Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements

PLoS One. 2015 Jun 1;10(6):e0128065. doi: 10.1371/journal.pone.0128065. eCollection 2015.


Background: Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP).

Methods: Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays.

Findings: The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.

Conclusion: Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Dietary Supplements*
  • Epitopes, T-Lymphocyte / chemistry*
  • Epitopes, T-Lymphocyte / immunology
  • Gliadin / chemistry*
  • Gliadin / immunology
  • Humans
  • Peptide Hydrolases / chemistry*
  • Peptide Hydrolases / immunology
  • Proteolysis*


  • Epitopes, T-Lymphocyte
  • Gliadin
  • Peptide Hydrolases

Grant support

This work was carried out in the framework of the Celiac Disease Consortium, an Innovative Cluster approved by The Netherlands Genomics Initiative, and the Dutch government (grant BSIK03009). FK received the funding ( This research was partly funded by DSM Food Specialties. FK received the funding ( CC, LE, and DS are employed by DSM Food Specialties. DSM Food Specialties paid part of the salary of GJ. Neither GJ, YK-W, PvV, FK nor the LUMC has commercial interest in the marketing of the AN-PEP enzyme or digestive enzyme supplements for gluten degradation. DSM did not have a role in study design, decision to publish, or preparation of the manuscript. The specific roles of authors are articulated in the 'author contributions' section.