Enhancing gluten modification for celiac patients: harnessing microbial transglutaminase, ε-poly-l-lysine, and prolyl endopeptidase

The immunogenicity of gluten poses a significant challenge for individuals with celiac disease. This study aimed to reduce gluten immunogenicity using a dual treatment strategy: microbial transglutaminase and ε-poly- l -lysine (ε-PL) for structural modification, followed by enzymatic degradation with prolyl endopeptidase. Structural characterization of gluten proteins treated with transglutaminase and ε-PL revealed substantial modification of gliadins, with higher ε-PL concentrations showing more pronounced effects. Low-molecular-weight glutenins were less affected, and no deamidation or impact on digestibility was observed. Although an 80% reduction in R5 epitopes was achieved, gluten levels still exceeded the gluten-free threshold. The application of prolyl endopeptidase successfully reduced gluten content below this threshold, particularly in modified samples. When prolyl endopeptidase was used as a digestive adjuvant, only modified samples reached gluten-free levels. These findings show that combining structural modification and enzymatic degradation can achieve safe gluten-free levels, enabling the development of alternative diets for people with gluten-related disorders. • Pre-treatment with transglutaminase and ε-PL significantly modified gliadins. • Covalent modification of glutamine with ε-PL reduced R5 epitopes by 80%. • No deamidation or digestibility impact was observed during structural modification. • Prolyl endopeptidase reduced flour's gluten levels below the gluten-free threshold. • Prolyl endopeptidase as digestive adjuvant was effective only in modified samples.