Food Science: Testing for Microbes, Pesticides, and Fakes
Food contaminants and adulterants fall under many categories, all with significant potential to adversely affect brands, profits, and health
Food contaminants may be chemical, microbiological, or broadly classified adulterants.
Foods vary so broadly in composition that nearly every analytical method has the potential to provide some bit of information, but selecting wisely is essential to achieve meaningful, timely answers to the myriad, multidimensional questions of safety, quality, and authenticity.
Molecular Methods
Rapid, reliable first-pass assays for pathogen testing are an initial step usually followed by culturing or molecular techniques. By targeting an organism’s genes, a polymerase chain reaction (PCR) detects harmful and non-pathologic bacteria and viruses as well. Viruses represent an interesting avenue for food pathogen investigation, as they may affect the health and productivity of raw material sources (e.g., trees, animals), and sometimes infect humans as well. bioMérieux (Paris, France) recently introduced GENEUP ®, a PCR platform suitable for identifying pathogens at every point in the ingredient and product supply chain. The system employs PCR technology developed at Bio- Fire Diagnostics, which specializes in medical diagnostics. bioMérieux acquired BioFire in 2013. Also in play is PCR virus detection know-how from Ceram, another recent addition to the bioMérieux corporate family.
GENE-UP tests for Salmonella, Escherichia coli O157:H7, Listeria, and enterohemorrhagic E. coli at levels as low as one organism per 25 g of food sample. “These pathogens are among the most commonly tested in food because they pose a risk to human health,” says Pascal Montes, senior global product manager for food safety (at bioMérieux). Since it is based on highly sensitive fluorescence resonance energy transfer (FRET), food producers can manage risk from raw materials to finished product on the same instrument, Montes adds.
Neogen (Lansing, MI) uses DNA amplification technology similar to PCR but which works through isothermal reaction rather than temperature cycling. Target genes polymerize from the ends of “nicks” created in double- stranded DNA by the action of an endonuclease. Amplified target sequences are detected in real time using fluorescent molecular probes. The firm’s ANSR (Amplified Nucleic Single-Temperature Reaction) tests for E. coli O157:H7, two Listeria pathogens, and Salmonella. “Back in the day, lengthy pathogen culturing was the only recourse, and some very good applications that rely on culturing are still in use,” explains James Topper, Neogen’s market development manager.
But for the production of foods, nutraceuticals, and other high-value products, rapid testing has become the norm. “The longer you go not knowing you have a problem, the more of your production you are forced to waste or the greater your rework burden,” Topper tells Lab Manager.
Food pathogen detection is unique among PCR applications in the need to grow target bacteria to detectable levels. “The presence of a single pathogen in a product can cause concern,” Topper says. Thus all pathogen detection platforms require a growth enrichment stage, by far the most time-consuming part of the assay.
Interestingly, how companies deal with a positive test is a matter of policy and not regulation. “There is no universal response,” Topper says. Companies commonly confirm positive pathogen results with plates or dry culture media and set aside product pending confirmation. A positive allergen test might involve cleaning cross-contaminated product surface areas. As with any diagnostic, food safety tests carry a small but finite percentage of false positives.
Similarly, neither the U.S. Dept. of Agriculture nor the Food and Drug Administration require specific tests. Instead they specify levels for certain adulterants, and leave it to manufacturers to decide on the assay. Companies can also justify testing on the basis of genuine concern for consumer health or for their own peace of mind. So while regulation is a significant motivation for safety testing, it is not the only one.
That is not meant to imply that the agencies are unconcerned. The Food Safety Modernization Act (FSMA) of 2011, which is just now coming into effect, was hailed as the first update to food safety laws in 70 years. By providing guidance on preventing contamination rather than simply detecting it after the fact, the law seeks to implement quality by design into food production rather than “testing it in.”
Food Fraud
Food fraud compromises authenticity and usually involves adulteration. For example, olive oil producers may claim an oil comes from Italy while the product might originate from another country. Adulteration might involve adding some fraction of another edible oil which, although usually perfectly safe, is not genuine. Adulterants may range from components of questionable taste or healthfulness that stretch the product (e.g., sugar or salt in canned goods) to the downright dangerous (methanol in distilled spirits).
The Grocery Manufacturers Association estimates the cost of economic adulteration and counterfeiting of food and consumer products at $10 to $15 billion per year. The expense of one adulteration incident averages between two and 15 percent of a company’s yearly revenues. This translates to a $60 million loss for a modestly sized company, and $400 million for a $10 billion food giant. Analysis for chemical contaminants, such as pesticides, veterinary drugs, dioxins, and food packaging contaminants, is highly regulated worldwide and monitored throughout the food value chain, from raw ingredients to finished products.
“From an analyst’s perspective, adulteration is often a question of whether something exists in a product that shouldn’t be there or has been added without declaration. Adulteration is the addition of something,” says John Lee, global food marketing manager at Agilent (London, UK).
Authenticity is a related but somewhat different issue. “A product doesn’t need to be adulterated or dangerous to be non-authentic; extra virgin olive oil from Italy, which is not extra virgin and not from Italy, may be unadulterated olive oil but clearly is still non-authentic, therefore fraudulent,” Lee adds.
Adulteration takes on a dangerous course when the product is an herbal or over-the-counter supplement, where pharmacologically active ingredients affecting putative potency may be added to enhance the product’s performance. “There’s often a grey area between due diligence around accurate labeling and intentional misrepresentation,” Lee notes. “We can help suppliers test for labeling accuracy but I leave determinations of intent to lawyers.”
Analysis by stand-alone liquid chromatography for authenticating olive oil or dietary supplements includes reference methods from standards or industry organizations, e.g., the International Olive Council. Generally, these are targeted methods that look for known components. Interest in mass spectrometry (MS), combined with either liquid chromatography (LC) or gas chromatography (GC), has been increasing as well.
“Sometimes the red flag is beyond the capabilities of reference methods,” Lee admits. “These often include unexpected ingredients, dilution, or adulteration.” To obtain a fuller picture, he recommends full-scan MS, like Q-TOF, for finding and identifying markers of adulteration or other misrepresentation, though this is a process that requires a lab to also pay sufficient attention to the natural variability in a commodity. Such profiling work with MS is certainly within the capabilities of most food analysis labs, but adulteration and authenticity work has so far been primarily developed within academia and government organizations. “However we’re now seeing more and more private labs get involved,” says Lee.
Isotope ratio analysis is an emerging MS technique popular for profiling food. It relies on subtle, natural geographical differences in ratios of stable isotopes of atoms most abundant in foods, for example, 13C/12C and 1H/2H, which are unique for geographic regions. Combustion front-end methods provide ratios of isotopically divergent water and carbon dioxide—a measure of isotope ratio. Similarly, inductively coupled plasma-MS can be used to profile a wide range of trace minerals, where the ratios between them can also act as markers of authenticity. Chinese regulators have developed an ICPMS method for the elemental fingerprinting of honey. “This was accomplished with an Agilent instrument,” Lee informs. The University of California, Davis, has employed similar methods to characterize wine varieties by grape species, vineyard, and even processing facility.
The basic food profiling with MS is, as Lee puts it, “at the interface of chemistry and statistics. Companies and governments are working hard to perfect such methods, and address the challenge of separating expected commodity variation from fraudulent practice.”
Evolving Standards
“Food safety standards change and evolve, which often means that what was once at the lower end of a permissible pesticide residue level may be at the upper end tomorrow,” says Khalil Divan, PhD, senior director for the food and beverage market with Thermo Fisher Scientific’s chromatography and mass spectrometry business. Thermo Fisher specializes in systems and methods for analyzing foods’ physical, chemical, and microbiological attributes. The goal of suppliers of instruments and test kits is to stay ahead of the curve.
Because foods consist of sample matrices varied in chemical, mechanical, and contaminant composition, sample preparation methods are in great demand, as are automated analysis systems. Thermo Fisher, for example, sells QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) dispersive solid phase extraction (SPE) products and consumables for manual, semiautomated, and automated workflows.
But at the bottom line, successful contaminant detection, identification, and quantitation begin with successful separations. Some contaminant classes are more amenable to separation by gas, liquid, or ion chromatography, while others can be separated using multiple orthogonal methods. GC, which often requires derivatization, has slowly given way to LC, although established reference methods for GC persist. Depending on the level of detail required and the prior characterization of specific samples, LC may be followed by common detection modes like ultraviolet. When molecular weight determination or unknown identification is required, mass spectrometry is the detector of choice.
As pesticide residue standards evolve, foodborne pathogens often become topical for a while, then fade. “If you’d asked me two years ago for an emerging pathogen, it would have been non-015787, called Shiga toxin E. coli or STEC,” Topper says. The 1993 Jack in the Box E. coli contamination issue, which involved E. coli 015787, led to further investigations that found that non-015787 STECs also cause illness. “It looked for a while like this would be an emerging testing market. The USDA even came out with a guidance implying need for testing this agent,” Topper says. “We and other vendors developed tests for these bacteria but they never took off.”