Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties

Abstract

Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten.

Natural Variation of Hazelnut Allergenicity: Is There Any Potential for Selecting Hypoallergenic Varieties?

“…The mixture was incubated at 4 ℃ with stirring for 2 h. Then, the mixture was centrifuged at 30,000× g for 20 min at 4 ℃ and the supernatant collected. In the case of two-dimensional electrophoresis (2-DE), defatted hazelnut samples were suspended (1:10, w / v ) in extraction solution containing 4% ( w / v ) CHAPS (7 mol/L urea, 2 mol/L thiourea, 1% ( v / v ) immobilized pH gradient (IPG) buffer), 20 mmol/L of DTT in milliQ ultrapure water, as described elsewhere [ 13 ]. The mixture was stirred for 1 h at 4 ℃, disrupted by sonication with an ultrasonic homogenizer (Vibra-CellTM VCX130, Sonics & Materials Inc., Newtown, CT, USA) in three bursts of 10 s at 30% of full power [ 14 ], centrifuged at 30,000× g for 20 min at 4 ℃ and the supernatant collected.…”

Triticum aestivum ssp. vulgare and ssp. spelta cultivars. 1. Functional evaluation

“…For quantification of glutenin and gliadin subfractions, prolamins were separated on the basis of hydrophobicity by reversed phase-high performance liquid chromatography (RP-HPLC) as described [17].…”

In Situ Gluten–Chitosan Interlocked Self‐Assembled Supramolecular Architecture Reduces T‐Cell‐Mediated Immune Response to Gluten in Celiac Disease

“…Gliadin and glutenin proteins were extracted from the control wheat flour and gluten, and wheat flours and glutens containing the supramolecular assemblies with chitosan at different protein:chitosan weight ratios, and separated according to hydrophobicity by RP‐HPLC as previously described . Development of a standard curve for the RP‐HPLC semi‐quantitative procedure was conducted through the use of increasing BSA concentrations.…”

“…Therefore, several strategies have been developed in order to detoxify the gluten and make it potentially safe for celiac patients such as the degradation of immunodominant peptides by specific proteases, the development of genetically modified low‐gliadin wheat lines and the previous transamidation of glutamine residues present in gluten proteins in order to hamper its deamidation by human tissue transglutaminase . However, in general these strategies result in a decrease in gluten functionality and in particular for the genetically modified low‐gliadin wheat lines there are still doubts associated with dealing with genetically modified organisms …”