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Mechanism of Degradation of Immunogenic Gluten Epitopes from Triticum turgidum L. var. durum by Sourdough Lactobacilli and Fungal Proteases
- De Angelis, Maria, Cassone, Angela, Rizzello, Carlo G., Gagliardi, Francesca, Minervini, Fabio, Calasso, Maria, Di Cagno, Raffaella, Francavilla, Ruggero, Gobbetti, Marco
- Applied and environmental microbiology 2010 v.76 no.2 pp. 508-518
- durum wheat, chromatography, digestion, mass spectrometry, interferons, sourdough, Triticum turgidum, epitopes, fermentation, pasta, interleukin-2, enzyme-linked immunosorbent assay, cooking quality, cultivars, Lactobacillus, gluten, semolina, hydrolysis, membrane alanyl aminopeptidase, free amino acids, proteinases, Cucurbita, electrophoresis, fungi
- As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected sourdough lactobacilli, in combination with fungal proteases, hydrolyzed gluten (72 h at 37°C) of various cultivars of Triticum turgidum L. var. durum to less than 20 ppm. Complementary electrophoretic, chromatography, and mass spectrometry techniques were used to characterize the gluten and epitope hydrolysis. Nine peptidases were partially purified from the pooled cytoplasmic extract of the sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the most immunogenic peptide generated during digestion of Triticum species. At least three peptidases (general aminopeptidase type N [PepN], X-prolyl dipeptidyl aminopeptidase [PepX], and endopeptidase PepO) were necessary to detoxify the 33-mer without generation of related immunogenic epitopes. After 14 h of incubation, the combination of all or at least six different peptidases totally hydrolyzed the 33-mer (200 mM) into free amino acids. The same results were found for other immunogenic epitopes, such as fragments 57-68 of α9-gliadin, 62-75 of A-gliadin, and 134-153 of γ-gliadin. When peptidases were used for fermentation of durum wheat semolina, they caused the hydrolysis of gluten to ca. 2 ppm. The in vivo digestion was simulated, and proteins/peptides extracted from pepsin-trypsin (PT) digestion of durum wheat semolina fermented with selected sourdough lactobacilli induced the expression of gamma interferon and interleukin 2 at levels comparable to those of the negative control. Durum wheat semolina fermented with sourdough lactobacilli was freeze-dried and used for making Italian-type pasta. The scores for cooking and sensory properties for this pasta were higher that those of conventional gluten-free pasta.