February 2003, Volume 57, No. 2 ›
Microbial Testing of Produce
‹ February 2003, Volume 57, No. 2
James Giese
Human illnesses associated with the consumption of fresh produce and sprouts have increased during the past two decades. According to research by Larry R. Beuchat and Jee-Hoon Ryu at the University of Georgia (see "Produce Handling and Processing Practices" at www. cdc. gov/ ncidod/ eid/vol3no4/ beuchat.htm), changes in farm practices, harvesting, distribution, processing, and consumption patterns have contributed to the increase.
Pathogens of concern include Listeria monocytogenes, Clostridium botulinum, Bacillus cereus, Salmonella, Escherichia coli O157:H7, Campylobacter jejuni, Vibrio cholerae, parasites, and viruses. The first three are naturally present in the soil and often found on produce. The others are more likely to contaminate fresh produce through vehicles such as raw or improperly composted manure, irrigation water containing untreated sewage, or contaminated wash water.
As a general rule, the amount of microorganisms present on fruit and vegetables reflects the initial conditions at the time of harvest, as well as the sanitary quality of the handling and processing steps taken afterward.
Currently, there are no microbiological standards for fresh produce. Because the microflora of fresh produce varies widely, it is unlikely that a uniform set of standards could be developed.
Responding to concerns about the increased incidence of illnesses associated with produce, Congress authorized an appropriation of $6.23 million for fiscal year 2001 to fund a microbiological monitoring program for foodborne pathogens on domestic and imported fruits and vegetables. The program received an additional $6.23 million for continuing activities in 2002. The program, part of the broader 1997 Presidential Food Safety Initiative, seeks to establish a microbiological baseline to assess the risks of contamination in the domestic food supply. Another goal of the project is to get a better idea of the microbial ecology of fruits and vegetables. The U.S. Dept. of Agriculture's Agricultural Marketing Service (AMS) was appointed to undertake the creation and implementation of the program. The Microbiological Data Program (MPD) began collecting and analyzing samples in April 2001. More information about the program is available at www.ams.usda.gov/science/MPO).
According to USDA, the MDP is designed to establish a microbial baseline to assess the risks of contamination, if any, in the domestic food supply. The data will be used to establish benchmarks by which to evaluate the efficacy of procedures to reduce or eliminate harmful foodborne microorganisms. The data will also be provided to stakeholders for decision-making purposes. The stakeholders include federal and state public health agencies, growers, processors, retail stores, food handlers, and consumers.
There are a variety of microbiological tests that can be done on produce, depending on the need. For a general overview of pathogen testing for produce, see the Produce Marketing Association's fact sheet, "Q&A on Microorganisms and Lab Testing for Produce," prepared by Michael P. Doyle, Robert E. Brackett, and Larry R. Beuchat at the University of Georgia's Center for Food Safety and Quality Enhancement (www.pma.com/ii/mem_only/fact_labtesting.cfm).
Aerobic plate counts provide a good general estimate of the total number of microorganisms on produce. E. coli counts can indicate the presence of fecal contamination. Since E. coli is commonly present in feces, its occurrence in foods suggests that feces may be present. Coliform and fecal coliform assays were originally developed to indicate the presence of E. coli and consequently fecal contamination. However, for produce, these assays are questionable indicators of fecal contamination because they detect many bacteria other than E. coli that do not originate from feces but are commonly present on produce. According to Doyle, Brackett, and Beuchat, coliform and fecal coliform assays are not recommended for produce unless there is evidence that the results have a useful purpose.
Testing Procedures
AMS uses several screening tests to gather information for the MDP. These tests were chosen to obtain results as simply and quickly as possible (i.e., in 48 hr or less) on the presence or absence of two major pathogenic bacteria, namely, Salmonella and E. coli O157:H7.
The screening tests are AOAC approved or have been used by the Food and Drug Administration. They are also the tests that FDA plans to use as screening tests to monitor spent irrigation water at sprouting facilities.
• For E. coli O157:H7, USDA uses the VIP EHEC test and the Reveal E. coli O157:H7 test.
The VIP EHEC test (AOAC Official Method 996.09) from Biocontrol Systems, Inc., Bellevue, Wash., is said to be the first method for the detection of E. coli O157:H7 to successfully complete validation. The test is said to be accurate and simple and gives results in 18 hr from sample enrichment to reading results.
The Reveal E. coli O157:H7 test system from Neogen Corp., Lansing, Mich., allows the screening of samples for this pathogen following as little as 8 hr of sample enrichment. The system was designed for quick product turnaround. The test is said to be easy to use and requires only a relatively minor investment in equipment and training.
To perform the test, a 120-mL portion of the enrichment culture is placed into the round sample port of the test device. The sample is wicked through a reagent zone which contains specific anti-E. coli O157:H7 antibodies conjugated to colloidal gold particles. If antigens are present in the sample, they will bind to the gold-conjugated antibodies. This antigen–antibody complex then leaves the reagent zone and travels through the nitrocellulose membrane which contains a zone of anti-E. coli O157:H7 antibody. The immune complex with gold conjugate is captured and aggregates in this zone, thus displaying a visible line. The remainder of the sample continues to migrate to the end of the membrane, where it is eventually deposited into a waste reservoir.
The reagent zone also contains a gold conjugate of a proprietary antigen (color indicator), which is eluted by the sample solution regardless of the presence of E. coli O157:H7 antigen. The color indicator migrates through the membrane to the negative-control capture zone (antibody to the proprietary antigen), where it is captured and aggregated to form a visible line. Regardless of the presence or absence of the E. coli O157:H7 antigen, the control line will form in the control zone, ensuring that the test is working properly.
• For Salmonella detection, USDA uses BioControl Systems' Assurance Gold Salmonella EIA test (AOAC Official Method 999.08) and Visual Immunoprecipitate (VIP) Assay for Salmonella (AOAC Official method 1B 999.09).
A variety of articles about the microorganisms on fresh fruit and vegetables are readily available. For example, the Institute of Food Technologists' report to FDA entitled "Analysis and Evaluation of Preventive Control Measures for the Control and Reduction/Elimination of Microbial Hazards on Fresh and Fresh-Cut Produce" can be found at www.cfsan.fda.gov/~comm/ift3-toc.html. A review article by Larry Beuchat entitled "Surface Decontamination of Fruits and Vegetables Eaten Raw" is available through the World Health Organization Web site at www.who.int/fsf/fos982~1.pdf. A detailed description of USDA's QTV program can be found at www.ams.usda.gov:80/fv/ PPBfilecodes/qtv-rm8b.pdf. And the joint FDA/USDA document, "Guide to Minimize Microbial Food Safety Hazards for Fresh Foods and Vegetables," is available at www.fda.gov/ohrms/dockets/98fr/ 97N0451.pdf.
Additional information can be obtained from the University of Georgia's Center for Food Safety and Quality Enhancement (CFSQE), a research center specializing in microbiological food safety and related food quality issues. The Center has several researchers with specific expertise in the microbiology of fresh and minimally processed produce. For more information, contact CFSQE, University of Georgia, Griffin, GA 30223-1797 (phone 770-228-7284; fax 770-229-3216).
Online LIMS System allows clients to access results of their microbiological testing at any time. According to William L. Brown, President of ABC Research Corp., the new online Laboratory Information Management System can provide clients submitting food or beverage samples for testing the ability to get results as the tests are completed and posted, without waiting for fax, phone, or e-mail notification. This means that quality control and quality assurance directors at food production facilities will know quickly that their product is free of pathogens and safe to ship. The system can be accessed 24 hr/day, 7 days/week. Even the status of a particular sample can be checked. Companies using this system, which is protected through a firewall with a login procedure, need no additional software. For more information, contact ABC Research, Corp., 3437 S.W. 24th Ave, Gainesville, FL 32607 (phone 352-372-0436; fax 352-378-6483).
pH System, the IQ400, is both a pH meter and handheld computer. The meter accepts both glass and non-glass electrodes. The meter features touch screens, pop-up windows, on-screen help tips, and troubleshooting guides for each function. The "eDoodle," a virtual sticky note, allows users to save handwritten notes and sketches with their pH readings. The system allows the user to run all Palm OS applications, such as advanced scientific calculators and spreadsheets, and saved records can be downloaded to a computer. Features include hi/ low pH level alerts, recalibration alarms, automatic buffer recognition, save/recall of up to 9,999 records, automatic buffer recognition, and user selectable buffer values. For more information, contact IQ Scientific Instruments, Inc., 11021 Via Frontera, Suite 200, San Diego, CA 92127 (phone 858-673-1851; 800-276-0723; fax 858-673-1853).
Potentiometer Titrator is said to offer accurate quantitative analysis for a variety of needs. The KEM Automatic Potentiometer Titrator is suitable for testing acid base, COD, water hardness, redox, and Karl Fisher moisture. The instrument is said to make even complicated titrations with multiple or difficult-to-read endpoints or multiple reagent dosing easy. For more information, contact CSC Scientific Co., Inc., 2810 Old Lee Hwy., Fairfax, VA 22031-4304 (phone 800-458-2558; fax 703-280-5142).
Viscometers and Rheometers are described in a new 74-p catalog. It features several new and updated products including the CAP 1000+ and the CAP 2000+ viscometers, the new YR-1 yield stress rheometer, and a new process inline viscometer. The catalog also has information on services available, including viscosity testing, methodology consulting, viscosity test development, instrument calibration and certification, and other services. The catalog has a "Help Me Choose a Viscometer" section and a selection chart with features and benefits for each instrument. For a copy of the catalog, contact Brookfield Engineering Laboratories, Inc., 11 Commerce Blvd., Middleboro, MA 02346 (phone 800-628-8139; fax 508-946-6200; www.brookfieldengineering.com).
Nano-Particle Measurement System, the Zetasizer Nano series, may be used for particle measurement in food applications. It uses advanced technology for the simplest, most repeatable, and accurate measurement of particle size, zeta potential, and molecular weight of molecules and nano-sized particles. Applications include the measurement of suspensions, emulsions, solutions, and microencapsulated products. The series includes the Nano-ZS for measurement of all three parameters, the Nano-S for combined particle size and molecular weight, and the Nano-Z for measurement of zeta potential. The latter is a measure of the stability of a solution or a suspension and is an important parameter in the food industry for products such as edible films, milk, flavorings, and oil-in-water suspensions. For more information, contact Malvern Instruments Ltd., Enigma Business Park, Grovewood Rd., Malvern, Worcestershire, UK WR14 1XZ (phone +44 (0)1684 892456; fax +44 (0)1684892789).
Pre-weighed Dehydrated Culture Media, RapiDCM™, aids microbiologists in the preparation of culture media. The product is prepared in three steps: Add product to water, boil or autoclave, and dispense. A variety of media are available in both 1.0 and 3.0 L sizes. According to the company, all media are manufactured in an ISO 9002 Certified facility. All animal product components used in the manufacture of the products are of United States origin or are imported in compliance with USDA/APHIS regulations. Imported components are certified by the suppliers to be from countries that are BSEfree. For more information, contact Remel Laboratory Products, 12076 Santa Fe Dr., Lenexa, KS 66215-3594 (phone 800-255-6730; 913-888-0939; fax 913-888-5884).
by JAMES GIESE
Internet Editor