Food Safety Magazine

INSIDE MICROBIOLOGY | December 2003/January 2004

Advances in Biotechniques Used in the Quality Assurance of Food Products

By Sanjeev K. Anand, Ph.D., and Mansel W. Griffiths, Ph.D.

Advances in Biotechniques Used in the Quality Assurance of Food Products

In contrast to quality control (QC), which is a reactive system that focuses on legal requirements and emphasizes statistically relevant measurements, quality assurance (QA) is a preventive approach that emphasizes operational procedures. These procedures must be robust, regularly reviewed and focused on the consumer. To establish QA/QC parameters, the food microbiologist uses two approaches. The first sets out to determine the total load of microbes in a sample, and the second attempts to determine the presence or absence of a particular microbial species, usually a pathogen or related type used as their indicators. Thus, while the first type of microbiological quality assurance test aims to establish that food products meet statutory requirements, the second type of analysis is focused on public health impacts with regulatory requirements as an integral part of the testing procedure.

In addition to the general testing requirements under QC programs, there has been an added element of quality assurance that is being pursued vigorously under the implementation of quality management systems such as Hazard Analysis & Critical Control Points (HACCP) plans. To further enhance the utility of these systems, there is a need to develop rapid microbiological detection techniques that are sensitive and accurate. Accordingly, much effort has been devoted to shortening assay times and to replacing the visible endpoints with alternative measurements.

Several biotechniques are employed by microbiologists for assuring the safety of food products. These include electrical methods such as impedance/conductance; chemical methods such as direct epifluorescent filter technique (DEFT); bacterial adenosine triphosphate (ATP) bioluminescence, flow cytometry, biosensors, and agglutination/immunological assays; and nucleic acid technologies such as polymerase chain reaction (PCR), ribotyping and microarrays. A review of those used today in food company safety and QA programs highlights many improved and innovative technologies available to the microbiologist.

Impedance/Conductance
The detection of microbial growth using electrical systems is based on the measurement of ionic changes occurring in culture media caused by the metabolism of microorganisms. The electrical changes are detected at the threshold of detection and the time taken to reach this point is called the detection time. These assays have been widely accepted for QA programs. In most cases, the net metabolic activity of the culture, measured as detection time, correlates well with—and is inversely proportional to—the microbial load in the sample. Thus, the higher the initial microbial load, the shorter the assay time. Results can be obtained within 2 to 4 hours.

For the detection and counting of specific pathogenic bacteria, innovative substrates used solely by the target bacteria or inhibitors to which all but the target bacteria are sensitive are included in the medium to increase the response by the pathogen and to minimize the growth and contribution by other microbes.

The automated instruments on the market—Malthus (IDG, Bury, UK), RABIT (Don Whitney Scientific, Yorkshire, UK), Bactometer (Biomérieux, Basingstoke, UK) and BacTrac (Sylab, Austria)—operate at frequencies between 2 and 10 kHz, with the capacitance changes and microbial numbers more pronounced at 2 kHz. All of these instruments have similar basic components: an incubator system, a monitoring unit that measures the conductance and/or capacitance and a computer-based data handling system. Up to 500 samples can be examined simultaneously on the current machines.

ATP Bioluminescence
All living cells contain adenosine triphosphate, the high energy intermediate that powers most energy-consuming reactions. The test depends on the reaction between ATP and the enzyme luciferase, producing light, which is measured photometrically, with a claimed sensitivity down to 10-16mol ATP l-1. It is a rapid test, taking less than one hour to complete. The light-yielding reaction is efficient, producing a single photon of light for every luciferin molecule oxidized and thus every ATP molecule used.

The problem with most foods is that they also contain ATP from non-microbial sources, and that the ATP content of microbial cells is variable depending upon their nature, the type of microorganism (e.g., bacterium, yeast) and their physiological state. In addition, the assay has no specificity. It is possible to destroy the somatic (food) cell ATP by using detergents to lyse the cells and ATPase to destroy the ATP or to remove microbial cells from the sample by filtration prior to extracting their ATP. However, the most widespread application of ATP measurement is as a hygiene test to monitor the cleanliness of food production areas. For this, the surface is swabbed and the total ATP on the swab is extracted and measured. In this application, it does not matter whether the ATP is derived from food residues or from microorganisms because both are indicative of an inadequately cleaned surface.

Some ATP systems come in the form of automated luminometers (Hygienia systemSure, BioControl Lightning MVP, BioTrace Unilite) and some are swab methods (3M QuickSwab) that provide rapid results on-site. Some ATP bioluminescence systems use adenylate kinese present in a sample to recycle phosphate and make ADP into ATP (Biotrace AKRAPID).

There is interest in developing ATP bioluminescence for the detection of specific microorganisms. Current research is focused on combining ATP bioluminescence with techniques such as immunomagnetic separation for specifically removing target organisms from food, or by specifically lysing target cells with bacteriophage.

Direct Epifluorescent Filter Technique
Automated microscopy methods have been widely applied. Due to the relatively low density and small size of microbes, they must be treated to stand out from the background mass. One such example is the direct epifluorescent filter technique, which depends on the uptake of acridine orange by the cells. Viable cells fluoresce orange under ultraviolet light (UV) due to their ribonucleic acid (RNA) content, whereas dead or non-growing cells fluoresce green due to interactions of the dye with deoxyribonucleic acid (DNA). Samples are concentrated by filtration, stained and viewed microscopically. Image analysis equipment can be used to automate counting. The first fully automated instrument based on fluorescence microscopy was the Bactoscan (Foss Electric, Denmark).

These tests have been employed primarily on products that have not undergone a heat treatment (to prevent the possibility of misleading results) and food products that can be easily filtered. The sensitivity is only ~104 cells/mL.

Cytometric Techniques
Flow cytometry based techniques have been reported recently. The sample containing microorganisms is injected into a stream of fluid, which then passes a sensor where each particle is detected. The cells under investigation are inoculated into the center of a stream of fluid (known as sheath fluid). This constrains them to pass individually past the sensor and enables measurements to be made on each particle in turn, rather than average value for the whole population. The sensing point consists of a beam of light (either UV or laser) that is aimed at the sample flow, and one or more detectors that measure light scatter or fluorescence as the particles pass under the light beam.

Fluorescent probes based on enzyme activity, nucleic acid content, membrane potential and pH have been developed and examined. Use of antibody-conjugated fluorescent dyes confers specificity to the system. Perhaps the most successful application of flow cytometric methods to food products has been the use of the D-Count (AES Chemunex, Maisons-Alfort, France) system to detect yeast contamination in dairy and fruit products. The technique is still in its infancy and although good results are being reported for some foods, sensitivity for specific detection of a particular organism in different foods is rather less developed. The success of the system depends on the development and use of suitable staining systems and the protocols for the separation of microorganisms from food debris that would otherwise interfere with the detection system.

A relatively new solid phase cytometric technique, Fluorassure, also has been developed by AES Chemunex. In this procedure, samples are passed through a membrane filter that captures contaminating microorganisms. A stain is applied to the filter to fluorescently mark metabolically active microbial cells. After staining, the membrane is transferred to a Chemscan RDI instrument, which scans the whole membrane with a laser and counts the fluorescing cells. The complete procedure takes about 90 minutes to perform and can detect single cells in filtered samples.

Immunological Methods
Immunochemicals have been used for several decades to detect microflora in foods. Microorganisms are antigenic and thus stimulate the production of antibodies when injected into animals. Antibodies are protein molecules that are produced by animal white blood cells in response to contact with a substance causing an immune response. The area to which an antibody attaches on a target is known as the antigen.

Two types of antibodies can be employed in immunological tests. These are known as monoclonal and polyclonal antibodies. Polyclonal antibodies react with a broad range of antigens, whereas monoclonal antibodies are highly specific to particular antigenic structures. Both types of antibodies have been used in reagent kits for the detection and identification of specific type of bacteria, their surface structure and toxins. The antibodies are tagged to assist in the measurement of the antigen-antibody complexes. The most sensitive labels are radioactive isotopes but these can not be used in food production environments; hence, fluorescent antibodies labeled with fluorescein or umbelliferones are most common. There have also been reports of the incorporation of antibodies, produced in plants, in packaging films. They show visible changes on reaction with the target microbes.

A number of latex agglutination reactions have been commercialized by manufacturers and have been successfully used within the food industry. They offer a relatively fast test time, are easy to use and require no specialist equipment, making them ideal for QA applications. Good examples are latex agglutination kits for Salmonella confirmation, such as the Oxoid Salmonella Latex kit, RIM for E. coli O157 (Remel), Micro-screen Latex Slide (Microgen Bioproducts), Wellcolex Color Salmonella Test (Wellcolex), and Spectate Salmonella test (May and Baker Diagnostics). The latter two can even differentiate between serogroups. Agglutination kits have been also developed for the detection of microbial toxins.

The enzyme immunoassay (EIA) and the enzyme-linked immunosorbent assay (ELISA) are the most commonly used techniques for rapid detection of pathogen and toxins. They have the advantage of specificity conferred by the use of a specific antibody, coupled with colored or fluorescent end-points that are easy to detect, either visually or with a spectrophotometer or fluorimeter. Auto-mation of enzyme immunoassays has taken a number of forms, including the standard microplate ELISAs. The automated VIDAS system (Biomérieux, Hazelwood, MO) is based on enzyme-linked fluorescent assay (ELFA) technology in a solid phase receptable (SPR) and pipetting device. VIDAS, which can be used to detect Salmonella, Listeria, E. coli O157:H7, Campylobacter and staphylococcal enterotoxins, can handle up to four analytical modules at once for a total testing capacity of 240 tests per hour. The company has released a second generation of VIDAS assays, the LMO2 and SET2 (for Listeria monocytogenes and staphyloccocal enterotoxin, respectively), in which complementary monoclonal antibodies are directed to different antigenic sites of a specific protein.

The EIAFOSS (Foss Electric, Denmark) is another fully automated ELISA system. In this case, the instrument transfers all of the reagents into tubes containing the sample in which all of the reaction occurs. The EIAFOSS procedure is novel as it uses antibody-coated magnetic beads as a solid phase. Immunomagnetic reagents are increasingly used for capturing and concentrating pathogenic bacteria, so much so that they have become essential steps in detection protocols for some potential pathogens, including Salmonella, Listeria and E. coli O157.They permit larger volumes of extract to be screened and about a nine-fold increase in assay sensitivity has been achieved by such means.

More recently, Matrix Microscience (Newmarket, UK) has developed the PATHATRIX system, which allows capture of target bacteria from an entire enrichment culture (250 mL) by directing flow of the sample across immobilized antibody-coated beads. PATHATRIX products for the detection of Salmonella, Listeria and E. coli O157, and a dual Salmonella-Listeria test, are marketed.

Immunoassay-based diagnostic test kits come in a variety of forms—membranes, tubes or dipsticks—and these rapid tests have become well-established in the QA toolbox because many of them provide results in 24 to 48 hours and provide the ability to test many samples simultaneously. Some are based on lateral flow technique, such as the Reveal for E coli, Salmonella and Listeria (Neogen Corp.); the RapidCheck assay for Salmonella (Strategic Diagnostics International); and VIP for Listeria, EHEC and Salmonella (BioControl). ELISA/EIA-based assays, which provide visual indicators within minutes, include automated systems such as the TECRA Unique Plus (International BioProducts); tests that can be read visually such as PetriFilm (3M Microbiology), Assurance Listeria and EHEC EIA (BioControl Systems), and TECRA Salmonella or Listeria Immunoassays (International BioProd-ucts), PATHIGEN for E. coli O157:H7, Listeria, Salmonella and Campylobacter (IGEN International) and MICRO-ID Listeria (Remel); and those that can be read visually or instrumentally, such as the Assurance Gold for Salmonella and Campylobacter (BioControl). Also interesting is the introduction of chromogenic culture media that detect and differentiate species in 24-48 hours (Bio-Rad Laboratories RAPID’L.Mono agar).

Alternative immunoassay formats of interest to the QA microbiologist also have been developed. One example is immunochromatography, which operates on a dipstick composed of an absorbent filter material containing colored particles coated with antibodies to a specific organism. The particles are on the base of the dipstick, which, when dipped into a microbiological enrichment broth, results in movement of the particles by capillary action along the filter material. At a defined point along the filter material lays a line of immobilized target-specific antibodies. If present in the sample, the target organism binds to the coloured particle and the resulting cell/particle conjugate moves along the filter dipstick by capillary action until it meets the immobilized antibodies where it is bound. The buildup of colored particles results in a clearly visible colored line, indicating a presumptive positive test result. Some of the commercial kits based on this technology are Oxoid Listeria Rapid test and the Celsis Lumac Pathstik.

Another newer alternative method is the Biosys (MicroFoss) automated system, which is based on an optical technique. This system, used for the detection of Listeria in environmental samples, can provide presumptive positive results in two days and test 128 samples simultaneously.

Nucleic Acid Hybridization
Nucleic acid technologies are being used increasingly for quality assurance purposes. The specific characteristics of any organism depend on the particular sequence of the nucleic acid contained in its genome. The sequence of bases of nucleic acids makes different organisms unique. Nucleic acid probes are small segments of single-stranded nucleic acid that can be used to detect specific genetic sequences in test samples. Probes can be developed against DNA or RNA sequences. The attraction for the use of gene probes is that a probe consisting of only 20 nucleotide sequences is unique and can be used to identify an organism accurately. In order to identify the binding of a nucleic acid probe to DNA or RNA from a target organism, it must be attached to a label that can be easily detected. These labels can be radioisotopic (32P), or more recently non-radiolabelled probes, such as DIG-labels, have been routinely used.

Probes directed towards cell DNA attach to only a few sites on the chromosome of the target cell. Work on increasing the probe sensitivity has centered on the use of RNA as a target, especially ribosomal RNA (rRNA) that is present in very high copy numbers within cells. The first commercially available nucleic acid probe-based assay, GENE-TRAK, was introduced in 1985 for food analysis. This used Salmonella-specific DNA probes (32P labeled) directed against chromosomal DNA to detect Salmonella in enriched foods. Later, in 1988, they introduced non-isotopically labeled probes for Salmonella, Listeria and E. coli based on targeting the ribosomal RNA. This type of colorimetric hybridization assay is based on a liquid hybridization reaction between the target rRNA and two separate DNA oligonucleotide probes (the capture probe and reporter probe) that are specific for the organism of interest. The capture probe molecules are extended enzymatically with a polymer of about 100 deoxyadenosine monophosphate residues. The reporter probe molecules are labeled chemically with hapten fluorscein. The GENE-TRAK microwell tests available today are used to detect Salmonella and Listeria (Neogen Corp.). Other commercially available nucleic acid probes exist for the confirmation of Campylobacter, Staphylococcus aureus and Listeria and are marketed by Genprobe.

In recent years, several genetic amplification techniques have been developed, with polymerase chain reaction and its variants—nested PCR, reverse transcriptase (RT) PCR and multiplex PCR—emerging as a biotechnique of choice in the food industry. PCR involves detection of specific gene fragments by in-vitro enzymatic amplification of the target DNA, followed by detection of the amplified DNA molecule by electrophoresis, ELISA or other techniques. The PCR method is a highly specific and sensitive method allowing the detection of low numbers of microorganisms. In the past, the general limitations of the technique have included difficulty in obtaining specific DNA primers and production of nonspecific PCR products. Also, organisms that have been killed during processing were not recognized as dead if their DNA was still present, thus giving false-positive reactions.

Automation and improvements in PCR systems have addressed some of these problems and made the technique an extremely attractive option. Currently, there are several manufacturers producing PCR-based test methods for the detection of foodborne pathogens, including the BAX System Assays for Screening (DuPont Qualicon, USA) for Salmonella, Listeria, and E.coli O157; Roche Diagnostics LightCycler (Roche Diagnostics, USA); Probelia (Sanofi-Pasteur, France) for Salmonella and Listeria, Genevision for Listeria monocytogenes, Salmonella and E. coli O157:H7 (Warnex, Canada), and TaqMan (Perkin Elmer, USA) for Salmonella.

Biosensors and Biomarkers
Using chemo- or biosensors as QA tools to detect pathogens or to assess quality attributes such as shelf-life is a relatively new development. The most promising targets for biosensor-based techniques in food analysis are free radicals and DNA. Scil Diagnostic GmbH and GeneScan Europe AG have commercialized a microarray reader family, BioDetect 645, which can be used for the detection of food pathogens, as well as genetically modified foods. The DNA chips have an active area of 2 cm2 and the indication of DNA hybridization takes place via fluorescence detection by a CCD camera. The sensitivity is <10 fluorophors/mm2. These Nutri-Chips can detect Lactobacillus lactis, L. brevis, L. plantarum, Salmonella spp., Listeria spp., Campylobacter spp., E. coli and Shigella spp.

In addition, there are gas sensor arrays and fingerprint mass spectra (MS) systems (FMS), based on quadrupole mass spectrometer combined with headspace sampler and a computer, like the 4440B Chemical Sensor (Agilent Technologies), based on pattern recognition technique (PARC). A technique known as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry has been used to analyze crude cellular fractions or cellular suspensions and this has led to the rapid development of a number of MALDI-TOF methods involving bacteria. Applications include the analysis of bacterial RNA and DNA, the detection of virulence markers, and the very rapid characterization of bacteria at the genus, species and strain levels. The demonstrated ability of taxonomic classification at the strain level, using unprocessed cells, opens the possibility that MALDI-TOF and similar MS approaches may result in the development of near real-time methods for the characterization of bacteria.

The future technologies using biomarkers look very promising. In this category special mention should be given to bioluminescent markers, popularly known as lux-bioluminescent systems. The bacteriophage-bioluminescent reporter system for foodborne pathogens is rapidly finding acceptance. A recent application is the AK-phage assay (Alaska Food Diagnostics) that is based on immunomagnetic separation of target cells, phage lysis and adenylate kinase assay. Current research in our lab on real-time monitoring of pathogenesis in animal hosts and the linking of cell signals to virulence expression may well form the basis of a single-step detection system for virulent pathogens using bioluminescent-bioreporters in the future.

Conclusions
The food industry has the responsibility to produce safe and wholesome food and providing this assurance to consumers is the ultimate goal of food microbiologists. What is needed is a microbiological test that is able to analyze a batch of food non-destructively, on-line and with the required accuracy, sensitivity and specificity. Although our current technical capabilities fall short of this ideal situation, the rapidly expanding array of biotechniques at our disposal are playing a key role in helping microbiologists assure food safety and quality.

Sanjeev K. Anand, Ph.D., is a Senior Scientist and in charge of the Quality Assurance Lab, Dairy Microbiology Division, National Dairy Research Institute (NDRI) in Karnal, India. He has been working in the area of food safety and quality assurance for more than 18 years, including 10 years as a scientist-in-charge with the Food Microbiology Laboratory in the Poultry Products Technology Division of India’s Central Avian Research Institute. An author of more than 75 research papers in peer-review journals, Anand recently completed a DBT Overseas Associateship involving advanced research in pathogens at the Canadian Research Institute for Food Safety, Guelph, Canada, where he conducted studies on cell-to-cell signaling and virulence expression of pathogens.

Mansel W. Griffiths, Ph.D., holds an Industrial Research Chair in Dairy Microbiology in the Food Science Department at the University of Guelph. Griffiths’ position is funded jointly by the Dairy Farmers of Ontario and the Natural Science and Engineering Re-search Council of Canada. He is program chair for the M.Sc. in Food Safety and Quality Assurance and is the Director of the Canadian Research Institute for Food Safety, a research collaboration between federal and provincial government and the University of Guelph. He is a member of the International Dairy Federation working group on milk-borne pathogens and serves on the Expert Scientific Advisory Committee for Dairy Farmers of Canada. He is the recipient of the IAFP Maurice Weber Laboratorian of the Year for 2002.

Categories: Regulatory: HACCP; Testing and Analysis: Methods, Microbiological