Allergen Detectives

Food allergies are adverse immune responses to food proteins. An estimated 9 million adults and 6 million children in the United States have food allergies. Eight foods—peanut, milk, egg, wheat, tree nuts, soy, fish, and shellfish—account for 90% of all food allergies. In addition, approximately one in 133 consumers in the United States suffers from celiac disease. Their consumption of gluten, found in wheat, barley, and rye, triggers the production of antibodies that damage the small intestine and limits their ability to absorb nutrients, putting them at risk of other serious health problems.

Avoidance is difficult since trace amounts of allergens in food can trigger allergic reactions and even death. Undeclared allergens can inadvertently appear in processed foods as a result of incorrect labeling, cross-contact during and after processing, and insufficient or ineffective equipment sanitation procedures. Foods served in foodservice operations can similarly be contaminated.

The U.S. Food and Drug Administration (FDA) requires that the presence of these eight allergens be declared in the ingredient list of processed foods. The agency also issued a final rule on August 2, 2013, defining the term “gluten-free” for voluntary food labeling. To be labeled gluten-free or similar, a food must contain less than 20 ppm of gluten. Food manufacturers will have a year to bring their labels into compliance. In addition, the FDA Food Code  was revised in 2013 to include requirements to improve awareness of food allergen concerns in foodservice and retail settings. The National Restaurant Association (NRA) offers an interactive ServSafe Allergens Online Course (www.servsafe.com/allergens), which provides information that foodservice employees and managers need to know about food allergies. The course covers such topics as identifying allergens, communicating with customers, preventing cross-contact, food labeling, and other topics. Moreover, several symposia and numerous technical papers on allergens and detection methods were presented at the Institute of Food Technologists’ 2013 IFT Annual Meeting.

Symposia Address Challenges
In the symposium “Challenges and Approaches for Controlling Allergens in Foodservice Operations,” Kevin Smith of the FDA discussed control of allergens in foodservice establishments from a regulatory perspective, specifically how food safety regulations and guidance address the control of allergens in foodservice operations. Dojin Ryu of the University of Idaho discussed results of a survey on menu labeling of common food allergens and the knowledge, attitudes, and practices of owner-operators of independent restaurants. Almost nine out of 10 owner-operators agreed that it is the customer’s responsibility to order food that is allergen-free, but there were  statistically significant differences in opinions about the responsibility of the restaurant to warn customers about food containing common allergens and providing allergen-free food. Hal King of Chick-Fil-A discussed management control systems in supply chain and retail operations that can be used to reduce allergen risk. He said that food-allergen risk can be more challenging to manage in foodservice operations than in a food manufacturing facility because of lack of dedicated equipment in food preparation, continual change in ingredient use, difficulty in training employees to prevent cross-contact of allergens during food preparation, and lack of ingredient labeling. David Crownover of the NRA discussed results of a survey to determine what the food-service industry thinks about food allergens and described what the NRA is doing to help the industry.

In the symposium “Detection of Allergens in Processed Food: Challenges and Solutions,” Xi Zhang of the Institute for Food Safety and Health discussed the limitations of commercial enzyme-linked immunosorbent assay (ELISA) test kits for the quantitative analysis of allergens in processed foods and ways to improve detection. Ronald Niemeijer of R-Biopharm AG discussed DNA-based methods for detection of allergens in processed foods. While the ELISA has been the most frequently applied and accepted test method, he said, a different approach to specifically detect an allergenic food component is to detect a specific sequence of the DNA. Christine Hebling Parker of the FDA studied application of a comprehensive liquid chromatography-mass spectrometry method for simultaneous allergen detection in thermally processed food samples containing milk, egg, and peanut. She said that global proteomics analysis of a wide variety of food samples will ultimately be used to improve the performance of quantitative methods for allergen detection in  complex food systems. Balunkeswar Nayak of the University of Maine discussed the impact of nonthermal processing methods on the ELISA detection of allergens. He said that changes in the structure, solubility, and digestibility of allergens may occur during processing as a result of degradation, denaturation, aggregation, cross-linking, fragmentation, and reassembly.

In the symposium “Gluten-Free Product Development: Replacing Wheat,” Joe Baumert at the University of Nebraska’s Food Allergy Research & Resource Program (FARRP) provided an overview of celiac disease and non-celiac sensitivity and said that analytical methods such as quantitative and qualitative ELISAs are available for detection of gluten in raw ingredients and finished products and for assessment of adequate cleaning of shared equipment to ensure that cross-contact does not occur. Dilek Uzunalioglu of Ingredion Inc. discussed how specialty flours and starches can be used to produce gluten-free bakery products with taste, texture, and nutrient profile similar to those of gluten-containing products. Mehmet C. Tulbek of Alliance Grain Traders discussed how pulse ingredients can be used in gluten-free formulations. Bruce Hamaker of Purdue University’s Whistler Center for Carbohydrate  Research said that non-wheat cereal proteins can be made viscoelastic and provide functionality in dough and bread systems but that no protein so far provides the same functionality as wheat gluten. He provided an overview of the protein structure/function relationship and discussed strategies for making non-wheat cereal proteins as viscoelastic as gluten.

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Technical Papers
• Peanuts. Binu Bedford and others at the FDA measured the effectiveness of filtration methods on removing residual protein from peanut-contaminated oil. They found that substantial concentrations of peanut protein transfer into oil used to roast peanuts and recommended that the oil, even after filtration, not be reused.

Xi Zhang and others at IFSH investigated the effectiveness of processing equipment cleaning regimens on removing allergenic food residue. Cereal bars containing peanut flour, nonfat dry milk, and egg powder were manufactured on a pilot-scale processing line followed by four different cleaning regimens. They swabbed the equipment and analyzed the swabs with lateral-flow devices specific for peanut, milk, and egg. They found differences in effectiveness by regimen and said that the results illustrate the importance of validated cleaning protocols for preventing allergen cross-contact on shared processing lines.

Ben Remington and others at FARRP said that three peanut-allergic individuals experienced severe reactions to a high-protein beverage in the same month. The beverage contained soy protein and cow’s milk but no peanut protein, and the individuals had previously consumed soy products without adverse reactions. Analyzing the sera from the individuals for specific immunoglobulin E (IgE) levels, the researchers concluded that individuals with very high specific IgE scores to peanut may need to avoid certain types of soy milk.

Balunkeswar Nayak at the University of Maine and others at FARRP studied the effectiveness of six commercial ELISA kits to detect peanut residues in a liquid model food processed by boiling, ultrahigh-temperature processing, microwaving, and ultrasonic processing and found that certain kits recovered higher peanut residues than others.

• Fish. Yi-Tien Chen and Yun-Hwa Hsieh of Florida State University (FSU) developed a polyclonal-antibody-based sandwich ELISA for the detection of fish protein in food products. The method was able to detect both raw and cooked fish samples of 64 common food fish species without any cross-reaction with land animals, shellfish, and food additive samples. It was also able to detect fish products processed by salting, smoking, and canning.

Ye Wang and Yun-Hwa P. Hsieh of FSU used a fish-specific polyclonal antibody and indirect ELISA to study the effects of white vinegar on the immunoreactivity and allergenicity of whiting, cod, and red grouper. They found that vinegar dramatically reduced the immunoreactivity of raw and cooked fish samples.

Yuhong Wang and Yun-Hwa P. Hsieh of FSU developed a simple, rapid, and sensitive nanoparticle-labeled lateral-flow biosensor that uses gold nanoparticles to detect finfish proteins in foods. The sensitivity and specificity of the biosensor were evaluated by using 63 common fish species and 10 non-fish proteins.

Yaozhou Zhu and Yun-Hwa P. Hsieh of FSU studied the effect of storage and processing on the immune response detectability of fish proteins using ELISA and dot blot methods. They investigated the effects of frozen storage length, cooking time, and smoking and salting methods on the detectability of nine species of fish, using indirect ELISA and dot blot. All stored fish tissue and protein extracts could be clearly detected after storage for at least 11 months except amberjack, which became undetectable after six months of frozen storage.

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• Gluten. Prasad Rallabhandi and others at the FDA characterized the prolamin and glutelin fractions of gluten from wheat, rye, barley, oats, and corn, using commercially available ELISA kits. The ELISA methods showed high variability in gluten quantification depending on the grain source and gluten hydrolysis.

• Buckwheat. Chaeyoon Lee and others at Ewha Womans University in South Korea hydrolyzed buckwheat proteins with proteases and analyzed them by SDS-PAGE, using sera from buckwheat-allergic patients, and found that proteins hydrolyzed with papain did not show any allergenicity whereas proteins hydrolyzed with pepsin and chymotrypsin did.

• Milk. Katherine Ivens and others at FARRP evaluated commercial ELISA kits for quantitative detection of bovine milk residues. Milk contains many proteins that can serve as ELISA targets; not all ELISAs target the same protein fractions, so comparison among kits is challenging. The researchers concluded that appropriate selection and understanding of ELISA kits is critical for accurate quantification of milk residues.

Brigitta Santha and others at FARRP studied the effect of thermal and nonthermal processes on detection of milk residues in a model liquid food system, using commercially available ELISA kits. Santha recommended that food companies should compare various kits to select the kit that might be most appropriate for their specific product.

• Eggs. Alyssa Pagel and others at the University of Minnesota investigated combined immunomagnetic separation and surface-enhanced Raman spectroscopy for the detection of ovalbumin in dairy products and on processing equipment. The combined method was able to detect ovalbumin in milk, yogurt, and ice cream as well as on stainless-steel surfaces within 30 minutes. The results demonstrate superior detection times and similar detection levels to the ELISA method.

• Almonds. Santosh Dhakal and others at Ohio State University evaluated the effect of high-pressure processing on the allergenicity of almond milk. Processed samples were analyzed using ELISA, Western blot, and dot blot methods, using two monoclonal antibodies as detection antibodies. The results demonstrated that high-pressure processing may reduce the allergenicity of almond milk.

Ying Zhang and others at FSU conducted electrophoretic and immunological analyses to determine the prevalence and immunoreactivity of the allergen amandin in almond, peach, and related Prunus species. The results indicated that amandin is an excellent marker for the robust detection of trace amounts of almond.

• Pecans. Denise Tran and others at FARRP developed a sandwich ELISA to detect and quantify pecan residues in processed foods. The method was highly specific with no cross-reactivity to more than 100 foods and food ingredients except for walnut. The researchers concluded that the method demonstrates high specificity toward both roasted and raw pecans and can serve as a useful tool to detect pecan residues in processed foods and to validate the effectiveness of allergen-control programs for pecan.

• Soy. Jordan Walter and others at the University of Minnesota combined two current methods to reduce the allergenicity of soy protein. One of the methods, extensive hydrolysis, leads to loss of functionality and formation of bitter peptides, and the other, Maillard-induced glycosylation, adversely affects digestibility, functionality, flavor, and color. The researchers hydrolyzed soy protein isolate and subjected it to mild Maillard-induced glycosylation and determined the immunoreactivity of the samples. They concluded that soy protein allergenicity can be decreased with limited enzymatic hydrolysis and glycosylation.

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Weili Xu and others at Harbin Institute of Technology in China developed a method for rapid detection of soybean milk adulteration of cow’s milk using a duplex polymerase chain reaction (PCR). The method could detect up to 50% soybean milk in raw, pasteurized, and sterilized cow’s milk.

Chern Lin Koh and Christina A. DeWitt of Oregon State University studied the effect of food matrices and thermal processing on soy detection using DNA and ELISA methods. Four food matrices (protein, fat, carbohydrate, and water) were spiked with soy protein isolates and heated in a water bath. Two methods of DNA recovery—column and magnetic beads—were used, and real-time PCR was performed on recovered DNA. The researchers found that heat treatment can significantly reduce the ability of the ELISA method to detect soy in all food matrices and that, depending on the food matrix, DNA methods can be used to detect soy in heated foods.

• Mango. Wen-Che Tsai and Hsiao-Wei Wen at Taiwan’s National Chung Hsing University investigated the protein profile of four major mango allergens and found the protein profile to be similar among the species. Using sera from mango-allergic subjects, the researchers found no obvious difference in immunore-activity among the species.

• Other Papers. Ben Remington and others at FARRP discussed the value of using proactive quantitative risk assessment to predict the number of allergic reactions due to past and future production efforts and to determine if product recalls and/or labeling changes were necessary.

What’s Ahead
Stephen L. Taylor, Professor, Dept. of Food Science & Technology, University of Nebraska, and Director of FARRP, said that the newest developments in allergen tests are mass spectrometry (MS) methods for confirmation of allergen results, but he added that this is still in the realm of research and development. It will probably become routine in 5–10 years. Both MS and ELISA suffer from the same limitation, he said: extraction of analyte from food. It’s the crudest part of the technique but the hardest to do efficiently.

Taylor said that when the FDA’s proposed rulemaking on preventive controls is finalized, allergen control will be part of the food safety plans of the food manufacturing industry, almost on balance with microbial pathogen testing. This will mean that a lot more food companies will look at analytical testing for allergens to validate their allergen-control plans. In addition, lateral-flow kits are increasingly popular, he said, because they can produce results in a processing facility within a few minutes of sampling.

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Allergen Test Manufacturers
• ELISA Systems, www.elisas.com.au
• ELISA Technologies Inc., www.elisa-tek.com
• GlutenTox, http://glutentoxpro.com
• Morinaga Institute of Biological Science Inc., www.miobs.com
• Neogen Corp., www.neogen.com
• R-Biopharm AG, www.r-biopharm.com
• Romer Labs, www.romerlabs.com

Neil H. Mermelstein, a Fellow of IFT, is Editor Emeritus of Food Technology
[email protected]