Temperature-treated gluten proteins in Gluten-Friendly™ bread increase mucus production and gut-barrier function in human intestinal goblet cells

Abstract The effects of a control bread (CB) and a Gluten Friendly™ bread (GFB) on intestinal epithelium mucus production and barrier function in healthy human mucus-secreting goblet cells HT-29-16E were investigated. Mucus production in cells exposed to digested breads (GFB and CB) was preliminarily investigated using staining techniques, Periodic Acid-Schiff (PAS) and Alcian blue (AB), and MUC2 and MUC3 were also quantified by ELISA assay. The barrier function of the cell monolayer was evaluated by trans-epithelial electrical resistance (TEER) measurements. GFB increased the secretion of mucins, expressed as the level of PAS and AB staining in comparison with the control. MUC3 levels were not affected, whereas higher MUC2 concentrations (

An In Vitro Fermentation Study on the Effects of Gluten FriendlyTM Bread on Microbiota and Short Chain Fatty Acids of Fecal Samples from Healthy and Celiac Subjects

Recently, an innovative gluten detoxification method called Gluten FriendlyTM (GF) has been developed. It induces structural modifications, which abolish the antigenic capacity of gluten and reduce the in vitro immunogenicity of the most common epitopes involved in celiac disease, without compromising the nutritional and technological properties. This study investigated the in vitro effects of GF bread (GFB) on the fecal microbiota from healthy and celiac individuals by a three-stage continuous fermentative system, which simulates the colon (vessel 1, proximal colon; vessel 2, transverse colon; and vessel 3, distal colon), as well as on the production of short chain fatty acids (SCFA, acetate, propionate, butyrate). The system was fed with GFB and the changes in microbiota through fluorescence in situ hybridization and in SCFA content were assessed. GFB exerted beneficial modulations such as bifidogenic effects in each compartment of the model both with healthy- and celiac-derived samples, as well as growth in Clostridium clusters XIVa+b in celiac-derived samples. Furthermore, increased levels of acetic acid were found in vessel 1 inoculated with the fecal microbiota of healthy individuals, as well as acetic and propionic in vessel 1 and 2 with celiac-derived samples. In addition, the use of multivariate approaches showed that the supplementation of GFB could result in a different modulation of the fecal microbiota and SCFA, as a function of initial equilibrium

Impact of gluten-friendly bread on the metabolism and function of in vitro gut microbiota in healthy human and coeliac subjects

The main aim of this paper was to assess the in vitro response of healthy and coeliac human faecal microbiota to gluten-friendly bread (GFB). Thus, GFB and control bread (CB) were fermented with faecal microbiota in pH-controlled batch cultures. The effects on the major groups of microbiota were monitored over 48 h incubations by fluorescence in situ hybridisation. Short-chain fatty acids (SCFAs) were measured by high-performance liquid chromatography (HPLC). Furthermore, the death kinetics of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Staphylococcus aureus, and Salmonella Typhimurium in a saline solution supplemented with GFB or CB were also assessed. The experiments in saline solution pinpointed that GFB prolonged the survival of L. acidophilus and exerted an antibacterial effect towards S. aureus and S. Typhimurium. Moreover, GFB modulated the intestinal microbiota in vitro, promoting changes in lactobacilli and bifidobacteria members in coeliac subjects. A final multivariate approach combining both viable counts and metabolites suggested that GFB could beneficially modulate the coeliac gut microbiome; however, human studies are needed to prove its efficacy

Study of Chemical, Biochemical and Technological characteristics of Gluten Friendly™ grains of cereals and derived products

La tecnologia "Gluten Friendly ™" è un metodo per la detossificazione delle proteine del glutine dalla granella dei cereali, in particolare dai semi di grano, finalizzato all'ottenimento di farine detossificate per la preparazione di pane e prodotti a base di grano, preferibilmente adatti all'alimentazione di pazienti con malattia celiaca, ma adeguati anche per le proprie caratteristiche organolettiche, per l'alimentazione di tutta la popolazione. Questo è un metodo brevettato italiano n °: 0001414717, anch'esso depositato in base al Trattato di cooperazione in materia di brevetti, la domanda n. PCT / IB2013 / 000797 e pubblicato in Europa come EP 2903453 A1 e intitolato "Metodo di detossificazione delle proteine del glutine dai semi di cereali". Questa tecnologia implica l'applicazione di energia a microonde per alcuni secondi ai semi di grano idratati prima della molitura per raggiungere una temperatura elevata per un breve periodo di tempo in modo da indurre cambiamenti nelle proteine che riducono l'immunogenicità in vitro degli epitopi più comuni coinvolti nella malattia celiaca. (Lamacchia et al., 2015a Metodo Brevetto Italiano N. 0001414717 (2015) .Modelli: Lamacchia, C., Di Luccia, A., Gianfrani, C (Lamacchia et al. ., 2015b; Lamacchia, C., Di Luccia, A. & Gianfrani C. (2015) .Metodo per la detossificazione delle proteine del glutine dai cereali dei cereali. Congresso BioVaria, le migliori tecnologie europee, Munchen, Germania, https: // www .biovaria.org / passato-evento / tecnologie /) preservando le proprietà tecnologiche della farina. La tecnologia "Gluten Friendly ™" si basa sull'analisi di recenti studi in cui Lamacchia e altri (2010) hanno riferito che, quando le alte temperature si applicano alla cariosside del grano, le proteine subiscono cambiamenti che non sono simili a quelli visti nei sistemi modello, che consistono solo di glutine (Schofield et al., 1983; Singh e MacRitchie, 2004), né a quelli visti nella pasta durante i cicli di essiccazione.. In un recente studio, Tosi e altri (2009) hanno confermato, infatti, che le HMW sono particolarmente abbondanti nello strato più interno della cariosside del frumento (endosperma) e praticamente assente nello strato subaleuronico, che tuttavia è ricco di gliadine e LMW. Questo modello di deposizione viene mantenuto durante tutta la fase di sviluppo della cariosside del frumento e continua anche dopo la fusione dei corpi proteici e la formazione della matrice amidacea. Pertanto, la segregazione delle proteine del glutine nei corpi proteici quando sono nella cariosside e l'applicazione di alte temperature in questa fase prima dell molitura, consentirebbe a tali proteine di sperimentare cambiamenti strutturali tali da non renderli più riconoscibili dalla transglutaminasi intestinale, bloccando quindi la cascata di citochine infiammatorie. Da allora la tecnologia è stata ulteriormente migliorata (brevetto prioritario italiano n_: 102015000084813). Metodo per la disintossicazione delle proteine del glutine dai cereali dei cereali e relativi usi medici presentato il 17 dicembre 2015. Inventore: Lamacchia C.).“Gluten Friendly™” technology is a method for the detoxification of gluten proteins from grains of cereals, in particular from the grains of wheat, aimed to obtain detoxified flours for the preparation of bread and pasta products, from wheat, preferably suitable for the alimentation of patients with celiac disease, but also adequate for its organoleptic characteristics and for its aspect, for the alimentation of the whole population. This is an Italian patented method n°: 0001414717, also filed under the Patent Cooperation Treaty, application no. PCT/IB2013/000797 and published in Europe as EP 2903453 A1 and titled “Detoxification method of gluten proteins from cereal grains”. This technology implies the application of microwave energy for a few seconds to hydrated wheat kernels before milling to reach a high temperature for a short amount of time so to induce changes in proteins that reduce the immunogenicity in vitro of the most common epitope involved in cealic disease. (Lamacchia et al., 2015a Italian Patented Method N. 0001414717 (2015). Metodo per la detossificazione delle proteine del glutine dalle granaglie dei cereali. Inventors: Lamacchia, C., Di Luccia, A., Gianfrani, C (Lamacchia et al., 2015b; Lamacchia, C., Di Luccia, A. & Gianfrani C. (2015). Method for the detoxification of gluten proteins from grains of cereals. BioVaria Congress, Europe’s top technologies, Munchen, Germany, https://www.biovaria.org/past-event/ technologies/) preserving the technological properties of the flour. The “Gluten Friendly™” technology is based on the analysis of recent studies in which Lamacchia and others (2010) have reported that, when the high temperatures are applied to the caryopsis of wheat, the proteins undergo changes that are not similar to those seen in model systems, consisting only of gluten (Schofield et al., 1983; Singh and MacRitchie, 2004), nor to those seen in the pasta during the drying cycles. The researchers Lamacchia and others (2010) explained this phenomenon on the basis of the fact that in the caryopsis of wheat, gluten is not yet formed and gluten proteins are deposited in different protein bodies (Rubin et al., 1992, Krishnan et al., 1986; Lending et al., 1989). In a recent study, Tosi and others (2009) confirmed, in fact, that the HMW are particularly abundant in the innermost layer of the caryopsis of wheat (endosperm) and practically absent in the subaleuronic layer which, however, is rich in gliadins and LMW. This pattern of deposition is maintained throughout all the development phase of the caryopsis of wheat and continues even after the merger of protein bodies and the formation of the starchy matrix. Therefore, the segregation of gluten proteins in protein bodies when they are in the caryopsis and the application of high temperatures in this stage before the milling, would allow such proteins of experiencing structural changes such as not to make them recognizable anymore by intestinal transglutaminase, thereby blocking the waterfall of inflammatory cytokine. The technology has since been further improved (Italian priority patent n_: 102015000084813. Method for the detoxification of gluten proteins from grains of cereals and related medical uses filed on 17th December 2015. Inventor: Lamacchia C.)

Changes in wheat kernel proteins induced by microwave treatment

Wheat kernels were subjected to microwave treatment, and the proteins were characterized by size exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this process, the proteins polymerize, forming intermolecular bonds among the same classes of proteins. Furthermore, the polymerization occurs only through disulphide bonds. Although SDS-PAGE did not show any differences for either the number or intensity of protein bands between flour samples before and after microwave treatment, gliadins from treated flours showed significantly reduced cross-reactivity with the R5 antibody. Moreover, the gluten became soluble in an aqueous saline solution, and it was not possible to isolate it using the Glutomatic apparatus. However, the treated flour, in the presence of water, was able to form dough and leaven and produce brea

Impact of Gluten-FriendlyTM technology on wheat kernel endosperm and gluten protein structure in seeds by light and electron microscopy

The main aim of this paper was to assess the impact of Gluten-FriendlyTM (GF) technology (Italian priority patent n 102015000084813 filed on 17th December 2015) on wheat kernel endosperm morphology and gluten protein structure, using SEM, light and immunofluorescent microscopy. Microscopy was combined with immunodetection with specific antibodies for gliadins, c-gliadins, LMW subunits and antigenic epitopes to gain a better understanding of the technology at a molecular level. The results showed significant changes to gluten proteins after GF treatment; cross-reactivity towards the antibodies recognizing almost the entire range of gluten proteins as well as the antigenic epitopes through the sequences QQSF, QQSY, PEQPFPQGC and QQPFP was significantly reduced. The present study confirms the results from our previous work and shows, for the first time, the mechanism by which a chemical-physical treatment abolishes the antigenic capacity of gluten

One-way ANOVA (P<0.05) and homogeneous groups based on propionate after 24 and 48 h of fermentation in pH-controlled batch culture systems.

<p>Increases/decreases refer to the inoculum of the negative control. Samples: A, negative control healthy donors; B, healthy donors + CB; C, healthy donors + GFB; D, negative control coeliac donors; E, coeliac donors + CB; F, coeliac donors + GFB. CB, control bread; GFB, gluten-friendly bread.</p

Conditions used to assess the effect of control bread and gluten friendly bread towards <i>L</i>. <i>acidophilus</i>, <i>B</i>. <i>animalis</i>, <i>Salmonella</i> sp. and <i>S</i>. <i>aureus</i> in saline solution.

<p>Conditions used to assess the effect of control bread and gluten friendly bread towards <i>L</i>. <i>acidophilus</i>, <i>B</i>. <i>animalis</i>, <i>Salmonella</i> sp. and <i>S</i>. <i>aureus</i> in saline solution.</p

One-way ANOVA (P<0.05) and homogeneous groups based on butyrate after 24 and 48 h of fermentation in pH-controlled batch culture systems.

<p>Increases/decreases refer to the inoculum of the negative control. Samples: A, negative control healthy donors; B, healthy donors + CB; C, healthy donors + GFB; D, negative control coeliac donors; E, coeliac donors + CB; F, coeliac donors + GFB. CB, control bread; GFB, gluten-friendly bread.</p