHODS TO DETERMINE ADEQUATE THERMAL PROCESSING OF GROUND
BEEF PATTIES
By

Amie Sair

The goal of this study was to determine whether triose phosphate isomerase (TPI)
could be used as a marker protein to verify adequate thermal processing Of ground beef
patties. The Objectives were 1) to determine the effect of muscle source, composition and
storage on TPI activity and concentration in ground beef, and 2) to compare the use of
TPI activity, lactate dehydrogenase (LDH) concentration and color to verify processing
adequacy of ground beef patties. TPI activity differed between muscles in cows and
steers. TPI activity and concentration fluctuated in cooked beef during storage at -13°C
for 5 months. A maximum TPI activity of 6.2 U/kg meat indicated that ground beef (20%
fat) was adequately cooked to 66.1°C/4l sec in a model system. In pilot studies, TPI
activity of beef patties (24% fat) decreased between 65.6 and 7l.l°C to a value of 6.28
U/kg meat. Lactate dehydrogenase (LDH) concentration decreased between 71.1 to
767°C. While color measurements were able to detect differences between undercooked
and firlly cooked beef patties, past research has shown color to be a poor indicator of
adequate thermal processing. TPI could possibly be used as an indicator to verify the
adequate processing of ground beef patties cooked to 7l.l°C and above with no holding

time.

This thesis is dedicated to my father and mother for providing me with the opportunities to
excel in achieving my goals and to my sisters, for being the best sisters in the world.

iii

ACKNOWLEDGEMENTS

The author would like to thank his major professor, Dr. Denise Smith, for her
guidance during the two years of graduate work at Michigan State University. Both the
training and knowledge that was received will tremendously help during upcoming
graduate studies.

Sincere appreciation is expressed to the guidance committee members: Dr. Alden
Booren for helping coordinate the studies within this project, and Dr. James Pestka for
helping during the planning stages of this project and spurring my interests in food
microbiology.

Special thanks are extended to Dr. Brad Berry at the United States Department of
Agriculture - Agricultural Research Service Meat Science Research Lab in Beltsville, MD
for his involvement in the cooking and color measurements of the beef patties which
significantly contributed to the quality of this research; and Dr. Ann Hollingsworth for
providing the beef patties.

Thanks to Dr. Robert Tempelman for his tremendous help during the statistical
analysis of the data, and introducing me to SAS sofiware which will inevitably help me in
my fixture studies.

Thanks to Tom Forton for helping me obtain beef samples and prepare them for

model system studies.

iv

Thanks to all of my lab mates for assisting me within the lab and keeping the
working environment an enjoyable one to work in: Dr. Ivy Hsu, Dr. Virginia Warner, Dr.
Stephanie Smith, Alicia Orta-Ramirez, Manee Vittayanont, Tammy Zielinski, and Dr. Giri

Veeramuthu.

This research was supported by the Michigan State University Crop and Food
Bioprocessing Center and the Cooperative State Research, Education and Extension
Service, United States Department of Agriculture under agreement number 96-35201-
3343. Any opinions, findings, conclusions, or recommendations expressed in this
document are those of the author and do not necessarily reflect the view of the United

States Department of Agriculture.

TABLE OF CONTENTS

List of Tables ................................................................................. x
List of Figures ................................................................................ xii
Chapter 1. Introduction .................................................................. 1
Chapter 2. Literature Review ........................................................... 7

2.1 United States Department of Agriculture-Food Safety Inspection
Service Thermal Processing Regulations ................................. 7

2.2 Proposed Performance Standards for the Production of Certain
Meat Products ............................................................... 7

2.3 Current USDA Methods for Endpoint Temperature

Determination ......................................... . ...................... 10
2.3.1 Bovine Catalase Activity Test .................................... 10
2.3.2 Protein "Coagulation Test" ....................................... 11
2.3.3 Acid Phosphatase Activity Test .................................. 11
2.4 Alternative Methods for Endpoint Temperature Determination ...... 12

2.4.1 Color of Cooked Ground Beef Patties as an Indicator of
EPT .................................................................. 12

2.4.2 Enzymatic Methods for EPT Determination in Beef
Products ............................................................. 15

2.4.3 Immunoassays for EPT Determination in Meat Products... . . 17
2.4.3.1 Ground Beef Products .................................... 18

2.5 Thermal Death Time (TDT) Studies ...................................... 19

2.6

2.7

2.8

Chapter 3.

3.1
3.2

3.3

2.5.1 Definitions ........................................................... 19
2.5.2 TDTStudiesinGroundBeef.....................................19

Time-Temperature Integrators as Indicators of Adequate

Thermal Processing ......................................................... 21
2.6.1 Methods Of Thermal Process Evaluation ........................ 21
2.6.2 Time-Temperature Integrators ................................... 22

2.6.3 Time-Temperature Integrators to Determine Thermal

Adequacy of Ground Beef Processing ............................ 24
Biochemical Properties of Triose Phosphate Isomerase ................ 25
2.7.1 Triose Phosphate Isomerase Activity Assay .................... 27
Beef for Model System Studies ........................................... 28
2.8.1 Carcass Selection ................................................... 28
2.8.2 Muscle Selection ................................................... 29
Factors Affecting the Activity and Concentration of Triose
Phosphate Isomerase in Ground Beef .................................... 30
Abstract ...................................................................... 30
Introduction .................................................................. 3 1
Materials and Methods ..................................................... 33

3.3.1 Source and preparation of beef for model system studies... . . 33

3 .32 Initial TPI in raw cow and steer muscles ........................ 35
3.3.3 Model system cooking of steer muscles ........................ 36

3 .3 .4 Effect of fat on triose phosphate isomerase activity in
cooked ground beef ................................................ 37

3.3.5 Ground beef cooking for storage studies ........................ 39

vii

3.4

3.3.5.1 Frozen storage stability of TPI in cooked ground
beef ......................................................... 39

3.3.5.2 Refrigerated storage of meat extracts from cooked

ground beef ................................................ 40

3.3.6 Effect of five freeze-thaw cycles on TPI activity and

concentration of raw and cooked ground beef ................. 40
3.3.7 Effect of phosphate and salt content on TPI activity

in ground beef ...................................................... 41
3.3.8 Extraction of protein fiom cooked beef ......................... 42
3.3.9 TPI activity ......................................................... 42
3.3.10 TPI concentration .................................................. 43
3.3.11 Bradford extractable protein determination .................... 44
3.3.12 Proximate composition ............................................ 44
3.3.13 Statistical analysrs 45
Results and Discussion .................................................... 45
3.4.1 Effect of muscle type and maturity on TPI activity in

muscle ............................................................... 45
3.4.2 Frozen storage of steer muscles ................................. 49
3.4.3 Thermal inactivation of triose phosphate isomerase in

selected steer muscles ............................................. 51
3.4.4 Effect of fat on triose phosphate isomerase activity in

ground beef ......................................................... 55
3.4.5 Effect of frozen storage of cooked ground beef ................ 58
3.4.6 Effect of refrigerated storage of protein extracts .............. 60
3.4.7 Effect of freeze-thaw cycles on TPI activity and

concentration of raw and cooked ground beef ................. 62

viii

3.4.8 Effect of phosphate and salt content in ground beef on

TPI activity ......................................................... 64

3 .5 Conclusions .................................................................. 66
Chapter 4. Indicator Proteins and Color Measurements as Methods to
Determine Adequate Thermal Processing of Ground Beef

Patties ........................................................................ 68

4.1 Abstract .......................................... . ........................... 68

4.2 Introduction ................................................................. 69

4.3 Materials and Methods ..................................................... 71

4.3.1 Patty formulation .................................................. 71

4.3.2 Cooking procedure ................................................ 72

4.3.3 Color ................................................................ 73

4.3.4 Extract preparation ................................................ 74

4.3.5 LDH concentration ................................................ 74

4.3.6 Proximate composition ........................................... 75

4.3.7 Statistics ............................................................ 76

4.4 Results and Discussion ..................................................... 76

4.4.1 TPI activity and LDH concentration ............................ 76

4.4.2 Internal color ....................................................... 79

4.4.3 Variability in degree of doneness scores ........................ 81

4.5 Conclusions .................................................................. 85

Chapter 5. Conclusrons 86

Chapter 6. Future Research ............................................................. 88

References ..................................................................................... 9O

ix

Table

2.1

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

4.1

LIST OF TABLES

Permitted heat-processing temperature/time combinations for firlly-
cooked patties ........................................................................ 8

Thermal processing schedules used to prepare adequately and
inadequately processed ground beef in a model system ......................... 38

Proximate composition and pH of cow and steer muscles ...................... 46

Triose phosphate isomerase activity (U/kg meat) of muscles from cows
and steers ............................................................................. 48

Stability of triose phosphate isomerase activity and extractable protein
concentration in selected steer muscles during frozen storage at -13°C.. . . . .. 50

Triose phosphate isomerase activity (U/kg meat) of ground chuck of

differing fat contents processed using two USDA approved schedules for
ground beef and inadequately processed by reducing the holding time by

0.5 and 1.0 log cycles ................................................................ 56

Effect of frozen storage (-13°C) on triose phosphate isomerase in
cooked (628°C) ground beef ....................................................... 59

Effect of five freeze-thaw cycles on triose phosphate isomerase and
extractable protein concentration in raw and cooked (628°C) ground
beef ..................................................................................... 63

Effect of NaCl, tetrapotassium diphosphate (TPDP) and heating
temperature on triose phosphate isomerase activity (U/kg meat) in
ground beef. ........................................................................... 65

Influence of temperature on triose phosphate isomerase (TPI) activity
and lactate dehydrogenase (LDH) content of cooked beef patties
(24.4% fat) ............................................................................ 77

4.2

4.3

Instrumental color values of ground beef patties (24.4% fat) cooked to
several endpoint temperatures ..................................................... 80

Degree of doneness (d/d) scores of beef patties cooked to 5 endpoint
temperatures .......................................................................... 82

LIST OF FIGURES

Figure

3.1 Effect Of heating temperature on triose phosphate isomerase activity
(U/kg meat) of selected steer muscles ............................................ 52

3 .2 Effect of heating temperature on triose phosphate isomerase content
(ug TPI/g meat) of selected steer muscles ........................................ 53

3.3 Effect of refrigerated storage (4°C) of protein extracts on triose
phosphate isomerase activity (U/kg meat) ............ . ........................... 61

CHAPTER 1

INTRODUCTION

Heat processing is commonly used to ensure the destruction of pathogens in meat
products. Salmonella, hemorrhagic Escherichia coli, Campylobacter, and
Staphylococcus are the most prevalent pathogenic bacteria found in foodbome illness
outbreaks associated with domestic and imported precooked meats. Inadequate cooking,
or survival of the pathogen during cooking is a common cause of foodbome disease
outbreaks associated with meat products (Bean and Griffin, 1990; Mermelstein, 1993).
Salmonella has been the traditional pathogenic microorganism of concern in meat
products, however, E. coli 0157:H7 has emerged as a significant cause of foodbome
disease. While most types of E. coli are normally found in the intestinal tract of humans
and other animals, there are a limited number of pathogenic strains of E. coli. In 1982, E.
coli 0157:H7 was associated with two outbreaks of hemorrhagic colitis where ground
beef was epidemiologically linked as the vehicle of transmission. Since 1982,
undercooked ground beef has been identified as the most frequent vehicle of E. coli
0157:H7 infection (CDC, 1996). Cattle have been identified as a major reservoir of E.
coli 0157:H7. An outbreak of foodbome disease in several western states fi'om late 1992
to early 1993 resulted in more than 475 cases and several children’s deaths. It was caused
by the consumption of undercooked enteropathogenic E. coli 01572H7-contaminated

ground beef patties (Mermelstein, 1993; Ahmed et al., 1995). There are an estimated

10,000 to 20,000 cases of E. coli 01572H7 infection in the United States each year (CDC,
1996). Infection often leads to bloody diarrhea and occasionally to kidney failure.

F oodbome illness is recognized as a significant public health problem in the United
States (USDA-FSIS, 1997a). Data from varied sources suggests that each year, 24 to 81
million people become ill from pathogens in food resulting in an estimated 10,000 deaths.
Of the foodbome illnesses and deaths resulting from microbial pathogens in food, 37
million illnesses and more than 5700 deaths may be associated with meat and poultry
products. The annual cost of foodbome illness is estimated to be between $7.7 and $23
billion (FDA, 1995).

On May 12, 1997, a five-point Administrative plan was unveiled by Vice President
Gore to strengthen and improve food safety for the American people (FDA, 1997). The
plan detailed how President Clinton planned to use the $43.2 million in new funds he
requested in his 1998 budget to supplement the food safety precautions already in place.
Among the measures developed by the Administration to reduce foodbome illness from
microbial contaminants was increased research to develop new tests to detect foodbome
pathogens and to assess risks in the food supply. An accurate and rapid method is
currently needed to verify that meat products have received adequate thermal treatment to
prevent foodbome disease.

The US. Department of Agriculture - Food Safety Inspection Service (U SDA-
FSIS) requires that meat products be cooked to safe endpoint temperatures (EPT) under
Title 9 of the Code of Federal Regulations to ensure destruction of pathogenic bacteria

and viruses. The USDA-FSIS (1997b) has required that patties labeled as "fully cooked"

be cooked to any one of seven time/temperature processing schedules that range from
661°C for 41 sec to 694°C for 10 see.

On May 2, 1996, the USDA-FSIS proposed to amend the processing regulations
of cooked beef, roast beef, and cooked corned beef; fiilly cooked, partially cooked, and
char-marked uncured meat patties; and certain fully and partially cooked poultry products
into performance standards (U SDA—F SIS, 1996). The three performance standards which
meat processing establishments would be required to meet are lethality, stabilization, and
handling. To meet the lethality standard, establishments would be required to achieve a 5
log (SD) or 7 log (7-D) reduction in Salmonella, depending on the product. While E.
coli 0157:H7 has recently emerged as a significant pathogen of concern in meat products,
Salmonella is more resistant to heat than E. coli 0157:H7. Therefore, a 7-D reduction in
Salmonella in cooked beef products would eliminate all pathogenic microorganisms and
provide a significant margin of safety. For cooked beef, roast beef, and cooked corned
beef products, the USDA-FSIS has proposed a 7-D reduction in Salmonella.

A 5-D reduction in Salmonella has been proposed for fully cooked uncured meat
patties. Any of the seven time/temperature processing schedules currently being used for
the cooking of beef patties generate a 5-D lethality. Therefore, an establishment using one
of the time/temperature combinations could continue to produce beef patties which meet
the proposed lethality standards. However, in order to have an overall lethality standard,
the USDA-FSIS may reduce the proposed lethality performance standard for roast beef,
cooked beef, and cook corned beef to a 5-D reduction in Salmonella as well (Anderson,

1997). The performance standards would dictate the level of reduction in pathogenic

microorganisms needed to be achieved by the processing establishments, while allowing
the use of processing procedures other than those prescribed in the current regulations.

A time-temperature integrator (TTI) is needed to verify whether fiIlly cooked beef
patties have been processed to meet the lethality standard. A TTI is a measuring device
which undergoes a time/temperature dependent, irreversible change after being subjected
to a variable temperature exposure (Van Loey et al., 1996). The irreversible change in the
TTI should parallel that of the target attribute, such as Salmonella, also being exposed to
the time/temperature process. A TTI should: 1) quantify the impact of a thermal process
on a target attribute, 2) be easy to recover from the food after processing, and 3) be
quickly and easily prepared for monitoring (Hendriclor et al., 1995). Through the use of a
TTI with a similar 2 value as that of Salmonella in ground beef patties, the monitoring of
adequate thermal processing can be achieved.

Current USDA methods to monitor endpoint temperature (EPT) are difficult to
interpret, subjective, impractical in that they require the use of sophisticated scientific
equipment, trained operators, and are time consuming. The methods currently in use by
the USDA include the Bovine Catalase Test (USDA, 1989), the Coagulation Test (USDA,
1986a), and the Acid Phosphatase Activity Method (USDA, 1986b). The greatest
disadvantage of the USDA methods for EPT determination is that none of them utilize an
indicator with a 2 value that is similar to that of Salmonella, the microbial pathogen
selected by the USDA-F SIS to define the proposed lethality standards. Therefore, it is not
currently possible to determine whether beef products have been adequately processed
after being cooked to one of the seven time/temperature processing schedules established

by the USDA-FSIS.

In previous work (Orta-Ramirez et al., 1997; Wang et al., 1996b; Hsu, 1997),
triosephosphate isomerase (TPI) was identified as a potential TTI in ground beef. Orta-
Rarnirez et a1. (1997) found TPI to have a similar thermal inactivation rate as E. coli
0157:H7 and S. senfienberg between 53 and 66°C. Hsu (1997) determined TPI activity in
roast beef after processing to three adequate and three inadequate (decreased cook times)
USDA processing schedules. The activity of TPI was found to be the same when
compared within adequate cooking treatments and increased as cooking time decreased.
The above results suggested that TPI could be used as a TTI in beef products to verify
compliance to USDA processing schedules.

Lactate dehydrogenase (LDH) concentration, as quantified by ELISA, has been
identified as a means for determining the EPT of cooked ground beef (Orta-Ramirez et al.,
1996; Wang et al., 1996a). Wang et al. (1996a), using polyclonal antibodies against
bovine muscle LDH, tested a sandwich ELISA to monitor the EPT of cooked ground
beef. In a model system, the ELISA was able to detect differences in LDH concentration
in ground beef at 2°C intervals within the temperature range of 66 to 74°C, suggesting it
could be used to verify cooking adequacy of beef patties processed using the USDA-FSIS
time/temperature combination of 694°C for 10 sec.

Orta-Ramirez et a1. (1996) produced monoclonal antibodies to bovine muscle
LDH and a sandwich ELISA was developed to determine the EPTs of cooked ground
beef. The LDH content of commercial patties, cooked from 54.4 to 656°C, 68.3 to
71. 1°C and 73.9 to 822°C decreased as cooking temperature was increased. Results of

Orta-Ramirez et a1. (1996) suggested that a maximum concentration of about 3 ug of

LDH/g meat might indicate that ground beef was processed to the USDA recommended
EPT of 71°C.

The overall goal for this study was to determine whether TPI could be used as a
marker protein to verify adequate thermal processing of ground beef patties. The first
study was designed to determine the effect of several factors that may have an effect on
TPI activity and concentration in ground beef, including; 1) muscle type and gender of the
animal; 2) fat content of ground beef; 3) frozen storage of raw steer muscles; 4) frozen
storage of cooked ground beef; 5) refrigerated storage of meat extracts; 6) repeated
freeze-thaw cycles of raw and cooked ground beef; and 7) salt and phosphate
concentration of beef. The second study was designed to determine whether the EPT of
ground beef could be differentiated using residual TPI activity and LDH concentration and
to compare the results to the internal cooked color of the beef patties. This study was
completed in collaboration with Dr. Brad Berry at the United States Department of

Agriculture - Agricultural Research Service Meat Science Research Lab in Beltsville, MI).

CHAPTER 2

LITERATURE REVIEW

2.1 UNITED STATES DEPARTMENT OF AGRICULTURE-FOOD SAFETY
INSPECTION SERVICE THERMAL PROCESSING REGULATIONS

To assure the adequate cooking and safety of various cooked and partially cooked
meat and poultry products, the US. Department of Agriculture - Food Safety Inspection
Service (U SDA-FSIS) has issued specific cooking regulations to meat processing
establishments since 1972 (USDA-FSIS, 1996). The regulations ensured that all
establishments were subject to the same rules and were using similar methods to kill
harmful bacteria. The USDA-FSIS (USDA-F SIS, 1997b) requires that beef patties be
cooked to any of seven time/temperature processing schedules that range from 661°C for
41 sec to 694°C for 10 sec (Table 2.1). These regulations do not take into account an
establishment's individual processing procedures nor the distinct needs of the
establishment. While large scale businesses are able to spread the costs of the command-
and-control regulations over their high production volumes, the smaller businesses
producing meat and poultry products often pay a high cost per unit when required to

employ a specific process.

2.2 PROPOSED PERFORMANCE STANDARDS FOR THE PRODUCTION
OF CERTAIN MEAT PRODUCTS

The USDA-F SIS has proposed to convert the command-and-control regulations to

performance standards (USDA-FSIS, 1996). Establishments would be granted the

Table 2.1-Permitted heat-processing temperature/time combinations for fully-cooked

 

 

patties
Minimum internal temperature (° C (°F)) Minimum holding time (sec)
at the center of after temperature is
each patty reached
66.1 (151) 41
66.7 (152) 32
67.2 (153) 26
67.8 (154) 20
68.3 (155) 16
68.9 (156) 13
69.4 (157) 10

 

Code of Federal Regulations, Title 9 (USDA-F SIS, 1997b).

freedom to employ individually suitable processing procedures to produce safe products,
while not being obligated to follow any of the specific time/temperature processing
requirements established by the USDA-FSIS. Three performance standards are being
proposed by the USDA-F SIS: lethality, stabilization, and handling. The lethality standard
is to ensure that establishments eliminate pathogenic microorganisms from the food
product. Reduction of pathogenic microorganisms is measured in terms of decimal
reduction time, or D value. The D value (rrrinutes) is defined as the time at constant
temperature required to reduce the initial microbial population by 90% (Van Loey et al.,
1996)

While in recent years Escherichia coli 0157:H7 has been recognized as the
pathogenic microorganism of concern in cooked beef products, the traditional
microorganism of consequence has been Salmonella. Salmonella is generally more
resistant to heat than E. coli 0157:H7 (Doyle and Schoeni, 1984; Line et al., 1991; Orta-
Rarnirez, 1994). Listeria monocytogenes is more resistant to heat than Salmonella (Fain
et al., 1991), however the presence of Listeria monocytogenes in a finished product is
indicative of recontamination. Therefore, the expected levels of Listeria monocytogenes
afier heat treatment are much lower than that of Salmonella. Based on the above data,
the destruction of Salmonella in beef products would be indicative of the destruction of
the other two pathogens. For cooked beef, roast beef, and cooked corned beef products,
the USDA-F SIS is proposing a 7-D reduction in Salmonella, while for fiilly cooked,
uncured meat patties the lethality performance standard is proposed to be a 5 log, or 5-D
reduction in Salmonella (Goodfellow and Brown, 1978; USDA-FSIS, 1996). The lower

proposed lethality performance standard that would be required for fully cooked, uncured

10

meat patties would be sufficient from a food safety standpoint while also maintaining
product flavor and texture. However, in order to have an overall lethality standard, the
USDA-FSIS may reduce the proposed lethality performance standard for roast beef,
cooked beef, and cook corned beef to a 5-D reduction in Salmonella as well (Anderson,
1997). The time/temperature guidelines for beef patties, developed by the USDA, were
based on the destruction of E. coli 0157:H7. When utilized, any of the seven
time/temperature combinations generate a 5-D lethality. Therefore, an establishment
currently applying one of the time/temperature combinations, could continue to produce
beef patties that meet the proposed lethality standards.

2.3 CURRENT USDA METHODS FOR EN DPOINT TEMPERATURE
DETERMINATION

Currently, the USDA-FSIS is using the protein "Coagulation Test", developed 30-
35 years ago, to monitor the endpoint temperatures (EPT) of beef and pork products heat
processed to temperatures lower than 65°C (Townsend and Blankenship, 1989). A
residual “Acid Phosphatase Activity Method” for determining the EPT of canned hams,
canned picnics and canned luncheon meat, and the “Bovine Catalase Test”, for the
determination of catalase which gives a pass/fail indication at a cooking temperature of
628°C for rare roast beef and cooked beef, are also in use. The Phosphatase Activity
Method was developed 30-25 years ago, and the Bovine Catalase Test was developed
approximately 10-11 years ago (Townsend and Blankenship, 1989).
2.3.1 Bovine Catalase Activity Test

The Bovine Catalase Test (USDA, 1989) is subjectively performed by observing

the appearance of foam when a meat sample is immersed in a solution of hydrogen

11

peroxide and sodium lauryl sulfate (shampoo). Strong, medium, weak and no activity

must be inferred from the quantity of foam produced. Errors can occur if the peroxide
reagent is weak or inactive, if the analyst does not carefully observe the appearance of
extra foam formed by the catalase reaction, or if the catalase activity is low.

2.3.2 Protein "Coagulation Test"

The “Protein Coagulation Test” is based on measurable loss in protein solubility as
temperature of the product is increased (USDA, 1986a). The soluble muscle proteins are
extracted with 0.9% saline, filtered, and heated. The temperature at which the first signs
of cloudiness or turbidity appear in the filtrate is recorded, and is considered to be the
maximum internal cooking temperature of the product. Because visible observation is
involved in this test, the test is considered to be empirical and subjective in nature
(Townsend and Blankenship, 1989). Operator experience and several testing steps are
also required.

2.3.3 Acid Phosphatase Activity Test

The FSIS residual acid phosphatase activity method (USDA, 1986b) is based on
the residual enzyme activity after cooking, and is expressed as the micromoles of phenol
formed per 1,000 g sample, when the sample is allowed to react with the substrate,
disodium phenylphosphate, for 60 min at 37°C and pH 6.5. The residual acid phosphatase
activity method demonstrated poor correlation with actual internal temperature
(Townsend and Blankenship, 1989; Korrnendy et al., 1987; Cohen, 1969). Recently,
Korrnendy et a1. (1987) reported that the residual activity of acid phosphatase in hams
does not depend directly on temperature alone, but also on a combined time and

temperature effect. The acid phosphatase test requires trained personnel, expensive

12

equipment, several reagents, and takes over one hour to perform. Recently, a new and
more rapid fluorometric assay of acid phosphatase activity in cooked poultry meat was
developed and field tested by the USDA (Davis and Townsend, 1994).

2.4 ALTERNATIVE METHODS FOR ENDPOIN T TEMPERATURE
DETERMINATION

The methods described above do not provide sensitive, rapid, and accurate results
that are necessary when testing meat products to verify processing adequacy and to ensure
safety. Therefore, alternative methods have been evaluated for testing the endpoint
temperature (EPT) of meat and poultry products. These methods include the internal
cooked patty color and color of expressible juices (Berry, 1994; Hague et al., 1994), the
electrophoresis of meat extracts to monitor changes in protein composition (Lee et al.,
1974; Caldironi and Bazan, 1980; Steele and Lambe, 1982), differential scanning
calorimetry of muscle proteins (Parson and Patterson, 1986; Ellekjaer, 1992), near
infrared spectroscopy (Ellekjaer and Isaksson, 1992), and residual enzyme activity
(Townsend and Blankenship, 1989). It has been reported that lactate dehydrogenase
(LDH) activity could be used as an endpoint heating indicator in turkey products (Wang et
al., 1992, 1993; Abouzied et al., 1993), beef muscle model systems (Collins et al., 1991a;
Stalder et al., 1991), and beef patties (Wang et al., 1996b).

2.4.1 Color of Cooked Ground Beef Patties as an Indicator of EPT

To assure that a safe internal temperature has been reached in cooked beef and
poultry, the USDA-FSIS recommends that consumers use a meat thermometer when
cooking meat and poultry. However, focus group research has shown that consumers are

not likely to use a meat thermometer on products as small as hamburgers (USDA-FSIS,

I3

1997c). The USDA-PSIS advises consumers who choose to not use a meat thermometer
to evaluate the doneness of ground beef patties by several factors including: (1) the cook
out juices should have no trace of pink, red, or cloudiness, (2) the cooked ground beef
should be brown in the center, (3) the cooked ground beef should have a firm or flaky
texture, as compared to the raw meat which has a soft, mushy texture, regardless of the
color (U SDA—F SIS, 1997d).

Two problems have recently been identified by the USDA-F SIS regarding its
advice to consumers about using a color index, to test for doneness of ground beef, used
to ensure the destruction of pathogenic microorganisms. One problem is that some
ground beef loses all pink color before firlly cooked (Hague et al., 1994). This
preforrnation of cooked color could be due to storing meat for long periods of time,
storing above recommended temperatures, or if the meat was ground from carcasses of
older animals (Hague et al., 1994; USDA-F818, 1997d).

The second problem is related to a pink color that persists in beef patties cooked
to 71°C (Mendenhall, 1989; Troutt et al., 1992; Hunt et al., 1995; Van Laack et al., 1996).
Van Laack et al. (1996) cooked 17 different commercially prepared patty formulations to
an internal temperature of 71°C and characterized the cooked color of the patties. Large
differences in color were found among the different formulations afier cooking. The a*-
values, a measure of redness in the cooked patty, ranged from 5.3 to 14.2. Eight of the 17
formulations were classified as red or pink after reaching 71°C (a*-values 2 11.4).
Spectral analysis of the cooked products showed undenatured myoglobin (Mb) to be
responsible for the red color. Research has shown that the oxidative state of the

myoglobin at the time of cooking plays a role in the final cooked color (Hunt et al., 1995).

14

The more metmyoglobin (meth), as opposed to oxyrnyoglobin (oxyMb), that the raw
product has, the more the cooked product will appear to be done. Mendenhall (1989)
demonstrated that the characteristic pink color of fresh meat remained in beef cooked to
716°C if the pH was 6.0 or higher. The higher the pH of the meat, the longer the cooking
time and/or higher the final internal temperature required for the denaturation of
myoglobin to be complete. The cooking times of the 17 patty formulations used by Van
Laack et a1. (1996) ranged from 4.1 to 7.7 min.

Van Laack et a1. (1996) also examined the effect of fi'ozen storage (-27°C) on the
color of patties cooked to 71°C. After one year of frozen storage, 16 of the 17 patty
formulations were red/pink when cooked to 71°C. Internal temperatures of 81 to 87°C
were needed for complete disappearance of the red/pink color. The use of such high
cooking temperatures to attain the cooked brown color can negatively affect the sensory
qualities of the beef patties.

The amount of fat in beef patties can also affect the cooked ground beef color.
Due to lower heat conduction in low-fat beef, low-fat beef patties require longer cooking
times to reach equivalent internal temperatures of higher-fat patties (Troutt et al., 1992).
Low-fat beef patties have maintained a pink color at temperatures of 71.1 to 745°C
(Berry, 1994).

Many studies continue to use similar cooking times based on the false assumption
that all beef patties cooked for the same amount of time have reached the same EPT. Liu
and Berry (1996) documented the variability that exists in cooking properties of beef
patties even when the cooking process is controlled. In the first study of Liu and Berry

(1996), beef patties were processed in the same facility in two separate trials using similar

15

raw materials containing 10% fat by cooking to internal temperatures of 68, 71 or 74°C.
Beef patties in the second trial required longer cooking times and had higher a*-values
than the first replication. In the second study, constant times were used to cook beef
patties to target EPTS of 68 and 71°C. There was considerable variation found in the
degree of doneness and final EPTS of the beef patties. The third study evaluated the
variability of internal temperatures of beef patties of different formulations when cooked
for constant times to attain an internal temperature of 71°C. Nine percent of the patties
did not achieve an internal temperature of 68°C and 1.3% did not reach 60°C. The fourth
study evaluated the temperature changes that beef patties undergo for a 1-min period after
being removed from a griddle. The internal temperature of the ground beef patties did not
change greatly within 40 sec of cooking. Forty seconds was determined to be was
determined to be available after cooking to assess the EPT. All of the studies showed
variability in cooking properties of beef patties due to cooking conditions. Further
research was deemed necessary to determine mechanisms to control the cooking
conditions of beef patties.
2.4.2 Enzymatic Methods for EPT Determination in Beef Products

Several enzymes have been evaluated as possible means for determining EPTS of
beef products (Collins et al., 1991a; Stalder et al., 1991; Townsend and Davis, 1992;
Wang et al., 1996b). Townsend and Davis (1992) investigated the use of a glutamic
oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) combination test
kit to assess the thermal treatment that had been applied to ground beef products. While
substantial enzyme activity remained in the beef when cooked to 71 . 1°C, there was a

distinct loss of enzyme activity when processed to 794°C. It was concluded that the

l6

combination test kit could be used as a method to verify the adequacy of heat treatment
given to imported cooked beef which must be heat processed to 794°C, but could not be
a used as an enzyme indicator for determining the EPT of domestic-type meat products
heat processed to temperatures lower than 71 . 1°C.

Collins et al. (1991a) analyzed LDH activity in beef top round muscles that were
processed using thermal treatments similar to those found within the meat industry.
Results showed that a significant amount of LDH activity was lost upon heating to 63°C
and there was minimal activity present at 66°C. Beef top rounds that underwent one
freeze-thaw cycle had the same LDH activity as fresh beef muscle.

Stalder et al. (1991) reported the potential for LDH to indicate the minimum
heating endpoint in precooked beef. The muscle-to-muscle variations, the influence of
gender, age, pH, salt, phosphate, and cooking temperature on LDH activity was
investigated. Significant differences were found in LDH activity between the 15 major
muscles sampled from three bovine carcasses. The largest disparity in LDH activity was
found between the semimembranosus, with 1476 U activity/g muscle, and the vastus
intermedius, with 283 U activity/g muscle. Gender was found to have no effect on LDH
activity, but as the maturity of the carcass increased, activity decreased. LDH activity was
reduced from greater than 1000 U/g muscle in unheated samples to nearly undetectable
levels at 66°C, regardless of pH. Between 57 and 63°C, at pH 5.6, LDH activity in the
sample slurries decreased. There was a sharp decrease in LDH activity above 60°C at pH
6.4 in the sample slurries. Increasing the salt concentration (0 to 4.5% NaCl) reduced
LDH activity at pH 5.6, but to a lesser degree at pH 6.4. The addition of NaCl and 0.3%

sodium tripolyphosphate (STP) decreased LDH activity between 57 and 63°C at pH 5.6.

17

Wang et al. (1996b) examined five proteins that might be used as indicators to
verify that ground beef patties have been cooked to the proper EPT. An ideal protein
indicator was identified as one that was present in relatively large quantities at low internal
temperatures and then disappeared or decreased markedly in intensity at higher
temperatures. Ground beef patties were cooked to 63, 66, 68 and 71°C. Five proteins,
acid phosphatase, bovine serum albumin, glyceraldehyde—3-phosphate dehydrogenase,
LDH, phosphoglycerate mutase, and TPI were analyzed. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis, Western blotting and enzyme activity were used to
monitor the changes in protein composition of extracts of cooked patties. The
concentration or activity of each protein decreased as EPTs of the ground beef patties
were increased. Therefore, each of the five proteins showed possible usefirlness to verify
processing temperatures of cooked ground beef patties.

2.4.3 Immunoassays for EPT Determination in Meat Products

Immunoassays, specifically enzyme-linked immunosorbent assays (ELISAs), have
been used to determine the presence of pathogenic and toxigenic microorganisms in foods
(Samarajeewa et al., 1991). Immunoassay test kits, currently in use within the food
industry, have been developed for detecting antibiotics, pesticide residues,
microorganisms, mycotoxins, and indicator proteins to verify minimum EPTs of meat and
poultry products (Fukal, 1991; Samarajeewa et al., 1991; Wang et al., 1992; Abouzied et
al., 1993; Orta-Ramirez et al., 1996). ELISAS have high specificity and sensitivity, are
easy to use, and are rapid and adaptable to automation. An ELISA can be performed

rapidly on a large number of samples by minimally trained personnel with small quantities

18

of solvent and does not require major scientific equipment in the field or processing plant
(Smith, 1995).
2.4.3.1 Ground Beef Products

The use of irnmunoassays to determine the EPT of meat products (i.e. ground beef
and poultry products) has been well documented (Wang et al., 1992; Abouzied et al.,
1993; Wang et al., 1993, 1994, 1996a; Orta-Ramirez et al., 1996; Smith et al., 1996).
Lactate dehydrogenase concentration, as quantified by ELISA, has been investigated as a
means for determining the EPT of poultry products (Wang et al., 1992, 1993; Abouzied et
al., 1993; Smith et al., 1996) and ground beef (Wang et al., 1996a; Orta-Ramirez et al.,
1996). Wang et al. (1996a), using polyclonal antibodies against bovine muscle LDH,
tested a sandwich ELISA to monitor the EPT of ground beef. In a model system, LDH
content decreased (p<0.05) as ground beef was cooked to temperatures ranging from 62
to 74°C at 2°C intervals. The ELISA was able to detect differences in LDH concentration
at 2°C intervals within the temperature range of 66 to 74°C, suggesting it could be used to
verify cooking adequacy of beef patties processed using the USDA-FSIS minimum
time/temperature combination of 694°C for 10 sec.

Orta—Ramirez et al. (1996) produced monoclonal antibodies to bovine muscle
LDH and a sandwich ELISA was developed to determine the EPTs of cooked ground
beef. Fat concentrations and the effect of freeze-thaw cycles on LDH concentrations in
ground beef were also investigated. The sandwich ELISA was able to detect differences
in LDH content of ground beef between 64 and 74°C, at 2°C intervals, in a model system.
The LDH content of commercial patties, cooked from 54.4 to 656°C, 68.3 to 71. 1°C and

73.9 to 822°C also decreased as cooking temperature was increased. Similar

19

concentrations of LDH (3 ug/g meat) were detected in both the model system ground beef
and commercially cooked patties at a cooking temperature of 70°C. Fat content had no
effect on the concentration of LDH in ground beef cooked to 694°C. The LDH
concentration of raw ground beef remained the same through five freeze-thaw cycles. The
LDH concentration of cooked beef was decreased afier undergoing one freeze-thaw cycle
and was reduced by 70.4% after undergoing four freeze-thaw cycles.
2.5 THERMAL DEATH TIME (TDT) STUDIES

Due to the continued threat of foodbome illnesses caused by the consumption of
meat products, research has been undertaken to establish the effects of thermal processing
on the pathogenic microorganisms responsible for the outbreaks. The thermal inactivation
kinetics of Escherichia coli 0157:H7 in ground beef (Line et al., 1991), Listeria
monocytogenes Scott A in ground beef and turkey (Fain et al., 1991), and Salmonella in
beef products (Goodfellow and Brown, 1978) have been established.
2.5.1 Definitions

The decimal reduction time, or D value is the time (min) required to destroy 90%
of the organisms in a food system at a specified temperature. A thermal resistance curve
(TRC) can be constructed by plotting temperature against the D values for each
temperature on a log scale. The negative reciprocal of the slope of the TRC is the 2 value.
The 2 value is defined as the temperature change needed for the TRC curve to traverse
one log cycle (Jay, 1992).
2.5.2 TDT Studies in Ground Beef

Goodfellow and Brown (1978) determined the D values of Salmonella serotypes

in ground beef to establish time-temperature processes for manufacturers to use to insure

20

the elimination of 107 Salmonella/g in the center of beef roasts. Salmonella whimurium
strain TMI was a reference strain in the experiment as its D values had previously been
reported (N g et al., 1969). A mixture of six Salmonella serotypes including, Salmonella
newport, Salmonella agona, Salmonella bovis-morbificans and Salmonella muenchen
strains, all isolated from food poisoning outbreaks associated with meat, was inoculated
into the ground beef. The thermal resistance curves for the Salmonella strains inoculated
into ground beef were used to establish processing times necessary for a 7-D reduction of
Salmonella in cooked beef. The data presented by Goodfellow and Brown (1978) was
accepted by the USDA and was the agency's basis for the implementation of time-
temperature schedules to govern the cooking of roast beef.

Fain et al. (1991) determined the D values and 2 values for L. monocytogenes
Scott A in lean (2.0% fat) and fatty (30.5% fat) ground beef. The 2 value of L.
monocytogenes was higher in fatty ground beef than the lean beef. At all three
temperatures tested (52.0, 57.7, and 628°C), the D values for L. monocytogenes Scott A
exceeded those for Salmonella spp. The 2 value of L. monocytogenes in fatty ground
beef, 114°C, was higher than the 10°C reported for Salmonella spp. (Goodfellow and
Brown, 1978). The Scott A strain of L. monocytogenes showed a higher heat resistance
than that of Salmonella spp. in ground beef. Fain et al. (1991) proposed additional
research to include other strains of L. monocytogenes to determine if the cooking
procedures that were in use were sufficient to destroy populations of L. monocytogenes
normally present in meat and poultry products.

In recent years, E. coli 0157:H7 has been recognized as the pathogenic

microorganism of concern in cooked beef products. To the chagrin of the meat and

21

poultry industry, Doyle and Schoeni (1987) found E. coli 0157:H7 to be a common
contaminant of fresh meats and poultry after assaying a total of 896 samples. E. coli
0157:H7 was isolated from 6 (3.7%) of 164 beef, 4 (1.5%) of 264 pork, 4 (1.5%) of 263
poultry, and 4 (2.0%) of 205 lamb samples. Doyle and Schoeni (1984) compared the D
values of E. coli 0157:H7 to those of Salmonella spp. that were determined by
Goodfellow and Brown (1978). E. coli 0157:H7 in ground beef was more sensitive to
heat than salmonellae. Therefore, at the same temperature, less time is needed to
inactivate E. coli 0157:H7 than the same number of salmonellae. Consequently, any
processing schedule that is developed to inactivate Salmonella in beef products will
ultimately inactivate E. coli 0157:H7.

Recognizing E. coli 0157:H7 as a microorganism of concern in beef products, the
USDA-FSIS commissioned Line et al. (1991) to determine the thermal lethality of E. coli
0157:H7 in lean (2.0% fat) and fatty (30.5% fat) ground beef. The D values for fatty
ground beef exceeded those for lean ground beef at the temperatures tested (51.7, 57.2,
and 628°C). This indicated that E. coli 0157:H7 was killed more rapidly in the lean beef
than fatty beef. Increasing the fat concentration decreased the water content of the
product and protected the pathogen from heat.

2.6 TIME-TEMPERATURE INTEGRATORS AS INDICATORS OF
ADEQUATE THERMAL PROCESSING

2.6.1 Methods of Thermal Process Evaluation
Currently, there are three methods used to evaluate the adequacy of thermal

processes. The two most commonly used techniques are the in situ method and the

22

physical-mathematical method, while the use of time-temperature integrators (TTI) has
been a rather recent research development (Van Loey et al., 1996).

The in situ method is used to monitor the concentration of the actual quality or
safety attribute before and after thermal processing (Maesmans et a1, 1993). Quality or
safety attributes may include microorganisms, vitamins, or proteins. A disadvantage of the
in situ method includes not being able to detect the attribute being measured after the
thermal processing due its level not able to be detected with current analytical methods
(Hendrickx, et al., 1995). As data is collected before and after the thermal processing, the
in situ method can also be considered laborious, time-consuming, and expensive, making
routine checks impractical.

The physical-mathematical method combines prior knowledge of the kinetic
response of a food quality attribute with the time-temperature history curve imposed on
the food. This method can only be used if the time-temperature data is available. In many
thermal processes, i.e. rotating retort or a continuous aseptic system, time-temperature
data acquisition is not practical in that the thermocouples could disrupt the free movement
of the product, thereby leading to misinterpretations of the temperature history
(Maesmans et al., 1993). The use of models to reconstruct the time-temperature history
depends on the accuracy of the physical input parameters that are required (e.g., data on
viscosity or conductivity at high temperatures, fluid-to-particle heat transfer coefficients)
(Van Loey et al., 1996).

2.6.2 Time-Temperature Integrators
To overcome the limitations and disadvantages associated with the in situ and the

physical-mathematical methods, TTIs have been, and are being developed as a means to

23

evaluate the adequacy of thermal processing. A TTI is a measuring device which
undergoes a time/temperature dependent, irreversible change afier being subjected to a
variable temperature exposure (Van Loey et al., 1996). The irreversible change that the
TTI undergoes should parallel that of a target attribute also being exposed simultaneously
to the time/temperature process. The TTI should be able to quantify the impact of the
process on a target attribute. It must be easy to recover after the processing and must be
able to be incorporated into the food product without disturbing the heat transfer. A TTI
should be economical and easy to prepare (Hendrickx et al., 1995).

The major advantage to the use of TTIS is the ability to quantify the impact of a
time-temperature process on a target attribute without information regarding the actual
time-temperature history of the product. To be considered an ideal indicator of thermal
processing, an endogenous muscle protein should have a thermal inactivation rate constant
(2 value) that closely resembles the 2 value reported for the microorganism used for
establishing the thermal process used for monitoring the safety of the food (Van Loey et
al., 1996). The thermal inactivation rate constants of the TTI and the target should be
equal in the relevant temperature range (Taoukis and Labuza, 1989).

TTIS can be grouped into three major categories: (1) biological (microbiological
and enzymatic), (2) chemical, and (3) physical. The use of microbiological TTIS is the
most common within the food industry (e. g. the use of Bacillus spp. and Clostridium
sporogenes spores). TTIS can be further classified as either intrinsic or extrinsic to the
food system. Intrinsic TTIS are compounds present in the food, while extrinsic TTIS are

incorporated or added to the food prior to processing.

24

Mulley et al. (1975) added thiamin to pureed vegetables, beef and brine of canned
peas to act as a chemical TTI to monitor thiamin reduction. The use of the thiamin as a
TTI had advantages over typical quantitative microbiological techniques, such as enhanced
accuracy in the assay, ease of handling and absence of contamination.

To monitor sterilization processes, the heat inactivation kinetics of a-arnylase from
Bacillus amyloliquefaciens, a possible enzymatic TTI, were studied at low moisture
contents and in the presence of various organic solvents (De Cordt et al., 1992; Saraiva et
al., 1993; De Cordt et al., 1994). After promising results were published regarding the
development of TTIS based on a-amylase from Bacillus species, Van Loey et al. (1995)
used a-amylase from Bacillus subtilis to develop enzymatic TTI systems to monitor the
thermal death of C lostridium botulinum nonproteolytic type B in cod, Streptococcus
faecalis in fish, and the denaturation of chlorophyllase in spinach.

2.6.3 Time-Temperature Integrators to Determine Thermal Adequacy of Ground
Beef Processing

The current USDA methods cannot be used to verify whether beef products,
cooked using the USDA-FSIS time/temperature schedules, were adequately processed.
To verify adequate beef processing when any one of the seven USDA-F SIS time-
temperature processing schedules are used, the use of a TTI is necessary. If the proposed
USDA-FSIS lethality standard for beef patties (USDA-FSIS, 1996) is implemented, a new
method, utilizing TTIs should be developed to accurately verify proper processing.

The thermal inactivation of E. coli 0157:H7 and Salmonella senftenberg (S.
senftenberg) were compared to that of several previously proposed protein markers in

ground beef heated at four different temperatures (Orta-Ramirez et al., 1997). The 2

25

values for E. coli 0157:H7, S. senftenberg, and TPI were 5.59, 6.25, and 556°C,
respectively. To be considered an ideal indicator of thermal processing, an endogenous
muscle protein should have a thermal inactivation rate constant (2 value) that closely
resembles the 2 value reported for the microorganism used for monitoring the safety of the
food (Van Loey et al., 1996). To calculate the 2 value for the hamburger processing
schedule, Orta-Ramirez et al. (1997) plotted the log of the holding time vs. the
temperatures stated in the USDA regulations. The calculated 2 value for the USDA-FSIS
beef patty processing schedule was 548°C, therefore Orta-Ramirez et al. (1997) found
that TPI could be used as an indicator of the adequacy of thermal processing of beef
patties.

To identify a marker protein in ground and roast beef, Hsu (1997) determined the
enzyme activities of peroxidase, acid phosphatase and TPI afier roast beef was processed
using three adequate (high, medium and low temperatures were 622°C/5 min, 58.3°C/24
min, and 54.4°C/ 121 min, respectively) and three inadequate (0.5 and 1 log reductions in
adequate cook times at each processing temperature) processing schedules. The activity
of TPI was the same when compared within adequate cooking treatments and increased as
cooking time decreased. TPI was identified as the best marker protein in ground beef
when using the medium to high temperature processes.

2.7 BIOCHEMICAL PROPERTIES OF TRIOSE PHOSPHATE ISOMERASE

Triose phosphate isomerase (TPI, D-glyceraldehyde—3-phosphate ketol-isomerase,
EC 5.3.1.1) is a glycolytic enzyme that catalyzes the interconversion of dihydroxyacetone
phosphate (DHAP) and D—glyceraldehyde 3-phosphate (GAP) (Webb and Knowles,

1975). TPI is a homodimer (26,000 Da per monomer), however only the dimer is fiilly

26

active (W ierenga and Noble, 1992). TPI has been isolated and crystallized from calf
skeletal muscle (Beisenhertz, 1955), rabbit skeletal muscle (Czok and Buecher, 1960;
Norton et al., 1970), chicken breast muscle (Bonner et al., 1976) and human skeletal
muscle (Dabrowska et al., 1978). Recently, Hsu (1997) was successful in purifying TPI
from bovine semimembranosus muscle (top round choice muscle).

The molecular mass of TPI from human skeletal muscle, as determined by gel
filtration on a Sephadex G-100 column, was 57,400 i 3000 Da (Dabrowska et al., 1978).
This result was in agreement with molecular masses determined for TPI from other
species, i.e. 52,900 i 2000 Da in rabbit muscle (Norton et al., 1970), and 60,000 Da in
rabbit liver (Krietsch et al., 1970). Using sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, Hsu (1997) determined the subunit molecular weight of bovine TPI to be
23,000 Da. The subunit molecular weight of TPI from rabbit and human skeletal muscle
was 26,500 Da (Norton et al., 1970) and 28,300 Da (Dabrowska et al., 1978),
respectively.

Multiple forms of TPI have been documented (Eber and Krietsch, 1980). Rabbit
muscle TPI may have eight electrophoretic forms (Sawyer et al., 1972). Lee et al. (1971)
resolved rabbit muscle, horse liver, and human liver TPI into five, three and three
isozymes, respectively, by polyacrylamide gel electrophoresis. Using DEAE-cellulose
chromatography, Eber and Krietsch (1980) resolved purified human skeletal muscle TPI
into three principal forms (A, B and C). The three major forms of TPI accounted for 97%
of the total activity. The A and C forms of TPI were classified as homodimers, era and
[313, while the B form was grouped as a heterodimer, 043. All three forms (A, B and C)

were assumed to be immunologically identical after gel diffusion tests, and

27

immunotitration produced no observable differences. The amino acid composition,
molecular mass and antigenicity of the or and B polypeptide chains were determined to be
identical.

Using electrofocusing in a sucrose stabilized, pH 5-8 gradient, Norton et al. (1970)
resolved TPI into two major and two minor components. The isoelectric points (pl) of the
minor species were 5.55 and 5.95, and the major components had p15 of 6.75 and 6.85.
Sawyer et al. (1972) studied human TPI from erythrocytes. The human TPI was resolved
into three active forms (I, II, III) by isoelectric focusing. Variant I, with 1-5% of total
activity, had an isoelectric point of 6.7. Variants II and 111, 70-75% and 20-25% of total
activity, respectively, had isoelectric points of 6.5 and 6.1, respectively.

2.7.1 Triose Phosphate Isomerase Activity Assay
The TPI activity is determined through the following two coupled reactions:
Triose phosphate

isomerase
glyceraldehyde-3-phosphate 47‘ dihydroxyacetone phosphate (DHAP)

 

glycerol-3-phosphate dehydrogenase
DHAP + NADH > glycerol-3-phosphate + NAD

 

Glyceraldehyde-3-phosphate (GAP) is used as the substrate and the glycerol-3-
phosphate dehydrogenase (GDH) as the coupling enzyme (Beisenherz, 1955). The
activity of TPI is twice that in triethanolamine-HCI buffer as in bicarbonate-C02 buffer at
pH 7.5 (Beisenherz, 1955). Between the optimal pH range of 7 to 8, the activity of TPI

varies little, however, at pH 6.3, the activity is about half (Beisenherz, 1955). Triose

28

phosphate isomerase is inhibited by phosphate ions. A buffer containing 0.05 M
phosphate reduced the activity of TPI by 75% (Oesper and Meyerhof, 1950; Beisenherz,
1955)

2.8 BEEF FOR MODEL SYSTEM STUDIES

2.8.1 Carcass Selection

In 1995, the total production of ground beef in the United States was estimated at
3.22 billion kg (AMI, 1996). Seventy five percent of an average cow carcass and 25.8%
of an average steer carcass account for the trimmings plus whole muscle/cuts ground by
the meat packer. The "commercial" USDA quality grade is the classification that is
assigned to a cow of D/E maturity (Hale, 1994). Research of Smith et al. (1988)
supported claims by the USDA of declining palatability in beef with advancing maturity.
Meat from D/E maturity cows is not sold as "prime" cuts, but is used primarily as ground
beef. Meat packers report that approximately 49.4% of their trimmings sales are marketed
in a final form as ground beef, as is 54.9% of their whole muscle/cuts sales (AMI, 1996).

Steers of B maturity produce cuts of meat that are classified as "prime".
Therefore, the trimmings from the young steers are used in ground beef production.
Approximately one-third of the trimmings of what meat packers sell from steers is
marketed as ground beef. An average of 38.8 kg of ground beef is produced per steer
carcass, up from 30.5 kg in 1990 (AMI, 1996). The activity of several glycolytic enzymes
including, phosphorylase a and a+b, glycogen synthetase I and I+D, hexokinase, LDH and
glyceraldehyde-3-phosphate dehydrogenase, change very little after the age of 6 months in
bovine carcasses (Talmant et al., 1986). Therefore, the maturity of a carcass does not

influence the activity of the enzyme being investigated.

29

2.8.2 Muscle Selection

Most supermarkets sell ground beef from many sources, including ground chuck,
ground round, or ground sirloin (Evans and Greene, 1973). It is estimated that 30.3% of
retail ground beef, up from 21.3% five years ago, was labeled and marketed as a ground
out (ground chuck, ground round, ground sirloin) (AMI, 1996). The beef chuck from
both cows and steers contains many small muscles and large quantities of intramuscular fat
and connective tissue (Ruiz et al., 1993). Therefore, the chuck is used primarily for

ground beef, and lower-priced steaks and roasts.

CHAPTER 3
FACTORS AFFECTING THE ACTIVITY AND CONCENTRATION OF TRIOSE

PHOSPHATE ISOMERASE IN GROUND BEEF

3.] ABSTRACT

In previous research, triose phosphate isomerase (TPI, EC. 5.3.1.1) was identified
as a potential indicator to verify adequate thermal processing of ground beef. The
objective of this study was to investigate different factors that may affect TPI activity and
concentration in beef. TPI activity differed in four muscles obtained fi'om cows and steers,
although animal maturity had no effect. When beef was cooked, TPI activity and
concentration decreased with increasing temperatures. TPI activities were similar when
ground beef was inadequately processed by reducing the required USDA cook times at the
low (661°C) and high (694°C) temperatures by 1.0 log, but different when the ground
beef was fully cooked. Frozen storage (-13°C) of raw steer muscles for 10 months had no
effect on TPI activity. During storage at -13°C for 5 months, TPI activity increased after
two months, however returned to a level similar to the initial after 5 months. TPI activity
in raw and cooked beef extracts fluctuated during the first 8 hours of refrigerated (4°C)
storage, however remained constant from 24 to 48 hours. Five freeze-thaw cycles had no
effect on TPI concentration of raw beef. The TPI activity of raw beef increased by 10%
after five freeze-thaw cycles. The TPI activity of the cooked beef increased by 13% after

four freeze-thaw cycles and then decreased. Cooked beef (628°C) with 0.5%

30

31

tetrapotassium diphosphate had increased TPI activities and cooked beef (694°C) with
1.0% NaCl had lower TPI activities than the control. Further investigation into the factors
having a significant effect on TPI activity is warranted if TPI is to be used as an indicator
of adequate thermal processing of ground beef patties.

Key words: Ground beef, cooking temperature, frozen storage, triose phosphate
isomerase, endpoint temperature

3.2 INTRODUCTION

To assure the adequate cooking and safety of various cooked and partially cooked
meat and poultry products, the USDA-F SIS has issued specific cooking regulations to
meat processing establishments since 1972 (USDA-F SIS, 1996). The USDA-FSIS
(USDA-F818, 1997c) requires that patties labeled as "fully cooked" be cooked to any of
seven time/temperature processing schedules that range fi'om 661°C for 41 sec to 694°C
for 10 see. In May of 1996, the USDA-FSIS proposed to convert the command—and-
control regulations to performance standards (USDA-FSIS, 1996). The lethality standard
is to ensure that establishments eliminate pathogenic microorganisms from the food
product. Salmonella is generally more resistant to heat than E. coli 0157:H7 (Doyle and
Schoeni, 1984; Line et al., 1991; Orta-Ramirez, 1994) and was selected on that basis as
the organism to indicate the destruction of pathogens in beef products. For cooked beef,
roast beef, and cooked corned beef products, the USDA-FSIS is proposing that
processors use any thermal process to achieve a 7-D reduction in Salmonella, while for
fiJlly cooked, uncured meat patties the lethality performance standard is proposed to be a
5-D reduction in Salmonella (Goodfellow and Brown, 1978; USDA-FSIS, 1996).

However, in order to have an overall lethality standard, the USDA-FSIS may reduce the

32

proposed lethality performance standard for roast beef, cooked beef, and cook corned beef
to a 5-D reduction in Salmonella (Anderson, 1997).

Current USDA methods to monitor endpoint temperature (EPT) are difficult to
interpret, subjective, impractical in that they require the use of sophisticated scientific
equipment and trained operators, and time consuming (Smith and Orta-Ramirez, 1995).
The greatest disadvantage of the USDA methods for EPT determination is that none of
them are based on the thermal inactivation of Salmonella, the microbial pathogen selected
by the USDA-F SIS to define the proposed lethality standards. Therefore, it is not possible
to determine whether beef products have achieved adequate heat processing after being
cooked to one of the seven time/temperature processing schedules established by the
USDA-FSIS.

A time-temperature integrator (TTI) is a measuring device which undergoes a
time/temperature dependent, irreversible change after being subjected to a variable
temperature exposure (Van Loey et al., 1996). The irreversible change that the TTI
undergoes should parallel that of a target attribute also being exposed simultaneously to
the time/temperature process. To be considered an ideal indicator of the thermal process
in ground beef, an endogenous muscle protein should have a thermal inactivation rate
constant (2 value) that closely resembles the 2 value for Salmonella (Van Loey et al.,
1996). The thermal inactivation of E. coli 0157:H7 and Salmonella senftenberg (S.
senfienberg) were compared to that of several previously proposed protein markers in
ground beef heated at four different temperatures (Orta-Ramirez et al., 1997). The 2
values for E. coli, S. senftenberg, and TPI were 5.59, 6.25, and 556°C, respectively.

With the calculated 2 value for the USDA-FSIS beef patty processing schedule 548°C,

33

Orta—Ramirez et al. (1997) found that TPI could be used as a TTI to verify the adequacy
of thermal processing of beef patties.

To identify a TTI in ground and roast beef, Hsu (1997) determined the enzyme
activity of TPI after roast beef was processed using three adequate (high, medium and low
temperatures were 622°C/5 min, 58.3°C/24 min, and 54.4°C/121 min, respectively) and
three inadequate (0.5 and 1 log reductions in adequate cook times at each processing
temperature) USDA processing schedules. The activity of TPI was the same when
compared within adequate cooking treatments and increased as cooking time decreased
using the medium and high temperature schedules. TPI was identified as a possible TTI in
ground beef when using the medium to high temperature processes.

The objective of this study was to investigate different factors that may affect TPI
activity and concentration in beef. The effects of muscle type, maturity of the animal, fat
concentration, and frozen storage of beef on TPI activity were investigated. The thermal
inactivation of TPI in four different steer muscles heated from 489°C to 767°C was
characterized. Frozen storage of cooked ground beef, refiigerated storage of raw and
cooked beef extracts, repeated freezing and thawing of raw and cooked ground beef, and
the effect of salt and phosphate in ground beef were studied to determine how these
conditions affect TPI activity and concentration.

3.3 MATERIALS AND METHODS
3.3.1 Source and preparation of beef for model system studies

The effect of muscle type and maturity on TPI activity in fresh and frozen beef was

determined using four different muscles fi'om three cows (D/E maturity) and three steers

(B maturity) obtained on the day of slaughter from Ada Beef (Ada, MI 49301). The

34

thermal inactivation of TPI was also determined in selected steer muscles. The following
muscles were analyzed: 1) trapezius (T, predominantly red muscle); 2) longissimus dorsi
(LD, predominantly white muscle); 3) vastus intermedius (VI, predominantly red muscle);
and 4) semimembranosus (S, predominantly white muscle). The muscles were stored in
Ziploc bags (Gordon Food Service, Inc, Grand Rapids, MI 49501), packed in ice filled
coolers and transported to Michigan State University. In order for use in future studies,
each muscle sample was divided into three equal portions prior to freezing. The muscle
samples were vacuum packaged and frozen to -13°C the same day.

The effect of fat on TPI activity was determined using a 115 - two piece boneless
chuck (34.9 kg), purchased from a local wholesaler and vacuum packaged the same day.
The chuck (4°C) was transported to Michigan State University Food Stores and processed
within one hour in a cutting room at 14°C. The lean and fat portions of the chuck were
divided and ground through a 4.8 mm diameter grinder plate in a Hobart grinder (Model
4146, Hobart Mfg. Co., Troy, OH 45374). Each portion was then separately mixed in a
Keebler mixer (Type VAV-TF-GR TE, Keebler Mfg. Co., Chicago, IL) at speed setting
0.] (57 rpm) for 2 min and ground through a 3.2 mm diameter grinder plate in the Hobart
grinder. The fat content of the lean and fat portions was determined by AOAC method
991.36 (AOAC, 1990). The fat and lean beef were combined to achieve fat
concentrations of 10, 20 and 30%. The fat and lean portions were blended 3 times at each
fat concentration for a total of 9 replicates in a 4°C refiigerator using a Hobart mixer
(Model KS-A, Hobart Mfg. Co., Troy, OH, 45374) at speed 2 for 2 min. The ground beef

was vacuum packaged and frozen to -80°C.

35

Ground beef containing 10% fat was also utilized to determine the effect of the
phosphate and salt concentrations on TPI activity. The 10% fat ground beef underwent
one freeze-thaw cycle and was stored at -13°C for two weeks prior to this study.

All other experiments were completed using chuck roasts (choice roast, A/B
maturity) purchased from a local store within 6 hr of purchase. The longissimus dorsi
(LD) muscles were excised and trimmed of external connective tissue. The muscles were
ground twice through a 3.175 mm diameter grinder plate in a Hobart grinder (Model KS-
A, Hobart Mfg. Co., Troy, OH 453 74). The ground beef was then mixed within a Ziploc
bag for 5 min to obtain a homogeneous sample and aliquoted prior to cooking.

3.3.2 Initial TPI in raw cow and steer muscles

After storage at -l3°C for one week, the vacuum packaged raw cow and steer
muscles (3 replicates/muscle) were thawed overnight in a 4°C refrigerator. The muscles
that were not immediately used were assayed for TPI activity after 5 and 10 months of
storage at -13°C. Each muscle was sampled in triplicate. After trimming excess fat and
connective tissue, 25 g of beef was cut with a knife into 2.5 cm x 2.5 cm pieces and then
blended using 3 volumes (w/v) cold phosphate buffer saline (PBS, 0.15 M NaCl, 0.01 M
Na phosphate buffer, pH 7.2) in a Waring blenderTM (Model 1120, Winsted, CT 06057)
for a total of 90 sec (45 sec on, 45 see off, repeated two times). The extract was stirred
for 15 min using a magnetic stir bar. The homogenate was immediately centrifuged at
4,500 x g for 10 min at 4°C. The supernatant was collected after filtration (Whatman No.

1) and stored on ice until assayed for TPI activity as described below.

36

3.3.3 Model system cooking of steer muscles

Due to the small sample size (30 - 60 g) of each frozen steer muscle, dry ice was
used to grind the meat (Bunch et al., 1995). Each muscle was sampled in triplicate. Meat
was frozen overnight at -80°C. Meat was then hammered into pieces weighing about 5 g.
Dry ice was chiseled into 20 g portions. The 5 g pieces of meat were ground with about
40 g dry ice in an Oster Blender for 15 sec (Model 869-18, Schaumburg, IL 60173). The
meat/dry ice mixture was transferred to aluminum foil and the dry ice was allowed to
evaporate for 45 min.

The ground beef was packed into a 60 mL syringe (Becton Dickinson and Co.,
Franklin Lakes, NJ, 07417). Two grams of beef were compressed through 7 cm of plastic
tubing into 10 x 75 mm borosilicate glass thermal death time (TDT) tubes (Fisher
Scientific®, Pittsburgh, PA 15219). The tubes were sealed with Teflon tape and heated in
a Polystat circulator bath (Model 1268-52, Cole-Parmer Instrument Company, Chicago,
IL) connected to a temperature programmer (Model 1268-62, Cole-Parmer). To monitor
the temperature of the meat, a resistance temperature detector (RTD, 1.6 mm diameter,
+/- 0.5°C) connected to a Solomat MPM 2000 Modumeter (Solomat Partners LP,
Glenbroon Industrial Park, Stanford, CT), was inserted into the center of a control tube
containing 2.0 g of meat. For each of the three replicates of the four steer muscles, three
tubes packed with meat were heated to internal temperatures of 489°C (120°F), 544°C
(130°F), 60°C (140°F), 656°C (150°F), 711°C (160°F), and 767°C (170°F). The
temperature of the water bath was set to 822°C (180°F). Once the control tube reached

the target temperature, the tubes were removed and immediately placed in an ice-water

37

bath. Protein was extracted from the meat and assayed for TPI activity and concentration
as described below.

3.3.4 Effect of fat on triose phosphate isomerase activity in cooked ground beef
Ground beef was cooked using two adequate USDA processing schedules
including a low and high temperature cook [661°C (151°F) for 41 sec, 694°C (157°F) for

10 sec]. Inadequate processing schedules were designed by reducing the adequate
processing time at each temperature by 0.5 log and 1.0 log (Table 3.1).

For each fat concentration, two grams of meat were packed into eighteen 10 x 75 mm
glass thermal death time (TDT) tubes and placed on ice. Nine of the test tubes were
cooked to the low temperature processing schedules (3 test tubes at each processing time)
and nine tubes were cooked to the high temperature schedules (3 test tubes at each
processing time). Type-T (copper-constantan) thermocouples (Part # TJ48-SCPSS-
O32G—325-OST-M, Omega Engineering, Inc, Stamford, CT 06907) were used to
monitor ground beef temperature during cooking and cooling. Thermocouples were
inserted into the first 9 test tubes to be cooked. The thermocouples were connected to 9
channels of a DanookT'fI Data acquisition system equipped with a DBK 19 Thermocouple
Card (Omega Engineering, Inc, Stamford, CT 06907). The DanookTM was connected to
a Zenith Data Systems 466X+ computer (Zenith Data Systems, St. Joseph, MI 49085)
loaded with Omegasoft® Daqview software, version 4.1 (Omega Engineering, Inc,
Stamford, CT 06907). Temperatures of the thermocouples were scanned and data was
acquired at a rate of 1 scan/sec. To increase the effective accuracy of a noisy signal the
averaging function was set to 50 (Omega Engineering, Inc, 1994). Once the starting

temperature of 1.2 i 03°C was attained, the test tube connected to the first channel being

38

Table 3.1-Thermal processing schedules used to prepare adequately and inadequately

processed ground beef in a model system

 

 

Low temp process High temp process
Adequate cooka 661°C (151°F)/41 sec 694°C (157°F)/10 sec
Inadequate cook 661°C (151°F)/13 sec 694°C (157°F)/3 sec
(0.5 log reduction in time)
Inadequate cook 661°C (151°F)/4 sec 694°C (157°F)/1 sec

(1.0 log reduction in time)

 

a Adequate cook schedules were based on the USDA-FSIS fiJlly-cooked patty

processing schedules (USDA-F SIS, 1997b).

39

utilized was submersed into the circulating water bath, as previously described. After
undergoing the desired thermal process, the test tube was immediately placed in an ice-
water bath. The procedure was continued for all remaining test tubes in both the low and
high temperature processes. Protein was extracted and assayed for TPI activity and
protein concentration as described below.
3.3.5 Ground beef cooking for storage studies

Thirty two 15 mL polypropylene centrifuge tubes (430052, Corning Costar Corp,
Oneonta, NY 13820) were each packed with 10 g of meat for the frozen cooked storage
study. Ten grams of meat were packed in four 15 mL centrifuge tubes for the refrigerated
storage studies. The tubes containing beef were placed in a test tube rack and were held
in an ice-water bath until cooked. A resistance temperature detector (RTD, 1.6 mm
diameter, +/- 0.5°C) connected to a Solomat MPM 2000 Modumeter (Solomat Partners
LP, Glenbroon Industrial Park, Stanford, CT) was inserted into the center of a control
tube containing meat which was placed in the middle of the rack. The test tube rack was
submersed in the Polystat circulator bath (Model 1268-52, Cole-Partner Instrument
Company, Chicago, IL) connected to a temperature programmer (Model 1268-62, Cole-
Parmer) and was removed and placed in an ice-water bath once the control tube reached
628°C (145°F). The temperature of the water bath was set to 71 . 1°C (160°F). Protein
was extracted from the meat and assayed for TPI activity and concentration as described
below.
3.3.5.1 Frozen storage stability of TPI in cooked ground beef

After cooking, ground beef was removed fiom the 32 centrifiige tubes, combined

and placed into a Ziploc bag. The meat, about 320 g, was hand mixed for 5 min to obtain

40

a homogeneous mixture. Approximately 10 g of cooked meat was aliquoted into each of
24 vacuum packaging bags. Meat from three of the bags was immediately assayed for TPI
activity and concentration. The concentration of protein was also determined. The 21
remaining bags were stored in a -13°C freezer to be assayed over time. Three samples
were thawed at 4°C and were assayed for TPI and protein concentration, and TPI activity
after four days of frozen storage as an initial check. Each month, for a total of five
months, three samples were removed and meat assayed for extractable protein
concentration, TPI concentration, and TPI activity as described below.
3.3.5.2 Refrigerated storage of meat extracts from cooked ground beef

After cooking, ground beef was removed from 4 centrifiige tubes and placed into a
Ziploc bag. The meat was hand mixed for 5 min to obtain a homogeneous mixture.
Protein was immediately extracted, as described in section 3.3.8, from three 5 g aliquots of
cooked meat and three 5 g aliquots of raw meat. Triose phosphate isomerase activity and
protein concentration were assayed and the results were used as the zero time point. Raw
and cooked meat extracts were assayed for TPI activity and protein concentration during
storage at 4°C for 1, 2, 3, 4, 8, 24, and 48 hr.

3.3.6 Effect of five freeze-thaw cycles on TPI activity and concentration of raw
and cooked ground beef

Raw ground meat was stuffed into 48 TDT tubes. The tubes were sealed with
rubber stoppers and Parafilm® (American National Canm, Chicago, IL 60631) and were
frozen at -13°C. The following day, all tubes were removed from the freezer and the meat
was thawed in a 25°C reciprocal shaking water bath (Model 25, Precision Scientific,

Chicago, IL 60647) for 45 min. Five tubes of meat were cooked to a temperature of

41

628°C (no holding time) using the DanookT“ Data acquisition system equipped with a
DBK 19 Thermocouple Card (Omega Engineering, Inc, Stamford, CT 06907) as
previously described, and then cooled in an ice—water bath. Three tubes containing thawed
raw meat and the tubes containing the cooked meat were placed on ice, while the
remaining thawed tubes were refrozen. The fi'eeze-thaw cycles were repeated four more
times at 24 hr intervals. After each freeze-thaw cycle, the raw and cooked meat were
assayed for extractable protein concentration, TPI concentration, and TPI activity as
described below.
3.3.7 Effect of phosphate and salt content on TPI activity in ground beef

Beef was thawed overnight in a 4°C refrigerator. Two hundred and fifty grams of
ground beef (9.3% fat) were blended in a Hobart mixer (Model KS-A, Hobart Mfg. Co.,
Troy, OH, 453 74) to obtain mixtures of either 1.0 % NaCl (2.5 g NaCl), 0.5%
tetrapotassium diphosphate (TPDP, 50% Food Grade Liquid Potassium Diphosphate,
Butcher & Packer Supply Co., Detroit, MI 48207) (2.5 mL TPDP), or 1% NaCl and 0.5%
TPDP. A control sample was prepared the same way by adding 2.5 mL H20 before
blending. Ground beef was blended 3 times for each mixture at 4°C.

Two grams of meat from each ground beef mixture were packed into six 10 x 75
mm TDT tubes and placed on ice as previously described. Three of the test tubes were
cooked to 628°C and three test tubes were cooked to 694°C and monitored using the
DanookTM Data acquisition system equipped with a DBK 19 Thermocouple Card
(Omega Engineering, Inc, Stamford, CT 06907) as previously described, and then cooled
in an ice-water bath. Protein was extracted and assayed for TPI activity and protein

concentration as described below.

42

3.3.8 Extraction of protein from cooked beef

The 2.0 g cooked beef was transferred from the TDT tubes into scintillation vials
(Research Products International Corp, Mount Prospect, IL 60056). Meat was blended
with 3 volumes (w/v) of PBS buffer by mixing for 30 sec on a Fisher Vortex Genie2TM
(Scientific Industries, Inc, Bohemia, NY 11716). Extracts were then stirred on a
magnetic stir plate, without foaming, for 15 min at 4°C. Extracts were immediately
centrifuged at 4,500 x g for 10 min at 4°C. Supematants were collected after filtration
(Whatman No. 1) and were held on ice until assayed for TPI activity and concentration.
3.3.9 TPI activity

Triose phosphate isomerase activity was determined as described by Bergmeyer
(1984) except that 1.0 mL triethanolarnine buffer (TEA, 0.2M, pH 8.0), 0.2 mL 15mM
glyceraldehyde—3-phosphate (GAP), 10 uL B-nicotinamide adenine dinucleotide (NADH,
10 mg/mL), 10 BL glycerol-3-phosphate dehydrogenase (GDH, 1.5 mg/mL), and 10 BL
of protein extract were used in the assay solution. The change in absorbance was read at
340 nm for 1 min at 25°C. Meat extracts were diluted in water so that the change in
absorbance per min was less than 0.2 (Beisenherz, 1955; Norton et al., 1970). One unit
(U) of activity was defined as the conversion of 1 umol GAP into dihydroxyacetone
phosphate per minute (Norton et al., 1970). TPI activity was calculated based on the

following formula:

43

IOOOxAxV

 

Activity (U/L) =
t x e x d x ((p/D)

A = absorbance

V = assay volume, mL

t = time (min)

a = extinction coefficient (6.310), L x mmol'l x mm1

d = distance of light path, 10 m

(p = sample volume, mL

D = dilution of sample
3.3.10 TPI concentration

The TPI concentration was quantified using a sandwich ELISA (Hsu, 1997). The

sandwich ELISA was performed by coating Immulon® 2 Removawell Strips (Dynatech
Laboratories, Inc, Chantilly, VA 22021) with 100 1.11.. of bovine muscle TPI polyclonal
antibody (PAb), the capture antibody, diluted (1/250) in 0.1 M carbonate buffer, pH 9.6,
and drying overnight at 37°C. Plates were washed four times with PBS containing 0.05%
Polyoxyethylene sorbitan monolaurate (Tween 20) (PBS-T). Nonspecific binding sites
were blocked by adding 300 BL of PBS containing 0.5% casein and 0.1% Tween (PBS-
CT) to each well and incubating at 37°C for 30 min. Plates were washed four times and
50 ILL of serially diluted bovine muscle TPI or diluted ground beef extracts was added to
each well and incubated at 37°C for 30 min. After four washings, biotin-labeled bovine
muscle TPI PAb, for detection, was diluted (1/100) in PBS-CT, and 50 BL was added to
each well and incubated at 37°C for 30 min. After incubation, the plate was washed four

times with PBS—T and 100 uL of avidin-horseradish peroxidase (HRP) conjugate

44

(Sigma®) diluted (1/8000) in PBS-CT was added to each well. Plates were incubated for
30 min at 37°C and washed eight times with PBS-T. Peroxidase binding was determined
with ABTS (2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) substrate (Pestka,
1982). After 30 min, absorbance was read at 405 nm using a microplate reader
(THERMOmax, Molecular Devices Co., Menlo Park, CA). On each plate, purified bovine
muscle TPI (Hsu, 1997) was used to prepare a standard curve ranging from 0 to 40 pg
TPI/mL. Results were expressed as micrograms of TPI per gram of meat.

3.3.11 Bradford extractable protein determination

Protein concentration of meat extracts was determined using the Bradford method
with bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, MO 63178) as the
standard (Bradford, 1976). The BSA standard curve ranged from 10 to 913 pg
protein/mL. Ten microliters of diluted meat extract and 200 uL of Coomassie blue G-250
dye reagent (500-0006, Bio-Rad°’) were added to each microwell (Immulon® 1
Removawell Strips, Dynatech Laboratories, Inc, Chantilly, VA 22021) and incubated at
room temperature for 5 min. Absorbance was read at 590 nm using a microplate reader
(THERMOmax, Molecular Devices Co., Menlo Park, CA).

3.3.12 Proximate composition

The moisture, fat, and protein contents of the beef samples were determined
according to AOAC methods 950468, 991.36, and 981.10, respectively (AOAC, 1990).
For pH determination, 10 g meat was homogenized with 90 g deionized water using a
Polytron homogenizer (Model PT 10/35, Brinkmann Instrument, Co., Westbury, NJ

11590) equipped with a PTA 10TS generator. The sample was stirred for two periods of

45

30 sec at 5.0 speed setting and the pH was measured while the solution was continuously
stirred over a magnetic stirrer.
3.3.13 Statistical analysis

Data were analyzed using analysis of variance in the General Linear Model
Program of the Statistical Analysis System (SAS Institute, Inc, 1996). Least square
means procedures were used to separate means at P<0.05. Animal was a random effect
when analyzing the effects of muscle type and maturity of the animal on TPI activity.
Analysis of covariance was used to determine if TPI concentration was an accurate
predictor of TPI activity. The linear or curvilinear change in TPI activity in different fat
contents of beef was determined using the orthogonal polynomial contrast test by SAS
software (Release 6.12). Tukey's test was used to test significant differences among
means at P<0.05 for storage and freeze-thaw studies.
3.4 RESULTS AND DISCUSSION
3.4.1 Effect of muscle type and maturity on TPI activity in muscle

The proximate composition and the pH of 4 muscles from cows and steers varied
by muscle and gender. The cow longissimus dorsi (LD) contained 8.4% fat, the highest
among the four cow muscles (Table 3 .2). The vastus intermedius (VI) in steer had 7.6%
fat, the highest among the four steer muscles. The V1 muscle in cow contained the highest
moisture content of 74.2%. All four steer muscles contained similar moisture
concentrations, ranging from 70.6 to 71.8%. The semimembranosus (S) in steer had the
highest percent protein of 24.6%. The trapezius (T) muscle in cow had the highest
percent protein of 24.8%. The steer and cow VI had the highest pH of 6.2 and 6.8,

respectively.

46

Table 3.2-Proximate composition and pH of cow and steer musclesa

 

 

 

 

 

Muscleb Cow Steer
Fat (% w/w)
Longissimus dorsi 8.4 a: 1.0 5.2 :t 0.8
Semimembranosus 5.2 i 4.5 3.7 d: 1.4
Trapezius 3.7 :t 2.0 6.3 i 3.3
Vastus intermedius 4.5 i 0.6 7.6 i 1.7
Moisture (% w/w)
Longissimus dorsi 69.9 i 0.6 71.8 :1: 1.4
Semimembranosus 73.1 i 1.2 71.3 i 1.2
Trapezius 70.5 i 0.9 70.6 i: 2.5
Vastus intermedius 74.2 d: 0.8 71.1 d: 1.2
Protein (% w/w)
Longissimus dorsi 21.4 i 0.4 22.6 :t 1.4
Semimembranosus 24.4 i 1.9 24.6 :t 1.6
Trapezius 24.8 :h 1.1 22.3 i 1.1
Vastus intermedius 20.3 :t 1.3 20.1 :t 2.0
pH

Longissimus dorsi 6.2 :t 0.4 —— 5.4 d: 0.1
Semimembranosus 5.3 :1: 0.1 5.1 :t 0.1
Trapezius 6.1 i 0.3 5.7 :1: 0.3
Vastus intermedius 6.8 i 0.1 6.2 :t 0.0

 

° Means i standard deviation of 3 replicate values.

b . . . . . .
Longrssrmus dorsr and senumembranosus are white muscles. Trapezrus and vastus

intermedius are red muscles.

47

The above results are in agreement with others who reported the proximate
composition and pHs of muscles from cow and steer carcasses. Browning et al. (1990)
reported the percent moisture, fat and protein of a composite of 10 muscles from eight
typical steers to be 72.69, 5.89 and 20.80%, respectively. Talmant et al. (1986) reported
the pHs of trapezius, longissimus dorsi and semimembranosus muscles from cows (D/E
maturity) and steers (B maturity) to be 5.8, 5.6 and 5.6, respectively. The proximate
composition and pHs of the selected cow and steer muscles used in this study were found
to be similar to those found in typical beef carcasses.

Differences (P<0.05) in TPI activity were found between muscles in both the cows
(D/E maturity) and steers (B maturity) (Table 3.3). The cow trapezius had a TPI activity
of 2213 U/kg meat, the highest among the four cow muscles. The steer trapezius,
semimembranosus, and Iongissimus dorsi muscles all had greater TPI activities than the
vastus intermedius. The cow and steer vastus intermedius had the lowest TPI activities of
595 and 796 U/kg meat, respectively. In both cows and steers, the largest difference in
TPI activity was in the round muscles. The steer and cow semimembranosus had TPI
activities of 3704 and 1985 U/kg meat, respectively, while the steer and cow vastus
intermedius had TPI activities of 796 and 595 U/kg meat, respectively. Stalder et al.
(1991) found a large difference in LDH activities in the round muscles with the bovine
semimembranosus having 1476 U activity and the vastus intermedius having 283 U
activity. This data is in agreement with Talmant et al. (1986) who reported that muscle
type has a large influence on glycolytic enzyme activity.

Differences in TPI activity were nonsignificant for carcasses differing by maturity.

Talmant et al. (1986) reported that glycolytic enzyme activities do not change after the

48

Table 3.3-Triose phosphate isomerase activity (U/kg meat) of muscles from cows

 

 

and steers

Muscle Cow Steer
Chuck trapezius 2213.4 :1: 7924° 1950.3 :t 282.3a
Round semimembranosus 1984.6 4: 667.6° 3703.6 a: 945.23
Chuck longissimus dorsi 17544 d: 398.4° 2926.9 :1: 417.93
Round vastus intermedius 595.0 i 231.0° 796.3 at 154.3b

 

H Mean :1: standard deviation in the same column followed by the same

letter are not different (P>0.05).

49

age of six months. Previous research indicates that gender of the animal has no effect on
the activities of glycolytic muscle enzymes (Talmant et al., 1986; Stalder et al., 1991).
Therefore, the effect of gender on TPI activity was not investigated.

To use TPI as a TTI to monitor the adequate thermal processing of beef patties,
the TPI activity within the raw beef should initially be high so that a measurable decrease
can be detected after cooking. High levels of TPI activity were found within both the
round and chuck muscles, ones commonly used for the production of ground beef. The
average TPI activity was 1636 and 2344 U/kg meat for the cow and steer muscles,
respectively. Due to a variety of muscles used for the production of ground beef, TPI
activity of raw ground beef should be similar to the average activity from a number of
muscles.

3.4.2 Frozen storage of steer muscles

Triose phosphate isomerase activity did not change (P>0.05) in the raw steer
muscles during frozen storage at -13°C for 10 months (Table 3.4). Frozen storage had no
effect (P>0.05) on the TPI specific activity of the longissimus dorsi and vastus
intermedius muscles during 10 months of frozen storage. The TPI specific activity in the
trapezius and semimembranosus muscles did not change after the initial 5 months of
storage, but increased in the trapezius and decreased in the semimembranosus after an
additional 5 months of frozen storage. The discrepancy between the constant activity and
changing specific activity of these two muscles can be attributed to the change in
extractable protein content during frozen storage. Between 5 and 10 months of frozen
storage, the trapezius showed a decrease (P<0.05) in extractable protein, which increased

the ratio of activity to milligrams of extracted protein, which resulted in the increased

50

Table 3.4-Stability of triose phosphate isomerase activity and extractable protein

concentration in selected steer muscles during fiozen storage at -l3°C
Steer muscle
Trapezius Semimembranosus Vastus

Time Longissimus
intermedius

(months) dorsi
TPI activity (U/kg meat)

2926.8 :1: 417.9a 1950.3 3: 265.7”I 3703.6 i 854.63 796.3 :1: 141.03
2381.4:t307.1° 4513.0i 1131.0a 893.41 151.6a

5 2870.1 3: 587.93
2366.8 :t 971.3a 3771.5 :1: 996.8° 953.1:h123.8°

10 3349.7 i 862.4a

 

TPI specific activity (U/g protein)

 

 

 

86.6 4 11.3“ 59.4 4 10.7“b 105.2 4 27.2“ 26.9 4 5.7“

5 66.3 416.8“ 53.74 7.9b 90.04154“b 25.94 5.5“

10 81.74198“ 71.24244“ 72.24224b 31.34101“
Extractable protein concentration (mg/mL)

0 11.3407“ 11.0407” 11.8405“ 9.9405“

5 14.6 419" 14.9 41.8b 16.6 4 2.5b 11.6 41.1“

10 13.7 41.3“b 11.14 2.1‘2 17.7 4 2.3b 10.9 4 2.8“

 

°'° Mean :1: standard deviation in the same column followed by the same letter are not
different (P>0.05).

51

specific activity. The extractable protein in the semimembranosus increased during 10
months of frozen storage, which decreased the ratio of activity to milligrams of extracted
protein and resulted in the decreased specific activity.

The changes in the extractable protein concentration of the steer muscles could
have been caused by the microstructural alterations that the bovine muscles underwent
during frozen storage (Awad et al., 1968). Ice crystal formation within the muscle tissue
could have led to the denaturation of the beef muscle proteins. The changes in the
extractability of the muscle proteins would be expected if protein denaturation had
occurred.

3.4.3 Thermal inactivation of triose phosphate isomerase in selected steer muscles

TPI activity in the four steer muscles decreased as cooking temperature was
increased from 48.9 to 769°C (Fig. 3.1). There were significant effects on TPI activity
due to muscle, animal and cooking temperature. Muscle x animal and muscle x
temperature were significant interactions, however animal x temperature was not. TPI
activity in the two chuck muscles, trapezius and longissimus dorsi, decreased from 48.9 to
713°C. The TPI activity in the round muscles was more heat labile as the
semimembranosus and vastus intermedius had their lowest TPI activities when heated to
657°C and above. The semimembranosus muscle had the greatest decrease in TPI
activity of 861 U/kg meat between 48.9 to 545°C. In all four muscles, there was no
difference in TPI activity between the 71.3 and 769°C cooking temperatures.

The TPI concentration of the four steer muscles decreased as the cooking
temperature of the beef was increased (Fig. 3.2). There were differences in TPI

concentration due to muscle and cooking temperature, however animal had no effect

52

 

1 400

  
   
 
  

 

 

 

 

 

 

 

 

1200 - .
+Trapezrus
-l- Longissimus dorsi
1': 1°00 ‘“ + Vastus intermedius
a
‘5’ + Semimembranosus
is" 800 -
a
.E,
E 600 --
U
4:
~
on
E"
400 ~
200 -~
0 % ‘
48.9 54.5 60.1 65.7 71.3 76.9

Endpoint Temperature (0 C)

Fig. 3.1-Effect of heating temperature on triose phosphate isomerase activity
(U/kg meat) of selected steer muscles. Standard error of the means is at 120.9.

53

 

  
   
   

 

 

 

 

 

 

1400 «~
1 .
l +Trapezrus l
1200 . l . . .
[ +Longrssrmus dorsr
l +Vastus intermedius
g 1000 “ l + Semimembranosus
Q A
'4': E!
g ‘5'
5 on 800 ,
g D
o E:
U on
a 3 600 ~
[-
200 +
0 T . + .
48.9 54.5 60.1 65.7 71.3 76.9

Endpoint Temperature (° C)

Fig. 3.2-Effect of heating temperature on triose phosphate isomerase content
(pg TPI/g meat) of selected steer muscles. Standard error of the means is :t 34.8.

54

(P>0.05). Muscle x animal and animal x temperature were significant interactions,
however muscle x temperature was not. At 769°C, the lowest concentration of TPI was
173.7 ug/g meat in the semimembranosus muscle. No differences were found in TPI
concentrations in all four steer muscles between the cooking temperatures of 48.9 to
545°C. The trapezius showed a decrease in TPI concentration fi'om 48.9 to 601°C. TPI
concentration decreased only in the longissimus dorsi from 65.7 to 769°C.

Triose phosphate isomerase concentration was not a good predictor (P>0.75) of
TPI activity in the ground beef model system. Both the TPI activity and concentration
decreased as the cooking temperature was increased, however, the difference in TPI
activity was more evident. The average decrease in TPI activity among all four muscles
was 99.4%, while only a 73.5% decrease in TPI concentration on heating from 48.9 to
769°C was observed. The vastus intermedius, while having among the lowest TPI
activities of the four steer muscles assayed, had the highest concentration of TPI over all
cooking temperatures. The TPI activity of the semimembranosus muscle decreased by
1227 U/kg meat as the temperature was increased from 48.9 to 769°C, the largest
decrease in activity seen among all four muscles. However, the TPI content of the
semimembranosus only decreased by 361 ug TPI/g meat from 48.9 to 769°C, the smallest
decrease in TPI content among all four muscles. When Hsu (1997) measured the TPI
activity and concentration of ground beef after heating to EPTs ranging from 48.9 to
769°C, the change in TPI activity with cooking temperature was more apparent than the
change in TPI concentration.

Hsu (1997) produced anti-bovine TPI PAb to be used in the sandwich ELISA.

The poor specificity related to the use of PAb could have contributed to the less apparent

55

change in TPI content vs. TPI activity. The development of monoclonal antibodies that
recognize an epitope related to the conformational changes that TPI undergoes during
heating might enhance the specificity of the assay.

3.4.4 Effect of fat on triose phosphate isomerase activity in ground beef

The fat content of ground beef was found to have an effect on TPI activity of the
cooked product (Table 3.5). At both the low and high temperature processing schedules,
the TPI activities in the cooked ground beef containing 9.3 and 20.0% fat did not differ,
but activities were higher than cooked beef containing 30% fat. TPI activity varied due to
the batch of meat used, although identical procedures were maintained from batch to
batch. The temperature and time of cook were significant effects on TPI activity, as was
the interaction (temperature x time) of the two.

Within all three fat contents, the TPI activity was similar at 66.1 and 694°C when
the ground beef was inadequately processed by reducing cook time by 1.0 log, but
different at the hill cook and 0.5 log reduction in holding time processes. At 661°C, TPI
activity linearly decreased as the cook time was increased. Time had a quadratic effect on
TPI activity in beef cooked to 694°C. Smith et al. (1991) reported TPI activity when
ground beef was heated to 70°C, but not at 80°C. Orta-Ramirez et al. (1997) reported
that TPI was inactivated in ground beef before reaching 68°C. Research showing TPI to
be close to its thermal inactivation point at 694°C may explain the quadratic, rather than
linear decrease in TPI activity in beef cooked to 694°C.

The results in the model system show that TPI could not be used as a TTI to
determine the adequate thermal processing of ground beef because activities were different

at the lowest and highest temperatures of the USDA-FSIS processing schedules for beef

56

Table 3.5-Triose phosphate isomerase activity (U/kg meat) of ground chuck of differing
fat contents processed using two USDA approved schedules for ground beef and
inadequately processed by reducing the holding time by 0.5 and 1.0 log cycles

Low temperature cookA

(661°C; 151°F)

High temperature cookB

(694°C; 157°F)

 

 

 

 

 

 

9.3% Fat
Full cook 7.3 4 2.0“ 9.7 4 4.6b
Inadequate cook 9.1 :t 16° 12.4 :1: 31°
(0.5 log reduction in time)
Inadequate cook 10.5 4 26° 10.5 d: 19°|
(1.0 log reduction in time)

20.0% Fat
Full cook 6.2 4 1.7“ 9.6 4 1.9b
Inadequate cook 8.9 4 22° 9.8 4 38°
(0.5 log reduction in time)
Inadequate cook 9.6 4 24° 9.5 :1: 33"
(1.0 log reduction in time)

294% Fat
Full cook 5.1 4 2.4“ 6.6 4 2.0b
Inadequate cook 6.1 4 29° 8.2 4 1.9b
(0.5 log reduction in time)
Inadequate cook 6.1 i 29° 6.2 i 26°

(1.0 log reduction in time)

A Means i standard deviation in the same column for each fat content showed a linear
response to temperature (P<0.05).
B Means i standard deviation in the same column for each fat content showed a quadratic
response to temperature (P<0.05).
°’° Means in the same row followed by the same letter are not different (P>0.05).

57

patties. In order for TPI to function as a TTI, TPI activity should be Similar within
equivalent adequately and inadequately schedules at the lowest and highest temperatures.
Results of Wang et al. (1996b), Orta-Ramirez et al. (1997) and Hsu (1997) were used in
selecting TPI as a possible TTI to determine the adequate thermal processing of ground
beef. Wang et al. (1996b) reported that several proteins including, TPI, LDH,
glyceraldehyde-3-phosphate dehyrogenase (GAPDH), phosphoglycerate mutase (PGAM),
and acid phosphatase (AP) might be usefirl in assays to verify the adequate cooking of
ground beef patties. Hsu (1997) investigated several enzymes as possible indicators of the
adequate thermal processing of roast beef and reported TPI activity to be the same in roast
beef when equivalent processes were used at low and high temperatures. Orta-Ramirez et
al. (1997) showed the temperature dependence of TPI to be similar to those of E. coli
0157:H7 and S. senftenberg. TPI was found to have a similar 2 value to that of
Salmonella, the indicator organism used by the USDA-FSIS to create the lethality
performance standards (Orta-Ramirez et a1, 1997). The 2 value of TPI was determined
over the temperature range of 53 to 66°C, while the USDA-FSIS beef patty
time/temperature processing schedule covers 66.] to 694°C. The temperature range from
which the 2 value for TPI was determined may have contributed to the lack of similarity
between the low and high temperature TPI activities within the model system (Taoukis
and Labuza, 1989; Van Loey et al., 1996).

The beef in the model system was cooked to the lowest and highest temperatures
of the USDA approved processing schedules for ground beef. The firlly cooked ground
beef had lower TPI activities when cooked to the lowest temperature of 661°C for 41 sec

(equivalent to 71°C instantaneously) at all fat contents. While TPI was determined to be

58

unsuitable for use as a TTI in a ground beef model system, TPI might be a suitable EPT
indicator in ground beef. The residual TPI activities at 661°C for 41 sec determined for
each fat content of ground beef could be proposed as the maximum allowable activities to
verify the adequate thermal processing of beef patties. For example, the 20.0% fat ground
beef cooked to the USDA-F SIS low temperature schedule, 661°C (151°F) for 41 sec, had
a TPI activity of 6.2 U/kg meat. The TPI activity of the 20% fat ground beef processed to
the high temperature schedule, 694°C (157°F) for 10 sec, was 9.6 U/kg meat. Therefore,
it is suggested that any 20% fat beef patty having a TPI activity less than 6.2 U/kg meat
could be considered fully cooked.

Further studies are needed to establish a maximum TPI activity for ground beef
patties cooked in a commercial setting to the recommended USDA endpoint processing
temperature of 71 . 1°C. Once a maximum TPI activity is established for fully cooked beef
patties, TPI activity can possibly be used as an indicator of the adequate thermal
processing of ground beef patties.

3.4.5 Effect of frozen storage of cooked ground beef

The effect of frozen storage (-13°C) on TPI activity and concentration of ground
beef (9.5% fat) cooked to 628°C was studied for 5 months (Table 3.6). After four days
of frozen storage, the TPI activity increased (P<0.05), while the TPI concentration
decreased as compared to the unfrozen cooked beef. Both the TPI activity and
concentration increased after two months of frozen storage, and then decreased after an
additional month of storage. After three months of fi'ozen storage, TPI concentration did
not change. TPI activity did not change from three to four months of frozen storage, but

increased after an additional month. A TPI activity similar to that of the unfrozen cooked

59

Table 3.6-Effect of frozen Storage (-13°C) on triose phosphate isomerase in cooked

 

 

(628°C) ground beef

TPI activity TPI conc

(U/kg meat) (11g TPI/g meat)
Initial cook 21.5 4 1.9“b 1582.2 4 544.7“
4 Day 34.1 4 09C 881.4 4 122.4bc
One month 26.8 4 1.0d 987.7 4 111.4bd
Two month 33.1428c 1373.3 4492.6“l
Three month 17.6 4 4.7b 486.1 4 1893““
Four month 17.2 4 1.9b 312.0 4 1247'“
Five month 22.8 4 2.5“d 432.3 4 1888““

 

°'° Mean 3: standard deviation in the same column followed by the same letter are not

different (P>0.05).

6O

beef was seen after five months of frozen storage. The changes in activity and
concentration could have been caused by the microstructural and chemical alterations that
the bovine muscle underwent during frozen storage (Awad et al., 1968). These changes
can cause the leakage of drip, containing amino acids, salts, proteins, and peptides, from
thawed muscle (Forrest et al., 1975).
3.4.6 Effect of refrigerated storage of protein extracts

Due to time constraints, TPI activity was not always assayed immediately
following protein extraction. As the meat extracts were held at 4°C until assayed, the
effect of refrigerated (4°C) storage of raw and cooked beef protein extracts on TPI
activity was investigated. The TPI activity in the raw extracts did not change (P>0.05)
during the first 4 hr of storage (Fig. 3 .3). After one hr of refiigerated storage, the TPI
activity of the cooked beef extract decreased as compared to the TPI activity observed at
the zero time point. The cooked beef extract increased in TPI activity after 3 hr of
storage. The lowest TPI activities of both the raw and cooked beef extracts, 1502 and 7.2
U/kg meat, respectively, were after 8 hr of refiigerated storage. During 8 to 24 hr of
refrigerated storage, both the TPI activity of raw and cooked beef extracts increased to
levels similar to that of the zero time point and did not change thereafter. These results
suggest that TPI activity may fluctuate during the initial hours of storage in refrigerated
(4°C) conditions. Therefore, the TPI activity of meat extracts was determined after an 8

hr holding period throughout our studies.

61

 

 

 

 

 

 

 

 

 

 

 

 

3000 14

a 12 ‘3
*5 2500 a
g a
u 10 kl
E A 2000 « A 95
3 a ,_ 8 3' a:
““ “ “é a
G

a é .. 5 é o
u- : D U
G v v .-
4 1000 ‘~ 0
u 4. .
< ‘ t:
E + Raw Extract <
I. 500 -~ . 2 E
-l- Cooked Extract “ I-

0 4 t 4 t t : t t 4 t i 0
0 4 8 12 16 20 24 26 32 36 40 44 48
Storage Time (hr)

Fig. 3.3-Effect of refrigerated storage (4°C) of protein extracts on triose phosphate
isomerase activity (U/kg meat). Standard error of the means for raw beef extracts
is :t 119.5. Standard error of the means for cooked beef extracts is i 0.4.

62

3.4.7 Effect of freeze-thaw cycles on TPI activity and concentration of raw and
cooked ground beef

Five freeze-thaw cycles had no effect (P>0.05) on TPI concentration or extractable
protein concentration of raw ground beef (Table 3.7). Orta-Ramirez et al. (1996)
reported that five freeze-thaw cycles had no effect on the LDH concentration of raw
ground beef. The TPI activity of the raw and cooked beef decreased after one freeze-
thaw cycle. After the second freeze-thaw cycle, TPI activity increased in the raw beef and
did not change thereafter. Collins et al. (1991b) reported one freeze-thaw cycle to
decrease LDH activity in raw ham muscles.

Three freeze-thaw cycles decreased TPI activity by 57.2% in the cooked beef.
However, after a fourth freeze-thaw cycle, TPI activity in the cooked beef increased.
After two freeze-thaw cycles, the TPI concentration of the cooked meat was greater than
the unfrozen cooked meat, but returned to concentrations similar to the unfrozen meat
after additional freeze-thaw cycles. The changes in TPI activity and concentration of the
cooked beef can be attributed to the damage brought upon by the formation of ice crystals
within the muscle tissue. Ice crystal formation within the meat can damage cell
membranes allowing TPI to be lost into the drip (Collins et al., 1991b; Orta-Ramirez et al.,
1996). The location of the drip, as well as the amount of drip within the test tube, will
dictate how heat susceptible TPI will be when cooking the beef in the water bath. The
ground beef in test tubes containing greater amounts of drip loss, and therefore more TPI
in the drip, would be expected to have lower TPI activities after cooking. Another factor

contributing to changes in TPI activity and concentration of the cooked beef may be

63

Table 3.7-Effect of five freeze-thaw cycles on triose phosphate isomerase and extractable

protein concentration in raw and cooked (628°C) ground beef

 

 

 

 

 

 

 

Cycle Raw meat Cooked meat
TPI activity Percent of TPI activity Percent of
(U/kg meat) initial activity (U/kg meat) initial activity
Initial 2131.1 4 368.2“b 100.0 68.4 4 15.6“b 100.0
1 1774.4 4 224.2b 89.2 54.0 4 21.2b 79.4
2 2235.3 4 238.3“ 109.0 56.4 4 11.0““ 82.4
3 1902.1 4 1699““ 90.8 29.3 4 8.5c 42.8
4 1916641546“b 92.5 77.44258“ 113.2
5 2289.9 4 250.9“ 110.5 53.5 4 10.3b 78.2
TPI concentration Percent of TPI concentration Percent of
(pg/g meat) initial conc. (ug/g meat) initial conc.
Initial 510.7 4 262.3“ 100.0 313.4 4 64.5““ 100.0
1 478.8 4 216.4“ 93.9 384.4 4 81.8“b 122.3
2 520.6 4 137.8° 102.1 778.5 4 241.1c 248.3
3 250.6 4 188.8“ 49.0 302.4 4 122.8b 96.5
4 486.3 4 248.7“ 95.3 500.5 4 2625°° 159.6
5 552.8 4 2982° 108.2 524.5 4 258.2“ 167.3
Extractable protein Percent of Extractable protein Percent of
(mg/mL) initial conc (mg/mL) initial cone.
Initial 11.4 4 0.5“ 100 5.3 4 0.3“ 100
1 11.5408“ 100.9 5.9406“b 111.3
2 11.8403“ 103.5 6.0401“b 113.2
3 12.3 40.7“ 107.9 5.4404'“ 101.9
4 12.7409“ 111.4 6140.4b 115.1
5 111401“ 97.4 5.5403“b 103.8

 

°'° Means 1 standard deviation in the same column followed by the same letter are not
different (P>0.05).

64

changes in the extractability of the ground beef muscle proteins caused by the denaturing
of the proteins during each freeze-thaw cycle.
3.4.8 Effect of phosphate and salt content in ground beef on TPI activity

The cooking temperature (62.8 and 694°C), phosphate and salt content had
significant effects on TPI activity of the ground beef (9.3% fat), as did the temperature x
ingredient (phosphate and salt) interaction. Wang et al. (1996b) and Hsu (1997) reported
cooking temperature to have a significant effect on TPI activity in ground beef. At
694°C, the ground beef with 1.0% NaCl had a lower TPI activity than the control (Table
3.8). Stalder et al. (1991) reported a decrease in LDH activity in semimembranosus
slurries as the salt concentration was increased. At 628°C, ground beef with 0.5%
tetrapotassium diphosphate (TPDP) and 1.0% NaCl, and the ground beef with only 0.5%
TPDP, had higher TPI activities than the control. The addition of TPDP increased the pH
ofthe beeffrom 5.9 to 6.2.

The increased extractability of the beef muscle proteins in the presence of the
TPDP might explain the increased TPI activities as compared to the control beef.
Extractable protein increases as the pH of the beef is increased above the isoelectric point
of the muscle proteins (Saflle and Galbreath, 1964; Prusa and Bowers, 1984). Van Den
Cord and Wesdorp (1978) found an increase in extractability of pork muscle proteins in
the presence of increasing pyrophosphate (0.01-1%) concentrations. Similarly, Fukazawa
et al. (1961) showed that protein extracted from beef and rabbit muscle myofibrils
increased in the presence of sodium pyrophosphate, tripolyphosphate, and

hexametaphosphate.

65

Table 3.8-Effect of NaCl, tetrapotassium diphosphate (TPDP) and heating temperature

on triose phosphate isomerase activity (U/kg meat) in ground beef

 

 

628°C 694°C
Control 31.9 i 110° 8.4 4 32°
1.0% NaCl 5941.2“ 2,740.5b
0.5% TPDP 841.2 4 211.9b 7.2 4 0.9“
1.0% NaCl and 0.5% TPDP 135.5 4 507C 8.4 4 2.4“

 

°’° Means :1: standard deviation in the same column followed by the same letter are not

different (P>0.05).

66

At 694°C, the ground beef with 0.5% TPDP and 1.0% NaCl, and the ground beef
with 0.5% TPDP had similar TPI activities to the control. Past studies showing TPI to be
close to its thermal inactivation point at 694°C may explain why the addition of 0.5%
TPDP had no effect on the TPI activity of the ground beef cooked to 694°C (Smith et al.,
1991; Orta-Ramirez et al., 1997). Current USDA-F SIS regulations require that beef
patties be cooked to 694°C for 10 sec. The addition of phosphate salts and NaCl to beef
should not affect the TPI activity of ground beef patties cooked to an internal temperature
of 694°C.

3.5 CONCLUSIONS

Muscle type had a significant effect on TPI activity. However, the assaying of TPI
activity from a ground beef patty would be similar to taking an average TPI activity from a
number of muscles. Therefore, the significant muscle effect would not negatively inhibit
the use of TPI activity as an indicator of the adequate thermal processing of ground beef
patties. During storage at -13°C for 5 months, the TPI activity in cooked ground beef
increased and decreased from the initial cook levels. As the fat content of ground beef
was increased, TPI activity decreased. TPI was found not to be able to be used as a TTI
to verify the adequate thermal processing of ground beef in this model system because
activities were different at the lowest and highest temperatures of the USDA-FSIS
processing schedules for beef patties. However, TPI might be a suitable indicator to
determine the EPT of cooked ground beef. The residual TPI activities at 661°C for 41
sec (equivalent to 71°C instantaneously) for each fat content of ground beef could be
proposed as the maximum allowable activities to verify the adequate thermal processing of

beef patties. The USDA advises consumers to cook beef patties to an internal EPT of

67

71 .1°C. Further study is needed to detemrine a maximum allowable TPI activity for
ground beef patties that are cooked in a commercial setting to the recommended USDA

processing temperature.

CHAPTER 4
INDICATOR PROTEINS AND COLOR MEASUREMENTS AS METHODS TO
DETERMINE ADEQUATE THERMAL PROCESSING OF GROUND BEEF

PATTIES

4.1 ABSTRACT

The objective of this study was to determine whether the EPT of ground beef
could be differentiated using TPI activity and LDH concentration and to compare the
results to the internal color of the beef patties. Ground beef patties (24.4% fat) were
cooked to 5 endpoint temperatures (EPT) ranging from 60 to 822°C. Triose phosphate
isomerase (TPI) activity, lactate dehydrogenase (LDH) concentration and the internal
color of the beef patties were quantified to determine if these assays could be used to
differentiate between adequately and undercooked beef patties. TPI activity of beef
patties decreased from 20.26 to 6.28 U/kg meat between 65.6 and 71 . 1°C. LDH
concentration decreased from 3.93 to 0.02 ug/g meat from 71.1 to 767°C. Beef patties
cooked to 65.6 and 71 . 1°C had different primary degree of doneness scores, as determined
by a sensory panel. Both the a* and b* values of the ground beef patties decreased as the
EPT was increased, however temperature had no effect on the L“ values. While the
primary degree of doneness scores, a* and b* values were able to detect differences
between undercooked and fully cooked beef patties in this study, past research has shown

visual and instrumental color measurements to be poor indicators of adequate thermal

68

69

processing. Using TPI activity, undercooked ground beef patties were able to be
distinguished from those that had been adequately cooked. TPI may be usefiil as an EPT
indicator to verify the adequate thermal processing of ground beef patties.

Key words: Ground beef, endpoint temperature, triose phosphate isomerase, lactate
dehydrogenase, internal color

4.2 INTRODUCTION

Since 1982, undercooked ground beef has been identified as the most frequent
vehicle of E. coli 0157:H7 infection (CDC, 1996). An outbreak of foodbome disease in
several western states from late 1992 to early 1993, which resulted in more than 475
people becoming seriously ill and several children’s deaths, was caused by the
consumption of undercooked enteropathogenic E. coli 0157:H7-contaminated ground
beef patties (Merrnelstein, 1993; Ahmed et al., 1995).

The USDA-F SIS (1997b) has required that meat processing establishments cook
beef patties to any one of seven time/temperature processing schedules that range from
661°C for 41 sec to 694°C for 10 see. In May of 1996, the USDA-FSIS proposed to
convert the current meat and poultry processing regulations into performance standards
(USDA-FSIS, 1996). A thermal process sufficient to cause a 5 log, or 5-D reduction in
Salmonella was proposed by the USDA-FSIS as the lethality performance standard for
fillly cooked, uncured meat patties. When utilized, any of the seven currently used USDA
time/temperature combinations generate a 5-D lethality.

The USDA-FSIS recommends that consumers use a meat thermometer to cook
beef patties to an internal EPT of 71 . 1°C. If a meat thermometer is not available,

consumers are advised to evaluate the doneness of ground beef patties by one of several

70

factors including: ( 1) the cook out juices should have no trace of pink, red, or cloudiness,
(2) the cooked ground beef should be brown in the center, (3) the cooked ground beef
should have a firm or flaky texture, as compared to raw meat that has a soft, mushy
texture, regardless of the color (USDA-F SIS, 1997d).

Two problems have recently been identified by the USDA-FSIS regarding its
advice to consumers about using a color index to test for doneness of ground beef. One
problem is that some ground beef loses all pink color before it is fully cooked.
Alternatively, the pink color may persist in some adequately cooked beef patties
(Mendenhall, 1989; Hague et al., 1994; Van Laack et al., 1996). The characteristic pink
color of flesh meat can remain in beef cooked to 716°C if the pH is 6.0 or higher. The
amount of fat in beef patties can also afi‘ect the cooked ground beef color.

Triose phosphate isomerase (TPI) was identified as a potential time-temperature
indicator (TTI) in beef (Hsu, 1997; Orta-Ramirez et al., 1997). TPI was found to have
similar thermal inactivation kinetics as that of Salmonella, the microbial pathogen selected
by the USDA-FSIS to define the proposed lethality standards in ground beef.

Sair (Chapter 3) determined TPI activity in 9.3, 20.0, and 29.6% fat ground beef
after cooking to the USDA-F SIS time/temperature schedules in a water bath. The 20.0%
fat ground beef cooked to the USDA-FSIS low temperature schedule, 661°C (151°F) for
41 sec, had a TPI activity of 6.2 U/kg meat. The TPI activity of the 20% fat ground beef
processed to the high temperature schedule, 694°C (157°F) for 10 sec, was 9.6 U/kg
meat. Therefore, it was suggested that any 20% fat beef patty having a TPI activity less

than 6.2 U/kg meat could be considered fully cooked.

71

Wang et al. (1996b) recognized LDH as a potential protein indicator for ground
beef patties. Orta-Ramirez et al. (1996) prepared monoclonal antibodies (MAb) to bovine
muscle LDH and used them to develop an enzyme-linked immunosorbent assay (ELISA)
to determine the endpoint cooking temperature of ground beef. Commercially cooked
ground beef patties with an EPT ranging from 68.3 to 71 . 1°C averaged 3.38 ug LDH/g of
meat. It was suggested that ground beef patties with a maximum concentration of 3 ug
LDH/g meat might indicate that the ground beef was processed to 70°C or above.

The objective of this study was to determine whether the EPT of ground beef
could be differentiated using TPI activity and LDH concentration and to compare the
results to the internal color of the beef patties. The subjective and objective color
evaluation of the cooked beef patties was completed in collaboration with Dr. Brad Berry
at the United States Department Of Agriculture - Agricultural Research Service Meat
Science Research Lab in Beltsville, MD.

4.3 MATERIALS AND METHODS
4.3.1 Patty formulation

Ground beef patties for this study were obtained from a meat supplier to fast food
restaurants. US. cutter and canner grades of beef were used as the lean source and US.
choice and select grades of beef as the fat source. Beef was formulated to contain 18 to
24% fat. Lean and fat sources were initially ground through a 0.95 cm plate, followed by
grinding through a 0.24 cm plate. All processing operations were completed at
approximately 4°C or below. The meat temperature at forming was -2.2°C. The ground
beef was processed into patties (113.4g) using a Forrnax (Model 24, Formax, Inc,

Mokena, IL) patty machine. Patties were individually quick frozen in a mechanical spiral

72

freezer, boxed, and immediately shipped by overnight courier to the Meat Science
Research Lab at the Beltsville Agricultural Research Center - East (Beltsville, MD 20705-
2350)

4.3.2 Cooking procedure

Dr. Brad Berry at the United States Department of Agriculture - Agricultural
Research Service - Meat Science Research Lab at the Beltsville Agricultural Research
Center - East cooked the patties and evaluated the internal color. Patties were
immediately placed in a -18°C freezer. Forty patties were cooked the next day. The
remaining 35 patties were cooked the following day. Immediately prior to cooking, each
patty was removed from the freezer and weighed. Patties were individually cooked on
electric griddles (Farberware Model 350A, Walter Kidde and Co., Bronx, NY) at a
temperature of 163°C. Patties were turned after 2 min, turned again at 4 min and then at 1
min intervals until the desired temperature was reached. Fifteen patties were cooked to
endpoint temperatures of 60, 65.6, 71 . 1, 76.7, or 822°C.

Preliminary cooking tests at all five temperatures were conducted to determine
approximate cooking times to establish times for thermocouple insertion. An
iron/constantan thermocouple, made by welding TT-J-36 special limits of error iron-
constantan wire (Omega Engineering, Inc, Stamford, CT 06907) inside 19 gauge syringe
needles, was connected to a recorder (Model 202, Honeywell Co., Ft. Washington, PA).
During cooking, the thermocouple was inserted into the side of the patty to the thickest
portion without any wiggling or pressure from the top of the patty (USDA, 1993). The

thermocouple was inserted into the patty about 90 see before the endpoint temperature

73

was attained. Cooking times were recorded. Immediately following cooking, patties were

blotted with paper towels, reweighed to determine cooking yields and evaluated for color.

Patties were placed in the open air for 10 min to allow surfaces to reach room
temperature, after which they were placed in freezer bags. Patties were placed in a 3°C
cooler before shipping overnight, on ice, to the Department of Food Science and Human
Nutrition at Michigan State University. The temperature of the patties after shipping to
Michigan State University was 4°C.
4.3.3 Color

Cooked patties were immediately divided into two portions (perpendicular to the
flat surface of the patty) and their interior cut surfaces were assessed for degree of
doneness by two trained evaluators using the Kansas State University Ground Beef Patty
Cooked Color Guide; A score of 1 = medium rare, 3 = medium, and 5 = well done
(Marksberry et al., 1993). Secondary degree of doneness scores were recorded for beef
patties having substantial areas of color beyond the primary score. Patty degree of

doneness evaluations were performed using GE. Deluxe Warm White lighting set to be

100 foot candles at the surface of the exposed patties. A Minolta Chroma Meter (Minolta

Camera Co., Ltd. Osaka, Japan) CR-200 (illuminant C, calibrated with brown plate,
C=26.5, X=0.366 and Y=0.340) was used for determining instrumental color values of
L*, a*, b*, saturation index (chroma) and hue angle (C.I.E., 1976; Van Laack et al.,
1996). Three readings were obtained from different locations on each cut surface of the
patty. The time from removal of patties from the grill to initiation of instrumental color

measurement was approximately 30 sec.

 

{4.1“

 

74

4.3.4 Extract preparation

Patties were removed from the freezer bags and the center of the patty was
removed using a 7 cm diameter Cookie & Donut Cutter (Norpro®, Everett, WA 98203).
Three volumes of PBS buffer (0.15M NaCl, 0.01 M sodium phosphate buffer, pH 7.2)
were used to extract the protein. The beef samples were blended for 30 sec (15 sec on, 10
sec off, 15 sec on) in an Oster Blender (Model 869-18, Schaumburg, IL 60173) and then
stirred on a magnetic stir plate for 15 min at 4°C. Extracts were immediately centrifuged
at 4,500 x g for 10 min at 4°C. Supernatants were collected after filtration (Whatman No.
1) and were held on ice until assayed for TPI activity and LDH concentration (Orta-
Ramirez et al., 1996). Triose phosphate isomerase activity was determined as described
by Bergmeyer (1984) except that 1.0 mL triethanolarnine buffer (TEA, 0.2M, pH 8.0), 0.2
mL 15mM glyceraldehyde-3-phosphate (GAP), 10 BL B-nicotinarnide adenine
dinucleotide (NADH, 10 mg/mL), 10 uL glycerol-3-phosphate dehydrogenase (GDH, 1.5
mg/mL), and 10 BL of protein extract were used in the assay solution (Sair, Chapter 3).
4.3.5 LDH concentration

Lactate dehydrogenase concentration was quantified using a sandwich ELISA
(Orta-Ramirez et al., 1996). Lactate dehydrogenase monoclonal antibodies (MAb) were
produced by Orta-Ramirez et a1. (1996) following the procedures of Abouzied et al.
(1990). Polyclonal antibodies (PAb) against beef muscle LDH were prepared as described
by Wang et al. (1996a). The sandwich ELISA was performed by coating Immulon® 2
Removawell Strips (Dynatech Laboratories, Inc, Chantilly, VA 22021) with 100 uL of
LDH MAb, the capture antibody, diluted ( 1/3 000) in 0.1 M carbonate buffer, pH 9.6, and

drying overnight at 37°C. Plates were washed four times with PBS containing 0.05%

75

Polyoxyethylene sorbitan monolaurate (Tween 20) (PBS-T). Nonspecific binding sites
were blocked by adding 300 uL of PBS containing 2% ovalbumin (PBS-0A) to each well
and incubating at 37°C for 30 min. Plates were washed four times and 50 ILL of serially
diluted bovine muscle LDH or ground beef patty extracts was added to each well and
incubated at 37°C for 30 min. After four washings, 50 11L of LDH specific PAb in 2%
PBS-0A (1:500) was added to each well and incubated at 37°C for 30 min. After
incubation, the plates were washed four times with PBS-T and 100 11L of goat anti-rabbit
IgG peroxidase conjugate diluted in 2% PBS-0A (1:500) was added to each well. Plates
were incubated for 30 min at 37°C and washed eight times with PBS-T. Peroxidase
binding was determined with ABTS (2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid)
substrate (Pestka, 1982). After 30 min, absorbance was read at 405 nm using a microplate
reader (THERMOmax, Molecular Devices Co., Menlo Park, CA). Results were
expressed as micrograms of LDH per gram of meat.
4.3.6 Proximate composition

The moisture, fat, and protein contents of raw ground beef patties were
determined according to AOAC methods 950.46B, 991.36, and 981.10, respectively
(AOAC, 1990). For pH determination, 10 g meat was homogenized with 90 g deionized
water using a Polytron homogenizer (Model PT 10/35, Brinkmann Instrument, Co.,
Westbury, NJ 11590) equipped with a PTA 10TS generator. The sample was stirred for
two periods of 30 sec at 5.0 speed setting and the pH was measured while the solution

was continuously stirred over a magnetic stirrer.

76

4.3.7 Statistics

Data were analyzed using analysis of variance in the General Linear Model
Procedure of the Statistical Analysis System (SAS Institute, Inc, 1996). Least square
means procedures were used to separate means at P<0.05. Fifteen ground beef patties
were cooked at each EPT. The linear or curvilinear change in selected variables was
determined using the orthogonal polynomial contrast test by SAS software (Release 6.12).
Tests revealed that the assumptions of the general linear model were not met for TPI
activity and LDH content of cooked beef patties. Triosephosphate isomerase activity and
LDH content data underwent a log transformation prior to analyses.
4.4 RESULTS AND DISCUSSION
4.4.1 TPI activity and LDH concentration

The raw ground beef patties contained 24.4 i 0.3% fat, 62.5 i 5.7% moisture and
17.0 i 0.6% protein. The pH of the raw patties was 5.8 i 0.1. Raw ground beef patty
extracts contained 2203 U TPI/kg meat. The TPI activity of the cooked beef patties
linearly decreased (P<0.05) as the processing temperature was increased from 60.0 to
822°C (Table 4.1). Triose phosphate isomerase activity decreased from 52.50 U/kg meat
at 600°C to 5.36 U/kg meat at 822°C. Differences were detected in TPI activity from
60.0 to 656°C, however no difference (P>0.05) in TPI activity was detected between 71 . 1
and 822°C. Ground beef patties had 6.28 U TPI/kg meat when processed to 71. 1°C; the
endpoint temperature recommended by the USDA to consumers to ensure the safe
preparation of cooked ground beef (USDA, 1994). Wang et al. (1996b) reported a TPI

activity of 5.2 U/g meat in ground beef patties processed to 71°C. Ground beef cooked to

77

Table 4.1-Influence of temperature on triose phosphate isomerase (TPI) activity and

lactate dehydrogenase (LDH) content of cooked beef patties (24.4% fat)

 

 

Temp (°C) LDH content TPI activity
(pg/g meat) (U/kg meat)
60.0 2.96 4 066° 52.50 4 12.69“
65.6 3.80 4 1.03“ 2026 4 6.40b
71.1 3.93 4 1.40“ 6.28 4 0.26c
76.7 0.02 4 0.01b 5.27 4 028°
82.2 000 4 000'“ 5.36 4 0.38“

 

°'° Means i standard error of the mean in the same column with the same letter were not

different (P>0.05).

78

661°C for 41 sec (equivalent to 71 . 1°C instantaneously) in model system studies had a
TPI activity of 6.2 U/kg meat (Sair, Chapter 3). Results in both the model system and
ground beef patties suggest that a maximum TPI activity of about 6 U/kg meat (mean
minus one standard deviation) might indicate that ground beef was processed to 71°C or
above.

Three cooked ground beef patties (113.4 g raw weight) were purchased from a
local fast food restaurant. After removing the center of each patty with the Cookie &
Donut Cutter (7 cm diameter), the protein was extracted and TPI activity determined, as
previously described. The TPI activity of the cooked ground beef patties averaged 1.8 i
0.3 U/kg meat, suggesting they were cooked above the recommended EPT of 71°C.

The LDH content decreased (P<0.05) as cooking temperature of ground beef
patties was increased from 60.0 to 822°C (Table 4.1). No differences (P>0.05) were
detected in the LDH concentration of ground beef patties processed from 60.0 to 71 . 1°C
or 76.7 to 822°C. The LDH concentration of patties processed to 71 . 1°C averaged 3 .93
ug/g meat. The LDH content decreased from 3.93 jig/g meat at 71. 1°C to 0.02 ug/g meat
at 767°C. Orta-Ramirez et al. (1996) reported LDH concentrations of 3.38 and 0.30 jig/g
meat for commercially cooked beef patties processed within the temperature ranges of
68.3 to 71 . 1°C and 73.9 to 822°C, respectively. Wang et al. (1996a) reported a LDH
concentration of 0.48 ug/g meat for commercially cooked beef patties cooked within the
temperature range of 73 .9 to 822°C. In our study, the LDH concentration of the cooked
beef patties was similar at 65.6 and 71 . 1°C. However, the LDH concentration of the beef
patties cooked to 767°C was different fiom those cooked to 71. 1°C. Beef patties had

0.02 ug LDH/g meat when processed to 767°C, an EPT above that recommended to

79

consumers by the USDA. As the EPT of cooked beef patties is increased, reduced
palatability results (Liu and Berry, 1996). To determine the lowest EPT above 71 . 1°C
needed to significantly decrease the LDH concentration in cooked beef patties, LDH
concentrations need to be established in beef patties cooked to EPTs between 71.1 and
767°C.

4.4.2 Internal color

The primary degree of doneness scores increased (less red) as the EPT of beef
patties was increased (Table 4.2). Differences were seen in the primary degree of
doneness scores of patties cooked to 60.0 and 656°C. Patties cooked from 71.1 to
822°C had similar primary degree of doneness scores. At 656°C, patties were evaluated
as moderately pink and could be visually distinguished (P<0.05) fi'om patties cooked to
711°C, the temperature on which the USDA (1994) cooking recommendations are based.
Therefore, using primary degree of doneness scores, undercooked ground beef patties
could be distinguished from those that had reached the desired endpoint cooking
temperature of 71 . 1°C.

However, temperature had no effect (P>0.05) on the L* values (measure of
lightness) of the ground beef patties. No difference in D“ values was detected between the
656°C (undercooked) and 822°C (overcooked) EPTs. Hague et a1. (1994) reported no
difference in the L* values of ground beef patties cooked to 60 and 77°C. The use of the
L* values as a measure of adequate cooking could potentially lead to the consumption of
undercooked patties.

As expected, both the a* and b* values decreased (less red and less yellow,

respectively) as the EPT of the patties was increased. These color changes agreed with

80

Table 4.2-Instrumental color values of ground beef patties (24.4% fat) cooked to several
endpoint temperatures
Endpoint temperature (° C)

 

 

60.0 65.6 71.1 76.7 82.2
Degree of
doneness° 23° 2.8b 39° 41° 41°
L* value 50.2“ 504““ 51.4“ 51.5“ 51.3““
a* value 10.7“ 8.3“ 6.4“ 6.0“ 5.9“
15* value 14.1“ 13.7“ 13.1“ 12.9“d 128°
Chromaf 17.8“ 16.1b 14.6“ 14.2“ 14.2“
Hue angle“ 53.3“ 59.2“ 64.2“ 65.2“ 65.3“
a*/b* ratio 076° 06“ 0.49“ 0.46“ 0.46“

 

“I Means within a row with the same letter are not different (P>0.05).
° Degree of doneness score: 1 = medium rare, 3 = medium, 5 = well done.
fChroma = (a°+b°)°'°.

g Hue angle = arctan(b/a).

81

the results of Hague et al. (1994) who reported decreasing a* and b* values in ground
beef patties as the EPT was increased. The beef patties became less red and less intense in
color, illustrated by a decreasing chroma and a*/b* ratio and an increasing hue angle, as
the EPT was increased. Through the use of these instrumental color measurements,
patties cooked to 65.6 could be distinguished from those cooked to 71 . 1°C. However, the
chroma, a*/b* ratio and hue angle values were all similar for the patties cooked from 71 . 1
to 822°C. Consumers probably would not be able to differentiate the minor differences
detected in the instrumental color measurements of the cooked beef patties at similar
EPTs

Hague et al. (1994) reported that assigning visual scores to the internal color of
cooked beef patties as a method to determine adequate processing was not accurate. No
difference was found between the internal colors of beef patties cooked to 66 and 71°C
(Hague et al., 1994). Using internal color visual scores, some ground beef patties can be
perceived to be well-done at internal temperatures as low as 55°C (Hunt et al., 1995).
Van Laack et a1. (1996) reported a pink color to persist in eight out of seventeen
commercially prepared patty formulations when cooked to an internal temperature of
71°C. Therefore, the use of color values to detect differences in cooked patties is not a
practical option for consumers or the food industry to ensure food safety.
4.4.3 Variability in degree of doneness scores

Larger standard errors were seen with TPI activity and LDH concentration at the
lower endpoint processing temperatures (Table 4.1). Similarly, larger variations in degree
of doneness scores were observed when beef patties were processed to lower

temperatures (Table 4.3). At 600°C, 14 out of the 15 patties received secondary degree

82

Table 4.3-Degree of doneness (d/d) scores° of beef patties cooked to 5 endpoint
temperatures

 

 

 

Temp (°C) Patty # Degree of doneness
Primary Secondary

60.0 1 2 3
2 3 4
3 3 4
4 2 1,3
5 3
6 2 1,3
7 3 2,4
8 3 2,4
9 2 3,4
10 2 1,3
1 1 2 3,4
12 2 1,3,4
13 2 I
I4 I 2
15 2 3

65.6 1 3 2,4
2 3 4
3 2 3
4 3 4
5 3 4
6 3
7 3 4
8 3 4
9 3 4
10 3
1 I 3 4
12 3 4
I3 3 4
14 2 1,3,4
15 2 3

71 . l 1 4 3
2 4
3 4 3
4 3 4
5 4 3
6 4 3
7 4

83

Table 4.3-(cont'd).

 

 

 

Temp (°C) Patty # Degree of doneness
Primary Secondary
71.1 8 4 3
9 4
10 4
11 4 3
13 4 3
14 4 3
15 4 3
76.7 1 4
2 4
3 4
4 4
5 4
6 4
7 4
8 4
9 4
10 4 5
11 4 5
12 4
13 4 5
14 4 5
15 5 4
82.2 1 4
2 4
3 4
4 4
5 4 5
6 4 5
7 4 5
8 4
9 4 5
10 4
1 1 5
12 4 5
13 5
14 4 5
15 4 5

 

° d/d score: 1 = medium rare, 3 = medium, 5 = well done.

84

of doneness scores which indicates another color, different from the primary degree of
doneness score, was detected within the processed patty. Eight of the patties processed to
600°C received two secondary degree of doneness scores suggesting there were three
areas of substantial color in the cooked beef patties. One patty received three secondary
degree of doneness scores indicating large variations in color throughout the patty.
Different regions within a single ground beef patty can be cooked to distinct EPTs, as
illustrated by the variation within the degree of doneness scores at the lowest EPT.

Liu and Berry ( 1996) documented the wide variability in the EPTs of beef patties
following cooking. Beef patties were cooked to 2 EPTs using constant cooking times.
There was considerable variability in the degree of doneness scores and internal
temperatures of the cooked beef patties when constant cooking times were used. The TPI
activity and LDH concentration will have larger standard errors when multiple
temperature gradients occur within individual beef patties. As the EPT was increased, the
number of secondary degree of doneness scores assigned to the ground beef patties
decreased. At 71 . 1°C, 10 of the 15 ground beef patties, and at 767°C only 5 of the
ground beef patties received one secondary degree of doneness score. Smaller standard
errors were reported for TPI activities and LDH concentrations for the ground beef patties
that were given fewer secondary degree of doneness scores at the higher EPTs. The
smaller standard errors indicate more uniform cooking at the higher EPTs. Methods need
to be developed to achieve more uniform cooking of beef patties at all EPTs in order to
establish, with the greatest accuracy and precision, TPI activities and LDH concentrations

to be used as indicators of adequate processing.

85

4.5 CONCLUSIONS

Lactate dehydrogenase concentration in cooked beef patties decreased between
71 . 1 to 767°C and could possibly be used to verify the adequate processing of ground
beef patties. The primary degree of doneness scores showed a difference between patties
cooked to 65.6 and 71 . 1°C, however the L* values were not an accurate indicator of patty
doneness as those cooked to 656°C had similar values to those cooked to 822°C. The a*
and b* values both showed differences between patties cooked to 65.6 and 71 . 1°C. While
the primary degree of doneness scores, a* and b* values were able to detect differences
between undercooked and fillly cooked beef patties in this study, past research has shown
visual and instrumental color measurements of cooked beef patties to be poor indicators of
adequate thermal processing (Hague et al., 1994; Hunt et al., 1995; Van Laack et al.,
1996). Triose phosphate isomerase activity decreased from 20.26 to 6.28 U/kg meat in
ground beef patties between the EPTs of 65.6 and 71 . 1°C; the EPT recommended by the
USDA to consumers to ensure the safe preparation of cooked ground beef. In this study,
TPI activity proved to be the most effective method for the determination of the adequate
thermal processing of beef patties. In order to use TPI as an EPT indicator to verify the
adequate thermal processing of beef patties, additional factors that may have an effect on
TPI activity in cooked beef patties need to be investigated. These factors include: the
effects of patty size, whether the beef patties were cooked fi'om the frozen or raw state,

cooking method, cooking rate, and other formulation and processing variables.

CHAPTER 5

CONCLUSIONS

In previous research, triose phosphate isomerase (TPI) and lactate dehydrogenase
(LDH) were identified as possible means for determining the adequate thermal processing
of cooked ground beef. Current recommendations of the United States Department of
Agriculture - Food Safety Inspection Service (U SDA-FSIS) are to evaluate the doneness
of beef patties by the cooked ground beef color.

The frozen storage (-13°C) of raw steer muscles for 10 months had no effect on
TPI activity, however TPI activity and concentration changed in cooked (628°C) beef
during frozen storage. Ground beef had different TPI activities when processed to the low
(66.1°C/41 sec) and high (694°C/ 10 sec) temperatures of the required USDA processing
schedules in a model system. While, TPI was found not to be a suitable time-temperature
integrator in ground beef, TPI was determined to be a possible endpoint temperature
(EPT) indicator.

In pilot studies, lactate dehydrogenase (LDH) concentration decreased in cooked
ground beef patties (24.4% fat) between 71.1 to 767°C. Beef patties had 0.02 11g LDH/g
meat when processed to 767°C, an EPT above that recommended to consumers by the
USDA. LDH concentrations need to be established in beef patties cooked to EPTs
between 71.1 and 767°C to determine the lowest EPT above 71 . 1°C needed to

significantly decrease the LDH concentration in cooked beef patties.

86

87

TPI activity of beef patties was less when cooked to 711°C as compared to
656°C. TPI activity was 6.28 U/kg meat when processed to 71 . 1°C; the EPT
recommended by the USDA to consumers to ensure the safe preparation of cooked
ground beef. A maximum TPI activity of about 6 U/kg meat might indicate that ground
beef was processed to 71°C or above.

The primary degree of doneness scores showed a difference between patties
cooked to 65.6 and 71 . 1°C, however the D“ values were not an accurate indicator of patty
doneness as those cooked to 656°C had similar values to those cooked to 822°C. The a*
and b* values both showed differences between patties cooked to 65.6 and 71 . 1°C. While
the primary degree of doneness scores, a“ and b* values were able to detect differences
between undercooked and fully cooked beef patties in this study, past research has shown
visual and instrumental color measurements of cooked beef patties to be poor indicators of
adequate thermal processing.

Therefore, TPI and LDH could possibly be used as indicator enzymes to
determine the adequate thermal processing of ground beef patties. Sair (Chapter 3)
examined factors that might have an effect on TPI activity in ground beef. The fat content
of ground beef had an effect on TPI activity. The fi'ozen storage of cooked (628°C)
ground beef for 5 months caused changes in TPI activity. Cooked beef (694°C) with
1.0% NaCl had lower TPI activities than the control. These factors could influence the
use of TPI as an indicator enzyme in ground beef patties. Further research needs to be
undertaken to study the effects of the above factors and additional ones that may have an

impact on the TPI activity and LDH concentration in cooked ground beef patties.

CHAPTER 6

FUTURE RESEARCH

This study was designed to determine whether triose phosphate isomerase (TPI)
could be used as a time-temperature integrator (TTI) to determine adequate thermal
processing of ground beef patties. Further investigation into this line of research is
warranted and is described below.

The thermal inactivation rates of TPI, E. coli 0157:H7 and Salmonella
senftenberg were determined in ground beef by Orta-Ramirez et al. (1997) using
semitendinosus muscle from the eye of the round. The muscles used in the production of
ground beef primarily include those from the chuck (trapezius and Iongissimus dorsi). A
similar thermal inactivation study to determine the D and 2 values of enzymes and
microorganisms in chuck muscles should be completed.

The USDA-FSIS time/temperature processing schedule for fillly cooked beef
patties ranges fiom 661°C for 41 sec to 694°C for 10 sec. The D and z values of six
protein markers in ground beef were determined over the range of 53°C to 66°C (Orta-
Ramirez et al., 1997). In order to be used as a TTI, an endogenous enzyme should have a
similar thermal inactivation rate as that of the target microorganism over a similar
temperature range. The D and 2 values of the previously identified marker proteins should
be re-deterrnined over the temperature range established by the USDA-FSIS to assure

adequate processing of beef patties.

88

89

The come-up times associated with the cooking of ground beef in thermal death
time (TDT) tubes are about 2.5 to 4 min. As means to achieve instantaneous heating and
to avoid the thermal inactivation of the enzymes during come up times that are incurred
when using the 10 x 75 mm TDT tubes, the use of smaller diameter tubes should be
considered.

The sandwich ELISA used to measure the TPI concentration in beef products used
polyclonal antibodies for both capture and detection of bovine muscle TPI. The sensitivity
and specificity of the sandwich ELISA may be improved through the use of monoclonal
antibodies specific to native TPI.

Both TPI and lactate dehydrogenase (LDH) were found to be possible endpoint
temperature indicators in ground beef patties. Additional factors that may have an effect
on TPI activity and LDH concentration in cooked ground beef need to be investigated.
These factors include: effects of patty size, cooking the beef patties from the frozen or
thawed state, cooking method, heating rate, and other formulation and processing

variables.

 

REFERENCES

Abouzied, MM, Asghar, A., Pearson, A.M., Miller, ER, Gray, J .I., and Pestka, J .J .
1990. Hybridoma based enzyme-linked immunosorbent assay for C19 A°°-steroids
in sera ofboars. J. Agric. Food Chem. 38:331-335.

Abouzied, M.M., Wang, C.H., Pestka, II, and Smith, OM. 1993. Lactate
dehydrogenase as safe endpoint cooking indicator in poultry breast rolls:
development of monoclonal antibodies and application to sandwich enzyme-linked
immunosorbent assay (ELISA). J. Food Protec. 56: 120-124, 129.

Ahmed, N.M., Conner, DE, and Huffman, BL. 1995. Heat-resistance of Escherichia
coli 0157:H7 in meat and poultry as affected by product composition. J. Food
Sci. 60:606-610.

AMI. 1996. Relative ground beef contribution to the United States beef supply. Am.
Meat Institute Foundation, Arlington, VA.

Anderson, WL. 1997. Lethality performance standards may change in F818 final rule.
Food Chem. News. 39(7):9.

AOAC. 1990. Oflicial Methods of Analysis, 15th ed. Association of Official Analytical
Chemists, Arlington, VA.

Awad, A., Powrie, W.D., and Fennema, O. 1968. Chemical deterioration of frozen
bovine muscle at -4°C. J. Food Sci. 33:227-234.

Bean, NH, and Griffin, PM. 1990. Foodbome disease outbreaks in the United States,
1973-1987: Pathogens, vehicles, and trends. J. Food Protec. 53:804-817.

Beisenherz, G. 1955. Triosephosphate isomerase from calf muscle. Ch. 57, In Methods
in Enzymology, S.P. Colowick and NO. Kaplan (Ed), p. 387-391. Academic
Press, Inc. New York, NY.

Bergmeyer, H.U. 1984. Methods of Enzymatic Analysis, Vol. 1, 2“° ed. Verlag Chemie
International, Deerfield Beach, FL.

Berry, B.W. 1994. Fat level, high temperature cooking and degree of doneness affect
sensory, chemical and physical properties of beef patties. J. Food Sci. 59:10-14.

90

91

Bonner, D.W., Bloomer, A.C., Petsko, G.A., Phillips, DC, and Wilson, IA. 1976.
Atomic coordinates for triose phosphate isomerase from chicken muscle.
Biochem. Biophys. Res. Commun. 72:145-155.

Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72:248-254.

Browning, M.A., Huffman, D.L., Egbert, W.R., and Jungst, SB. 1990. Physical and
compositional characteristics of beef carcasses selected for leanness. J. Food Sci.

5519-14.

Bunch, E.A., Altwein, D.M., Johnson, L.E., Farley, J .R., and Harnmersmith, AA. 1995.
Homogeneous sample preparation of raw shrimp using dry ice. J. AOAC 78:883-
887.

Caldironi, H.A., Bazan, NC. 1980. Quantitative determination of low-salt soluble
protein patterns of bovine muscles cooked at different temperatures, by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. J. Food Sci. 45:901-904.

CDC. 1996. "Preventing Foodbome Illness: Escherichia coli 0157:H7."
<http://www.cdc.gov/ncidod/diseases/foodborn/e__coli.htm> (7 June. 1997).

CIE. 1976. International Commission on Illumination, Colorimetry: Official
Recommendations of the International Commission on Illumination. Publication
CIE No. 15 (E-1.3. 1.), Bureau Central de la CIE, Paris.

Cohen, EH. 1969. Determination of acid phosphatase activity in canned hams as an
indicator of temperature attained during cooking. Food Technol. 23(961): 101-
104.

Collins, 88., Keeton, J .T., and Smith, SE. 1991a. Lactate dehydrogenase activity in
bovine muscle as a potential heating endpoint indicator. J. Agric. Food Chem. 39:
1291-1293.

Collins, S.S., Keeton, J .T., and Smith, SE. 1991b. Lactate dehydrogenase enzyme
activity in raw, cured, and heated porcine muscle. J. Agric. Food Chem. 39:1294-
1297.

Czok, R., and Buecher, T. 1960. Crystallized enzymes from the myosin of rabbit skeletal
muscle. Adv. Protein Chem. 15:315.

Dabrowska, A., Karnrowska, 1., and Baranowski, T. 1978. Purification, crystallization
and properties of triosephosphate isomerase from human skeletal muscle. Acta.
Biochim. Polonica. 25:247-255.

 

92

Davis, C .E., and Townsend, W.E. 1994. Rapid fluorometric analysis of acid phosphatase
activity in cooked poultry meat. J. Food Protec. 57: 1094-1097.

De Cordt, S., Hendriclor, M., Maesmans, G., and Tobback, P. 1992. Immobilized 0t-
amylase from Bacillus licheniformis: a potential enzyrnic time-temperature
integrator for thermal processing. Int. J. Food Sci. Technol. 27:661-673.

De Cordt, S., Hendrickx, M., Maesmans, G., and Tobback, P. 1994. The influence of
polyalcohols and carbohydrates on the therrnostability of (ll-amylase. Biotechnol.
Bioeng. 43:107-114.

Doyle, MP, and Schoeni, J .L. 1984. Survival and growth characteristics of Escherichia
coli associated with hemorrhagic colitis. Appl. Environ. Microbiol. 48:855-856.

Doyle, MP, and Schoeni, IL. 1987. Isolation of Escherichia coli 0157:H7 fiom retail
fi'esh meats and poultry. Appl. Environ. Microbiol. 53 :2394-2396.

Eber, S.W., and Krietsch, KG. 1980. The isolation and characterization of the multiple
forms of human skeletal muscle triosephosphate isomerase. Biochim. Biophys.
Acta. 614: 173-184.

Ellekjaer, MR. 1992. Assessment of maximum cooking temperatures of previously heat
treated beef. Part 2: Differential scanning calorimetry. J. Sci. Food Agric.
60:255-261.

Ellekjaer, M.R., and Isaksson, T. 1992. Assessment of maximum cooking temperatures
in previously heat treated beef. Part 1: Near infrared spectroscopy. J. Sci. Food
Agric. 59:335-343.

Evans, T.M., and Greene, D. 1973. Chopped beef - everyone's favorite. Ch. 7, In The
Meat Book, p. 69-76. Charles Scribner's Sons, New York, NY.

Fain, Jr., A.R., Line, J.E., Moran, A.B., Martin, L.M., Lechowich, R.V., Carosella, J.M.,
and Brown, W.L. 1991. Lethality of heat to Listeria monocytogenes Scott A: D-
value and Z-value determinations in ground beef and turkey. J. Food Protec.
54:756-761.

FDA. 1995. Recommendations of the US. Public Health Service Food and Drug
Administration, Food Code. Preface. Food and Drug Administration,
Washington, DC.

FDA. 1997. "Food Safety Initiative Fact Sheet."
<http://www.fda.gov/opacom/foodsafety/fsfact.htm> (16 May. 1997).

93

Forrest, J.C., Aberle, E.D., Hedrick, H.B., Judge, M.D., Merkel, RA. 1975. Principles
of Meat Science, lst ed. Plenum, New York.

Fukal, L. 1991. Modern immunoassays in meat-product analysis. Die Nahrung 35:431-
448.

Fukazawa, T., Hashimoto, Y., and Yasiu, T. 1961. The relationship between the
components of myofibrillar protein and the effect of various phosphates that
influence the binding quality of sausage. J. Food Sci. 26:550.

Goodfellow, SJ, and Brown, W.L. 1978. Fate of Salmonella inoculated into beef for
cooking. J. Food Protec. 41 :598-605.

Hague, M.A., Warren, K.E., Hunt, M.C., Kropf, D.H., Kastner, C.L., Stroda, S.L., and
Johnson, DE. 1994. Endpoint temperature, internal cooked color, and
expressible juice color relationships in ground beef patties. J. Food Sci. 59:465-
470.

Hale, DS 1994. Grading. Ch. 7, In Muscle Foods: Meat, Poultry, and Seafood
Technology, Kinsman, D.M., Kotula, A.W., and Breidenstein, B.C. (Ed), p. 186-
223. Chapman and Hall, New York.

Hendrickx, M., Maesmans, G., De Cordt, S., Noronha, J., Van Loey, A., and Tobback, P.
1995. Evaluation of the integrated time-temperature effect in thermal processing
of foods. Crit. Rev. Food Sci. Nutr. 35:231-262.

Hsu, Y.C. 1997. Identification and verification of an endogenous time-temperature
indicator to determine processing adequacy of roast beef. Ph.D. Dissertation.
Michigan State University, East Lansing, MI.

Hunt, M.C., Warren, K.E., Hague, M.A., Kropf, D.H., Waldner, C.L., Stroda, S.L., and
Kastner, CL. 1995. Cooked ground beef color is unreliable indicator of
maximum internal temperature. Presented to Amer. Chem. Soc, Manhattan, KS,
April 6.

Jay, J .M. 1992. High-temperature food preservation and characteristics of
thermophilic microorganisms. Ch. 14, In Modern Food Microbiology. 4th ed., p.
335-3 55. Chapman & Hall, New York.

Korrnendy, L., Rehasi, E., and Fetter, I. 1987. Determination of the extent of heat
treatment in canned hams by use of the phosphatase test. Meat Sci. 19:77-79.

94

Krietsch, W.K.G., Pantchew, P.G., Klingenberg, H., Hofstetter, T., and Buecher, T.
1970. The isolation and crystallization of yeast and rabbit liver triose phosphate
isomerase and a comparative characterization with rabbit muscle enzyme. Eur. J.
Biochem. 14:289-300.

Lee, B.W., Barriso, J .A., Pepe, M., and Snyder, R. 1971. Purification and properties of
liver triose phosphate isomerase. Biochim. Biophys. Acta. 242: 261-266.

Lee, Y.B., Rickansrud, D.A., Hagberg, EC, and Briskey, EJ. 1974. Application of
SDS-acrylamide gel electrophoresis for determination of the maximum
temperature to which bovine muscles have been cooked. J. Food Sci. 39:428-
429.

Line, J .E., Fain, Jr., A.R., Moran, A.B., Martin, L.M., Lechowich, R.V., Carosella, J.M.,
and Brown, W.L. 1991. Lethality of heat to Escherichia coli 0157:H7 : D-value
and Z-value determinations in ground beef. J. Food Protec. 54:762-766.

Liu, M.N., and Berry, B.W. 1996. Variability in color, cooking times, and internal
temperature of beef patties under controlled cooking conditions. J. Food Protec.
59:969-975.

Maesmans, G., Hendrickx, M., De Cordt, S., and Tobback, P. 1993. Theoretical
considerations on design of multicomponent time temperature integrators in
evaluation of thermal processes. J. Food Proc. Pres. 17:369-389.

Marksberry, C.L., Kropf, D.H., Hunt, M.C., Hague, M.A., and Warren, KB. 1993.
Ground beef patty cooked color guide. Kansas Agric. Exp. Sta, Manhattan, KS.

Mendenhall, VT 1989. Effect of pH and total pigment concentration on the internal
color of cooked ground beef patties. J. Food Sci. 5421-2.

Mermelstein, NH. 1993. Controlling E. coli 0157:H7 in meat. Food Technol.
47(4):90-91.

Mulley, A., Stumbo, C., and Hunting, W. 1975. Thiamine: a chemical index of the
sterilization efficacy of thermal processing. J. Food Sci. 40:993-996.

Ng, H., Bayne, HQ, and Garibaldi, IA. 1969. Heat resistance of Salmonella: the
uniqueness of Salmonella seftenberg 775W. Appl. Microbiol. 17:78-82.

Norton, I.L., Pfilderer, P., Stringer, CD, and Hartman, PC. 1970. Isolation and
characterization of rabbit muscle triose phosphate isomerase. Biochem. 9:4952-
4958.

95

Oesper, P., and Meyerhof, O. 1950. The determination of triose phosphate isomerase.
Arch. Biochem. Biophys. 27:223.

Omega Engineering, Inc. 1994. Data Acquisition Hardware & Software for Notebook
and Desktop PCs, Omega Engineering, Inc, Stamford, CT.

Orta-Ramirez, A. 1994. Lactate dehydrogenase as indicator of proper heat processing
and death of Escherichia coli 0157:H7 and Salmonella in ground beef patties.
MS. Thesis. Michigan State University, East Lansing, MI.

Orta-Ramirez, A., Wang, C.H., Abouzied, M.M., Veeramuthu, G.J., Price, J.F., Pestka,
J .J ., and Smith, D.M. 1996. Lactate dehydrogenase monoclonal antibody
sandwich ELISA to determine cooking temperature of ground beef. J. Agric.
Food Chem. 44:4048-4051.

Orta-Ramirez, A., Price, J .F ., Hsu, Y.C., Veeramuthu, G.J., Cherry-Merrit, IS, and
Smith, D.M. 1997. Thermal inactivation of Escherichia coli 0157:H7,
Salmonella senftenberg and enzymes with potential as endpoint temperature
indicators in ground beef. J. Food Protec. 60:471-475.

Parson, SE, and Patterson, R.L.S. 1986. Assessment of previous heat treatment given
to meat products in the temperature range 40-90°C. Part 2: Differential scanning
calorimetry, preliminary study. J. Food Technol. 21 :123-131.

Pestka, J.J., Lee, Y.K., and Chu, PS. 1982. Reactivity of aflatoxin 8;. antibody with
aflatoxin B1 modified DNA and related metabolities. Appl. Environ. Microbiol.
44: 1 159-1 165.

Prusa, K.J., and Bowers, IA. 1984. Protein extraction fi'om frozen, thawed turkey
muscle with sodium nitrite, sodium chloride, and selected sodium phosphate salts.
J. Food Sci. 49:709-713.

Ruiz, C.F., Higginbotham, D.A., Carpenter, J .A., Resurreccion, A.V.A., and Lanier, TC.
1993. Use of chuck muscles and their acceptability in restructured beef/surimi
steaks. J. Anim. Sci. 71 :2654-2658.

Saffle, KL, and Galbreath, J .W. 1964. Quantitative determination of salt-soluble protein
in various types of meat. Food Technol. 18(12):]19-120.

Samarajeewa, U., Wei, C.I., Huang, TS, and Marshall, MR. 1991. Application of
immunoassay in the food industry. Critical Rev. Food Sci. Nutr. 29:403-434.

96

Saraiva, J ., De Cordt, S., Hendriclot, M., Oliveira, J ., and Tobback, P. 1993. Inactivation
of (it-amylase fi'om Bacillus amyloliquefaciens at low moisture contents. 459-466.
Cited in van den Tweel, W.J.J., Harder, A., and Buitelaar, RM. Stability and
Stabilization of Enzymes. Elsevier Science Publishers, Amsterdam.

SAS Institute, Inc, 1996. SAS User's Guide: Basic Statistical Analysis, Version 6.12,
SAS Institute Inc, Cary, NC.

Sawyer, T.H., Tilley, BE, and Gracy, R.W. 1972. Studies on human triosephosphate
isomerase - nature of the electrophoretic multiplicity in erythrocytes. J. Biol.
Chem. 247: 6499-6504.

Smith, D.M. 1995. Immunoassays in process control and speciation of meats. Food
Tech. 49(2): 1 16-1 19.

Smith, D.M., and Orta-Ramirez, A. 1995. Enzyme-linked immunosorbent assay
technology to verify endpoint cooking temperatures of meat products. J. Clin.
Ligand Assay. 18:161-165.

Smith, D.M., Desrocher, L.D., Booren, A.M., Wang, C.H., Abouzied, M.M., Pestka, J.J.,
and Veeramuthu, G]. 1996. Cooking temperature of turkey ham affects lactate
dehydrogenase, serum albumin and immunoglobulin G as determined by ELISA.
J. Food Sci. 61:209-212, 234.

Smith, G.C., Berry, B.W., Savell, J .W., and Cross, HR. 1988. USDA maturity indices
and palatability of beef rib steaks. J. Food Qual. 11:1-13.

Smith, G.L., Alford, ES, and Keeton, J .T. 1991. Screening of endogenous enzyme
activity as a means of assessing previous heat treatment in bovine muscle. Paper
no. 454, presented at the 51st Annual Meeting of the Inst. of Food Technologists,
Dallas, TX, June 2-5.

Stalder, J .W., Smith, G.L., Keeton, IT, and Smith, SB. 1991. Lactate dehydrogenase
activity in bovine muscle as a means of determining heating endpoint. J. Food Sci.
56: 895-898.

Steele, P., and Lambe, W.J. 1982. SDS-gradient gel electrophoresis separation of muscle
polypeptides for the estimation of maximum cooking temperatures in meat. J.
Food Protec. 45:59-62.

Talmant, A., Monin, G., Briand, M., Dadet, M., and Briand, Y. 1986. Activities of
metabolic and contractile enzymes in 18 bovine muscles. Meat Sci. 18:23-40.

Taoukis, PS, and Labuza, TR 1989. Applicability of time-temperature indicators as
shelf life monitors of food products. J. Food Sci. 54:783-788.

 

97

Townsend, W.E., and Blankenship, LC. 1989. Methods for detecting processing
temperatures of previously cooked meat and poultry products - a review. J. Food
Protec. 52: 128-135.

Townsend, W.E., and Davis, CE. 1992. Transaminase (AST/GOT and ALP/GPT)
activity in ground beef as a means of determining end-point temperature. J. Food
Sci. 57:555-557.

Troutt, E.S., Hunt, M.C., Johnson, DE, Claus, J .R., Kastner, C.L., Kropf, DH. 1992.
Characteristics of low-fat ground beef containing texture-modifying ingredients. J.
Food Sci. 57:19-24.

USDA. 1986a. Determination of internal cooking temperature (Coagulation). In Revised
Basic Chemistry Laboratory Guidebook No. 3.019, pp. 3-55. Science Chemistry
Div., Food Safety and Inspection Service, US. Dept. of Agriculture, Washington,
DC.

USDA. 1986b. Determination of internal cooking temperature (Acid phosphatase
activity). In Revised Basic Chemistry Laboratory Guidebook No. 3.018, pp. 3-49.
Science Chemistry Div., Food Safety and Inspection Service, US. Dept. of
Agriculture, Washington, DC.

USDA. 1989. Performing the catalase enzyme test. A self-instruction guide. Technical
Services Training Div., Food Safety and Inspection Service, US. Dept. of
Agriculture, Washington, DC.

USDA. 1993. Instructions for verifying internal temperature and holding time of meat
patties. FSIS Directive 7370.1, Food Safety Inspection Service, US. Department
of Agriculture, Washington, DC.

USDA. 1994. A quick consumer guide to safe food handling and labels. Home Garden
Bull. No. 254. Food Safety Inspection Service, US. Department of Agriculture,
Washington, DC.

USDA-F SIS. 1996. Performance standards for the production of certain meat and
poultry products. Federal Register. 61(86), 19564. US. Department of
Agriculture, Food Safety Inspection Service, Washington, DC.

USDA-FSIS. 1997a. "Protecting the Public from Foodbome Illness: The Food Safety
and Inspection Service." <http://www.usda.gov/agency/fsis/mission.htm> (16
May. 1997).

 

98

USDA-F SIS. 1997b. Heat-processing procedures, cooking instructions, and cooling,
handling and storage requirements for uncured meat patties. In Code of Federal
Regulations, Food Safety & Inspection Service (Meat and Poultry), Ch. 111, Part
318.23. Office of the Federal Register, National Archives and Records,
Washington, DC.

USDA-F SIS. 1997c. "Addendum to Color of Cooked Ground Beef and Juices as it
Relates to Doneness: Technical Information from F SIS, April 1996."
<http://www.usda.gov/agency/fsis/befcolr2.htm> (8 May. 1997).

USDA-FSIS. 1997d. "Color of Cooked Ground Beef and Juices as it Relates to
Doneness." <http://www.usda.gov/agency/fsis/befcolor.htm> (8 May. 1997).

Van Den Oord, A.H.A., and Wesdorp, J .J . 1978. The specific effect of pyrophosphate on
protein solubility of meat. 24th European Meeting of Meat Research Workers,
Zevenaar, Netherlands, D 13:1(Abstract).

Van Laack, R.L.J.M., Berry, B.W., and Solomon, MB. 1996. Variations in internal
color of cooked beef patties. J. Food Sci. 61:410-414.

Van Loey, A., Hendrickx, M., Ludikhuyze, L., Weemaes, C., Haentjens, T., De Cordt, S.,
and Tobback, P. 1995. Potential Bacillus subtilis or-amylase-based time-
temperature integrators to evaluate pasteurization processes. J. Food Protec.
59:261-267.

Van Loey, A, Hendriclor, M., De Cordt, S., Haentijens, T., and Tobback, P. 1996.
Quantitative evaluation of thermal processes using time-temperature integrators.
Trends Food Sci. Tech. 7(1): 16-26.

Wang, C.H., Abouzied, M.M., Pestka, J.J., and Smith, D.M. 1992. Antibody
development and enzyme-linked immunosorbent assay for the protein marker
lactate dehydrogenase to determine safe cooking end-point temperatures of turkey
rolls. J. Agric. Food Chem. 40:1671-1676.

Wang, C.H., Booren, A.M., Abouzied, M.M., Pestka, J.J., and Smith, D.M. 1993.
ELISA determination of turkey roll endpoint temperature: effects of formulation,
storage, and processing. J. Food Sci. 58:1258-1261, 1264.

Wang, C.H., Pestka, J .J ., Booren, A.M., and Smith, D.M. 1994. Lactate dehydrogenase,
serum protein, and immunoglobulin G content of uncured turkey thigh rolls as
influenced by endpoint cooking temperature. J. Agric. Food Chem. 42: 1829.

Wang, C.H., Abouzied, M.M., Pestka, J.J., and Smith, D.M. 1996a. Lactate
dehydrogenase polyclonal antibody sandwich ELISA for determination of endpoint
heating temperatures of ground beef. J. Food Protec. 59:51-55.

 

99

Wang, S.F., Abouzied, M.M., and Smith, D.M. 1996b. Proteins as endpoint temperature
indicators for ground beef patties. J. Food Sci. 61 :5-7.

Webb, M.R., and Knowles, IR. 1975. The orientation and accessibility of substrates on
the active site of triosephosphate isomerase. Biochem. 14:4692-4698.

Wierenga, R.K., and Noble, M.E.M. 1992. Comparison of the refined crystal structures
of liganded and unliganded chicken, yeast and trypanosomal triosephosphate
isomerase. J. Mol. Biol. 224: 1 1 15-1 126.

 

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