title: Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease.
abstract: Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization-time of flight, strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-electrospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration. Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced gamma interferon production at levels comparable to the negative control. None of the iTCLs demonstrated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal proteases may be considered an efficient approach to eliminate gluten toxicity.
authors.affiliation: Department of Plant Protection and Applied Microbiology, University of Bari, Bari, Italy.
authors.forename: Carlo G
authors.initials: CG
authors.lastname: Rizzello
authors.forename: Maria
authors.initials: M
authors.lastname: De Angelis
authors.forename: Raffaella
authors.initials: R
authors.lastname: Di Cagno
authors.forename: Alessandra
authors.initials: A
authors.lastname: Camarca
authors.forename: Marco
authors.initials: M
authors.lastname: Silano
authors.forename: Ilario
authors.initials: I
authors.lastname: Losito
authors.forename: Massimo
authors.initials: M
authors.lastname: De Vincenzi
authors.forename: Maria D
authors.initials: MD
authors.lastname: De Bari
authors.forename: Francesco
authors.initials: F
authors.lastname: Palmisano
authors.forename: Francesco
authors.initials: F
authors.lastname: Maurano
authors.forename: Carmen
authors.initials: C
authors.lastname: Gianfrani
authors.forename: Marco
authors.initials: M
authors.lastname: Gobbetti
mesh_terms: D015153:Blotting, Western; D001939:Bread / Q000382:microbiology*; D002446:Celiac Disease / Q000178:diet therapy*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005285:Fermentation; D005658:Fungi / Q000201:enzymology* / Q000378:metabolism; D005983:Glutens / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D007422:Intestines / Q000276:immunology; D007778:Lactobacillus / Q000201:enzymology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D005983:Glutens; D007371:Interferon-gamma; D010447:Peptide Hydrolases