For a long time, food microbiologists operated with tools that were considerably outdated when compared to the advanced instruments available in fields like analytical chemistry.
Their foundational technology, which remained mostly unchanged for decades, posed significant limitations on their capabilities. However, the ongoing revolution in molecular biology that has already transformed many microbiology sectors is poised to usher food microbiology into a modern era.
Equipped with the latest tools, food microbiologists stand on the precipice of groundbreaking discoveries, offering them a broader and crucial role in ensuring the safety and quality of our food supply.
In contrast, if one were to step into a food chemistry lab in the past two decades, it would be evident that automation and computer-controlled equipment were increasingly becoming the norm, with technicians often glued to their screens, and traditional instruments like test tubes and Bunsen burners becoming a rarity.
While some level of automation has seeped into microbiology labs, most processes still necessitate culturing microorganisms. This need to culture is a time-consuming step, taking anywhere from 24 hours to several days, thus limiting microbiological testing’s immediacy and scope in ensuring food safety and quality.
Moreover, this dependency on culturing also confines the scope of microbial study. An illustrative example is the human gut, where it’s believed that a significant proportion of microbial species remain unidentified, primarily because current methods can’t cultivate them.
Consequently, while chemical testing has advanced in leaps and bounds, the need to culture organisms has restricted microbiologists’ capabilities.
An expanding market
The market for microbiological testing in the food sector has been experiencing robust growth, as reported by Strategic Consulting Inc. (SCI) in their recent report, Food Micro-2008 to 2013. The report details a consistent annual growth of almost 9% since 1998, with the food sector accounting for about half of the total industrial microbiology market.
The factors propelling this growth encompass an upsurge in food production, rising food safety concerns, increasing demands from retailers, and stricter regulatory requirements. By 2013, it’s estimated that close to 970 million tests will be conducted globally, a significant increase from 740 million in 2008.
The nature and purpose of testing have evolved over the past two decades. Previously, the focus was predominantly on quality control, with food products often held back for clearance by in-house microbiology laboratories.
With the widespread adoption of the HACCP approach to food safety, prolonged microbiological testing has lost its primary utility. Traditional testing methods, though still significant, are gradually being outpaced as they can’t provide timely results for HACCP’s critical control points.
The discrepancy between an expanding market and outdated technology that doesn’t address users’ core needs has amplified the demand for innovative techniques. The SCI report forecasts that the market for “rapid methods” of testing will grow at double the rate of the overall testing market in the coming years.
Tom Weschler, president of SCI, anticipates a shift by 2013; while traditional methods will remain predominant, they will constitute only 50.7% of all tests. Over the last few decades, there’s been a surge in attempts to develop faster methods for microbial counting, from impedance measurements to ATP measurements and dye-reduction tests.
While some have gained traction, they typically require a significant number of live microbial cells, necessitating the culturing of these organisms. Even with the rise in rapid testing methods, the ISO methods used for enforcement predominantly remain rooted in traditional, culture-based methods.
The rise of PCR
The roots of the challenge to traditional methodologies in studying microorganisms lie in the 1980s with the advent of molecular biology techniques centered on microbial genes. This shift was primarily driven by the discovery of the polymerase chain reaction (PCR) in 1983 by Dr. Kary Mullis, who was later awarded the Nobel Prize for this contribution.
PCR, essentially a method to rapidly duplicate specific DNA segments, revolutionized molecular biology by allowing for the rapid detection of minuscule DNA amounts. To conduct PCR, four essentials are required: a DNA template specific to the target, primers designed meticulously to border the target sequence, a heat-resilient DNA-polymerase enzyme (usually Taq polymerase), and free nucleotides that serve as building blocks in the reaction.
Executing PCR necessitates an initial step of DNA extraction from a given sample, traditionally done by using heat alongside a detergent to break bacterial cells and free the DNA. Extracting DNA from food samples can be intricate due to potential interferences from various food components, such as fats and polyphenols, collectively termed as ‘PCR inhibitors’.
While there are commercially available DNA purification kits tailored for food materials, the prevalent method in food microbiology still necessitates an enrichment culture step before the extraction.
Once the DNA is extracted and purified, the actual PCR begins by cycling through three temperatures to denature the DNA, allow primers to bind, and enable the DNA-polymerase enzyme to duplicate the target sequence. This process, cycled 30-40 times, can amplify the DNA enough for detection in just a few hours.
The amplified DNA is then detected using one of two primary methods. The first, end-point PCR detection, is executed post-amplification and employs gel electrophoresis followed by staining to identify the amplified DNA fragments.
This method, however, is more time-consuming and prone to contamination, rendering only qualitative results. Conversely, real-time PCR detection fuses the amplification and detection phases, offering continuous monitoring and a more precise quantifiable result. The majority of tests for foodborne pathogens utilize real-time PCR detection with commercial kits often employing ‘fluorescent reporter probes’.
These probes, specifically binding to the target DNA, are equipped with dyes at both ends, and during the extension phase, the probe disintegrates, emitting fluorescence which is proportional to the number of target sequence copies. This fluorescence can be quantified, permitting calculations of the DNA amount in the sample, and even detection of multiple DNA targets simultaneously.
Developing practical products
Real-time PCR assays, developed for food microbiology, have revolutionized the process of detecting foodborne pathogens. These commercial products are highly automated, reducing manual operations and contamination risks. Conducted inside a thermocycler combined with a fluorescence detection instrument, they utilize pre-prepared reagents, typically in dried tablet forms.
By standardizing test conditions, multiple pathogen assays can run simultaneously, and dedicated software controls the entire process, offering results within 20-30 hours. This swift detection system offers advantages over other methods, including saving technician time and providing the potential for simultaneous multiple pathogen tests.
PCR assays also outpace even rapid immunoassay-based detection methods, which often require up to 48 hours for a conclusive result. The precision of PCR further minimizes repeat tests, and the flexibility of this method enables simultaneous testing for multiple pathogens. Another edge of PCR is its quantitative output, which is not always possible with other methods.
However, the implementation of PCR assays in food microbiology is not without challenges, primarily the cost factor. Automated PCR systems, both in equipment and consumables, can be prohibitively expensive, especially when compared to alternative methods. This might deter smaller labs from adopting the technology. But, larger laboratories can benefit from economies of scale and reduced labor expenses, making PCR cost-effective in scenarios where rapid results are paramount.
A study from the German Federal Institute for Risk Assessment (BfR) even indicated that real-time PCR might be more economical than traditional labor-intensive methods for certain tests. The marketplace now offers an array of commercial PCR systems for food microbiology, like the Bax(r) system by Dupont Qualicon, TaqMan(r) kits by Applied Biosystems, and foodproof(r) kits by Biotecon Diagnostics.
These are designed primarily to detect pathogens such as Salmonella, Listeria, E. coli O157, and Campylobacter, but also extend to quality screening tests like those for the brewing industry. PCR’s adaptability is showcased in its ability to swiftly pivot to new tests by merely designing relevant primers, like the recent Bax test for Vibrio species by Dupont Qualicon.
The potential impact of PCR (Polymerase Chain Reaction) on food microbiology is substantial, with the belief that we have merely glimpsed its possible influences. As the technology continues to evolve, there’s an expectation that the array of PCR-based tests will expand, potentially replacing traditional microbiological examinations.
With advancements in DNA extraction, cleanup techniques, and more sensitive assays, there’s potential to expedite results and possibly detect food pathogens without the typically time-intensive enrichment stage. Additionally, as the demand for PCR grows and competition intensifies, costs are anticipated to decline, making the technology more accessible.
Already, PCR boasts capabilities beyond many traditional methods, such as identifying non-cultivable and damaged cells, and pinpointing specific pathogenic strains. Future advancements in this field might instigate revisions in food-related regulations, like the EU Microbiological Criteria Regulation, which could become outdated as testing methods progress and specificity enhances.
The versatility of PCR might also expand the scope of microbiology labs, enabling them to test for aspects like GMOs, allergens, and ingredient authentication. As PCR becomes more central, food microbiology labs might increasingly resemble molecular biology diagnostic centers. However, smaller labs might face challenges, especially if they can’t adapt to or invest in PCR.
There’s a possibility that this will push a trend towards outsourcing tests to larger, well-equipped contract labs that provide a wide range of services. Undoubtedly, the integration of molecular biology, especially PCR, is set to revolutionize food microbiology, ushering it into a promising new epoch filled with potential.