Recognizing the unique problems associated with the accurate detection and quantitation of gluten in fermented foods, a regulation regarding the use of gluten-free label for fermented, hydrolyzed, and distilled foods was proposedin 2015 ( 11). In 2013, the FDA issued a regulation defining and allowing the use of the term gluten-free for food that вЂњdoes not contain an ingredient that is a gluten-containing grain (e.g., spelt wheat) an ingredient that is derived from a gluten-containing grain and that has not been processed to remove gluten (e.g., wheat flour) or an ingredient that is derived from a gluten-containing grain and that has been processed to remove gluten (e.g., wheat starch), if the use of that ingredient results in the presence of 20 parts per million (ppm) or more gluten in the food or inherently does not contain gluten and that any unavoidable presence of gluten in the food is below 20 ppm gluten (i.e., below 20 mg gluten per kg of food).вЂќ It was further вЂњrecognized that some food matrices, such as fermented or hydrolyzed foods, may lack currently available scientifically valid methods that can be used to accurately determine if these foods contain в‰Ґ20 ppm glutenвЂќ ( 10). The abundance of proline residues in gluten makes them highly resistant to complete proteolytic degradation in the human gastrointestinal track ( 6, 7).ДЄpproximately 1 in 141 people in the US are affected by CD and adherence to a strict gluten-free diet is the only option to prevent inflammatory symptoms in sensitive individuals ( 8, 9).

A typical feature of gluten T cell stimulating peptides is their high proline content. The wheat glutelins, glutenins, can be divided into high molecular weight (HMW) glutenins with molecular weights of 66вЂ“88 kDa, and low molecular weight (LMW) glutenins with molecular weights falling in the range of the gliadin proteins, ~32вЂ“45 kDa ( 4, 5). The wheat prolamins, gliadins, are monomeric proteins with molecular weight ranging from 30 to 50 kDa and can be classified into О±/ОІ, Оі, and П‰-type. Gluten can be fractionated into alcohol soluble prolamins and the alcohol insoluble glutelins. Subsequently, these peptides interact with human leukocyte antigen (HLA)-DQ2 or -DQ8 molecules evoking a T cell response, resulting in inflammation in the small intestine ( 2, 3). Upon ingestion, proteases in the gastrointestinal tract degrade gluten proteins into peptides, which undergoes deamidation by transglutaminase. These complimentary antibody-based technologies provide insight into the extent of proteolysis resulting from various fermentation processes and have the potential to aid in the selection of appropriate hydrolytic calibration standards, leading to accurate gluten quantitation in fermented-hydrolyzed foods.Д¬eliac disease (CD) is an immune mediated enteropathy triggered by the interaction of the prolamin and glutelin fractions of proteins from wheat, barley, and rye with the intestinal mucosa of sensitive individuals ( 1). Utilizing gluten-specific antibodies, a recently developed multiplex-competitive ELISA along with western blot analysis provides a potential path forward in this direction. This is a severe limitation that makes accurate quantitation and, ultimately, a detailed evaluation of any potential health risk associated with consuming the food difficult. The commercially available antibody-based methods routinely used to detect and quantitate gluten are not able to distinguish between different hydrolytic patterns arising from differences in fermentation processes. The recent uses of proteases (e.g., proline endopeptidases or PEP) to hydrolyze immunopathogenic sequences of gluten proteins further complicates the quantitation of immunopathogenic gluten. Accurate quantitation of gluten in fermented-hydrolyzed foods by antibody-based methods is a challenge due to the lack of appropriate reference materials and variable proteolysis. The FDA requires food bearing the labeling claim вЂњgluten-freeвЂќ to contain <20 ppm gluten. The Codex Standard and the European Commission states that gluten level of gluten-free foods must not exceed 20 ppm.

Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, MD, United StatesД¬eliac disease (CD) affects ~1 in 141 individuals in the United States, requiring adherence to a strict gluten-free diet.

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