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Gluten is responsible for adverse immune effects and causes wheat allergy, celiac disease, and other wheat-related disorders to genetically predisposed individuals. Wheat flour protein immunogenicity can be reduced by proteolytic degradation enzymes. The present study aims to utilize Aspergillus niger prolyl endoprotease (EC 3.4.21.26) to specifically cleave the proline-rich sequences and degradation of gluten proteins in the wheat flour. The Triticum aestivum (HD-2851, NIAW-917), Triticum durum (UAS-428), Triticum dicoccum (DDK- 1025) were taken for proteolytic degradation using AN-PEP. The enzymatic modification was carried out in an optimum condition (pH-4.0, at 40 °C) for 5 h. The wheat flour gliadin content was reduced up to 90–95% in HD-2851, DDK-1025, NIAW-917 and UAS-428 shown up to 74% reduction of gliadins. The complete hydrolysis of gliadins (α, β, ω, ϒ) and glutenin subunits were confirmed by using immunoblot, ELISA, RP-HPLC chromatograph, anti-PEP-I (PSQQQQQPK), anti-PEP-II (LGQQQPFPPQQPYPQPQPFK) and anti-HMW-GS antibodies. The modified wheat flour was taken for the processing of pasta with the replacement of 30% chickpea flour. The gluten content of MWF pasta was found to be 48 ppm which is 99.95% lesser than the control pasta. Sensory evaluation revealed that the pasta was acceptable with overall quality when compared with control pasta.

Wheat is one of the major cereals consumed throughout the world; there has been a radical increase in the population suffering from many wheat-related disorders. The present study was conducted to screen low immunogenic hexaploid and tetraploid wheat varieties. A total of 34 different wheat varieties were tested for its total protein content, gliadin content and immunoreactivity with immunoglobulins of celiac (IgA) and wheat allergy patients (IgE). The wheat varieties HD-2851 and NIAW-917 (hexaploid) and NIDW-295, UAS-428, PDW-291, MPO-1215 and DDK-1025 (tetraploid) were found to be less immunoreactive varieties for both celiac and wheat allergy patients. The immunoblot assays for IgE reactivity have revealed that gliadin subunits present in all varieties are found to be allergic proteins. The principal component analysis biplot showed that tetraploid wheat varieties are less immunoreactive than hexaploids. The low immunogenic wheat varieties are suitable for plant breeding and preparation of low gluten or hypo-immunogenic products.

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.

Gluten-related diseases such as wheat allergy, celiac disease, and gluten intolerance are widespread around the globe to genetically predisposed individuals. The present study aims to develop a wheat-gluten induced BALB/c murine model for addressing wheat-gluten related disorders by sensitizing the wheat gluten through the route of intraperitoneal and oral challenge in prolonged days. During the sensitization, the sera were collected for specific antigliadin antibodies response and proinflammatory markers quantification. Ex vivo primary cells and organs were collected for subsequent analysis of inflammatory profile. Prolonging sensitization of gluten can moderate the antigen-specific inflammatory markers such as IL-1β, IL-4, IL-15, IL-6, IFN-γ and TNF-α levels in mice sera. However, ex vivo primary cells of splenocytes (SPLs) and intestinal epithelial lymphocytes (IELs) significantly increased the IL-6, IL-15, IL-1β, and IL-4 levels in G+ (gliadin and gluten) treated cells. Histopathology staining of jejunum sections indicates enterocyte degeneration in the apical part of villi and damage of tight junctions in G+ (gliadin and gluten) sensitized murine model. Immunohistochemistry of embedded jejunum sections showed significant expression of positive cells of IL-15, tTG and IL-4 in G+ sensitized murine model. In contrast, all markers of gluten-related disorders are expressed exclusively such as tTG, ZO-1, IL-15, IL-6, IL-4, and intestinal inflammation was mediated by iNOS, COX-2, TLR-4 and NF