Tag Archives: ELISA

Jens Brockmeyer
Allergen Alley

HPLC-MS: Advancing Routine Food Allergen Testing

By Jens Brockmeyer, Eva-Maria Niehaus
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Jens Brockmeyer

In the U.S., allergens are the sixth leading cause of chronic illness with over 50 million people affected annually.[1] This is just one country among many that has seen the steady increase of the prevalence and severity of food allergies.[2]With increased media attention on this growing issue, food product labeling and allergen testing have never been under such scrutiny.

In food product labeling the stakes are high. Consumers need to know that the ingredient lists of the products they buy are accurate, and manufacturers need assurance that their foods are allergen-free, especially as current labeling guidelines lack clarity.

Food testing methods, which have hitherto been deficient in detecting and identifying unknown allergens and cross-contamination within the food manufacturing process, must be further developed to become quicker and more precise, cost effective, and user-friendly. New analytical tests can identify a broader range of potential allergens and offer food manufacturers a way to detect emerging allergens.

The Pressure for Improved Regulation

With the number of people suffering from food allergies in the U.S. doubling in each of the last two decades,[3] there is a high demand for food manufacturers to improve the allergen information they provide for consumers.

However, there is a lack of uniformity across different regions. While European Union law stipulates that allergens must be listed in bold on product ingredient lists, only 14 of the 200 foods that could potentially cause allergic reactions are prioritized[4] and, in the U.S., the FDA lists only nine key allergens.[5] Furthermore, while organizations such as Anaphylaxis UK agree that the most severe allergic reactions are caused by the consumption of a certain quantity of an allergen, there is no universal agreement on what such threshold levels should be.[6]

The allergens regulated by the FDA and EU.
Figure 1: The allergens regulated by the FDA and EU.

Single ingredients are not the only cause of allergic reactions: contamination and cross-contamination can occur at various stages of the food manufacturing process and put consumers at even greater risk.[7] Many manufacturers voluntarily use Precautionary Allergen Labeling (PAL) on their products to mitigate the risk of undeclared allergens, which may be used when there is a risk of allergen cross-contamination in the supply chain.[8] An example of PAL is ‘may contain milk’. This is not a legal requirement, however, and PAL protects the manufacturer more than the consumer as it is unlikely to be based on an assessment of the risk of cross-contamination for each of the 14 regulated allergens.[9]

Current Allergen Testing Options

Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are the two main analytical methods presently used to detect and quantitate allergens in routine food testing.[10]

Highly sensitive, the ELISA method allows for the quantitative and qualitative analysis of antigens, proteins, hormones, and antibodies in biological samples. It is most commonly used in the identification of foodborne microorganisms, such as Salmonella and L. monocytogenes, across a range of food products.[11]

Although ELISA can identify specific analytical targets, it cannot detect unknown allergens in contaminated food supplies. Moreover, the overall structure of proteins and their extractability is often altered during food processing, which can affect assay test results. Other factors, including poor comparability of results between test kits that use different antibodies, can also impact each test, and therefore make reproducibility between methods difficult.

A comparison of ELISA, PCR and MS.
Figure 2: A comparison of ELISA, PCR and MS.

PCR testing, which uses deoxyribonucleic acid (DNA) as a genetic marker, is popular because of its sensitivity and specificity, and also because sample preparation is standardized. A limitation of PCR testing is that, as an indirect indicator, it lacks sensitivity for foods that could contain high quantities of allergenic protein, but little to no DNA.[12]

Liquid chromatography-mass spectrometry (LC-MS) is not commonly used in current food testing. Unlike ELISA, LC-MS has multiplexing capabilities, allowing for the detection of multiple allergens in a single run. It can provide precise separation, identification, and quantification of the specific peptides rather than proteins in samples, which not only increases test accuracy, but also improves upon traditional testing methods by allowing for the differentiation of closely related allergens.

However, LC-MS, too, has limitations. The complex matrices of some biological samples can cause issues, and the various sample techniques needed for specific analytes mean that implementing a LC-MS workflow in routine testing laboratories is difficult. Professor Jens Brockmeyer and his team at the University of Stuttgart have been working on further advancing LC-MS to mitigate such drawbacks.

The Solution for Comprehensive Allergen Testing

A research team at the University of Stuttgart is investigating the influence of food processing on allergenic potential. The team is seeking to improve the methods used to screen for allergens primarily through the use of mass spectrometry (MS) and has developed a new workflow for allergen testing that delivers results quickly and efficiently.

There are three components to the novel method: sample preparation, analysis by high-performance liquid chromatography (HPLC) coupled to MS, and data analysis software. The approach can be used in the analysis of food products and allergens, making sample preparation easier and simplifying laboratory workflows.

The workflow for the allergen screening method.
Figure 3: The workflow for the allergen screening method.

Protein extraction, manual or automated enzymatic digestion, and the cleanup of peptides takes place to prepare the sample. Protein digestion takes three hours, a considerable time saving in comparison to the usual proteomic procedures.[13] HPLC-MS is used to generate data; the detection of precursor masses of the specific peptides resulting from the allergenic proteins of the given ingredient verifies the presence of each allergenic element.

The new multiplexed method removes the need to run multiple tests to identify each allergen individually; just one run is required for the detection of several allergens. This multiplexing capability and the analytical software’s ability to evaluate measurements retrospectively significantly accelerates experiment time.

Improved Visibility of Unknown Allergens

Current food testing methods, such as ELISA and PCR, are valued for their sensitivity but both lack multiplexing capabilities and produce results that are affected by food processing and thermal processing, respectively. HPLC-MS is an innovative method that offers multiplexed analysis of complex samples, producing standardized results in an accelerated timeframe suited to the high throughput needs of food testing laboratories.

With undisclosed allergens the cause of 42% of food product recalls in the U.S. in 2022,[14] the precision and speed of HPLC-MS offers exciting potential for the future of food allergen testing, paving the way for the feasible implementation of clearer and more stringent regulations.

References

[1] American College of Allergy, Asthma, & Immunology. Allergy Facts. Accessed at: https://acaai.org/allergies/allergies-101/facts-stats/. Last accessed: November 17, 2023.

[2] The Guardian. ‘It’s one of the great mysteries of our time’: why extreme food allergies are on the rise – and what we can do about them. Accessed at: https://www.theguardian.com/society/2023/jul/15/its-one-of-the-great-mysteries-of-our-time-why-extreme-food-allergies-are-on-the-rise-and-what-we-can-do-about-them. Last accessed: November 17, 2023.

[3] Food Allergy & Anaphylaxis Connection Team. Food allergy & anaphylaxis. Accessed at: https://www.foodallergyawareness.org/food-allergy-and-anaphylaxis/prevention/food-allergies-on-the-rise/#:~:text=The%20number%20of%20people%20with,identified%20food%20allergy%20(source). Last accessed: December 21, 2023.

[4] European Union. Annex 2 – allergen labelling. Accessed at: https://food.ec.europa.eu/system/files/2018-07/codex_ccfl_cl-2018-24_ann-02.pdf. Last accessed: November 15, 2023.

[5] U.S. Food and Drug Administration. Allergic to sesame? food labels now must list sesame as an allergen. 2023. Accessed at: https://www.fda.gov/consumers/consumer-updates/allergic-sesame-food-labels-now-must-list-sesame-allergen. Last accessed: November 8, 2023.

[6] Anaphylaxis UK. Allergen thresholds. Accessed at: https://www.anaphylaxis.org.uk/fact-sheet/allergen-thresholds/. Last accessed: November 16, 2023.

[7] Food Allergy Canada. Avoiding cross-contamination. Accessed at: https://www.foodallergycanada.ca/living-with-allergies/day-to-day-management/avoiding-cross-contamination/. Last accessed: November 16, 2023.

[8] Food StandardsAgency. Precautionary allergen labelling. Accessed at: https://www.food.gov.uk/business-guidance/precautionary-allergen-labelling. Last accessed: November 16, 2023.

[9] Food Standards Agency. Precautionary allergen labelling. Accessed at: https://www.food.gov.uk/business-guidance/precautionary-allergen-labelling. Last accessed: November 16, 2023.

[10] Allergen Bureau. Food allergen analysis. Accessed at: https://allergenbureau.net/food-allergens/food-allergen-analysis/. Last accessed: November 16, 2023.

[11] Law JW-F, Ab Mutalib N-S, Chan K-G, Lee L-H. Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations. Frontiers in Microbiology. 2015; 5.

[12] Stoyke M, Becker R, Brockmeyer J, et al. German government official methods board points the way forward: Launch of a new working group for mass spectrometry for protein analysis to detect food fraud and food allergens. Journal of AOAC International. 2019;102(5):1280-1285.

[13] Switzar L, Giera M, Niessen WM. Protein digestion: An overview of the available techniques and recent developments. Journal of Proteome Research. 2013;12(3):1067-1077.

[14] U.S. PIRG Education Fund. Food for thought part 2: An analysis of food recalls for 2022. Accessed at: https://pirg.org/edfund/resources/food-for-thought-an-analysis-of-food-recalls-for-2022/. Last Accessed: December 21, 2023.

Guangtao Zhang, Ph.D., director of the Mars Global Food Safety Center

Complexity of Food Allergen Management Requires Global Collaboration

By Maria Fontanazza
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Guangtao Zhang, Ph.D., director of the Mars Global Food Safety Center

Undeclared allergens continue to be a big cause of food recalls. For allergen management practices to be effective within food companies, there must be a shared responsibility between food manufacturers, government agencies, regulators and consumers, says Guangtao Zhang, Ph.D., director of the Mars Global Food Safety Center. In a Q&A with Food Safety Tech, Zhang discussed key concerns related to undeclared allergens in food as well as the research that Mars is conducting to improve allergen management.

Food Safety Tech: The presence of undeclared allergens continues to be a hazard in the food safety space. Specific to peanut detection, what challenges is the industry facing?

Guangtao Zhang, Ph.D., director of the Mars Global Food Safety Center
Guangtao Zhang, Ph.D., director of the Mars Global Food Safety Center. All images courtesy of Mars.

Guangtao Zhang, Ph.D.: As food materials become more varied and complicated, food allergen management becomes increasingly complex. Robust, accurate and sensitive detection methods are essential to ensure consumer safety as well as compliance with regulatory standards for allergens in the food supply chain.

When you look at the regulatory aspects, detection methods go hand in hand. Firstly, there is a need to ensure that current standard detection methods used in regulatory control of consumer goods are validated for a range of complex food matrices to ensure neither over- nor under-estimation of allergen content occurs within a food supply chain. This is important because underestimation of allergen poses a significant food safety hazard to consumers, while overestimation of allergen can result in unnecessary product recalls, driving up product costs and food waste.

Secondly, validation and monitoring of the effectiveness of cleaning and handling practices in areas of potential cross contamination with allergen containing materials depend on reliable and robust quantitative food allergen test methods for their success. The more robust the testing protocols, the more we can improve our understanding of the risks associated with cross contamination of food allergens, potentially reducing the frequency of accidental contamination events.
It is also important to note that whilst the most common cause of undeclared allergen in the global food supply chain is through accidental contamination in raw materials or finished products, this is not the only method by which undeclared allergen may be found in a product.

For example, peanut flour may be used in economically motivated adulteration (EMA) food fraud cases. In 2018 the European Commission estimated that the cost of food fraud for the global food industry is approximately €30 billion every year. Due to its high protein content, peanut flour has been used as a bulking agent to raise the overall protein content of e.g., wheat flour, thus raising the ‘quality’, and therefore price, of lower value goods. The ability to effectively quantify peanut traces within complex products therefore has the potential to enable consumers of food products to further trust the safety of the food they eat.

ELISA (Enzyme linked immunosorbent assay) is the method used most frequently for peanut allergen detection in the food manufacturing industry because of its sensitivity and ease of use. However, it has disadvantages in certain settings. It is not currently validated for complex food matrices, as it is believed that the effects of both food matrices and food processing could result in an underestimation of peanut concentrations in thermally processed foods, leading to false negatives, as well as overestimation in complex food matrices, leading to false positives which are a potential food safety hazard to consumers.

Food Safety Tech: Tell us about the research that the Mars Global Food Safety Center is doing to help the industry with effective methods for peanut quantification.

Zhang: At the Mars Global Food Safety Center (GFSC) we believe that everyone has the right to safe food and that we have a responsibility to generate and share insights to help solve for global food safety challenges. We also know we can’t tackle these alone, which is why we collaborate with external partners. One of our focus areas is advancing understanding and knowledge sharing in peanut allergen detection. As part of that work, we are exploring methods of improving food safety via the development of advanced analytical methods to detect peanut allergen content, in the hopes that it will enable the food industry to expand on current preventative management protocols, including early detection methodologies, for faster response to future food allergen contamination events.

As part of our latest published research, we investigated the accuracy and sensitivity of ELISA-based test methods on raw and cooked wheat flour, wheat flour-salt and wheat flour-salt-oil matrices, which are common ingredients in the food industry. 10 ppm peanut was doped into each matrix during sample preparation. Recovery testing demonstrated that in all matrices the current industry standard ELISA method overestimated results with recoveries ranging from 49.6 to 68.6 ppm.
These findings prompted the development of a new confirmatory method based on liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for peanut quantification. When subjected to the same validation testing programme the HPLC-MS/MS technique was demonstrably more accurate and sensitive, with a limit of quantification of 0.3 ppm and the detected peanut concentration ranging from 6.8 to 12.8 ppm for samples doped with 10 ppm peanut.

This work is a first step in the development of a new standard method for peanut detection in complex food matrices and could ultimately inform safer manufacturing Quality & Food Safety (Q&FS) processes across global supply chains to help ensure safe food for all.

Mars GFSC Lab Food Integrity Team
The Lab Food Integrity Team at the Mars Global Food Safety Center.

Food Safety Tech: What projects are researchers at the Center working on to enhance allergen management as a whole?

Zhang: A successful allergen management program depends on rigorous control of allergenic foods and ingredients from all other products and ingredients at every step of the food production process, from raw material development to the delivery of final products. This means that for allergen management practices to be effective, they must be a shared responsibility between food manufacturers, government agencies, regulators and consumers.

At the Mars GFSC, we take a precompetitive approach to research, knowledge sharing and collaborations—this means we openly share insights and expertise to help ensure safe food for all. This is important in driving forward innovations, helping unlock solutions that may not have previously been possible.

We have shared our latest work both through an open access publication in Food Additives & Contaminants: Part A but also directly with regulatory bodies such as the FDA in the hopes of advancing knowledge in both food safety risk management and allergen management in complex flour-based media within global supply chains. In addition to this, this research contributes to a wider Food Safety Best Practice whitepaper focused on food allergen risk management currently under draft by the Mars GFSC, which will be published in collaboration with Walmart Food Safety Collaboration Center and the Chinese Institute of Food Science and Technology (CIFST) later this year.

We believe that global collaborations such as this are essential to improving food allergen management and mitigating food safety risks. Communication, training and knowledge sharing are core principles of the Mars GFSC and as such form a large part of our ongoing activities in this space. For example, we have hosted Food Allergen Management workshops in collaboration with Danone and Romer Labs focused on helping to raise awareness of current and future food allergen trends. At one such event in 2019, 100 participants from 16 food companies came together to promote food allergen management in the industry and ensure that the next generation of food integrity testing capability is relevant, practical, and directly applicable to the real-world problems experienced by manufacturers and processors throughout the supply chain.

Representatives of the Mars GFSC have also shared our insights externally at a number of international conferences as well as during a Food Enterprise Food Allergen Management Seminar on topics including effective allergen management procedures, our guiding principles for allergen managements at Mars, and shared our approach to encourage and share knowledge with other manufactures in this area.

We continue to support requests for technical insights, for example providing insights during a global consultation session on General Principles for Labeling of Prepackaged Food. This resulted in the addition of characterization requirements for possible allergenic substances, promoting the use of a recognizable naming system in ingredient lists that contain allergen warnings.

Food Safety Tech: Can you comment on additional work your team is doing in the area of food fraud?

Zhang: Food allergen risk management forms only one part of our wider food integrity focus at the Mars GFSC. We are committed to helping ensure food authenticity in an increasingly complex, global food supply chain through collaboration with global partners to develop new and improved tools and analytical methods that help protect the integrity of raw materials and finished products.

We have collaborated with researchers at Michigan State University to develop a Food Fraud Prevention Cycle roadmap (Introducing the Food Fraud Prevention Cycle (FFPC): A dynamic information management and strategic roadmap) which answered questions such as how to detect food fraud, how to start a food fraud prevention program, what to do in terms of testing, how much testing is enough, and how to measure success. Our intention in publishing this research was that the adoption of a holistic and all-encompassing information management cycle will enable a globally harmonized approach and the continued sharing of best practices across industry partners.

More recently, we completed an international collaboration tackling rice adulteration together with Queen’s University Belfast (QUB), Agilent Technologies, International Atomic Energy Agency (IAEA), China National Center for Food Safety Risk Assessment (CFSA), and Zhejiang Yangtze Delta Institute of Tsinghua University (Yangtze Delta). This work successfully developed a two-tier testing program, capable of rapidly screening the geographical origins of rice within the global supply chain (Food Fingerprinting: Using a two-tiered approach to monitor and mitigate food fraud in rice). By developing a tiered system, we could ensure that manufacturers use the right techniques for the right occasion, to maximize the information available in investigating food fraud at the best value. As part of this work, we have helped develop hands-on training in Ghana and inform best practice guidance to help build the foundations of a strong food safety culture in rice authenticity across the global supply chain.

Susanne Kuehne, Decernis
Food Fraud Quick Bites

Today’s Pig Is Tomorrow’s…Beef?

By Susanne Kuehne
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Susanne Kuehne, Decernis
Pig, cow, food fraud
Find records of fraud such as those discussed in this column and more in the Food Fraud Database, owned and operated by Decernis, a Food Safety Tech advertiser. Image credit: Susanne Kuehne

Balkan countries are enduring their share of adulterated foods. In Kosovo, commercial samples of meat labelled as beef or chicken were investigated with ELISA (enzyme-linked immunoassay test) and PCR (polymerase chain reaction) in order to detect pork mitochondrial DNA. The test series looked into the efficiency and cost of different methods and showed a preference for commercial ELISA combined with real-time PCR. Almost a third of beef was adulterated with pork, as were 8% of the chicken samples.

Resource

  1. Gecaj, R.M., et al. (August 2021). “Investigation of pork meat in chicken and beef based commercial products by ELISA and real-time PCR sold at retail in Kosovo”. Czech Academy of Agricultural Science, Open Access CAAS Agricultural Journals.
Allergens

Key Trends Reinforce Food Allergen Testing Market Across North America

By Saloni Walimbe
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Allergens

The food allergen testing industry has garnered considerable traction across North America, especially due to the high volume of processed food and beverages consumed daily. Allergens are becoming a significant cause for concern in the present food processing industry worldwide. Food allergies, which refer to abnormal reactions or hypersensitivity produced by the body’s immune system, are considered a major food safety challenge in recent years and are placing an immense burden on both personal and public health.

In 2019, the most common reason behind recalls issued by the USDA FSIS and the FDA was undeclared allergens. In light of this growing pressure, food producers are taking various steps to ensure complete transparency regarding the presence of allergenic ingredients, as well as to mitigate risk from, or possibly even prevent contact with, unintended allergens. One of these steps is food allergen testing.

Allergen detection tests are a key aspect of allergen management systems in food processing plants and are executed at nearly every step of the process. These tests can be carried out on work surfaces, as well as the products, to detect any cross contamination or allergen presence, and to test the effectiveness of a food processing unit’s cleaning measures.
There has been a surge in awareness among consumers about food allergies and tackling the risk of illnesses that may arise from consuming any ingredient. One of the key reasons for a higher awareness is efforts to educate the public. In Canada, for example, May has been designated “Food Allergy Awareness Month”. It is estimated that more than 3 million people in Canada are affected by food allergies.

The size of the global food allergen testing market is anticipated to gain significant momentum over the coming years, with consistent expansion of the dairy, processed food and confectionary segments.

Understanding the Prevailing Trends in Food Allergen Testing Industry

Food allergies risen nearly 50% in the last 10 years, with a staggering 700% increase observed in hospitalizations due to anaphylaxis. Studies also suggest that food allergies are a growing health concern, with more than 250 million people worldwide estimated to be affected.

Although more than 170 foods have been identified as causing food allergies in sensitive consumers, the USDA and the FDA have identified eight major allergenic foods, based on the 2004 FALCPA (the Food Allergen Labeling and Consumer Protection Act). These include eggs, milk, shellfish, fish, peanuts, tree nuts, soybean, and wheat, which are responsible for 90% of allergic reactions caused due to food consumption. In April 2021, the FASTER (Food Allergy Safety, Treatment, Education, and Research) Act was signed into law, which categorized sesame as the ninth major food allergen.

This ever-increasing prevalence of allergy-inducing foods has presented lucrative opportunities for the food allergen testing industry in recent years since food processing business operators are placing a strong emphasis on ensuring transparency in their products’ ingredient lists. By testing for allergens in food products, organizations can accurately mention each ingredient, and thereby allow people with specific food allergies to avoid consuming them.

Several allergen detection methods are used in the food processing industry, including mass spectrometry, DNA-based polymerase chain reaction (PCR) as well as ELISA (enzyme-linked immunosorbent assay), to name a few. The FDA, for instance, created a food allergen detection assay, called xMAP, designed to simultaneously identify 16 allergens, including sesame, within a single analysis, along with the ability to expand for the targeting of additional food allergens. Such industry advancements are improving the monitoring process for undeclared allergen presence in the food supply chain and enabling timely intervention upon detection.

Furthermore, initiatives, such as the Voluntary Incidental Trace Allergen Labelling (VITAL), created and managed by the Allergen Bureau, are also shedding light on the importance of allergen testing in food production. The VITAL program is designed to support allergen management with the help of a scientific process for risk assessment, in order to comply with food safety systems like the HACCP (Hazard Analysis and Critical Control Point), with allergen analysis playing a key role in its application.

ELISA Gains Prominence as Ideal Tool for Food Allergen Testing

In life sciences, the detection and quantification of various antibodies or antigens in a cost-effective and timely manner is of utmost importance. Detection of select protein expression on a cell surface, identification of immune responses in individuals, or execution of quality control testing—all these assessments require a dedicated tool.

ELISA is one such tool proving to be instrumental for both diagnostics as well as research). Described as an immunological assay, ELISA is used commonly for the measurement of antibodies or antigens in biological samples, including glycoproteins or proteins.

While its utility continues to grow, ELISA-based testing has historically demonstrated excellent sensitivity in food allergen testing applications, in some cases down to ppm (parts per million). It has a distinct advantage over other allergen detection methods like PCR, owing to the ability to adapt to certain foods like milk and oils, where its counterparts tend to struggle. The FDA is one of the major promoters of ELISA for allergen testing in food production, involving the testing of food samples using two different ELISA kits, prior to confirming results.

Many major entities are also taking heed of the growing interest in the use of ELISA for food allergen diagnostics. A notable example of this is laboratory analyses test kits and systems supplier, Eurofins, which introduced its SENSISpec Soy Total protein ELISA kit in September 2020. The enzyme immunoassay, designed for quantitative identification of soy protein in swab and food samples, has been developed by Eurofins Immunolab to measure residues of processed protein in various food products, including instant meals, chocolate, baby food, ice cream, cereals, sausage, and cookies, among others.

In essence, food allergens continue to prevail as high-risk factors for the food production industry. Unlike other pathogens like bacteria, allergenic proteins are heat resistant and stable, and cannot easily be removed once present in the food supply chain. In this situation, diagnostic allergen testing, complete segregation of allergenic substances, and accurate food allergen labeling are emerging as the ideal courses of action for allergen management in the modern food production ecosystem, with advanced technologies like molecular-based food allergy diagnostics expected to take up a prominent role over the years ahead.

Gabriela Lopez, 3M Food Safety
Allergen Alley

Method Acting: Comparing Different Analytical Methods for Allergen Testing and Verification

By Gabriela Lopez-Velasco, Ph.D.
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Gabriela Lopez, 3M Food Safety

Every day, food industries around the world work to comply with the food labeling directives and regulations in place to inform consumers about specific ingredients added to finished products. Of course, special attention has been placed on ensuring that product packaging clearly declares the presence of food allergens including milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, soy, sesame and mustard. (Additional food allergens may also be included in other regions.)

But labeling only covers the ingredients deliberately added to foods and beverages. In reality, food manufacturers have two jobs when it comes to serving the needs of their allergic consumers:

  1. Fully understand and clearly declare the intentional presence of allergenic foods
  2. Prevent the unintended presence of allergenic foods into their product

Almost half of food recalls are the result of undeclared allergens, and often these at-fault allergens were not only undeclared but unintended. Given such, the unintended presence of allergenic foods is something that must be carefully considered when establishing an allergen control plan for a food processing facility.

How? It starts with a risk assessment process that evaluates the likelihood of unintentionally present allergens that could originate from raw materials, cross-contact contamination in equipment or tools, transport and more. Once the risks are identified, risk management strategies should then be established to control allergens in the processing plant environment.
It is necessary to validate these risk management strategies or procedures in order to demonstrate their effectiveness. After validation, those strategies or procedures should then be periodically verified to show that the allergen control plan in place is continually effective.

In several of these verification procedures it may be necessary to utilize an analytical test to determine the presence or absence of an allergenic food or to quantify its level, if present. Indeed, selecting an appropriate method to assess the presence or the level of an allergenic food is vitally important, as the information provided by the selected method will inform crucial decisions about the safety of an ingredient, equipment or product that is to be released for commercialization.

A cursory review of available methods can be daunting. There are several emerging methods and technologies for this application, including mass spectroscopy, surface plasmon resonance, biosensors and polymerase chain reaction (PCR). Each of these methods have made advancements, and some of them are already commercialized for food testing applications. However, for practical means, we will discuss those methods that are most commonly used in the food industry.

In general, there are two types of analytical methods used to determine the presence of allergenic foods: Specific and non-specific methods.

Specific tests

Specific methods can detect target proteins in foods that contain the allergenic portion of the food sample. These include immunoassays, in which specific antibodies can recognize and bind to target proteins. The format of these assays can be quantitative, such as an enzyme-linked immunosorbent assay (ELISA) that may help determine the concentration of target proteins in a food sample. Or they can be qualitative, such as a lateral flow device, which within a few minutes and with minimum sample preparation can display whether a target protein is or is not present. (Note: Some commercial formats of ELISA are also designed to obtain a qualitative result.)

To date, ELISA assays have become a method of choice for detection and quantification of proteins from food allergens by regulatory entities and inspection agencies. For the food industry, ELISA can also be used to test raw ingredients and final food products. In addition, ELISA is a valuable analytical tool to determine the concentration of proteins from allergenic foods during a cleaning validation process, as some commercial assay suppliers offer methods to determine the concentration of target proteins from swabs utilized to collect environmental samples, clean-in-place (CIP) final rinse water or purge materials utilized during dry cleaning.

ELISA methods often require the use of laboratory equipment and technical skills to be implemented. Rapid-specific methods such as immunoassays with a lateral flow format also allow detection of target specific proteins. Given their minimal sample preparation and short time-to-result, they are valuable tools for cleaning validation and routine cleaning verification, with the advantage of having a similar sensitivity to the lowest limit of quantification of an ELISA assay.

The use of a specific rapid immunoassay provides a presence/absence result that determines whether equipment, surfaces or utensils have been cleaned to a point where proteins from allergenic foods are indiscernible at a certain limit of detection. Thus, equipment can be used to process a product that should not contain a food allergen. Some commercial rapid immunoassays offer protocols to use this type of test in raw materials and final product. This allows food producers to analyze foods and ingredients for the absence of a food allergen with minimum laboratory infrastructure and enables in-house testing of this type of sample. This feature may be a useful rapid verification tool to analyze final product that has been processed shortly after the first production run following an equipment cleaning.

Non-Specific Tests

While non-specific testing isn’t typically the best option for a cleaning validation study, these tests may be used for routine cleaning verification. Examples of non-specific tests include total protein or ATP tests.

Tests that determine total protein are often based on a colorimetric reaction. For example, commercial products utilize a swab format that, after being used to survey a defined area, is placed in a solution that will result in a color change if protein is detected. The rationale is that if protein is not detected, it may be assumed that proteins from allergenic foods were removed during cleaning. However, when total protein is utilized for routine verification, it is important to consider that the sensitivity of protein swabs may differ from the sensitivity of specific immunoassays. Consequently, highly sensitive protein swabs should be selected when feasible.

ATP swab tests are also commonly utilized by the food industry as a non-specific tool for hygiene monitoring and cleaning verification. However, the correlation between ATP and protein is not always consistent. Because the ATP present in living somatic cells varies with the food type, ATP should not be considered as a direct marker to assess the removal of allergenic food residues after cleaning. Instead, an analytical test designed for the detection of proteins should be used alongside ATP swabs to assess hygiene and to assess removal of allergenic foods.

Factors for Using One Test Versus Another

For routine testing, the choice of using a specific or a non-specific analytical method will depend on various factors including the type of product, the number of allergenic ingredients utilized for one production line, whether a quantitative result is required for a particular sample or final product, and, possibly, the budget that is available for testing. In any case, it is important that when performing a cleaning validation study, the method used for routine testing also be included to demonstrate that it will effectively reflect the presence of an allergenic food residue.

Specific rapid methods for verification are preferable because they enable direct monitoring of the undesirable presence of allergenic foods. For example, they can be utilized in conjunction with a non-specific protein swab and, based on the sampling plan, specific tests can then be used periodically (weekly) for sites identified as high-risk because they may be harder to clean than other surfaces. In addition, non-specific protein swabs can be used after every production changeover for all sites previously defined in a sampling plan. These and any other scenarios should be discussed while developing an allergen control plan, and the advantages and risks of selecting any method(s) should be evaluated.

As with all analytical methods, commercial suppliers will perform validation of the methods they offer to ensure the method is suitable for testing a particular analyte. However, given the great diversity of food products, different sanitizers and chemicals used in the food industry, and the various processes to which a food is subjected during manufacturing, it is unlikely that commercial methods have been exhaustively tested. Thus, it is always important to ensure that the method is fit-for-purpose and to verify that it will recover or detect the allergen residues of interest at a defined level.

Eurofins Technologies, Gold Standard Diagnostics

Eurofins Technologies, Gold Standard Diagnostics Enter Strategic Partnership

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Eurofins Technologies, Gold Standard Diagnostics

This week Eurofins Technologies announced a strategic partnership with Gold Standard Diagnostics (GSD), a developer and manufacturer of fully-automated diagnostic instruments and assays for various test methods. The agreement unites Gold Standard’s ELISA-based instruments and Eurofins Technologies rapidly-expanding diagnostic test kit portfolio for  food, environmental and animal health testing.

Gold Standard Diagnostics will be the standard platform for Eurofins Technologies ELISA-based food testing kits including food pathogens, allergens, mycotoxins, veterinary drug residues, and animal health kits.

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Pathogens Drive More Than Half of $12 Billion Global Food Safety Testing Market

By Maria Fontanazza
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The importance of food safety testing technologies continues to grow, as companies are increasingly testing their products for GMOs and pesticides, and pathogens and contamination. Last year the global food safety testing market had an estimated value of $12 billion, according to a recent report by Esticast Research & Consulting. Driven by pathogen testing technologies, the global food safety testing market is expected to experience a 7.4% CAGR from 2017–2024, hitting $21.4 billion in revenue in 2024, said Vishal Rawat, research analyst with Esticast.

With a CAGR of 9.3% from 2017–2024, rapid testing technologies are anticipated to lead the market. Testing methods responsible for this growth include immunoassays (ELISA), latex agglutination, impedance microbiology, immune-magnetic separation, and luminescence and gene probes linked to the polymerase chain reaction, said Rawat, who shared further insights about the firm’s market projections with Food Safety Tech.

Food Safety Tech: With the GMO food product testing market expected to experience the highest growth in the upcoming future, can you estimate the projected growth?

Vishal Rawat: The GMO food product testing market is estimated to generate a revenue of approximately $5.2 billion in 2016. The market segment is expected to witness a compound annual growth rate of 8.3% during the forecast period of 2017–2024. This is a global market estimation.

FST: What innovations are occurring in product testing?

Rawat: Nanomaterials and nanobased technologies are attracting interest for rapid pathogen testing. Sustainable technologies such as edible coatings or edible pathogen detection composition can attain a trend in the near future. Also, new rapid allergen testing kits are now emerging out as the latest food testing technology in the market, which are portable and easy to use.

FST: Which rapid pathogen detection testing technologies will experience the most growth from 2017–2024?

Rawat: New and emerging optical, nano-technological, spectroscopic and electrochemical technologies for pathogen detection, including label-free and high-throughput methods would experience the highest growth.

FST: What pathogen testing technologies are leading the way for meat and poultry in the United States?

Rawat: The presence of a microbial hazard, such as pathogenic bacteria or a microbial toxin, in ready-to-eat (RTE) meat or poultry products is one basis on which these products may be found adulterated. The FSIS is especially concerned with the presence of Listeria monocytogenes, Salmonella, Escherichia coli O157: H7, and staphylococcal enterotoxins in RTE meat and poultry products. Rapid pathogen testing for E. coli O157:H7 and Salmonella, for ground beef, steak and pork sausages is going to lead the U.S. market.

An overview of the report, “Food Safety Testing Market By Contaminant Tested (Pathogens, GMOs, Pesticides, Toxins), By Technology (Conventional, Rapid), Industry Trends, Estimation & Forecast, 2015– 2024” is available on Esticast’s website.

Suresh Neethirajan, University of Guelph
In the Food Lab

Identifying Peanut and Other Allergens Outside the Lab

By Suresh Neethirajan, Ph.D
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Suresh Neethirajan, University of Guelph

Judging the nature and suitability of items we put in our mouths is a task we perform daily, whether it’s due to different taste preferences, being on a diet, or from particular foods not agreeing with our metabolisms. Some foods can trigger mild reactions such as an upset stomach, or more serious skin rashes and outbreaks, from shortness of breath to even death.

Many of us have been somewhere where someone with a peanut allergy has been brought to everyone’s attention. The situation may have been publicized before boarding a plane, at a school where parents are asked to refrain from giving their children any food containing peanut products, or restaurants that clearly indicate which dishes are peanut-free on their menu, or that the kitchen is absent of the legume.

The number of people with food allergies continues to rise, and although many theories have been provided for the increase, the exact cause is unknown. Many foods are documented as being able to produce an allergic reaction—milk, eggs, soy and shellfish, to name a few—but peanuts and gluten are highlighted as major offenders. Canadian government regulations require that manufacturers label products that contain certain allergens, even if they are made in a facility where allergens are in another product.

The Threat of Gluten and Peanuts

Gluten contained in wheat has become a widely avoided food substance, although the reason for this might has more to do with health concerns than allergies. The American College of Allergy, Asthma and Immunology (ACAAI) estimates that 400,000 U.S. school children have a peanut allergy, with many of those also having other food allergies. According to the ACAAI, many children will eventually outgrow most food allergies, but only 20% of those who have a peanut tolerance will outgrow it.

The charity organization Food Allergy Canada states that 2.5 million people suffer from a food allergy in Canada, while 2 in 100 children are susceptible to peanuts causing a reaction. There isn’t a cure for food allergies, so governments and food inspectors have the weighty task of ensuring that commercially produced products are packaged or served with proper labeling and information to protect consumers. This requires constant checking and testing of products that may have come in contact with peanuts or gluten.

New Tool for Food Inspectors

To provide regular analysis, the procedure has been lengthy and expensive, but scientific researchers at Canada’s University of Guelph have developed an apparatus that can identify allergens in a much shorter time span while being considerably more cost effective. The new allergen detector could expedite allergen reporting and possibly reduce the number of allergic reactions through more timely results.

Biosensor, University of Guelph
Schematic of the biosensor for the rapid detection of food allergens. Image courtesy of BioNanoLab, University of Guelph.

Based on the ELISA (enzyme-linked immunosorbent assay) platform that is widely used in diagnostic labs to identify allergens, the new apparatus provides comparable accuracy. The technology has been miniaturized so that equipment is portable, about the size of an audiocassette case, and tests can be conducted on location instead of relying on a lab that may be far away.

An Allergen that Glows

In the case of peanuts, the scientists focused on a prominent allergen named Ara h 1, because it can be identified through non-radioactive fluorescence. Although there are other allergens in peanuts, they don’t share the same property by which they can be identified, as does Ara h 1.

The process requires a small amount of the suspected food to be liquefied in a suspension so that it can be injected using a filter syringe into a silicon-based plate, or chip, of microcapillaries. As the sample passes through tiny tubes of the microfluidic chip using capillary action, it travels through a beam of light from a LED source that is monitored by a specialized camera, which is also a product of the scientists’ work.

The image captures Ara h 1 protein particles that fluoresce when they come in contact with the chemical properties of the suspension. Currently, the camera records the data and sends it to a computer to be analyzed and deciphered with a result being provided within 20 minutes, compared to a conventional lab test that takes up to four hours after a sample has been received.

In a modification to provide an extremely portable system, research is underway to develop an app to enable results via a smartphone. Testing foods in the near future will be as convenient and prompt as holding the detector in one hand and a smartphone in the other so that a restaurant owner, for example, will be assured that dishes are allergen-free before being served to customers.

Imitating the Human System for Detection

To enable the allergen to fluoresce, the compound graphene oxide (GO) was utilized in combination with a bio-sensing component, known as an aptamer. The aptamer acts similarly to antibodies that identify and attach themselves to foreign and hostile elements that enter our blood system. Once a GO-aptamer mixture is attached to the allergen, the light source allows the protein particle to be detected and its image captured electronically.

By altering an aptamer’s composition to identify other allergens, such as gluten, the detector is a versatile piece of scientific equipment for identifying potentially hazardous food ingredients. The developers of the technology are confident that their discovery will change the future of identifying potentially hazardous food components. The final step in the allergen detector’s development seems to be fine tuning the detection process for certain processed foods, such as roasted peanuts, that can alter the composition of Ara H 1 making it less obvious to be identified.