The Center for Food Safety EngineeringThe Center for Food Safety Engineering
Purdue UniversityPurdue University2001-2002 Research Report2001-2002 Research Report
2
CFSE Research ReportsCenter for Food Safety Engineering
The Center for Food Safety Engineering Logo
The logo of Purdue University’s Center for Food Safety Engineering (CFSE)
represents the center in many more ways than one. Understanding the iconography
of its images is one way to understand and appreciate CFSE.
The Gear—Engineering
One of the primary components of the CFSE logo is a gear, symbolizing the
engineering component of the center. CFSE engages many of the different branches
of engineering, and the gear calls to mind the basics of that discipline.
The teeth of the gear bear the insignia of the five Purdue schools that contribute to the
center, organized alphabetically in a counterclockwise arc starting from the upper right.
• The first insignia represents the School of Agriculture;
• The second insignia represents the School of Consumer and Family Sciences;
• The third insignia represents the Schools of Engineering;
• The fourth insignia represents the School of Science; and
• The fifth insignia represents the School of Veterinary Medicine.
The Circuit Board—Progress
The circuit board in the center is actually part of the revolutionary biochip developed
by Purdue researchers. In the CFSE logo, the biochip symbolizes the combination of
engineering and food safety for technological progress.
The Petri Dish—Food Safety
The petri dish in the lower portion represents food safety, as epitomized by traditional
microbial identification methods. One of the center’s goals is to develop more rapid
methods of identifying microbes.
The Circle—Synthesis
The circle bounding the petri dish and the biochip inside the gear represents the
synthesis of old and new technologies for improved food safety.
CFSE logo rendering courtesy of Kellen Maicher, Purdue University Department of Computer Graphics Technology
3
The safety of our food supply has never been so
important. The Centers for Disease Control and
Prevention estimate that 76 million cases of foodborne
illness, 325,000 hospitalizations, 5000 deaths, and costs
of 7.7-23 billion dollars occur each year in the United
States. One of the keys to preventing foodborne illness
is effective measures to detect and reduce risk of
contaminant presence, survival, and growth. Pathogens
such as Listeria monocytogenes and Escherichia coli
O157:H7 have been targets for detection and
prevention because of their low infective dose,
dangerous disease characteristics, and widespread
occurrence in foods. At the Center for Food Safety
Engineering we direct our efforts toward detecting
problems and protecting consumers.
The events of September 11, 2001, raised heightened
concern about food biosecurity and about harmful
agents that could pose a threat to plant and animal
systems and ultimately to our food supply. There is an
even more imminent need for improved systems for
detection of microbial and chemical contamination in
food. Current detection systems are not sensitive or
accurate enough, involve too many days for
contaminant identification, are too costly, and are not
easily usable by the industry or regulatory agencies.
Our approach at Purdue has been to combine
engineering technologies and food safety expertise to
find solutions to these problems. The Center for Food
Safety Engineering, or CFSE, is a newly formed center
at Purdue University operating through the Office of
Agricultural Research Programs. The center focuses on
developing better methods for hazard detection and
developing better ways to control hazards in our food
system. We accomplish our goals by building research
teams through collaborative efforts of five Schools,
including Agriculture, Consumer and Family Sciences,
Engineering, Science, and Veterinary Sciences.
Currently, 37 research faculty, and 39 staff (students,
technicians, and support personnel) are involved in 11
CFSE research projects. We offer updated information
The Center for Food Safety Engineeringat Purdue University –
Collaborating to Make Our Food Safer
on CFSE activities, funded projects, and research
progress on our Web site at: <http://www.cfse.
purdue.edu/>.
The focus of the CFSE is to develop and improve the
efficiency of detection and control of biological and
chemical hazards in foods. Our current objectives
include:
1. Developing diagnostic tools for rapid identification
of biological and chemical foodborne contaminants;
2. Developing models to predict and track foodborne
contaminants;
3. Identifying, designing, and evaluating alternative
processing, handling, packaging, transport, and
storage systems to minimize and/or reduce food
contaminants; and
4. Enhancing technology transfer of information and
knowledge related to food safety for the food
industry, government agencies, academia, and the
public.
The CFSE is a wonderful example of research success
through multi-disciplinary collaboration. Engineers and
food microbiologists are working on developing
postage stamp-sized biochips to detect pathogens more
quickly and more accurately. Animal scientists, food
scientists, and microbiologists are working on
developing a post-pasteurization procedure to reduce
the risk of Listeria monocytogenes contamination in
ready-to-eat luncheon meats. Plant pathologists and
food mycologists are developing rapid screening
methods for mold identification and isolation that will
help in grain storage and food processing operations.
The development of the CFSE has been very
rewarding. Through CFSE, Purdue University positions
itself as a national leader in multi-disciplinary food
safety research. Our multi-disciplinary approach,
including a strong engineering component, makes
Purdue University truly unique. I am extremely excited
about the future possibilities of the CFSE. I invite you
to share that excitement.
Introduction
Dr. Richard H. Linton,
Director of the Center
for Food Safety
Engineering
4
CFSE Research Reports
What major problem or issue are
you resolving, and how are you
resolving it?
The goal of this research is to harness the power of
bacterial phage display with affinity chromatography in
order to develop a biological amplifier for the detection
of small numbers of pathogenic organisms in foods.
The research will generate and purify phages that are
designed to selectively detect Salmonella spp. Projects
on biochips, antibody-based assays coupled with
impedance based spectroscopy, fluorescence micros-
copy, and enzyme-linked immuno-assays (ELISA’s) are
addressing the diverse needs of detection methods for
food pathogens.
Research underway in our laboratories that involves
cooperative efforts with investigators in Food Science,
Electrical and Computer Engineering (ECE), Biomedi-
cal Engineering, Agricultural and Biological Engineer-
ing, Biology, and the Laboratory of Renewable
Resources Engineering (LORRE) is addressing the
processing of food samples, amplification of organisms
using culture or rapid separation techniques, and
selective capture of pathogenic organisms from a
complex background of other organisms, protein
macromolecules, and other substances.
The proposed project complements these other efforts.
We will be examining the biological fundamentals of
applying a phage display method with the use of
affinity (liquid) chromatography to obtain purified
phages for infecting Salmonella spp. and also separat-
ing phages that would be generated in the presence of
Salmonella spp., thereby indicating the presence of this
food pathogen even if other (non-pathogenic) bacteria
are present. If successful, this technology platform
could be directly coupled to the existing antibody-based
projects mentioned above.
How serious is the problem? Why
does it matter?
Microbial contamination of meat and, more important,
of fresh fruits and vegetables has become a mounting
concern during the last decade due to an increased
emphasis on the importance of these products in a
healthy diet and the recognition of new foodborne
pathogens such as Campylobacter jejuni, Escherichia
coli O157:H7, and Listeria monocytogenes. In response
to this rising concern, President Clinton announced a
Food Safety Initiative in 1997. A joint agency guide on
approaches to minimize microbial contamination of
fresh fruits and vegetables was recently released as part
of this initiative. While the recommended practices
provide a framework for minimizing risk, availability
of rapid methods for detection of pathogens in the
production, processing, and distribution systems could
provide real-time assessment of risks.
What was your most significant
accomplishment in this past year?
We have successfully modified the GIII protein of M13
so that we can create the lacZ-gIII transcriptional
fusion.
What do you expect to accomplish,
year by year, over the duration
of the project?
Year 1
• Construct and propagate modified bacteriophages
(MB) in host cells consisting of E. coli and Salmo-
nella spp.
• Purify modified bacteriophages using affinity
chromatography
Bioamplification Using Phage Display for the Detection ofSalmonella spp. and Its Evaluation as a Technology Platform for
Simultaneous Detection of Multiple Pathogens in the Same SampleInvestigators: Bruce M. Applegate, Michael R. Ladisch
5
Year 2
• Develop an assay using the modified bacteriophages
to detect food samples infected with Salmonella
and/or non-pathogenic organisms, capture, concen-
trate, and detect using affinity chromatography and
other forms of liquid chromatography
Science/Technology Transfer
A Provisional Patent Application was filed in March
2002 covering the current technology of the
Bioamplification/Phage Display technology.
Presentation
An oral presentation, “Bioamplification Mechanisms to
Increase Sensitivity of Biosensors,” was given at the
Fifth Workshop on Biosensors and Biological Tech-
niques in Environmental Analysis, May 31-June 4,
2002, organized by the International Association of
Environmental Analytical Chemistry.The goal of this research is to harness thepower of bacterial phage display with affinitychromatography in order to develop abiological amplifier for the detection of smallnumbers of pathogenic organisms in foods.
Applegate, Ladisch
Using bioluminescence to detect Salmonella
6
CFSE Research Reports
Detection of Specific Foodborne Pathogens Using a TwoComponent Bacteriophage/Bioluminescent Reporter
System in Conjunction with a Hand-Held LuminometerInvestigator: Bruce M. Applegate
What major problem or issue are
you resolving, and how are you
resolving it?
Food safety is an increasing concern. The desire for
rapid, specific methods for the detection of viable
potential pathogens has grown into a necessity. Our
long-term research goals are to develop bioluminescent
sensors for the detection of food pathogens and
integration of these bioreporters with emerging light
detection technologies. Advancements in optical
detection technologies will permit development of
small, portable, and highly sensitive optical transducers
capable of measuring bioluminescent responses from
bioreporter organisms. Integration of these transducers
with bioluminescent reporters will provide detection
technology to ensure food safety.
The primary research objective is development of a
suite of bacteriophage-based bioluminescent
bioreporters for detection and monitoring of pathogenic
bacterial species in raw or processed meats, fruits, and
vegetables. Bacteriophage bioluminescent bioreporters
are capable of infecting specific host cells, resulting in
production of visible light. Our specific aims are to:
• Genetically construct pathogen-specific bacterioph-
age capable of inducing bioluminescence from
bioluminescent bioreporter cell populations for
quantitative sensing of microbial pathogenic species
in raw or minimally processed meats, fruit, and
vegetables.
• Analyze bacteriophage/bioluminescent bioreporter
system with portable field-based photomultiplier
unit for the development of a suite of simple, rapid,
real-time, on-site testing mechanisms for bacterial
pathogens in meats, fruit, and vegetables.
How serious is the problem?
Why does it matter?
Microbial contamination of meat, fresh fruits, and
vegetables has become a mounting concern during the
last decade due to emphasis on their importance in a
healthy diet and the recognition of new foodborne
pathogens such as Campylobacter jejuni, Escherichia
coli O157:H7, and Listeria monocytogenes. In
response, President Clinton announced a Food Safety
Initiative in 1997. A joint agency guide on approaches
to minimize microbial contamination of fresh fruits and
vegetables was recently released as part of this
initiative. While the recommended practices provide a
framework for minimizing risk, availability of rapid
methods for detection of pathogens in the production,
processing, and distribution systems could provide real-
time assessment of risks.
Of the nearly 2300 identified serovars of Salmonella,
300 have been related to human illness. Salmonella
commonly cause gastroenteritis, but can result in severe
systemic infections. Salmonella-related illness has been
linked to cantaloupe, alfalfa sprouts, tomatoes, and
watermelon. Salmonella has also been isolated from a
variety of fresh vegetables, including artichoke,
cabbage, cauliflower, celery, eggplant, endive, fennel,
lettuce mustard cress, parsley, and spinach.
Listeria monocytogenes causes serious human disease
manifested by sepsis and meningitis. It is commonly
found on vegetables, including lettuce, tomatoes,
asparagus, broccoli, and cauliflower. A documented
listeriosis outbreak has been associated with cabbage.
Growth of L. monocytogenes can occur at cool (5-
15o C) storage temperatures.
E. coli 0157:H7 is an emerging human pathogen first
linked to food-illness (i.e., fast food hamburgers)
outbreaks in 1982. It produces enterohemorrhagic
toxins that can lead to death, particularly in the very
young and old. Cattle appear to be a primary reservoir,
but E. coli 0157:H7 has been linked to outbreaks from
cantaloupe, broccoli, and, potentially, lettuce. The
organism can survive and grow on cubed melon and
watermelon, and has been isolated from cabbage,
celery, cilantro, and coriander.
Campylobacter is an emerging pathogen causing acute
gastroenteritis and has been identified as a common
antecedent to Guillan-Barre syndrome. Illness is most
7
commonly associated with contaminated poultry and
raw milk, although Campylobacter has been linked to
raw fruits and vegetables.
What were your most significant
accomplishments this past year?
• Constructed bioluminescent reporter strain for acyl-
homoserine lactone detection and evaluated its
performance characteristics.
• Constructed genetically modified M13 bacteriophage
containing the luxI gene in a lacZ-luxI fusion.
• Evaluated M13/bioreporter model system.
• Developed technology for rapid insertion of luxI into
phage genomes, which will allow rapid construction
of bacteriophage/bioluminescent reporter systems.
What do you expect to accomplish,
year by year, over the duration of
the project?
Year 1
• Genetically engineer a lux-based bioreporter cell line
responsive to acyl-homoserine quorum sensing signal
induction.
• Develop four luxI-based bacteriophage specific for
infection of L. monocytogenes, E. coli O157:H7,
Salmonella spp., and Campylobacter spp.
• Test bacteriophage/bioreporter systems in pure
culture studies, emphasizing hand-held luminometer
format.
• Lyophilize bacteriophage/bioreporter systems for
long-term storage assessment.
Year 2
• Comprehensively test bioreporter systems on lettuce
and tomato with introduced pathogens.
• Compare and contrast pathogen detection parameters
utilizing hand-held photomultiplier units and
microtiter plate format.
• Continue evaluating lyophilized bacteriophage/
bioreporter systems for long-term storage assessment.
Science/Technology Transfer
A United States Patent Application was filed in July
2001 covering the current technology of the Bacte-
riophage/Bioluminescent Reporter System. The patent
will be held jointly by Purdue University and the
University of Tennessee (Knoxville).
Presentations
A poster presentation, “Detection of Specific
Foodborne Pathogens Using a Two Component
Bacteriophage/Bioluminescent Reporter System”
(Abstract # 99), was given at the American Society of
Microbiology conference on Cell-Cell Communication
in Bacteria, July 6-9, 2001, Snowbird, Utah.
A poster presentation, “Filter Based Assay for Pathogen
Detection Using a Two Component Bacteriophage
Bioluminescent Reporter System” (Abstract # M-38),
was given at the 102nd General Meeting of the
American Society of Microbiology, 2002 in Salt Lake
City, Utah.
A poster presentation, “Biological Bioluminescence
Amplification Using Quorum Sensing Molecules for
Increasing the Sensitivity of Light Detection Systems,”
was given at the Fifth Workshop on Biosensors and
Biological Techniques in Environmental Analysis,
organized by the International Association of Environ-
mental Analytical Chemistry, May 31-June 4, 2002.
An oral presentation, “Bioamplification Mechanisms to
Increase Sensitivity of Biosensors,” was given at the
Fifth Workshop on Biosensors and Biological Tech-
niques in Environmental Analysis, May 31-June 4,
2002, organized by the International Association of
Environmental Analytical Chemistry.
Applegate
E. coli O157:H7 is an important target for
detection for the CFSE.
8
CFSE Research Reports
What major problem or issue are
you resolving, and how are you
resolving it?
Presence of even a few Listeria monocytogenes cells in
processed, ready-to-eat products could be a serious
threat to susceptible consumers with compromised
immune systems. Therefore, sensitive and specific
detection of this pathogen is essential. Conventional
microbiological methods take too long (2-7 days) to
detect and identify pathogens in food. A biosensor-
based approach is a promising and sensitive alternative
to detect bacterial cells in hours instead of days.
After initial capture and concentration of Listeria cells
from food using immunomagnetic beads or other
immunobeads, our goal was to use biosensor-based
probes to detect viable L. monocytogenes cells. We are
currently pursuing three approaches to measure Listeria
interaction with animal cells: (a) antibody-coupled fiber
optic biosensor, (b) interdigitated microsensor electrode
(IME)-chip, and (c) enzyme-fluorescence assay.
How serious is the problem? Why
does it matter?
Listeria monocytogenes has caused several recent
outbreaks of foodborne infections. CDC estimates there
are more than 2500 cases of foodborne Listeria
infections, with mortality ranging from 20-28%. An
investigative report of a recent major outbreak indi-
cated that L. monocytogenes at levels below one colony
forming units per gram of meat was fatal to some
consumers. Therefore, technology should be sophisti-
cated and sensitive, and should be able to detect very
low levels of offending bacterial contaminants in foods.
Biosensor-Based Approaches for Rapid and SensitiveDetection of Listeria monocytogenes from Food
Investigators: Arun K. Bhunia, Mark T. Morgan, Rashid Bashir
What were your most significant
accomplishments this past year?
Single Most Significant Accomplishment
During FY 2001
Our most significant accomplishment was development
of a sensitive and rapid two-step method to detect
Listeria monocytogenes at concentrations less than
1 CFU/ml of food extract. We used immunomagnetic
beads along with a newly developed antibody coated
protein-A Immunobeads to capture bacterial cells from
naturally contaminated or spiked hotdog samples after a
selective enrichment step, and directly tested bead-
captured bacterial cells in a cytotoxicity assay. Results
showed that L. monocytogenes at a concentration of
about 1-100 CFU/100 ml of food extract could be
captured in 12-16 hours starting with the food sample
and subsequently be detected in an additional 1-2
hours. Currently, we are in the process of evaluating
this two-step detection method extensively with
naturally contaminated hotdog samples, and we believe
this method will help to detect the presence of any
viable, virulent L. monocytogenes cells in test samples
in less than 24 hours.
Other Significant Accomplishments
• We developed an interdigitated microsensor
electrode (IME)-chip based cytotoxicity assay (cell-
based sensor) for detection of Listeria
monocytogenes from food. We placed mammalian
cells (Ped-2E9) exposed to L. monocytogenes for 1-2
hours on the IME-chip and measured Ped-2E9 cell
damage with an Impedance Analyzer. The data
showed that the IME-chip is capable of distinguish-
ing Listeria-induced damaged Ped-2E9 cells from
healthy ones. This IME-chip-based cytotoxicity
assay could be used in conjunction with the
immunobead separation method (see above) for
direct detection of L. monocytogenes from food
products in less than a 24-hour period.
9
• We developed a fiber optic biosensor for
detection of L. monocytogenes. We used
polystyrene wave-guides immobilized with
anti-Listeria polyclonal antibody to capture
Listeria cells and subsequently detected
them by fluorescein-labeled anti-Listeria mono-
clonal antibody and a laser detector (Analyte 2000).
Using a pure culture of L. monocytogenes, we were
able to detect as low as ~38 CFU/ml with the fiber
optic probe. We will evaluate this probe with
Listeria isolates obtained from naturally contami-
nated hotdogs using immunobeads, and we antici-
pate developing a very sensitive and rapid fiber optic
assay for this pathogen.
• We developed a L. monocytogenes-specific fluores-
cence-based cytotoxicity assay using mammalian
Ped-2E9 cells as the target. We examined various
potential foodborne contaminants such as E. coli
O157:H7, Salmonella, Citrobacter, Bacillus,
Staphylococcus, and Corynaebacterium along with
L. monocytogenes with Ped-2E9 cells separately in a
fluorescence-based cytotoxicity assay. Results
showed that Citrobacter, Bacillus, and
Corynaebacterium species produced positive
cytotoxicity, whereas other test organisms did not.
In order to make this assay a specific one for
L. monocytogenes, we are currently using selective
enrichment media containing antibiotics to inhibit
non-Listeria organisms prior to testing with
Ped-2E9 cells.
What major accomplishments do you
anticipate over the life of your
project, and what do you predict
their impact will be?
Our overall goal is to develop rapid and sensitive
methods (two-step detection methods) for low levels
(1-100 CFU) of L. monocytogenes contamination in
ready-to-eat food in a relatively short period of time.
We developed a two-step method to detect viable cells
and to reduce false-positive or false-negative results.
The first step of this detection method involves capture
and concentration of Listeria cells from food products.
For that, we have developed an immunobead separation
method that is capable of capturing cells from food
with initial cell populations of approximately 1-100
CFU/100 ml in 12-16 hours. In the second step, the
viable captured Listeria cells are detected by the
cytotoxicity-based assay in 1-2 additional hours. Thus,
our current two-step method could detect viable
L. monocytogenes cells as few as <1 CFU/ml of
product extract in 14-16 hours.
Soon, in the second step of the two-step method, we
will use biosensor tools such as interdigitated
microsensor electrode-chip, fiber optic immunosensor,
and fluorescence-based assays, which are designed for
specific detection of L. monocytogenes. Currently these
probes are showing promising results when tested with
pure laboratory Listeria strains; however, we are
continuing our efforts to refine the technology for
desirable response.
Once completed, we will use these two-step detection
systems for their ability to detect L. monocytogenes
from naturally contaminated food products for valida-
tion. We anticipate that, once fully developed, this
method could be used by meat processors to detect the
presence of any viable harmful Listeria monocytogenes
in processed, ready-to-eat products in less than 24
hours before the products are sold for consumption.
Bhunia, Morgan, Bashir
Cell-based biochip to detect Listeriamonocytogenes
10
CFSE Research Reports
What do you expect to accomplish,
year by year, for the duration of your
project?
With our first two years of support, we believe we have
reached a major milestone with the capture of low
numbers of viable Listeria cells from hotdogs in 12-16
hours and the specific detection by cytotoxicity assay.
Once captured, we could test these cells for pathogenic
L. monocytogenes by biosensor probes. In the third
year, if funded, we will focus on:
1. Refining our biosensor tools so that they could be
highly sensitive and could provide a much more
stronger signal than what is currently obtained;
2. Designing and configuring the probes in such a
fashion that they could be readily used by the end
users;
3. Testing the efficiency of this method to detect viable
but stressed Listeria cells (heat, cold, salt, and acid
induced stress) from food products; and
4. Validating the biosensor probes by testing with large
numbers of naturally contaminated food products.
Science/Technology Transfer
It will be at least another 2 years before this promising
technology will be available for end users.
Scientific Publications
Bhunia, A., Z.W. Jaradat, K. Naschansky, M. Shroyer,
M. Morgan, R. Gomez, R. Bashir, M.R. Ladisch.
2001. Impedance spectroscopy and biochip sensor
for detection of Listeria monocytogenes. Proceed-
ings of the Society for Photo Optical Engineers.
4206: p32-39.
Nebeker, B.M., B. Buckner, E.D. Hirleman, A. Lathrop,
A.K. Bhunia. 2001. Identification and characteriza-
tion of bacteria on surfaces using polarized light
scattering. Proceedings of the Society for Photo
Optical Engineers. 4206: p224-234.
Singh, R.K., A. K. Bhunia, A. Singh. 2001. Light
emission based biosensors for detection of food
pathogens: a review. Proceedings of the Society for
Photo Optical Engineers. 4206: p7-12.
Singh, N., R.K. Singh, A.K. Bhunia, R.L. Stroshine,
J.E. Simon. 2001. Sequential disinfection of
foodborne pathogens by ozone, chlorine dioxide,
and natural plant extracts. Proceedings of the Society
for Photo Optical Engineers. 4206: p159-166.
Gomez, R., R. Bashir, A. Sarikaya, M.R. Ladisch, J.
Sturgis, J.P. Robinson, T. Geng, A. K. Bhunia, H. L.
Apple, S.T. Wereley. 2001. Microfluidic biochip for
impedance spectroscopy of biological species.
Biomedical Microdevices 3 (3): 201-209.
Jaradat, Z.W., G.E. Schutze, A.K. Bhunia. 2002.
Genetic homogeneity among Listeria
monocytogenes strains from infected patients and
meat products from two geographic locations
determined by phenotyping, ribotyping and PCR
analysis of virulence genes. Int. J. Food Microbiol.
76:1-10.
Menon, A., M.L. Shroyer, J.L. Wampler, C.B. Chawan,
A.K. Bhunia. 2002. In vitro study of Listeria
monocytogenes infection to murine primary and
human transformed B cells. Comp. Immunol.
Microbiol. Infect. Dis. (accepted).
Gomez, R., R. Bashir, A.K. Bhunia. 2002. Microscale
electronic detection of bacterial metabolism. Sensors
and Actuators B: Chemical. (accepted)
Abstracts
Geng, T., R. Gomez, R. Bashir, M.R. Ladisch, A.K.
Bhunia. 2001. Reaction patterns of monoclonal
antibodies C11E9 and EM-7G1 to stressed or injured
Listeria monocytogenes cells for use in the biochip.
American Society for Microbiology General
Meeting, Orlando, FL, May 20-24, 2001.
Abstr. P-33, p563.
Presence of even a few Listeria monocytogenes cells in processedready-to-eat products could be a serious threat to susceptibleconsumers with compromised immune systems.
11
Gomez, R., T. Geng, R. Bashir, M.R. Ladisch, A.K.
Bhunia. 2001. Micro-fabricated biochip for the
electronic detection of Listeria cells. American
Society for Microbiology General Meeting, Orlando,
FL, May 20-24, 2001. Abstr. P-91, p575.
Naschansky, K.M., M. Morgan, A.K. Bhunia, Interdigi-
tated microsensor electrode-chip for detection of
cytotoxicity effect of Listeria monocytogenes from
food. American Society for Microbiology General
Meeting, Orlando, FL, May 20-24, 2001. Abstr. P-
32, p563.
Shroyer, M.L., A. Menon, A.K. Bhunia. 2001. A
sensitive fluorescence-based cytotoxicity assay to
differentiate Listeria monocytogenes from other
common foodborne bacteria. American Society for
Microbiology General Meeting, Orlando, FL, May
20-24, 2001, Abstr. P-11, p559.
Lathrop, A.L., Z.W. Jaradat, A.K. Bhunia. 2002.
Increased antibody specificity for Listeria
monocytogenes by partially masking of antigen
binding site in MAb C11E9 with L. innocua
antigens. American Society for Microbiology
General Meeting, Salt Lake City, UT. May 19-23,
2002. Abstr. P-42, p370.
Jaradat, Z.W., A.K. Bhunia. 2002. Variation in adhesion
and invasion behavior among different serotypes of
Listeria monocytogenes to Caco-2 cells. American
Society for Microbiology General Meeting, Salt
Lake City, UT. May 19-23, 2002. Abstr. P-76, p376.
We anticipate that, once fully developed, this method could be used bymeat processors to detect the presence of any viable harmful Listeriamonocytogenes in processed ready-to-eat products in less than 24 hoursbefore the products are sold for consumption.
Bhunia, Morgan, Bashir
Kim, K.P., T. Geng, F. Soyer, A.K. Bhunia. 2002.
Increased transcription of lepA homologue, a GTP
binding protein, in Listeria monocytogenes during
heat stress. Institute of Food Technologist Annual
Meeting, Anaheim, CA. June 15-19, 2002. Abstr.
100A-4.
Naschansky, K.M., A.K. Bhunia. 2002. Detection of
low levels of pathogenic Listeria monocytogenes in
14 - 20 h using immunoseparation and cytotoxicity
techniques Institute of Food Technologist Annual
Meeting, Anaheim, CA. June 15-19, 2002.
Abstr.100A-22.
12
CFSE Research Reports
What major problem or issue are
you resolving, and how are you
resolving it?
Most Fusarium species produce one or more mycotox-
ins in cereal grains and foods. Three major mycotoxins
produced by Fusarium species, fumonisins,
trichothecenes, and zearalenone, are linked to human
health concerns. These mycotoxins are produced in
grains grown in the Midwest and worldwide during
growth and storage. Also, Fusarium species can grow
during some food processing operations and produce
mycotoxins. Once the toxins are in the grain or food,
they are difficult to destroy because they resist most
food processing operations.
There is a need to detect Fusarium species in grains
and foods to prevent their growth and subsequent
production of mycotoxins. Currently, there are no rapid
methods to detect Fusarium species before they
produce mycotoxins. Our project was designed to
develop rapid methods to detect Fusarium species in
grains and foods by using techniques based on enzyme-
linked immunosorbent assay (ELISA) and polymerase
chain reaction (PCR). The objectives are to develop
methods that could detect Fusarium species as a
general group and also to detect the major species of
Fusarium that produce the three major mycotoxins
(fumonisins, trichothecenes, and zearalenone).
How serious is the problem? Why
does it matter?
The exposure of humans to mycotoxins produced by
Fusarium species is not fully understood because few
studies have been done on dietary intake and risk
assessment. There are some published reports that low
levels of Fusarium mycotoxins are in many ready-to-
eat foods, especially those made from barley, corn, and
wheat. The USDA in the published “Food Guide
Pyramid” suggests that 6 to 11 servings of cereal or
grain-based foods be consumed daily.
It is not known whether these recommendations will
pose greater health risks from long-term consumption
of low levels of Fusarium mycotoxins. From a safety
standpoint, the less exposure to mycotoxins, the better
will be the health of all consumers. Because there are
no rapid methods to detect Fusarium species in grains
or foods, it is difficult for grain storage operators or
food processors to rapidly detect Fusarium species
and alter conditions to prevent the production of
mycotoxins.
What was your most significant
accomplishment this past year?
Single Most Significant Accomplishment
During FY 2001
The goal of this part of the research was to develop a
PCR-based (polymerase chain reaction) technique for
detecting and distinguishing between trichothecene-
and fumonisin-producing Fusarium species. A multi-
plex PCR assay was developed to detect Fusarium
species as a group and those that produce either
fumonisins or trichothecenes. The primer to detect the
genus level Fusarium species that was developed from
the internal transcribed spacer regions (ITS1 and ITS2)
of the rDNA detected as little as 10 pg template/
reaction of Fusarium species. The primer to detect
trichothecene-producing Fusarium species that was
made from the TRI6 gene involved in trichothecene
biosynthesis detected 100 pg template/reaction of
Fusarium graminearum. The primer to detect
fumonisin-producing Fusarium species detected 1 ng
template/reaction of F. verticillioides. This part of the
research shows that a PCR assay can be developed to
detect Fusarium species and specific mycotoxin-
producing Fusarium species.
Other Significant Accomplishment
The second goal of the research is to develop an ELISA
(enzyme-linked immunosorbent assay) for detecting
mycotoxin-producing Fusarium species in grains and
Detection of Fusarium Species in Grainsand Foods by ELISA and PCR
Investigators: Maribeth A. Cousin, Charles P. Woloshuk
13
foods. We used antibodies that were produced to
Fusarium graminearum and Fusarium verticillioides to
produce an indirect ELISA to detect these species in
foods. In competitive assays with 70 molds from 22
genera, these two antibodies detected Fusarium species
as well as Monascus species and Phoma exigua;
however, these two non-Fusarium fungi are not
common in foods. This research suggests that an ELISA
assay can be developed to detect Fusarium species in
foods or grains within a short time and at low spore
levels.
What major accomplishments do you
anticipate over the life of the project,
and what do you predict their impact
will be?
We developed three primer sets to detect Fusarium
species in grains and foods. These primer sets were to
the ITS1 and ITS2 of the rDNA for the general detec-
tion of Fusarium species, to the TRI6 gene involved in
trichothecene biosynthesis by Fusarium graminearum,
and to the FUM5 gene involved in fumonisin biosynthe-
sis by Fusarium verticillioides.
These primers detected 10 pg template/reaction of
Fusarium species, 100 pg template/reaction of
F. graminearum and 1 ng template/ reaction of
F. verticillioides, respectively. When genomic DNA was
isolated from 43 fungal species representing 14 genera,
these primers detected only Fusarium species. Detec-
tion and differentiation of DNA of F. graminearum and
F. verticillioides that was extracted from inoculated
cornmeal occurred at < 105 CFU/g. This level of
detection is similar to that for PCR and ELISA methods
used by the food industry to rapidly detect bacteria.
We produced antibodies to F. graminearum and
F. verticillioides and used them to develop an indirect
ELISA to detect Fusarium species in foods. In competi-
tive assays with 70 molds from 22 genera, these two
antibodies detected all Fusarium species as well as two
uncommon molds in foods, Monascus species and
Phoma exigua. The indirect ELISA detected a mini-
mum of 0.1 mg of Fusarium verticillioides/ml of buffer
but 1 mg/ml cornmeal. For F. graminearum, the
detection limit in buffer was 1 ng/ml but 0.1 mg/ml for
cornmeal. The lower sensitivity in food compared to
buffer suggests that the food may affect binding to the
microtiter plate. Live spores of F. graminearum and
F. verticillioides were detected by the indirect ELISA in
a cornmeal slurry at concentrations between 102 and 103
CFU/ml.
Because both the PCR and ELISA showed promise in
developing assays to rapidly detect Fusarium species in
foods, the next phase of the research will be to use the
antibodies that were produced for the ELISA to capture
the molds from grains and foods. These molds could
then be used to extract the DNA for use in PCR
analysis. In essence, this would result in an
immunocapture-real time PCR assay for the rapid
detection of both Fusarium species and specific
mycotoxin-producing Fusarium species.
Maribeth Cousin using the CFSE-developed
ELISA detection system for foodborne molds
Cousins, Woloshuk
14
CFSE Research Reports
What do you expect to accomplish,
year by year, for the duration of your
project?
This project was completed on December 31, 2001;
however, we have started a new project to combine the
antibodies with PCR to produce an immunocapture-real
time PCR assay for the rapid detection of both
Fusarium species and specific mycotoxin-producing
Fusarium species in grains and foods.
Science/Technology Transfer
This project is not at a point where the technology can
be transferred to diagnostic companies; however, it is
anticipated that, with further research, companies that
manufacture test kits based on immunoassays and PCR
will be able to use this information. Purdue University
has signed a licensing agreement with a diagnostic
company to transfer technology from other research
done on the development of antibodies to molds, and
similar transfer of this technology is anticipated after
further research on this project.
Presentations
A report was presented to the Microbial Food Safety
Research Unit at the USDA Agricultural Research
Service, Eastern Regional Research Center in
Wyndmoor, PA on October 25, 2000.
A report was presented in Indianapolis, IN on October
3, 2002 in a symposium entitled “Detection of Micro-
bial and Chemical Contaminants in Foods.”
A report was presented at the North Central Regional
Research Project (NC129) on April 8, 2002, in
Peoria, IL.
A report, “Immunological detection of Fusarium
species in a model food system,” was presented by
M. S. Iyer and M. A. Cousin at the Institute of Food
Technologists Annual Meeting in Anaheim, CA, June
15-19, 2002.
A report, “PCR detection of fumonisin- and
trichothecene- producing Fusarium species,” was
presented by B. H. Bluhm, J. E. Flaherty, and
C. P. Woloshuk at the Annual Meeting of the
American Pathological Society in Milwaukee, WI,
July 27-31, 2002.
Scientific Publications
Bluhm, B. H., J. E. Flaherty, M. A. Cousin,
C. P. Woloshuk. 2002. A multiplex polymerase chain
reaction (PCR) assay for the differential detection of
trichothecene- and fumonisin-producing species of
Fusarium in cornmeal. Journal of Food Protection.
Submitted.
Iyer, M.S., M. A. Cousin. 2002. Immunological
detection of Fusarium species in cornmeal. Journal
of Food Protection. Submitted.
Because there are no rapid methods to detect Fusarium species in grains orfoods, it is difficult for grain storage operators or food processors to rapidlydetect Fusarium species and alter conditions to prevent the production ofmycotoxins.
15
Haley, Campanella, Gerrard, Bhunia, Cornillon
What major problem or issue are
you resolving, and how are you
resolving it?
Our research focuses on the development of a post-
package pasteurization process to eliminate Listeria
monocytogenes from sliced bologna. Under current
manufacturing practice, surface contamination can
occur before final packaging via aerosols or handling
after the product has been pasteurized. Our research is
developing one possible processing method to elimi-
nate this potential health hazard. The approach being
investigated is the application of a high-temperature-
short-time (HTST) process to pouches containing one
or two meat slices. The process will be designed to
pasteurize the surface of the slice(s) where contamina-
tion may have occurred sufficient to destroy low levels
of the pathogen. The objectives of our research are to:
• Determine a death rate kinetic model for low levels
of L. monocytogenes on sliced bologna.
• Develop and validate a numerical heat transfer
model for post-package pasteurization of sliced
bologna.
• Develop the control system to implement the
process.
How serious is the problem? What
does it matter?
Low levels of L. monocytogenes on the surface of
ready-to-eat (RTE) meats can be life threatening,
particularly to immune-compromised individuals, the
elderly, and unborn fetuses. In 1999, the Centers for
Disease and Prevention estimated that over 2500 cases
occur each year, with a corresponding death rate of
20%. The infective dose is still unknown, but it is
thought that a very low number of cells may lead to
illness, especially in the immunocomprimised popula-
tions (where the death rate has been reported between
34-70%). Because of these concerns, a “zero tolerance”
Development and Evaluation of a “Virtual” Sensor for Use inPost-Package Pasteurization to Eliminate Low Levels of
Listeria monocytogenes on Surfaces of Sliced BolognaInvestigators: Tim A. Haley, Osvaldo H. Campanella, David E. Gerrard, Arun K. Bhunia, Paul C. Cornillon
has been established for processed and RTE products,
including dairy foods and processed meats. Within the
past year alone, there have been several product recalls
and illnesses associated with foods contaminated with
L. monocytogenes.
An emerging concern has been for RTE processed meats.
In 1999, the CDC reported a multi-state outbreak of
L. monocytogenes in RTE frankfurters occurred,
resulting in 21 deaths, 5 miscarriages, and more than 15
million pounds of product recalled. The source of the
organisms was thought to be from post-process contami-
nation by air in the meat packaging area. More recent
The post-pasteurization process for ready-
to-eat luncheon meats
16
CFSE Research Reports
recalls of RTE processed meats, pasteurized milk, and
chicken burritos have also been initiated.
While prevention of contamination is important in all
areas of food production, special precautions must be
taken after foods have been heat processed and before
final packaging. Many foods that are contaminated with
L. monocytogenes are contaminated by post-processing
handling. For ready-to eat foods, control of the
environment is very important to prevent contamination
by air, equipment, processing water, or food handler
contamination. Alternative methods, such as post-
process heating treatments, should also be considered
as an intervention strategy. In a recent USDA-FSIS
survey, L. monocytogenes was found in 5.7% of RTE
luncheon meat samples, and an incidence level from
5.1 - 30% was reported in raw unprocessed meats. This
information, combined with unknown infective dose,
high illness and death rate, and challenging prevention,
generates a public heath problem of immediate
concern. Intervention must be developed to reduce and
control this organism for RTE foods.
What were your most significant
accomplishments in the past year?
We determined a kinetic model to describe quality
degradation in packaged, sliced, full-fat, and low-fat
bologna.
The application of a pasteurization process to pack-
aged, sliced bologna results in degradation of bologna
quality in terms of moisture loss and color change. This
portion of the research was conducted because there
have been no studies in the research literature that
indicate quality degradation of packaged, sliced
bologna in a pasteurization process. We conducted this
portion of the research in the animal science laborato-
ries at Purdue University, where we developed a kinetic
model that describes the quality degradation of
packaged bologna during pasteurization. This kinetic
model will be instrumental in developing a suitable
HTST process for post-packaging pasteurization that
minimizes quality damage to the product.
We determined a thermal-death-time model for
L. monocytogenes. Prior research literature indicates
that the thermal death characteristics of L. mono-
cytogenes vary widely depending on the food involved
in contamination. We conducted this portion of the
research because no data exists for thermal death
characteristics of this organism in bologna. We
conducted this research in the food safety laboratories
at Purdue University, where we developed a thermal-
death-time (TDT) model for L. monocytogenes. This
TDT model will be instrumental in developing a
suitable HTST process for post-packaging
pasteurization.
We determined a heating-rate model for packaged,
sliced, full-fat and low-fat bologna. We conducted this
research because there have been no studies in the
research literature that indicate heat transfer character-
istics of packaged, sliced bologna. We conducted this
portion of the research in the food engineering labora-
tories at Purdue University, where we developed a heat
transfer model for packages containing one or two
slices of bologna. This heat transfer model will be
instrumental in developing a suitable HTST process
for post-packaging pasteurization.
The results of the science and technology developed in this project will betransferred to the meat processing industries that produce ready-to-eatmeat products for retail sale.
17
What major accomplishments do you
anticipate over the life of your
project, and what do you predict
their impact will be?
The major accomplishment of this project will be to
define a methodology that can be employed to pasteur-
ize packaged, sliced, full-fat, and low-fat bologna. The
methodology can be used as a protocol for developing
post-packaging pasteurization processes for other
ready-to-eat meat products to eliminate potential
contamination with L. monocytogenes.
What do you expect to accomplish,
year by year, over the duration of
the project?
In year 2, we expect to combine the thermal-death-time
model, the kinetic model that describes quality
degradation, and the heat transfer model to determine a
post-packaging pasteurization process. The resulting
models will allow prediction of accomplished tempera-
tures and lethality on the surfaces of bologna. We will
use them to develop a process control strategy to
optimize the process.
Science/Technology Transfer
The results of the science and technology developed in
this project will be transferred to the meat processing
industries that produce ready-to-eat meat products for
retail sale. All new information derived from this
research will be published as scientific articles in
reputable, peer-reviewed journals.
Haley, Campanella, Gerrard, Bhunia, Cornillon
Papers
Corvalan, C., Y.R. Kim, G. Chen, T.A. Haley, O.H.
Campanella. 2002. Development and validation of a
numerical heat transfer model for post-packaged
pasteurization of sliced bologna. Paper 58-2.
Institute of Food Technologist Meeting, Anaheim,
California.
Corvalan, C., T.A. Haley, O.H. Campanella. 2002.
Design and development of a post-package pasteur-
ization process to inactivate Listeria monocytogenes
in ready-to-eat meat products. Paper 13-11. Institute
of Food Technologist Meeting, Anaheim, California.
Selby, A. Berzins, A.L. Grant, T.A. Haley, A.K. Bhunia,
D.E. Gerrard, R.H. Linton. 2002 Thermal inactiva-
tion kinetics of listeria monocytogenes in ready-
to-eat bologna. Paper 97-10. Institute of Food
Technologist Meeting, Anaheim, California.
18
CFSE Research Reports
What major problem or issue are
you resolving, and how are you
resolving it?
Rapid identification and isolation of bacteria such as
pathogenic Listeria monocytogenes and Escherichia
coli in foods are imperative to reduce the health hazards
to the general population. Development of selective
enrichment and plating methods has significantly
reduced time needed for identification, but quicker
methods are highly sought after by the industry. Our
objective is to develop a rapid light scattering sensory
method for the identification of strains of Listeria and
enterohemorrhagic E. coli.
How serious is the problem? Why
does it matter?
In recent years, there have been multiple outbreaks,
product recalls, illnesses, and loss of lives resulting
from the association of pathogens in processed, ready-
to-eat food products. Bacterial contamination in
products not only puts the public at risk, it is costly to
companies due to the loss of production time, product
recalls, and liability.
What was your most significant
accomplishment this past year?
Single Most Significant Accomplishment
during FY 2001
We adopted a scatterometer (an instrument to measure
the angle-resolved light scattering characteristics of
surfaces) for applications to bacterial detection. The
scatterometer, which can use optical wavelengths from
the ultraviolet into the visible region of the spectrum,
is located in the Mechanical Engineering
Micro/Nanosystems Diagnostics Laboratory at
Purdue University.
Other Significant Accomplishments
We tested 10 different bacterial cultures to determine if
the scatterometer could differentiate the bacteria. We
diluted pure cultures of Listeria monocytogenes,
Listeria innocua, Escherichia coli, Streptococcus
mutans, Bacillus subtilis, Bacillus cereus, Pseudomo-
nas aerugin, Staphylococcus aureus, Salmonella
enteritidus, and Citrobacter fruendil in the Molecular
Food Microbiology Laboratory at Purdue, plated them
onto BHI agar, and then transported them to Purdue’s
Micro/Nano Diagnostic Laboratory. We illuminated the
colonies and recorded the light scattering characteris-
tics. We noted some differences amongst the bacterial
colony angle scans, but further analysis is necessary to
determine their significance.
We tested Listeria innocua at different time intervals to
determine if the scatterometer could detect micro
colonies. In the Molecular Food Microbiology Labora-
tory, we plated Listeria innocua at 2-hour time intervals
ranging from 2 hours to 20 hours. We ran samples on
the scatterometer in the Micro/Nano Diagnostic
Laboratory and recorded the light scattering character-
istics. The scatterometer appeared to detect bacteria as
early as 6-10 hours after initial inoculation. This is
earlier than the human eye can detect the colony.
What major accomplishments do you
anticipate over the life of the project,
and what do you predict their impact
will be?
In year 2 we will continue collection and analysis of
different bacterial light scattering characteristic and
determine if the characteristics are unique to bacteria
genus and or species. We will make comparisons
between experimentally attained data and the numeri-
cally predicted light scattering characteristics. From
Light-Scattering Sensory Method for RapidAssessment of Foodborne Bacterial Contaminants
Investigators: E. Dan Hirleman, Arun K. Bhunia
19
this information, we will determine the level of
modeling accuracy of the light scattering codes. We
will apply a light scattering sensor for detection and
characterization of Listeria species and E. coli in
artificial or naturally contaminated meat samples. The
objective of these experiments is to determine if viable
target organisms can be identified in the presence of
background microflora, which may grow on the
selective agar plates. To determine the accuracy of the
light scattering method, we will confirm the bacterial
colonies by standard microbiological and molecular
biology based (RiboPrinter) assay methods.
Scientific Publication
Nebeker, B.M., B. Buckner, E.D. Hirleman, A. Lathrop,
A. Bhunia. 2001. Identification and characterization
of bacteria on surfaces using light scattering.
Proceedings of SPIE. p. 224-234.
The scatterometer appeared to detectbacteria as early as 6-10 hours afterinitial inoculation. This is earlierthan the human eye can detect thecolony.
Hirleman, Bhunia
Electron micrograph for foodborne
pathogens on produce
20
CFSE Research Reports
What major problem or issue are
you resolving, and how are you
resolving it?
Pathogenic bacteria in foods cause a high percentage
of reported foodborne illnesses. Of these, Listeria
monocytogenes has emerged as one of the most
important food pathogens, with a “zero tolerance” for it
in ready-to eat processed (lunch) meats and dairy foods.
This bacterium not only causes serious illness but is
also lethal in infants, people over 60, and immune-
compromised individuals.
Current methods of detecting this bacterium rely upon
enrichment in the numbers of bacteria present in a
sample by incubating a food or food extract in special
growth media for 12 to 24 hours. The resulting culture
is tested for L. monocytogenes using analytical
procedures that require an additional 3 to 24 hours to
complete.
The food industry includes many small food processors
and producers that do not have in-house microbiologi-
cal laboratories for the purpose of testing for food
pathogens. Many companies therefore send out samples
for analysis. This adds up to another 24 hours to the
time that elapses between when the food is sampled and
the bacterium, if present, is detected. An overall time of
2 to 3 days is typical of the time that elapses between
when the food is sampled and the test results are
available. The elapsed time, referred to as time to result
or TTR, is problematic because some foods are
consumed before test results would be available.
Rapid and affordable technologies to detect low
numbers of L. monocytogenes cells directly from food,
and which distinguish living from dead cells, are
therefore needed. Our multi-disciplinary, multi-
departmental research project is addressing the
fundamental engineering and science required for
development of microchip, bio-based assays that are
transportable to the field, useable in a manufacturing
plant environment, and capable of rapidly detecting
L. monocytogenes at the point of use. Our research has
the goal of microscale detection of Listeria
monocytogenes on a real-time or near real-time basis.
Structures that are the diameter of a hair carry fluid
obtained from a food sample to a postage-stamp sized
device that distinguishes Listeria monocytogenes from
other organisms. Biological-based recognition mol-
ecules fixed to the surface of the chip identify the
bacterium (i.e., the target) if present. The chip sends out
an electronic signal if target cells are present and bind
to the biorecognition molecules on the chip.
Our multi-disciplinary research team is addressing the
development, engineering, and validation of such a
microchip system. We believe our approach will result
in a technology platform capable of detecting other
types of foodborne and medically relevant pathogens,
although the focus of the research is on rapid detection
of Listeria monocytogenes by a combination of
technologies that will ultimately give a time to result of
hours. Our team approach has allowed us to pool our
resources to maximize our progress.
How serious is the problem? Why
does it matter?
The Centers for Disease Control and Prevention
estimates that there are 76 million cases of foodborne
illness per year in the U.S., resulting in hospitalization
of 325,000 people, 5000 deaths, and an annual cost of
$7 to 23 billion. E. coli O157:H7 and Listeria
monocytogenes are the pathogens of most concern.
Ground meat containing E. coli O157:H7 is now
considered to be an adulterated food, while Listeria
monocytogenes has emerged as one of the most
important food pathogens, and there’s a “zero toler-
ance” for it in ready-to-eat processed (lunch) meats and
dairy foods.
Early and rapid detection of pathogenic bacteria in
foods is a necessary condition for preventing the food
Engineering of Biosystems for the Detection ofListeria monocytogenes in Foods
Investigators: Michael R. Ladisch, Stephen F. Badylak , Rashid Bashir,
Arun K. Bhunia , J. Paul Robinson, Rakesh K. Singh
21
from reaching the consumer, being eaten, and causing
illness. A rapid and facile detection system that can be
used by scientists and non-scientists alike is needed to
ensure a safe food supply. Our research targets the
pathogenic bacterium Listeria monocytogenes.
The genus Listeria is comprised of six species,
L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua,
L. welshimeri, and L. grayi. However, only
L. monocytogenes is harmful to humans. Consumption
of contaminated food may cause meningitis, encephali-
tis, liver abscess, headache, fever, and gastroenteritis
(diarrhea) in immunologically challenged individuals
and abortion in pregnant women. L. monocytogenes is
ubiquitous in nature and can be found in meat, poultry,
seafoods, and vegetables. Occurrence of this organism
in food could be as high as 32%. In a food sample,
L. monocytogenes is often present in close association
with other nonpathogenic Listeria species, thereby
complicating the specific detection procedures.
Biochips that are affordable, capable of rapid detection
of food pathogens, and easy to use by small food
processors as well as major food companies are needed.
Achieving such devices requires research on:
• Interfacing biological molecules (i.e., biomolecules)
with electronic components;
• Electronically detecting and amplifying
biomolecular interactions between target the
biomolecules that form the biorecognition compo-
nents of the chip;
• Achieving in vitro biospecificity for the target
molecule;
• Sampling and conditioning biological fluids while
maintaining their information;
• Content (i.e., the molecules or cells that represent
possible targets of the chip);
• Transporting sample fluids on and/or off the chip;
and
• Interfacing biochip systems with electronic reading
devices.
Research in these areas will contribute to 1) improved
food safety through better diagnostic technology for
food pathogens and 2) the development of a fundamen-
tal knowledge base and a technology plat-form for
application of this approach to detection of multiple
foodborne pathogens and other targets.
What was your most significant
accomplishment in the past year?
Single Most Significant Accomplishment
During FY 2001
The “zero-tolerance” goal for L. monocytogenes in
foods translates to detecting as few as 10 organisms and
distinguishing living from dead organisms. The
cooperators on this project team in Electrical and
Computer Engineering, Food Science, Biomedical
Engineering, Agricultural and Biological Engineering,
and the Laboratory of Renewable Resources Engineer-
ing have carried out multi-disciplinary research on
sample concentration, on-chip processing of samples,
electronic detection, and validation using microscopy
and biological means. Our combined efforts culminated
in demonstration of the electronic, on-chip detection of
10 organisms in Bashir’s laboratory and validation of a
bioreceptor and an electronic method, respectively,
capable of distinguishing living from dead cells in
Bhunia’s, Robinson’s, and Bashir’s laboratories. While
we obtained these results for relatively clean culture
broths of the organism, this first proof of sensitive, bio-
electronic detection is a key step on the road to a
functional biochip.
Ladisch, Badylak, Bashir, Bhunia, Robinson, Singh
CFSE’s multidisciplinary biochip team
22
CFSE Research Reports
Other Significant Accomplishments
Because all foods contain nonpathogenic bacteria that
are not harmful, a viable test must distinguish patho-
genic from nonpathogenic bacteria. We showed
detection of pathogenic L. monocytogenes in the
presence of a non-pathogenic form of Listeria spp. in
test-tube experiments carried out in Food Science. We
identified a monoclonal antibody that is biospecific and
binds L. monocytogenes much more strongly than
L. innocua, therefore giving a ten-fold higher response
of L. monocytogenes over L. innocua. Biospecificity is
needed to distinguish harmful from harmless bacteria
and avoid false positive results that would lead to
unnecessary, expensive actions for addressing a threat
that does not really exist.
The viability of the antibody to be used on the chip for
detecting L. monocytogenes must be confirmed by
independent assays. In Badylak’s facility, we developed
an enzyme-linked immuno-sorbent assay (ELISA)
using an antibody provided by Bhunia’s laboratory. A
sandwich format in which antibody fixed to small
beads binds the target (L. monocytogenes) while
secondary antibodies amplify the colorimetric response
due to the capture of the target cells on the beads
was able to detect between 107 to 108 cells of
L. monocytogenes per mL. This is an important test for
validating biochip operation because the ELISA result
corresponds to a 1 to 10 cells per 100 nanoliters (nL),
where 100 nL is the volume of fluid characteristic of a
single well on the chip, and 10 cells represent a number
that is readily detectable on the chip by electronic
means.
The biologically and chemically complex sample
obtained from meat must be processed to concentrate
bacterial cells and to remove fats, oils, sugars, salts, and
proteins before the fluid is presented to the chip. We
have developed a compositional fingerprint of hotdog
sample, and we have systematically examined
adsorbents, bioaffinity ligands, and methods for rapidly
handling the samples in Ladisch’s and Bhunia’s
laboratories. The result is bioseparations techniques
capable of rapidly concentrating bacterial cells and
removing proteins and other components before the
fluid is presented to the chip. Off-chip sample process-
ing removes components that would otherwise plug the
micron-sized channels, foul the biorecognition mol-
ecules, and obscure electronic sensing surfaces on the
chip before a measurement can be made.
The transport of the fluid sample to the chip is a critical
component of the chip’s operation. We have fabricated
microscale structures in Ladisch’s and Bashir’s
laboratories that are capable of efficiently moving
fluids to a chip with the potential of simultaneously
concentrating the cells in the fluid. Testing of these
structures and direct observation of their flow charac-
teristics using fluorescence microscopy in Robinson’s
laboratory confirm their operation and feasibility. This
device provides a way of coupling off-chip sample
processing with sample introduction onto the chip using
a pipettor or syringe.
What major accomplishments do you
anticipate over the life of your
project, and what do you predict
their impact will be?
Our project’s goal is to contribute to the fundamental
engineering knowledge base of microscale technology
for the rapid detection of pathogenic strains of Listeria
monocytogenes in foods. Real-time or near real-time
detection of foodborne bacterial pathogens directly
from food is a major challenge. Current methods rely
upon the initial selective enrichment of the food extract,
which may require at least 12 to 24 hours. Specific
detection or identification of L. monocytogenes may
take an additional 3 to 24 hours to complete. Thus,
rapid and affordable technologies to detect low
numbers of L. monocytogenes cells directly from food
with minimal enrichment steps are needed.
23
Our proposed research will address the fundamental
engineering and science required for development of
micro-chip, bio-based assays that are transportable to
the field and can rapidly assess whether or not viable
Listeria monocytogenes contamination is present. The
availability of such a test will enable rapid testing for
food pathogens so that the time to result is a matter of
hours or less. This would make corrective actions more
practical and help to ensure food safety. The microchip
technology that results from this project will also
provide a platform for development of protein based
chips for detecting other types of pathogens for uses in
medical, environmental, and other food applications.
What do you expect to accomplish,
year by year, for the duration of
your project?
The goal of our project is to engineer a biosystem to
rapidly detect Listeria monocytogenes and other
pathogens in foods. The biosystem being developed by
our multi-disciplinary team combines protein chemistry
with a computer chip at a micron scale. We anticipate
developing a postage-stamp-sized biosensor that will
detect the presence of Listeria monocytogenes in a
matter of minutes. This biosystem will have the
capability to be coupled to a hand-held device capable
of reading and interpreting the signal from the chip and
will thereby facilitate rapid, in-plant detection of a
pathogenic organism. Such a configuration will enable
rapid transmission of the results to a computer and
ultimately through the Internet to remote locations, as
needed.
This biosystem is based on the concept of binding a
protein (antibody) on electrically conductive surfaces
placed on a non-conducting surface (such as silicon
dioxide or plastic) of a microchip. The protein is chosen
so that it will selectively bind with the pathogenic
organism, if present, in a liquid sample passed over the
chip. The goal is also to detect binding, and therefore
the presence of a pathogenic organism, by a change in
an electronic signature when the antibody on the
surface of the chip binds with its antigen (a protein on
the surface of the cell). Electronic detection is intended
to supplant the more expensive optical methods that are
currently available and in use (including in our
laboratories).
The biosystem will incorporate bioseparations technol-
ogy in order to condition the sample by rapidly
removing potential interfering compounds (including
other proteins) before the sample is presented to the
chip. The quantities of sample that will need to be
processed are expected to be small because the volume
of the channels on the chip are on the order of micro-
liters or less.
Objectives of our proposed four-year, USDA-Purdue
University cooperative project are to:
• Characterize binding of polyclonal and monoclonal
antibodies with Listeria monocytogenes cells at
silicon dioxide/platinum surfaces that will be
representative of microchip based sensing systems;
• Carry out fundamental research on detecting the
binding and state of cells (living or dead) using
electrically based sensing on a micro-chip; and
• Develop methods for sampling, cell concentration,
and sample delivery for a microchip-based system.
We have initiated research to address all three objec-
tives. Our specific aims, on a year-by-year basis, are
as follows.
Year 1
• Prove the concepts of protein receptors (monoclonal
antibodies) on the surface of the biochip, verify
binding of cells to receptors by both optical and
electronic means, and then combine all of the
elements into functioning concept prototype(s) that
would be used to detect Listeria monocytogenes.
This approach is intended to identify fundamental
Our multi-disciplinary, multi-departmental research project is addressing thefundamental engineering and science required for development of microchip,bio-based assays that are transportable to the field, useable in a manufacturingplant environment, and capable of rapidly detecting L. monocytogenes at thepoint of use.
Ladisch, Badylak, Bashir, Bhunia, Robinson, Singh
24
CFSE Research Reports
scientific issues that we will need to address in
greater depth to achieve a functioning biochip, as
well as to identify key unresolved technology
(engineering) issues that we must address to obtain a
device that is designed for use in detecting food
pathogens.
• Improve methods for generating monoclonal
antibodies against L. monocytogenes. Increase the
volumes that can be generated. Carry out fundamen-
tal studies on the specificity of binding of antibodies
to their targets, as well as on culture conditions that
are associated with pathogen propagation.
• Fabricate and test several different chip designs,
based on preliminary results. Probe tradeoffs among
chip structural features, fluid transport, and elec-
tronic detection.
Year 2
• Continue research on monoclonal and polyclonal
antibodies from year 1.
• Carry out fundamental research on the binding
chemistry of protein receptors at platinum (or other
metal) and silicon dioxide surfaces for the purpose
of directing the binding to specific surfaces or
surface structures. Characterize receptor density
(concentration) at the surface of the chip. Character-
ize rates and strengths of binding of the target
molecule to the receptor that is at the chip’s surface.
Define selectivities of binding that are achievable
when L. monocytogenes is presented to the antibod-
ies on the chip in the presence of other bacteria.
Characterize receptor binding with respect to other
proteins. Calibrate sensitivity of the chip against
sampling and measuring conditions.
• Optimize design of the chip to maximize signal to
noise ratio when cells are bound to the receptor.
Design and characterize microfluidic features on the
chip to enable transport of sample on and off the
chip. Correlate electronic (impedance) measure-
ments with loading of cells and with respect to
electrode design and chip design. Assemble func-
tional prototypes to test different design features
with respect to fluids obtained from hotdogs. Make a
limited number of chips for preliminary testing in
industry or USDA food laboratories.
• Define types of bioseparations and extent of removal
of extraneous proteins and other substances (e.g.,
salts) that will be needed to achieve the desired
selectivity of binding. Study fundamental character-
istics of binding of food proteins (in hotdogs) to ion
exchange, hydrophobic, or reversed phase
adsorbents that can be packaged on the surface of
the chip, or in a separate sampling system for
conditioning the sample before it is presented to
the chip.
• Interface chip with handheld device. (There are a
number of devices already on the market for
measurement of pH, temperature, etc., that we
expect to be suitable for modification for this
purpose).
Year 3
• Continue experiments from year 2 on the generation
of monoclonal antibodies and the study of cell
membrane proteins of L. monocytogenes, as well as
the interactions between these proteins and the
antibodies. Continue fundamental research on
preparing samples obtained from hotdogs for
presentation to the chip. Examine fractionation and
concentration of cells. Initiate work on sampling
other types of meats, as well as a range of biological
fluids that might be tested for pathogens. Study
microfluidic (fluid flow on the chip) for improving
throughput (number of samples handled per unit
time).
• Combine optimized designs of chips obtained from
laboratory research in addition to tests carried out in
off-site food laboratories with previously determined
25
adsorption protocols (from research in years 1 and 2)
to obtain an operational prototype chip. Interface
chip with prototype handheld instrument. Test chip
prototypes with respect to detection of pathogenic
organisms in hotdog samples in a laboratory setting,
and design key (wet) processing steps for sub-
systems that would be needed to place receptors on
large numbers of biochips at reasonable cost.
Combine chip with hand-held device for field
testing. Begin reliability testing (false positives vs
false negatives). Use protocols that were developed
in year 2. Sample chips to other laboratories in the
food industry for further testing and for generation
of data needed for validating the test for use in food
processing plants.
Year 4
• Use techniques and technology developed in years 1
to 3 to fix other receptors to the chip to detect
different pathogenic organisms. Look at samples
from other types of meats and food products.
Examine possibilities for detecting selected types of
agricultural pests (plant pathogens or animal
pathogens) as an early warning strategy, using
receptors derived from plants or other organisms that
specifically bind to these predators.
• Undertake testing to determine statistical variability
of assays carried out with the prototype chips.
Develop statistical models for interpreting data
obtained from chips. Examine the selectivity of the
chips for the specific pathogen (L. monocytogenes in
this case) relative to large numbers of other contami-
nating organisms. Examine automation or concerted
assays to detect multiple antigens or organisms on
the same chip, or within the same system.
Science/Technology Transfer
There has been no transfer yet. A start-up company in
West Lafayette (Purdue Research Park) is currently
negotiating with the Office of Technology Commercial-
ization to license the technology for purposes of
commercializing the technology is resulting from our
research. The timing of the availability of this technol-
ogy in terms of devices and biochips for the food
industry will depend on the progress of the research,
the rapidity with which suitable licensing and other
technology transfer issue can be worked out, and the
raising of capital to build manufacturing, service, and
support capabilities.
Aside from business issues, constraints to the adoption
of the technology are likely to be cost of goods (current
assays range from about $2.30 to 3.00 per sample) and
time to result. Our goal is to keep this cost below $3.00
and to minimize the time to result.
In addition, one US patent application and associated
PCT filing has been done. Additional patent disclosures
have been submitted or are in the process of being
submitted to the Office of Technology
Commercialization.
Reports, Presentations, and
Popular Articles
Gómez, R., T. Geng, A.K. Bhunia, M.R. Ladisch, R.
Bashir. 2001 “Micro-fabricated Biochip for the
Electronic Detection of Listeria monocytogenes,”
American Society of Microbiology, Annual Meeting,
May 2001.
Geng ,T., R. Gomez, R. Bashir, M.R. Ladisch, A. K.
Bhunia. 2001.”Reaction Patterns of Monoclonal
Antibodies C11E9 and EM-7G1 to Stressed or
Injured Listeria monocytogenes Cells for Use in the
Biochip,” American Society of Microbiology, May
2001.
Ladisch, M.R., A. Bhunia, R. Bashir. 2001. “Biochips:
Integrating Biological Receptors with Computer
Chips, Foods and Nutrition Department, 695
Seminar, Purdue University, January 26, 2001.
Biochips that are affordable, capable of rapid detection of foodpathogens, and easy to use by small food processors as well as majorfood companies are needed.
Ladisch, Badylak, Bashir, Bhunia, Robinson, Singh
26
CFSE Research Reports
Ladisch, M.R., A. Bhunia, R. Bashir, R. Robinson, S.
Badylak, R. Singh, 2000. Biosparations: from
Diagnostics to Therapeutics, Boilermaker Block-
buster Event, Presented at Conseco Fieldhouse on
behalf of Biomedical Engineering Department,
Indianapolis, December 16, 2000.
Ladisch, M.R., R. Bashir, A. Bhunia. 2000. Biochips:
It’s a Small, Small World, TTI Vanguard (Executive
Seminar), Atlanta, GA., Nov 6, 2000.
Tally, S. 2000. “Biochemistry on a Microchip.”
Agricultures Magazine, Purdue University, Summer,
2000.
Scientific Publications
Gomez, R., R. Bashir, A. Sarikaya, M.R. Ladisch, J.
Sturgis, J. P. Robinson, T. Geng, A.K. Bhunia, H.L.
Apple, S.T. Wereley. 2001. “Microfluidic Biochip
for Impenednce Spectroscopy of Biological
Species.” Biomedical Microdevices: BioMEMS
and Biomedical Nanotechnology J.
Bashir, R., M.R. Ladisch, R. Gomez, A. Sarikaya,
J. Sturgis, and J.P. Robinson. 2001. Adsorption of
Avidin on Micro-fabricated Surfaces for Protein
Biochip Applications, Biotechnol. Bioeng.
Bhunia, A.K., Z.W. Jaradat, K. Naschansky, M.
Shroyer, M. Morgan, R. Gomez, R. Bashir, M.R.
Ladisch. 2001. Impedance Spectroscopy and
Biochip Sensor for detection of Listeria
monocytogenes, Proceedings of SPIE-Society of
Photo-Optical Instrumentation Engineers -The
international society for optical engineering,
Bellingham, Washington, Vol 4206.
A rapid and facile detection system that can be used by scientists andnon-scientists alike is needed to ensure a safe food supply.
27
What major problem or issue are
you resolving, and how are you
resolving it?
The cost for health departments to develop fish
consumption advisories is prohibitive. We are testing
new technologies that will reduce or eliminate the need
for direct sampling of fish for determination of residue
levels, and we are developing new methods for
measuring contaminants at a lower cost and with a
rapid throughput. When we reduce the cost for analysis,
federal and state agencies will have the ability to collect
and analyze a greater number of samples and improve
the accuracy of their advisories or surveys.
In addition, we are attempting to determine whether a
total polychlorinated biphenyls (PCB) measurement
correlates with toxicity. Because PCBs in fish include a
possible 209 congeners, current estimates suggest that
toxicity for the congeners may vary by a factor of 5,
and it appears that reporting of total PCB concentration
is not predictive of toxicology. Thus, we are testing fish
tissue samples to see if a total PCB measure correlates
with a standardized toxicity index.
Our research efforts are also attempting to improve the
process by which fish consumption advisories are
developed by using existing data collected over the past
decade and statistical analysis to generate regression
equations for predicting residue levels. We hope that a
simplified advisory will increase angler compliance and
reduce exposure to mercury and PCBs in the diet.
Our objectives are to protect consumers, especially
pregnant and lactating women who can act as a vehicle
to pass contaminants to the fetus or infant. Specifically,
our research objectives are to:
• Develop extraction and cleanup methods to allow
measurement of PCBs in fish tissue prior to analysis
using a commercial enzyme-linked immunosorbent
assay (ELISA),
• Use a “fake fish” device for predicting contaminant
levels in fish, and
Santerre, Miller, Dorworth, Stahl
Rapid Detection of PCBs and Toxicity EquivalenceQuotient (TEQ) in Fish Tissue from Indiana Waters and
Use of a Novel Device to Predict Contaminant Load in FishInvestigator: Charles R. Santerre, Brian k. Miller, Leslie Dorworth, James R. Stahl
• Determine statistical relationships between a given
species and contaminant residues.
How serious is the problem? Why
does it matter?
The major route of exposure to PCBs and mercury is
through fish consumption. The population most at risk
from PCBs is developing fetuses and infants who can
receive PCBs from the mother through the placenta or
breast milk. A meal of contaminated fish consumed by
the mother can result in a larger dose of contaminants
being passed to the fetus or infant. PCBs are believed to
alter reproductive function and delay neurobehavioral
and physical development in perinatal and school-aged
children. It has been estimated that a two- to eight-point
reduction in IQ results from in-utero exposure to PCBs.
In addition, PCBs are believed to adversely affect the
liver, thyroid, and immune systems and to increase
cancer risk from non-Hodgkin’s lymphoma.
Another aspect of this problem is that science is
continually demonstrating the need for omega-3 fatty
acids in the diet of pregnant and nursing mothers. Fish
is the primary source of these healthy fatty acids in the
diet. Therefore, recommendations must be carefully
crafted to inform at-risk populations (e.g., pregnant and
Detection of PCBs in catfish
28
CFSE Research Reports
nursing mothers) of the sources of these lipids that are
also low in contaminants. Many believe that contami-
nants in fish is a problem isolated in the Great Lakes
region because of the significant effort there to measure
contaminant residues in fish and develop strategies to
protect residents. However, EPA (1997) estimates that
35 states have issued fish consumption advisories
related to PCB levels, and 2299 waterbodies in the U.S.
are under some type of statewide health advisory.
Due to the high cost and slow turn-around time for
sample analysis, states are limited in the amount of data
that they can generate when developing fish consump-
tion advisories. For instance, in the 10 years that
Indiana has been analyzing fish, fewer than 700 carp
and 200 largemouth bass composite samples have been
analyzed. Other species were analyzed even fewer
times, with samples being collected over a wide
geographical range.
Another problem with fish consumption advisories is
compliance by residents. It has been estimated that 38%
of anglers do not follow fish consumption advisories in
Indiana. This extrapolates to a potential 10% of the
population, or around 600,000 people who may be
exposed to elevated levels of PCBs and mercury
because they do not follow the advisories. Attempts
should be made to inform anglers and their families of
the importance of the advisories and to simplify the
advisories to encourage greater compliance.
What were your most significant
accomplishments this past year?Single Most Significant Accomplishment
During FY 2001
Our objectives for this year were to validate a lower
cost, rapid assay for the measurement of PCBs in fish
tissue that could be used by states when developing fish
consumption advisories. This would allow states to
analyze more fish samples and develop a better
advisory. Furthermore, we attempted to use a “fake
fish” to simplify the process for determining PCB
residues in fish from a given location.
Our research group was able to validate the extraction
and cleanup methodology prior to analysis with a
commercial ELISA kit for the measurement of PCBs in
fish tissue. This technique reduces analysis time,
produces less solvent waste, and can be performed at a
lower cost than the conventional GC/ECD method.
We analyzed PCBs in fish tissue using the ELISA. We
obtained standard curves for Aroclor 1248, 1254, and
1260 by spiking catfish tissue samples in the ranges of
0.05 to 0.5 ppm and 0.5 to 5.0 ppm. We used a sulfuric
acid/silica gel column for the extraction and cleanup of
the fish tissue sample. Thereafter, we obtained 40 wild
fish samples (0.05 to 0.5 ppm total PCBs) and 12 wild
fish samples (0.5 to 5.0 ppm total PCBs). The concen-
tration of PCB residues we obtained from the ELISA
were not significantly different (p=0.05) when com-
pared with the residues obtained using GC/ECD.
We carried out the analysis at the Department of Foods
and Nutrition, Purdue University. Other collaborators
included J.L Zajicek and D.E. Tillit from U.S. Geologi-
cal Survey, Columbia Environmental Research Center,
Columbia, MO and D.C. Deardoff of Strategic Diag-
nostics Inc., Newark, DE.
Our research group attempted to validate the use of
SPMDs to predict the contaminant load in fish. We
developed SPMDs, also known as “fake fish,” at
various water depths at three locations along the St.
Joseph River in northern Indiana for 30 days. We
analyzed PCB residues in the triolein oil that is placed
in the semipermeable bags using ELISA. We then
compared residues measured in the SPMDs to PCB
residues measured in various fish species from the
same locations.
Our results suggest that measurement of PCBs in the
waterbody is not necessarily a good predictor of
residues in fish tissue. In fact, measurement of PCBs
residues in one species of fish is not necessarily a good
We are testing new technologies that will reduce or eliminate the need for directsampling of fish for determination of residue levels, and we are developing newmethods for measuring contaminants at a lower cost and with a rapidthroughput.
29
predictor of residues in other species. Our results
demonstrate the importance of dietary intake (which
cannot be predicted using SPMDs) on concentrations of
PCBs in the edible tissue of fish.
Other Significant Accomplishments
We analyzed polychlorinated biphenyls (PCBs) in fish
tissue using enzyme-linked immunosorbent assay
(ELISA) and gas chromatography/electron capture
detector (GC/ECD) methods. We collected fish tissue
samples in 2000-2001 during an Indiana fish survey.
We analyzed extracts derived from fish tissue samples
by the GC/ECD. We then divided the extracts and fish
tissue samples into two groups such that they fell into
either one of two ranges 0.05 to 0.5 ppm total PCB and
0.5 to 5.0 ppm total PCB. We exchanged the extracts
into methanol and ELISA diluent, and analyzed them
by ELISA. We analyzed fish tissue samples by ELISA
after a sulfuric acid/silica gel extraction and clean-up.
We compared the data we obtained in the two ranges
from the analysis of extracts and fish tissue by ELISA
with the GC/ECD data. From 0.05 to 0.5 ppm range
(n=40), ELISA analysis of fish tissue was as precise as
the GC/ECD, but they were significantly different from
the Soxhlet’s extracts (p<0.05). However, from 0.5 to
5.0 ppm range (n=12), the results we obtained from the
GC/ECD of fish tissue and ELISA analysis of the
extracts were not significantly different, but were found
to be different from the ELISA of fish tissue (p<0.05).
We immersed Triolein-filled semipermeable membrane
devices (SPMD) at three locations along the St. Joseph
River in northern Indiana for 30 days to see if the PCB
content of fish from the same location could be
predicted with this model device. We analyzed Triolein
from the SPMD’s for PCB using enzyme-linked
immunosorbent assay (ELISA) and compared it to
residues detected in fish collected from the same
locations. There was a significant difference (p<0.05)
in total PCB concentrations between SPMD samples.
However, due to variability in PCB residues between
species and low PCB residues in SPMDs, we could not
determine a direct correlation between PCBs in fish
and SPMD.
Other research groups have reported the successful use
of SPMDs to predict PCB concentrations in fish. Due
to their experimental designs, their results suggested a
good correlation between PCB residues in fish tissue
and SPMDs. We have demonstrated that SPMDs are
not sensitive enough to predict PCB levels in fish tissue
due to contributions from the fish’s diet which cannot
be predicted using this device.
What major accomplishments do you
anticipate over the life of the project,
and what do you predict their impact
will be?
Our project has shown that it would be necessary to
standardize the ELISA kit each time an analysis in fish
tissue is carried out, using Aroclor-spiked fish samples.
Between 0.05 to 0.5 ppm, the three mixtures are not
statistically different, therefore either one of the three
Aroclor mixtures can be used to standardize the ELISA
kit. In the range of 0.5 to 5.0 ppm, Aroclor 1254 is the
best predictor of total PCB in the fish tissue samples.
So far, this method has proven to be a rapid, feasible
and economically viable method for the analysis of
PCBs in fish tissue samples.
We anticipate that the impact that our project could
have is that the fish consumption advisories could be
built on the analysis of larger number of fish samples.
Thus, advisories would be more accurate and helpful to
anglers and their families.
What do you expect to accomplish,
year by year, over the duration of the
project?
The objective of the project is to show that the ELISA
is a feasible method to replace the GC/ECD method for
Santerre, Miller, Dorworth, Stahl
30
CFSE Research Reports
the analysis of total PCB in fish tissue samples. The
next step will be to show that the ELISA is a suitable
method for the determination of total PCB even in aged
fish samples as opposed to fish tissue samples that have
been analyzed with freshly spiked Aroclors. We will
compare the method with the conventional GC/ECD
method to determine if they are significantly different.
We are investigating other methods that can be used to
measure total PCB and specific PCB congeners with
reduced sample preparation. Techniques that have come
of age in recent years may offer analytical solutions
that reduce cost and increase sample throughput. One
technique that we are currently investigating uses solid
phase micro-extraction (SPME) prior to GC/MS/MS
analysis. Our efforts to develop simplified extraction
and clean-up methods prior to ELISA may be useful
when taking this new approach.
We are attempting to use statistical reduction of data
collected by state agencies over the past decade to build
regression equations for predicting PCB residues in fish
tissue. We have had some success developing a
regression equation to predict PCBs in carp.
We will use the ELISA and a PCB congener method to
measure PCBs in fish oil supplements. Additionally, we
will measure the omega-3 fatty acids to determine the
quality of individual products. Because these products
may be taken by at-risk populations, it is important that
they be low in residues and appropriately labeled for
fatty acid profile.
Science/Technology Transfer
In addition to technical presentations, we have devel-
oped a Web site (http://www.cfs.purdue.edu/extension/
foodsafety/anglingindiana/) to inform anglers and their
families of the Indiana fish consumption advisories.
The Web site provides information on fishing locations
that may have contaminated fish, the safety of farm-
raised fish, the proper preparation techniques for
reducing contaminants, the nutritional value of fish, and
the dangers of consuming fish that are contaminated
with PCBs or mercury.
We are working closely with staff at the Indiana State
Department of Health and the Indiana Department of
Environmental Management. We hope to demonstrate
to these agencies the benefits of using a rapid assay in
the development of fish consumption advisories.
Presentations and Popular Articles
Lasrado, J.A., Santerre C. R., Deardorff, D.C., Stahl,
J.R., Noltenmeyer, T. 2002. Measurement of PCBs
in Fish Tissue Using GC and ELISA. Institute of
Food Technologists Annual Meeting, Anaheim, CA.
Paper #46I-1, Technical Poster Session.
Shim, S.M., Santerre, C.R., Dorworth, L.E., Miller,
B.K., Stahl, J.R. 2002. Prediction of PCB Concen-
tration in Fish Using Semipermeable Membrane
Devices (SPMD). Institute of Food Technologists
Annual Meeting, Anaheim, CA. Paper #46I-2,
Technical Poster Session.
An article in the May 2001 issue of Food Technology
(page 200) describes our outreach efforts in the
following excerpt: “Newsmakers would like to
extend hearty congratulations to Purdue University
professor Charles Santerre for his proactive use of
the news media in circulating to consumers data
from his recent study on farm-raised fish.”
Scientific Publication
Lasrado, J.A., C.R. Santerre, J.L. Zajicek, D.E. Tillitt,
D.C. Deardorff, ELISA analysis of PCBs in fish
tissue. 2001 IFT Annual Meeting Book of Abstracts.
44G-5, p.108.
We anticipant that the impact that our project could have is that the fishconsumption advisories could be built on the analysis of larger number of fishsamples. Thus, advisories would be more accurate and helpful to anglers andtheir families.
31
What major problem or issue are
you resolving and how are you
resolving it?
Owing to the current fast-paced life-style of most
people and their preference for “nutritious yet natural”
food, consumer-buying trends lean towards ready-to-eat
fruits and vegetables (i.e., fresh produce with high
quality). The fresh-cut produce market has been
projected to contribute to 25% of the retail food market
(Garret, 1994).
One major problem with fresh-cut (i.e., minimally
processed) fruits and vegetables is illness caused by
pathogens that may be present on the surfaces of the
produce. The most common method used today to
eliminate microorganisms in fruits and vegetables is
heat. Although thermal processing is an effective
method of elimination, it destroys original sensory
attributes (i.e., flavor, texture, color, and some heat-
sensitive nutrients) of these food products to a certain
degree. Therefore, the food industry, especially the food
distribution and catering sector, has shown an interest
in developing alternative processing technologies that
reduce or eliminate the initial microbial loads and
pathogens in fruits and vegetables and yet preserve
their natural sensory qualities.
We are investigating alternative techniques of non-
thermal processing methods such as using rinsing
solutions and high-pressure coupled mild heat treat-
ments in this project to determine the effectiveness of
each treatment and of a combination of these methods
of eliminating food-borne pathogens in fruits, veg-
etables, and sprouts. We are studying such sanitizer
solutions as chlorine dioxide solution (with residual
chlorine dioxide not exceeding 3 ppm, FDA
21CFR173.300) and ozonated water (GRAS) and a
solution containing essential oils such as clove, thyme,
and oregano oils (GRAS). We are treating such fruit
items as strawberry and grape, such vegetable items as
lettuce and baby carrots, and such sprouts items as
alfalfa and mung bean.
Alternate Technologies for Elimination of FoodbornePathogens in Fruits, Vegetables, and SproutsInvestigators: Rakesh K. Singh, Arun K. Bhunia, Richard L. Stroshine, James E. Simon
We are studying the treatments specifically to target
elimination of food-borne pathogens such as, E. coli
O157:H7, Listeria monocytogenes, and Salmonella
enteritidis. We are also conducting sensory evaluations
on the fruits, vegetables, and sprouts that have been
subjected to these treatments, in order to evaluate
whether such treatments impart adverse sensory
qualities to the produce.
How serious is the problem? Why
does it matter?
The Centers for Disease Control and Prevention
reported that microorganisms are the most common
cause of food-borne illness, contributing to more than
60% of annual food-borne outbreaks (Sofos et al.,
1998). Microbial pathogens in food cause an estimated
76 million cases of human illness and claim approxi-
mately 5000 deaths in the United States each year. Six
of the most common bacterial pathogens incur the cost
of approximately $9.3 to $12.9 billion annually due to
human illness. In 1995, more than 20,000 people in
North America were reported to have contracted
salmonellosis caused by eating contaminated alfalfa
Singh, Bhunia, Stroshine, Simon
Rakesh Singh’s work is helping make fruit
and vegetables safer.
32
CFSE Research Reports
sprouts. Van Beneden et al. (1999) concluded that
current sprouting methods are inadequate to protect
consumers from such events. Other recent outbreaks of
food-borne illness associated with fresh produce
include E. coli O157:H7 in lettuce.
These outbreaks have raised concerns regarding the
potential safety of fruits and vegetables that are not
processed to reduce or eliminate pathogens. Develop-
ment of alternate methods of minimal processing
techniques such as those outlined in our report will
provide safer products with minimum loss in nutritional
and sensory attributes to the consumers; that is, such
alternative methods will preserve the natural qualities
of the produce.
What were your most significant
accomplishments this past year?
We conducted the research to evaluate the efficacy of
washing treatments (aqueous chlorine dioxide,
ozonated water, and thyme essential oil) by using
different inoculation methods, population load, and
repeated washing procedures against E. coli O157:H7
on shredded lettuce. We also tested the efficacy of the
sanitizers in 0.1 % peptone water against test micro-
organisms. We tried a combination of different sanitizer
treatments to see the synergistic effect of washing
treatments for reduction of microbial population on
lettuce and baby carrots. And we have completed
studies related to washing of alfalfa seeds before
sprouting with different sanitizers against E. coli
O157:H7 and S. enteritidis. Sequential washing of
alfalfa seeds during and after sprouting to inactivate
pathogenic microorganisms with different sanitizers is
in progress, as are experiments involving high-pressure
treatment of alfalfa seeds and sprouts. We also intend to
try a combination of different sanitizers treatments with
mild heat treatment on other fruits and vegetables,
mung bean seeds, and sprouts.
What major accomplishments do you
anticipate over the life of the project,
and what do you predict actual
impact will be?
We anticipate developing methods or a combination of
methods to ensure higher reduction in bacterial load on
fresh produce. This will mean safer product for
consumers.
What do you expect to accomplish,
year by year, for the duration of the
project?
In year 2 we expect to couple high pressure with mild
heat treatment and combine that method with the
previously mentioned technologies.
Science/Technology Transfer
The technologies we develop will be evaluated in local
industries for their efficiency in commercial settings.
Several industries dealing in fresh produce have shown
interest in our research. Once the project has reached
completion stage (probably at the last quarter), the
techniques that produce the more promising results will
be tested at the industrial scale.
33
Scientific Publications
Singh, N., R.K. Singh, A.K. Bhunia, R.L. Stroshine,
J.E. Simon. 2000. Sequential disinfection of E. coli
O157:H7 on shredded lettuce leaves by aqueous
chlorine dioxide, ozonated water and thyme essential
oil. Proceedings of SPIE-Photonic Detection and
Intervention Technologies for Safe Food. v. 4206. p.
159-166.
Singh, N., R.K. Singh, A.K. Bhunia, R.L. Stroshine.
2001. Effect of inoculation methods for evaluating
the efficacy of different sanitizers against E. coli
O157:H7 on lettuce. Institute of Food Technologists.
Abstract p. 143.
Singh, N., R.K. Singh, A.K. Bhunia, and R.L.
Stroshine. 2002. Efficacy of different sanitizer
treatments for eliminating Escherichia coli O157:H7
from alfalfa seeds prior and during sprouting.
Institute of Food Technologists Meeting. Paper
#91E-30.
Ariefdjohan, M.W., R.K. Singh, P.E. Nelson, A.K.
Bhunia, R.L. Stroshine. 2002. The efficacy of high
hydrostatic pressure in eliminating Escherichia coli
O157:H7 and Listeria monocytogenes from alfalfa
seeds. Institute of Food Technologists Meeting.
Paper #15D-17.
Singh, Bhunia, Stroshine, Simon
The food industry, especially the food distribution and catering sector, has shownan interest in developing alternative processing technologies that reduce oreliminate the initial microbial loads and pathogens in fruits and vegetables,and yet preserve their natural sensory qualities.
References
CFR21. 2000. Code of Federal Regulations. Title 21:
Food and Drugs. Section 173.300. US Printing
Office, Washington D.C.
Garrett, E. 1994. Challenges and opportunities in
marketing fresh-cut produce. In: Modified Atmo-
sphere Food Packaging. Ed. : A.L. Brody. Institute
of Packaging Professional, Herndon, VA, USA.
P. 31-34.
Sofos, J.N., L.R. Beuchat, Davidson, E.A. Johson.
1998. Naturally occurring antimicrobials in food.
Council for Agricultural Science and Technology,
Ames, Iowa, USA.
Van Beneden, C.A., W.E. Keene, R.A. Strang, D.H.
Werker, A.S. King, B. Mahon, K. Hedberg, A. Bell,
M.T. Kelly, V.K. Balan, W.R. Mackenzie, D.
Fleming. 1999. Multinational outbreaks of Salmo-
nella enterica serotype Newport infections due to
contaminated alfalfa sprouts. Journal of American
Medical Association, 281(2): 158-162.
34
CFSE Research Reports
Introduction
Guaranteeing a safe food supply in a rapidly changing
marketplace of transgenic food plants and industrial
agricultural technologies is a challenge for the up-
coming century. To meet this challenge, improved
efficiency in the agricultural industry is necessary,
including understanding how food plants transport and
store food and potentially harmful non-food compo-
nents. From the development of genetically synthesized
biocides to production enhanced enzymes/growth
products, many concerns are being raised over food
safety. Similarly, modification of food crops to produce
non-renewable industrial resources, such as modified
lipids to replace dwindling petroleum, will further
threaten the safe quality of food.
Understanding the kinetic and thermodynamic inter-
actions of synthetic/transport pathways for plant
materials is critical to food safety. As we start to rely
more on transgenic crops for both food and industrial
products, the issue of cross-contamination within the
plant becomes increasingly relevant. For example, can
a new crop be designed to produce hydrocarbons for
fuel as well as food oils, or do the risks and additional
processing safeguards necessitate strictly different
crops for different uses? The answer to this and similar
questions lies in a deeper understanding of how
biological systems transport and store these materials.
Rationale
In recent years we have seen genetically modified
plants affect environmental issues by implementation of
biocides and increased resistance to disease and
herbicides. While work continues in this area, research
has now developed relative to the genetic improvement
of food quality and shelf life. Over 60 million tons of
edible oil are generated each year from plants (Murphy,
2000). Because plant lipids are a key component of
both foods and industrial products, it seems logical to
start here when addressing the needs of tomorrow’s
society. This thought is further supported by USDA-
projected surpluses for agricultural commodities that
hurt the farm economy and induce federal subsidies.
The goal of the project reported here is to examine
engineering principles that can help explain the
mechanisms for storage of lipids within soybeans.
General Review of Literature
Currently, less than 15% of total vegetable oil produc-
tion is attributed to industrial purposes; however, this
number can be expected to rise with the depletion of
petroleum reserves (Murphy, 2000). Plants segregate oil
body production designating oils for membrane
incorporation or storage. Although the mechanism for
this segregation has not been completely elucidated
(Lacey et al., 1999; Murphy, 1999), several models
exist to explain storage oil production.
First, storage oil bodies are produced at the endoplas-
mic reticulum (ER) and then bud off with a monolayer
of phospholipids surrounding them (Loer and Herman,
1993). Study of these oil bodies in the past two decades
shows that the protein oleosin can also be found on the
surface of these storage vesicles (Tzen et al., 1993;
Herman, 1987). Oleosins probably evolved as a way to
allow excess triacylglycerols to be removed from the
ER and thus maintain normal function of this organelle
(Huang, 1996). These hydrophobic proteins are
believed to either deter coalescence in seed desiccation
(Tzen and Huang, 1992) or provide stability throughout
imbibition (Leprince et al., 1998). Leprince and
colleagues suggest that the high viscosity of the
cytoplasm and the rate of drying actually prevent
coalescence (1998).
Physiologically, this implies that the oleosins help to
provide the ideal surface area for lipases to attach
during post-germinate growth and shield the PL from
hydrolysis by phospholipases (Huang, 1994; Tzen and
Huang, 1992). However, the mechanism that maintains
consistent size of oil bodies at 0.5-2.0 mm (Huang,
1994; Tzen et al., 1993) remains unknown.
Development and Characterization ofOil Body Proteins
Investigator: Douglas K. Allen
CFSE Student Research Reports
35
In essence, there remain many questions as to the
biological phenomena that explain the partitioning of
different hydrophobics, including toxic components
within living organisms. Fundamentally, proteins such
as oleosin are key to developing an understanding of
these systems. Additionally, future transgenic crops
must be able to correctly partition “unusual oils” and
genetically abnormal products away from plant
membranes, where they can be more easily processed.
But an equally challenging question remains: How will
the current food supply be affected?
Certainly, geographic separation of similar species will
help prevent outcrossing, but even with sterile male
species lines, some cross contamination is probable.
Because our society as a whole is so dependent upon
petroleum, the plants will become bigger producers of
industrial products, creating concerns about the foods
produced within these plants and about how to use the
plant most efficiently. In many cases, the food compo-
nent may even become a byproduct. As the utility of
feedstocks change, the ways in which we process plants
will change and lead to more food safety concerns.
Objectives
One focus of our project is to explore expression
systems to obtain recombinant proteins at desirable
levels and then study the effects of oil modification
through these systems, starting with E.coli production.
The growth characteristics, expression localization and
levels, degree of biological activity and proper folding,
regulatory issues, and post-translational modifications,
for example,can all significantly affect this process.
Unlike other prokaryotes and most eukaryotes, E. coli
does not require complex preformed organic molecules,
thus growth can be easily controlled with a simple
feeding substrate that is most cost effective.
As a model system, the complete genome of E. coli is
known and therefore is readily available for recombi-
nant gene expression work. Unfortunately, hydrophobic
and membrane-bound proteins are often expressed in
miniscule amounts, if at all, and may lead to cell
apoptosis. Because eventual goals include the examina-
tion of oleosin in a near natural oil body environment,
we will eventually transform other hosts, such as yeast,
as well. However, the simplicity and breadth of
knowledge about E. coli systems dictates their use for
initial production needs of protein characterization. As
part of this effort, we must also consider methods to
quantify protein production and establish localization.
Additionally, we may measure fusion partners for
solubilization and rapid detection of different compo-
nents by use of green fluorescent proteins, although this
is more likely to occur in work with eukaryotic or
more advanced systems that have better partitioning
capabilities.
Results and Discussion
E. coli Transformation and Thiofusion
Due to the technical difficulties expected with recombi-
nant E. coli production and yeast systems, a fusion
partner, we considered thioredoxin to help solubilize
the protein and prevent cell apoptosis. We prepared
purified plasmids to be used as a PCR template from
the original cDNA E. coli transformation. The presence
of the plasmid as a template necessitated excising
purified bands of PCR product form agarose gel
electrophoresis, which were used in cloning reactions
and then subsequently transformed in to E.coli using
Invitrogen’s pBAD/Thio Expression System. Colony
PCR for positive clones and the correct orientation
using restriction digestion ensued to produce three
clones with the desired properties.
Optimization of Recombinant Production
Successful transformation has led to experimental
development and optimization of E. coli growth and
protein expression. First, we considered the time period
of induction. Western blots showed no significant
improvement of protein concentration for the time
periods considered of 2, 4, and 6 hours of induction
(Figures 1 and 2). Additionally, concerns of proteolysis,
that we are currently considering may make a shorter
Because our society as a whole is so dependent upon petroleum, plants willbecome bigger producers of industrial products, creating concerns about thefoods produced within these plants and about how to use the plant mostefficiently.
Allen
36
CFSE Research Reports
time period optimum for current production. Clearly,
the lower set of bands could be an effect of proteolysis,
as they are reactive to antibody detection.
We varied arabinose concentration, which is the source
of induction, between 0.00002% and 0.2% levels and
found it to be most optimized by 0.002%, which
showed nearly twice the amount of protein as the next
best arabinose concentration of 0.02.
The necessity of visualization on western blot is an
indication of relatively low protein yield and further
supports this optimization work, because standard
coomassie blue staining could not distinguish the
product from E. coli native proteins. Therefore, we
considered further optimization of pH, temperature, and
oxygen demand individually. We noticed no significant
differences over a range of 28-37˚C temperatures,
and pH seems to be optimum at 7.5-8.0, although
differences are still small in magnitude. Finally, for 250
ml flasks used, we varied the amount of LB medium
from 20-50 ml to change surface to volume ratio and
improve the oxygen demand, with a small improvement
for decreased volumes.
Protein Localization
Low protein expression levels created concern about
the localization of the protein. Although all previous
westerns employ a standard SDS page gel sample
buffer that should disrupt inclusion bodies and mem-
branes, understanding the relative magnitude of
proteins located within the membrane fraction, soluble
fraction or as inclusion bodies will shed light on
production levels, hydrophobic component storage
mechanisms, the amount of purified material that is
recoverable and possible areas for yield improvement.
Thus, using ELISA to quantitate relatively the magni-
tude of each fraction, we created samples. “Wild type”
represents a non-transformed E. coli sample; “basal”
represents a transformed fraction that has not been
induced with arabinose; “glucose-repressed” is the
result of adding glucose to the LB medium, which is
postulated to strongly repress any basal transcription;
“extracellular” is the fraction that is located within the
broth; “soluble” is the fraction that is within the cell but
not located in a specific membrane or inclusion body
fraction; and the membrane and inclusion body
fractions are self-explanatory.
Table 1. Production of Protein Within E.coli
Description Percentage
Wild Type Negligible
Basal Negligible
Glucose repressed Negligible
Extracellular Negligible
Soluble fraction 58 %
Inclusion Body fraction 27 %
Membrane fraction 13 %
The high level comparatively of soluble protein
signifies the ability of the thiofusion to adequately
solubilize at least to some extent, even very hydropho-
bic proteins such as oleosin. This high degree of soluble
protein also provides some light for future experiments,
because solubilization and aggregation continue to be
substantial hindrances. We suspect that the inclusion
body fraction, although relatively concentrated, may be
difficult to utilize and study for this reason.
Optimization of Recombinant
Oleosin Purification
We are also optimizing nickel column purification for
production of oleosin. The largest protein loss appears
to be in the cell lysis stage because oleosin is only 50%
soluble, and remaining fractions that exist as inclusion
bodies and membrane fractions are not in acceptable
forms for batch binding to columns. Preliminary results
have shown limited purification (Figure 3). A western
blot of the identical samples shows that the contami-
nants do not contain the poly-His epitope and are
therefore non-specific binders that may be effectively
removed with repeated columns or better washing
Figures 1 and 2. Optimization of Inducer Concentrations
CFSE Student Research Reports
37
techniques (Figure 4). Unfortunately, a nickel affinity
column should be capable of much better separations.
We hypothesize that either his tail is not located on the
exterior structure, or not enough protein is being loaded
to achieve the desired concentration and purity.
Concluding Remarks
Exploration of P. pastoris-based production and
modification of clones with green fluorescent protein to
track these storage systems is ongoing. The rich
diversity of lipids naturally occurring makes them an
ideal area for metabolic engineering. Nature has proven
that the variation of oilseed composition has little
consequence on plant morphology and physiology
(within reason) because the primary function of storage
oil is as a carbon and energy source. The future demand
for plastics, fuels, and other petro-based feedstocks can
be partially offset by advances in this area, reducing
U.S. dependence on international suppliers and
improving the overall farm economy of the Midwest.
However, these same crops serve to provide us with
many valuable food products, and their safe production
for human consumption must not be jeopardized in the
process.
Major hurdles lie in the lack of understanding of lipid
metabolic regulation, which appears to be multi-
faceted. The safe production of food and industrial
products within the same plant can only come as a
result of increased knowledge of plant systems. As our
understanding of these systems and how best to
engineer them to suit our needs evolves, so will the
quality of life we enjoy.
References
Herman, E.M., “Immunogold–Localization and
Synthesis of an Oil-body Membrane Protein in
Developing Soybean Seeds.” Planta 172: 336-345,
1987.
Huang, A.H.C., “Oleosins and Oil Bodies in Seeds and
Other Organs.” Plant Physiol. 110: 1055-1061,
1996.
Huang, A.H.C., “Structure of Plant Seed Oil Bodies.”
Curr. Op. Str. Biol. 4: 493-498, 1994.
Huang, A.H.C., “Oil Bodies and Oleosins in Seeds.”
Ann. Rev. Plant Physiol. Plant Mol. Biol. 43: 177-
200, 1992.
Lacey, D.J., F. Beaudoin, C.E. Dempsey, P.R. Shewry,
J.A. Napier, “The Accumulation of Triacylglycerols
within the Endoplasmic Reticulum of Developing
Seeds of Helianthus Annus.” Plant J., pp. 397-405,
1999.
Leprince, O., A.C. van Aelst, H.W. Pritchard, D.J.
Murphy, “Oleosins Prevent Oil body Coalescence
During Seed Imbibition As Suggested by a Low-
temperature Scanning Electron Microscope Study of
Desiccation-tolerant and –sensitive Oilseeds.”
Planta 204: 109-119, 1998.
Loer, D.S., E.M. Herman, “Cotranslational Integration
of Soybean (Glycine max) Oil Body Membrane
Protein Oleosin into Microsomal Membranes.” Plant
Physiol. 101: 993-998, 1993.
Murphy, D.J., “Development of New Oil Crops in the
21st Century.” Inform. 11: 112-117, 2000.
Murphy, D.J., J. Vance “Mechanisms of Lipid-body
Formation.” TIBS. 24: 109-115, 1999.
Tzen, J.T.C., Y. Cao, P. Laurent, C. Ratnayake, A.H.C.
Huang, “Lipids, Proteins, and Structure of Seed Oil
Bodies from Diverse Species.” Plant Physiol. 101:
267-276, 1993.
Tzen, J.T.C., A.H.C. Huang, “Surface Structure and
Properties of Plant Seed Oil Bodies.” J. Cell Biol.
117: 327-335, 1992
Figures 3 and 4. Stained and Western Blot of Oleosin Nickel Column Purification
Allen
38
CFSE Research Reports
Introduction
Listeria monocytogenes is a rod-shaped, Gram-positive
foodborne pathogen. The Centers for Disease Control
and Protection (CDC) estimate that as many as 2500
people in the United States become infected by Listeria
every year. Approximately 500 of those infected die
from the infection or from complications relating to
the infection, the majority of whom are immuno-
compromised. Listeriosis is an especially serious health
threat to pregnant women, newborns, the elderly, as well
as to those who are ill, such as people with AIDS or
cancer. A few Listeria cells may be enough to cause very
serious illness in these individuals (Mead et al., 1999).
Due to the ability of Listeria to survive in diverse
environments, it is found virtually everywhere,
although the CDC estimates that as high as 99% of
Listeria infections are through foodborne transmission.
L. monocytogenes has been reported to be associated
with milk and dairy products, raw vegetables, poultry,
meats, fish, and ready-to-eat foods such as lunch-
meats. Much of this is caused by post-processing
contamination.
Conventional L. monocytogenes detection methods are
time consuming, requiring a 24- to 48-hour enrichment
followed by a variety of tests and incubations, resulting
in a total identification time of 5 to 7 days. These
detection methods may not even be sufficiently sensitive
for detection of low concentrations of cells from food.
Further, these test also do not indicate the pathogenicity
of the organism (Hitchins et al., 1995).
Several alternative methods have been developed for the
isolation of Listeria, including immunoseparation by
immunomagnetic bead systems. Bacteria-specific
antibodies are conjugated to the surfaces of the beads,
which are able to capture target bacteria from a sample
suspension. These beads can then be plated for enumera-
tion or used directly in other assays. Commercial beads
for the collection of Listeria cells have been found to
detect <10 CFU/g following a 24-hour enrichment
(Uyttendaele et al., 2000).
Recently, biosensor-based methods have been discussed
for use in the detection of foodborne pathogens as an
alternative to the conventional methods (Ivnitski et al.,
1999). These systems detect and quantify biological
reactions with the test organism. Biosensor systems
were also developed for monitoring mammalian cell
growth and propagation (Pancrazio et al., 1999). Many
of these mammalian cell culture biosensors are based
on impedance spectroscopy, which involves passing a
current of varying frequencies through an object to
determine its impedance values (Kyle et al., 1999).
Individual mammalian cells can be equated to simple
circuits (Kyle et al., 1999), so changes in their mem-
brane potential can be monitored.
Several cytotoxicity assays have been developed to
detect virulent strains of L. monocytogenes (Van
Langendonck et al., 1998; Pine et al., 1991; Dallas et
al., 1995; Siddique, 1969). A hybridoma, Ped-2E9 cell-
based cytotoxicity assay has been developed, which
measures L. monocytogenes-induced cell damage in 1.5
to 2 hours (Bhunia and Westbrook, 1998).
Rationale for the Research
Listeria monocytogenes is a dangerous pathogen, as is
evident from the yearly rate of 2500 illnesses and 500
deaths. Therefore, steps must be taken to control its
emergence in the food supply. With the increasing
number of foodborne outbreaks of L. monocytogenes in
recent years, it is obvious that the time-consuming and
often tedious conventional methods for detection of
Listeria are not sufficient. In light of the current
situation, it is apparent that more sensitive and rapid
methods of detection are necessary. This may be
accomplished through the promising introduction of
biosensors into the field of pathogen detection.
Development of an Immunoseparation and ImpedanceSpectroscopy Based Cytotoxicity Assay to Isolate andDetect Listeria monocytogenes from Hotdog Samples
Investigator: Kristen M. Naschansky
CFSE Student Research Reports
39
Research Objectives
The overall objective of this research was to develop
tools, techniques, and rapid methods for assaying L.
monocytogenes in order to further promote the food
safety initiative. Specific objectives were to capture and
concentrate Listeria cells by immunoseparation,
following a brief selective enrichment step, and to
detect viable cytopathogenic L. monocytogenes strains
by analyzing interaction of isolates with mammalian
cells (Ped-2E9) by electrical impedance spectroscopy.
Materials and Methods
Bacterial Cultures and Mammalian
Cell Lines
The bacterial cultures used are Listeria monocytogenes
F4244 and Scott A and L. innocua F4248. The mamma-
lian cell culture used is a murine hybridoma Ped-2E9
cell line developed by Bhunia and Johnson (1992).
Preparation of Beads with Antibody and
Immunoseparation
For initial capture and concentration of Listeria cells,
we developed an immunobead that includes conjuga-
tion of anti-Listeria polyclonal Ab to agarose beads.
Listeria capture efficiency of immunobeads was
compared with that of Anti-Listeria Dynabeads
(magnetic beads) from Dynal® (Dynal A.S., Oslo,
Norway).
We diluted an 18-hour culture of Listeria mono-
cytogenes to a concentration of approximately 105 CFU/
ml and aliquoted in volumes of 1 ml to microcentrifuge
tubes. We added Dynal® Anti-Listeria Dynabeads or
immunobeads at 20, 50 or 75 µl/ml of bacterial
suspension and allowed them to react for 30 minutes in
a rotary-shaker at room temperature. We also gave
bacterial samples without the beads the same treatment
and plated them to determine the presence of any
background bacteria. We collected and plated both
beads on Oxford agar.
Analysis of Naturally Contaminated or
Spiked Hotdog Samples by
Immunoseparation
We serially diluted an 18-hour culture of L. mono-
cytogenes (10-5 to 10-9) and added 1 ml of each dilution,
plus a dH20 control, to a stomacher bag containing a
hotdog in 100 ml half-Fraser broth. We incubated bags
at 30ºC, removing a sample at 0, 6, 12, 18 and 24 hours
for analysis. For each sampling period, we massaged
the stomacher bag and removed 1 ml from the filtered
side of the stomacher bag to a microcentrifuge tube.
Bacteria were captured by Dynabeads or immunobeads,
and we plated them directly (without
immunoseparation) onto Oxford agar plates and
incubated them at 37ºC for 24 hours. We picked natural
isolates obtained from uninoculated hotdogs and
transferred them to Brain-Heart Infusion (BHI) agar
stabs for later confirmatory tests.
Cytotoxicity Assay
We suspended the hybridoma Ped-2E9 cells (2 x 106
cells/ml) in 500 µl or 1 ml of 0.15 M cell- and infected
them with Listeria spp. (Bhunia et al., 1994) in either a
multiplicity of infection (MOI) of 100:1 (bacteria:
hybridoma) or with 100 µl of concentrated beads
following the immunoseparation procedure. In some
experiments, we added dithiothreitol (DTT) at a
concentration of 0.5 mM to all samples (Westbrook and
Bhunia, 2000). We incubated cell suspensions at 37ºC
and made viable Ped-2E9 cell counts at hourly intervals
by staining with 0.4% Trypan blue and observing under
the microscope.
L. monocytogenes Confirmation
CAMP Assay
We streaked isolates on a blood agar plate perpendicu-
lar to a streak of ß-lysin producing Staphylococcus
aureus to distinguish between Listeria spp. Enhanced
lysis of red blood cells in the junction of S. aureus
streak and the Listeria streak is indicative of a positive
CAMP test for L. monocytogenes (Christie et al., 1944).
Naschansky
40
CFSE Research Reports
RiboPrinter® Analysis
We analyzed isolates by using an automated
RiboPrinter® (Qualicon, Inc., Wilmington, DE). Briefly,
we picked isolates from BHI agar plates and suspended
them in the sample buffer and transferred 30 µl of each
suspension to the sample carrier wells for analysis. We
obtained genomic fingerprint patterns of isolates after 8
hours.
Impedance Spectroscopy-Based
Cytotoxicity Assay
Impedance Probes and Accessories
Several probes were designed and constructed by Dr.
Mark Morgan (Purdue University, Department of
Agricultural and Biological Engineering), including a
tetrapolar probe and a copper-chip (both used with a 1
KHz circuit box), a conduction-based cup, a copper-
chip, a stainless steel impedance-based cup (used with
an impedance analyzer at 200 KHz), and a gold pin
probe (used with an impedance analyzer in the range of
199-201 KHz).
Interdigitated Microsensor Electrode
(IME)-Chip
We micro fabricated an IME 1550.5 M-Au-P by
ABTECH Scientific, Inc. (Richmond, VA) from
magnetron sputtered gold onto an insulating substrate
chip in a monolithic configuration. The interdigitated
fingers were spaced 15 µm apart, and the apparatus was
packaged with attached lead wires and encapsulated.
We used a Hewlett-Packard 4194 Impedance/Gain
Phase Analyzer to determine impedance, and we
analyzed data by LabVIEW™ 5.1 Software (National
Instruments™, Austin, TX).
Buffers. We evaluated various buffers for use with the
impedance-based cytotoxicity assay. We suspended
hybridoma Ped-2E9 cells in varying ratios of 5 mM
Tris-glycine (pH 7.2) and 0.15 M cell-PBS buffers,
containing various levels of additives which we
believed to have some potential for altering the osmotic
conditions of the buffer. We determined viability of
Ped-2E9 cells by staining with 0.4% Trypan blue and
Figure 1. Immunoseparation
comparison of Dynal® Anti-Listeria
Dynabeads and Immunobeads from
Spiked (L. monocytogenes F4244) or
Naturally Contaminated Commercial
Hotdog Samples. The percent captured
was calculated based on the number of
bacteria captured by the beads divided
by the number of bacteria in the sample
as per Oxford plating. Data are the
average of two experiments and the
average capture rate for all three
dilutions at each hour were found to be
statistically similar. The control values
are not shown; there was no detectable
direct count at any of the 6 h intervals,
however there were cells recovered by
both types of beads at 18 and 24 h,
resulting in yields greater than 100%.
With the increasing number of foodborne outbreaks of L. monocytogenesin recent years, it is obvious that the time-consuming and often tediousconventional methods for detection of Listeria are not sufficient.
CFSE Student Research Reports
41
observing under the phase contrast microscope. We
selected a combination of 4 mM Tris-glycine and 30
mM cell-PBS (TGCP), and the combination appeared
to be osmotically favorable for Ped-2E9 survival.
IME-Chip Protocol
We attached the IME-chip to the electrical lead, which
was attached to the HP-impedance analyzer. We placed
a rubber O-ring (approx. 5 mm in diameter) on the
IME-chip surface, encircling the interdigitated finger
area of IME-chip. We added a 20-µl volume of Ped-
2E9 cell suspensions, with or without bacterial
treatments, to the center of the O-ring. We collected
impedance data after a predetermined 3-minute settling
period. We made all readings in the frequency range of
1 – 10,000 KHz. In some experiments, we added
dithiothreitol (DTT) at a concentration of 0.5 mM to all
samples (Westbrook and Bhunia, 2000). We also made
viable Ped-2E9 cell counts at hourly intervals by
staining with 0.4% trypan blue. Following use, we
cleaned the IME-chip with acetic acid, rinsed it with
dH2O, cleaned it with 100% ethanol, and allowed it to
air dry.
Statistical Analysis
We analyzed data by ANOVA and calculated the least
significant difference (LSD) method to determine
statistical differences.
Results and Discussion
Listeria capture by Dynabeads from spiked or naturally
contaminated hotdog samples, following a 0 – 24 hours
enrichment in half-Fraser broth, was often inconsistent.
Due to the inconsistency of the results, as well as the
high cost of the Dynabeads, we developed another
method of immunoseparation.
We developed an alternative immunoseparation method
using immunobeads in response to the limitations of the
Dynabeads and tested both in a 24-hour spiked and
naturally contaminated hotdog immunocapture study.
Immunocapture analysis of hotdog samples during
enrichment, as shown in Figure 1, showed that an
inoculation of approximately 100 CFU/100 ml of
L. monocytogenes could be accurately detected after a
12-hour incubation, whereupon an incoluation of
approximately 1 CFU/100 ml could be detected at 18
hours. Comparison of the immunobeads to the Dynal
beads indicated that these two methods had very similar
recovery of Listeria cells from hotdog samples, which
were not significantly different at p≤0.01.
Following immunoseparation, the beads could be used
directly in a cytopathogenicity assay, where interaction
of Listeria isolates with murine hybridoma B-lympho-
cyte, Ped-2E9 cells could be determined by Trypan blue
viability staining. As seen in Figure 2, the immuno-
Figure 2. Comparison of Cytotoxicity of L. Monocytogenes Captured by Dynabeads or
Immunobeads at Different Levels of Initial Inoculation after 24 h of Growth in the
Enrichment Broth. All samples were tested with 2 x 106 hybridoma cells/ml and 0.5 mM
DTT. Controls were inoculated at a MOI of 100:1. Listeria cell suspensions were
inoculated with immunoseparated beads resuspended in 100 µl PBS.
Naschansky
42
CFSE Research Reports
captured bacteria were able to cause cytotoxicity in 1-2
hours.
This study indicates that data obtained by the
immunobeads are comparable to the Dynal
immunomagnetic beads; therefore, immunobeads could
possibly be used as an alternative to the Dynal
immunomagnetic beads for concentration of Listeria
from food samples. Furthermore, the captured Listeria
cells could be detected directly with the cytotoxicity
assay with initial contamination levels of 1 – 100 CFU/
100 ml of hotdog extracts in 12-18 hours. We estimated
the approximate price of immunobeads. They appeared
to be less expensive than the immunomagnetic beads.
We observed inconsistent recovery of Listeria cells
during immunoseparation procedures. This may be due
to formation of bead-bacteria aggregates, or multiple
bacteria bound to one bead, and these situations would
result in only one colony on an agar plate, not giving an
accurate picture of how many bacteria were captured.
However, this situation would not affect the cytotoxic-
ity results because the bacteria do not need to be
removed from the bead to cause cytotoxicity in the
Ped-2E9 cell culture.
Based on the positive cytotoxicity response, we
presumptively identified the natural isolates recovered
through the immunoseparation method to be
L. monocytogenes. Positive CAMP and RiboPrinter
results for representative colonies further confirmed
that the bacteria captured through immunoseparation
methods are L. monocytogenes (Table 1).
We designed and tested a variety of impedance- and
conductance-based biosensor apparatuses to measure
the inherent electrical properties of the Ped-2E9 cells in
the presence or absence of Listeria cells. We used a
monolithic, interdigitated microsensor electrode (IME)-
chip to detect L. monocytogenes-induced Ped-2E9 cell
membrane damage by impedance spectroscopy over the
frequency range of 1 – 10,000 KHz
We developed a buffer mixture of 4 mM Tris-glycine
and 30 mM cell-PBS (pH 7.2) for use in the IME-chips
system for its low conductivity and its ability to support
the Ped-2E9 cell viability. The IME-chip was able to
distinguish varying concentrations of Ped-2E9 cells in
the low conductivity buffer, as seen in Figure 3. Data
from the IME-chip for the cytotoxicity assay produced
an average change in impedance values for the
L. monocytogenes-infected cells, following a 1-hour
incubation, to be approximately 21 ohms (magnitude)
and –6.57º (phase angle). Upon analysis, we found the
average change in magnitude for the L. monocytogenes-
infected sample was found to be significantly different
from either the control or nonpathogenic samples
values.
Cytotoxicity data indicate that the immunobeads
containing the captured Listeria cells could be used
directly in the cytotoxicity assay with the IME-chip to
not only detect Listeria in a food sample in 1–2 hours,
but also confirm pathogenicity of the organism. The
effect the beads may have in the IME-chip system is
currently unknown although the use of the
immunobeads would appear more favorable than the
Dynabeads due to their metallic properties. These data
indicate that the use of immunoseparation possibly in
conjunction with electrical impedance spectroscopy
with an IME-chip should prove to be a rapid (14-20
hours) and sensitive method for isolation and detection
of L. monocytogenes from food samples.
Table 1. Summary of Identification and Confirmation of Natural Isolates Obtained Through Immunomagnetic
Separation of Commercial Hotdog Samples
Samplea Total Isolates CAMP Testb Cytotoxicityc Analysis DuPont IDc
PUR-A 5 + + DUP-1044
PUR-B 5 + + DUP-1044
PUR-C 5 + + DUP-1044
PUR-E 5 + + DUP-1044
PUR-F 5 + + NTd
PUR-G 5 + + NTd
a Letter designations (A, B, C, …, etc.) represent different hotdog samples purchased at different times.b All five isolates wee tested by CAMP test (Christie, et al., 1944).c Cytotoxicity and RiboPrinter® analysis was performed on one isolate from each sample group.d NT, Not Tested.
CFSE Student Research Reports
43
References
Bhunia, A.K., D.G. Westbrook. 1998. Alkaline
phosphatase release assay to determine the virulence
of Listeria species. Lett. Appl. Microbiol. 26:305-
310.
Dallas, H.L., D.P. Thomas, A.D. Hitchens. 1995.
Virulence of Listeria monocytogenes, Listeria
seeligeri, and Listeria innocua assayed with in vitro
murine macrophagocytosis. J. Food Protect.
59(1)24-27.
Hitchins, A.D. 1995. Listeria monocytogenes .p. 10.01-
10.13 In Bacteriological analytical manual, 8th ed.
AOAC International, Arlington, VA.
Ivnitski, D., I. Abdel-Hamid, P. Atanasov, E. Wilkins.
1999. Biosensors for detection of pathogenic
bacteria. Biosens. Bioelectron. 14:599-624.
Jaradat, Z.W., F. Soyer, A.K. Bhunia. 2001. Develop-
ment of polyclonal antibodies to Listeria
monocytogenes surface protein. (Unpublished).
Kyle, A.H., C.T.O. Chan, A.I. Minchinton. 1999.
Characterization of three-dimensional tissue cultures
using electrical impedance spectroscopy. Biophys. J.
76:2640-2648.
Mead, P.S., L. Slutsker, V. Dietz, L.F. McCaig, J.S.
Bresee, C. Shapiro, P.M. Griffen, R.V. Tauxe. 1999.
Food-related illness and death in the United States.
Emerg. Infect. Dis. 5(5):607-625.
Pancrazio, J.J., J.P. Whelan, D.A. Borkholder, W.Ma,
D. A. Stenger. 1999. Development and application
of cell-based biosensors. Ann. Biomed. Eng.
27:697-711.
Pine, L., S. Kathariou, F. Quinn, V. George, J.D.
Wenger, R.E. Weaver. 1991. Cytophatogenic effects
in enterocytelike Caco-2 cells differentiate virulent
from avirulent strains. J. Clin. Microbiol. 29(5):990-
996.
Siddique, I.H. 1969. Cytotoxic activity of hemolysin
from Listeria monocytogenes on L-M strain of
mouse cells. Can. J. Microbiol. 15:955-957.
Uyttendaele, M., I., Van Hoorde, J. Debevere. 2000.
The use of immuno-magnetic separation (IMS) as a
tool in a sample preparation method for direct
detection of L. monocytogenes in cheese. Int. J.
Food Microbiol. 54:205-212.
Van Langendonck, N., E. Bottreau, S. Bailly, M.
Tabouret, J. Marly, P. Pardon, P. Velge. 1998. Tissue
culture assays using Caco-2 cell line differentiate
virulent from non-virulent L. monocytogenes strains.
J. Appl. Microbiol. 85:337-346.
Westbrook, D.G. and A.K. Bhunia. 2000. Dithiothreitol
enhances Listeria monocytogenes mediated cell
cytotoxicity. Microbiol. Immunol. 44(6):431-438.
Figure 3. Change in impedance values due to varying concentrations of Ped-2E9
cells. Magnitude and phase angle differences between Tris-glycine-cell-PBS (TGCP)
buffer and varying cell concentrations, averaged over the frequency range of 1000 –
10,000 KHz.
Naschansky
44
CFSE Research Reports
Scientific Publications
Bhunia, A.K., Z.W. Jaradat, K.M. Naschansky, M.
Shroyer, M. Morgan, R. Gomez, R. Bashir, M.
Ladisch. 2000. Impedance spectroscopy and biochip
sensor for detection of Listeria monocytogenes.
Proceedings of SPIE. 4206:32-39.
Presentations
Naschansky, K.M., M. Morgan, A.K. Bhunia. 2001.
Poster: Interdigitated Microsensor Electrode-Chip
for Detection of Cytotoxicity Effect of L.
monocytogenes from food. American Society for
Microbiologists 101st General Meeting. Orange
County Convention Center in Orlando, Florida.
P-32.
Naschansky, K.M. 2001. Master’s Defense. Develop-
ment of an immunoseparation and impedance
spectroscopy-based cytotoxicity assay to isolate and
detect Listeria monocytogenes from hotdog samples.
Purdue University, Department of Food Science.
July 11, 2001.
Naschansky, K.M., A.K. Bhunia. 2002. Detection of
low levels of pathogenic Listeria monocytogenes in
14 - 20 h using immunoseparation and cytotoxicity.
2002 Institute of Food Technologists Meeting.
Paper #100A-22.
These data indicate that the use of immunoseparation possibly inconjunction with electrical impedance spectroscopy with an IME-chipshould prove to be a rapid (14-20 hours) and sensitive method for isolationand detection of L. monocytogenes from food samples.
CFSE Student Research Reports
45
CFSE Administration
Name Phone Fax Email
Dr. Richard H. Linton* (765) 494-6481 (765) 494-7953 [email protected] of the Center for
Food Safety Engineering
Dr. W. R. (Randy) Woodson* (765) 494-8362 (765) 494-0808 [email protected] of Agricultural Research
Programs
CFSE Researchers
Dr. Barbara A. Almanza (765) 494-9847 (765) 494-0327 [email protected]
Dr. Bruce Applegate* (765) 496-7920 (765) 494-7953 [email protected]
Dr. Stephen Badylak* (765) 494-2994 (765) 494-1193 [email protected]
Dr. Susan J. Barkman (765) 494-8423 (765) 496-1152 [email protected]
Dr. Rashid Bashir* (765) 496-6229 (765) 494-6441 [email protected]
Dr. Darrell Bayles* (215) 233-6400 (215) 233-6581 [email protected]
Dr. Arun K. Bhunia* (765) 494-5443 (765) 494-7953 [email protected]
Dr. James Binkley (765) 494-4261 (765) 494-9176 [email protected]
Dr. Ernest R. Blatchley, III (765) 494-0316 (765) 494-0395 [email protected]
Dr. Jeff Brewster* (215) 233-6447 (215) 233-6400 [email protected]
Dr. Osvaldo Campanella* (765) 496-6330 (765) 496-1115 [email protected]
Dr. Natalie Carroll (765) 494-8433 (765) 496-1152 [email protected]
Rjichard J. Carroll (765) 496-6171 (765) 494-7953 [email protected]
Dr. John P. Cherry* (215) 233-6595 (215) 233-6777 [email protected]
Dr. R. Graham Cooks (765) 494-5263 (765) 494-9421 [email protected]
Dr. Carlos Corvalan* (765) 494-7357 (765) 496-1356 [email protected]
Dr. Maribeth A. Cousin* (765) 494-8287 (765) 494-7953 [email protected]
Dr. C. Gerald Crawford* (215) 233-6628 (215) 233-6581 [email protected]
Dr. Laszlo N. Csonka (765) 494-4969 (765) 494-0876 [email protected]
Dr. Otto C. Doering, III (765) 494-4226 (765) 496-1224 [email protected]
Leslie Dorworth* (219) 989-2726 (219) 989-2130 [email protected].
purdue.edu
Dr. Robert G. Elkin (765) 494-4820 (765) 494-6816 [email protected]
Dr. Okan K. Ersoy (765) 494-6162 (765) 494-3358 [email protected]
Dr. Stephen Feairheller* (215) 233-6610 (215) 233-6777 [email protected]
Dr. Bill Fett* (215) 233-6418 (215) 233-6406 [email protected]
Dr. John C. Forrest (765) 494-8283 (765) 494-6816 [email protected]
Dr. Pina Fratamico* (215) 233-6525 (215) 233-6581 [email protected]
Dr. David Gerrard* (765) 494-8280 (765) 494-9346 [email protected]
Center StaffPersons whose names are marked with an asterisk (*) are also part of the
USDA-ARS Food Safety Engineering Project.
46
CFSE Research Reports
Dr. Lawrence T. Glickman (765) 494-6301 (765) 494-9830 [email protected]
Dr. Jayavant P. Gore (765) 494-1452 (765) 494-0539 [email protected]
Dr. Alan L. Grant (765) 494-8282 (765) 494-6816 [email protected]
Dr. Kamyar Haghighi (765) 494-1182 (765) 496-1115 [email protected]
Dr. Tim Haley (865) 588-7685 [email protected]
Dr. Kevin Hicks* (215) 233-6579 (215) 233-6406 [email protected]
Dr. E. Dan Hirleman (765) 494-5688 (765) 494-0539 [email protected]
Dr. Peter Irwin* (215) 233-6400 (215) 233-6581 [email protected]
Dr. Brad Joern (765) 494-9767 (765) 496-2926 [email protected]
Dr. Allan E. Konopka (765) 494-8152 (765) 494-0876 [email protected].
purdue.edu
Dr. Michael R. Ladisch* (765) 494-7022 (765) 494-7023 [email protected]
Dr. Mickey Latour (765) 494-8011 (765) 494-6816 [email protected]
Dr. Tsang Long Lin (765) 494-0244 (765) 494-7927 [email protected]
Dr. James A. Lindsay* (301) 504-4674 (301) 504-5467 [email protected]
Dr. Stephen B. Lovejoy (765) 494-4245 (765) 494-9176 [email protected]
Dr. John B. Luchansky* (215) 233-6620 (215) 233-6581 [email protected]
Dr. Jay S. Marks* (765) 494-8261 (765) 494-7953 [email protected]
Dr. Marshall A. Martin (765) 494-8365 (765) 494-0808 [email protected]
Dr. April Mason (765) 494-8252 (765) 496-1947 [email protected]
Dr. Linda Mason (765) 494-4586 (765) 496-2295 [email protected]
Dr. Brian K. Miller* (765) 494-3586 (765) 496-2422 [email protected]
Dr. Rabi H. Mohtar (765) 494-1791 (765) 496-1115 [email protected]
Dr. Mark Morgan* (765) 494-1180 (765) 496-1115 [email protected]
Dr. Cindy H. Nakatsu (765) 496-2997 (765) 496-2926 [email protected]
Dr. Douglas Craig Nelson (765) 496-2498 (765) 494-0327 [email protected]
Dr. Philip E. Nelson (765) 494-8256 (765) 494-7953 [email protected]
Dr. S. Suzanne Nielsen* (765) 494-8328 (765) 494-7953 [email protected]
Dr. Martin R. Okos (765) 494-1211 (765) 496-1115 [email protected]
Dr. John Patterson (765) 494-4826 (765) 494-9347 [email protected]
Dr. Fred E. Regnier (765) 494-3878 (765) 494-0239 [email protected]
Dr. Brian Richert (765) 494-4837 (765) 494-9347 [email protected]
Dr. Paul J. Robinson* (765) 494-6449 (765) 494-0517 [email protected]
Dr. Charles R. Santerre* (765) 496-3443 (765) 494-0674 [email protected]
Dr. Gerald Sapers* (215) 233-6417 (215) 233-6406 [email protected]
Dr. Allan P. Schinckel (765) 494-4836 (765) 494-9347 [email protected]
Dr. Gregory E. Shaner (765) 494-4651 (765) 494-0363 [email protected]
Name Phone Fax Email
Center Staff (continued)
Persons whose names are marked with an asterisk (*) are also part of the
USDA-ARS Food Safety Engineering Project.
Center Staff
47
Dr. Rakesh K. Singh* (706) 542-2286 (706) 542-1050 [email protected]
Dr. James R. Stahl (317) 308-3187 (317) 308-3219
Dr. Gregory W. Stevenson (765) 494-7473 (765) 494-9181 [email protected]
Dr. Richard L. Stroshine* (765) 494-1192 (765) 496-1115 [email protected]
Dr. Mark L. Tamplin* (215) 836-3794 (215) 233-6581 [email protected]
Dr. Bernard Y. Tao* (765) 494-1183 (765) 496-1115 [email protected]
Dr. Shu-I Tu* (215) 233-6466 (215) 233-6581 [email protected]
Dr. Mark J. Volkmann (765) 494-5679 (765) 496-1622 [email protected]
Dr. Bruce A. Watkins (765) 494-5802 (765) 494-7953 [email protected]
Dr. Connie M. Weaver (765) 494-8231 (765) 494-0674 [email protected]
Dr. M. Randall White (765) 494-7456 (765) 494-9181 [email protected]
Dr. Charles P. Woloshuk* (765) 494-3450 (765) 494-0363 [email protected]
Dr. Ching-ChingWu (765) 494-7459 (765) 494-9181 [email protected]
Dr. Linda Yu* (215) 233-6400 (215) 233-6581 [email protected]
CFSE Staff
Name
Ann Guentert* (765) 496-3827 (765) 494-7953 [email protected]
Equipment Manager
Cathy Moss (765) 494-8360 (765) 494-0808 [email protected]
Secretary, Agricultural
Research Programs
CFSE Students
Douglas K. Allen* (765) 496-3837 (765) 494-7953 [email protected] H. Blum* (765) 496-2428 (765) 494-7953 [email protected] G. Bright* (765) 496-6171 (765) 494-7953 [email protected] Chen* (765) 496-3825 (765) 494-7953 [email protected] E. Flaherty* (765) 497-2428 (765) 494-7953 [email protected] Guo* (765) 494-7057 (765) 494-0539 [email protected] Lasrado* (765) 494-8249 (765) 494-0674 [email protected] Naschansky* (765) 496-3828 (765) 494-7953 [email protected] Selby* (765) 494-8280 (765) 494-9346 [email protected] Shim* (765) 494-8249 (765) 494-0674 [email protected] L. Shroyer* (765) 496-6171 (765) 494-7953 [email protected]
Name Phone Fax Email
Center Staff (continued)
Persons whose names are marked with an asterisk (*) are also part of the
USDA-ARS Food Safety Engineering Project.
48
CFSE Research Reports
February 7, 2002
WEST LAFAYETTE, Ind. – In an effort to protect the
nation's food supply from biological and chemical
contaminants, Purdue University and U.S. Department
of Agriculture engineers and food scientists have
teamed up to develop faster, more exact ways to detect
possibly deadly substances.
With research grants and a partnership with the USDA's
Agricultural Research Service, Purdue has launched the
Center for Food Safety Engineering focused on
developing methods to find, identify and eradicate
microbes or chemicals.
“The Purdue Center for Food Safety Engineering is
utilizing a multidisciplinary team to contribute to the
science and technology needed to enhance food safety,”
said Michael Ladisch, a scientist in the center that
includes work by nearly 90 university researchers.
The Centers for Disease Control and Prevention (CDC)
estimates that 76 million cases of foodborne illness
occur in the United States annually and claim approxi-
mately 5,000 lives and cost $7.7 billion or more.
Although disease-causing bacteria accidentally can
contaminate meat, fruit and vegetables at any stage,
from the field through processing and storage, concern
over food contamination has heightened since the Sept.
11 terrorist attacks on New York City and the Pentagon.
Health officials have long viewed the safety of the
country's food as a prime concern, Ladisch said.
Foodborne pathogens cause 325,000 hospitalizations
yearly, according to the CDC. In fact, the Clinton
Administration issued a "no tolerance" edict for
Listeria monocytogenes in processed and ready-to-eat
foods, such as hot dogs. Listeria is one of the most
deadly of the biological food contaminants, with a
fatality rate of about 20 percent.
One aspect of the task with which researchers must
cope is the difficulty of tracing the source of foodborne
illness. In addition, a minuscule amount of some
pathogens, such as listeria, can cause illness. So the
center's scientists are investigating detection methods
that not only are faster and more exact, but also require
smaller bacteria-containing food samples to make an
analysis.
Food science Associate Professor Richard Linton, as
center director, leads the biochemists; molecular
biologists; physicists; and biomedical, electrical,
computer, agricultural and biological engineers. Their
quest is to prevent microbial organisms such as
Salmonella enteritidis, Listeria, Escherichia coli (E.
coli) O157:H7, Campylobacter and Fusarium from
entering the food chain at any point, whether it's the
farm gate, the processing plant or the consumer's table.
The investigators come from five schools within the
university – Agriculture, Consumer and Family
Sciences, Engineering, Science, and Veterinary
Medicine, and team with the USDA Agricultural
Research Service scientists.
“The multidisciplinary center provides an important
platform for bringing different scientific expertise
together,” said Linton, a microbiologist. "With this
collection of creative minds working together, new and
exciting research approaches are being developed and
studied. This is an important step for solving complex
food safety problems and, most importantly, for
protecting the health of consumers."
A five-year, $7 million USDA grant provides funding
for cooperative projects between the center and the
Agricultural Research Service, while other funding has
allowed creation of the center for expansion of the
university researchers' work.
“We need long-term research to develop and improve
techniques and to engineer methods in systems that are
readily usable in the plant and the laboratory,” said
Ladisch, who also is director of Purdue's Laboratory of
Renewable Resources Engineering and a distinguished
professor of agricultural and biological engineering and
biomedical engineering. “The partnership between the
USDA and the university allows us to carry out
Purdue Center Aims at Preventing,Detecting Food Contamination
Recent CFSE News Release
49
cooperative research that facilitates achieving results
that would be difficult otherwise."
The projects currently under way by Purdue scientists
at the Center for Food Safety Engineering focus on the
following areas:
• Fusarium is a fungus that infests grains and then
produces carcinogenic mycotoxins that can affect
both people and animals. It often appears in moldy
corn, but also has been found in sorghum, rice,
cottonseed meal, legumes, wheat and barley.
Maribeth Cousin and Charles Woloshuk are working
on a method to detect various Fusarium species in
grain and foods. This research may give grain storage
operators information enabling them to alter storage
conditions in order to prevent Fusarium growth.
• Nanotechnology and magnetic beads are integral to
investigations by Arun Bhunia and Mark Morgan to
develop more sensitive, less expensive, faster
methods of detecting Listeria. This bacterium can
contaminate almost any food, from vegetables to
ready-to-eat bologna, and even grow in the refrigera-
tor. Listeria is responsible for about 2,500 cases of
foodborne diseases each year. In the first of the two-
step method, microscopic beads separate Listeria
cells from food. Then the researchers use a test called
a cytotoxicity assay that differentiates between
disease-causing Listeria cells and those that are
harmless.
• Molecular biology and tiny chips with electronic
signaling and other possible Listeria detecting
procedures are being investigated by Rashid Bashir,
Bhunia, Paul Robinson, Stephen Badylak, Ladisch
and their graduate students. Bashir's group is leading
the fabrication and design of microscopic, hair-thin
structures that will carry fluid from a food sample
across a postage stamp-sized device called a biochip.
The chip will electronically detect Listeria and
distinguish it from other organisms. Molecules on the
chip target and capture Listeria. When the targeted
microorganism is captured, an electrical response
from the biochip will signal its presence.
• Charles Santerre is developing ways to rapidly and
accurately detect PCBs in fish and is investigating a
rapid test for predicting the toxicity of PCBs in fish.
The full effect of the common environmental
contaminant polychlorinated biphyenyls, or PCBs, is
unknown, but anecdotal evidence indicates they
adversely impact fetal neural and physical develop-
ment as well as the function of the liver and thyroid,
and the immune and reproductive systems. PCBs
Ying Chang Han, a Purdue food
research scientist, tests a way to
sanitize a processing vat.
Researchers in the Purdue Center
for Food Safety Engineering are
finding new ways to eliminate
biological and chemical
contaminants at all stages of food
production.
50
CFSE Research Reports
also likely play a role in some forms of cancer.
Humans are most commonly exposed by eating PCB-
contaminated fish from rivers, streams and lakes. The
contaminants can pass through the placenta or
through breast milk at levels that are higher than in
the original fishmeal.
• Measuring the characteristics of light scattered
across a surface is another possible method to
quickly detect and identify biological contaminants
and distinguish between virulent and non-virulent
bacteria. E. Dan Hirleman and Bhunia are investigat-
ing this using an instrument called a scatterometer.
This technique may meet one research objective: to
detect bacterial pathogens quickly; the scatterometer
has found them as early as six to 10 hours after they
were introduced into food.
• Fruits and vegetables can carry a number of illness-
causing bacteria including E. coli O157:H7, Salmo-
nella and Listeria. A number of the center's research-
ers including Linton, Rakesh Singh, Bhunia and
Richard Stroshine are testing alternate processes to
eliminate these potentially deadly pathogens.
Sanitizing methods they are investigating include use
of chlorine dioxide, ozonated water and a solution
that contains clove, thyme and oregano oils.
• Listeria monocytogenes can invade commercially
processed and vacuum-packed ready-to-eat meats
from contaminated air, equipment and water, as well
as from food handlers in the processing plant. Tim
Haley, Bhunia, Osvaldo Campanella, David Gerrard
and Linton have designed a post-packaging pasteur-
ization process for sliced bologna. The high-
temperature, short-time method is applied to pouches
containing one or two meat slices. This extends shelf
life for ready-to-eat meat products and may provide
food processors and consumers with another weapon
in the fight to prevent Listeria-caused illness and
death.
Listeria can be found on all types of food
and can even grow in the refrigerator.
Purdue University Center for Food Safety
Engineering researchers have developed a
method to pasteurize ready-to-eat meat
after processing.
• Certain viruses called bacteriophages attack only
specific bacteria. Using that knowledge, Bruce
Applegate is genetically engineering bacteriophages
that detect and identify specific bacterial pathogens.
When bacteriophages infect the target pathogen, they
cause it to produce a signal compound. The com-
pound triggers a bioluminescent chain reaction in a
second type of bacteria causing it to glow. This
indicates the presence of pathogens. Eventually this
two-component detection system will include use of
a hand-held device called a luminometer that would
enable those in the field or the plant to test for such
bacteria as Salmonella, Campylobacter jejuni, E. coli
O157:H7 and Listeria on meat, fruit and vegetables.
Writer: Susan A. Steeves, Department of Agricultural
Communication, Purdue University
Recent CFSE News Release
51
CFSE Annual Report editing and design by Purdue Agricultural Communication
Center for Food Safety Engineering
The Center for Food Safety Engineering2001-2002
Research Report
It is the policy of the Purdue University Center for Food Safety Engineering, that all persons shall have equal opportunity and access to the programs and facilities
without regard to race, color, sex, religion, national origin, age, marital status, parental status, sexual orientation, or disability.
Purdue University is an Affirmative Action employer.
8/02