Food Safety and Beyond Jianrong (Janet) Zhang, Ph. D
Food safety background n n 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. ) n 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. ) n Food safety will continue to be important issue in the future, because: n n n 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 n n n 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 food safety and applied nutrition(CFSAN) USDA Food Safety and Inspection Service(FSIS)
Codex Alimentarius Commission n n Implements the joint FAO/WHO Food Standards Program To protect the health of consumers and to ensure fair practices in trade
Codex Alimentarius Commission n n 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 n n CDC published Preliminary Food. Net 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 n n There was 16 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 n n 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 n 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 n n n n 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 n A biological, chemical or physical agent in a food with the potential to cause an adverse health effect
Current Hazard n Biological n n Chemical n n n Bacteria Viruses Parasites Pesticide residues Veterinary drugs Physical n n Contaminated raw material Poorly designed or maintained equipment
Microbial growth, survive and death in food n n n n p. H Water activity aw Oxygen absence Temperature Nutrient content Antimicrobial constituents Biological structures
p. H 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 n Food can be divided into two major categories: low acid (p. H <4. 6) and acid (p. H > 4. 6). These were established based upon the growth of C. botulinum, whose minimum growth p. H requirement is generally accepted as 4. 8.
Water Activity Bacteria 0. 90 -0. 91 S. aureus – 0. 83 Halophilic bacteria – 0. 75 n Yeasts 0. 87 -0. 94 Osmotolerant yeasts – 0. 60 n Molds 0. 70 -0. 80 Xeromyces – 0. 60 n
Water activity of foods n n n 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 n n 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 n n n 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 n n n Campylobacter jejuni Clostridium botulinum E. coli O 157: H 7 Listeria monocytogenes Salmonella n n n Staohylococcus aureus Shigella Toxoplasam gondil Vibrio vulnificus Yersinia eneterocolitica
Microbial detection n n 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 n "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 n n First made available in the early 1980 s 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 n n n Biochemical test kits Antibody assay DNA-based assay
Partial list of miniaturized biochemical kits and automated systems for identifying foodborne bacteria n n n n n APIb Cobas IDA Micro-IDb Enterotube. II Spectrum 10 Rap. ID BBL Crystal Minitek Microbact Vitekb n n n n 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 n n ELISA - Enzyme-Linked Immunosorbent Assay LA – Latex agglutination IMS – Magnetic beads Major manufacturers: Bio. Merieux, Foss, Microgen, Biocontrol, TECRA, Elcatech, etc.
Partial list of commercially-available, nucleic acid-based assays used in detection of foodborne bacterial pathogens n n BAX Probelia n n n Based on PCR assay Accu. Probe GENE-TRACK n n Based on Probe assay Bindb n Based on Phage assay
Some other rapid methods n n n 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®(Bio. Merieux) n n 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.
®(Bio. Merieux) cards VITEK n n n 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 n n 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 o. C to be fresh sample
® work (cont. ) How does VITEK n n n 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? n n 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) n n n Fully automated ribotyping system that provides a genetic "fingerprint" of any bacterium in about eight hours. The system extracts a Ribo. Print® 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.
® work How does Riboprinter Getting a sample n n A colony is picked manually from the plate and introduced into the Ribo. Printer system, where the colony is suspended in a buffered solution and then heated Preparing the DNA n n 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. ) n Separating and transferring DNA n n 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 n 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. ) n Detection and extraction n The glow of the DNA fragments is not visible to the naked eye. However the Ribo. Printer 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 Ribo. Printer pattern that can be compared with other such patterns from other images.
Riboprinter®
PCR n n n 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 n n 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 n n n 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 n n n 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 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 n n 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
® (Dupont Qualicon) BAX n n n 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 O 157: H 7, L. monocytogenes, etc.
® (Dupont Qualicon) BAX n n 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 n n 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 n Biosensor n n 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: n n Electrochemical Optical Piezoelectric Thermal
Future trend n DNA biochip n A miniature silicon surface containing thousands of gene probes in a thumbnail size area
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