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Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to alleviate symptoms and to normalize the intestinal mucosa. We previously found that transamidation reaction by microbial transglutaminase (mTG) was effective in down-regulating the gliadin-specific immune response in CD patients. In this study, the two-step transamidation protocol was adopted to treat commercial wheat semolina on a pilot scale. The effectiveness of the enzymatic reaction was tested by means of consolidated biochemical and immunological methods on isolated prolamins. We found that water-insoluble gliadin and glutenin yields decreased in wheat semolina to 5.9% ± 0.3% and 11.6% ± 0.1%, respectively, after a two-step transamidation reaction. Using DQ8 transgenic mice as a model of gluten sensitivity, we observed a dramatic reduction in IFN-γ production in spleen cells challenged in vitro with the residual insoluble gliadin from transamidated semolina ( N = 6; median values: 850 vs. 102; control vs. transamidated semolina, p &lt; 0.05). The technological properties of treated wheat semolina were then tested by manufacturing classical pasta (spaghetti). Notably, the spaghetti manufactured with transamidated semolina had only minor changes in its features before and after cooking. In conclusion, the two-step transamidation reaction modified the immunogenic epitopes of gliadins also on a pilot-scale level without influencing the main technological properties of semolina. Our data shed further light on a detoxification strategy alternative to the current gluten-free diet and may have important implications for the management of CD patients.

Effects of Two-Step Transamidation of Wheat Semolina on the Technological Properties of Gluten