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THESlS

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This is to certify that the
thesis entitled
R-Phycoerythrin as a time-temperaure integrator

and Triose phosphate isomerase as a temperature
indicator of thermal processing in beef patties

presented by

Sarah E. Smith

has been accepted towards fulfillment
of the requirements for

 

 

Masters of Science degree in Food Science

MM 44%0

 

 

Major professor
%/ Major Professor D.M. Smith
Datef / 7’6! U Department Chair M. Uebersax
0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

LIBRARY
Michigan State
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1100 mm.“

 

R-PHYCOERYTHRIN AS A TIME-TEMPERATURE INTEGRATOR, AND TRIOSE
PHOSPHATE ISOMERASE AS A TEMPERATURE INDICATOR OF THERMAL
PROCESSING IN BEEF PATTIEs
By

Sarah E. Smith

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirments
for the degree of

MASTER OF SCIENCE
Department of Food Science and Human Nutrition

2000

ABSTRACT
R—PHYCOERYTHRIN AS A TIME-TEMPERATURE INTEGRATOR AND

TRIOSE PHOSPHATE ISOMERASE AS A TEMPERATURE INDICATOR OF
THERMAL PROCESSING IN GROUND BEEF PATTIES

By
Sarah E. Smith

The D values for an 8-snain Salmonella cocktail in log phase were 16.34, 2.72,
0.44, and 0.15 min at 55, 58, 61, and 63°C, respectively, with a 2 value of 3.90°C in
ground beef. Frozen storage of inoculated beef and starvation conditions in media prior
to inoculation decreased the thermal resistance of the Salmonella cocktail. The D values
of the thermally injured Salmonella cocktail were not different (P<0.05) from the
Salmonella cocktail in log phase. Similar results were seen with the heat shocked
Salmonella cocktail except at 58°C where it had a lower (P<0.05) thermal resistance.

Encapsulated R—Phycoerythrin (PE) was inserted into the center of a beef patty
that had been inoculated with a Salmonella cocktail. In 113g patties, a normalized PE
fluorescence less than 0.29 was correlated to a 5 logm decrease in Salmonella. At this cut
off value, PE was able to correctly categorize 93% of the high and low fat beef patties as
undercooked or properly cooked according to current USDA regulations.

Five 1g samples within each patty were assayed for triose phosphate isomerase
(TPI) enzyme activity after cooking. Large variability in activity was seen within each
patty and internal temperatures were estimated to differ by as much as 25.7°C. TPI
activity was affected by the heterogeneous nature of ground beef, but was able to identify

temperature variability within patties.

To Mom and Dad

I could never be where I am today without all of your love and support.

ACKNOWLEDGMENTS

I would like to thank my major advisor, Dr. Denise Smith, for all of her support
and guidance throughout not only my Masters Degree but also in my decision to choose a
career in Food Science. I would also like to thank my committee members Dr. James
Steffe, Dr. Robert Ofoli, Dr. James Merrill, and Dr. Elliot Ryser for all of their insight.
My lab mates offered support and fiiendship that one I only dreamed of, thank you Dr.
Alicia Orta-Ramirez, Dr. Manee Vittayanont, Jennifer Maurer, Carolyn Ross, and J in
Shan Shie.

I would like to thank my Sisters Hilary and Lisa for their support and
unconditional love. Finally I would like to thank Ryann Simpson, who made it possible

for me to continue my education and still live a wonderfully full life.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ viii
LIST OF FIGURES ................................................................................. ix
CHAPTER 1: INTRODUCTION ................................................................... 1
CHAPTER 2: LITERATURE REVIEW ......................................................... 4
2.1 Evolution of thermal inactivation regulations ....................................... 4
2.2. Thermal inactivation of Salmonella strains and Escherichia coli 0157:.H7
in meat ........................................................................................ 7
2.2.1. Salmonella ................................................................... 8
2.2.2 Escherichia coli 01571H7 ................................................ 13
2.3. Time-temperature integrators ....................................................... 15
2.3.1. In situ methods ............................................................ 16
2.3.2. Physical-mathematical models .......................................... 17
2.3.3. Time-temperature integrators (TTIS) ................................... 17
2.4. R-Phycoerythin ....................................................................... 21
2.4.1. R-Phycoerythin as a TTI to verify adequate processing of beef
products ............................................................................ 22
2.5. Triose phosphate isomerase ......................................................... 25
2.5.1 Triose phosphate isomerase as a TTI for thermal processing of
ground beef ........................................................................ 25
CHAPTER 3: Thermal inactivation of Salmonella Typhimurium DT104 and an
8-strain Salmonella cocktail under different conditions ................................. ..... 28
3.1. ABSTRACT ........................................................................... 28
3.2. INTRODUCTION .................................................................... 29
3.3. MATERIALS AND METHODS ................................................... 31
3.3.1. Bacterial strains ............................................................ 31
3.3.2. Culture preparation ........................................................ 31
3.3.3. Ground beef preparation .................................................. 32
3.3.4. Inoculation of ground beef ................................................ 33
3.3.5. Thermal inactivation procedure .......................................... 33
3.3.6. Enumeration of Salmonella .............................................. 34
3.3.7. Calculation of D values and 2 values .................................... 34
3.4. RESULTS AND DISCUSSION .................................................... 35
3.4.1. Composition ............................................................... 35
3.4.2. Heat resistance of Salmonella Typhimurium DT104 ................. 35
3.4.3. Salmonella cocktail ........................................................ 37
3.4.4. 2 Values ................................................................ 1 ...... 41
3.5. CONCLUSIONS ..................................................................... 44

CHAPTER 4: Evaluation of phycoerythrin as a time-temperature integrator in beef

patties ................................................................................................. 45
4.1. ABSTRACT ........................................................................... 45
4.2. INTRODUCTION .................................................................... 46
4.3. MATERIALS AND METHODS .............................................. - ..... 48

4.3.1. Ground beef patties ........................................................ 48
4.3.2. Bacterial strains ............................................................ 48
4.3.3. Culture preparation ........................................................ 49
4.3.4. Inoculation of ground beef ................................................ 49
4.3.5. Preparation of phycoerythrin ............................................. 50
4.3.6. Ground beef cooking preparations and procedures ................... 50
4.3.7. PE fluorescence measurements... . ....; ................................. 53
4.3.8. Enumeration of surviving bacteria ....................................... 53
4.3.9. Calculation of process lethality .......................................... 54
4.4. RESULTS AND DISCUSSION .................................................... 54
4.4.1. Effect of maximum center temperatures on Salmonella in high fat
ground beef patties ............................................................... 54
4.4.2. Effect of lethality on Salmonella in high fat ground beef
patties ............................................................................... 58
4.4.3. Relationship between normalized PE fluorescence and Salmonella
in high fat ground beef patties ................................................... 59
4.4.4. Effect of maximum center temperature on Salmonella in low fat
ground beef patties ................................................................ 64
4.4.5. Effect of lethality on Salmonella in low fat ground beef
patties ............................................................................... 66
4.4.6. Relationship between normalized PE fluorescence and
Salmonella in low fat ground beef patties ...................................... 68
4.4.7. Effect of maximum center temperature on Salmonella in 60g high
fat ground beef patties ............................................................ 71
4.4.8. Effect of lethality on Salmonella in 60g high fat ground beef
patties .............................................................................. 73
4.4.9. Relationship between normalized PE fluorescence and Salmonella
in 60g high fat ground beef patties .............................................. 75
4.5 CONCLUSIONS ...................................................................... 80

CHAPTER 5: Triose phosphate isomerase as an indicator of thermal processing of

ground beef patties ................................................................................. 81
5.]. ABSTRACT ........................................................................... 81
5.2. INTRODUCTION .................................................................... 82
5.3. MATERIALS AND METHODS ................................................... 84

5.3.1. Ground beef patties ........................................................ 84
5.3.2. Patty cooking procedures ................................................. 84
5.3.3. Protein extraction for TPI activity ....................................... 85
5.3.4. TPI activity determination ................................................ 87
5.3.5. Calculation of process lethality .......................................... 87

vi

5.4. RESULTS AND DISCUSSION ................................................. 88
5.4.1. Effect of maximum center temperature on TPI activity

in ground beef patties ......................................................... 88

5.4.2. Effect of lethality of the cooking processes on TPI activity

in ground beef patties ......................................................... 93

5.4.3. Temperature variation within ground beef patties ................. 95

5.4.4. Effect of location on the TPI activity of ground beef patties. . . 100

5.5. CONCLUSIONS ................................................................. 103

CHAPTER 6: CONCLUSIONS .............................................................. 104
CHAPTER 7: FUTURE RESEARCH ....................................................... 106
REFERENCES ................................................................................... 108

vii

LIST OF TABLES

Table 2.1. D values of an 8-strain Salmonella cocktail in ground beef ..................... 10
Table 2.2. D values for Salmonella in chicken breast meat .................................. 10
Table 2.3. D values for E. coli 01572H7 in ground beef ..................................... 14

Table 2.4. D values of E. coli 01572H7 in ground beef as affected by fat content ....... 14

Table 3.1 D values and regression parameters of three strains of Salmonella
Typhimurium DT104 in high (19.1%) and low (4.8%) ground beef ........................ 36

Table 3.2 D values and regression parameters of an 8-Stain Salmonella cocktail
in stationary phase after freezing inoculated ground beef (19.1% fat) ...................... 38

Table 3.3 D values and regression parameters of an 8-Strain Salmonella cocktail after
thermal injury (56°C for 30 min) or heat shock (48°C for 30 min) .......................... 40

Table 3.4 The 2 value and regression parameters of Salmonella Typhimurium
DT104 and an 8-strain Salmonella cocktail in stationary phase after freezing in high
(19.1%) and low (4.8%) fat ground beef .......................................................... 43

viii

LIST OF FIGURES

Figure 4.1. (A) Attachment of the capillary tube containing phycoerythrin to a
thermocouple, (B) Insertion position of the capillary tube and thermocouple into a
beef patty ........................................................................................... 52

Figure 4.2. Representative time-temperature cooking profiles of a 113g high fat
(19.1%) ground beef patty. (A) Illustrates a patty cooked to 564°C and total

lethality of 5.258; (B) Illustrates a patty cooked to 71 .9°C and total lethality

of14203 ............................................................................................ 55

Figure 4.3. Effect of maximum internal center temperature on the log decrease

in Salmonella cocktail of ground beef patties (19.1% fat). Patties were categorized

as undercooked or properly cooked according to the time-temperature regulations

of the USDA (2000) .............................................................................. 57

Figure 4.4. Effect of process lethality on the log decrease in Salmonella in ground
beef patties (19.1% fat). Patties were categorized as undercooked or properly
cooked according to the time-temperature regulations of the USDA (2000) .............. 60

Figure 4.5. Correlation between normalized fluorescence and log decrease in
Salmonella cocktail in ground beef patties (19.1% fat) ....................................... 61

Figure 4.6. Relationship between normalized phycoerythrin fluorescence and log
decrease in Salmonella in ground beef patties (19.1% fat). Patties were categorized as
undercooked or properly cooked according to the time-temperature regulations

of the USDA (2000) ................................................................................ 63

Figure 4.7. Effect of maximum internal center temperature on log decrease in
Salmonella in ground beef patties (4.8% fat). Patties were categorized as

undercooked or properly cooked according to the time-temperature regulations
of the USDA (2000) ................................................................................ 65

Figure 4.8. Effect of process lethality on log decrease in Salmonella ground beef
patties (4.8% fat). Patties were categorized as undercooked or properly cooked
according to the time-temperature regulations of the USDA (2000) ........................ 67

Figure 4.9. Correlation between normalized fluorescence and log decrease in
Salmonella cocktail in ground beef patties (4.8% fat) ......................................... 69

Figure 4.10. Relationship between normalized phycoerythrin fluorescence and log
decrease in Salmonella in ground beef patties (4.8% fat). Patties were categorized as

undercooked or properly cooked according to the time-temperature regulations
of the USDA (2000) ............................................................................... 70

Figure 4.11. Representative time-temperature heating profile of a 60g high fat

(19.1%) ground beef patty. (A) Illustrates a patty cooked to 63.1°C and a total

lethality of 24.93 at 65°C; (B) Illustrates a patty cooked to 751°C and a total

lethality of 23305 at 65°C ......................................................................... 72

Figure 4.12. Effect of maximum center temperature on log decrease in Salmonella
in 60g ground beef patties (19.1% fat). Patties were categorized as undercooked or
properly cooked according to the time-temperature regulations of the USDA (2000) ...74

Figure 4.13. Effect of process lethality on the log decrease in Salmonella in 60g
ground beef patties (19.1% fat). Patties were categorized as undercooked or
properly cooked according to the time-temperature regulations of the USDA (2000) ...76

Figure 4.14. Correlation between normalized fluorescence and the log decrease in
Salmonella in 60g ground beef patties (19.1% fat) ............................................ 78

Figure 4.15. Relationship between log decrease in Salmonella and normalized
phycoerythrin fluorescence in 60g ground beef patties (19. 1% fat). Patties were
categorized as undercooked or properly cooked according to the time-temperature
regulations of the USDA(2000) .................................................................... 79

Figure 5.1. Location of 5 meat cores removed from each ground beef patty for triose
phosphate isomerase enzyme activity analysis ................................................. 86

Figure 5.2. Effect of center point temperatures on the triose phosphate isomerase
activity in the center of a beef patty. Patties were categorized as undercooked or
properly cooked according to the USDA-F SIS time-temperature safe harbor

guidelines (1996) ................................................................................... 90

Figure 5.3. Effect of center point temperature on the average triose phosphate isomerase
activity at five locations in the beef patty. Patties were categorized as undercooked or
properly cooked according to the USDA-FSIS time-temperature safe harbor guidelines
(2000) .................................................................................................. 92

Figure 5.4. Effect of lethality of the cooking process on the triose phosphate isomerase
activity of the center point of a beef patty. Patties were categorized as undercooked or
properly cooked according to the USDA-FSIS time-temperature safe harbor guidelines
(2000) ................................................................................................. 94

Figure 5.5. The effect of lethality of the cooking process on the average triose phosphate
isomerase activity at five sampling points within a beef patty. Patties were categorized as
undercooked or properly cooked according to the USDA-FSIS time-temperature safe

harbor guidelines (2000) .......................................................................... 96

Figure 5.6. Standard curve showing the effects of maximum internal temperature in
a ground beef patty on the of triose phosphate isomerase activity from Sair et
al. (1999) ............................................................................................. 98

Figure 5.7. The effect of process lethality on the calculated temperature differences
within ground beef patties ......................................................................... 99

Figure 5.8. The effect of maximum measured internal temperature of the center
point of ground beef patties on the average calculated internal temperature error bars

indicate standard deviation ...................................................................... 101

Figure 5.9. Location of the heating coil and ground beef patty on the cooking surface
Of the griddle ....................................................................................... 102

xi

EA

FTref

ko

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LIST OF SYMBOLS AND UNITS

activation energy of a process

processing value

processing value at the reference temperature
reaction rate constant

pre-exponential factor

reaction order

universal gas constant

time

temperature

concentration of a target attribute at time t

concentration Of a target attribute at time zero

xii

-1
kJ mole
min

min

- -1
min
- -1
min
J mole K‘l
min

°C

CHAPTER 1

INTRODUCTION

Inadequate cooking or survival of pathogens during cooking is a common cause
of foodborne disease outbreaks associated with meat products (Bean and Griffin, 1990).
Between 1988 and 1992 a total of 2,423 foodborne outbreaks were reported, with
bacterial pathogens causing 79% of all outbreaks (Bean et al., 1997). This group of
pathogens consists mainly of Salmonella, Campylobacter, and Escherichia coli 01572H7.
all of which have been found in ground beef. It is well known that ground beef is one of
the most common vectors for bacterial pathogens and that thermal processing is the main
solution to inactivation of these microbes. One of the best known outbreaks involving E.
coli 01572H7 resulted from the consumption of undercooked ground beef.

The United States Department of Agriculture-Food Safety and Inspection Service
(USDA-F SIS) published meat and poultry regulations in Title 9 of the Code of Federal
Regulations. Processors are required to follow published time-temperature cooking
schedules for ground beef. Recently, lethality performance standards have been added to
allow flexibility in processing procedures (USDA-F818, 1999). In cooked beef, roast
beef, and cooked corned beef the regulations state that any processing schedule may be
used, as long as it is equivalent to a 6.5-log10 reduction in Salmonella. A 5-log10
reduction in Salmonella has been proposed for cooked, uncured, meat patties. These
processing standards are based on microbial thermal death time studies performed in a

laboratory and have not been verified using actual meat products.

Processing compliance should be documented with challenge studies using a
cocktail of Salmonella serotypes. Particular serotypes are not specified by the USDA.
However, the cocktail should contain Salmonella strains that exhibit relatively high heat
resistance and have been previously implicated in foodborne outbreaks (USDA-F818,
1999).

The emergence Of pathogens with the ability to resist antibiotics and traditional
heat treatments has prompted research in the area of microbial thermal inactivation.
Recent research has investigated the effects of sub-lethal heat treatment prior to thermal
inactivation. Studies have been completed with single strains of Salmonella and Listeria
and a cocktail of E. coli 0157:H7, but not with a Salmonella cocktail. Many different
environmental conditions alter the thermal inactivation parameters Of pathogens. The
effect of fat concentration of meat, starvation conditions, and freezing of inoculated
ground beef on the thermal inactivation parameters of a Salmonella cocktail are
unknown.

Even though current USDA regulations are based on the thermal destruction Of
bacterial pathogens, current methodologies for monitoring adequacy of heat treatments
are not. Time-temperature integrators (TTIS) are devices capable of quantifying a time-
temperature impact on a quality parameter without acquiring the actual thermal history.
A TTI that correlates to a 5 or greater loglo decrease in Salmonella could assist
processors in proving process lethality. Processors are limited by the inability to take
pathogens into production facilities to conduct challenge studies on actual processing
equipment. An extrinsic TTI could be brought into a plant environment to assess the

thermal process, while an endogenous TTI would already be present for monitoring.

Thermal inactivation parameters were determined for phycoerythrin (PE) in a
buffer system. PE with a 2 value of 5.99 was able to distinguish between undercooked,
properly cooked, and overcooked thermal processing schedules for ground beef (Orta—
Ramirez, 1999). The inactivation of PE did not follow a first order reaction, yet the
lethalities calculated for PE and Salmonella, assuming non-linear kinetics, were very
predictable. A mathematical model could be developed to correlate the thermal
destruction of PE fluorescence to the destruction of Salmonella in meat products. More
work is needed to evaluate this extrinsic fluorescence based TTI in beef patties.

Triose phosphate isomerase (TPI) is an endogenous enzyme in ground beef that
has shown potential as a time-temperature integrator for thermal processing. The 2 value
of TPI was reported as 556°C, within the range given for Salmonella in ground beef
(Orta-Ramirez et al., 1997). A difference was detected in TPI activity between ground
beef patties cooked to 65.6 and 71 .1°C. More research is needed to evaluate the potential
of TPI as a temperature indicator in ground beef patties during cooking.

The objectives of this project were (1) to investigate the effect of fat content of
meat, growth phase of microorganism, starvation conditions, and sub-lethal heat
treatment on thermal inactivation parameters of S. Typhimurium DT104 and a
Salmonella cocktail, (2) to verify the ability of PE to indicate proper thermal inactivation
of Salmonella in a ground beef patty, and (3) investigate the potential of TPI to indicate

temperatures within ground beef patties.

CHAPTER 2

LITERATURE REVIEW

2.1. Evolution of thermal inactivation regulations

Title 9 of the Code of Federal Regulations has traditionally been used to outline
the USDA meat processing requirements needed for the destruction Of pathogens that
cause foodborne disease (USDA-F SIS, 2000). In 1990, the USDA-FSIS finalized
regulations for cooked beef, roast beef, and corned beef. These regulations consisted of
16 time/temperature schedules (USDA-FSIS, 1990) to ensure a 7-log reduction in
Salmonella. There were no federal regulations concerning fully cooked, uncured meat
patties until 1996.

The regulations were reviewed after the 1993 outbreak of Escherichia coli
0157:H7 caused by the consumption of undercooked ground beef patties. Schedules for
beef patties were established based on the thermal destruction of Salmonella and E. coli
0157:H7 (Goodfellow and Brown, 1978; Doyle and Shoeni, 1984). Meat processors,
subjected to federal inspection, were required to use one of the following
time/temperature protocols for fully cooked, uncured meat patties: 66.1°C (151°F)/4l
sec, 66.7°C (152°F)/32 sec, 672°C (153°F)/26 sec, 67.8°C (154°F)/20 sec, 683°C
(155°F)/l6 sec, 689°C (156°F)/l3 sec, or 694°C (157°F) (and up)/10 sec (USDA-FSIS
1996). The time-temperature regulations for cooked beef, roast beef, and corned beef
were also altered to reflect a 6.5-log decrease in Salmonella, a reduction from previous

regulations. Thirty different schedules are allowed for these products ranging from a

minimum internal temperature of 544°C (130°F) with a holding time of 112 min to
71.1°C (160°F) for 103.

Guidelines for cooking meat products in foodservice establishments are published
in The Food Code of the Food and Drug Administration (FDA, 1999). The Food Code
recommends that roast beef and corned beef be cooked to one of the following
time/temperature combinations: 54°C (l30°F)/121 min, 56°C (l32°F)/77 min, 57°C
(l34°F)/47 min, 58 (136°F)/32 min, 59°C (138°F)/ 19 min, 60°C (l40°F)/ 12 min, 61°C
(l42°F)/8 min, 62°C (144°F)/5 min, or 63°C (145°F)/3 min. Whereas, hamburger patties
are recommended to be cooked to 70°C (158°F) for < ls, 68°C (155°F) for 153, 66°C
(150°F) for 1 min, or 63°C (145°F) for 3 min.

Recently the USDA-FSIS, in addition to new time/temperature schedules, has
allowed processors to establish processes based on lethality standards (USDA-F SIS,
1999). Lethality performance standards are based on the destruction of a specific number
of Salmonella microorganisms.

Lethality performance standards are outlined in terms Of decimal reductions or
logo reductions in Salmonella. A D value is defined as the time (min) required to reduce
a target population by one log at a given temperature. The larger the D value, the greater
the heat resistance of a culture. A 2 value is defined as the temperature increase necessary
to reduce a D value by one log. The 2 value is an indication of temperature sensitivity.
The lower the 2 value the more temperature sensitive the organism. Therefore, for
accurate process control, thermal inactivation parameters for Salmonella and other

pathogens must be established in the meat product Of interest.

In addition to lethality performance standards, ready-tO-eat products also must
meet stabilization and handling standards. Stabilization standards are designed to prevent
growth of spore-forming bacteria that can cause food borne illness. It was anticipated
that most processors would meet this standard by rapidly cooling products following
cooking. The third standard, handling, was introduced to prevent infectious pathogens
from being introduced into the product following processing, stabilization, or final
packaging.

Performance standards tell establishments what degree of effectiveness their
HACCP plans will be expected to achieve while allowing processors the flexibility of
incorporating individualized processing procedures. If an establishment chooses to use
an alternative treatment or intervention, such as organic rinses or curing, in combination
with heat treatment, it is the responsibility of the establishment to demonstrate a
cumulative equivalency in microbial reduction. The process Should not only Show
Specific reductions in Salmonella, but also the reduction of all other foodborne pathogens
of concern.

Although E. coli 0157:H7 has been identified in beef products with greater
frequency, it has been shown to be less heat resistant than Salmonella (Doyle and Shoeni,
1984; Line et al., 1991). Listeria monocytogenes, another pathogen of concern in beef
products, has shown to exhibit a higher heat resistance than Salmonella. Yet its incidence
is much lower and Often is the result of post-cooking contamination. Therefore
monitoring the thermal reduction Of Salmonella in cooked products would indicate

destruction of most other pathogens.

Compliance to lethality performance standards must be verified using challenge
studies. Challenge studies are laboratory studies designed to mimic commercial
processes to verify the destruction of pathogens. The USDA-FSIS is recommending the
use of multiple serotypes of Salmonella for challenge studies. The use of a combination
of serotypes is referred to as a cocktail. The pathogenic strains comprising the cocktail
should be relatively heat resistant and also should be among those historically implicated
in food borne outbreaks. Yet the USDA-FSIS does not specify each strain that should be
included in a cocktail to be used in challenge studies. Thus, different cocktails will
render varying kinetic parameters, with different processors using different standards to
verify processing schedules. These variations could be eliminated if one universal
cocktail was defined for use.

A proposed lethality performance standard for fully cooked, uncured meat patties
includes a 5 IOgIO reduction in Salmonella (USDA-FSIS, 1996). However, in review, the
FSIS has concluded that a higher lethality standard may be needed to produce safe
ground beef patties. It appears that the lethality performance standard for meat patties
will be increased to 6.5 loglo (Hasiak, 2000). While this is under further investigation,
the time/temperature schedules published in 1996 remain in effect. Although these
thermal processing guidelines are available, there is still a need to verify adequate
cooking once a product has been processed.

2.2. Thermal inactivation of Salmonella strains and Escherichia coli 0157:H7 in
meat

In 1999 the USDA-F SIS finalized the approved use of lethality performance

standards for beef processing. Quantitative information on lethality Of pathogens is

required to establish effective schedules. Thermal processing has evolved into the most
common means of preventing food borne disease. Variables affecting heat resistance of
pathogens include species, muscle type, pH, and fat content (Murphy et al., 2000).
Thermal inactivation parameters can also be influenced by initial population of
pathogens, age of the cultures, and heat shock injury (Murphy et al., 1999)

2.2.1. Salmonella

The first regulations for roast beef processing were based upon results Obtained by
Goodfellow and Brown in 1978. Thermal death time studies were performed on a
Salmonella cocktail comprised of S. Typhimurium, S. Newport, S. Agona, S. Bovis-
Morbificans, and S. Muenchen in ground meat. The resulting D values were 61-62 min at
516°C (125°F), 3.8-4.2 min at 572°C (135°F), and 0.6-0.7 min at 627°C (145°F),
depending on the recovery method used. The 2 value Of Salmonella was found to be
556°C. From these D values, processing time/temperature requirements were
established to achieve a 7 logm reduction in Salmonella in ground beef ranging from 195
min at 128°F (533°C) to 5 min at 144°F (622°C) (USDA-FSIS, 1990).

More recently, in response to new recommendations by the USDA-F SIS, thermal
death time studies have been completed utilizing an 8-strain Salmonella cocktail in
ground beef (Juneja et al., 2000). Juneja et al. (2000) attempted to identify the most
representative and appropriate strains of Salmonella to include in a cocktail used to
quantify heat resistance in meat products. NO correlation between the heat resistance and
the origin of the Salmonella spp. could be established due to significant variation in the
heat resistance among strains. The D values reported in meat were higher at 58 and 60°C

than those calculated in chicken broth. The D values of the Salmonella cocktail in

ground beef are presented in Table 2.1. The D values found by Juneja et a1. (2000) were
lower than those reported by Goodfellow and Brown (1978) and Orta Ramirez et al.
(1997) in ground beef. The 2 values were higher, thus indicating the 8-strain Salmonella
cocktail was less temperature sensitive. These differences could be explained by the
choice of strains used.

Thermal death time studies using another Salmonella cocktail were performed in
ground chicken (Murphy et al., 2000). Six Salmonella strains were inoculated into
ground chicken breast patties prior to thermal processing. The D values were higher in
ground chicken than those in peptone-agar solution at all temperatures tested. Juneja et
a1. (2000) also found higher D values in beef as compared to chicken broth (Table 2.2).
This cocktail was less heat resistant than S. Senftenberg in ground turkey (Veeramuthu et
al., 1998). The D values reported here were higher than Juneja et al. at all connnon
temperatures. The higher D values observed might be due to the inclusion of S.
Senftenberg in the cocktail. Early studies have demonstrated the unusual heat resistance
of S. Senftenberg in buffer (D57oc = 31 min) (N g et al., 1969) and poultry (D66oc = 3.5
min) (Milone and Watson, 1970).

Orta-Ramirez et al. (1997) tested the thermal resistance of S. Senfienberg in
ground beef using 10 by 75 mm thermal-death-time tubes. D values were 53.00, 15.17,
2.08, and 0.22 min at 53, 58, 63, and 68°C, respectively, with a 2 value of 624°C. These
results are in good accordance with those reported by Murphy et al. (2000), which also
included S. Senftenberg.

Thermal resistance Of pathogens is influenced not only by the strain(s) chosen, but

also the microbial growth phase of the organism during testing. Heddleson et a1. (1991)

Table 2.1 D values3 of Salmonella cocktail in ground beef (Juneja et al., 2000)

 

 

Temperature (°C) D value (min)
58 8.65 i 0.03
60 5.48 i 0.04
62.5 1.50 i: 0.01
65 0.67 i 0.04

 

3‘D values are expressed as the mean of two replicate experiments i standard
deviation.

Table 2.2. D valuesa for Salmonella in chicken breast meat (Murphy et al., 1999)

 

 

Temperature (°C) D value (min)
55.0 30.1
57.5 12.9
60.0 5.88
62.5 2.51
65.0 1.16
67.5 0.287
70.0 0.175

 

aD values are expressed as the mean of three triplicate thermal death time studies.

10

reported that maximum heat resistance occurred during stationary phase of Salmonella
spp. in phosphate buffer. Freezing of Salmonella cultures in liquid media also can lower
thermal resistance (Smith, 1995). Enumeration of S. Typhimurium strain 55 cultures that

were frozen for 24h at -30°C during log phase exhibited a 2 loglo decrease in comparison

to cultures that were chilled for 24h at 0°C. Greater recovery of S. Typhiumurium strain
55 was observed using cells frozen in stationary rather than log phase.

Environmental stresses and their effects on pathogen heat resistance have received
a lot of attention in recent years. Heat shock is defined as the exposure of cultures to sub-
lethal heat treatments. The stress associated with sub-lethal heat shock induces the rapid
synthesis of specific proteins, known as heat-Shock proteins (Juneja et al., 1998).
Increased thermal tolerance to a second heat challenge, which would normally be lethal,
has been observed in microbial cells that have synthesized these heat-shock proteins.

Bunning et a1. (1990) reported that heat shocking or exposing Salmonella cultures
to temperatures just slightly above their normal growth temperature added a degree of
protection against lethal effects. Heat Shocking S. Typhimurium at 48°C for 30 min
increased the D value from 21.3 min to 96.1 min at 52°C in broth. Yet, when the cultures
were heat shocked at 42°C and 52°C, no significant difference was seen in D values at an
inactivation temperature of 578°C. Mackey and Derrick (1987) heat shocked S.
Thompson cultures at 48°C for 30 min before performing thermal inactivation studies in
minced beef. The D values at 54°C and 60°C were increased by factors of 2.4 and 2.7,
respectively.

Juneja et a1. (1998) reported that heat shocking a four-strain cocktail of E. coli

0157:H7 at 46°C for 15 to 30 minutes increased the heat resistance in ground beef. In

11

inoculated ground beef, the non-heat-Shocked cells decreased by 2.17 logs colony
forming units (CFU)/g within 4 min and by 7.8 logs CFU/g after 15 min at 60°C.
Whereas the heat shocked cocktail decreased 0.35 and 6.38 loglo CF U/g, respectively
under the same inactivation conditions. Thermal death time studies were performed on
E. coli 0157:H7 in tryptic soy broth (TSB) and a ground beef slurry after heating at a
sub-lethal temperature of 45°C for 30 min (Williams and lngham, 1997). The heat shock
increased the D values by 37, 68, and 50%, respectively, at 54, 58, and 62°C in tempered
TSB. Yet, the D values at 58°C in a ground beef slurry did not differ from those of the
non-heat-shocked cultures.

These results are similar to those from a heat shock study with L. monocytogenes.
Farber and Brown (1990) reported that after heat shocking L. monocytogenes in a
sausage mix at 48°C for 30 min before heating at the final test temperature, D values at
62 and 64°C were not significantly different from non-heat-shocked values. No
differences in D values were seen until after 120 min of heat shocking at 48°C, which
produced a culture 2.4 times as heat resistant at 64°C.

In addition to heat-Shocked cultures, thermally injured cells have also been
studied. Thermally injured cultures are defined as microbial cells that are recoverable on
non-selective media but not detected on selective media. Therefore, thermally injured
Salmonella would grow on a tryptic soy agar, but not on bismuth sulfite. The difference

between survivor counts on the two media would give an indication of the percentage of
cells that have been injured. Ahmed et al. (1995) heated E. coli 0157:H7 at 50, 55, and

60°C for differing times to produce injured cells. L. monocytogenes cells were

thermally injured after being heated at 52°C for 1h (Smith and Archer, 1988). The

12

degree of thermal injury was determined by plating the survivors on tryptose phosphate
agar with and without sodium chloride. Thermally injured cells are of concern if present
in a food product, but remain undetected with normal enumeration procedures.
Therefore, thermal inactivation of Salmonella has been shown to be influenced by the
freezing history of the meat product, growth phase and previous thermal treatment of the
cultures.
2.2.2 Escherichia coli 0157 :H7

Thermal inactivation of a four-strain cocktail Of E. coli 0157:H7 was determined
in ground beef (Table 2.3) (Juneja et al., 1997). The 2 value was reported as 6.0°C.
Thermal death time studies of E. coli 0157:H7 in ground beef were also conducted in
thermal death time tubes (10 by 75 mm) by Orta-Ramirez et al. (1997). The D values
ranged from 46.10 min at 53°C to 0.12 min at 68°C. The 2 value was reported as 560°C.

At comparable temperatures, Juneja et a1. (1997) reported that E. coli 0157:H7
was less heat resistant at lower temperatures (58°C ), yet more resistant at higher
temperatures (63°C). Differences in D values could be attributed to the use of different
E. coli 0157:H7 strains. Juneja et al. (1997) performed the thermal inactivation studies
on a 4-Strain mixture of E. coli 0157:H7, whereas Orta-Ramirez et a1. (1997) used a
single strain.

Thermal resistance of E. coli 0157:H7 in ground beef is influenced by the fat
content in meat. D values were higher in high fat as compared to low fat ground beef
(Table 2.4). D values reported by Line et al. (1991) for E. coli 0157:H7 in ground beef

at 575°C were in good agreement with Juneja et a1. (1997). Juneja et al. (1997) reported

a D value Of 4.95 min at 575°C in ground beef containing 10% fat. Line et a1. (1991)

Table 2.3. D values" for E. coli 0157:H7 in ground beef (Juneja et al., 1997)

 

Temperature (°C) D value (min)

 

55 21.13 i 0.25
57.5 4.95 i 0.16
60 3.17 i 0.18
62.5 0.93 i 0.01
65 0.39 i 0.00

 

aD values are expressed as the mean Of two replicate experiments i standard

deviation.

Table 2.4. D values (min)“‘ of E. coli 0157:H7 in ground beef as affected by fat content

 

Line et al., 1991

Ahmed et al., 1995

 

 

 

 

Fat (%) Fat (%)
Temp Temp
(°C) 2.0 30.5 (°C) 7 10 20
51.6 78.2 115.5 50 55.34 80.66 92.67
57.2 4.1 5.3 55 11.40 15.30 19.26
62.7 0.30 0.47 60 0.45 0.46 0.47

 

“D values are expressed as mean value from three experiments

14

performed thermal inactivation studies in ground beef containing 2.0% and 30.5% fat and
found D values at 572°C to be lower and higher, respectively, than Juneja et a1. (1997).
These results further confirm higher thermal resistance in higher fat ground beef. The D
values reported by Ahmed et al. (1995) were 1.4 and 6.9 magnitudes lower at 55 and
60°C, respectively, than those reported by Juneja et a1. (1997) in 10% fat ground beef.
Differences in results could be attributed to Juneja et a1. (1997) using a 4-strain mixture
of E. coli 0157:H7, whereas Ahmed et al. (1995) performed studies with a single strain.
Differences could also be explained by recovery methods used in each of the studies.
Ahmed et a1. (1995) also found that higher fat content decreased the 2 value of E. coli
0157:H7 in beef products.
Juneja et al. (1998) assessed the influence of storage conditions on thermal resistance of
E. coli 0157:H7 in ground beef. After 48h of frozen storage, numbers of E. coli
0157:H7 decreased 5.05 logs after 16 min at 60°C in comparison to E. coli 0157:H7 in
fresh ground beef that decreased 6.7 logs under the same conditions. Thus thermal
inactivation of E. coli 0157:H7 is dependent on the strain chosen, the fat content and the
storage conditions of the beef product.
2.3. Time-temperature integrators

Thermal processing is one of the major methods of food preservation. The
variability in factors contributing to the effectiveness of a thermal process Often require
the Specific determination of appropriate heat treatments. Variables that affect thermal
processing of meat products include species, muscle type, pH, fat content, and storage
conditions. Even with reliable heat treatment recommendations it is still essential to

monitor the cooking process to assure that the desired effects are achieved.

Consumers and food handlers have accepted the fact that visible signs, such as color
changes in food, are indicators of safe processing. However, recent research has shown
that color and texture indicators are not reliable. Sufficient numbers of ground beef
patties were found to turn brown well before reaching the internal target temperature of
160°F (Hunt et al., 1995). The USDA-FSIS continues to advise consumers to use food
thermometers when cooking meat and poultry.

It is very difficult to use a thermometer and thermocouple in commercial processing
plants to verify that the time-temperature requirements have been met for a ground beef
patty. Berry et al. (1999) found that the internal temperature of beef patties varied as

much as 18°C when the center temperature was 71°C. Current regulations state that

commercial establishments must verify the time and temperature within i 1°F on one
beef patty on a production line each hour. Yet with the large temperature gradients in a
patty, these regulations may not be adequate.

The enforcement of Hazard Analysis Critical Control Point (HACCP) programs for
meat processors has decreased the incidence of microbial contamination, yet methods to
verify efficacy of thermal processes are still needed. Methods to evaluate the accuracy of
thermal processes are classified into three different approaches: (1) in situ methods, (2)
physical-mathematical methods, and (3) time-temperature integrators (TTIS) (Hendrickx
et al., 1995; Van Loey et al., 1996).

2.3.1. In situ methods
In the in situ methods, changes in a food component are evaluated before and
after thermal processing. Attributes frequently measured are microbial contaminants,

sensory factors, or nutrients. In practice, however, monitoring often proves laborious,

l6

time-consuming, and expensive. In addition, the concentration of the attribute must be
large enough to be detected. Therefore, other more convenient and successful methods
have been and must be developed.

2.3.2. Physical-mathematical models

Physical-mathematical models combine previous knowledge fiom the kinetic
parameters of a food quality attribute with a time-temperature profile to calculate the
impact of the thermal treatment on the parameter of interest. Limitations of these
methods are attributed to the need for time-temperature data. These temperature profiles
can be collected by either direct physical measurement or from constructive computation.
Problems arise when direct recording is not possible due to processing conditions or
when inaccuracies occur with time-temperature histories that are reconstructed by
mathematical modeling. Conservative estimates used in these models Often result in
over processing which in turn leads to significant nutrient loss and quality reduction of
the product. Another limitation with physical-mathematical models is the lack of data for
kinetic models for new technological advances in cooking, including ohmic and
microwave heating.

2.3.3. Time-temperature integrators (TTIS)

TTIs can overcome the limitations created by the in situ and physical-
mathematical modeling approaches. A TTI has been defined as a device that undergoes
an irreversible and precisely measurable change in response to a time-temperature history
that mimics the change in a target attribute exposed to the same thermal history
(Hendrickx et al., 1995). A major advantage of a TTI is the quantification of the

integrated time-temperature impact on a quality attribute without acquiring the actual

time-temperature history Of the product. Ideal TTIs would be simple and inexpensive to
prepare, easily recoverable with an accurate and user-fiiendly read-out. TTIs should be
incorporated into a food product without disrupting the heat transfer within the product
while quantifying the thermal process on a target attribute.

TO quantify the treatment that was performed on the target attribute, the TTI must
Show the same time-temperature dependent response as that Of the target attribute when
the temperature is the only rate determining factor. Mathematically, this can be expressed
as:

(FTref)target = (FTrcf)TTI
Assuming an nth order TTI system, the rate equation can be expressed as:
dX/dT = - kxX"
where k)( is the rate constant and n the reaction order. For a first order reaction under
isothermal conditions, the integrated equation is:
In (Xo/X) = kxt
where X0 is the initial value of the target attribute. In many cases, according to the
Arrhenius equation the rate constant can be expressed as:
kx = ko exp (-EA/RT)

where k0 is the rate constant at a reference temperature, EA is the activation energy, R is
the universal gas constant and T is the absolute temperature.

In a general case where the reaction order is different than unity, with the reaction
rate constant and independent of the order of reaction, the F value can be written as

(Hendrickx et al., 1995):

(Frrcrh = Itexp [EA (L - 1)] dt
0 R T... T

which represents the equivalent heating time at a reference temperature resulting in the
same lethality as the time-varying temperature profile. This equation is valid for the
target attribute only when the activation energies of both the TTI and the target parameter
are the same. In the simplest form of TTI, 2 values of the target attribute and TTI should
be equal. An overestimation of the actual processing impact by the use of a TTI with a
higher 2 value should be avoided, as this could lead to undercooking of food products,
and a public health risk. A TTI with a lower 2 value than the target attribute would lead
to overprocessing and decreased quality Of the cooked product.

TTIS can be classified into biological, chemical or physical systems. In the
biological TTI category, measuring devices detect a change in biological activity, such as
numbers of microorganisms or level of enzyme activity after heating. Chemical and
physical systems are based on a chemical or physical response, respectively, toward time
and temperature (Hendrickx et al., 1995). A TTI can be extrinsic or intrinsic (Van Loey
et al., 1996). An extrinsic TTI is incorporated into a food, whereas intrinsic TTIS are
naturally present in the food. Three approaches can be distinguished with regard to the
application of a TTI: dispersed, permeable, or isolated. In a dispersed system, the TTI is
homogeneously distributed throughout the food. Permeable systems allow for some
diffusion of a TTI through a food product, whereas isolated systems allow for well
defined positioning of a TTI within a food product.

Most biological TTIS are based on microbiological or enzymatic assays.

Microbiological TTIS can be used to monitor thermal processing through the use of

count-reduction systems and survivor-kill systems (Van Loey et al., 1996). Utilizing
count-reduction systems, the impact of the thermal process can be quantified from the
survivor counts. The survivor-kill system indicates the presence or absence of microbial
growth (an indication of sufficient thermal impact).

For low-acid foods, Bacillus stearothermophilis, B. subtilis 5230, B. coagulans,
and Clostridium sporogenes are used for monitoring wet heat sterilization (Hendrickx et
al., 1995). Pflug (1980) used a spore solution Of B. stearothermophilis in a plastic rod to
verify the sterilization of green beans, corn, and peas. Upon comparison of the results
with values determined using the physical-mathematical model, it was concluded that the
plastic rods could be used effectively to validate sterility. An overview of other
applications with microbial TTIS has been published by Hendrickx et a1. (1995). The
main disadvantages of microbiological TTIS include the length of incubation, low level of
analytical precision, the need to properly calibrate the Spores to determine their thermal
resistance and the possibility of contamination.

Protein-based systems, particularly enzyme systems, have some advantages as
TTIS over microbial systems. The relative ease of assaying remaining enzyme activity
make protein-based systems very applicable. In addition, enzymatic TTIS are less
expensive to prepare, while being rapid and accurate. (It-Amylase of Bacillus
licheniformis immobilized on glass beads and Bacillus amyloliquefaciens in the presence
of polyolic alcohols or carbohydrates have been studied as possible TTIS (De Cordt et al.,
1992; 1994). Application of these TTIS is limited by the temperature of enzyme

inactivation.

20

Very few chemical or physical indicators are currently developed. Initially
compounds within food products, for which analytical methods of detection were already
established, were studied as potential TTIS. These compounds included thiamin,
pantothenic acid, vitamin C, and methylmethionine sulfonium salt (Kim and Taub, 1993).
The limitations of these methods included lengthy procedures for recovery and post
treatment assay. Kim and Taub (1993) also studied compounds that form during thermal
processing of foods. Difficulties arose when they could not find a compound that was
easily assayed and whose concentration after processing could be used to verify sterility.
2.4. R-Phycoerythin

R-Phycoerythin (PE), phycocyanin, (PC) and allophycocyanin (APC) are three
major proteins that comprise a class of fluorescent compounds named phycobiliproteins
(PBP) (MacColl and Guard-Friar, 1987). Phycobiliprotiens are photosynthetic antenna
complexes that provide characteristic colors to cyanobacteria, red algae, and
cryptomonads. They are located on the cytoplasmic face of the thykaloid membrane of
the chloroplasts (Glazer, 1994). These compounds harvest solar energy in regions of the
visible spectrum that show low chlorophyll absorption and transfer the excitation energy
to chlorophyll in the photosynthetic membrane.

The PBP structure is comprised of apoproteins and chromophores called bilins,
which are linear tetrapyrrole groups covalently attached to the apoprotein. The bilins
fluoresce in the visible range. The two major chromophores are phycoerythrobilin and
phycocyanobilin, at least one of which will be present in all phycobiliproteins. Three

minor bilins have been identified: phycourobilin, criptobviolin, and the 697-nm bilin.

21

PE, red in color, has absorption bands at 498—568 nm and fluorescent emission
bands at 570-620 nm. The blue proteins, PC and APC, have absorption bands at 550-630
nm and 598-680 nm and fluorescent emission bands at 630-650 nm and 660-680 nm,
respectively.

R-phycoerythin (R-PE) contains three polypeptide chains: or, B, and y or y’. These
subunits are usually organized as (16136}! or (Xéfifi'Y,. The subunit structure has a diameter
of 12.7 nm with a ratio of or + B : y of 12: 1. The or subunit contains two

phycoerythrobilins, the [3 subunit contains three subunits, and the y subunit contains two
phycoerythrobilins and two phycourobilins for a total of thirty-four chromophores
(Glazer, 1994). The molecular mass has been reported to range between 240 and 260
kDa. The entire amino acid sequence of the on and [3 subunits of PE from Porphyridium
cruentum has been identified (Glazer and Hixon, 1977; Sidler et al., 1989). Heat or
chemical denaturation can cause conformational changes in the native structure of PBP
resulting in partial or complete loss of optical properties (Ogawa et al., 1991; Creighton,
1993)
2.4.1. R-Phycoerythin as a TTI to verify adequate processing of beef products
Orta-Ramirez (1999) investigated the effectiveness Of PE as an extrinsic, single
component, fluorescence-based TTI to monitor thermal processing in beef products. PE
was easily extracted from edible algae, abundant (up to 20% dry weight) and had a wide
range of thermal tolerance under different chemical conditions. In addition, after thermal
processing, the fluorescent response of PE was readily detectable without intermediate

recovery steps.

22

The effect of pH on the spectral properties Of R-PE was investigated (Orta-
Ramirez, 1999). Fluorescence intensity was similar at pH values from 5.0-9.0. At pHs
below 4.0 or above 10.0 R-PE did not fluoresce. In addition, it was shown that a solution
of R-PE exhibited a bright pink-red color at neutral pH values, and shifted to purple at
pHs above and below neutrality.

It was Shown by Ogawa et al. (1991) that at pH near 4.0, R-PE exists in the form
of aggregates. At pH 7.0, PE has a compact, native conformation with the chromophores
tightly packed in the interior. The protein is partially unfolded at pH 10.0. The addition
of sucrose, SDS, NaCl, or ME at pH 4.0 resulted in a complete loss of R-PE fluorescence
even before heating. Similarly, at pH 10.0, the protein structure was very sensitive to the
addition of solvents due to being partially unfolded.

The effects of pH on thermal inactivation of R-PE were also studied (Orta-
Ramirez, 1999). D values were calculated at temperatures from 60-90°C for pH values
from 4.0-10.0. The R-PE was most thermally resistant at pH 6.0 and 7.0 (pH 6.0: D70 =
12258.72 min, D30 = 239.74 min, D90 = 1.43 min; pH 7.0: D70 = 2204.75, D30 = 461.87
min, D90 = 1.21 min). The 2 values increased from pH 4.0 (2 = 459°C) to pH 9.0 (2 =
915°C) and then decreased once again at pH 10.0 (2 = 444°C).

A large range of 2 values was Obtained by manipulating the environment Of R—PE,
thus prompting the study of combined effects of pH and additives. Overall, sucrose and
B-mercaptoethanol (ME) had stabilizing effects, while the addition of sodium dodecyl
sulfate (SDS), sodium chloride (N aCl), and urea caused protein denaturation of PE.

It was concluded from these preliminary experiments that by modifying the

environment, the 2 value of PE can be adjusted to that of a target pathogen in meat

23

products. The D values of R-PE in 0.012 M borate buffer, pH 9.0 ranged from 512.82
min at 60°C to 10.57 min at 70°C. The 2 value of 599°C fell within the range given for 2
values of Salmonella of 56°C to 62°C (Goodfellow and Brown, 1978; Orta-Ramirez et
al., 1999). This range of 2 values was chosen for comparison due to the fact that
Salmonella is the target microorganism in thermal processing of beef products (USDA-
FSIS, 2000).

Non-isothermal experiments were also completed to verify the adequacy of R-PE
as a TTI for thermal processing of meat products (Orta-Ramirez, 1999). Results
indicated that R-PE could be used to distinguish between undercooked, properly cooked,
and overcooked samples. Thus this protein could be used to verify proper processing of
meat products once a cut-off value of residual fluorescence was determined for thermal
processing. Though the R-PE was able to differentiate between adequate and inadequate
heat processes, the PE did not respond accurately to the time temperature combinations
tested. The General Method (Pflug, 1997) was used to calculate predicted fluorescence
values corresponding to the heating protocols. A weak correspondence (R2< 0.80) was
seen between the predicted and Observed values, indicating that non-isothermal
inactivation Of PE did not follow first-order kinetics.

When normalized fluorescence (fluorescence at time t/ initial fluorescence) was
plotted against calculated lethalities for each process, an exponential relationship was
found. Thus it was concluded that even though the thermal inactivation of PE did not
follow first order kinetics, it could be used as a TTI to predict the thermal destruction of
Salmonella during thermal processing of beef products. Therefore, even though the 2

value of PE will not be equal to the 2 value of Salmonella, results Show that the PE

24

destruction is very predictable. PE will be able to distinguish between undercooked and
properly cooked beef products.
2.5. Triose phosphate isomerase

Triose phosphate isomerase (TPI) is an enzyme that catalyzes the interconversion of
dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) (Webb
and Knowles, 1975). TPI has been isolated and crystallized from many different sources
including 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).

TPI activity is determined through the following coupled reactions:

Triose phosphate

isomerase
Glyceraldehyde-3-phosphate * dihydroxyacetone phosphate (DHAP)

 

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

 

Glyceraldehyde-3-phosphate is the substrate of the reaction and the glycerol-3-phosphate
dehydrogenase (GDH) acts as the coupling enzyme (Beisenhertz, 1955). It has been
shown that TPI activity is affected by choice of buffer and pH during the assay
procedures. In a neutral pH range (7 to 8) there is little variation in activity, but at pH
6.3, the activity is reduced by half (Beisenherz, 195 5). It has also been shown that the
activity of TPI is doubled in triethanolamine-HCI buffer in comparison to bicarbonate-
C02 at pH 7.5 (Beisenherz, 1955). Oesper and Meyerhof (1950) reported that a buffer
containing 0.05 M phosphate reduced TPI activity by 75%.

2.5.1 Triose phosphate isomerase as a TTI for thermal processing of ground beef

25

Endogenous enzymes have been suggested as potential indicators of proper
thermal processing in beef products. Orta-Ramirez et al. (1997) conducted thermal.
inactivation studies on several enzymes, E. coli 0157:H7 and S. Senftenberg. For an
endogenous protein to be an ideal indicator Of thermal processing, it should exhibit the
same temperature sensitivity (2 value) of the target microorganism. The 2 value reported

for TPI in ground beef Of 556°C, (Orta—Ramirez et al., 1997) was close to the value for

Salmonella (z = 564°C) (Goodfellow and Brown, 1976), the pathogen used to establish
processing guidelines for ground beef patties.

Sair et al. (1999) investigated the relationship between TPI activity and cooking
endpoint temperature in ground beef patties. TPI activity decreased with increasing
cooking temperatures in ground beef patties (24.4% fat) from 60.0 to 71 .1°C. Yet no
differences in TPI activity could be distinguished when patties were cooked to internal
temperatures of 71.1 to 822°C. Enzyme activity in beef patties decreased from a raw
value of 2,303U/ g meat to 6.3 U/g meat or less at temperatures greater than or equal to
71 .1°C.

Larger standard deviations were seen in TPI activity at lower processing
temperatures suggesting that large temperature variations were present within a ground
beef patty during processing (Sair et al., 1999). This hypothesis was supported by Berry
et al. (1999) who reported temperature differences up to 18°C using infrared
therrnography on cross sections Of patties cooked to a final internal temperature of
71 .1°C.

The use of TPI to verify that roast beef has been properly processed was

investinged by Hsu et al. (2000). Ground semimembranous muscle was cooked to

26

achieve a 7 logm reduction using either low, medium, or high temperature roast beef
processing schedules (USDA, 1996). TPI activities were similar (P<0.05) in ground
meat cooked using three equivalent time-temperature regulations. The lowest TPI
activities in adequately processed roast beef model systems and pilot plant studies were
2.1 and 1.56 U/g, respectively. It was suggested that a residual TPI value less than or
equal to about 2 U/ g could be used to indicate proper thermal processing of roast beef

cooked under any of the time-temperature regulations.

27

CHAPTER 3
THERMAL INACTIVATION OF SALMONELLA TYPHIMURIUM DT104 AND

AN 8-STRAIN SALMONELLA COCKTAIL UNDER DIFFERENT CONDITIONS

3.1. ABSTRACT

Thermal death time studies using three strains of Salmonella Typhimurium
DT104 in ground beef (4.8% fat) revealed that the D values of each strain were not
different (P>0.05) at 55 or 58 °C. At 61 °C, the D value of strain 10127 (0.57 min) was
higher (P<0.05) than those of strains 01071 (0.43 min) and 10601 (0.41 min). The D
values for strain 10127 in ground beef were similar at 58, 61, and 64 °C, regardless Of fat
content. However, at 55 °C the D value in high fat ground beef (21 .98 min) was more
than two fold greater than that in the low fat ground beef (9.05 min).

The heat resistance of the Salmonella cocktail in stationary phase decreased after
inoculation and frozen storage of ground beef at 55, 58 and 61°C. Thermal resistance of
the cocktail decreased (P<0.05) at 55, 61 , and 63°C after 14d of starvation in 0.1%
peptone water at 4°C. The D values for the thermally injured (56°C for 30 min) and heat
shocked (48°C for 30 min) cocktails were not different (P<0.05) from one another at any
of the test temperatures. The D values Of the thermally injured cocktail were also not
different (P<0.05) from the Salmonella cocktail in log phase. The heat shocked cocktail
was less heat resistant than the non-heat shocked cocktail at 58°C. The 2 value of the
thermally injured cocktail was the highest recorded, thus these cultures were the least

temperature dependent.

28

3.2. INTRODUCTION

Current command and control regulations require that beef patties be cooked to a
certain temperature for a Specified amount of time to eliminate pathogens. Proposed
regulations (USDA-F SIS, 1999) would supplement Specific time temperature
requirements with lethality performance standards. A 5 loglo reduction in Salmonella has
been proposed as the lethality performance standard for fully cooked, uncured mea‘.
patties (USDA-F SIS, 1999). The FSIS has not finalized this standard due to concerns
that a process equivalent tO a 5 log 10 reduction in Salmonella may not be sufficient to
protect public health. The lethality performance standard for roast beef requires the use
of a combination of thermal and non-thermal processes sufficient to achieve a 6.5 log
reduction in Salmonella (USDA-FSIS, 1999).

The USDA-F SIS (2000) recommends the use Of a cocktail or a combination Of
Salmonella serotypes to verify compliance to the performance standard. This cocktail
should consist of Salmonella strains that exhibit relatively high heat resistance and have
been previously implicated in foodborne outbreaks (UDSA-F SIS, 2000). The D and 2
values for different Salmonella serotypes were determined in chicken broth and meat
products in order to identify the most appropriate strains to use for defining heat
resistance in meat products (Juneja et al., 2000). A comparison of these strains to E. coli
0157:H7 and other Salmonella strains would be useful to verify that the
recommendations are appropriate.

Salmonella Typhimurium DT104 has emerged as a particularly dangerous
Salmonella phage type that is resistant to ampicillin, chloramphenicol, streptomycin,

sulfonarnides, and tetracycline (Hollingsworth et al., 1997). In 1996, 32% of the S.

29

Typhimurium isolates tested at the Centers for Disease Control and Prevention
demonstrated the ACSSuT resistant pattern associated with Definitive Type 104
(Morbidity and Mortality Weekly Review, 1997). D values for a clinical isolate of
Salmonella Typhimurium DT104 in chicken broth ranged from 4.16 min at 55°C to 0.27
min at 62°C (Juneja et al., 2000).

Several factors influence the thermal resistance of Salmonella, including age of
the culture, nutrient content of the growth medium, heat treatment history, and initial
population. According to Heddleson et al., (1991) maximum heat resistance of
Salmonella spp. occurred in cells that had reached stationary phase in tryptic soy broth
after 24h of incubation. The numbers of viable pathogens also can be affected by the
nutrients available in the growth medium. Dykes and Withers (1999) examined sub-
lethal damage in Listeria monocytogenes cells starved at 4°C vs. fresh cultures. Growth
of fresh cells was minimally affected by selective agar. In contrast, numbers of starved
cells were greatly reduced after direct plating on a selective agar. Potential public health
risks could arise if starved cultures within raw food products remain undetected on
selective media.

The history of heat treatment and its effect on thermal resistance of pathogens has
been studied intensely in recent years. Mackey and Derrick (1987) reported that the D
values of S. Thompson in minced beef increased from 10.5 to 25.0 min at 54°C and from
0.46 to 1.26 min at 60°C after a previous heat treatment at 48°C for 30 min. Thermal
resistance of E. coli 0157:H7 in ground beef at 60°C increased following thermal
treatment at 46°C for 15 to 30 min. Muscle type, pH, fat and moisture content of the

heating menstruum also influence thermal resistance of microorganisms. Therefore,

30

thermal inactivation parameters of a microorganism must be established in the food of
interest with controlled environmental conditions for accurate process control.

The objectives of this study were to determine (1) the thermal resistance of S.
Typhimurium DT104 in ground beef of two fat contents and (2) to assess the effects Of
microbial growth phase, freezing, starvation conditions and non-lethal heat treatment on
thermal resistance of a Salmonella cocktail.

3.3. MATERIALS AND METHODS
3.3.1. Bacterial strains

Three human isolates of Salmonella Typhimurium DT104 (10601, 10127, 01071)
were obtained from B. Swaminathan (Centers for Disease Control and Prevention,
Atlanta, GA). The following eight Salmonella strains were Obtained from V.K. Juneja
(Agricultural Research Service, Eastern Regional Research Center, USDA-ARS,
Philadelphia, PA): S. Thompson FSIS 120 (chicken isolate), S. Enteriditis H3527 and
H3502 (clinical isolates phage type 13A and 4, respectively), S. Typhimurium H3380
(human isolate DT104), S. Hadar MF60404 (turkey), S. Copenhagen 8457 (pork), S.
Montevideo FSIS 051 (beef), and S. Heidelberg F5038BG1 (human isolate). All strains
were preserved in vials containing tryptic soy broth (TSB) (Difco, Detroit, MI) with 10%
glycerol at —80 °C.

3.3.2. Culture preparation

TO propagate the cultures, one loopful of frozen culture was transferred to 9 mL
Of TSB in 20 mL culture tubes. Cultures were maintained by daily transfers in TSB
(37°C, 18-24h), with a minimum of two consecutive transfers before use. Each inoculum

was prepared from either an 18-24h (log phase) or 48h (stationary phase) culture

31

containing approximately 109 CF U/ml. The eight Salmonella strains received from
Juneja were grown in separate culture tubes and then combined in equal volumes just
before centrifugation to produce a cocktail for thermal death time studies.

On the day of each experiment, cultures were pelleted by centrifugation at 6,000 x
g for 10 min at 4 °C and resuspended in sterile 0.1% buffered peptone water to a
concentration of about 1010 CFU/ml. Following centrifugation, the starved Salmonella
cocktail was resuspended in 5 mL of0. 1% peptone water and stored at 4 °C for 14 days.
The cultures were enumerated by serially diluting in 0.1% peptone water and plating on
Petrifilm® aerobic count plates (3M, St. Paul, MN) in duplicate.

To produce thermally injured and heat shocked Salmonella cocktails, 4 x 1012
CFU/mL were added to 200 mL of tempered TSB in a 3L Erlenmeyer flask and heated
for 30 min at 56 or 48°C, respectively (Bunning et al., 1990; Mackey and Derrick, 1987).
Preliminary studies showed that a 1-log decrease in growth was observed on bismuth
sulfite agar (selective agar) when compared to tryptic soy agar with 0.9% yeast extract
(non-selective agar), thus producing a 90% thermally injured culture after heating at 56°C
for 30 min.

3.3.3. Ground beef preparation

Low fat beef was Obtained from the Michigan State University Meat Laboratory.
High fat beef was received from a local processor. The meat was ground twice through a
4 mm diameter plate in a Kitchen Aid grinder (Model K5-A, Hobart, Troy, OH), vacuum
packaged in 60g portions, and frozen at -12 °C. The frozen meat was transported on dry

ice to Iowa State University and irradiated (10 kGy) to eliminate indigenous microflora.

32

The irradiated beef was tested for sterility by plating a 1:10 dilution onto Petrifilm®
Aerobic count plates.

Moisture, fat, and protein contents were determined by AOAC (1996) methods
950468, 991.36, and 981.1, respectively. For determination of pH, 10g of ground beef
were homogenized with 90 mL of distilled water using a Polytron homogenizer (Model
PT 10/35, Brinkman Instruments, Westbury, NJ) at Speed setting 3 for 303. The pH of
the homogenized mixture was measured using a combination electrode (Model 145,
Coming, Medfield, MA). Analyses were done in triplicate.

3.3.4. Inoculation of ground beef

Microorganisms were added dropwise, using aseptic procedures, to obtain a target
concentration Of 108 CFU/g ground beef. The meat was mixed in a sterile bowl using
sterile gloves to ensure even distribution. Inoculated meat was transferred into a 60 mL
syringe (Becton Dickinson and Co., Franklin Lakes, NJ) after which 1 g aliquots were
extruded into 5 x 25.5 cm sterile polyethylene laminated nylon bags (Butcher and Packer,
Detroit, MI). Sterility of the bags was confirmed by mixing 9 mL Of 0.1% peptone water
in random bags and plating the resultant liquid on Petrifilm® Aerobic count plates. Bags
containing the meat were rolled to a thickness of 1 mm using a template and then heat
sealed.

For the freezing studies, the inoculated meat was held at —9 °C for 7 days. Meat
was tempered to 4 °C under cold running water prior to thermal death time studies.

3.3.5. Thermal inactivation procedure
The bags containing meat were placed in a rack and completely submerged in a

Polystat circulating water bath (Model 1268-52, Cole-Palmer Instrument Co._. Chicago,

33

IL) set at 55, 58, 61, 64 or 66 °C. The temperature was monitored using a thermocouple

(Type T, length 8.6 cm, width 1.0 mm, accuracy i 0.1 °C, Omega Engineering, Stamford,
CT) connected to a datalogger system (Daqbook 100, Omega) inserted and sealed in the
pouches. The heating lag time was 83. Time zero was defined as the time when the meat
reached the target temperature. Preliminary studies were conducted to determine
appropriate time-temperature combinations to achieve 2 a 5 logo reduction in pathogens.
Meat was removed at specific time intervals, held on ice at 4 °C and used within 4h.
Three replicate batches of ground beef were heated at each temperature.
3.3.6. Enumeration of Salmonella

Cooked ground beef was transferred to sterile WhirlpakT“ bags (18 oz, Nasco, Ft.
Atkinson, WI) and manually homogenized for 1 min with 9 mL Of 0.1% sterile peptone
water. Bacterial counts were determined by decimal dilution of the ground beef in sterile
0.1% peptone water and enumerated using Aerobic Petrifilm Count Plates (3M, St. Paul,
MN). All plates were incubated at 37 °C for at least 24 hr.
3.3.7. Calculation of D values and 2 values

Bacterial counts were converted to logarithms. D values were estimated assuming
linear inactivation kinetics. D values (in minutes) were calculated by linear regression of
surviving bacteria vs. time from at least 4 data points with a correlation coefficient i>0.80
using Microsoft Excel Version 5.0a (Microsoft Corp., Redmond, WA). Thermal
resistance curves were determined by plotting log D values vs. temperature. The 2 value
was determined as the negative reciprocal of the slope of the thermal resistance curve.
Significant differences of means were determined using paired t-tests (P<0.05) (JMP,

SAS Institute, 1998)

34

3.4. RESULTS AND DISCUSSION
3.4.1. Composition

Low fat ground beef (pH 5.65 i 0.01) contained 4.8% i 1.1% fat, 72.4% i 0.2%
moisture and 15.8% i 2.6% protein. High fat ground beef (pH 5.72 i 0.01) contained
19.1% i 0.7% fat, 63.4 % :t 0.6% moisture, and 14.4% i 1.9% protein. NO
microorganisms were detected in the irradiated meat.
3.4.2. Heat resistance of Salmonella Typhimurium DT104

Thermal death time studies were performed using three strains of S. Typhimurium

DT104 in ground beef (4.8% fat) (Table 3.1). The D values of the three strains were not
different (P>0.05) at 55 or 58 °C. At 61 °C, the D value of strain 10127 (0.57 min) was
higher (P<0.05) than those of strains 01071 (0.43 min) and 10601 (0.41 min). At 64 °C
the D value for strain 10601 (0.07 min) was about half that of the other two strains
(P<0.05). The D values calculated for the three strains at 58 °C were similar to those
reported by Humphrey et al., (1997) for three wild strains of S. Typhimurium DT104 on
pork muscle (D53 cc = 2.61 min).

Maurer (2000) reported D values of 16.34 min, 2.72 min, 0.44 min and 0.15 min
at 55, 58, 61, and 63°C, respectively, for the log phase 8-strain Salmonella cocktail in
19.1% ground beef. Thus, the S. Typhimurium DT104 strain 10127 was more thermally
resistant at 55 and 61°C. If beef was cooked according to the processes established for
this cocktail, a 5D reduction in S. Typhimurium DT104 might not occur. Thus, this

cocktail would underestimate thermal processing schedules needed to accomplish a 5D

reduction if more heat resistant strains of Salmonella were present.

35

Table 3.1 D values‘ and regression parameters of three strains of Salmonella
Typhimurium DT104 in high (19.1%) and low (4.8%) ground beef. ‘

 

 

Bacterium Fat Temp y- Slope R‘ D value
Content (°C) intercept (min)
S. Typhimurium High 64 6.30 -6.20 0.97 0.16 :I: 0.01
DT104-10127
61 6.32 -1.54 0.97 0.65 i 0.03
58 6.57 -0.38 0.95 2.63 i 0.19
55 6.49 -0.05 0.95 21.98 i 2.15
S. Typhimurium Low 64 7.13 -6.70 0.98 0.15 i 0.01
DT104-10127
61 6.97 -1.75 0.96 0.57 i 0.01
58 7.01 -0.44 0.95 2.26 i 0.10
55 6.97 -0.11 0.95 9.05 -_r 0.13
S. Typhimurium Low 64 7.03 -l4.43 0.99 0.07 i 0.01
DT104-10601
61 6.67 -2.47 0.98 0.41 i 0.01
58 7.87 -0.49 0.95 2.15 :r 0.07
55 6.40 -0.10 0.93 10.55 i 1.04
S. Typhimurium Low 64 6.97 -7.20 0.95 0.14 i 0.01
DT104-01071
61 7.49 -2.33 0.94 0.43 i 0.02
158 7.24 -049 0.97 2.06 i 0.13
55 7.09 -0.10 0.96 10.27 i 0.78

 

aD value is expressed as the mean i standard error Of the mean of three replicate

determinations.

36

D values for Escherichia coli 0157:H7 in high fat (19.1%) ground beef were
reported as 22.47 min, 2.05 min, 0.32 min, and 0.18 min at 55, 58, 61, and 63°C,
respectively (Maurer, 2000). Thus, E. coli was more heat resistant at 55°C, than S.
Typhimurium DT104 strain 10127.

The D values for strain 10127 in ground beef were similar at 58, 61, and 64°C,
regardless Of fat content. However, at 55°C the D value in high fat ground beef (2'. .98
min) was more than two fold greater than that in the low fat ground beef (9.05 min).
Similar effects due to fat content were seen with E. coli 0157:H7 in ground beef. (Line
et al., 1991; Ahmed et al., 1995; Maurer, 2000).

3.4.3. Salmonella cocktail

The combination of the 8-strain Salmonella cocktail in stationary growth phase
with freezing of inoculated meat was also investigated. D values for the 8-strain
Salmonella cocktail in stationary phase without freezing were previously reported as
18.66 min, 3.39 min, 0.57 min, and 0.20 min at 55, 58, 61, and 63°C, respectively in high
fat (19.1%) ground beef (Maurer, 2000). Thermal resistance of the Salmonella cocktail
in stationary phase decreased (P<0.05) after inoculation and frozen storage of ground
beef at 55, 58 and 61°C (Table 3.2). Formation of ice crystals during freezing which
disrupt the cell membrane is likely responsible for the observed decrease in viability
(Doyle and Cliver, 1990; Smith, 1995). Given these findings, greater total lethality may
be achieved when cooking previously frozen raw products as compared products that

have not been frozen.

37

Table 3.2 D valuesa and regression parameters of an 8-stain Salmonella cocktail in
stationary phase after freezing inoculated groundAbeef (19.1% fat).

 

 

Temp (°C) y-intercept Slope R1 D value (min)
63 6.47 -5.13 0.90 0.20 i 0.01
61 7.08 -3.54 0.94 0.28 i 0.02
58 6.74 -0.42 0.85 2.36 i 0.14
55 6.87 -0.08 0.85 12.52 i 1.27

 

aD value is expressed as the mean i standard error of the mean of three replicate
determinations.

The thermal stability of E. coli 0157:H7 was increased due to cold-shock protein
synthesis after freezing (-18°C) in comparison to fresh (3°C) hamburger patty storage
(Jackson et al., 1996). Heat resistance of the Salmonella cocktail decreased in ground
beef after freezing. Juneja et al., (1997) reported that freezing of E. coli 0157:H7
cultures (-18°C) in ground beef did not affect the heat resistance. Differences could be
due to storage conditions including sample size, length of storage, and storage
temperature. During studies completed here, 1 g of ground beef was frozen at -9°C for 7
days. Juneja et al., (1997) and Jackson et al., (1996) held 3g samples for 48h and 113g
samples for 9h at -18°C. Sample size could have an impact cooking rates. In addition,
cold-shock protein induction and biochemical changes in the bacterial membrane might
be affected by the time and temperature of storage.

Storing the Salmonella cocktail in 0.1% peptone water at 4°C for 14 days to
produce starved cells resulted in D values of 5.85 min, 2.93 min, 0.22 min, and 0.11 min

at 55, 58, 61 , and 63°C, respectively, thus reducing the heat resistance at all four

38

temperatures tested. Differences (P<0.05) were seen between the D values of the starved
Salmonella cocktail and the cocktail in log phase at 55, 61, and 63°C.

No literature could be found on the effect of starvation on thermal inactivation,
yet starvation at 4°C reportedly reduced the growth Of L. monocytogenes and E. coli
0157:H7 cultures on selective media. According to Dykes and Withers (1999) growth of
fresh L. monocytogenes cultures showed a reduction in growth numbers of 8% or less
when plated on a selective compared to a non-selective medium. However, populations
of cells that had been stored in phosphate-buffered saline (pH 5.5) for 4 weeks were
reduced by more than 2 log (99%) on selective medium.

In addition, when E. coli 0157:H7 cultures were starved in phosphate buffered
saline, Arnold and Kasper (1995) reported that viable counts decreased 100 fold after 48
hr. Although it appears that extended freezing may decrease thermal resistance, the
presence sub-lethally damaged strains may constitute in a substantial health risk, since
these pathogenic strains could remain undetected in foods.

D values for the thermally injured and heat Shocked Salmonella cocktail were not
different (P< 0.05) from one another at any of the temperatures tested (Table 3.3).
Maurer (2000) reported similar D values for the Salmonella cocktail in log phase the
same temperatures tested. The D values for the heat shocked cocktail were also not
different from the non-heat Shocked cocktail at 63, 61, or 55°C. However, at 58°C, the D
value of the heat shocked cocktail was lower (P< 0.05).

Studies have shown that therrnotolerance of bacteria is greatly affected by the
heating menstuum during heat shock and subsequent thermal inactivation (Farber and

Brown, 1990; Juneja et al., 1998). Thermal inactivation studies were performed with

39

Table 3.3. D values and regression parameters of an 8-strain Salmonella cocktail
after thermally injury (56°C for 30 min) or heat shock (48°C for 30 min).

 

 

Treatment Temp y-intercept Slope R‘ D value (min)
(°C)

56°C - 30min 63 5.69 -3.86 0.82 0.26 i 0.01a
61 7.58 -247 0.88 0.41 i 0.05"
58 5.68 -034 0.88 2.98 i 0.22c
55 7.13 -0.09 0.88 11.14 i 1.75d

48°C — 30 min 63 7.71 -6.03 0.90 0.17 i 0.02a
61 8.20 -2.84 0.97 0.35 i 0.01b
58 7.73 -053 0.92 1.90 i 0.12c
55 7.53 -009 0.91 11.71 i 2.62d

 

75°" Means i the standard error of the mean followed by the same letter were not different
(P<0.05).

E. coli 0157:H7 in tryptic soy broth (TSB) and a ground beef slurry after heating at a
sublethal temperature of 45°C for 30 min (Williams and lngham, 1997). Heat Shock
increased the D values by 37, 68, and 50%, respectively, at 54, 58, and 62°C in tempered
TSB. However, the D values at 58°C in a ground beef slurry were not different.

These results are in good accordance with a heat shock study involving L.
monocytogenes. F arber and Brown (1990) reported that after heat shocking L.
monocytogenes at 48°C for 30 min, D values in a sausage mix were not different (P <
0.05) at 62 and 64°C. D values did not change until the cultures were heat shocked for
120 min at 48°C.

Mackey and Derrick (1987) heat shocked S. Thompson cultures at 48°C for 30
min before performing thermal inactivation studies in minced beef. The D values at 54°C

and 60°C increased by factors of 2.4 and 2.7, respectively. Xavier and Ingham (1997)

40

reported that heat shocking at 42°C for 60 min produced in higher D values in casein
peptone-soymeal peptone broth for S. Enteriditis at 52, 54, and 56°C, but not at 58°C. In
addition, Bunning et a1. (1990) reported that heat shocking S. Typhimurium at 48°C for
30 min increased the D value from 21.3 min to 96.1 min at 52°C in broth. However,
when the cultures were heat shocked at 42°C and 52°C, no difference was seen in D
values at an inactivation temperature of 578°C. These results suggested that 42°C is tOO
low to initiate the synthesis of heat-shock proteins, while 52°C led to a progressive loss
of bacterial viability.

Heat shocking a four-strain cocktail of E. coli 0157:H7 at 46°C for 15 to 30 min

increased thermal resistance in ground beef (Juneja et al., 1998). Using the untreated
cocktail populations decreased by 2.17 and 7.8 loglo CFU/g, respectively, after 4 and 15
min of heating at 60°C. Populations in the heat shocked cocktail decreased 0.35 and 8.5
loglo CF U/ g under the same inactivation conditions. Differences in the results reported

here could be due to the use Of a cocktail rather than a homogenous culture.

3.4.4. 2 Values

Thermal inactivation of all three Salmonella Typhimurium DT104 strains
(z=4.13-5.07°C) was less temperature dependent than that of E. coli 0157:H7 (z = 3.60)
and the Salmonella cocktail in log phase (z = 3.90) in ground beef with 19.1% fat as
indicated by the higher 2 values (Table 3.4) (Maurer, 2000). The 2 value for S.
Typhimurium DT104 strain 10127 was higher in low as compared to high fat ground
beef. Ahmed et a1. (1995) reported similar findings in which the 2 value for E. coli
0157:H7 increased from 4.35 to 478°C in ground beef as the fat content decreased from

20 tO 7%.

41

The 2 value of the Salmonella cocktail in stationary phase after freezing was not
different from the cocktail in stationary phase (2 = 408°C) (Maurer, 2000) (P<0.05).
There was no difference between the 2 values for the starved cocktail and the cocktail in
log phase. In addition, the 2 values for the heat shocked and thermally resistant cocktail

were not different (P<0.05) from the non-heat shocked cocktail in log phase.

42

Table 3.4 The 2 valuesa and regression parameters of Salmonella Typhimurium
DT104 and an 8-strain Salmonella cocktail in stationary phase after freezing in high
(19.1 %) and low (4.8%) fat ground beef.

 

 

Bacterium Fat y-intercept Slope R2 2 value (°C)
content

S. Typhimurium High 14.11 -0.23 0.99 4.28 i 0.05

DT104-10127

S. Typhimurium Low 11.80 -0.20 1.00 5.07 i 0.02

DT104-10127

S. Typhimurium Low 14.38 -0.24 1.00 4.13 i 0.07

DT104-10601

S. Typhimurium Low 12.50 -0.21 0.99 4.77 i 0.06

DT104-01071

Salmonella cocktail High 14.18 -0.24 0.97 4.20 i 0.06

(48h frozen)

Salmonella cocktail - High 13.75 -0.23 0.95 4.28 i 0.01

starved

Salmonella cocktail - High 12.92 -0.22 0.98 4.64 i 0.09

(56°C for 30 min)

Salmonella cocktail - High 13.86 -0.23 0.99 4.28 i 0.27

(48°C for 30 min)

 

8The 2 value is expressed as the mean 1*: standard error of the mean of three replicate
determinations.

43

3.5. CONCLUSIONS

The new performance standards require meat processors to validate the efficacy
of their processes to reduce microbial contamination if cooking procedures vary from
published compliance and safe harbor guidelines. Results stated here indicate that the
particular pathogen, the state of the meat (fresh or frozen), and the fat content must be
considered when designing thermal processes. Based on our findings, this 8-strain
Salmonella cocktail may not be able to adequately predict a 5-log reduction for some
pathogens including more heat resistant strains of S. Typhimurium DT104 at low
temperatures (555°C). In addition, great care should be taken during thermal processing
to reduce the lag time Of heating meat products, so as not to produce therrnotolerant cells

that may require greater process lethalities to ensure a safe meat product.

44

CHAPTER 4
EVALUATION OF PHYCOERYTHRIN AS A TIME-TEMPERATURE

INTEGRATOR IN BEEF PATTIES

4.1. ABSTRACT

R-phycoerythrin encapsulated in glass capillary tubes was inserted into ground
beef patties of varying fat content and size to evaluate its applicability as a time-
temperature integrator. The normalized fluorescence of PE was 0.31 or below when 113g
high fat (19.1%) patties were processed to meet USDA-FSIS time-temperature
regulations or lethality performance standards. All of the patties exhibited at least a 5-log
decrease in a Salmonella cocktail or met one of the safe harbor guidelines when the PE
fluorescence value was lower than 0.31.

Similar results were seen in 113g low fat (4.8%) ground beef patties. Analysis of
the normalized fluorescence of PE revealed that a value of 0.29 was indicative of a
properly cooked patty. Of the 41 patties tested, only one sample which met the USDA-

F SIS thermal processing guidelines had a PE value greater than 0.29, meaning that the PE
did not correctly identify this patty as being properly cooked.

A normalized PE fluorescence of 0.21 or below was found when 60g ground beef
patties were properly cooked according to the USDA-F SIS safe harbor guidelines. This
endpoint fluorescence value was lower than in the 113g patties. Improper application of
methodologies designed for larger patties limited the collection of accurate results.
Further studies will be needed to validate phycoerythrin as a time-temperature integrator

for proper thermal processing of ground beef patties.

45

4.2. INTRODUCTION

Title 9 of the Code of Federal Regulations has traditionally been used to outline
the USDA processing requirements to destroy food borne pathogens in meat products
(USDA-F SIS, 1999). Recently the USDA-F SIS, in addition to time/temperature
schedules, has allowed processors to establish thermal processes based on lethality
standards (USDA-FSIS, 1999). Lethality performance standards are based on the
destruction of a specific, significant number of Salmonella microorganisms.

One proposed lethality performance standard for fully cooked, uncured meat
patties allows the use of any thermal process to achieve a 5 logm reduction in Salmonella
(USDA-F SIS, 1999). Although these thermal processing guidelines are available, there is
still a need to verify adequate cooking.

A time temperature integrator (TTI) is a device that undergoes an irreversible and
precisely measurable change in response to time-temperature history that mimics the
change in a target attribute exposed to the same thermal history (Hendrickx et al., 1995).
A major advantage of a TTI is the quantification of the integrated time-temperature
impact on microbial destruction without acquiring the actual time-temperature history of
the product. Ideal TTIS would be simple, inexpensive to prepare and easily recoverable
with an accurate and user-friendly read-out. TTIS Should be incorporated into a beef
patty without disrupting heat transfer while quantifying the thermal process on microbial
destruction.

Phycoerythrin (PE), a phototosynthetic antenna complex protein in cyanobacteria,

red algae, and cryptomonads, was investigated as an extrinsic, single component,

46

fluorescence-based TTI to monitor thermal processing in beef products (Orta-Ramirez,
1999). PE, red in color, has absorption bands at 498—568 nm and fluorescent emission
bands at 570-620 nm. In earlier experiments, the 2 values of PE increased from pH 4.0 (2
= 459°C) to pH 9.0 (2 = 915°C) and then decreased at pH 10.0 (2 = 444°C). A large
range of 2 values was obtained by manipulating the environment of R-PE. It was
concluded from these experiments that the 2 value of PE could be adjusted to that of a
target pathogen in meat products by modifying the content, pH, and concentration of the
buffering solution. The 2 value of PE in 0.012 M borate buffer, pH 9.0, of 599°C fell
within the range given for 2 values of Salmonella of 56°C to 662°C (Goodfellow and
Brown, 1978; Orta-Ramirez et al., 1997, Juneja et al., 2000). This range Of 2 values was
chosen for comparison since Salmonella is the target microorganism in thermal
processing of beef products based on the new lethality standards (U SDA-F SIS, 2000).

In non-isothermal studies, residual PE fluorescence in a buffer system could
distinguish between undercoooked, properly cooked, or overcooked thermal processes for
ground beef. It was concluded that inactivation of PE did not follow first order kinetics.
A non-linear model was developed to fit the thermal inactivation of S. Senftenbergand
fluorescence loss of PE. In both cases, lethality was very predictable, suggesting that a
mathematical relationship could be established between the lethality of S. Senftenberg
and the fluorescence loss of R-PE.

The objectives of this study were to (1) verify the ability of PE to distinguish
between cooking schedules Of varying time and temperature combinations within a beef
patty and (2) to identify a minimum fluorescence reduction to indicate proper cooking of

a ground beef patty.

47

4.3. MATERIALS AND METHODS
4.3.1. Ground beef patties

Low fat ground beef was obtained from the Michigan State University Meat
Laboratory. High fat ground beef was received from a local processor. The meat was
ground through a 3.175 mm diameter plate in a grinder (Tor Rey, Model M-32-5,
Monterrey, Nuevo Leon, Mexico), vacuum packaged in 1135g portions, and frozen at
-12°C. The frozen meat was transported on dry ice to Iowa State University and
irradiated (10 kGy) to eliminate indigenous microflora. The irradiated beef was tested for
sterility by plating a 1:10 dilution on Petrifilm® Aerobic count plates. Ground beef was
thawed at 4°C for 24h prior to use.

Moisture, fat and protein contents were determined by AOAC (1996) methods
950468, 991.36, and 981 .1, respectively. For determination of pH, 10g of ground beef
plus 90 mL of distilled water were homogenized using a Polytron homogenizer (MOdel
PT 10/35, Brinkman Instruments, Westbury, NJ) at speed setting 3 for 303. A
combination electrode (Model 145, Coming, Medfield, MA) was used to measure the pH
of the ground beef slurry. Analyses were done in triplicate.

4.3.2. Bacterial strains

Eight Salmonella strains were obtained from V.K. J uneja at the Agricultural
Research Service, Eastern Regional Research Center of the USDA. The strains were S.
Thompson F SIS 120 (chicken isolate), S. Enteriditis H3 527 and H3502 (clinical isolates
phage type 13A and 4, respectively), S. Typhimurium H3380 (human isolate DT104), S.

Hadar MF60404 (turkey), S. Copenhagen 8457 (pork), S. Montevideo FSIS 051 (beef),

48

and S. Heidelberg F5038BG1 (human isolate). The strains were preserved at —80 °C in
tryptic soy broth (TSB) (Difco, Detroit, MI) containing 20% glycerol.
4.3.3. Culture preparation

To propagate the cultures, one loopful of frozen culture was transferred to 100 mL
of TSB in a 200 mL culture bottle. Cultures were maintained by daily transfers in TSB,
with a minimum of two consecutive 24h transfers before use. Each inoculum was
prepared from a l8-24h (log phase) culture containing approximately 109 CF U/ml. A
preliminary growth curve study was conducted to determine the times of incubation
needed to achieve log phase (Maurer, 2000). The eight cultures used in the Salmonella
cocktail were each grown in separate culture tubes and combined in equal volume before
centrifugation just prior to thermal death time studies.

On the day of experimentation, cultures were pelleted by centrifugation at 6,000 x
g for 15 min at 4 °C and resuspended in sterile 0.1% buffered peptone water to a
concentration of about 1010 CFU/ml. The inoculums were serially diluted in 0.1%
peptone water and plated in duplicate on tryptic soy agar (TSA) (Difco, Detroit, MI)
containing 0.6% yeast extract to determine initial cell numbers.
4.3.4. Inoculation of ground beef

The 10 mL inoculum was added dropwise to thawed ground beef to obtain a
target concentration of 108 CF U/g meat. The meat was mixed using a sterilized Kitchen
Aid mixer (Model K5-4, Hobart, Troy, OH) at speed setting 3 for 6 minutes. Inoculated
meat (113 or 60g) was transferred to a 100 x 15 mm sterile polystyrene Petri dish (VWR
Scientific Products, West Chester, PA) using a sterile spatula and pressed into beef

patties. The inoculated patties were kept at 4°C and used within 6h.

49

4.3.5. Preparation of phycoerythrin

Phycoerythrin (PE) was previously isolated from fresh cultures of Porphyra
yezoenis (Orta-Ramirez 1999). The protein was stored in 60% ammonium sulfate in a
light impermeable glass bottle at 4°C. Each sample of PE was dialyzed overnight against
distilled deionized water. The protein was then diluted in 0.025 M borate buffer, pH 9.0,
to achieve an absorbance of 0.1 at 565 nm. .

Capillary tubes (Accu Fill 90 Micropet, Cat. NO. 4624, Becton-Dickenson, and
Co., Parsippany, NJ) were filled with PE solution (200uL) by capillary action. To allow
for even headspace at both ends, 50 uL were removed using a micropipet. One end was
sealed using a gas/oxygen flame and the other with Teflon tape.
4.3.6. Ground beef cooking preparations and procedures

A thermocouple (Type T, length 8.6 cm, width 1.0 mm, accuracy i 0.1°C, Omega
Engineering, Stamford, CT) was attached in parallel to the capillary tube with dental floss
near the tip and tape at the other end (Figure 4.1A). Dental floss was used to introduce
the least disruption to heat transfer within the patty while binding the thermocouple and
capillary tube together. The thermocouple and capillary tube were inserted horizontally
into the beef patty (Figure 4. 18). The thermocouple was connected to a datalogger
system (Daqbook 100, Omega Technologies Inc.) to record the temperature at the center
of the patty every second throughout the cooking process.

Ground beef patties were cooked on a griddle (Model 07032, Presto Jumbo
griddle, National Presto Industries, Inc., Eau Claire, WI) set to 177°C (3 50°F).
Preliminary studies revealed the griddle cycled i 7°C around the target surface

temperature. The griddle was preheated for a minimum of 30 min prior to use. During

50

the cooking process, patties were heated for 2 min, flipped, and heated for another 2
minutes, then flipped every minute until the process was completed based on the
procedures of Berry and Bigner—George (1999). Each beef patty was cooked in the same
location on the griddle.

Cooking times for the 113g patties ranged from 6 to 13 min producing maximum
internal temperatures Of 54.2 to 75.9°C. The 60g patties were cooked for 4 to 8 min
producing internal temperatures of 60.7 to 75.9°C. The patties were immediately
transferred to sterile WhirlpakTM bags (18 oz, Nasco, Ft. Atkinson, WI) and placed in an
ice water bath for quick cooling. Ten 113g patties, either high or low fat, were cooked
each day for 10 days for a total of 50 low fat and 50 high fat patties. The high fat and low
fat patties were grilled Over 43 and 9 day periods, respectively. Eighteen high fat 60g
patties were cooked on consecutive days for a total of 54 patties.

Following the cooling period, patties were sliced open vertically with a sterile
knife and measurements were taken to determine the vertical placement of the
thermocouple and capillary tube containing PE. Patties were accepted if the
thermocouple and capillary tube were located within i5% from the center point of the
cooked patty thickness. The capillary tubes were removed, sterilized, kept on ice, and
analyzed within 6h. Each heated patty was weighed following the cooling period to
calculate the percentage of cook yield (Equation 1).

% Cook yield = (Cooked weight / Raw weight) x 100 (1)
A 25g sample Of the ground beef was removed from the center Of the patty using a sterile
50 mL beaker as a template and placed into a 7 by 12 cm sterile stomacher bag (Seward

Medical, London, UK). The beef samples were kept at 4°C and analyzed within 6h.

51

(A)

 

Capillary tube Phycoerythrin Thermocouple

(B)

 

 

 

 

 

[ L/J l/\
Beef patty Capillary tube and thermocouple

Figure 4.1. (A) Attachment of the capillary tube containing phycoerythrin to a
thermocouple, (B) Insertion position of the capillary tube and thermocouple into a beef

patty

52

4.3.7. PE fluorescence measurements

PE was measured at maximum emission wavelength using a CytoFluor II
Microwell Fluorescence reader equipped with CytoFluor 11 software, Version 2.0c
(Biosearch Incorporated, Bedford, MA). Heated and unheated PE solutions were
transferred from capillary tubes into a 96-well fluorescence plate reader (Cat. no.
DGSSIS, Life Science Products Inc., Denver, CO). The CytoFluor lamp was allowed to
warm-up for a minimum of 10 min prior to use. The excitation setting used was 485/20
nm and emission setting 590/35 nm. These settings allowed for excitation readings at
485 i 20nm and emission readings at 590 i 35 nm. After a mixing time of 203, 10
readings were taken on each plate. PE fluorescence measurements were expressed as
normalized values calculated by dividing the fluorescence at time t (F) by the initial
fluorescence before heating (F0).
4.3.8. Enumeration of surviving bacteria

Cooked ground beef (25 g) was diluted in 225 mL of lactose broth (Difco, Detroit,
MI) and stomached for 905 in a masticator (Model 0410, IUL Instruments USA, Inc.
Cincinnati, OH). Bacterial counts were determined by decimal dilution of the ground
beef in sterile 0.1% peptone water and plated on TSA containing 0.6% yeast extract. All
plates were counted after 48h of incubation at 37°C for. Destruction Of the 8-strain
Salmonella cocktail was expressed as the log of the ratio between the count at time t (N)
and the initial count (No). This was calculated by subtracting the log of the final count
afier heating from the log of the initial count before cooking (log No). The resulting
values gave the log numbers of Salmonella colonies per gram of beef destroyed by the

heat treatment.

53

4.3.9. Calculation of process lethality

Lethality values were calculated using the General Method (Pflug, 1997) with a
reference temperature of 65°C (Equation 2). The 2 value used for R-PE was 5.99°C
(Orta-Ramirez, 1999).

Lethality = l
10"((65-T) / 5.99) (2)

 

where T = temperature (°C) at time t. Each temperature recorded over time by the
datalogger system was converted into an equivalent lethality. Individual lethalities were
then summed to give the total lethality of the process.

4.4. RESULTS AND DISCUSSION

4.4.1. Effect of maximum center temperatures on Salmonella in high fat ground beef

patties.
High fat ground beef (pH 5.72 i 0.01) contained 63.4 % i 0.6% moisture, 19.1%
i 0.7% fat, and 14.4% i 1.9% protein. No microorganisms were detected in the

irradiated meat. Typical center temperature histories during cooking for ground beef

patties (19. 1% fat) are given in Figure 4.2. Inserting a thermocouple into the center of a

54

(A)

 

 

 

 

 

 

60
50-
Q 401
e 30.
E
33 20.
On
8 10
[—4
0- fl .
0 200 400 600 800 1000
Time(s)
(B)
70-
6 6° -
E 50.
g 40
g 30-
.5. 20.
I—
10
0

 

 

0 2CD 400 600 800 1000
Time (s)

Figure 4.2. Representative time-temperature cooking profiles of a 113g high fat (19.1%)
ground beef patty. (A) Illustrates a patty cooked to 564°C and total lethality of 5.25s;
(B) Illustrates a patty cooked to 71 .9°C and total lethality of 14203.

beef patty allowed for measurement of the maximum temperature achieved at the center
of each patty. The maximum center temperature of the 41 high fat patties ranged from
56.8 to 75.9°C. Regulations provided by the USDA-FSIS (1996) Offer processors time-
temperature guidelines for proper thermal processing of ground beef patties. These
regulations vary from an endpoint temperature Of66.1°C (151°F) for 413 to 694°C
(157°F) (and up) for 103. These safe harbor guidelines were designed to produce a 5
logo reduction in Salmonella in ground beef patties. I

As center temperature of the beef patties was increased, the number of surviving
Salmonella (R2 = 0.83) decreased (Figure 4.3). From the time-temperature data recorded
during each cooking process, it was possible to categorize each patty as undercooked or
properly cooked according to the safe harbor guidelines. Out of 41 patties with
acceptable thermocouple placement, 19 patties did not meet the required time-
temperature regulations provided by the USDA. Therefore 22 patties met or exceeded
the time-temperature safe harbor guidelines.

Four patties met at least one of the time-temperature guidelines provided by the
USDA, but the Salmonella cocktail did not decrease by 5 logo. This same trend was
seen by Orta-Ramirez (2000) in non-isothermal experiments with the Salmonella cocktail
in ground beef and could have serious implications on the safety of meat products.
Ground beef patties cooked according to safe harbor guidelines may not provide a 5 loglo
decrease in the number of surviving Salmonella.

There was also a patty that did not meet any of the safe harbor guidelines but
Salmonella decreased by greater than 5 logo. The lowest temperature stated in the

processing regulations is 66. 1°C. A patty may be cooked at a temperature below 661°C

56

 

 

 

 

Log Decrease in Salmonella (CFU)

0" O Undercooked
I Properly Cooked

 

 

 

o—xmootscncnxrooco
o
r

 

 

 

55 a) 65 70 75 3)
Maximum Center Temperature (°C)

Figure 4.3. Effect of maximum internal center temperature on the log decrease in
Salmonella cocktail of ground beef patties (19.1% fat). Patties were categorized as

undercooked or properly cooked according to the time-temperature regulations of the
USDA (2000).

57

for a longer time to achieve a 5 loglo decrease in Salmonella. Yet according to the safe
harbor guidelines this patty would be classified as undercooked. The advantage of the
proposed lethality performance standard is that a beef patty may be cooked according to
any time-temperature combination as long as a 5 log”) decrease in Salmonella is verified.
4.4.2. Effect of lethality on Salmonella in high fat ground beef patties.

In addition to time-temperature guidelines, the USDA is in the process of
establishing lethality performance standards for ready-to-eat ground beef patties.
Lethality standards allow processors to cook meat products according to any time-
temperature combination if validation of a significant decrease in Salmonella is proven.
A proposed lethality performance standard for fully cooked, uncured meat patties
includes a 5 logo reduction in Salmonella (USDA, 1999). Lethality values were
calculated from the thermal process each patty received.

Lethalities of the 41 patties ranged from 5.25 to 49803 at 65°C. The lethality
equivalent to the time-temperature combinations given by the USDA-F SIS for ground
beef patties processed using safe harbor guidelines was calculated using the General
Methods (Pflug, 1997). The lethality for all time-temperature combinations was 665 at
65°C. Any patty with a lethality greater than 663 at 65°C could be considered properly
processed by USDA definitions. The range of processing lethality used in this study
resulted in undercooked and properly cooked beef patties. An increase in process
lethality resulted in a decrease in cooking yield in beef patties (R2 = 0.86). This suggests
that the time and temperature data used to calculate process lethality were accurately
collected. Accurate process lethalities offer a balance between undercooked patties that

could lead to a public health risk and overcooked patties with decreased quality.

58

A correlation was seen between the log decrease in Salmonella and lethality of the
cooking process (R2 = 0.86) (Figure 4.4). This was a higher correlation than between
Salmonella and maximum center temperature, suggesting that using process lethalities to
predict destruction of Salmonella in beef patties may be more accurate than using center
temperature. Twenty-two patties had lethalities greater than 665 at 65°C and met one of
the time-temperature regulations of the USDA. There were 7 patties with lethalin
greater than 665 at 65°C that did not meet either the time-temperature regulations or the
5-log10 decrease in Salmonella. Lethality is calculated using the entire time-temperature
history. A total lethality may be greater than 665 at 65°C if a beef patty were held at a
temperature below 66. 1°C for an extended length of time. Time-temperature regulations
do not extend below 66. 1°C, therefore patties with lower maximum endpoint
temperatures would be considered undercooked. A 5 log 10 decrease was consistently
seen when process lethality was greater than 3008 at 65°C.

4.4.3. Relationship between normalized PE fluorescence and Salmonella in high fat
ground beef patties.

Normalized PE fluorescence in cooked patties ranged from 0.05 to 0.86.
Normalized fluorescence was compared to the log decrease in the Salmonella cocktail in
forty-one 113g high fat ground beef patties. A linear trend was seen between normalized
fluorescence and the number of surviving Salmonella (R2 = 0.74) in cooked beef patties.
Using linear regression, it was possible to calculate the normalized PE fluorescence that

corresponded to a 5 logo decrease in Salmonella (Figure 4.5). Normalized fluorescence

59

 

 

 

 

 

 

 

 

 

9
8 . I I
E 7 - I I
% 6 .I I I $
0 5 -_o—' L
8 °. '
(4 I
.E 4 22‘
g 3
0
8 2 Q Undercooked
CD
3 1 I Properly Cooked
0 - . , . .
0 1000 2000 3000 4000 5000
Lethality (s)

Figure 4.4. Effect of process lethality on the log decrease in Salmonella in ground beef patties
(19.1% fat). Patties were categorized as undercooked or properly cooked according to the time-
temperature regulations of the USDA (2000).

60

 

0.9 .
n y = -0.089lx + 0.7662

R2 = 0.7355

0.8 . I I

 

 

Normalized Fluorescence

 

 

 

Log Decrease in Salmonella (CFU)

Figure 4.5. Correlation between normalized fluorescence and log decrease in Salmonella
cocktail in ground beef patties (19.1% fat).

61

of PE was reduced to 0.31 (CI 99%) or below inside patties with a 5-log10 decrease in
Salmonella. A normalized PE fluorescence of 0.31 or below may be used to verify
proper thermal processing of ground beef.

The relationship between normalized fluorescence and log decrease in Salmonella
was used to evaluate this endpoint fluorescence value (Figure 4.6). A line was drawn
horizontally across the graph to indicate a 5 logo decrease in Salmonella. Cooked patties
above this line indicated a 5 logo decrease in Salmonella, patties below indicated less
than a 5 loglo decrease. A vertical line indicates a normalized PE fluorescence Of0.31.
Patties plotted to the left of this line indicated a normalized fluorescence less than 0.31
and to the right of the line greater than 0.31. With the addition of these lines the graph
was divided into 4 quadrants.

Patties in the upper left quadrant had a 5 or greater loglo decrease in Salmonella
and were correctly categorized by PE as being properly cooked with a normalized
fluorescence value below 0.31. Sixteen patties were observed in the upper left quadrant.
Patties in the top right quadrant had a 5 or greater logm decrease in Salmonella, yet the
PE incorrectly categorized the patties as being undercooked with a normalized value
above 0.31. Three patties were categorized in the upper right quadrant. Beef patties in
this quadrant were processed correctly, yet PE indicated otherwise, therefore safe product
would be disposed of thus incurring unnecessary financial losses.

Patties categorized in the lower right quadrant had less than a 5 loglo decrease in
Salmonella and were properly distinguished by the PE, with a normalized fluorescence

value greater than 0.32. Twenty patties were observed in the lower right quadrant.

62

 

 

 

 

 

 

 

 

 

 

8 I I
Q Undercooked
A
:3 I I Properly cooked
LL: 7 4 '
U a
V . i I
a
'Q 6 I
s - r - '
Q o .
S 5 fl T
Q 0
f4 . o I
O O

.5 4 o .
Q)
a ° o
8 3 .
Q)
Q 0
go 2 O O
._1 0 °

1 o O

O
O
0 , LL ,
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Normalized Fluorescence

Figure 4.6. Relationship between normalized phycoerythrin fluorescence and log
decrease in Salmonella in ground beef patties (19.1% fat). Patties were categorized as
undercooked or properly cooked according to the time-temperature regulations of the
USDA (2000).

63

Eighteen of these did not meet any of the USDA time-temperature guidelines.
Two patties met the guidelines but without a 5 logm decrease in Salmonella. PE correctly
classified the patties as undercooked following lethality standard guidelines, but the
patties were improperly categorized according to the time-temperature regulations.

Finally, patties in the lower left quadrant had less than a 5 log 10 decrease in
Salmonella, but the PE indicated a safe product with a normalized value less than 0.32.
These two patties suggest a problem with PE as a TTI. Both of these patties met one of
the time-temperature safe harbor guidelines of the USDA without a 5 logo decrease. PE
correctly categorized the patties according to either time-temperature guidelines or
lethality performance standards. Overall, 88% of the cooked patties were correctly
identified by a normalized PE fluorescence of 0.3 1.

4.4.4. Effect of maximum center temperature on Salmonella in low fat ground beef
patties.

Low fat ground beef (pH 5.65 i 0.01) contained 4.8% i 1.1% fat, 72.4% i 3.2%
moisture and 15.8% i 2.6% protein. No microorganisms were detected in the irradiated
meat. Typical center temperature histories during cooking of low fat (4.8%) ground beef
patties were very similar to that given in Figure 4.2.

The maximum center temperature inside the 41 low fat patties ranged from 54.2
to 756°C. AS the center temperature of the beef patties increased, the number of
surviving Salmonella (R2 = 0.87) decreased (Figure 4.7). As with the high fat patties, the
time-temperature schedules recorded during each cooking process were used to

categorize each patty as undercooked or properly cooked according to the time

64

 

 

 

 

 

 

 

 

 

 

7 O Undercooked I
I Properly cooked I I
I
A I
E 6 I
I I

23 o' -
a I I I I
E 5 I—r
S
D
5- .
53 4 o
.S o
it 3 0°
“.3.
0
<1) 0
E. 2- . °

1 oo o

m 0
o 0 °
0 . .
5) 55 60 $ 70 75 80

Maximum Center Temperature (°C)

Figure 4.7. Effect of maximum internal center temperature on log decrease in Salmonella
in ground beef patties (4.8% fat). Patties were categorized as undercooked or properly
cooked according to the time-temperature regulations of the USDA (2000).

65

temperature regulations for ready-to-eat ground beef patties.Out of 41 patties with
acceptable thermocouple locations, 22 patties did not meet the required time-temperature
regulations provided by the USDA. Thus 19 patties met or exceeded the time-
temperature safe harbor guidelines. Three patties met at least one of the time-temperature
guidelines provided by the USDA but without a 5 loglo decrease in the Salmonella
cocktail. This suggests that the number of Salmonella survivors in beef patties cooked
according to the time—temperature regulations from the USDA-F SIS may not decrease by
5 logm in all cases. One patty did not meet any of the safe harbor guidelines and resulted
in greater than 5 loglo decrease in Salmonella cocktail. This patty may have been cooked
at a temperature below 661°C for a longer time to achieve a 5 logm decrease in
Salmonella, but it would not be classified as properly cooked under the USDA
regulations.

4.4.5. Effect of lethality on Salmonella in low fat ground beef patties.

The lethalities of the 41 patties ranged from 1.51 to 3980s at 65°C. The range of
processing lethality used in this study resulted in undercooked and properly cooked.
patties. An increase in lethality resulted in a decreased cooking yield for the 113g beef
patties (R2 = 0.70).

A correlation was seen between the log decrease in Salmonella and lethality of the
cooking process (R2 = 0.88) (Figure 4.8). There was a higher correlation coefficient with
lethality and log decrease in Salmonella than with maximum center temperature.
Lethality was able to successfully predict appropriate reductions in the number of
Salmonella survivors better than maximum center temperature. Twenty-four patties had

lethalities greater than 65S at 65°C and met one of the time-temperature regulations. Four

66

 

 

 

 

 

 

 

 

 

3 I I
5
E3 7 1 I I
N 6
g - I . I I I
g 5 -_9_' l I
CO O.
.9. w I
a 4‘ °-
“3 8
g 3 -
o 3
go 2 1’ O Undercooked
...i
1 I Properly cooked
0 fi . A f
0 1000 2000 3000 4000 5000

Lethality (s)

Figure 4.8. Effect of process lethality on log decrease in Salmonella ground beef patties
(4.8% fat). Patties were categorized as undercooked or properly cooked according to the
time-temperature regulations of the USDA (2000).

67

patties with lethalities greater than 663 did not meet either the safe harbor guidelines or
lethality performance standards. A patty may have been cooked for an extended period
of time at a temperature below those listed in the safe harbor guidelines. These patties
would be considered properly cooked when performance lethality standards are finalized
for ground beef patties. Lethality greater than 1053 at 65°C consistently reduced the
number of Salmonella survivors by 5 logm.

4.4.6. Relationship between normalized PE fluorescence and Salmonella in low fat"
ground beef patties.

Normalized PE fluorescence in cooked patties ranged from 0.03 to 0.90.
Normalized fluorescence was compared to the log decrease in the Salmonella cocktail
within forty-one 113g low fat ground beef patties. A linear relationship was seen
between the normalized fluorescence and the number of surviving Salmonella (R2 = 0.82)
in cooked beef patties. This was a higher correlation than seen with the high fat patties.
A normalized fluorescence of 0.29 (CI 99%) was calculated from linear regression to
correspond with a 5 loglo reduction in the number of surviving Salmonella (Figure 4.9) in
cooked patties. This value was very similar to the normalized PE fluorescence that
indicated 5 logo reduction in Salmonella in high fat ground beef patties. The relationship
between normalized PE fluorescence and log decrease in Salmonella was used to evaluate
the endpoint fluorescence value (Figure 4.10).

The sixteen patties in the upper left quadrant had a 5 or greater logm decrease in
Salmonella and were properly identified by PE with a normalized fluorescence below
0.29. Fifteen of these patties also met at least one of the time-temperature regulations.

One patty was classified into the upper right quadrant which indicated greater than a 5

68

 

0.9- I
' . y=-0.1104x+0.8511

0-3 1 . ' R2 = 0.8217

 

Normalized Fluorescence

 

 

 

 

0 1 2 3 4 5 6 7 8
Log Decrease in Salmonella (CFU)

Figure 4.9. Correlation between normalized fluorescence and log decrease in Salmonella
cocktail in ground beef patties (4.8% fat).

69

 

 

 

 

 

 

 

 

 

 

7 1. O Undercooked
S . I Properly Cooked
LL: . I I
U 6 I -
‘5 I I o
a I - I I
3 5 I
6
§ .
U: 4 O
.E: o
9}: 3 o 0
£6
9?.
O O
Q) 2 O
Q ‘2) o
g” 0o o 0
u—I 1 0 63
O O O o
o o
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Normalized Fluorescence

Figure 4.10. Relationship between normalized phycoerythrin fluorescence and log
decrease in Salmonella in ground beef patties (4.8% fat). Patties were categorized as

undercooked or properly cooked according to the time-temperature regulations of the
USDA (2000).

70

loglo reduction in Salmonella. However, PE incorrectly identified this patty as
undercooked with a normalized fluorescence greater than 0.29. The lower right quadrant
contained 24 cooked patties. Twenty-two patties did not meet any of the time-
temperature regulations and were properly categorized by a normalized PE fluorescence
of greater than 0.29. The remaining patty did meet one of the time-temperature
regulations without a 5 logo decrease in Salmonella. This patty was properly cooked
according to current regulations, but improperly categorized by PE. There were no
patties categorized into the lower left quadrant indicating that normalized PE
fluorescence of 0.29 was able to correctly categorize undercooked patties. Overall 98%
of the cooked patties were correctly categorized.

Smith et al. (2000) reported that S. Typhimurium DT104 cultures were less heat
resistant in ground beef of lower fat content. Maurer (2000) found that E. coli 0157: H7
had lower D values in 4.8% fat ground beef when compared to 19.1% fat. It was
expected that normalized PE fluorescence values for low fat ground beef would have
been greater than for high fat patties due to this decreased thermal resistance, but the two
fluorescence cutoff points were very Similar.

4.4.7. Effect of maximum center temperature on Salmonella in 60g high fat ground beef
patties.

Grilling methodologies were applied to 60g ground beef patties to verify the
applicability of PE as a time-temperature integrator in different size patties. The typical
center temperature history during cooking of 60g ground beef patty is given in Figure

4.11. The maximum center temperature of the 42 patties ranged from 60.9 to 75.9°C. As

the maximum center temperature of the cooked patty increased, the number of surviving

71

(A)

 

 

Temperature (C)
o 8 B 8 E 8 8 3

 

 

4CD 6CD 8m 1(11)
Tine (s)

g -

(B)

80
70
60 -

 

Temperature (C)
8

20-
10.

 

 

0 200 400 600 800 1000
Time (s)

Figure 4.11. Representative time-temperature heating profile of a 60g high fat (19.1%)
ground beef patty. (A) Illustrates a patty cooked to 63.1°C and a total lethality of 24.95 at
65°C; (B) Illustrates a patty cooked to 751°C and a total lethality of 2330s at 65°C.

72

Salmonella (R2 = 0.50) decreased. In addition to differences in cooking time at each
temperature of the cooking process, the low correlation could be due to the difficulty
encountered with methodologies. When attempting to insert the thermocouple and *‘
capillary tube into a lower weight patties the thermocouple was frequently found to be
closer to one surface or the other instead of in the center of the patty. Given that large
decreases in the width of the patty were observed during cooking, the changing Shape of
the patty during cooking had a major impact on the final position of the thermocouple
after cooking. Thus even though the thennocouple’s final location was within the i 5%
of the center point, it may not have been in the proper position for the entire cooking
time.

Twenty patties did not meet the required time-temperature regulations provided
by the USDA. Therefore 20 patties met or exceeded the time-temperature regulations of
the USDA. Although four patties met at least one of the time-temperature guidelines
provided by the USDA, Salmonella decreased less than 5 loglo (Figure 4.12). This trend
was seen in both the high and low fat 113g patties. Three patties did not meet the time-
temperature safe harbors and yet the Salmonella cocktail was reduced by greater than 5
log“). The time-temperature history of these patties revealed extended cooking at
temperatures below 61 . 1°C, the lowest endpoint temperature contained in the USDA
time-temperature regulations.

4.4.8. Effect Of lethality on Salmonella in 60g high fat ground beef patties.

Lethalities for the forty 60g cooked beef patties ranged from 11.4 to 2780s at

65°C. Therefore our range of lethality included processes for undercooked as well as

73

 

 

 

 

 

 

 

 

 

 

7 . I
I
5 ° '
. I I
Lg 3 o I I I I. I
.3 ° ' '
v?) 5 #fi
8 o o 0% '
s , ° -
53
.5 ° 0 °
‘9 3 O
18 o o
3 o
o o
5 2 o
w o
,3 1 ° 0 Undercooked
I Properly cooked
0 9 . ,
55 60 65 70 75 80

Maximum Center Temperature (°C)

Figure 4.12. Effect of maximum center temperature on log decrease in Salmonella in
60g ground beef patties (19. 1% fat). Patties were categorized as undercooked or properly
cooked according to the time-temperature regulations of the USDA (2000).

74

properly cooked beef patties. There was a low correlation between the increase in
lethalin and the decrease in cooking yield of the 113g beef patties (R2 = 0.69).
Methodology weaknesses could again explain the low correlation seen.

A slight correlation was seen between log decrease in Salmonella and lethality Of
the cooking process (R2 = 0.58) (Figure 4.13). This correlation was higher than the
endpoint temperature correlation, suggesting that lethality measurement may be a more
successful indicator of Salmonella destruction. The R2 values for both relationships are
low in comparison to the 113g patties suggesting that the placement of the thermocouple
may not have been as accurate in patties of smaller size.

Eight patties with lethalities greater than 665 did not meet either time-temperature
regulations or result in a 5-log10 decrease in Salmonella. When reviewing the time-
temperature profiles of the cooking process, large time intervals at temperatures below
61 .1°C were observed. The time at low temperatures contributes to the total lethality, but
the temperatures are not included in the safe harbor guidelines and were not high enough
to result in a 5 logo reduction of Salmonella. A 5 loglo decrease was consistently
Observed in cooked ground beef patties with lethality greater than 1355 at 65°C.

4.4.9. Relationship between normalized PE fluorescence and Salmonella in 60g high fat
ground beef patties.

Normalized PE fluorescence in cooked patties ranged from 0.05 to 0.58. Alinear
trend was seen between the decrease in normalized fluorescence and the decrease in
Salmonella survivors (R2 = 0.60) in cooked beef patties. Using linear regression it was

possible to calculate the normalized PE fluorescence that corresponded to a 5 logo

75

 

 

 

 

 

 

 

 

 

A 7 I I
I
E 6‘ ° ' I I l l
E 'u—I—I 1
I

g 5 - . I I .
a
a; o
.s 4 '
Q)
8
E 3
Q 0
go 2 ° 0 Undercooked
._I I Properly cooked

1

0 . . . .

0 5(1) 1111) 1500 2Q!) 2500 3GB

Lethality (s)

Figure 4.13. Effect of process lethality on the log decrease in Salmonella in 60g ground
beef patties (19.1% fat). Patties were categorized as undercooked or properly cooked
according to the time-temperature regulations of the USDA (2000).

76

decrease in Salmonella in the patties (Figure 4.14). The normalized fluorescence of the
PE was 0.21 (CI 99%) or below inside patties with a 5 loglo or greater decrease in
Salmonella. A normalized PE fluorescence of 0.21 or below may be used to verify
proper thermal processing of 60g ground beef patties. This endpoint normalized PE
fluorescence was lower than that reported for the high and low fat 113g patties.

Unusually low PE values were noted throughout the studies with 60g patties. It
has been hypothesized that the PE was exposed directly to the heat of the griddle surface
due to the narrow width of the beef patty. Therefore the lethality received by the PE was
not representative of the lethality received by the patty.

A plot of log decrease in Salmonella against normalized fluorescence was used to
evaluate the endpoint fluorescence value (Figure 4.15). Eighteen patties were classified
into the upper left quadrant. These patties were correctly identified with a normalized PE
fluorescence below 0.21 as being properly cooked. Two of the 18 patties did not meet at
least one of the time-temperature guidelines, but Salmonella was reduced by 5 logo or
greater.

Two of the patties were incorrectly classified by the PE as properly cooked only
because of the current time-temperature guidelines. Based on lethality performance
standards these two patties were properly cooked. Two patty was observed in the upper
right quadrant. The patty did not meet any of the time-temperature regulations, but the
number of Salmonella survivors was reduced by at least 5 loglo. Eighteen patties were
classified into the lower right quadrant. One of the eighteen met time-temperature

regulations, but did not exhibit a 5 logo decrease in Salmonella.

77

Normalized Fluorescence

O7

 

 

 

 

 

 

y=-0.0667x + 0.544
06 _ R2=O.5899
05.

I
04. - -
0.34 I I I I
I I I I
02 ' ' ..
‘ 'I
01. l'. .r.
I II .
o . ,
o 2 3 4 5 6 7 8

Log Decrease in Salmonella (CFU)

Figure 4.14. Correlation between normalized fluorescence and the log decrease in _
Salmonella in 60g ground beef patties (19. 1% fat).

78

 

 

 

 

 

 

 

 

 

 

 

7 . O Undercooked
I
A . o I Properly cooked
:3 I
LL ' I I
8 6 l I C o
:3
s ' - ' o
2 5 I - l
o I .
E 0 O o O
B o
to 4 '
.S
3’: 0 ° °
g 3 00
Q 0
Q)
Q 0 °
2.0 2 o
._] O
O
1
0 O
o o 1 o 2 o 3 0.4 o 5 o 6 o 7

Normalized Fluorescence

Figure 4.15. Relationship between log decrease in Salmonella and normalized
phycoerythrin fluorescence in 60g ground beef patties (19.l% fat). Patties were
categorized as undercooked or properly cooked according to the time-temperature
regulations of the USDA (2000).

79

Two patties were categorized into the lower left quadrant. They both met at least
one of the safe harbor guidelines but without a 5 logm reduction in Salmonella. Overall a
normalized PE fluorescence of 0.21 correctly identified 93% of the cooked beef patties.
These results are positive, but due to the low correlation seen between normalized
fluorescence and log decrease of Salmonella, new methodologies may need to be
developed to avoid exposure of PE to the griddle surface.
4.5. CONCLUSIONS

Although thermal processing guidelines are available, there is still a need to verify
adequate cooking of ground beef patties. Studies completed here reveal that PE has great
potential as a TTI for verifying proper thermal processing of ground beef patties. In
patties of two fat levels, PE was able to correctly categorize 93% of the ground beef
patties as properly cooked or undercooked with a normalized fluorescence value less than
0.29. However, in 60g high fat beef patties, methodologies limited the applicability of

PE. Future studies will need to address the encapsulation and delivery system for PE, as

well as the varying size of beef patties and its affect on PE applicability.

80

 

 

CHAPTER 5
TRIOSE PHOSPHATE ISOMERASE AS AN INDICATOR OF THERMAL

PROCESSING OF GROUND BEEF PATTIES

5.1. ABSTRACT

The maximum temperatures reached at the center point of 50 patties tested ranged
from 62.5 to 72.7°C during cooking. Triose phosphate isomerase activities did not
decrease with increasing maximum center temperature as expected. A large range in TPI
activity was seen within each individual patty. The 18 patties that achieved lethality equal
to or greater than 2005 at 65°C exhibited a TPI activity of less than 20 U/g of meat.
Maximum internal temperatures were calculated from TPI activity values for 5 locations
within the patty. The estimated temperature differences within ground beef patties
ranged from 3.11 to 25.68°C, but decreased with increasing process lethality. These
results suggest that the lethality performance standard should be raised from the proposed
5.0 logm to a 6.5 loglo reduction in Salmonella to ensure the safety of cooked ground beef

patties.

81

5.2. INTRODUCTION

Recently the USDA-FSIS, in addition to new time/temperature schedules, has
allowed processors to establish thermal processes for meat products based on lethality
standards. A proposed lethality performance standard for fully cooked, uncured meat
patties includes a 5 loglo reduction in Salmonella (USDA, 1999). However, in review,
the F SIS has concluded that a higher lethality standard may be needed to produce safe
ground beef patties. Although these thermal processing guidelines are available, there is
still a need to verify adequate cooking.

Current regulations state that commercial establishments must verify the time and
temperature within i 1°F on one beef patty on a production line each hour. It is very
difficult to use a thermometer or thermocouple in commercial processing plants to verify
that the time-temperature requirements have been met in a ground beef patty. None of
the established USDA methods to determine endpoint temperature are based on thermal
inactivation of Salmonella, the microbial pathogen selected to define proposed lethality
standards.

Berry et al. (1999) found that the internal temperature of beef patties varied as
much as 18°C when the center temperature was 71°C. Liu and Berry (1996) reported that
of 96 ground beef patties cooked for a predetermined time to a target temperature of
71°C, 16.7% did not reach 68°C. The lowest temperature measured within patties with
10% and 20% fat were 61 and 63 °C, respectively. In addition, considerable variability in

the mean time required for beef patties to reach a specific temperature was observed. The

standard deviations from the means ranged from 0.22 to 0.59 min. Thus, while the

82

average time for patties to cook to a certain internal temperature may be determined by
initial studies, there is a high probability that some patties will be over- or undercooked.
To overcome these difficulties in monitoring the thermal processing of ground beef, time-
temperature integrators (TTIS) have been studied.

A TTI has been defined as a device that undergoes an irreversible and precisely
measurable change in response to a time—temperature history that mimics the change in a
target attribute exposed to the same thermal history (Hendrickx et al., 1995). A major
advantage of a TTI is the quantification of the integrated time-temperature impact on
microbial destruction within a beef patty can be determined without the actual time-
temperature history of the product. Ideal TTIS would be simple, inexpensive to prepare,
and easily recoverable with an accurate and user-friendly read-out. To be considered an
ideal endogenous TTI, a protein should have a thermal inactivation rate constant (zavalue)
that closely resembles the 2 value of the microorganism used to establish the processing
schedules (Hendrickx et al., 1995; Van Loey et al., 1996).

The 2 value reported for TPI in ground beef, 556°C, (Orta-Ramirez et al., 1997)
was very close to the value for Salmonella (z = 56°C) (Goodfellow and Brown, 1976),
the pathogen used to establish processing guidelines for ground beef patties. Sair et al.
(1999) reported that TPI activity decreased as cooking temperatures in ground beef
patties (24.4% fat) increased from 60.0 to 71 .1°C. No differences in TPI activity could
be distinguished when patties were cooked to internal temperatures of 71.1 to 822°C.
Larger standard deviations were seen in TPI activity at lower processing temperatures in
ground beef patties, thus illustrating the large variations in internal temperatures that

occur in beef patties during cooking.

83

The objectives of this study were to (1) investigate TPI activity within ground
beef patties during cooking and (2) to evaluate TPI activity as an indicator of cooking

temperature at discrete locations in ground beef patties.

5.3. MATERIALS AND METHODS
5.3.1. Ground beef patties

Frozen ground beef patties (1 13 g) were acquired from J & B Meats (Lot #
113664, Coal Valley, IL) and held at -22°C until used. The frozen patties were stacked
in layers separated by wax paper inside a plastic bag and then placed inside a cardboard
box. The patties were thawed at 4°C for 24h prior to use. The raw patties were
approximately 11 cm in diameter and 1 cm in height.

Moisture, fat, and protein contents were determined by AOAC (1996) methods
950.46B, 991.36, and 981.1, respectively. For determination of pH, 10g of ground beef
were homogenized in 90 mL of distilled water using a Polytron homogenizer (Model PT
10/35, Brinkman Instruments, Westbury, NJ) at speed setting 3 for 303. The pH of the
homogenized mixture was measured using a combination electrode (Model 145, Coming,
Medfield, MA). Analyses were done in triplicate.

5.3.2. Patty cooking procedures

A thermocouple (Type T, length 8.6 cm, width 1.0 mm, accuracy i O.1°C, Omega
Engineering, Stamford, CT) connected to a datalogger system (Daqbook 100, Omega
Technologies Inc.) was inserted horizontally into the beef patty. The temperature at the
center of the patty was recorded every second during the cooking process.

Fifty ground beef patties were cooked during a 5 day period. The beef patties

were cooked on a griddle (Model 07032, Presto Jumbo griddle, National Presto

84

Industries, Inc., Eau Claire, WI) set at 177°C. Preliminary studies showed that the
griddle cycled i 7°C. The griddle was preheated for a minimum of 30 min prior to use.
During the cooking process, patties were heated for 2 min, flipped, and heated for another
2 minutes, then flipped every minute until the process was completed. Patties were
removed from the griddle when an internal target temperature between 60 and 75°C was
reached. Times of removal varied from 6 to 13 min. The patties were immediately
transferred to WhirlpakTM bags (18 oz, Nasco, Ft. Atkinson, WI) and placed in an ice
water bath for quick cooling.
Following the cooling period, patties were sliced vertically in half to reveal the cross
section of the patty and measurements were taken to determine the placement of the
thermocouple. Patties were accepted if the thermocouple and capillary tube were located
within i5% from the center point of the cooked patty thickness. A 50g sample of ground
beef was removed from the center of each patty by coring with a plastic lOOmL beaker
and then vacuum packaged in plastic bags. The beef samples were frozen and held at -
18°C until the enzyme assays were performed.
5.3.3. Protein extraction for TPI activity

A 1.0g sample of cooked ground beef was cored from the patty with a 16 x 150
mm disposable culture tube (VWR Scientific Products, West Chester, PA) in 5 locations.
One sample was removed from the center (C) of thewith four additional samples removed
from the top (T), bottom (B), left (L), and right (R) (Figure 5.1). The 1.0g samples were

transferred to scintillation vials (Research Products Intl. Corp. Mount Prospect, IL),

85

I
,’ |
I I
' I
I I
‘ I
‘ I
‘ I

 

Figure 5.1. Location of 5 meat cores removed from each ground beef patty for tn'ose
phosphate isomerase enzyme activity analysis. Dashed line indicates the central 50g

core.

86

and combined with 3 volumes of 0. 1 5 M NaCl, 0.01 M sodium phosphate buffer, pH 8.0
(PBS). The samples were mixed for 1 min using a Fisher Vortex Genie2TM (Scientific
Industries, Inc., Bohemia, NY). The extracts were then stirred, without foaming, for 15
min at 4°C followed immediately by centrifugation at 6000 x g for 10 min at 4°C. The
supematents were filtered through Whatman no. 1 filter paper and then held on ice until
assays were completed within 12h.
5.3.4. TPI activity determination

TPI activity was determined as described by Sair (1997). The change in
absorbance was measured at 340 nm for 1 min at 25°C. The protein extract was diluted

with PBS buffer as needed to achieve a change in absorbance of less than 0.2 per minute
(Beisenherz, 1995; Norton et al., 1970). TPI activity was calculated using the following

equation:

Activity (U/L) = 1000 x A x V

 

T x s x d x ((p/d)
A = Absorbance
V = Assay volume (mL)
T = time (min)
8 = extinction coefficient (6.310 L x mmol'l x cm)
(I = distance of the light path (1 cm)
(p = sample volume (0.01 mL)
D = dilution of sample
Enzyme activities were expressed as units (U) per gram of meat.

5.3.5. Calculation of process lethality

87

Lethality was calculated using the General Method (Pflug, 1997) with a reference
temperature of 65°C (Equation 2).

Lethality = 1
10"((65-T) / 5.44) (2)

 

where T = temperature (°C) at time t

The time-temperature data recorded by the datalogger system for each patty was
converted into an equivalent lethality. Individual lethalities were then summed to give
the total lethality of the process. Many different cooking schedules were used to achieve
a range of lethalities. Utilizing the time-temperature safe-harbor guidelines for proper
ground beef processing, an equivalent lethality was calculated to be 663 at 65°C using a 2
value of 544°C. This 2 value resulted in the smallest range of lethalities across all the
time-temperature regulations. Thus any cooking process that resulted in a lethality of 66s
at 65°C should be equivalent to the time-temperature regulations of the USDA.

5.4. RESULTS AND DISCUSSION
5.4.1. Effect of maximum center temperature on TPI activity in ground beef patties

Ground beef (pH 5.94 i 0.04) contained 23.6 i 5.2 % fat, 61.0 i 0.3% moiSture,
and 15.9 i 0.8% protein. The placement of a thermocouple in the center of each patty
made it possible to collect data on the maximum temperature at this location during each
cooking schedule. The maximum temperature at the center point of the 50 patties tested
ranged from 62.5 to 72.7°C. USDA regulations currently in effect for proper thermal

processing of ground beef patties include cooking schedules ranging from 66.1 °C for 415

to 694°C or up for 105. Under these regulations, thermal processes for each patty were

88

classified as having met the regulations and being properly cooked, or not having met any
of the time—temperature regulation and therefore were undercooked (Figure 5.2). Patties
may have reached a temperature included in the regulations, but if they did not meet the
time requirement, the patty was classified as undercooked.

Thirty-two patties were identified as being undercooked and 18 patties were
properly cooked according to the USDA-FSIS time-temperature regulations. The
maximum internal temperatures at the center point of patties that did not meet the USDA-
F SIS regulations ranged from 62.5 to 68°C, with TPI activities ranging from 1.5 to 139.3
U/ g of meat. The patties that met the time-temperature regulations achieved maximum
center temperatures that ranged from 67.6 to 72.7°C. The TPI activities of the center
point corresponding to these properly cooked patties ranged from 1.5 to 64.0 U/ g of meat.

TPI activities did not decrease with increasing maximum center temperature
(Figure 5.2). Sair et al. (1999) observed that TPI activity of beef patties decreased as the
maximum internal temperature increased. Discrepancies could be seen due to the small
sample size used to assay TPI activity. The enzyme assays reported here were completed
with a 1 g of ground beef, whereas Sair et al. (1999) used a much larger sample with a 7
cm diameter, that may have been more representative of an overall or average activity.

All ground beef patties that reached a maximum center temperature of 71 .1°C or
higher had TPI activities below 20 U/ g at the center point. Sair et a1. (1999) reported that
patties cooked to internal temperatures above 71 . 1°C had TPI activities below 6.3 U/ g of
meat. Lower TPI activities seen by Sair et al. (1999) could be due to the residual

temperature increase after the patty reached the target endpoint temperature and was

89

 

.1,, :-

 

w-r

TPI Activity at Center Point (U/ g meat)

160

140

120

100

80

60

40

20

 

 

 

0 O Undercooked
I Properly cooked

 

 

 

 

 

60

I
oo
o
oo
0o
'I
o o
0°00 o I
0 0' II
0 <9 0 . . I
06>
‘3 qb (3C) 'I) .l I'!! '. TIL
62 64 66 68 70 72

74

Maximum Measured Temperature at Center Point (°C)

Figure 5.2. Effect of center point temperatures on the triose phosphate isomerase
activity in the center of a beef patty. Patties were categorized as undercooked or properly
cooked according to the USDA-FSIS time-temperature safe harbor guidelines (1996).

90

removed from the grill. Maximum temperatures reported in this study were the highest
recorded within the patty during the cooking process. The lethality of different cooling
processes used in the two studies could also be a factor affecting TPI activity. Sair et al.
(1999) allowed patties to cool at room temperature, thus allowing the patties to remain at
higher temperatures for a longer time than our study where patties were placed directly
into an ice water bath.

When the TPI activities at all 5 locations were averaged, the values were different
from the center point TPI activity within the same beef patty (Figure 5.3). This suggests
that TPI activity of the center sample was not representative of the activity throughout the
patty. Large variations in average TPI activity were seen in patties cooked to the same
maximum center temperature. For example, patties undercooked to an internal
temperature of 65-66°C exhibited average TPI activities from 2.4 to 63.2 U/g meat.
Patties that were properly cooked to internal temperatures of 69-70°C had average TPI
activities from 2.96 to 57.36 U/g meat. Variations in average TPI activity at the
maximum center temperature did not decrease below 5 U/ g meat until 72°C.

The heterogeneous nature of ground beef leads to large differences in heat and
mass transfer and thus different heating rates within a patty (Pan and Singh, 1998).
Troutt et al.(l992) reported that rendering of fat during cooking resulted in air pocket
formation within a beef patty, which also has an effect on heat transfer. These effects on
heat transfer throughout a beef patty can result in large temperature differences. Berry et
al. (1999) reported internal temperature variations that exceeded 18°C on cut surfaces of
a ground beef patty cooked to 71 .1°C. TPI activity is very predictable, decreasing with

increasing maximum internal temperature. The large variation in activity observed

91

 

80

 

OUndercooked
O I Properly cooked

70

 

 

 

50

40

 

 

 

Average TPI Activity at 5 Locations (U/g meat)
0
o
I

I
o
30 o ' '
o
o
20 O
o o
oo o I
10 <9 O o ' I I
O o I
o I g I
0 O O O O 00 O . .I T-
60 62 64 66 68 70 72 74

Maximum Measured Temperature at Center Point (°C)

Figure 5.3. Effect of center point temperature on the average triose phosphate isomerase
activity at five locations in the beef patty. Patties were categorized as undercooked or
properly cooked according to the USDA-F SIS time-temperature safe harbor guidelines
(2000)

92

suggests that there were large temperature variations within the beef patties during

cooking.

5.4.2. Effect of lethality of the cooking processes on TPI activity in ground beef patties
Lethality was calculated to take into account the time-temperature history of the

patty during cooking. Overall it was observed that as lethality of the cooking process

increased, the TPI activity at the center point decreased. Beef patties with a lethality of

66s or greater at 65°C should be properly cooked according to the USDA safe harbor

guidelines. There were 15 patties that had lethalities less than 665 at 65°C and had TPI
activities greater than 20U/ g meat, thus exhibiting the behavior expected of the TPI. Yet
there were 13 patties that had lethality below 665 at 65°C and exhibited a TPI activity at
the center point less than 20 U/g meat (Figure 5.4). For example, the lethality for one of
the patties was calculated to be 22.3 s at 65°C and yet the TPI activity decreased from a
raw value of approximately 2000 U/g to 7.3 U/g of meat. Thus if TPI was being utilized
as an indicator of adequate thermal processing, this patty may be improperly identified as
safe for consumption.

Two of the eighteen patties that achieved lethalities greater than 663 at 65°C
failed to meet one of the time-temperature guidelines. The complete time-temperature
history of the cooking process was used in lethality calculations, including the gradual
increase from storage temperature to the maximum center temperature. Time-
temperature regulations are based upon endpoint monitoring, which only accounts for the
thermal processing time at the target temperature. The lethality of 66s at 65°C is
equivalent to only the holding time at each temperature. Therefore, when the heating lag

times are included, lethalities may be increased without an increase in maximum

93

140

 

 

 

 

 

 

 

 

A

*" o

g 20 O Undercooked

w I Properly cooked

E3, 00

in!

.E‘:

o

6.. 80

L.

I)

H

8 a

U 60 o o 0

E3 '1:

>.. O

3:: o

> 40 'I

'5 o

O 00

< 20 09’ '

E ' I

E— 0 Q o . .
0 o OQOQ o o U . I I -
o 50 100 150 200 250 300

Lethality (s)

Figure 5.4. Effect of lethality of the cooking process plotted on the triose phosphate
isomerase activity of the center point of a beef patty. Patties were categorized as
undercooked or properly cooked according to the USDA-FSIS time-temperature safe
harbor guidelines (2000).

94

center temperature, which explains why patties with lethalities greater than 663 at 65°C
did not meet one of the safe harbor guidelines.

Three patties with lethalities greater than 665 at 65°C had TPI values above 20
U/g meat. These patties were properly cooked based on the time-temperature regulations,
but TPI is indicating an undercooked patty. Once again, large temperature variations
within the patties and small sample size may explain these discrepancies.

All of the patties that had a cooking schedule with lethality equal to or greater
than 2005 at 65°C exhibited a TPI activity of less than 20 U/g of meat. There was a
better relationship between lethality and TPI activity than maximum internal temperature
and TPI activity. Lethality, which offers an expression of the time-temperature history,
may be a better indication of the magnitude of thermal processing than internal

temperature alone.

The average TPI activity of the 5 sampling locations of each patty was calculated
and compared to the TPI activities from Sair et al (1999) (Figure 5.5). Seventeen patties
had lethalities less than 663 at 65°C and also exhibited average TPI activity of less than
20 U/ g meat. Two patties had lethalities above 663 at 65°C, but did not meet any of the
time-temperature regulations of the USDA. Three patties with lethalities greater than 668
at 65°C had TPI values above 20 U/g meat. One patty which was cooked with a lethality
process less than 663 at 65°C met one of the time-temperature guidelines. Limitations in

heterogeneity of ground beef and internal temperature differences mentioned above

explain these variations.

5.4.3. Temperature variation within ground beef patties

95

 

 

 

 

 

 

c} 80

8

E 70 O Undercooked

:3)” o I Properly cooked

V O

m 60 o

8 u

'5 o

63

0 O

o 50

A 0

V3

... 40

N I

>. O 9

.t: O

.2. 30 o I

3 o

< O

a: 20 o 0

l--‘

<13 o I
0 0

3:30 10 8 o I . I

g 0 O O - I .

< o O O O 00 O O - .. I

 

 

0 25 50 75 100 125 150 175 200 225 250 275

Lethality (s)

Figure 5.5. The effect of lethality of the cooking process on the average triose phosphate
isomerase activity at five sampling points within a beef patty. Patties were categorized as
undercooked or properly cooked according to the USDA-F SIS time-temperature safe
harbor guidelines (2000).

96

Sair et al. (1999) reported TPI activities for beef patties cooked to four different
endpoint temperatures in the range tested in this study. These values were used to create
a standard curve to predict internal temperatures at the 5 locations within beef patties for
which TPI activity was known (Figure 5.6). A very good correlation was seen between
TPI activity and maximum internal temperature (R2 = 0.95). From the equation
describing this correlation, maximum internal temperatures were estimated from known
activities.

From the calculated internal temperatures at each sampling point it was possible to
estimate the temperature differences that occurred within the patties during cooking
(Figure 5.7). At lethalities less than 665, temperature differences within a patty ranged
from a low of 3.1 1 to a high of 25.68°C. At lethalities greater than 665, the temperature
differences ranged from 7.0 to 222°C. Overall, as process lethalities increased
temperature variations within beef patties decreased. This is in good agreement with Sair
et a1. (1999) who reported that the standard deviation of TPI activity decreased with
increasing maximum internal temperature.

Temperatures calculated from TPI activities at the 5 different patty locations were
averaged. A plot of the calculated average internal temperature 3:. standard deviations
against the maximum measured internal temperature at the center of the patty is given in
Figure 5.8. Large standard deviations were observed on this plot indicating that the range
of temperatures within beef patties was very large. These studies indicate the need to
determine mechanisms to accurately measure the cooking temperature in beef patties to
verify proper thermal processing. Overall the standard deviations for the average

calculated temperatures decreased as maximum internal temperatures increased.

97

 

75

7O

 

Maximum Internal Temperature (°C)

 

 

 

0 10 20 3O 40 50 60

TPI Activity (U/g meat)

Figure 5.6. Standard curve showing the effects of maximum internal temperature in a
ground beef patty on the of triose phosphate isomerase activity from Sair et al. (1999).

98

 

 

 

g) 25 'I
V
Q
(U I I
D...
(U 20 I
.s "'“ -' -
3“: . -
3 15 u '
(I)
5’!) ' ' I
E '-
0 I
S I . I ‘ I I I
*5 '|
I— 5 I
a I
E ' '
Q)
E‘ 0 .
o 50 100 150 200 250
Lethality (s)

Figure 5.7. The effect of process lethality on the calculated temperature differences

within ground beef patties.

99

 

No trend was seen between measured center temperature and the estimated average
temperature throughout the patty suggesting that the center temperature may not be a
good indicator of the thermal process employed on a patty (Figure 5.8). No correlation
was observed between lethality and the calculated average temperature within beef
patties suggesting that the small area measured by the thermocouple in the center of a
patty may not be representative of the temperatures within the overall patty.
5.4.4. Effect of location on TPI activity of ground beef patties

The cold spot of a hamburger has always been assumed to be in the geometrical
center of a patty. Therefore, it was predicted that the highest enzyme activity would be
observed at the center point. Yet, only 26% of the 50 patties exhibited the highest
activity at the center point. The majority (60%) of the patties tested had the highest TPI
activities to the left or right of the center point, while the remaining 14% had the highest
activity at the top or bottom point. The construction of the griddle used in this study
could offer some explanation for these findings. Placement of the heating coil on the
underside of the cooking surface can be seen from the diagram in Figure 5.9.

All patties were cooked individually in the center of the griddle. According to
Figure 5.9, the top and bottom points of the beef patty were in closer proximity to the
heating coils than the other three points. This may explain the lower TPI activities at
points T and B in comparison to the other three points in a majority of the patties tested.
Preliminary studies were performed to measure the temperature variations of the griddle
surface during cycling. Higher temperatures were recorded in the middle of the griddle
in comparison to the left and right sides. Again, explaining why the large number of

patties had the highest enzyme activity on either the left or right side of center. Thus it is

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Average Calculated Temperature in Patties (°C)

 

 

 

 

 

 

 

 

 

 

5 Y Y r v v y r 1 , T
62 63 64 65 ea 67 as as 7'0 71 72 7'3

Maximum Measured Temperature at Center Point (°C)

Figure 5.8. The effect of maximum measured internal temperature of the center point of
ground beef patties on the average calculated internal temperature error bars indicate
standard deviation.

101

 

 

 

 

 

 

,\
\

Ground beef patty Heating coil

 

 

Figure 5.9. Location of the heating coil and ground beef patty on the cooking surface of
the griddle.

102

 

reasonable that the maximum center temperatures did not correlate to the average
calculated temperatures within the patties.
5.5. CONCLUSIONS

The sampling method revealed large variations in TPI activity within ground beef
patties. Temperature differences as large as 25.7°C were estimated within a 113g patty.
Heat transfer within a beef patty is affected by fat content, initial temperature of the patty,
method of cooking, and air and fat pockets formed during cooking. Therefore, it would
be very difficult to develop methodologies to accurately measure temperatures inside
beef patties. A safety factor could be incorporated into thermal processing regulations to
account for temperature variation within patties. The lethality performance standard for
uncured, fully cooked beef patties has not been finalized. The results of this study would
recommend raising the lethality performance standard from the proposed 5.0 loglo to a

6.5 loglo reduction in Salmonella to ensure safely cooked ground beef patties.

103

 

 

CHAPTER 6

CONCLUSIONS

Lethality performance standards are in the process of being established for ground
beef patties to add flexibility to processing schedules allowed in the meat industry. The
USDA is recommending that lethality standards be verified through challenge studies
with a Salmonella cocktail comprised of heat resistant strains that have been previously
implicated in foodborne outbreaks.

Heat resistance of an 8-strain Salmonella cocktail in stationary growth phase
decreased after being inoculated into ground beef and frozen for 7 days. The thermal
stability of the cocktail also decreased (P<0.05) at 55, 61 , and 63°C after 14d of
starvation in 0.1% peptone water at 4°C. The D values of the thermally injured cocktail
were not different (P<0.05) from the Salmonella cocktail in log phase. The heat shocked
cocktail was not different (P<0.05) from the Salmonella cocktail in log phase except for
58°C at which it was less heat resistant. The 2 values of the Salmonella cocktail ranged
from 4.20 to 464°C. Based on our findings, this 8-strain Salmonella cocktail may not be
able to adequately represent a 5-log reduction for some pathogens including more heat
resistant strains of S. Typhimurium DT104 at low temperatures (355°C).

New lethality performance standard regulations require that processors validate
the efficacy of their processing schedules. Difficulties arise since Salmonella cocktails
cannot be used in commercial processing plants for verification. However, a time-
temperature integrator that could accurately predict reduction in the number of
Salmonella survivors, it could be used in a plant for validation. A normalized PE

fluorescence of 0.31 was correlated with a 5 or greater loglo decrease in Salmonella

104

within 113g ground beef patties (19.1% fat). A similar normalized fluorescence of 0.29
was determined as a cut-off point for proper thermal processing in low fat ground beef
patties (4.8%). A normalized PE fluorescence of 0.21 indicated proper thermal
processing of 60g beef patties. However, due to methodology limitations, the correlation
between normalized PE fluorescence and the log decrease in Salmonella was low. PE
was consistent in monitoring process lethalities in ground beef patties and does have
potential for actual use in the meat industry.

Many factors affect heat transfer in ground beef due to its heterogeneous nature,
resulting in large temperature differences throughout the patty during cooking. TPI, an
endogenous enzyme found in ground beef, was able to detect temperature differences
within a beef patty. Internal temperatures were estimated from activity levels measured
at 5 locations within a beef patty. Temperature differences within beef patties ranged
from 3.1 to 25.7°C. These temperature differences could have a large impact on final
endpoint temperature and lethality of beef patties. Lethality standards are currently being
determined for ready-to-eat ground beef patties. Results presented here indicate the need
to incorporate a safety factor into lethality performance standards. For example,
estimated temperature variations exceeded 25°C, meaning that an internal temperature of
40°C could be detected in a patty cooked to a target temperature of 65°C. Results
reported here support an increase in lethality performance standards from 5.0 loglo to 6.5

loglo decrease in Salmonella due to temperature variations within patties.

105

 

CHAPTER 7

RECOMMENDATIONS FOR FUTURE RESEARCH

The thermal inactivation parameters of a Salmonella cocktail were determined
under different environmental conditions. The cocktail was thermally injured and heat
shocked in tryptic soy broth before thermal death time studies. If sub-lethal heat
treatments were performed in ground beef, the resulting thermal inactivation parameters
could be used to evaluate effects of temperature abuse in meat products.

The experiments with PE were performed using glass capillary tubes. In a real
processing situation, this would be unacceptable for safety reasons. Additional research
is needed to find a suitable material to encapsulate PE for insertion into beef patties. The
encapsulating material should not change the heat transfer properties of the meat system
or interact with the PE.

Methodologies have been developed that accurately record lethality, maximum
center temperature and the destruction of PE in a patty system. Thus these
methodologies could be applied to other meat patty systems, including turkey and
chicken, whose target pathogen for proper thermal processing is also Salmonella.

Phycoerythrin is just one of three proteins in the class of phycobiliproteins.
Thermal inactivation parameters could be determined for the other two pigments to
investigate their possible application as a TTI in food systems.

Limitations were seen in the ability to accurately measure the internal center

temperature of the beef patty during cooking. Theoretical heat transfer calculations could

106

 

be used to predict the internal temperature without inaccuracies due to thermocouple

placement.

107

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108

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