center for food safety engineering · prevention estimate that 76 million cases of foodborne...

The Center for Food Safety EngineeringThe Center for Food Safety Engineering

Purdue UniversityPurdue University2001-2002 Research Report2001-2002 Research Report

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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

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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

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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

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Detection of Specific Foodborne Pathogens Using a TwoComponent Bacteriophage/Bioluminescent Reporter

System in Conjunction with a Hand-Held LuminometerInvestigator: Bruce M. Applegate



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

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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.


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.


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.

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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.


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



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.

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• 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



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.


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




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).


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.


accomplishment this past year?


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.


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



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.


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.


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



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


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.


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





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.


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.


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




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.


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.


accomplishment this past year?


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.


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.




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




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.


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.


accomplishment in the past year?


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



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.





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.


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.


24


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.


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.


26


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.


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



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.


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


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.


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.


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.




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.


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


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.


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


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.


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


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.



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.



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.


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.


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


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


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


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


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


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


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


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.


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


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.


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



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.


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


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]


Center Staff (continued)

Persons whose names are marked with an asterisk (*) are also part of the


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]


Center Staff (continued)

Persons whose names are marked with an asterisk (*) are also part of the


48


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


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

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