Prolyl endopeptidase-degraded low immunoreactive wheat flour attenuates immune responses in Caco-2 intestinal cells and gluten-sensitized BALB/c mice.

Targeted degrading Aspergillus niger-derived prolyl endopeptidase (AN-PEP) is promising in gluten hydrolysis
because it specifically cleaves the proline-rich sites in gluten. The current study aims to understand the safety
aspects of AN-PEP hydrolysed low immunoreactive wheat flours by testing immune responses using cell line and
animal models. In the AN-PEP hydrolysed wheat flour (AN-PEP HWF) gliadin extract, there was no increase in
the levels of zonulin-1 (Zo-1) and pro-inflammatory cytokines (IL-6 and IL-8) but a significant increase was noted
in the control wheat flour (CWF) gliadin-treated Caco-2 cells. The Zo-1 localization in Caco-2 cells was significantly noted in the reacted positive fluorescence cells that were treated with the control wheat flour. Further,
a safety evaluation of HWF was carried out in gluten-sensitized BALB/c mice. Mouse anti-gliadin (IgG, IgA and
IgE) antibodies were significantly generated in the CWF treated animals rather than the AN-PEP HWF groups.
The serum pro-inflammatory (IL-1β, IL-4, IL-6, IL-15, TNF-α and IFN-γ) markers were observed in significant
levels in CWF challenged mice and a similar trend was observed in ex-vivo splenocyte cells. A small intestine
histopathological sectioning revealed that there are no abnormalities or structural changes in AN-PEP HWF
challenged mice.