Food Safety and Beyond

1
Food Safety and Beyond

• Jianrong (Janet) Zhang, Ph.D

2
Food safety background

• 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 food-borne illnesses
  cause about 3 million deaths each year.

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Food safety background (cont.)

• 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.

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Food safety background (cont.)

• Food safety will continue to be important issue
  in the future, because
• 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.

5
Federal agencies

• 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)

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Codex Alimentarius Commission

• Implements the joint FAO/WHO Food Standards
  Program
• To protect the health of consumers and to ensure
  fair practices in trade

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Codex Alimentarius Commission

• 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

8
New CDC data

• 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 Agencys 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.

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HACCP

• 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.

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FDA HACCP

• 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

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USDA HACCP

• 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

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HACCP seven principles

• 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

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Food Hazard

• A biological, chemical or physical agent in a
  food with the potential to cause an adverse
  health effect

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Current Hazard

• Biological
• Bacteria
• Viruses
• Parasites
• Chemical
• Pesticide residues
• Veterinary drugs
• Physical
• Contaminated raw material
• Poorly designed or maintained equipment

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Microbial growth, survive and death in food

• pH
• Water activity aw
• Oxygen absence
• Temperature
• Nutrient content
• Antimicrobial constituents
• Biological structures

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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 lt4.6) and acid (pH gt 4.6). These
  were established based upon the growth of
  C.botulinum, whose minimum growth pH requirement
  is generally accepted as 4.8.

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Water Activity

• 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

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Water activity of foods

• 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

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Temperature effect

• 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., 335 ºC)

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Examples of food groups and their related
spoilage microorganism

• 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

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Ten least wanted foodborne pathogens

• Campylobacter jejuni
• Clostridium botulinum
• E.coli O157H7
• Listeria monocytogenes
• Salmonella

• Staohylococcus aureus
• Shigella
• Toxoplasam gondil
• Vibrio vulnificus
• Yersinia eneterocolitica

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Microbial detection

• 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

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Rapid methods

• "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

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Rapid methods

• 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

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Rapid methods

• Biochemical test kits
• Antibody assay
• DNA-based assay

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Partial list of miniaturized biochemical kits
and automated systems for identifying foodborne
bacteria

• APIb
• Cobas IDA
• Micro-IDb
• EnterotubeII
• Spectrum 10
• RapID
• BBL Crystal
• Minitek
• Microbact
• Vitekb

• Microlog
• MISb
• Walk/Away
• Replianalyzer
• Riboprinter
• Cobas Micro-ID
• Malthusb
• Bactometer

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Partial list of commercially-available,
antibody-based assay for the detection of
foodborne pathogens and toxins

• ELISA - Enzyme-Linked Immunosorbent Assay
• LA Latex agglutination
• IMS Magnetic beads
• Major manufacturers BioMerieux, Foss, Microgen,
  Biocontrol, TECRA, Elcatech, etc.

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Partial list of commercially-available,
nucleic acid-based assays used in detection of
foodborne bacterial pathogens

• BAX
• Probelia
• Based on PCR assay

• AccuProbe
• GENE-TRACK
• Based on Probe assay
• Bindb
• Based on Phage assay

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Some other rapid methods

• 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

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VITEK(BioMerieux)

• 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.

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VITEK(BioMerieux) cards

• ANI............. Anaerobes Micrococcus
• BAC............. Bacillus
• GPI.............. Gram Positives (Staph Strep)
• GNI/GNI... Gram Negatives (Oxidase Neg)
• NFC............. Gram Negatives (Oxidase Pos)
• YBC............. Yeast

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How does VITEK work

• 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

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How does 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

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How does ELISA work?

• 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

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Riboprinter Microbial Characterization System
(Dupont Qualicon)

• 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.

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How does Riboprinter work

• Getting a sample
• 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
• 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.

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How does Riboprinter work (cont.)

• Separating and transferring DNA
• 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
• 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

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How does Riboprinter work (cont.)

• Detection and extraction
• 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.

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Riboprinter
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PCR

• 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.

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PCRs requirement

• 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

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PCRs requirement

• 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

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PCRs three steps

• First, the target genetic material must be
  denatured-that is, the strands of its helix must
  be unwound and separated-by heating to 90-96C.
• 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.

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Commercialized PCR equipment

• 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

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BAX (Dupont 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 O157H7, L. monocytogenes, etc.  

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BAX (Dupont 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

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Limitation for rapid methods

• 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 cant detect the toxin
  occurrence

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Future trend

• Biosensor
• 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
• Electrochemical
• Optical
• Piezoelectric
• Thermal

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Future trend

• DNA biochip
• A miniature silicon surface containing thousands
  of gene probes in a thumbnail size area