Food Safety and Beyond
Jianrong (Janet) Zhang, Ph.D
Food safety background
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Safe, nutritious foods are essential to human
health and well-being. However, food-borne
diseases pose a significant problem
worldwide.
The World Health Organization (WHO)
estimates that 1.5 billion cases of foodborne illnesses cause about 3 million deaths
each year.
Food safety background (cont.)
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Although the United States produces the
safest foods in the world, food-borne
illnesses continue to threaten this country.
The Centers for Disease Control and
Prevention estimate that in the United States
more than 6 million cases of food-borne
illnesses occur annually -- causing 8,000
deaths and costing up to $13 billion in
health care and job-related absenteeism.
Food safety background (cont.)
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Food safety will continue to be important
issue in the future, because:
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First, the overall U.S. population is increasing
and changing;
The U.S. food system has increased in
complexity as our society has become more
urbanized;
Food-borne diseases will likely increase in light
of global economy.
Federal agencies
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The Centers for Disease Control and
Prevention (CDC) is recognized as the lead
federal agency for protecting the health and
safety of people
FDA Center for food safety and applied
nutrition(CFSAN)
USDA Food Safety and Inspection
Service(FSIS)
Codex Alimentarius Commission
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Implements the joint FAO/WHO Food
Standards Program
To protect the health of consumers and to
ensure fair practices in trade
Codex Alimentarius Commission
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North American Free Trade Agreement NAFTA(Canada, US, Mexico)
MERCOSUR (Argentina, Brazil, Paraguay,
Uruguay)
APEC (Asia-Pacific Economic
Cooperation)- 18 countries in Asia and the
Pacific
European Union
New CDC data
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CDC published Preliminary FoodNet Data on
Apr.2003, demonstrate a sustained decrease in
major bacterial foodborne illnesses caused by
Campylobacter and Listeria, indicating progress
toward meeting the Agency’s health objectives of
reducing the incidence of foodborne infections.
In addition, data from FSIS show a continuing
decline in the prevalence of Salmonella in
regulatory samples of meat and poultry.
HACCP
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There was16 percent decline in foodborne
illness over the last 6 years (1996-2002).
CDC attributes these results in part to the
implementation of the Hazard Analysis
Critical Control Point (HACCP) system in
all meat and poultry plants in the United
States.
FDA HACCP
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HACCP for the seafood industry in a final
rule December 18, 1995
For the juice industry, the final rule released
on January 19, 2001. It will take effect on
January 22, 2002 for large and medium
businesses, January 21, 2003 for small
businesses, and January 20, 2004 for very
small businesses
USDA HACCP
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In 1998, the U.S. Department of Agriculture
has established HACCP for meat and
poultry processing plants, as well. Most of
these establishments were required to start
using HACCP by January 1999. Very small
plants had until Jan. 25, 2000
HACCP seven principles
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Analyze hazards
Identify critical control points
Establish preventive measures with critical limits for
each control point
Establish procedures to monitor the critical control
points
Establish corrective actions to be taken when
monitoring shows that a critical limit has not been met
Establish procedures to verify that the system is
working properly
Establish effective record keeping to document the
HACCP system
Food Hazard
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A biological, chemical or physical agent in
a food with the potential to cause an adverse
health effect
Current Hazard
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Biological
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Chemical
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Bacteria
Viruses
Parasites
Pesticide residues
Veterinary drugs
Physical
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Contaminated raw material
Poorly designed or maintained equipment
Microbial growth, survive and death
in food
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pH
Water activity aw
Oxygen absence
Temperature
Nutrient content
Antimicrobial constituents
Biological structures
pH
Optimum Maximum Minimum
Bacteria
6.5-7.5
9.0
4.5
Molds
4.0-6.8
8.0-11
1.5-3.5
Yeast
4.5-6.5
8.0-8.5
1.5-3.5
 Food can be divided into two major categories:
low acid (pH <4.6) and acid (pH > 4.6). These
were established based upon the growth of
C.botulinum, whose minimum growth pH
requirement is generally accepted as 4.8.
Water Activity
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Bacteria
0.90-0.91
S.aureus –0.83
Halophilic bacteria – 0.75
Yeasts
0.87-0.94
Osmotolerant yeasts – 0.60
Molds
0.70-0.80
Xeromyces – 0.60
Water activity of foods
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Fruits/vegetables – 0.97-1.00
Meats – 0.95-1.00
Bread – 0.95-1.00
Cheese – 0.68 – 1.00
Jams/Jellies – 0.75-0.94
Honey – 0.54-0.75
Temperature effect
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Most pathogenic organisms are mesophilic (Min.,
5-15 ºC, Opt., 35 –37 ºC, Max, 30-45 ºC)
A number of foodborne pathogens are
psychrotrophic (Min., -5 to 5 ºC, opt., 12 –15 ºC,
Max., 15 –20 ºC)
Thermophiles (Min.40-45 ºC, Opt., 55 –75 ºC,
max. 60 – 90 ºC)
Psychorotrops (Min., -5 to 5 C, Opt. 25 –30 ºC,
Max., 3—35 ºC)
Examples of food groups and their
related spoilage microorganism
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Refrigerated foods – psychrotrophs
Juice concentrate – osmophilic yeasts
Fermented foods – acid tolerant lactic acid
bacteria and yeast
Meat products – psychotropic
pseudomonads
Hot – filled juices – heat resistance molds
Ten least wanted foodborne
pathogens
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Campylobacter jejuni
Clostridium botulinum
E.coli O157:H7
Listeria
monocytogenes
Salmonella
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Staohylococcus aureus
Shigella
Toxoplasam gondil
Vibrio vulnificus
Yersinia
eneterocolitica
Microbial detection
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Traditional methods to detect foodborne bacteria
often rely on time-consuming growth in culture
media, followed by isolation, biochemical
identification, and sometimes serology
Recent advances in technology make detection
and identification faster, more convenient, more
sensitive, and more specific than conventional
assays
Rapid methods
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"rapid methods", a subjective term used
loosely to describe a vast array of tests that
includes miniaturized biochemical kits,
antibody- and DNA-based tests, and assays
that are modifications of conventional tests
to speed up analysis
Rapid methods
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First made available in the early 1980s for
several groups of bacteria
Alternative approach besides convenient
methods, less time, labor and set up costs
Extensively evaluated
Now accepted by most microbiologists
Rapid methods
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Biochemical test kits
Antibody assay
DNA-based assay
Partial list of miniaturized biochemical kits
and automated systems for identifying
foodborne bacteria
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APIb
Cobas IDA
Micro-IDb
EnterotubeII
Spectrum 10
RapID
BBL Crystal
Minitek
Microbact
Vitekb
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Microlog
MISb
Walk/Away
Replianalyzer
Riboprinter
Cobas Micro-ID
Malthusb
Bactometer
Partial list of commercially-available,
antibody-based assay for the detection of
foodborne pathogens and toxins
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ELISA - Enzyme-Linked Immunosorbent
Assay
LA – Latex agglutination
IMS – Magnetic beads
Major manufacturers: BioMerieux, Foss,
Microgen, Biocontrol, TECRA, Elcatech,
etc.
Partial list of commercially-available,
nucleic acid-based assays used in detection
of foodborne bacterial pathogens
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BAX
Probelia
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Based on PCR assay
AccuProbe
GENE-TRACK
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Based on Probe assay
Bindb
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Based on Phage assay
Some other rapid methods
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To use disposable cardboards containing
dehydrated media, which eliminates the need for
agar plates, constituting savings in storage,
incubation and disposal procedures
To inncorporate specialized chromogenic and
fluorogenic substrates in media to rapidly detect
trait enzymatic activity
To measure bacterial adenosine triphosphate
(ATP) to rapidly enumerate the presence of total
bacteria
VITEK®(BioMerieux)
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The VITEK is a completely automated
instrument that offers rapid results (with an
average of 2-6 hour same-day turnaround).
It is used for bacterial and yeast
identification, antimicrobial susceptibility
testing and has a complete Data
Management System.
®
VITEK (BioMerieux)
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cards
ANI............. Anaerobes & Micrococcus
BAC............. Bacillus
GPI.............. Gram Positives (Staph & Strep)
GNI/GNI+... Gram Negatives (Oxidase Neg)
NFC............. Gram Negatives (Oxidase Pos)
YBC............. Yeast
How does VITEK® work
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Colony need to be isolated first
Isolates are subcultured to TSA Plates
A smear is prepared from original Tryptone
Soy Agar (TSA) plates for Gram Stain
Isolates are incubated at 35 oC to be fresh
sample
How does
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®
VITEK work
(cont.)
Observe subcultured plates
Perform preliminary testing (catalase,
oxidase, microdase, coagulase, etc.)
Set-up appropriate VITEK Card
Insert Card into VITEK Incubator/Reader
Come back to read the report
How does ELISA work?
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Antibody coated wells
Sample is added, target antigens, if present,
bind with antibodies
Reagent is added, antibodies sandwich the
antigen,, enzyme labeled antibody detectors
attach to the sandwich
Substrate is added, color change occurs
where the antigen is present
Riboprinter® Microbial Characterization
System (Dupont Qualicon)
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Fully automated ribotyping system that provides a
genetic "fingerprint" of any bacterium in about
eight hours.
The system extracts a RiboPrint® pattern from
image data, compares it to others in a database for
characterization and identification, and prints the
results in a report.
The system can process up to eight bacterial
isolates at one time, can accept new batches every
two hours, allowing up to 32 samples a day.
How does
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Riboprinter
work
Getting a sample
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A colony is picked manually from the plate and introduced into
the RiboPrinter system, where the colony is suspended in a
buffered solution and then heated
Preparing the DNA
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The sample is treated with a lysing agent, a chemical that
dissolves the bacterial cell walls to release the DNA. This
process is completed by adding a restricting enzyme that "cuts"
the DNA at specific points and creates identifiable fragments.
How does Riboprinter® work (cont.)
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Separating and transferring DNA
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The DNA fragments are put into eight small wells, and "markers" synthetic DNA of known weights - are placed in five other wells.
The DNA fragments are separated according to molecular size by a
process called gel electrophoresis. Through this process the
fragments are electrically drawn out of the gel and transferred
directly to a moving nylon membrane
Membrane processing
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At this point, the markers and samples are attached to the
membrane in 13 distinct "lanes." The membrane then goes through
a series of biochemical steps, including treatment with a
chemiluminescent agent that literally lights up the DNA fragments
of interest
How does Riboprinter® work (cont.)
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Detection and extraction
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The glow of the DNA fragments is not visible to the naked eye.
However the RiboPrinter system is equipped with a CCD camera
that can detect very low light levels. The camera takes a digital
picture of the membrane, resulting in an image of the DNA
fragments and markers. The system then uses a proprietary
algorithm to "understand" and normalize the image. The result is a
standard DNA pattern called a RiboPrinter pattern that can be
compared with other such patterns from other images.
Riboprinter®
PCR
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PCR stands for “Polymerase Chain Reaction”
First described only 10 years ago, in its short life
PCR has transformed the life sciences utterly.
It is far simpler and less expensive than previous
techniques for duplicating DNA, PCR has
democratized genetic research, putting it within
reach of all biologists, even those with no training
in molecular biology.
PCR’s requirement
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A template molecule - the DNA or RNA
you want to copy
two primer molecules (short chains of the
four different chemical components, named
as nucleotides or bases, that make up any
strand of genetic material - to get the
copying process started
PCR’s requirement
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DNA is double-stranded, consisting of two such
nucleotide chains that wind around each other in
the famous shape known as the double helix
Primers are single-stranded
Primers must be duplicates of nucleotide
sequences on either side of the piece of DNA of
interest, which means that the exact order of the
primers' nucleotides must already be known
PCR’s three steps
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First, the target genetic material must be denatured-that is,
the strands of its helix must be unwound and separated-by
heating to 90-96°C.
The second step is hybridization or annealing, in which the
primers bind to their complementary bases on the now
single-stranded DNA.
The third is DNA synthesis by a polymerase. Starting from
the primer, the polymerase can read a template strand and
match it with complementary nucleotides very quickly. The
result is two new helixes in place of the first, each
composed of one of the original strands plus its newly
assembled complementary strand.
Commercialized PCR equipment
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The key to PCR's automation has been Taq
polymerase. Taq is a nickname for Thermus
aquaticus, a bacterium that happily survives
and reproduces in an environment that is
lethal to other organisms: hot springs
So that it can stand rapidly fluctuating
temperatures of automated PCR
®
BAX (Dupont
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Qualicon)
Process up to 96 unique samples within four
hours after sample preparation.
Results are available as soon as the next day
and are clearly displayed on screen with a
simple positive or negative report
Provide reagents for screening Salmonella,
E. coli O157:H7, L. monocytogenes, etc.
®
BAX (Dupont
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Qualicon)
Samples are enriched according to standard protocols for
the food type.
Samples are then heated in a lysis reagent solution to
rupture the bacterial cell wall and release the DNA.
PCR tablets, which contain all the reagents necessary for
PCR plus fluorescent dye, are hydrated with lysed sample
and processed in the cycler/detector. Within a few hours,
the PCR amplifies a DNA fragment that is specific to the
target.
The amplified DNA generates a fluorescent signal, which
the BAX® system uses to analyze the findings. Results are
then displayed as simple positive or negative symbols
Limitation for rapid methods
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A positive result by a rapid method is only
regarded as presumptive and must be confirmed
by standard methods
Most rapid methods lack of sufficient sensitivity
and specificity for director testing, foods still need
to be culture-enriched before analysis
Rapid methods are food dependent
Can detect cell but can’t detect the toxin
occurrence
Future trend
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Biosensor
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A compact analytical device incorporating a biological
or biologically-derived sensing element(such as
enzyme, antibody, microbe or DNA) either integrated
with a physicochemical transducer.
Transducer:
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Electrochemical
Optical
Piezoelectric
Thermal
Future trend
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DNA biochip
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A miniature silicon surface containing
thousands of gene probes in a thumbnail size
area