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This is to certify that the

thesis entitled

Lactate Dehydrogenase as Indicator of Proper Heat
Processing and Death of Escherichia coli 0157:H7
and Salmonella in Ground Beef Patties

 

 

presented by

Alicia Orta-Ramirez ‘

has been accepted towards fulfillment
of the requirements for

Master of Science degree in Food Science

 

 

gW/fix

/ Major professor

Date //" /3’— f4

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

LIBRARY
Mlchlgan State
University

 

 

 

PLACE II RETURN BOXtomnavothbdnckmMmm yourncotd.
TOAVOID FINESMumonorbdonddoduo.

DATE DUE DATE DUE DATE DUE

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU IoAn Nam-two WM OM lm
W1

LACTATE DEHYDROGENASE As INDICATOR OF PROPER
HEAT PROCESSING AND DEATH OF Escherichia coli OlS7:H7
AND Salmonella IN GROUND BEEF PATTIES
by

Alicia Orta-Ramirez

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

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1994

McC
E .col
Colif.
inacfi
comp:
the mc
indical
13.6%
Grounc
tempera
P0lycl01
in LDH
uncooke.
found an
N0 Signif
and cookc
ConditionS

before after

ABSTRACT

LACTATE DEHYDROGENASE AS INDICATOR OF PROPER
HEAT PROCESING AND DEATH OF Escherichia call 0157 :H7 AND
Salmonella IN GROUND BEEF PATTIES

by
Alicia Orta-Ramirez

Three media (3M Petrifilm'“ Coliform Plate and E .coli Plate Counts, and
McConkey-Plate Count Agar, 50: 50) were compared for their ability to enumerate
E .coli 0157zH7. N 0 significant differences were found. For convenience
Coliform Plate Counts were chosen for heat inactivation studies. Thermal
inactivation of E.coli 0157:H7 in ground beef at 53°, 58°, 63° and 68°C was
compared to those of S .senftenberg and lactate dehydrogenase (LDH). LDH was
the most heat resistant at all temperatures suggesting that it could be used as
indicator of proper heat processing in ground beef. Ground beef patties containing
13.6% fat were cooked to internal temperatures of 62.8°, 65.6°, 68.3° and 71.1°C.
Ground beef patties containing 10.7% or 19.0% fat were cooked to an internal
temperature of 683°C. A sandwich ELISA using both anti-LDH monoclonal and
polyclonal antibodies was performed on extracts of patties. Significant differences
in LDH concentration were found between patties cooked to 628°C and both
uncooked patties and patties cooked to 65.6°C,- but no significant differences were
found among patties cooked tO internal temperatures of 656°, 683° and 71.1°C.
NO significant differences were found among patties containing different fat levels
and cooked to 683°C. High variation among patties cooked under same
conditions interfered with detection Of LDH. This problem should be addressed
before attempting further studies.

 

 

 

ant
per
mil

enc:

mak

iii

This is dedicated to my parents, Carlos Orta and Milagros Ramirez,
and my sisters, Nuria, Gemma and Teresa, without whom I would not be the
person I am. Also to my parents in-law, Enric Garcia and Rosa M“. Sirera,
without whom I would not be here, and to J osep for his love and
encouragement.

Finally, I would like to dedicate this work to the people in my life that
make me smile.

 

III
Ill

Ste

ACKNOWLEDGMENTS

I would like to express my appreciation to Dr. James F. Price, my
major professor, Dr. Denise M. Smith and Dr. James J. Pestka, members of
my committee, for all their guidance, counseling and patience throughout the
program.

Thanks also to Dr. Mohamed M. Abouzied for his help in producing
the monoclonal antibodies and for allowing me to use the polyclonal
antibodies that he produced.

I would specially like to thank Jamie Sue Cherry-Merritt for her help
during the thermal inactivation studies and also for her support,
encouragement and friendship.

It is also to be appreciated the help from Dr. Cheng-Hsin Wang,
Stephanie Smith-Dudra and Lisa Desrocher.

iv

 

 

II

1.1

1.1.

TABLE OF CONTENTS

List of Tables ............................................................................................. ix
List of Figures ............................................................................................ x
Introduction ................................................................................................ 1
1. Literature review .................................................................................... 3
1.1. Meat safety considerations .................................................................. 3
1.1.1. Current USDA methodology ............................................................. 5
1.1.2. Alternative methodogy ...................................................................... 6
1.2. Escherichia coli 0157:H7 ................................ 10
1.2.1. Characteristics Of the microorganism ................................................ 11
1.2.2. Clinical presentations ........................................................................ 12
l .2.3. Epidemiology ................................................................................... 14
1.2.4. Control and prevention ..................................................................... 16
1.2.5. Detection of E.coli 01572H7 in foods ............................................... 18
1.3. Lactate Dehydrogenase ....................................................................... 19
1.4. Immunoassays in Food Science ........................................................... 20

 

 

1.4.1. Types Of Antibodies ......................................................................... 21
1.4.2. Antigens ........................................................................................... 23
2. Comparison Of three media for enumeration of Escherichia call

01 57 :H7 .................................................................................................... 25
2.1 . Abstract ............................................................................................... 25
2.2. Introduction ......................................................................................... 25
2.3. Materials and methods ......................................................................... 26
2.3.1. Media ............................................................................................... 26
2.3.2. Cultures ............................................................................................ 26
2.3.3. Statistical Analysis ........................................................................... 27
2.4. Results and conclusions ....................................................................... 27

3. Thermal inactivation Of Escherichia coli 01572H7 as compared to

that of lactate dehydrogenase and Salmonella senflenberg .......................... 29
3.1 . Abstract ............................................................................................... 29
3 .2. Introduction ......................................................................................... 29
3.3. Materials and methods ......................................................................... 31
3.3.1 . Bacterial cultures .............................................................................. 3]
3.3.2. Preparation of meat ........................................................................... 31
3.3.3. Thermal inactivation ......................................................................... 32
3.3.4. Bacterial counts ................................................................................ 34

3.3.5. Extraction Of LDH from ground beef ................................................ 34

vii

3.3.6. Determination Of enzymatic activity .................................................. 34
3.3.7. Proximate analysis and determination of pH ..................................... 35
3.3.8. D and 2 values .................................................................................. 35
3.4. Results and discussion ......................................................................... 36
3.4.1. Raw ground beef .............................................................................. 36
3.4.2. D and 2 values .................................................................................. 36
3.5. Conclusions ......................................................................................... 49

4. Production Of monoclonal antibodies against bovine muscle lactate
dehydrogenase and verification Of endpoint temperature of ground beef

patties by sandwich ELISA ........................................................................ 51
4.1. Abstract ............................................................................................... 51
4.2. Introduction ......................................................................................... 51
4.3. Materials and methods ......................................................................... 53
4.3.1. Production Of Monoclonal Antibodies ............................................... 53
4.3.2. Indirect ELISA ................................................................................. 55
4.3.3. Sandwich ELISA .............................................................................. 56
4.3.4. Preparation of ground beef patties ..................................................... 57
4.3.5. Cooking of ground beef patties ......................................................... 57
4.3.6. LDH extraction ................................................................................. 58

4.3.7 . Proximate analysis and determination of pH ..................................... 58

 

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Apt
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App
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viii

4.3.8. Statistical analysis ............................................................................ 58
4.4. Results and discussion ......................................................................... 59
4.4.1. Production Of monoclonal antibodies ................................................ 59
4.4.2. Preparation and cooking of ground beef patties ................................. 63
5. Conclusions ............................................................................................ 66
Bibliography ............................................................................................... 69

Appendix A. Comparison Of three media for enumeration Of
Escherichia coli 0157:H7 .......................................................................... 78

Appendix B. Thermal inactivation Of Escherichia coli 01572H7 as
compared to that Of the enzyme lactate dehydrogenase and Salmonella
senflenberg ................................................................................................ 8O

 

Ur

Bi

5%

Apt?
corn;
sag/i

viii

4.3.8. Statistical analysis ............................................................................ 58
4.4. Results and discussion ......................................................................... 59
4.4.1. Production of monoclonal antibodies ................................................ 59
4.4.2. Preparation and cooking Of ground beef patties ................................. 63
5. Conclusions ............................................................................................ 66
Bibliography ............................................................................................... 69

Appendix A. Comparison Of three media for enumeration Of
Escherichia coli 0157:H7 .......................................................................... 78

Appendix B. Thermal inactivation Of Escherichia coli 0157:H7 as
compared to that Of the enzyme lactate dehydrogenase and Salmonella
senftenberg ................................................................................................ 8O

LIST OF TABLES

Table 2.1. Statistical comparison among mean log CFU/mL for both

regular and heat-shocked cultures of E.coli 0157:H7 across media. ........... 28
Table 3.1. Time and temperature schedules for thermal inactivation of

E.coli 0157:H7, S .senftenberg and LDH .................................................... 33
Table 3.2. Results of proximate analysis of raw ground beef samples ......... 37
Table 3.3. Summary Of D values and regression analysis for E.coli

0157:H7 in ground beef. ............................................................................ 43
Table 3.4. Summary Of D values and regression analysis for

S .senftenberg in ground beef. ..................................................................... 43
Table 3.5 . Summary of D values and regression analysis for LDH in

ground beef. ............................................................................................... 44
Table 3.6. Summary of 2 values and regression analysis for E.coli

0157:H7, S .senfrenberg and LDH in ground beef ....................................... 44
Table 3.7. Comparison Of D and 2 values for E.coli 0157:H7 and

S .senftenberg in ground beef. ..................................................................... 45
Table 4.1. Proximate analysis of ground beef patties ................................... 64
Table 4.2. Summary of ground beef patties cooking .................................... 65
Table A.1. Mean log CFU/mL for regular E.coli 0157:H7 culture .............. 78

Table A2. Mean log CFU/mL for heat-shocked E.coli 0157:H7
culture ........................................................................................ 79

LIST OF FIGURES

Figure 3.1. Survivor curves Of E.coli 0157:H7 and S.senftenberg in

ground beef at 53C. .................................................................................... 38
Figure 3.2. Effect Of heating on LDH in ground beef at 53C. ...................... 38
Figure 3.3. Survivor curves Of E.coli 0157 :H7 and S.senftenberg in

ground beef at 58C. .................................................................................... 39
Figure 3.4. Effect of heating on LDH in ground beef at 58C. ...................... 39
Figure 3.5. Survivor curves of E.coli 0157:H7 and S.senftenberg in

ground beef at 63C. .................................................................................... 41
Figure 3.6. Thermal inactivation of LDH in ground beef at 63C .................. 41
Figure 3.7. Survivor curves of E.coli 0157 :H7 and S.senftenberg in

ground beef at 68C ..................................................................................... 42
Figure 3.8. Thermal inactivation of LDH in ground beef at 68C. ................. 42

Figure 3.9. Thermal death time curves of E.coli 0157:H7,
S.senftenberg and LDH in ground beef. ...................................................... 46

Figure 4.1. Detection of bovine muscle LDH by competitive indirect
ELISA using sera of mice injected subcutaneously (sc). ............................. 60

Figure 4.2. Detection Of bovine muscle LDH by competitive indirect
ELISA using sera of mice injected intraperitoneally (ip). ............................ 61

Figure 4.3. Detection Of bovine muscle LDH by sandwich ELISA:
monoclonal antibodies (1: 250) as capture antibodies and polyclonal
antibodies (1: 500) as detector antibodies. .................................................. 62

Figure 4.4. Concentration Of LDH in ground beef patties cooked to
different internal temperatures. ................................................................... 67

Figure 4.5. Concentration of LDH in ground beef patties with different
fat content cooked to 68.3C ....................................................................... 67

 

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INTRODUCTION

Recent outbreaks Of Escherichia coli 0157:H7 (E. coli 0157:H7)
associated mainly with consumption of undercooked ground beef patties,
have led to a severe review Of the methodology used to ensure safety Of
cooked meat products. In January Of 1994, the Food and Drug
Administration (FDA) published new thermal processing recommendations
for meat products in a revised version Of the Food Code. Comminuted meats
should now be processed to an internal temperature of either 63°C (145°F)
for 3 min, 66°C (150°F) for 1 min or 68°C (155°F) for 15 sec. According to
the United States Department of Agriculture Food Safety Inspection Service
(USDA-FSIS) regulations, effective as Of September 1, 1993, Oflicial
establishments that manufacture fully-cooked patties will have to use any of
seven time/temperature schedules with holding time. Ofiicial establishments
that manufacture partially-cooked patties will have to raise the internal
temperature to a minimum Of 60°C (140°F) and then cool to a maximum
temperature of 4°C (40°F) within 2 h. (USDA-FSIS, 1993).

Current methods employed by USDA-F SIS to verify proper heat
processing Of meat products are considered subjective and do not provide an
adequate margin of safety. There is an urgent need for rapid and accurate
assays that ensure safety of cooked meat products.

Determination Of the endpoint temperature to which a product has been

cooked is a common way to verify compliance to regulations. The use of

 

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enzyme
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enzyme markers to assess the endpoint cooking temperature in meat products
has already been suggested. Once a potential marker has been identified, it
can be detected by different methods including residual enzymatic activity,
solubility and immunoassays. Among them, enzyme-linked irnmunosorbent
assays (ELISA) are gaining importance due to their specificity, sensitivity and
ease of application.

The purpose of this research was to develop an ELISA to verify
proper thermal processing Of ground beef patties. From previous work
(Abouzied et al., 1993; Wang et al., 1994), lactate dehydrogenase (LDH) had
already been identified as a potential marker. The first Obj ective was to
evaluate and compare three difierent media for enumeration of E. coli
0157:H7. The second Objective was to compare thermal inactivation of
LDH, E. coli 0157:H7 and Salmonella senflenberg in ground beef at four
different temperatures. A third Obj ective was to produce monoclonal
antibodies against bovine muscle LDH. The final Objective was to verify
endpoint cooking temperature Of ground beef patties by a sandwich ELISA
using polyclonal and monoclonal antibodies against LDH.

 

 

 

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1. LITERATURE REVIEW

Microbial contamination is considered the most important hazard
associated with foodbome illnesses. According to Todd (1989), microbial
diseases accounted for 84% Of all foodbome disease costs in the United
States, with a total estimated cost of $8.4 billion and an average of $670 per
case. More recently, Todd (1994) estimated the number of cases resulting
from foodbome infections and intoxications to be 5.5-6.2 million costing
$5.8-8.6 billion.

Inadequate cooking of meat products and improper storage or holding
temperatures seem to be the most common errors that lead to foodbome
outbreaks (Bean and Griffin, 1990). During the last decade, severe outbreaks
of food poisoning due tO E. coli 0157:H7 had become more frequent. The
number Of cases Of diarrhea caused by this microorganism is estimated to be
10,000-20,000 per year. Estimated costs Of medical treatment and lost
productivity account for $216-58O million per year (Anonymous, 1994).
Because Of the high correlation Of these outbreaks with consumption of
undercooked beef products and because Of the severity Of the clinical
presentations such as hemolitic-uremic syndrome and thrombotic
thrombocytopenic purpura, it has become a real concern for consumers, the
meat industry and government regulatory agencies. As a consequence, FDA
and USDA have reviewed the current regulations concerning cooking

temperatures of meat products.

1.1. Meat safety considerations
Title 9 Of the Code Of Federal regulations outlines the requirements for

the processing of meat products needed for destruction Of pathogenic

 

 

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microorganisms that can cause foodbome disease (U SDA-F SIS, 1990).
Cooked beef and roast beef are required to be heat processed in any of 16
time/temperature schedules with holding time or tO 63 °C (145°) with no
holding time. Pork products are required to be processed to an internal
temperature of 583°C (137°F) or subjected to freezing or drying to ensure
proper destruction Of the parasite T richinae. Uncured and cured/smoked
poultry products are required to be processed to internal temperatures Of 71.1
°C (160°F) and 68°C (155°F), respectively.

After the 1993 outbreak Of E. coli 0157:H7 associated with
consumption of undercooked ground beef patties that involved more than 500
people, including 4 deaths, the government regulatory agencies have reviewed
and updated the current thermal processing regulations.

In January 1994, the FDA published a new FOOd Code (FDA, 1993)
establishing new recommendations for cooking meat products. From now on,
pork products and conminuted meats shall be cooked to internal temperatures
Of either 63°C (145°F) for 3 min, 66°C (150°F) for 1 min or 68°C (155°F) for
15 sec. Roast beef and corned beef shall be cooked to any Of the following
time/temperature protocols: 54°C (130°F)/ 121 min, 56°C (132°F)/77 min, 57°
C (134°F)/47 min, 58°C (136°F)/32 min, 59°C (138°F)/ 19 min, 60°C (140°
F)/l2 min, 61°C (142°F)/8 min, 62°C (144°F)/5 min or 63°C (145°F)/3 min.
Poultry products shall be cooked to 74°C (165°F) or above for 15 see. In
addition, according to USDA-F SIS regulations, Official establishments that
manufacture fully-cooked patties shall use one of the following
time/temperature protocols: 661°C (151°F)/4l sec, 667°C (152°F)/32 sec,
672°C (153°F)/26 sec, 678°C (154°F)/20 sec, 683°C (155°F)/ 16 sec,
689°C (156°F)/13 sec, and 694°C (157°F) (and up)/ 10 sec. Also, Official
establishments that manufacture partially cooked patties are required to heat

42.4- V i [I

 

 

 

 

 

 

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patties to a minimum internal temperature 60°C (140°F) followed by cooling
to a maximum internal temperature Of 4°C (40°F) within 2 h (U SDA-FSIS,
1993)

Although the increase in temperatures requirements can help reduce the
incidence of foodbome disease, there is still the problem of confirming
adequate cooking once the product has been processed. In two outbreaks of
E.coli 0157:H7 (Michigan and Oregon, 1982) cooler spots on the grill were
detected during times Of peak use (Griffin and Tauxe, 1991), with subsequent
inadequate heating of some Of the hamburgers.

1.1.1. Current USDA methodology
The USDA-FSIS currently employs three different assays to verify

proper thermal processing Of meat products. The residual Acid Phosphatase
test (U SDA-FSIS, 1986a) is used for canned hams, picnics and luncheon
meats. This assay is based on the residual enzyme activity in water-soluble
protein extracts Of cooked samples. Positive reactions show a blue color that
can be read at 610 nm. One problem with this assay is that it has been
demonstrated that there is a great loss in acid phosphatase activity during
frozen storage (Townsend, 1989). This should be taken into consideration
when analyzing samples that have been kept under freezing conditions.

The Coagulation test (U SDA-FSIS, 1986b) is used for beef and pork
products. The method is based on loss Of protein solubility during heat
processing of the meat product. Protein extracts Of cooked samples are
heated until cloudiness appears. The temperature at which the turbidity
occurs is considered the internal temperature the product reached during
cooking. For products cooked to the 63°-71°C the assay can differ 8°—10°C

from the real temperature achieved during processing (Townsend and

 

 

 

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Blankenship, 1989). This test is thus very subjective and only gives a gross
estimation of the thermal process applied.

The Bovine Catalase test (U SDA-FSIS, 1989) is used to verify roast
beef and cooked beef. The assay is based on the production of foam when
catalase reacts with oxygen. Bovine catalase is destroyed in the 60.5°- 61 . 1°
C temperature range. With the increase Of cooking temperature requirements
a negative sample is likely to have been undercooked by at least 2°C. In
addition, the visual detection Of foam is very subjective. None Of these
assays are sensitive and reliable. There is urgent need for Objective and

accurate assays tO verify adequate thermal processing.

1.1.2. Alternative methodology
The need to assess proper heat processing of meat products to ensure

absence Of pathogenic bacteria and viruses has led to increased emphasis in
the search for a sensitive method that accurately determines the temperature
to which a meat product has been cooked. Several approaches have been
suggested which can be categorized in two groups: (1) those using an enzyme
system as indicator of internal temperature achieved during thermal
processing, and (2) those based on physical techniques.

Different enzymes have been tested for their potential as indicators Of
endpoint temperature (EPT) of cooking. Davis et a1. (1988) reported loss Of
pyruvate kinase activity in both a model system and a commercial canned
cured pork product. The enzyme became inactive between 695° and 70°C.
The method was based on the loss Of fluorescence when NADH is oxidized
which could be Observed under long-wave ultraviolet light. The assay,
however, requires visual determination, thus likely to be subjective.

McCormick et al. (1987) used a reverse phase high performance liquid

 

 

 

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chromatography to monitor the effect Of heating on porcine extracts.
Townsend and Blanckenship (1987) used the APIZYM enzyme system to
monitor decrease in enzymatic activity in heated aqueous and saline extracts.
The APIZYM system can detect 19 different enzymatic activities in a variety
of specimens. As a result, the authors suggested that the enzyme leucine
aminopeptidase had potential to act as indicator Of EPT. The method,
however, was not tested in whole muscle samples. Bogin et a1. (1992)
assayed the activity Of 12 enzymes in turkey breast samples heated to
different temperatures and they concluded that Of the 12, L-aspartate-Z-
oxoglutarate aminotransferase, creatine phosphotransferase, isocitric
dehydrogenase, aldolase, malate dehydrogenase, and lactate dehydrogenase
could be used as indicators for verification Of heat treatment. Townsend and
Davis (1992) evaluated the potential of glutamic oxalacetic transaminase
(GOT) and glutarnic pyruvic transarninase (GTP) as indicators of EPT in
ground beef using a commercial test kit. Low values of GTP activity in all
samples indicated that the enzyme could not be used as an indicator, whereas
because Of relatively high activity values for GOT in products cooked to 71.1
°C it was suggested that this enzyme might have potential in meat products
that have to be cooked to very high EPT. Kormendy et al. (1992) presented a
modified acid phosphatase assay and suggested that it could be successfully
used to verify heating in canned hams. Townsend et al. (1993) reported loss
in N-acetyl-B-D-glucosaminidase activity in samples Of beef, pork and poultry
heated fiom 40° to 70°C. Hsu (1993) screened enzymatic activity of 26
enzymes from extracts of turkey muscle for their suitability as indicators Of
proper heat processing. Of the 26, the author reported LDH and malate
dehydrogenase to have potential to be used as markers in poultry products.
Townsend et al. (1994) studied creatine phosphokinase activity as a possible

 

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indicator
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indicator Of EPT using a commercial test kit. Although enzymatic activity in
meat and poultry products was reduced with increasing processing
temperatures, the test was very product dependent, and the authors suggested
that it should not be used as a regulatory assay. Wang, Abouzied and Srrrith
(1994) screened different enzymes to be used as indicators of proper heat
processing in grOtmd beef patties, using sodium dodecyl sulfate-
polyacrylarnide gel electrophoresis (SDS-PAGE), Western blot and
enzymatic assays. They concluded that triosephosphate isomerase, LDH and
phosphoglycerate mutase showed potential tO be used as EPT indicators.
LDH is probably the most thoroughly studied enzyme for its potential
as indicator of adequate thermal processing. Collins et al. (1991a) measured
LDH activity in samples Of beef muscle cooked to different temperatures.
They Observed a sharp decline when samples were heated to 63 °C and only
marginal activity could be detected at 66°C. The same authors evaluated
LDH in cured and uncured pork products (Collins et al., 1991b). For uncured
products, activity decreased markedly at 63 °C and was almost undetectable at
68°C. For cured products no activity was detected at either 63° or 68°C.
Stalder et al. (1991) studied the effect of pH, salt, phosphate, cooking
temperature, muscle variation, carcass sex and maturity on LDH activity from
heated beef extracts. The authors concluded that LDH showed potential tO be
used as an EPT indicator. In a different approach, Wang et al. (1992)
developed an indirect competitive ELISA to verify proper cooking Of uncured
turkey breast rolls based on detection Of LDH using polyclonal antibodies.
The ELISA accurately distinguished EPT within i1.1-1 .2°C in the
temperature range Of 683° to 721°C. In a related study, Abouzied et al.
(1993) developed monoclonal antibodies to be used in a sandwich ELISA.
Minimum detection limits for turkey and chicken LDH were 1 ng/mL. Effects

 

 

 

 

of formul
studied (1
casing dit

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of meat p
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of formulation, storage and processing of poultry products on LDH were also
studied (Wang, 1993). Salt concentration, cooking protocol and type of
casing did not markedly influence LDH content.

Another approach tO monitor EPT reached during thermal processing
Of meat products is the use Of physical techniques. These include:
electrophoretic separation, differential scanning calorimetry (DSC), near
infrared spectroscopy (NIS), loss of protein solubility and determination of
color.

Caldironi and Bazan (1980) used SDS-PAGE to monitor disappearance
of low-salt soluble proteins when beef muscle samples were heated at
different temperatures. They Observed a gradual loss in some bands after
reaching 60°C until their complete disappearance above 80°C. Steele and
Lambe (1982) used SDS gradient gel electrophoresis tO separate polypeptides
formed during heating Of different muscle extracts. The intensity Of the bands
decreased with increasing heating temperatures. .

Isaksson et al. (1989) Observed changes in spectra Of samples Of beef
with increasing temperature when analyzed by DSC. In another study,
samples of beef muscle heated at five different temperatures were studied
using DSC (Ellekjaer, 1992). The method very accurately determined the
temperature to which the samples have been cooked ( i0.6°C). Ellekjaer and
Isaksson (1992) verified thermal processing Of beef samples that had been
cooked to nine different temperatures using near infrared reflectance and
transmittance. Both methods could detect EPT within 2.0°-2.1°C.

Davis et al. (1985) measured the amount Of water-extractable proteins
in pork and beef muscles heated following different time/temperature
schedules using the biuret method. They Observed that loss of solubility was

time/temperature dependent but concluded that more information was needed

 

 

 

to determin
model. In :
solubility if
gels. Agair
method to \

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suggested I
chicken leg
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10

to determine a extractable biuret-positive ratio to be used in a mathematical
model. In a related study, Davis et al. (1987) monitored loss Of protein
solubility in canned cured pork by isoelectric focusing on polyacrylamide
gels. Again the authors remarked about the difficulty Of establishing a
method to verify EPT based on a measure of extractable protein.

Determination Of color Of cooked meat products has also been
suggested by some authors. Ang and Huang (1994) studied color changes in
chicken leg patties cooked to different EPT. Although some differences
could be seen among patties, both packaging method and storage time
influenced markedly the results Of color values. Hague et al. (1994)
evaluated color Of ground beef patties cooked from 55° to 77°C. Internal
color lost redness with increasing temperature. Some patties, however,
showed premature browning. In addition, visual evaluation proved to be not
very accurate.

The main disadvantages in some Of these methods are that they require

expensive equipment and well trained personnel, or are very time consurrring.

1.2. Escherichia call 0157 :H7

Escherichia coli (E. coli) is a microorganism commonly found in the
intestinal tract Of humans and most mammals. A few strains, however, are
pathogenic and can cause gastrointestinal syndromes. These pathogenic
strains have been categorized according to their virulence, clinical
presentation, epidemiology and OH serogroups, and include:
enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and
enterohemorrhagic E. coli . Among them enterohemorrhagic E. coli , which
includes E.coli 0157:H7 and E.coli 0262H11 (Padhye and Doyle, 1992) has

 

 

 

ll

become very important due to its role in several foodbome disease outbreaks

during the last decade.

1.2.1. Characteristics of the microorganism
E. coli 0157:H7 shows most typical biochemical features Of E. coli

except sorbitol fermentation and B-glucuronidase activity. E. coli isolates
ferment sorbitol within 24 h, whereas E. coli 0157:H7 failed to ferment
sorbitol within 7 days (Wells et al., 1983). In addition, this microorganism
has been shown to be negative in the 4-methylumbelliferyl B-D-glucuronide
(MUG) assay. This test, used to detect E. coli, is based on the hydrolyzation
Of MUG which results in a fluorogenic compound (Doyle and Schoeni, 1984).

E.coli 0157:H7 grows well at 30-42°C with optimum growth at 37°C.
This microorganism grows poorly between 42° and 45°C and does not grow
at all at 4°, 10° and 455°C (Doyle and Schoeni, 1984). Common procedures
for fecal coliform enumeration use incubation temperatures of 44-45.5°C.
Therefore, being unable tO grow at these temperatures, E. coli 0157:H7
carmot be detected using standard coliform procedures (Doyle and Schoeni,
1984; Raghubeer and Matches, 1990).

Studies of thermal inactivation Of E. coli 0157:H7 in ground beef
containing 17-20% fat and heated to 544°, 572°, 589°, 60°, 622°, 628°
and 643°C resulted in D values Of 39.8, 4.5, 1.2, 0.8, 0.4 and 0.2 sec,
respectively (Doyle and Schoeni, 1984). In a similar study using ground beef
containing 2% fat, Line et al. (1991) reported D values of 78.2-80.1, 41-40
and 0.30-0.22 min when heated to 51.7°, 57.2° and 628°C, respectively,
using two different recovery methods.

When grOtmd beef patties inoculated with E. coli 0157:H7 were frozen
at -80°C and held at -20°C, there was little change in population after 9

12

months of storage, suggesting that this microorganism can survive well in
freezing conditions (Doyle and Schoeni, 1984). In studies of pH tolerance,
Abdul-Raouf et a1. (1993) found that E.coli 0157:H7 populations did not
change in beef slurries at pH 4.70 after 24 h Of storage at 5°C. In addition,
they Observed growth in slurries acidified tO 4.70 with either citric or lactic
acid after 24 h at 30°C. Weagant et al. (1994) reported more extended
survival times for E.coli 0157:H7 in mayonnaise (pH 3.65) held at 5° or 7°
than at 25°C. Thayer and Boyd (1993) studied the effect of irradiation on
E. coli 0157:H7 in ground beef and chicken meat. The microorganism was

sensitive to gamma radiation at the 1.5-3.0 kGy dose range.

1.2.2. Clinical presentations
Three major syndromes have been associated with E. coli 0157:H7:

hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic
thrombocytopenic purpura (TPP). .

Hemorrhagic colitis is characterized by severe abdominal pain
followed by watery diarrhea which progresses to a bloody discharge.
Vomiting can also occur but none or slight fever is Observed (Riley, 1987;
Cohen and Giannella, 1991; Padhye and Doyle, 1992). Clinical examination
reveals intestinal surface ulceration with marked edema in the lamina propia,
variable neutrophilic infiltration, and mucosal thickening in some cases (Neill
et al., 1987). The incubation period is approximately 3-4 days with an
average duration of 7 days. The microorganism can be detected in patients
feces within a week of the onset of symptoms, after which isolation becomes
more difficult or not possible. It is believed that some risk factors exist, such
as age (children and elderly are most susceptible), use of antibiotics and

previous gastrectomy (Griffm and Tauxe, 1991). The treatment Of HC is

13

based on alleviating dehydration and replenishing electrolyte abnormalities
and blood loss. The use Of antibiotics is controversial. According to Tarr et
al. (1988), antibiotic treatment can increase the risk Of HUS by two
mechanisms: (a) by depleting competitive gut microflora and (b) by lysis of
E. coli 0157:H7 bacterial cell with subsequent increase Of toxin liberation into
the intestinal lumen. However, as mentioned by Griffin and Tauxe (1991), in
other studies no correlation could be found between the administration of
antibiotics and progression to HUS.

Hemolytic uremic syndrome most commonly affects children between
1 and 4 years Old. Initial symptoms are those of enterocolitis followed by
pallor, vomiting, macroscopic hematuria or Oliguria. Clinical examination
reveals microangiopathic hemolytic anemia, thrombocytopenia and acute
renal failure. Additionally, hypertension hepatosplecnomegaly and purpura,
and leukocytosis may be seen. Complications include central nervous system
symptoms such as seizures, and gastrointestinal and cardiac symptoms. The
treatment Of HUS is based on maintenance Of a normal fluid and electrolyte
balance, and careful monitoring (Robson and Leung, 1990). Usually, patients
require dialysis and blood transfusions, even renal transplantation. Permanent
renal or neurologic damage can result. Death may occur in some cases
(Cohen and Giannella, 1991; Padhye and Doyle, 1992). As in HC,
administration Of antibiotics seems to be contraindicated.

Thrombotic thrombocytopenic purpura is similar to HUS because Of
the vascular histopathology involved, but it occurs more frequently in adults.
Clinical features include hemolytic anemia, severe thrombocytopenia,
neurologic symptoms such as seizures, drowsiness, confusion and fluctuating
levels Of consciousness, and fever (Kovacs et al., 1990). Blood clots can be
developed in the brain resulting in death (Padhye and Doyle, 1992).

l4

Reported risk factors for developing both HUS and TPP include age, female
sex, mental retardation, fever, and use of antibiotics (Griffm and Tauxe,
1991)

Less frequent clinical presentations are hemorrhagic cystitis and
balanitis (Gransden etal., 1985), convulsions and anerrria (Padhye and Doyle,
1992)

1.2.3. Epidemiology
E. coil 0157:H7 was first isolated in California in 1975 in a patient with

severe bloody diarrhea, but it was not until 1982, after two major outbreaks in
Michigan and Oregon, that it was recognized as a foodbome pathogen
microorganism (Padhye and Doyle, 1992). The people implicated in both
outbreaks had consumed ground beef hamburgers from the same fast-food
restaurant chain. Between 1984 and 1990, ten more outbreaks were reported
in Nebraska, North Carolina, Washington, Utah, Wisconsisn, Minnesota,
Missouri, North Dakota and Montana (Griffin and Tauxe, 1991). In six out
of the ten outbreaks, beef products were identified as the vehicle Of
contamination. Data from another source (Anonymous, 1994) points out an
estimated number of 2-3 outbreaks per year during the period 1982-1992.
Outbreaks have also been reported in Canada, United Kingdom, Mexico,
Argentina, China and Belgitnn. More recently, between November 15, 1992
and February 28, 1993, four states (Washington, California, Nevada and
Idaho) were involved in the largest outbreak of E. coli 0157:H7 in the US,
which affected a total Of 527 people and four children died (Dom, 1993).
Evidence was found that implicated undercooked contaminated hamburgers

from a fast-food restaurant.

IS

The mode of transmission is mostly by ingestion Of food contaminated
with E. coli 0157:H7. As mentioned before, the most common vehicle
implicated in all outbreaks was ground beef patties that had been
underprocessed. Borczyk et al. (1987) isolated E. coli 0157:H7 from fecal
samples Of healthy calves and cows after an outbreak due to consumption of
raw milk, providing evidence that dairy cattle may be a primary reservoir Of
this microorganism. During slaughter procedures or milking, meat and milk
can become contaminated, thus acting as vehicles of E. coli 0157:H7
poisoning. The microorganism has also been isolated from retail samples Of
beef, pork, lamb and poultry (Doyle and Schoeni. 1987), suggesting that it is
not a rare contaminant Of fresh meat and poultry. Other foods implicated in
other outbreaks were water, salad dressing, potatoes and apple cider.
Although E. coli 0157:H7 seems tO be mostly a foodbome pathogenic
microorganism, person-tO-person transmission, possibly through a direct
fecal-oral route, has also been described in some cases (Ratnam et al., 1986;
Griffin and Tauxe, 1991).

The mechanism Of pathogenesis is not yet fully understood. Virulence
seems to be due to adherence and attachment to intestinal mucosal cells and
the production Of toxins. Attachment to the mucosa allows the
microorganism to resist peristaltic movement and brings toxins into closer
contact with mucosal cells, causing damage to the intestinal wall (Cohen and
Giannella, 1991). E.coli 0157:H7 produces at least one or two toxins, called
Shiga-like toxins because of their resemblance in structure and activity to the
Shiga toxin produced by Shigella aysenteriae type 1 (Griffin and Tauxe,
1991). Shiga-like toxin I can be neutralized with antiserum against Shiga
toxin, whereas Shiga-like toxin H cannot (Doyle, 1991). The mechanism Of
action of Shiga toxin and both Shiga-like toxins is almost identical. They all

 

 

 

l6

interfere in protein synthesis in mammalian cells by cleaving an N-glycoside
bond at a specific adenine residue Of ribosomal RNA. The role Of these
toxins in the pathogenesis Of the disease is not that clear. There seems to be
both cytopathic action on the intestinal mucosa and systemic effects affecting
extraintestinal vasculature (Cohen and Giannella, 1991). Robson and Leung
(1990) reported that these toxins caused the endothelial damage Observed in
renal arterioles Of children with HUS. Both, Shiga-like toxin I and II, are
cytotoxic to VerO cells. Hence, another way to designate them is Verotoxin 1

and 2, respectively.

1.2.4. Control and prevention
After more than a decade since the first outbreak directly attributed to

E.coli 0157:H7, there is still insufficient epidemiological and ecological
information to outline control strategies to pursue for control and prevention
against this pathogenic microorganism. It is to be pointed out, however, the
position Of three different entities: the National Live Stock and Meat Board,
the American Gastroenterological Association Foundation and the USDA.

In 1994, the National Live Stock and Meat Board published the Final
Report Of the Blue Ribbon Task Force with the aim Of supplying directions
to improve meat safety based on information provided by experts from the
government, academia and industry. The general approach is based on a
thorough analysis Of nine critical points during the beef production process or,
as they called it, "from farm tO table". The nine critical areas include: (1) pre-
harvest, (2) beef carcass conversion process, (3) beef carcass break-up and
trim generation, (4) ground beef processing, (5) food service, (6) retail, (7)
public health/consumer education, (8) pathogen reduction/intervention

strategies, and (9) regulatory opportunities/challenges. The Task Force

 

 

 

 

17

suggested implementation and evaluation Of Hazard Analysis and Critical
Control Points (HACCP) system in each Of the nine steps Of the production
chain, promotion Of research to identify reservoirs Of E. coli 0157:H7 and
development Of pathogen reduction strategies, approval of antimicrobial
rinses for beef carcasses and beef irradiation, and design of consumer
education programs on food safety.

In July 11-13, 1994, a panel of experts convened by the American
Gastroenterological Association Foundation issued a consensus statement
providing recommendations for government, industry and medical
professionals for the control and prevention Of E. coli 0157:H7. The panel
recommended the implementation of HACCP by food service facilities and
retail operations, increase reporting Of outbreaks and funding of
epidemiological studies. They also recommended the inclusion Of E. coli
0157:H7 screening and detection in stools, besides the microorganisms
routinely examined; the implementation Of education programs in farms,
slaughter and production plants, food service and retail operations, nursing
homes and day care providers; education of the medical community on
diagnosis and treatment of the disease; and promotion Of research on E. coli
0157:H7 pathogenesis, reservoirs, detection, control and prevention. The
panel agreed that, at present, the complete elimination Of E. coil 0157:H7
would only be possible by thorough cooking or irradiation (Anonymous,
1994)

According to an interview with a USDA spokesperson published in the
Food Chemical News, July 18, 1994, USDA was about to implement the
HACCP systems in all meat and poultry plants. In addition, the agency is
following three different approaches: the development Of a rapid test for

E.coli 0157:H7 to be used in the plants, the use of a microbial kit so

l8

inspectors can check sanitation Of the equipment and facilities, and the

increase Of research studies of the meat and poultry production chain.

1.2.5. Detection of E.coli 0157:H7 in foods

As mentioned before, standard procedures for detection Of fecal
coliforrns are not likely to detect E. coli 0157:H7 since the test uses
incubation temperatures in the range Of 44°-45°C. It has been demonstrated
that this microorganism is not able to grow at these temperatures. For this
reason and because the number Of outbreaks due to E. coli 0157:H7
poisoning has markedly increased during the last decade, researchers have
been focusing in the development of methods for detection and isolation Of
this microorganism in foods. In 1992, Padhye and Doyle published a
complete review on the suggested procedures for detection and isolation Of
E.coli 0157:H7.

Published methods range from plating on differential media such as '
MacConkey-sorbitol agar, to the use of sophisticated Oligonucleotide probes
and colony blot hybridization techniques. With the advent Of immunoassays,
polyclonal and monoclonal antibodies have been produced against the 0157
antigen and two proteins associated with a plasmid. Other procedures include
latex agglutination, a specific coliphage, gene and DNA probes and a
polymerase chain reaction.

The major flaws in these techniques are the need for incubation period
and/or enrichment procedures which are very time consuming, the required
use Of sophisticated and expensive equipment, and high possibility Of cross-

reactions with other Enterobacteria and Shiga-like toxin producing E. coli .

19

Another approach could be indirect "detection" of absence Of E. coli
0157:H7 by a marker or indicator which could be somehow correlated with

the presence or absence of the microorganism.

1.3. Lactate Dehydrogenase

Lactate dehydrogenase (LDH) is the enzyme involved in the final step
of anaerobic glycolysis and reversibly catalyzes the conversion of pyruvate to
lactate in the presence Of NADH and NAD‘”, respectively, according to the

following reaction:

Glucose

l
Glycolysisl
I
l LDH
Pyruvate + NADH < > L-Lactate + NAD"

 

The enzyme consist Of a group Of five tetrameric isofonns which result
from the combination Of two different polypeptide subunits, H and M, as
follows: H4, H3M, H,M,, I-IM3, and M4 (HolbrOOk, 1975). The subunits
molecular mass is about 35 kD. All five isozymes can exist in most tissues.
The H-type subunit, however, predominates in aerobic tissues such as heart
muscle, whereas the M-type predominates in skeletal muscle and liver. The
H4 isoform has a low affinity for pyruvate and is inhibited by high levels of
this compound, while the M4 isoform has a much higher affinity and is not
inhibited by pyruvate. The other isofonns show intermediate properties

according to the proportion Of one subunit or the other. Therefore, it has been

20

suggested that H-type LDH catalyzes the transformation Of lactate to pyruvate
while the M-type catalyzes the reverse reaction (Voet and Voet, 1990).

LDH has been extensively reviewed for its potential as an indicator of
endpoint temperatures in beef (Collins et al., 1991a; Stalder et al., 1991;
Wang et al., 1994), pork (McCormick et al., 1988; Collins et al., 1991b) and
turkey (Wang et al., 1992; Abouzied et al., 1993; Wang et al., 1993;
Desrocher, 1994). From previous studies in the Department Of Food Science
and Human Nutrition at Michigan State University, LDH has been identified
as a possible indicator of proper heat processing Of ground beef patties
(Wang et al., 1994). However, a comparison between thermal inactivation
rates of LDH and those Of pathogenic microorganisms such as E. coli
0157:H7 and Salmonella senftenberg had never been done before.
1.4. Immunoassays in Food Science

In recent years, immunoassays have been replacing standard methods
Of food analysis due tO their sensitivity, specificity and ease Of application.
Immunoassays are now being developed to detect adulterants,
microorganisms and their toxins, pesticides, and other contaminants in foods
(Pestka, 1988; Martin et al., 1988; Sarnarajeewa et al., 1991). One great
advantage of immunoassays is that they can be optimized into a kit tO reduce
time and instrumentation, so they can be used in the field by non-trained
personnel and can be applied to a large number of samples. The two most
common immunoassays in the food area are ELISA and radioirnmunoassay
(RIA). ELISA, however, is more popular because Of ease and safety of
application.

An ELISA is based on the interaction between an antigen and an
antibody. Antibodies (ABS) are proteins secreted by specialized B-

lymphocytes, called plasma cells, during an immune response, that bind

 

 

 

 

21

specifically tO a foreign molecule or antigen (AG). ELISAs can be
categorized in three groups: the AB capture assay, the AG capture assay, and
the two-AB sandwich assay. In an AB capture assay the AG is immobilized
on a solid support and quantitation is done by measuring the amount Of
labeled AB that binds immobilized AG. In an AG capture assay the same
principle applies except that the AB is immobilized and free AG is added to
bind the AB. In a sandwich assay, one AB is immobilized on the solid
support and free AG is added. Quantitation is done by measuring the amount
Of a second labeled AB that binds the bound AG (Harlow and Lane, 1988).
When choosing an ELISA two factors should be considered: types of ABs
that are available, and purity of AG.

1.4.1. Types of Antibodies
As mentioned before, ABS or immunoglobulins (Ig) are proteins

synthesized in response to the presence Of a foreign substance. Each AB
consists Of at least four subunits, two identical heavy chains and two identical
light chains linked to form a Y shaped molecule. ABS can be categorized in
five groups, IgG, IgM, IgA, lgE and IgD, based on their biological activity
and type Of heavy chain (Burke, 1993).

Properties Of an AB are affinity, avidity, titer and specifity. Affinity
refers to the precision Of fit between AB and the antigenic determinant. The
affmity constant for an AB can range from 107 to 109 M", reaching values of
1012 M'1 in some cases. Since an AB has more than one binding site, avidity
refers to the likelihood Of a second antigenic determinant binding the AB after
the binding of a first site. This term reflects the strengh and stability of the
AG-AB complex. The titer is an arbitrary value defined as the dilution

showing an optical density that is at least twice the value Of the non-immune

22

serum at the same dilution. Finally, the specificity is the AB ability to
recognize more than one molecule.
According tO the production method two types Of ABS can be made:

polyclonal and monoclonal.

1) Polyclon_al ABS (PABS)

PABS are produced when different lymphocyte clones respond to the
same AG. The resulting immune serum will show a broad range Of ABS
directed against different antigenic determinants. In short, production Of
PABS requires immunization Of a suitable animal (usually rabbit) with the
desired AG, periodical reinjections tO induce immune response and collection
of immune serum.

Advantages Of using PABS are: versatility, stability in frozen or
lyophilized conditions, and lower production cost than monoclonals. They
are quicker to Obtain and can usually be used at higher dilutions (Peters and
Baumgarten, 1992). Disadvantages include limited supply, and the fact that
extremely pure AG is required to elicit high affinity PABS.

2) Monoclonal ABS (MS)
Each AB-producing cell has the ability to produce a specific AB

against one particular site or epitope Of an AG. ABS resulting from a single
cell or clone are homogeneous and called MABS. The method to produce
MABS involves immunization Of a suitable animal (usually mouse) and
periodic reinjections to elicit an immune response. Due to the limited life
span Of the AB-producing cells, it is necessary to find a way to "immortalize"
such cells. According to Galfre and Milstein (1981), if B-lymphocytes are
induced to fuse with a tumorous lymphoid cell (myeloma), the resulting

23

hybridoma will grow indefinitely in culture while secreting ABS. Hybridomas
can then be separated and cloned and different cell lines can be Obtained,
each one producing a different MAB against a specific epitope of the selected
AG. The MABS can be recovered and purified from the tissue culture
supernatant. Another way to produce MABS is by inducing tumor production
in the intraperitoneal cavity. MABS can be recovered from the ascites fluid in
higher concentration than culture supernatant.

The advantages Of using MABS over PABS are their specificity and
selectivity for individual antigenic determinants, and unlimited supply. They
dO not require highly purified AG to elicit a response and can also be used to
study functional domains in a molecule. The main disadvantages are: longer
time and higher cost of production, higher physicochemical variability and
biological activity than PABS and possibility Of cross-reactions (Peters and
Baumgarten, 1992). In addition, mutations can reduce AB production and

cell lines can be lost due to fungal contamination.

1.4.2. Antigens
For an AG to elicit an immune response it has to have a molecular

weight Of at least 5000 Daltons (Burke, 1993). In this case, the small AG is
called a hapten and it has to be conjugated to a large molecule such as bovine
serum albtmrin, to induce AB production.

The antigenic determinant Of a native protein must be at the surface of
the molecule to be able to be bound by the AB. Studies of antigenicity Of
native proteins suggest that many antigenic states depend on the secondary,
tertiary or even quaternary structure of the molecule, Since most Of the time
they are formed by discontinuous fragments Of the pOlipeptide chain and
brought together by folding (Burke, 1993). The same studies Show that

24

native proteins present only a few potential antigenic determinants, but short
peptide sequences derived from the native protein can be used to induce ABS

against that protein.

2. COMPARISON OF THREE MEDIA FOR ENUMERATION
OF Escherichia coli 0157 :H7

2.1. ABSTRACT

Three different media (3M PetrifilmTM Coliform Plate Counts, 3M
PetrifilmTM E. coli Plate Counts, and McConkey-Plate Count Agar, 50:50)
were compared for their ability to enumerate E. coli 0157:H7. Both, regular
and heat-Shocked cultures Of six strains Of E.coli 0157:H7 in Tryptic Soy
Broth were used in this study. NO significant differences in mean log
CFU/mL were found among the three media for both regular and heat-
shocked cultures. These findings indicated that the use Of the more
convenient dry plating media could be used instead Of conventional pre-

poured agar plates in thermal inactivation studies.

2.2. INTRODUCTION

Use of PetrifilrnTM Aerobic, Coliform and E. coli Plate Counts are well
recognized and convenient alternatives to standard pour plating methods for
enumerating specific bacterial cells. In a project tO evaluate the heat
resistance Of E.coli 0157:H7, we proposed to use PetrifilrnTM Coliform Plate
Counts tO enumerate cells. Since we were using pure cultures Of E. call
0157 :H7 to inoculate aseptically collected beef, the Coliform and E. coli Plate
Counts should yield the same result in estimating inoculum population and
surviving cells.

While it is recognized that some plating media such as Phenol red
sorbitol agar containing 1% pyruvate, appear to be superior for the recovery
of heat injured E.coli 0157:H7 cells (Ahmed and Conner, 1993), the main

25

26

Objective in this study was to compare a conventional fresh agar pour-plating
system employing nutrient media known to support growth Of E. coli 0157:H7
strains, with dry PetrifihnTM plates for use in heat resistance studies.

2.3. MATERIALS AND METHODS

2.3.1. Media

3M PetrifilmTM Coliform Count Plates and E. coli Count Plates were
purchased from 3M (St. Paul, MN). McConkey-Plate Count Agar (MC-
PCA), 50:50, was made by resuspending separately McConkey agar powder
(Becton Dickinson) and Plate Count agar powder (Difco) in distilled
deionized water following manufacturer instructions and mixing both in equal
amounts. The mixture was sterilized at 121°C for 15 min and “about 12 mL
were dispensed on each sterile Petri dishes (Baxter Health Care Corporation,
Mcgaw Park, IL). After dispensing 1 mL of each dilution, samples on MC-
PCA were covered with a layer Of MC-PCA.

2.3.2. Cultures

E.coli 0157:H7 (ATCC 43894) and strains 932, 204P, SOSB, CA-l
and E0019, provided by Dr. L. Beuchat, University Of Georgia, were
maintained by daily transfer in Tryptic soy broth (Difco Laboratories, Detroit,
MI ). The day before each trial one loopful Of each strain was transferred to
each Of two 16x150 mm culture tubes containing 10 mL Of Tryptic soy broth.
On the day Of the trial one of the tubes was submerged in a Polystat circulator
bath (Model 1268-52, Cole-Partner Instrument Company, Chicago, 11)
connected to a bath programmer (Model 1268-62, Cole-Farmer) and set at

584°C. The temperature in the tube was monitored using a thermocouple

27

inserted in the center of a control culture tube containing 10 mL of
uninoculated Tryptic soy broth. After 20 min at 58°C the inoculated tube was
removed and immediately placed in an ice bath until plated within 2 h. This
time/temperature treatment was chosen to give approximately a 50%
reduction in cell viability.

Both regular and heat-shocked cultures were plated in triplicate on
each of the three media. Bacterial counts were determined by decimal
dilutions in sterile 0.1% buffered peptone water (Benton Dickinson
Microbiological Systems, Cockeysville, MD). All plates were incubated at
35°C for 24 hr. Each study was performed in triplicate.

2.3.3. Statistical Analysis

Mean log CFU/mL comparisons were performed using Student-
Neuman- Keul's Multiple range test Of the MSTAT statistical program
(MSTAT, 1994) with the mean square error term at the 1% level of
probability.

2.4. RESULTS AND CONCLUSIONS

Cormts for regular E. coli 0157:H7 culture achieved 108 CFU/mL.
Counts for equivalent heat-shocked culture achieved 104 CFU/mL. When
comparing means for the three media, no significant differences were found at
the 1% level Of probability (Table 2.1) for both regular and heat-shocked
cultures. Either dry plating system was at least equal to the use Of the poured
agar overlayed system.

The use Of Petrifilrn Count Plates has two advantages over the use Of

MC-PCA: (1) no media preparation is needed, and (2) enumeration is easier

28

and more accurate. On the other hand, any Of the three media can be used for
identification of E.coli 0157:H7. Since Coliform Count Plates are much
cheaper than E. coli Count Plates the authors suggest that Coliform Count
Plates is the most convenient media for recovery of E. coli 0157:H7 among

the three tested.

Table 2.1. Statistical comparison among mean log CFU/mL for both regular
and heat-shocked cultures Of E.coli 0157:H7 across media.

 

 

 

 

 

 

 

 

 

 

Media / Culture Mean Log CFU/mL
Petrifilm Coliform / Regular 890“
Petrifilm E.coli / Regular ' 8.89“
MC-PCA / Regplar 8.82a
Petrifilm Coliform IHeat-Shocked 4.69b
Petrifilm E. coli/ Heat-Shocked 4.70b
MC-PCA / Heat-shocked 4.62b

 

Values with different superscript are statistically different (p < 0.01)

3. THERMAL INACTIVATION OF Escherichia coli 0157 :H7 AS
COMPARED TO THAT OF LACTATE DEHYDROGENASE
AND Salmonella senftenberg

3.1. ABSTRACT

Thermal inactivation Of Escherichia call 0157 :H7 (E. coli 0157:H7) in
ground beef at 53°, 58°, 63° and 68°C was compared to that of the enzyme
lactate dehydrogenase (LDH) and Of a more heat resistant microorganism,
Salmonella senfienberg (S.senfienberg). Inoculated and non inoculated
ground beef samples were placed in sterile thermal death time tubes and
immersed in a circulating bath then held at the above temperatures for
predetermined intervals of time. Bacterial counts were determined using
PetrifilmTM Coliform Count Plates and LDH was assayed for enzymatic
activity. The D values (in minutes) for E. coli 0157:H7 and S.senfienberg
were 46.1 and 53 at 53°; 6.44 and 15.17 at 58°; 0.43 and 2.08 at 63°; and
0.12 and 0.22 at 68°C; with 2 values of 560° and 624°C, respectively.
Inactivation rates or "D values" for LDH were 543.48, 19.61, and 1.40 min at
5 8°, 63° and 68°C, respectively, with a "2 value" Of 386°C. The D value for
LDH at 53°C could not be determined. The higher D values and lower 2
value for LDH as compared to either microorganism indicated that the
enzyme is less sensitive to heat inactivation than cells of either culture.

Indeed, S.senftenberg proved to be more heat stable than E. coli 0157:H7.
3.2. INTRODUCTION

Studies comparing thermal inactivation rates Of pathogenic
microorganisms to enzymatic inactivation rates have been done previously,

mostly in dairy products. Alkaline phosphatase has been used as an indicator

29

30

Of proper pasteurization Of milk based on its parallel inactivation to the
pathogen Mycobacterium tuberculosis (Kay and Graham, 1933). In addition,
Eckner (1992) demonstrated the relationship between alkaline phosphatase
inactivation and Listeria and Salmonella destruction in milk. More recently,
Patel and Wilbey (1994) compared the thermal inactivation Of the enzyme 7-
glutamyltranspeptidase to that Of Enlerococcusfaecium in different rnilk-
based systems and concluded that this enzyme could be used as an indicator
Of proper heat treatments more severe than pasteurization for milk products.

Because Of recent outbreaks of E. coli 0157:H7 due to insufficient
cooking of ground beef patties and because current US Department of
Agriculture (USDA) methodology is considered unreliable, there has been
increased interest in finding more accurate methods to assure safety Of
cooked meat products. Several enzymes have been suggested as indicators of
proper heat processing in meat products. Among them, the enzyme lactate
dehydrogenase (LDH) has been thoroughly studied as a potential end point
temperature indicator in pork (Collins et al., 1991b), turkey (Wang et al.,
1992; Abouzied et al., 1993; Wang et al., 1993; Desrocher, 1994) and beef
(Collins et al., 19913; Stadler et al., 1991; Wang et al., 1994). However, no
relationship between inactivation rates Of LDH and those Of pathogenic
microorganisms have yet been determined.

The Objective of this study was to determine the D and 2 values of
E. coli 0157:H7, Salmonella senftenberg and LDH in ground beef at four
different temperatures and to compare their respective thermal inactivation

rates.

31

3.3. MATERIALS AND METHODS

3.3.1. Bacterial cultures

Cultures of Escherichia coli 0157:H7 (ATCC 43894) and Salmonella
senfienberg (ATCC 43 845) were maintained by daily transfer in tryptic soy
broth (Difco Laboratories, Detroit, MI). Each inoculum was prepared from a
24 hr culture containing approximately 109 microorganisms/mL. On the day
Of the experiment each culture was sedimented by centrifuging at 4340 x g for
20 min at 4°C (Sorvall Superspeed RC2-B, Ivan Sorvall Inc, Norwallg CT)
and resuspended to its original volume using sterile 0.1 % buffered peptone
water (Becton Dickinson Microbiology Systems, Cockeysville, MD). The
inoculums were enumerated by serial dilution in sterile 0.1 % buffered
peptone water (Becton Dickinson Microbiology Systems) and plating on
Petrifilmm Coliform Count Plates (3M, St. Paul, MN).

3.3.2. Preparation of meat

Three eyes of the round (semitendinosus muscle), vacuum packaged
and less than six days old, were purchased from a local store. After trimming
fat and connective tissue, each piece was immersed in boiling water for 10
sec to reduce surface contamination. Using aseptic technique, the outer
surface Of meat was removed by cutting with a series Of sterilized knives and
discarded. Under a sterilized grinder and in a aseptic environment, the meat
was ground twice through a 3.175 mm diameter grinder plate in a Hobart
grinder (Model 84181D, Hobart Mfg. CO., Troy, OH) and then divided into
three portions of 200 g each. One portion was inoculated with E. coli
0157:H7 and one with S.senftenberg 24 hr cultures to achieve about 107

microorganisms/g meat. The cultures were added dropwise to the ground

32

meat and distributed thoroughly, kneading the mixture by hand, using sterile
latex gloves, for 5 min. The third portion was not inoculated and used for the

LDH studies.

3.3.3. Thermal inactivation

From each portion Of ground meat, one-gram samples were filled in
sterile 10 x 75 mm thermal death time (TDT) tubes using modified 60 cc
syringes. The samples were placed at the bottom Of each tube and the tubes
were sealed using a gas/oxygen flame. The number Of tubes varied according
to the time-temperature schedules (Table 3.1), based on preliminary results.

The tubes (one set Of each E. coli 0157:H7, S.senftenberg and LDH at
a time) were placed in a wire rack and immersed in a Polystat circulator bath
(Model 1268-52, Cole-Farmer Instrument Company, Chicago, 111.) connected
to a bath programmer (Model 1268-62, Cole-Partner). The temperature was
set 05°C above the target temperature and monitored using a thermocouple
inserted in the center of a control TDT tube containing 1 g of ground meat.
The thermocouple was connected tO a Solomat MPM 200 Modumeter
(Solomat Partners LP, Glenbrook Industrial Park, Stamford, CT). Time and
temperature were recorded using a Solomat MPM Logger (Solomat Partners
LP) connected to the modumeter. For 53° and 5 8°C trials, zero time was
when the internal temperature reached target temperature (less than 5 min).
For 63° and 68°C, zero time was when the intemal temperature reached 58°C
(less than 5 min).

Three tubes were removed at each time interval and immediately
placed in an ice bath. Tubes containing inoculated samples were kept at 4°C

and plated within 12 hr. Tubes containing uninoculated samples were kept at

33

Table 3.1. Time and temperature schedules for thermal inactivation of E. coli
0157:H7, S.senfienberg and LDH

 

 

 

 

 

 

T E.coli 0157:H7 S.senflenberg LDH
(°C)

53 0, 0.5, 1, 1.5, 2, 3, 4, 0, 0.5, 1, 1.5, 2, 3, 4, 0, 0.5, 1, 1.5, 2, 3, 4,
5,and6h 5,and6h 5,and6h

58 0, 2, 4, 8, 10, 15, 20, 0, 10, 20, 30, 40, 50, 0, 2, 4, 8, 10, 15, 20,
30, 35, and 45 min 60, 70, 80, and 90 30, 35, and 45 min

min '

63 0, 60, 75, 90, 100, 0, 60, 120, 150, 180, 0, 60, 75, 90, 100,
110, 120, 130, 140, 240, 270, 300, 360, 110, 120, 130, 140,
150, 160, 170, 180, 420, 480, and 600 150, 160, 170, 180,
210, and 240 sec sec 210, and 240 sec

68 0, 5, 10, 15, 20, 25, 0, 5, 10, 15, 20, 25, 0, 5, 10, 15, 20, 25,

 

30, 40, 50, and 60
sec

 

30, 40, 50, 60, 70,
80, and 90 sec

 

30, 40, 50, 60, 70,
80, and 90 sec

34

-20°C until analyzed within 8 weeks. Each thermal inactivation study was
performed in triplicate.

3.3.4. Bacterial counts

E. coli 0157:H7 and S.senflenberg counts were determined by decimal
dilution of the ground beef samples in sterile 0.1 % buffered peptone water.
The samples were homogenized using a Stomacher lab blender (Model 400,
Tekmar Company, Cincinnatti, OH) for 2 min and plated in duplicate on
PetrifilrnTM Coliform Count Plates (3M). Samples Of uninoculated ground
Meat were plated on both Petrifilmm‘ Aerobic Count Plates and Coliform
Count Plates for determination of possible initial contamination. All samples
were incubated at 37°C for 24 hr.

3.3.5. Extraction of LDH from ground beef ‘

After breaking the sealed TDT tubes, each sample was transferred to a
scintillation vial. To each sample, 2 mL Of cold phosphate buffer saline
(PBS, 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.2) were added
and the sample was mixed for 1 min using a vortex mixer. The mixture was
further stirred for 15 min at 4°C and then centrifuged at 4500 x g for 5 min
(Micro-centrifuge, Model 59A, Fischer Scientific, Itasca, IL). The
supematants were collected and kept at 4°C until analyzed within 4 hr.

3.3.6. Determination of enzymatic activity
The enzymatic activity Of LDH at 25°C was determined using a LDH
diagnostic kit (DG 1340-K, Sigma, St Louis, MO). The determination of

35

LDH activity is based on the reduction Of pyruvate to lactate and
simultaneous oxidation Of NADH to NAD which results in a decrease in the
absorbance at 340 nm. One unit (U) of LDH activity is expressed as the
amount of enzyme which catalyzes the formation Of lumol Of NAD/L per

minute under specified conditions.

3.3.7. Proximate analysis and determination of pH

Moisture and fat composition Of the ground beef were determined
using AOAC (1990) methods 950.46B and 960.39, respectively. For
determination Of the pH, 50 g ground beef were homogenized with 50 mL Of
distilled water in a Waring blender for 30 sec. The pH Of the homogenate
was measured using a Corning 145 pH meter (Fisher Scientific). Results are

the average value Of triplicate analysis Of the same sample.

3.3.8. D and 2 values ’

Plate counts (CF U/mL) and enzymatic activity (U/g) data were
converted to logarithms. Plate counts less than 10 were entered as zeros.
Linear regression analyses were conducted using LOTUS 1-2-3 for Windows,
Version 1.0 (Lotus Development Corporation, Cambridge, MA). In 53° and
5 8°C experiments all data points were used tO calculate regression analyses.
Since time zero was considered when internal temperature Of the samples
reached 58°C for the 63°C and 68°C experiments, values from the first 150
sec and 20 sec, respectively, were excluded from the regression analyses to
avoid the lag phase of the curve. D values (in minutes) were calculated
according to the Laboratory Manual for Food Canners and Processors (1968)
from the regression curves at each temperature. TDT phantom curves were

determined by plotting log of D values vs temperature. The 2 values were

36

determined as the absolute value Of the reciprocal Of the slope of the TDT

curve .

3.4. RESULTS AND DISCUSSION

3.4.1. Raw ground beef

Proximate analysis and pH Of the three semitendinosus muscles used in
this study are shown in Table 3.2. Percent Of moisture ranged from 70.8 to
74.7. Fat percent ranged from 1.7 to 5.8. The pH Of the samples ranged from
6.2 to 6.3.

Samples Of raw ground beef were plated tO determine bacterial load
after aseptic meat collection. NO E. coli or E. coll-like microorganisms were
detected. One sample showed total aerobic counts of approximately 102
CFU/g meat. The other two samples had less than 10 CFU/g meat. Analysis
Of LDH enzymatic activity in raw samples revealed mean initial values Of 886

U/g meat.

3.4.2. D and z values

Thermal inactivation or survivor curves for E. coli 0157:H7 and
S.senftenberg at 53°C are shown in Figure 3.1. D53 values for E.coli 0157:H7
and S.senftenberg were 46.1 and 53 min, respectively. Thermal inactivation
curve for LDH at 53°C is shown in Figure 3.2. There was no perceptible
change in enzymatic activity after 6 h. and the D value was not calculated.

Survivor curves for E.coli 0157:H7 and S.senftenberg at 5 8°C are
shown in Figure 3.3. D,8 values were 6.45 and 15.2 min for E.coli 0157:H7
and S.senftenberg, respectively. LDH thermal inactivation curve at 58°C is

shown in Figure 3.4. Inactivation rate or D58 value was 544 min. After 45

 

37

Table 3.2. Results Of proximate analysis Of raw ground beef samples”.

 

 

 

Sample # Moisture (%) Fat (%) pH
1 70.8 1.7 6.3
2 73.6 2.5 6.2
3 74.7 5.8 6.3
__A
Mean 73.0i2.01 3,312.17 63:0.06

 

 

 

 

 

 

 

a Values expressed as mean i SD.

 

 

38

 

+E.coli 0157:H7
' *S.sefntenbe

..(..r.r....vz—\... ,.,.,,,_ .1r~.... ..lfiTI
...........................................

 

 

 

 

 

 

 

Time (hours)

Figure 3.1. Survivor curves Of E.coli 0157:H7 and S.senftenberg in ground
beef at 53°C.

LDH Activity (U/g meat)

 

800

600 .. .. .. .

 

 

if\

400

I

 

 

 

Time (hours)

Figure 3.2. Effect of heating on LDH in ground beef at 53°C.

39

7 Log CFUs

 

+E.coli 0157:H7
. {S.sefntenberg

.‘.‘.‘.’.'.‘.‘.' ..................................

 

 

 

 

 

 

 

 

 

 

100

 

Time (minutes)

Figure 3.3. Survivor curves Of E.coli 0157 :H7 and S.senflenberg in ground
beef at 58°C.

LDH Activity (U/g meat)

 

1 000

 

 

 

 

0 l l 1 1 l l l
I l 1 I f 1 1

1f
0 5 10 15 20 25 30 35 40 45

Time (min)

Figure 3.4. Effect Of heating on LDH in ground beef at 58°C.

40

min at 58°C LDH enzymatic activity was reduced by 14% when compared to
the raw samples.

Survivor curves for E. coli 0157:H7 and S.senfienberg at 63°C are
Shown in Figure 3.5. D63 values were 0.43 min for E.coli 0157:H7 and 2.08
min for S.senftenberg, respectively. The thermal inactivation curve for LDH
at 63°C is shown in Figure 3.6. The D63 value was 19.6 min. The enzymatic
activity after 4 min at 63 °C had been reduced to a 40% of the initial activity.

Survivor curves for E.coli O1572H7 and S.senftenberg at 68°C are
shown in Figure 3.7 . At this temperature, D values were 0.12 and 0.22 min
for E. coli 0157:H7 and S.senftenberg, respectively. Thermal inactivation
curve for LDH at 68°C is shown in Figure 3.8. The D68 value was 1.40 min.
LDH activity after 90 sec at 68°C was reduced by 89 % Of the initial.

Summary Of D values and regression analysis at all temperatures for
E.coli 0157:H7, S.senftenberg and LDH are listed in Tables 3.3, 3.4 and
3.5, respectively. Summary of 2 values and regression analysis for E. coli
301571H7, S.senftenberg and LDH are listed in Table 3.6. The lowest 2
value, 386°C, corresponded to LDH. E.coli 0157 :H7 and S.senflenberg
showed 2 values of 560° and 6.24°C.,respectively. Phamtom thermal death
time curves for E. coli 0157:H7, S.senftenberg and LDH were Obtained by
plotting temperature vs. log D values (Figure 3.9).

Doyle and Schoeni (1984) and Line et al. ( 1991) reported D and 2
values for E. coli 0157:H7 in ground beef over a Similar range of
temperatures as in our study (Table 3.7). Doyle and Schoeni determined D
values at 544°, 572° and 628°C Of 39.8, 4.5 and 0.4 min, respectively, with
a 2 value of 4.1°C. Line et al. (1991) reported D values at 51.7°, 572° and
628°C based on two methods Of recovery. Their results were 78.2 and 80.1,
4.1 and 4.0, and 0.30 and 0.22 min, with 2 values Of4.61° and 433°C,

 

 

41

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8 Log CFUs
+E.coliO157:H7
7 -. - *S.sefntenbergw
5
4
o r r- r a i r r i
0 100 200 300 400 500 600 700 800 900
Trme(sec)

Figure 3.5. Survivor curves Of E.coli 0157:H7 and S.senfienberg in ground
beef at 63°C.

LDH Activity (U/g meat)

 

 

 

 

 

400 ~-
200 ~—
0 i 1 i i i l r
O 30 60 90 120 150 180 210 240
Time (see)

Figure 3.6. Thermal inactivation of LDH in ground beef at 63°C.

42

 

8 Log CFUs
+E.coli 0157:H7 g;

 

 

 

 

...............................................

 

 

 

 

 

 

Time (sec)

Figure 3.7. Survivor curves of E.coli 0157 :H7 and S.senftenberg in ground
beef at 68°C.

LDH Activity (U/g meat)

 

800

 

 

 

 

 

Time (see)

Figure 3.8. Thermal inactivation Of LDH in ground beef at 68°C.

 

43

Table 3.3. Summary Of D values and regression analysis for E.coli 0157 :H7

in ground beef.

D value
(min)

Constant

R squared

X coefficient

 

46.10

 

6.44

 

0.43

 

 

0.12

 

 

 

 

Table 3.4. Summary Of D values and regression analysis for S.senftenberg in

ground beef.

D value
(min)

Constant

R squared

X coefficient

 

53

 

 

 

 

 

 

 

 

 

Table 3.5. Summary of D values and regression analysis for LDH in ground

 

 

 

 

beef.
T D value Constant R squared X coefficient
(°C) (min)
58 543.48 2.88 0.55 -0.002
63 19.61 2.97 0.60 -0.001
68 1.40 3.15 0.90 -0.013

 

 

 

Table 3.6. Summary Of 2 values and regression analysis for E.coli 0157:H7,
S.senfienberg and LDH in ground beef.

2 value

EC)

Constant

R squared

X
coefficient

 

E.coli 0157 :H7

5.60

10.77

0.98

-0.17

 

S. senflenberg

6.24

10.32

0.99

-0.16

 

LDH

 

 

17.22

 

 

-0.25

 

 

 

45

Table 3.7 . Comparison Of D and 2 values for E.coli 0157:H7 and
S.senftenberg in ground beef.

E. coli 01 57 :H7 S. senflenberg

 

Doyle and Line et al. Goodfellow and
Schoeni (1984) (1991) Brown (1978)

 

T D value D value D value

(°C) (min) (min) Quin)

 

54.4 39.8 . 78.2“- 80.11) . 61°- 62d

 

57.2 . . 4.13- 4.0b . 3.8°- 4.2d

 

62.8 . . 0.30“- 0.22b . 0.6°- 07°

.1 4.1-...»
°C °C

 

 

 

 

 

 

" PCA

b 2-h indole test

° Plate Count Agar overlayed with XL Agar base
d Most probable number technique

46

 

5 Log D values
-°-E.coli 0157:H7
~ +S.senftenberg

 

 

 

...............................................

 

 

 

 

 

 

 

50 55 60 65 70
Temperature ('C)

Figure 3.9. Thermal death time curves of E. coli 0157:H7, S.senfienberg and
LDH in ground beef.

 

 

47

respectively for each method. When compared to our results, Doyle and
Schoeni Obtained lower D values at all three temperatures. Line and co-
workers Obtained greater D values at 53° and 63 °C and lower at 58°C.
Neither one calculated D values above 65°C. In both studies the 2 value was
1-1.5°C lower than ours.

Discrepancies among results could be due to the use Of different
recovery methods. Doyle and Schoeni used Tryptic Soy agar capped with
McConkey agar, and Line and co-workers used two different methods: Plate
Count agar containing 1% pyruvate and 2-h indole test. In addition, while
Line and co-workers used ground beef with a fat content (2%) Similar to ours,
Doyle and Schoeni used a much greater fat content (l7-20%).

In a similar manner, Goodfellow and Brown (197 8) calculated D
values for Salmonella spp in ground beef (Table 3.7). The D values
determined at 516°, 572° and 627°C were 61-62, 3.8-42, and 06-07 min,
respectively. The 2 value was found to be 18°C. When compared to our
results, our D53 was smaller while our D,8 and D63 were greater than theirs.
Again, discrepancies in the results could be due to use Of different
enumeration methods. Goodfellow and Brown used two different methods:
for the first one they used Plate Count agar overlayed with XL Agar base
supplemented with sodium thiosulfate and ferric ammonium sulfate, and for
the second method they used the most probable number technique according
to the Bacteriological Analytical Manual Of the FDA. The authors did not
mention the fat content of their meat.

Studies Of LDH heat inactivation have been reported in bovine, porcine
and poultry tissues. Collins et al. (19913) Observed a decrease in LDH
activity in samples of beef top round (semirnembranosus and abductor

muscles) that had been heated tO 545°C with a holding time Of 121 min or to

48

60°, 628° or 656°C without a holding time. Enzymatic activity decreased by
26.6, 33.7, 77.1 and 96.5%, respectively. Similar trends were reported by the
same authors in porcine muscle tissues (Collins et al.,1991b). Decreasing
LDH activity with increasing temperature was Observed when whole muscle
hams were heated to 65°, 68° or 71°C. Samples heated tO 65° and 68°C
showed a significant reduction in LDH activity when compared to unheated
samples, and almost undetectable levels were Observed in samples heated to
71 °C. Stalder et al. (1991) Observed a reduction in LDH activity when
extracts of bovine semirnembranosus muscle were heated to 57°, 60°, 63°
and 66°C. There was a large decrease in activity when internal temperature
of samples reached 63 °C and only marginal activity was detected at 66°C. In
poultry, Wang et a1. (1994) measured LDH activity in turkey breast rolls
cooked to 683°, 697°, 709° and 721°C. Activity decreased from
approximately 735 U/g meat in unheated samples to 22 U/g in samples heated
to 683°C. Negligible activity (< 2.5 U/g meat) was detected above this
temperature. Similarly, LDH activity in turkey thigh rolls decreased from 275
U/g meat to 44 U/g in samples processed to 722°C. Also, Desrocher (1994)
Observed a linear decrease in LDH activity in turkey hams with increasing
processing temperatures. Initial activity in uncooked hams was 210 U/g meat
and decreased to 39 U/g when cooked to 725°C.

While Collins and co-workers used semirnembranosus and abductor
muscles, Stalder and co-workers used semimembranosus muscle as the
source of meat. Semitendinosus muscle was used in our study. Differences
in LDH activity between bovine muscles have been described by Talrnant et
al. (1986) and Stalder et al. (1991). Muscles vary in their content of LDH
isozymes according to their content and distribution Of muscle fiber types.

Therefore, different muscles Show differences in LDH activity. Another

49

factor that can influence enzymatic activity of LDH is carcass maturity.
Stalder et al. (1991) Observed differences in LDH activity among A, BIG and
D/E maturity groups. LDH activity was greatest in A and the lowest in D/E.
Methodology employed in the experiment can also influence the results.
Stalder et al. (1991) performed the heat inactivation in muscle slurries
instead of whole muscle samples. Heating extracts results in higher
inactivation at lower temperatures since the enzyme is more exposed to the
heat stress. Collins and co-workers used whole muscle samples as we did,
but the sample Size as well as the TDT tubes, water bath setting and LDH
extraction method were different from ours, which can have an impact on the
results. In addition, after heating they stored the samples at 4°C for 3 weeks
prior to analysis. Such a long period, even at refiigeration temperatures, can
result in significant loss Of enzymatic activity. Finally, the general approach
in both Collins and Stalder studies was based on an endpoint temperature

while we attempted to cover a temperature range over time.

3.5. CONCLUSIONS

LDH has already been recognized as a potential indicator Of heat
processing in beef and poultry products. We attempted to confirm its
potential by comparing LDH thermal inactivation rate to those Of E. coli
0157:H7 and S.senftenberg using ground beef as a model. LDH was most
heat resistant, whereas E. coli 0157:H7 was the most heat sensitive. LDH
had a greater D value than both microorganisms but smaller 2 value, meaning
that the decrease in LDH enzymatic activity dming heating was more
pronounced than the reduction Of survivors for both E. coli 0157:H7 and

S.senftenberg. Therefore, careful consideration Of the time/temperature

50

relationship is of great importance when selecting an enzyme indicator to
monitor the adequacy Of previous heat treatments in meat and poultry
products. In our case, LDH seems to be more suitable to be used as indicator
of proper heat processing at the 65°-70°C range. At lower temperatures the
slopes Of the thermal death time curves Of LDH, E. coli 0157:H7 and
S.senftenberg are so different that any possible relationship is difficult to be
established.

4. PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST
BOVINE MUSCLE LACTATE DEHYDROGENASE AND
VERIFICATION OF ENDPOINT TEMPERATURE OF
GROUND BEEF PATTIES BY SANDWICH ENZYME-LINKED
IMMUNOSORBENT ASSAY

4.1. ABSTRACT

Monoclonal antibodies against bovine muscle LDH were produced
and a sandwich enzyme-linked irnmunosorbent assay (ELISA) was developed
using monoclonal and polyclonal antibodies. Ground beef patties containing
13.6% fat were cooked to internal temperatures Of 62.8°, 656°, 683° and
71 . 1°C. Additionally, ground beef patties containing either 10.7 or 19.0% fat
were cooked to an internal temperature Of 683°C. Extracts Of the ground
beef patties were tested using the sandwich ELISA to. detect differences in
LDH concentration. Significant differences were found between patties
cooked to 628°C and both uncooked patties and those cooked to 656°C. NO
differences were found among patties cooked to 656°, 683° and 71 .1°C.
Similarly, no significant differences in LDH content were found among

patties containing different fat levels and cooked to 683°C.
4.2. INTRODUCTION

E. coli 0157:H7 is an important cause of foodbome disease. Several
outbreaks have been reported in the USA, Canada, United Kingdom, Mexico,
China, Argentina and Belgium (Doyle, 1991). The severity of symptoms

including death in some cases, have made it a real concern for food

<1

52

processors, government regulatory agencies, and consumers. Most outbreaks
have been associated with consumption of undercooked ground beef
products.

Studies Of thermal inactivation in ground beef have Shown that E. coli
0157:H7 is heat sensitive (Doyle and Schoeni, 1984; Line et al., 1991).
Hence, one way to ensure its absence is to determine whether ground beef
has achieved the proper endpoint cooking temperature at the center Of the
product. The Food and Drug Administration (FDA) recommends cooking
ground beef patties by any Of the following time/temperature protocols: 63°C
(145°F)/3 min, 65°C (150°F)/1 min or 68°C (155°F)/15 sec (FDA, 1993). In
addition, the US Department Of Agriculture F OOd Safety Inspection Service
(U SDA-FSIS) requires Official establishments that manufacture fully-cooked
ground beef patties to cook patties using any Of seven time/temperature
schedules with holding time .

TO determine proper endpoint temperature of beef products, the USDA
currently employs a protein coagulation test (USDA, 1986), and a catalase
enzyme test (USDA, 1989). These methods, however, are considered
unreliable and do not provide enough margin of safety. There is a need for a
more accurate and rapid assay which could be used for routine verification Of
proper heat processing. Several authors have suggested using residual
enzyme activity as a way to detect the endpoint cooking temperature in beef
products (Townsend and Davis, 1990; Stalder et al., 1991; Collins et al.,
1991), but these assays are time consuming or require expensive equipment.
On the other hand, the use Of an enzyme indicator and further detection by
immunoassays has been used successfully for the determination Of endpoint
cooking temperature in poultry products (Wang et al., 1992; Abouzied et al.,
1993; Wang et al., 1993; Desrocher, 1994; Wang, 1994). The enzyme lactate

53

dehydrogenase (LDH) has already been Shown to be a potential indicator of
endpoint temperature of processing in beef (Collins et al., 1991; Stalder et al.,
1991; Wang et al., 1994) and turkey products (Wang et al., 1992; Abouzied
et al., 1993; Wang et al., 1993; Desrocher, 1994; Wang, 1994).

In recent studies we determined and compared the thermal inactivation
of LDH with those Of E. coli 0157:H7 and Salmonella senflenberg
(S.senftenberg) at four different temperatures (53°, 5 8°, 63° and 68°C). The
results showed that LDH inactivation rate is slightly lower than that for both
pathogens over the temperatures tested, confirming that this enzymecould be
used as an indicator Of proper thermal processing and, thus, ensure absence Of
pathogenic microorganisms in cooked ground beef products.

The purpose of this study was to develop a rapid and accurate assay to
verify proper heat processing Of ground beef patties. Monoclonal antibodies
against bovine muscle were produced and a sandwich enzyme-linked
immunosorbent assay (ELISA) was developed using the monoclonal
antibodies and polyclonal antibodies that had been previously produced
(Abouzied et al., 1994). The assay was used to detect changes in LDH
concentration in patties containing the same amount Of fat and cooked to four
different endpoint temperatures, or patties with different fat content but

cooked to the same internal temperature.

4.3. MATERIALS AND METHODS

4.3.1. Production of Monoclonal Antibodies

Two groups Of 6-8 week-Old female Balb\c mice (Charles River
Laboratories, Wilmington, MA) were injected either subcutaneously or
intraperitoneally (five mice each) with 75 pg Of bovine muscle LDH (Sigma

54

Chemical CO., St. Louis, MO) in saline (0.8%) and mixed (1:1) with Freund's
complete adjuvant (Sigma) for a total Of 0.2 mL/mouse. Two booster
injections were given at 2-week intervals as described above except that
incomplete Freund's adjuvant (Sigma) was used. One week after the last
injection, serum Obtained from the retrobulbar plexus Of each mice was tested
by indirect ELISA to determine antibody titer, sensitivity and specificity.
Three days before removal Of the spleen for fusion, an intraperitoneal
injection Of bovine muscle LDH in saline solution was given to those mice
with antisera showing the highest inhibition when tested by competitive
ELISA (Abouzied et a1, 1990).

Monoclonal antibodies were produced following the procedure Of
Galfre and Milstein (1981) as modified by Abouzied et al. (1990). Mouse
spleen cells (1x108) were fused with NS-l myeloma cells (1x107) (P3/N S
1/1-Ag4-1, ATCC TIB 18) using 50% polyethylene glycol (Sigma) as the
fusion agent. Following fusion, cells were suspended in Dulbecco's modified
medium (Sigma) containing 20% fetal bovine serum (FBS) (Gibco
Laboratories, Grand Island, NY), 1% NCTC medium (Gibco), 10 mM MEM
sodium pyruvate solution (Gibco), and penicillin/streptomycin solution (100
U/mL) (Gibco). The cell suspension was then distributed into Six 96-well flat
bottom tissue culture plates (Corning Laboratory Science CO., Corning, NY).
The plates were incubated at 37 °C in a humid atmosphere containing 8%
C02. After 24 h, half Of the supernatant was removed from each well and
replaced with an equal volume Of hypoxanthine, aminopterin and thynridine
(all three from Sigma) selective medium (HAT medium). Every three days
the cells were fed exactly as above. After 2 weeks HAT medium was
replaced by HT medium (same as HAT but without arrrinopterin). Those
wells showing cell growth and color change were tested for antibody

55

production by competitive ELISA. Hybridomas Showing continued
production of anti-LDH antibodies were expanded and cloned twice by
limiting dilution (Goding, 1980). Cloned cells were grown in HT medium
containing 15% FBS and 15% macrophage-conditioned medium (Abouzied et
a1, 1990; Sugasawara et al., 1985).

Supematants were collected and centrifuged to remove cells.
Antibodies were purified by precipitation with 50% ammonium sulfate
(Hebert et al., 1973), dialyzed for 3 days against 0.01M phosphate buffer
saline (PBS, pH 7.2) at 4°C, aliquoted, and frozen.

4.3.2. Indirect ELISA

The indirect ELISA (Abouzied et al., 1990) was performed by coating
microtiter plates (Immunolon-2 Removawells, Dynatech Laboratories Inc.,
Chantilly, VA) with 100 uL bovine LDH (10 ug/mL) in 0.1M carbonate
buffer, pH 9.6, following incubation overnight at 4°C. Plates were washed 4
times with PBS containing 0.05% Tween-20 (Sigma) (PBS-T). Next,
nonspecific binding sites were blocked by adding 300 uL of 1% ovoalbumin
(Sigma) in PBS (OA-PBS) to each well and incubating at 37°C for 30 min.
After washing 4 times with PBS-T, 50 uL Of serially diluted serum were
added to each well and incubated at 37°C for 1 h. After incubation, excess
antibodies were removed by washing 4 times with PBS-T. To each well 100
uL Of 1:500 goat-antimouse IgG peroxidase conjugate (Cappel Laboratories,
West Chester, PA) in OA-PBS were added and incubated at 37°C for 30 min.
After washing 8 times, peroxidase binding was determined by using 2, 2'-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma) (ABTS)-H202
substrate as described by Pestka et al. (1982). Absorbance at 405 nm was
read using a Vmax Kinetic Microplate Reader (Model 1234, Molecular

 

 

56

Devices Corporation, Menlo Park, CA). The titer Of each serum was
arbitrarily determined as the dilution showing an optical density reading that
was at least twice the reading Of the non-immrme serum at the same dilution.

The competitive indirect ELISA was identical as above except that
after blocking of non-specific binding Sites and washing, 50 uL Of standard
LDH were added to each well with 50 uL of the LDH antisera or 50 uL Of
cell culture supernatant.

4.3.3. Sandwich ELISA

The sandwich ELISA was performed by coating microtiter plates with
100 uL of monoclonal LDH antibodies in 0.1 M carbonate buffer, pH 9.6,
(1:250) and drying overnight at 40°C in a forced air oven. After washing 4
times with PBS-T, nonspecific binding Sites were blocked by adding 300 uL
Of 2% OA-PBS to each well and incubating at 37 °C for 30 min. Plates were
washed 4 times and 50 uL of serially diluted bovine muscle LDH or ground
beef patties extracts were added tO each well and incubated at 37°C for 30
min. After washing 4 more times, 50 uL/well Of polyclonal antibody in 2%
OA-PBS (1 :500) were added. Following incubation for 30 min at 37°C and
washing, 100 uL of 1:500 goat-antimouse IgG peroxidase conjugate in 2%
OA-PBS were added to each well and incubated at 37°C for 30 min. After
washing 8 times, bound peroxidase was determined by adding 100 uL K-Blue
substrate (ELISA Technologies, Lexington, KY) to each well. Color
development was stopped by addition Of 100 uL/well Of the Stopping reagent
(ELISA Technologies). Absorbance was read at 650 nm.

57

4.3.4. Preparation of ground beef patties

Three eyes Of the round (semitendinosus muscle) were trimmed,
combined and used to prepare all patties. Muscles were ground twice
through a 3.175 mm grinder plate in a Hobart grinder (Model 84181D, Hobart
Mfg. CO., Troy, OH) and a sample was taken to analyze for fat content. The
ground meat was separated into three batches. Ground beef kidney fat was
added to achieve different levels Of fat (10, 15 and 20%). Lean and fat were
mixed in a chopper (Hobart) and reground twice through a 3.175 mm grinder
plate. The ground meat was made into 113 g , 1.5 cm-thick patties using a
burger press (Robinson Company Inc., Foley, Alabama). The patties were
vacuum packaged and stored at 4°C until processed, within 5 days.

4.3.5. Cooking of ground beef patties

Patties were cooked using an electric frying pan (F aberware, Yonkers,
NY) set at 190.6°C. Each patty was placed on the center of the pan and
cooked on the first side for 3, 4 or 5 min, according to the internal
temperature to be achieved. Patties were pressed for 1 min intervals with a
Spatula held in place with a 500 g weight. The patties were then turned and
the temperature in the center Of the patty was monitored using a template with
a thermocouple connected to a control box (Model 660, Omega Engineering,
Inc.) When the target temperature was reached the patties were immediately
placed in an ice water bath. Total cooking time was recorded for each patty.
Each experiment was repeated three times for each temperature and fat level.

Three patties were cooked for each replicate for a total Of 9 patties.

58

4.3.6. LDH extraction

Five grams from the core of each patty in a replicate were combined
and extracted with 45 mL Of cold phosphate buffer saline (PBS, 0.15 M
NaCl, 0.01 M sodium phosphate buffer, pH 7.2) using a Tissue TearorTM at
maximum speed for 1 min. The homogenate was centrifuged at 4340 x g for
20 min (Sorvall Superspeed RC2-B, Ivan Sorvall Inc, Norwalk, CT) and
filtered through NO. 1 Whatrnan paper. The filtrate was collected and stored
at 4°C tmtil analyzed, within 8 h.

4.3.7. Proximate analysis and determination of pH

Moisture, protein and fat composition Of the ground beef were
determined using AOAC (1990) methods 950.463, 24.038 and 47.021, and
960.39, respectively. For determination Of pH, 25 g ground beef were
homogenized with 75 mL Of distilled water in a Waring blender for 30 sec.
The pH of the homogenate was measured using a Corning 145 pH meter
(Fisher Scientific, Itasca, IL). '

4.3.8. Statistical analysis

The analysis Of variance (ANOVA) was calculated using a randomized
complete block design. Mean LDH concentrations in ground beef patties
were compared using Student-Neuman- Keul's Multiple range test Of the
MSTAT statistical program (MSTAT, 1994) with the mean square error term
at the 5% level Of probability.

59
4.4. RESULTS AND DISCUSSION

4.4.1. Production of monoclonal antibodies

As a general rule, mice injected subcutaneously with bovine muscle
LDH Showed higher serum titers than those injected intraperitoneally (data
not shown). In addition, when tested by competitive indirect ELISA, sera Of
mice injected subcutaneously showed higher inhibition than those injected
intraperitoneally (Figure 4.1. and 4.2.).

A total of 360 wells were seeded with fused cells from the spleen Of
the mouse with the highest titer and NS-l myeloma cells, but only one
produced antibodies against LDH. A second fusion was performed using two
mice which resulted in two more positive hybridomas.

The three antibody-producing hybridomas, designated as 5E2, 4C5 and
5F8, were further expanded and cloned. Eight stabilized lines were Obtained
which showed high inhibition when tested by indirectcompetitive ELISA.

The cell lines were designated as:

-#3 B131-B6E6-11F6 -#15 A731-G3A2-11G5
-#5 Bl31-B6E6-11F10 -#16A73l-G3A2-1D10
-#7 B131-B6E6-11D3 -#17A731-G3A2-11D9
- #9 B131-B6E6-1 1D9 - #24 A731-G3A2-12C4

A sandwich ELISA was developed with monoclonal antibodies (B131-
B6E6-11D3) as the capture antibodies and polyclonal antibodies as the
detector antibodies. Detection Of LDH by sandwich ELISA is shown in
Figure 4.3.

 

 

60

 

 

 

 

 

 

 

 

 

1 10 100 1 000 10000 100000
Sera dilutions are: 1) 1: 10,000

2) 1: 12,000

3) 1: 10,000

4) r: 7,000
5) 1: 3,000

Figure 4.1. Detection of bovine muscle LDH by competitive indirect ELISA
using sera of mice injected subcutaneously (sc).

 

 

61

OD

 

 

Mouse No.

 

 

 

 

 

 

0.5 -‘--~ -

 

 

 

E
o F r ...... r . ...... . . .. .. ......«. . .......
1 10 100 1000 10000 100000
LDH (ng/mL)
Sera dilutions are: 1) 1: 8,000
2)]:10,000
4) 1: 8,000

Figure 4.2. Detection of bovine muscle LDH by competitive indirect ELISA
using sera of mice injected intraperitoneally (ip).

 

 

62

 

 

 

 

 

 

1 10 100 1000 . 10000
LDH (ng/mL)
Figure 4.3. Detection of bovine muscle LDH by sandwich ELISA:

monoclonal antibodies (1: 250) as capture antibodies and polyclonal
antibodies (1: 500) as detector antibodies.

63

4.4.2. Preparation and cooking of ground beef patties

Three different batches of ground beef patties containing three different
levels of fat were prepared. Results of proximate analysis and determination
of pH for the ground beef patties are shown in Table 4.1. Moisture ranged
from 64.3 to 67.6% and protein from 15.7 to 19.6%. Fat levels achieved
were 10.7, 13.6 and 19.0%. The pH ranged from 6.42 to 6.43.

Cooking schedules are Shown in Table 4.2. Nine patties were cooked
for each temperature and fat level. Ground beef patties containing 13.6% fat
were cooked tO internal temperatures Of 62.8°, 65 .6°, 683° and 71 .1°C.
Patties containing either 10.7 or 19.0% fat were cooked to an internal
temperature Of 683°C. Total cooking time ranged from 9 min in patties
cooked to 628°C to 18 min in patties cooked to 71 .1°C. Great variation was
Observed in terms Of cooking rate time among patties in a triplicate. The
maximum variation in total time was Observed in patties cooked to 683°C
independently of the fat content. .

LDH was extracted using a Tissue TearorT'“. This method Of extraction
was more efficient than homogenization in a blender, the common method Of
extraction employed in Similar studies (Collins et al., 1991; Stalder et al.,
1991; Wang and Smith, 1994). As a consequence, higher concentrations Of
LDH were Observed in both cooked and uncooked patties.

Extracts from the patties were tested using a sandwich ELISA. LDH
content in extracts from raw patties was 13.1 mg/g, 10.4 mg/g and 8.18 mg/g
meat, corresponding to fat concentrations Of 10.7, 13.6 and 19.0%,
respectively. The LDH content decreased Significantly (p < 0.05) in patties
cooked to 628°C (114 jig/g) when compared to the uncooked patties (10.4
mg/g). Significant differences (p < 0.05) were also Observed between patties
cooked to 628°C and those cooked to 656°(54 jig/g). NO

Table 4.1. Proximate analysis of ground beef patties“.

Moisture Protein
(%) (%)

 

67.6 19.6

 

66.1 17.6

 

 

 

 

 

‘ Average of three samples.

 

65

Table 4.2. Summary of ground beef patties cooking.

Internal

Temperature
(°C)

First Side
Cooking Time

(min)

Total
Cooking Timea

(min)

 

62.8

9.69 i 1.4

 

65.6

12.04 i 2.5

 

68.3

14.1 i 4.62

 

68.3

16.21: 3.7

 

68.3

16.31 :45]

 

 

'Expressed as mean :1: standard deviation.

 

 

 

18.55 i 3.2

:m in: n‘umm
- 1

 

 

66

significant differences (p > 0.05), however, were found among patties cooked
to 656° (54 jig/g), 683° (57.2 ug/g) and 71.1°C (21.8 jig/g) (Figure 4.4.).

In patties containing different levels Of fat and cooked to 683°C,
average LDH content was 57.5 ug/g and 21 .9ug/g, corresponding to 10.7 and
19.0% fat contents. NO Significant differences (p > 0.05) were found among
these values and the patties containing 13.6% fat (Figure 4.5.).

In a parallel experiment, the same grOtmd beef extracts were tested by
sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE),
Western blot and LDH enzymatic assay. Similar results tO those of sandwich
ELISA were Obtained by these three methods (data not shown).

Similar studies have been done in poultry products with successful
results. Antibodies against turkey LDH could detect differences in LDH
content of extracts between turkey thigh rolls that had been cooked to 711°
and 722°C (Wang, 1994). The same antibodies could detect differences in
LDH content of extracts from turkey breast rolls that had been cooked to
697°C and 709°C (Wang et al., 1993).

That the bovine muscle sandwich ELISA did not work in this
experiment does not mean that the approach was a complete failure. The
assay could detect different concentrations Of LDH even though the detection
limit was not as high as expected. Also, the variation among patties within a
replicate suggests that the same experiment should be repeated with patties

cooked under more controlled conditions.

5. CONCLUSIONS
Bovine muscle LDH had poor antigenic properties when injected into

mice for production Of monoclonal antibodies. A very low percentage

 

67

LDH (09/9)

 

 

 

 

62.8 65.6 68.3 71 .1
Temperature (C)

Figure 4.4. Concentration of LDH in ground beef patties cooked to different
internal temperatures.

LDH (us/9)

 

 

 

 

10.72 13.52 18.97
96 Fat

Figure 4.5. Concentration of LDH in ground beef patties with different fat
content cooked to 683°C.

68

(0.004%) of anti-LDH antibodies-producing hybridomas was Obtained after
two fusions. A sandwich ELISA using monoclonal and polyclonal antibodies
against bovine LDH has potential for determination Of LDH concentration in
beef products. If a relationship between LDH concentration and endpoint
temperature can be established, this method could be successfully used for
verification Of proper thermal processing of beef products.

Cooking of ground beef patties proved to be very inconsistent. High
variation in temperature rise as well as total cooking time among patties
cooked under exact conditions accounted for high variations in the final
product. This factor interferes with the accurate detection Of any protein
marker. Future research should include the development Of a more accurate
and consistent method of cooking. Once a controlled cooking method is

established, the LDH sandwich ELISA should be tested further.

 

BIBLIOGRAPHY

BIBLIOGRAPHY

Abdul-Raouf, U.M., Beuchat, LR. and Ammar, MS. 1993. Survival and
growth of Escherichia coli 0157:H7 in ground, roasted beef as
affected by pH, acidulants, and temperature. Appl. Environ. Microbiol.
59(8):2364-2368.

Abouzied, M.M., Asghar, A., Pearson, A.K., Miller, E.R., Gray, 1.1., and
Pestka, J .J . 1990. Hybridoma based enzyme-linked immunosorbent
assay for C19A‘6-steroids in sera of boars. J. Agric. Food Chem.
38:331-335.

Abouzied, M.M., Wang, C.H., Pestka, J .J ., and Smith, D.M. 1993. Lactate
dehydrogenase as safe endpoint cooking indicator in poultry breast
rolls: development Of monoclonal antibodies and application to
sandwich enzyme-linked immunosorbent assay (ELISA). J. Food
Protect. 56(2): 120-124, 129.

Abouzied, M.M., Wang, C.H., Pestka, J .J ., and Smith, D.M. 1994.
Unpublished data. Dept. of FOOd Science and Human Nutrition,
Michigan State University, East Lansing.

Ahmed, N. and Conner, DE. 1993. Evaluation of different media for
recovery Of therrnally-injured Escherichia coli 0157:H7. Paper NO.
133 presented at the 80th IAMFES meeting. Atlanta, GA, Aug. 1-4,
1 993.

Ang, C.Y.W. and Huang, Y.W. 1994. Color changes of chicken leg patties

due to end-point temperature, packaging and refrigerated storage. J.
Food Sci. 59(1):752-753.

69

70

Anonymous. 1994. Food and Chemical News, July 18, 1994.

AOAC. 1990. Ofi‘icial Methods of Analysis, 15th ed. Association Of Official
Analytical Chemists, Washington, DC.

Bean, NH. and Griffin, PM. 1990. Foodbome disease outbreaks in the
United States, 1973-1987: Pathogens, vehicles and trends. J. Food
Protect. 53(9):804-817

Bogin, E., Israeli, BA. and Klinger, I. 1992. Evaluation Of heat treatment of
turkey breast meat by biochemical methods. J. Food Protect.
55(10):787-791.

Borczyk, A.A., Karmali, M.A., Lior, H. and Duncan, L.M.C. 1987. Bovine
reservoir for verotoxin-producing Escherichia coli 0157:H7. Lancet.
Jan. 10, 1987.

Burke, B. 1993. Elementary immunology. Ch. 6, In Fine Structure
Immunocytochemistly, G. Griffiths (Ed.) 204-236. Springer-Verlag

Berlin, Germany.

Caldironi, HA. and Bazan, N.G. 1980. Quantitative determination Of low-salt

soluble proteins patterns Of bovine muscles cooked at different
temperatures. J. Food Sci. 45:901-904.

Cohen, ME. and Giannella, RA. 1991. Hemorrhagic colitis associated with
Escherichia coli 0157 :H7. Adv. Int. Med. 37:173-195.

Collins, 88, Keeton, J .T., and Smith, 8.8. 19913. Lactate dehydrogenase
activity in bovine muscle as a potential heating endpoint indicator. J.
Agric. Food Chem. 39:1291-1293.

Collins, 88, Keeton, J .T., and Smith, SE. 1991b. Lactate dehydrogenase
enzyme activity in raw, cured and heated porcine muscle. J. Agric.
Food Chem. 39:1294-1297.

Davis, GB, Lyon, B.G., Reagan, J .O. and Townsend, WE. 1985. Time

temperature heating effect on biuret-positive water-extractable porcine
and bovine muscle proteins. J. Food Protect. 48(3):215-220.

71

Davis, GB, Lyon, B.G., Reagan, J .O. and Townsend, WE. 1987. Effect of
heating on water soluble biuret-positive compounds of canned cured
pork picnic shoulder. J. Food Protect. 50(8):681-684.

Davis, GB, Searcy, G.K., Blankenship, LC. and Townsend, WE. 1988.
Pyruvate kinase activity as an indicator Of temperature attained during
cooking Of cured pork. J. Food Protect. 51(10): 773-777.

Desrocher, L.D. 1994. Enzyme-linked immunosorbent assay for rapid
endpoint processing temperature determination in turkey ham; and
effects Of delayed bleeding on residual serum proteins in turkey
muscles. MS. Thesis. Michigan State University, East Lansing.

Dom, CR. 1993. Review of foodbome outbreak Of Escherichia coli
0157:H7 infection in the western United States. JAVMA.
203(11):1583-1587.

Doyle, MP. 1991. Escherichia coli 0157:H7 and its significance in foods.
Int. J. Food Microbiol. 12:289-301.

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

Doyle, MP. and Schoeni, J .L. 1987. Isolation of Escherichia coli 0157:H7
from retail meats and poultry. Appl. Environ. Microbiol. 53(10):2394-
2396.

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

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

Eckner, KR 1992. Fluorometric analysis Of alkaline phosphatase inactivation
correlated tO Salmonella and Listeria inactivation. J. Food Protect.
55(12):960-963.

72

FDA. 1993. Recommendations Of the US Public Health Service Food and
Drug Administration, Food Code. Section 3-401.1 1. Food and Drug
Administration, Washington, DC.

Galfre, G. and Milstein, C. 1991. Preparation of monoclonal antibodies;
strategies and procedures. Methods Enzymol. 7321-40.

Goding, J .W. 1980. Antibody production by hybridoma. J. Immunol.
Methods. 39:285-308.

Goodfellow, SJ, and Brown, W.L. 1978. Fate Of Salmonella inoculated into
beef for cooking. J. Food Protect. 41(8):598-605

Gransden, W.R., Damm, M.A.S., Anderson, J .D., Carter, J .E. and Lior, H.
1985. Haemorrhagic cystitis and balanitis associated with verotoxin-
producing Escherichia coli 0157:H7. The Lancet, July 20, 1985.

Griflin, PM. and Tauxe, R.V. 1991. The epidemiology of infections caused
by Escherichia coli 0157:H7, other enterohemorrhagic E. coli, and the
associated hemolytic uremic syndrome. Epidemiol. Rev. 13:60-98.

Hague, M.A., Warren, K.E., Hunt, M.C., Kropf, D.H., Kastner, C.L., StrOda,
S.L. and Johnson, DE. 1994. Endpoint temperature, internal cooked

color, and expressible juice color relationships in ground beef patties.
J. Food Sci. 59(3):465-470.

Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold
Spring Harbor Laboratory, New York, NY.

Hebert, G.A., Pelham, PL. and Bittrnan, B. 1973. Determination Of the
optimal ammonium sulfate concentration for the fraction Of rabbit,
sheep, horse, and goat antisera. Appl. Microbiol. 25:26-36.

HolbrOOk, J .J ., Liljas, A., Steindal, SJ. and Rossmann, MG. 1975. The
Enzymes. Academic Press, New York, NY.

Hsu, Y.C. 1993. Characterization Of enzymes suitable as endpoint
temperature indicators in turkey muscle. MS. Thesis, Texas A&M
University, College Station.

Isakc

Kay

Kor

Kox

Line

M an

McC.

73

Isaksson, T., Ellekjaer, M.H.R. and Hildrum, KL 1989. Determination Of the
previous maximum temperature Of heat-treated minced meat by near
infrared reflectance spectroscopy. J. Sci. Food Agric. 49:385-387.

Kay, H.D., and Graham, W.R. 1933. Phosphorous compounds Of milk: the
effect Of heat on milk phosphatase. J. Dairy Res. 5:65-74.

Kormendy, L., Zsamoczay, G. and Mihalyi, V. 1992. A new, modified acid
phosphatase assay for determining the extent Of heat treatment in
canned hams. Food Chem. 44:367-375.

Kovacs, M.J., Roddy, J ., Gregoire, 8., Cameron, W., Eidus, L. and Drouirr,
J. 1990. Thrombotic thrombocytopenic purpura following hemorrhagic
colitis due to Escherichia coli 0157:H7. Am. J. Med. 88:177-179.

Line, J .E., Fain, A.R. JR, Moran, A.B., Martin, L.M., Lechowich, R.V.,
Carosella, J.M., and Brown, W.L. 1991. Lethality Of heat to
Escherichia coli 0157:H7: D-value and z-value determinations in
ground beef. J. Food Protect. 54(10):762—766.

Martin, R., Azcona, J 1., Cases, C., Hernandez, PE. and Sanz, B. 1988.
Sandwich ELISA for detection Of pig meat in raw beef using antisera to
muscle soluble proteins. J. Food Protect. 51(10):790-794.

McCormick, R.J., Reeck, GR, and Kropf, D.H. 1987Effect of heating
temperature and muscle type on porcine muscle extracts as determined
by reverse phase HPLC. J. FOOd Sci. 52:1481-1485.

National Canners Association. 1968. Laboratory manual for food canners and
processors. Vol. 1. AVI Publishing CO., Westport, CT.

National Live Stock and Meat Board. 1994. Solving the Escherichia coli
0157:H7 problem. A blueprint for industry action. Final report Of the
blue ribbon task force. Chicago, IL.

Neill, M.A., Tarr, P.I., Christie, D.L. and Clausen, CR. 1987. Clinical
findings in hemorrhagic colitis with Escherichia coli 0157:H7.
Gastroenterology. 92 (5 part 2): 1 551.

Padhye. NV.
Epiden
Food 1

Patel 55.. a1
i-gluta
systen

Pestka 1.1. I

immur

Pestka. J.J., ‘
antibo

Appl.

Peters, 111 .
ed. Sr

Ratnanr S,
Esche
.1. Me

Rhagubeer,
Eschr
medil

R113); Lu",
heme

Robson W
child
88(5:

Samariijeex

April
Nutr

74

Padhye, N.V. and Doyle, MP. 1992. Escherichia coli 0157:H7:
Epidemiology, pathogenesis, and methods for detection in food. J.
Food Protect. 55(7):555-565

Patel, SS, and Wilbey, RA. 1994. Thermal. inactivation Of
'y-glutamyltranspeptidase and Enterococcusfaecium in milk-based
systems. J. Dairy Res. 61:263-270.

Pestka, J .J . 1988. Enhanced surveillance Of foodbome mycotoxins by
immunochemical assay. J. Assoc. Ofi. Anal. Chem. 71(6): 1075-1081 .

Pestka, J .J ., Lee, Y.K., and Chu, ES. 1982. Reactivity Of aflatoxin B23
antibody with aflatoxin B1 modified DNA and related metabolites.
Appl. Environ. Microbiol. 44:1 159-1165.

Peters, J .H. and Baumgarten, H. (Eds). 1992. Monoclonal Antibodies. lst.
ed. Springer-Verlag Berlin, Germany.

Ratnarn, 8, March, SE. and Sprague, W.D. 1986. Are humans a source Of
Escherichia coli 0157:H7, the agent of hemorrhagic colitis. New Eng.
J. Med. 315(25):]612.

Rhagubeer, E.V. and Matches, J .R. 1990. Temperature range for growth of
Escherichia coli serotype 0157:H7 and selected colifonns in E.coli
medium. J Clin. Microbiol. 28(4):803-805.

Riley, L.W. 1987. The epidemiologic, clinical, and microbiologic features of
hemorrhagic colitis. Ann. Rev. Microbiol. 41 :383-407.

Robson, W.L.M. and Leung, A.K.C. 1990. Hemolytic-uremic syndrome in
children. A serious hazard of undercooked beef. Postgrad. Med.
88(5):]35-142

Samarajeewa, U., Wei, C.I., Huang, TS. and Marshall, MR. 1991.
Application of immunoassay in the food industry. Crit. Rev. Food Sci.
Nutr. 29(6):403-434.

Stalder. 1W» 5‘“
dehydroge
heating 6W

Steele. P. and La
of muscle
temperatu

Sugasawara, RJ
murme or:
antibody 1
Methods.

lalmant. A, M(
Actixities
Meat Sci.

Tan, Pl, Neill.
coli 015'

Thayer, DW. 3
in meats '
1034.

lodd,E.C.D. 1
the Unite

Todd 13.00. 1
seafoods
of Inst. c

Townsend W,

cured/c:
Food S I

 

 
  
  

Townsend, V;
treatmc
COmmo

75

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

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

Sugasawara, R.J., Cahoon, BE, and Karu, AE. 1985. The influence of
murine macrophage-conditioned medium on cloning efficiency,
antibody synthesis, and growth rate Of hybridomas. J. Immunol.
Methods. 79:263-275.

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

Tarr, P.I., Neill, M.A., Christie, D.L. and Anderson, DE. 1988. Escherichia
coli 0157:H7 hemorrhagic colitis. New Eng. J. Med. 318(25):]697.

Thayer, D.W. and Boyd, G. 1993. Elimination Of Escherichia coli 0157:H7
in meats by gamma irradiation. Appl. Environ. Microbiol. 59(4): 1030-
1034.

Todd, E.C.D. 1989. Preliminary estimates Of costs Of foodbome disease in
the United States. J Food Protect. 52(8):595-601.

Todd, E.C.D. 1994. Cost of foodbome illnesses caused by bacteria and
seafoods toxins. Paper NO. 27-1, presented at the 54th Annual Meeting
Of Inst. Of Food Technologists, Atlanta, GA, June 25-29.

Townsend, WE. 1989. Stability of residual acid phosphatase activity in
cured/caned picnic samples stored at -34°C for 15 and 36 months. J.
FOOd Sci. 54(3):752-753.

Townsend, WE. and Blankenship, LC. 1987 . Assessment Of previous heat
treatment of laboratory heat processed meat and poultry using a
commercial enzyme system. J. Food Sci. 52(6):]445-1448.

townsend.
pl’OCf
prodt

Tovmsend.
ALT
temp

Townsend
temp
beef

Townsend,
Eva]
dete
57(2

USDA-F S1
phos
Guit
and
USDA-F3
Rev
Scie
Was

USU/Hrs
F001
Divi

76

Townsend, WE. and Blankenship, LC. 1989. Methods for detecting
processing temperatures Of previously cooked meat and poultry
products-a review. J. Food Protect. 52: 128-135.

Townsend, WE. and Davis, CE. 1992. Transaminase (AST/GOT and
ALT/GTP) activity in ground beef as a means of detemrining end-point
temperature. J. FOOd Sci. 57:555-557.

Townsend , WE. Searcy, G.K., Davis, CE, Wilson, Jr. KL. 1993. Endpoint

temperature (EPT) affects N-acetyl-B-D-glucosaminidase activity in
beef, pork and turkey. J. Food Sci. 58:710-712.

Townsend, WE, Searcy, G.K., Davis, CE. and Wilson Jr., KL. 1994.
Evaluation of creatine phosphokinase activity as a means Of
determining cooking end-point temperature. J. Food Protect.

57(2):]59-162

USDA-F SIS. 1986a. Determination of internal cooking temperature (Acid
phosphatase activity). Revised Basic Chemistry Laboratory
Guidebook. NO. 3.01823-49. Science Chemistry Division. FOOd Safety
and Inspection Service, Washington, DC.

USDA-FSIS. 1986b. Determination Of cooked temperature (Coagulation).
Revised Basic Chemistry Laboratory Guidebook. NO. 3.019, p. 3-55.
Science Chemistry Division, Food Safety and Inspection Service,
Washington, DC.

USDA-F SIS. 1989. Performing the catalase test. A self-instruction guide.
Food Safety and Inspection Service. Technical Services Training
Division. Washington, DC.

USDA-F SIS. 1990. Code Of Federal Regulations, Title 9. Office Of the
Federal Register, National Archives and Records, GSA: Washington,
DC. Chapter 3. p. 491-492.

USDA-FSIS. 1993.Heat-processing procedures, cooking instructions and
cooling, handling and storage requirements for uncured meat patties.
Federal Register. 58 FR 41138, Docket NO. 86-041F. US Department
Of Agriculture, Food Safety Inspection Service, Washington, DC.

77

Voet, D. and Voet, J .G. 1990. Biochemistry. John Wiley & Sons, New York,
NY.

Wang, CH. 1994. Enzyme-linked immunosorbent assay for determination of
endpoint processing temperatures in uncured poultry products. Ph D.
Dissertation. Michigan State University, East Lansing.

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

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

Wang, S.F., Abouzied, M.M. and Smith, D.M. 1994. Identification Of
protein markers as potential endpoint temperature indicator in

hamburger patties. In Press.

Wells, J .G., Davis, B.R., Waschmuth, I.K., Riley, L.W., Remis, R.S.,
SOkOlow, R. and Morris, GK. 1983. Laboratory investigation of
hemorrhagic colitis outbreaks associated with a rare Escherichia coli
serotype. J. Clin. Microbiol. 18(3):512-520.

Weagant, S.D., Bryant, J .L. and Bark, DH. 1994. Survival of Escherichia
coli 0157:H7 in mayonnaise-based sauces at room and refrigerated
temperatures. J Food Protect. 57(7):629-631.

APPENDICES

APPENDIX A

Appendix A

Comparison of three media for enumeration of Escherichia coli 0157:H7

Table A.1. Mean log CFU/mL for regular E.coli 0157:H7 culture“.

 

Media lst Run 2nd Run 3rd Run

 

Coliform Count Plates 8.87:0.04 8.89i0.05 8.94:0.06

 

E.coli Count Plates 8.88i0.06 8.88:0.06 8.93:0.05

 

MC-PCA 87710.06 8. 82:0.06 8.88:0.1 8

 

 

 

 

 

 

“ Values expressed as mean i SD.

79

Table A2. Mean log CFU/mL for heat-Shocked E. coli 0157:H7 culture“.

 

Media 1st Run 2nd Run 3rd Run

 

Coliform Count Plates 4.88:1:0.05 4.76i0.06 4.44i0.1

 

E.coli Count Plates 4.87:0.03 4.78i0.02 4.45i0.09

 

MC-PCA 4.63i0.15 4.68i0.06 4.54:0.04

 

 

 

 

 

 

" Values expressed as mean i SD.

APPENDIX B

Thermal ii
the enzym

E
1

LI

 

Thermal inactivation of Escherichia coli 0157 :H7 as compared to that of

Appendix B

the enzyme lactate dehydrogenase and Salmonella senftenberg

5.6011 0157:"? It 53‘0
1) 1st Run
Time (In) 1
Raw 1.196406
0 MOE-+06
0.5 3.306+06
1 5.1OE+05
1.5 3.3OE+05
2 4.7OE+04
3 5.40sz
4 3.00E+01
5 0.00E+00
6 0.00E+00
2) 210 But
mm (In) 1
Raw 1.10506
0 7.00506
0.5 3.008406
1 2.00E+06
1.5 7.00E+05
2 2.60E+05
3 1.075104
4 0.00E+00
5 0.00E+00
6 0.00E+00
3) 3nd Run
11m. (his) 1
Raw 6.605506
0 2.40E+06
0.5 1.00E+06
1 1.765405
1.5 1.4OE+04
2 3.00E+03
3 5.00E+01
4 0.00E+00
5 0.006400
6 0.00E+00

2
1 .025+06
0.205406
2.605406
6.405405
2.505405
5.1 05404
3.005402
2.005401
0005.00
0.005400

1 .025406
5.005+06
3.605406
2.405406
6.605405
3.105405
9.005403
0.005400
0.005400
0.005400

6.605+06
2.905406
1 .065406
1 .765+05
5.005403
3.905403
6.005401
0.005400
0.005400
0.005400

80

Log 1
5.05
5.95
5.52
5.71
5.52
4.57

L09 2
6.01
6.06
6.45
5.61
5.40
4.71

Average

sum nun
Tim- (hr-l

mmaun'ar-a'

1
1 .01 5407
7.605406
3.105405
1 .205404
6.0054-03
2.005+02
3.005401
1 .005401
0.005400
0.005400

1
1 .41 5407
1 .645407
1 .605405
1 .505404
2.005403
3.005+02
2.705402
1 .005401
0.0054-00
0.005400

1 .075407
6.305406
3.505405
2.605404
2.005403
3.005402
1 .405402
0.005400
0.005400
0.005400

2
1 .165407
7.6054-06
3.205405
2.405404
6.005403
3.0054-02
2.005401
1 .005+01
0.005400
0.005400

2
1 .325407
1 .6254-07
1 .605405
1 .605404
1 .005403
3.005+02
3.405402
0.005400
0.005400
0.005400

2
1 .165407
6.605406
2.305+05
3.405404
2.005403
6.005402
1 .305+02
0.005400
0.005400
0.005400

LDH 63':
1) 1st Run
Time (In)

J
magenta-ciao

2mm hm
11m- (M)

cub
magnesia-duo

mam nun
11m- (m)

mmaun'ar-a'

1
MISSING
6.655402
6.365402
6.665402
6.635402
1 .055403
6.235402
6.755402
6.1 15402

4.725402
6.705402
4.605402
3.265402
6.605402
6.765402
7.575402
5.565402
2.745402

1
3.245402
4.655402
5.265402
3.665402
4.675402
3.345402
4.745402
4.605402

MISSING

82

2

MISSING
6.755402
6.365402
7.21 5402
6.61 5402
1 .01 5403
7.61 5402
7.665402
6.665402

2
5.1 25402
7.165402
4.365402
2.665402
7.31 5402
6.21 5402
5.665402
5.71 5402
2.445402

2
4.005402
4.765402
5.305402
3.465402
5.055402
3.435402
4.565402
4.51 5402

MISSING

888388°*~°

E. coli 0157:H7 It 501:
1)1ot RUN
Time (win) 1
0 6.50E406
2 1.40E406
4 1.50E406
8 7.605404
10 3.50504
15 1535403
20 3005-02
30 1.005401
35 MISSING
45 0.002400
212“ nun
11m- (uil) 1
0 3.308406
2 1.60E406
4 1.445406
8 1.56E405 -
10 4.005504
15 6.605403
20 8.60E403
30 0.008400
35 0.005400
45 0.006400
3M nun
Time (uh) 1

6.505406
2.505406
1 .61 5406
3.605405
1.1 1 5405
4.705403
1.255403
2.605402
2.005402
0.005400

83

2
6.605406
1 .605406
6.705405
7.205404
2.605404
1 .655403
4.605402
0.005400

MISSING
0.005400

3.305%
1 .565406
1.01 5406
2.005405
5.105404
7.405403
7.405403
2.005401
0.005400
0.005400

5.605406
2.305406
1 .605406
2.605405
1 .065405
3.105403
1 .1 254-03
3.605402
1 .205402
0.005400

W at we
1)1ot RUN
11m. (min) 1
o 1.91 E406
10 1.025405
20 2.50E404
30 4.505403
40 4.008402
50 5.208402
60 2.105402
70 8.005401
30 0.006400
00 0.00E400
2m BUN
Time (out) 1
0 2.605406
10 1.71 E405
20 1.305404
30 2.405403
40 1.00E402
50 6.00E402
60 2.306402
70 8.00E401
80 0.00E+oo
90 0.005400
313"! HUN

:1
3838388330;

2.605406
5.505405
3.405404
1 .005402
1 .005402
5.005402
6.005401
0.005400
1 .005401
0.005400

2
1 .765406
1 .265405
2.505404
2.605403
1 .005402
6.605402
1 .605402
6.005401
0.0054”
0.005400

2.505406
1 .525405
2.1 05404
4.205403
3.005402
4.605402
6.005401
2.005401
2.005401
0.005400

3.705406
5.305405
3.405404
1 .005402
1 .005402
6.105402
6.005401
0.005400
2.005401
0.005400

84

1.092

5.16
4.32
3.62

2.66
1 .60
1 .30
1 .30
0.00

38188518555450;

”MM

:1
i
ahnogs

10
15

88188

3311111011

:1
i
3

aseaaaa.nof

6.765402
6.745402
1 .205403
1.1 15403
6.61 5402
6.635402
5.525402
6.425402
6.605402
7.005402
6.655402

1
6.765402
7.565402
5.71 5402
6.61 5402
7.725402
7.375402
7.1 25402
7. 1 75402
6.325402
5.1 25402
6.425402

6.765402
6.325402
5.775402
7.265402
7.1 05402
6.305402
6.465402
6.665402
7.135402
7. 165402
5.575402

85

2
6.655402
6.745402
1 .205403
1.1 15403
6.61 5402
6.635402
5.625402
5.675402
6.605402
7.005402
6.655402

2
6.655402
6.565402
7.1 65402
6.055402
7.725402
6.675402
6.325402
7.425402
6.275402
5.1 75402
6.325402

6.655402
5.625402
5.605402
6.225402
6.025402
7.755402
6.165402
7.055402
7.725402
7.525402
5.475402

86

5.50" 0157:H7 at 636
mu Run
Time (sec) 1 2 Log 1
0 7.305406 6.205406 6.66
60 6.305406 6.105406 6.62
75 4.105406 4.005406 6.61
60 1.575406 1.725406 6.20
100 6.005405 5.605405 5.76
1 10 3.205405 5.005405 5.51
120 5.505405 4.505405 5.74
130 1.165405 1.155405 5.06
140 1.205403 3205403 3.06
150 5.605404 5.605404 4.77
160 66 66 2.00
170 0.005400 0.005400 0.00
160 0.005400 0.005400 0.00
210 MISSING MISSING
240 0.005400 0.005400 0.00
2m RUN
11mm») 1 2 Log 1
0 7.305406 6205406 6.66
60 5.305406 4.305406 6.72
75 2.705406 2.505406 6.43
100 1.565406 1.735406 6.16
1 10 6.405405 7.605405 5.62
120 2.115405 2.565405 5.32
130 1.565405 1.345405 5.20
140 3.765404 1.605404 4.56
150 4.605404 2.605404 4.66
160 7.705403 1.005404 3.66
170 4.305402 5.505402 2.63
160 1.065403 1.065403 3.04
210 0.005400 0.005400 0.00
240 0.005400 0.005400 0.00
3)3r0 BUN
Time (sec) 1 2 L06 1
0 7.305406 6.205406 6.66
60 4.405406 4.705406 6.64
75 4.605406 2.705406 6.66
60 1.165406 1.625406 6.07
100 6.205405 6.405405 5.66
1 10 6.705405 7.405405 5.63
120 1235405 1235405 5.06
130 6.605404 3.505404 4.64
140 1.655404 1.705404 4.22
150 7.505403 5.505403 3.66
160 6.005402 6.005402 2.65
170 1.315403 1.625403 3.12
160 30 20 1.46
210 2.405402 1.605402 2.36
240 6.005400 6.005400 0.65

 

the”
T111!

M
E ---p-‘t-‘nnm.a—n—o-

Tim

 

 

am at we
1)1ot RIIN
The. (sec) 1
0 3.605406
60 2.505406
120 2.405405
150 6.205404
160 1.665404
240 2.105403
270 6.005402
300 1.005402
360 2.405402
420 6.005401
460 1.005401
600 1.005401
2)2nd 11W
11m. (one) 1
0 3.605406
60 3.105406
120 6.605405
150 1.065405
160 1.155404
240 1.105403
270 7.005402
300 2.005402
360 1.105402
420 2.005401
460 1.005402
600 1.005401
3m RUN
Time (sec) 1
0 3.605406
60 1.765406
120 2.505405
150 1.365405
160 1.665404
240 1.105403
270 6.005402
300 5.005402
360 1.005402
420 4.005401
460 1.005401

1 .005401

87

2
3.705406
3.205406
3.705405
5.605404
1 .375404
6.005402
5.005402
0.005400
3.405402
7.005401
1 .005401
2.005401

3.705406
2.705406
3.205405
1 .155405
1 .125404
6.005402
3.005402
3.005402
6.005401
2.005401
6.005401
2.005401

3.705406
1.635406
1.605405
1.265405
1 .655404
5.005402
4.005402
6.005402
1.105402
2.005401
2.005401
2.005401

Average
6.57
6.27

5.12
4.27
2.67

2.74
2.02
1.45
1.15
1.15

110
120
130
140
150

170
160
210
240

2) 210 Run
Tim. (ace)

1
6.765402
1 .155403
6.665402
6.035402
1 .335403
7.765402
6.205402
6.045402
6.275402
5.665402
5.205402
6.675402
7.31 5402
6.165402
6.435402
6.065402

6.765402
1 .005403
7.665402
7.635402
7.625402
7.465402
7.455402
7.725402
6.135402
6.135402
7.635402
5.61 5402
7.755402
7.665402
5.665402
6.1 75402

6.765402
7.475402
7.565402
7.065402
7.465402
6.21 5402
6.61 5402
6.455402
7.665402
6.365402
6.245402
6.275402
6.635402
4.645402
5.205402
5.345402

88

2
6.655402
1 .1 65403
6.655402
6.255402
1.01 5403
6.265402
6.025402
4.545402
6.365402
5.605402
5.665402
7.1 65402
7.075402
5.61 5402
6.075402
5.365402

6.655402
6.655402
7.605402
7.635402
7.725402
7.425402
7.265402
6.025402
7.365402
7.635402
7.675402
6.505402
7.41 5402
7.565402
5.465402
6.545402

6.655402
7.545402
7.265402
7.755402
6.765402
6.005402
6.61 5402
6.455402
6.305402
6.135402
6.765402
6.425402
5.655402
4.61 5402
4.665402
5.365402

5.ch 0157:H7 It 66“:

1) 131 Run
11m- (one)
Raw
0
5

10
15

838888

2) ma nun
11m (see)

«a?

10
15

888888

3) am Run
11m- (soc)

...?

10
15

888888

1
3.205406
1 .675406
2.705406
1 .655406
1 .065406
1 .665405
4.405404
4.605403
1 .655403
1 .005402
0.005400

3.205406
4.305406
1 .765406
2.155406
1 .005406
1 .665405
3.055404
1 .665404

MISSING
0.005400
0.005400

3.205406
1 .355406
1 .605406
6.605405
7.405405
2.205405
7.605404
1 .51 5404
MISSING
0.005400
0.005400

89

2
2.605406
1 .475406
2.605406
1 .625406
1 .075406
1 .635405
6.m5404
6.605403
1 .505403
1 .705402
0.005400

2.605406
4.605406
1 .535406
2.265406
1 .145406
1 .605405
2.565404
1 .635404

MISSING
0.005400
0.005400

2.605406
1 .265406
2.125406
1 .165406
6.305405
2.405405
6.205404
1 .465404
MISSING
0.005400
0.005400

Ava-go
6.46
6.20

am
am
in
an

2.12
0.00

was“:

1) 161 Run
Tim- (ac)

ssassssasaamoi

2)“ Run
11m. (soc)

88388886883uo§

3) 3nd Run
11ml (one)

ssassssaaaamoi

1
1.01 5407
5.605406
3.605406
2.31 5406
1 .225406
6.305405
6.605405
4.705404
1.605404
6.605403
5.005401
3.005401
2.005401
0.005400

1
1 .41 5407
7.005406
2.605406
1 .605406
1 .605406
6.305405
1 .605405
7.605404
6.405403
6.605403
3.205402
6.005402
7.005402
0.005400

1 .075407
6.205406
7.405406
5.505406
2.365406
2.345406
1 .635406
6.005405
5.005404
MISSING
6.005402
1 .605402
5.005402
6.005401

90

2
1 .165407
5.405406
2.605406
2.055406
1.01 5406
6.505405
5.205405
3.605404
1 .505404
1 .035404
2.005401
1 .005401
3.005401
0.005400

1 .325407
6.605406
2.105406
2.105406
1 .665406
6.705405
4.005405
6.405404
5.305403
5.005403
4.605402
2.005402
3.005402
0.005400

2

1 .165407
6.605406
4.605406
6.205406
2.475406
2.475406
1 .665406
6.705405
7.205404

MISSING
6.005402
1 .205402
6.005402
6.005401

 

LDH we
1) 101 Run
11m- (soc)

88388888883mo§

2) 310 M
11m. (soc)

.. g
OIOUIO

88

8838888

3) 3rd Run
11m- (ace)

.9?

...—6
01°

883888888

1
6.765402
6.775402
6.51 5402
7.355402
6.355402
6.425402
4.635402
4. 125402
2.625402
2.325402

2.145402 -'

2.31 5402
1 .355402
1 .545402

6.765402
7.335402
6.675402
6.625402
6. 145402
6.665402
7.575402
7.465402
5.025402
4.21 5402
1 .645402
2.465402
2.365402
7.305401

6.765402.

6.145402
7.665402
6.665402
7.645402
7.125402
6.505402
5.555402
6.165402
3.61 5402
2.605402
3.165402
1 .135402
5.635401

2
6.655402
6.465402
6.355402
7.61 5402
6.505402
6.225402
4.455402
3.665402
2.455402
2.245402
2.1 55402
2.365402
1 .135402
1 .735402

6.655402
7.305402
6.605402
6.665402
6.135402
7.765402
7.675402
7.405402
4.565402
4.765402
2.1 25402
2.455402
2.325402
5.665401

2
6.655402
6.535402
6.035402
6.445402
7.625402
6.065402
6.405402
5.665402
5.545402
3.1 25402
2.635402
2.565402
1 .235402
5.725401

91