Investigating the Effect of Cloned AN-PEP Enzyme in Yeast on Gluten Degradation and Rheological Features of Dough

Document Type : Original Research

Authors
M. Hooshiyar 1
G. Ebrahimipour 1
K. Kaveh baghaei 2
M. I. Iravani Saad 3
1 Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
2 Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
10.52547/iem.7.4.337
Abstract
Backgrounds: Celiac disease (CD) is a common autoimmune disorder caused by intolerance to gliadin protein found in wheat, rye, and barley, which is prevalent among 1% of people in different parts of the world. Thus, in the last decades, the demand for gluten-free products has increased. The aim of the present study was to demonstrate the degradation of wheat gluten in laboratory.

Materials & Methods: Yeast colonies obtained from cloning were assessed for the presence of Saccharomyces cerevisiae with protease activity and then inoculated onto MSM (mineral salts medium) with 1% (w/v) gliadin. Aspergillus niger-derived prolyl endoprotease (AN- PEP ) production was also qualitatively examined on gliadin agar plates by determining yeast colony growth. Zones of clarification of gliadin around yeast colonies were regarded as the evidence of glutenase activity of AN- PEP . The qualitative effects of aspergillopepsin expressed in bakery yeast were studied on yeast gliadin and the rheological properties of wheat flour dough. The rheological properties of the dough were investigated by a rheometer.

Findings: In this survey, gluten was efficiently degraded into short fragments by the AN-PEP enzyme. The results of rheometer test showed that the use of AN-PEP could affect the rheological properties. The quality of dough and the ability of AN-PEP to degrade gluten in dough into smaller fragments were confirmed.

Conclusion: The current study gives evidence that in the future, the development of novel gluten-free products with high quality and taste is possible by degrading gluten protein into non-toxic peptides using a variety of AN-PEP enzymes.

Keywords

Subjects

References
[1] J. C. Martı and C. S. Martı, “A new microbial gluten-degrading prolyl endopeptidase : Potential application in celiac disease to reduce gluten immunogenic peptides,” pp. 1–22, 2019, doi: 10.1371/journal.pone.0218346.
[2] V. Montserrat, M. J. Bruins, L. Edens, and F. Koning, “Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation,” Food Chem., vol. 174, p. 440—445, 2015, doi: 10.1016/j.foodchem.2014.11.053.
[3] T. Walter, H. Wieser, and P. Koehler, “Degradation of gluten in rye sourdough products by means of a proline-specific peptidase,” Eur. Food Res. Technol., vol. 240, no. 3, pp. 517–524, 2015, doi: 10.1007/s00217-014-2350-5.
[4] T. Walter, H. Wieser, and P. Koehler, “SC,” J. Cereal Sci., 2014, doi: 10.1016/j.jcs.2014.02.012.
[5] I. Comino et al., “Fecal Gluten Peptides Reveal Limitations of Serological Tests and Food Questionnaires for Monitoring Gluten-Free Diet in Celiac Disease Patients,” Am. J. Gastroenterol., vol. 111, no. 10, pp. 1456–1465, 2016, doi: 10.1038/ajg.2016.439.
[6] S. STENMAN, Coeliac Disease-inducing Gluten. 2011.
[7] M. I. Pinto-Sánchez et al., “Bifidobacterium infantis NLS Super Strain Reduces the Expression of α-Defensin-5, a Marker of Innate Immunity, in the Mucosa of Active Celiac Disease Patients,” J. Clin. Gastroenterol., vol. 51, no. 9, pp. 814–817, 2017, doi: 10.1097/MCG.0000000000000687.
[8] R. Shetty et al., “Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles,” Enzyme Microb. Technol., vol. 107, pp. 57–63, 2017, doi: 10.1016/j.enzmictec.2017.08.002.
[9] N. G. Heredia-Sandoval, M. Y. Valencia-Tapia, A. M. Calderón de la Barca, and A. R. Islas-Rubio, “Microbial proteases in baked goods: modification of gluten and effects on immunogenicity and product quality,” Foods, vol. 5, no. 3, p. 59, 2016.
[10] T. Diefenthal, “Rapid purification of proline-specific endopeptidase from F / avobacte & m ~ e ~~~ gose ~ fic ~~ heteroiogously expressed in ~ sc ~ e ~~ c ~~ a co / i,” pp. 5–8, 1995.
[11] M. Lopez and L. Edens, “Effective prevention of chill-haze in beer using an acid proline-specific endoprotease from Aspergillus niger,” J. Agric. Food Chem., vol. 53, no. 20, pp. 7944–7949, 2005, doi: 10.1021/jf0506535.
[12] D. Stepniak et al., “Highly efficient gluten degradation with a newly identified prolyl endoprotease: Implications for celiac disease,” Am. J. Physiol. - Gastrointest. Liver Physiol., vol. 291, no. 4, pp. 621–629, 2006, doi: 10.1152/ajpgi.00034.2006.
[13] J. König, S. Holster, M. J. Bruins, and R. J. Brummer, “Randomized clinical trial: Effective gluten degradation by Aspergillus Niger-derived enzyme in a complex meal setting,” Sci. Rep., vol. 7, no. 1, 2017, doi: 10.1038/s41598-017-13587-7.
[14] L. Edens, P. Dekker, R. Van Der Hoeven, F. Deen, A. de Roos, and R. Floris, “Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates,” J. Agric. Food Chem., vol. 53, no. 20, pp. 7950–7957, 2005.
[15] M. Šebela et al., “Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics,” J. Mass Spectrom., vol. 44, no. 11, pp. 1587–1595, 2009, doi: 10.1002/jms.1667.
[16] M. Akeroyd et al., “AN-PEP, proline-specific endopeptidase, degrades all known immunostimulatory gluten peptides in beer made from barley malt,” J. Am. Soc. Brew. Chem., vol. 74, no. 2, pp. 91–99, 2016.
[17] H. Hamedi, A. Misaghi, M. H. Modarressi, and T. Z. Salehi, “Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production,” vol. 5, no. 1, pp. 29–34, 2013.
[18] R. Capparelli, I. Ventimiglia, L. Longobardo, and D. Iannelli, “Quantification of gliadin levels to the picogram level by flow cytometry,” Cytom. Part A, vol. 63, no. 2, pp. 108–113, 2005, doi: 10.1002/cyto.a.20109.
[19] Z. Fathi, L. R. R. Tramontin, G. Ebrahimipour, I. Borodina, and F. Darvishi, “Metabolic engineering of Saccharomyces cerevisiae for production of β-carotene from hydrophobic substrates,” FEMS Yeast Res., vol. 21, no. 1, pp. 1–11, 2021, doi: 10.1093/femsyr/foaa068.
[20] J. A. Lee and H. Y. Chee, “ In Vitro Antifungal Activity of Equol against Candida albicans ,” Mycobiology, vol. 38, no. 4, p. 328, 2010, doi: 10.4489/myco.2010.38.4.328.
[21] J. A. Tye-Din, H. J. Galipeau, and D. Agardh, “Celiac disease: A review of current concepts in pathogenesis, prevention, and novel therapies,” Front. Pediatr., vol. 6, no. November, pp. 1–19, 2018, doi: 10.3389/fped.2018.00350.
[22] J. A. Tye-din et al., “The effects of ALV003 pre-digestion of gluten on immune response and symptoms in celiac disease in vivo,” Clin. Immunol., vol. 134, no. 3, pp. 289–295, 2010, doi: 10.1016/j.clim.2009.11.001.
[23] C. Kang, X. W. Yu, and Y. Xu, “Gene cloning and enzymatic characterization of an endoprotease Endo-Pro-Aspergillus niger,” J. Ind. Microbiol. Biotechnol., vol. 40, no. 8, pp. 855–864, 2013, doi: 10.1007/s10295-013-1284-4.
[24] K. A. Scherf, H. Wieser, and P. Koehler, “Novel approaches for enzymatic gluten degradation to create high-quality gluten-free products,” Food Res. Int., vol. 110, pp. 62–72, 2018, doi: 10.1016/j.foodres.2016.11.021.
[25] M. Hooshiyar, G. Hossein, E. Pour, M. Rostami-nejad, and F. Sadat, “Journal of Chemical Health Risks Review of Recent Advances in Treatment of Celiac Disease Using Enzymatic Gluten Degradation,” vol. 11, 2021, doi: 10.22034/jchr.2021.1907514.1167.
Infection Epidemiology and Microbiology
Volume 7, Issue 4
November 2021
Pages 337-345