Study of biochemical mechanisms induced by Gluten Friendly™ in vitro and in vivo on coeliac patients

This thesis aimed to investigate the biochemical mechanisms induced by “Gluten FriendlyTM” in vitro and in vivo on coeliac patients. “Gluten Friendly™” is a patented technology (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”) that allows the reshaping of gluten proteins directly in the cereal grains, before milling. Thereby it is possible to obtain functional flours suitable for the preparation of bakery products made from wheat. Specifically, “Gluten Friendly™” technology involves 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. The method aims to combine the nutritional, organoleptic and rheological properties of wheat proteins with a beneficial role for the gut health and microbiota of both celiac and healthy subjects. This innovative technology relies on a wise combination of parameters such as temperature, humidity, resting, and evaporation, clearly described in the registered patent and quoted in this thesis. This temperature-based process does not cause gluten proteins denaturation, but changes in their conformation, even in the so-called antigenic sequences, that make them unrecognizable in vitro by a wide range of antibodies specific for gluten proteins. It has been postulated that “Gluten Friendly™” technology induces rearrangements of protein structure that involve the exposure of charges that may allow a new kind of aggregation among different classes of wheat endosperm proteins, only through hydrophobic and/or ionic interactions. The investigation of gluten proteins reshaping induced by Gluten Friendly technology requires tough efforts since the Gluten Friendly molecule shows difficulty in its isolation. However, the molecular structure could be investigated by using an approach based on integrated techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy and Infrared (IR) spectroscopy. While the temperature-treated gluten loses its immunogenic properties in vitro on celiac, gut-derived T-cells lines, it is able to act as a bioactive molecule with pleiotropic effects on human health, without losing its technological proprieties and viscoelasticity. The flour can still form dough, leaven and bake into bread. The sensory qualities of Gluten FriendlyTM bread (GFB) are comparable to that of the Control Bread (CB) in terms of appearance, taste, aroma, colour and bread texture. Furthermore, a previous in vitro study carried out on celiac and healthy fecal microbiota pinpointed that GFB prolonged the survival of L. acidophilus La-5 and exerted an antibacterial effect towards S. aureus and S. Typhimurium. GFB was able to positively modulate the composition of celiac gut microbiota in vitro, promoting changes in Lactobacilli and Bifidobacteria members. These results were confirmed in a following in vitro study, performed by a three-stage continuous gut model system, on the intestinal microbiota composition and metabolites production 3 of healthy and celiac subjects for a prolonged period of 30 days. The study showed the ability of GFB to restore even the microbiota of healthy individuals, increasing the number of bifidogenic bacteria and SCFAs levels. Based on this evidence, the technology has been further improved with the registration of a second patent: "Method for the detoxification of gluten proteins from cereal grains and related uses in the medical field" (Italian priority patent no: 102015000084813 also extended to European level PCT/EP2016/0815899). Further in vitro study showed that GFB, in comparison to CB, increased mucus secretion and gut-barrier function in human intestinal goblet cells. These data support the potential effect of GFB to maintain or improve gut homeostasis and cellular barrier integrity by maintaining higher mucin levels. Moreover, another study based on the preparation of synbiotic fermented milk, containing L. acidophilus La-5 and Gluten FriendlyTM flour (GFF), was carried out. The most important result was the positive effect of GFF on the viability of the probiotic, with a prolongation of the shoulder length to 20 days (12–13 days in the control). Based on this evidence, another in vitro study, aiming to produce a synbiotic yogurt containing Bifidobacterium infantis and GFF, was carried out. The results highlighted that GFF prolonged the viability of Bifidobacteria for 14 days instead of 4 days in the control and control flour (CF). Based on the reduced immunoreactivity and the beneficial effects of Gluten FriendlyTM molecule on the gut microbiota in vitro, an in vivo prospective, double-blind, randomized, placebo-controlled study of 12 weeks was performed on 48 celiac volunteers. After 12 weeks on a Gluten FriendlyTM diet, it induced i) symptoms relief in all patients; ii) a clustering of the mucosal histology and inflammation of celiac subjects in 3-zones, as follows: 35% black zone (cluster 1; increase of IELs and injury), 30% grey zone (cluster 2; intermediate trend), 35% white zone (cluster 3; normal serology, no injury and no increase of IELs, trend as the placebo group). Thus, suggesting positive effect in 35% of patients (white zone) and a possible dynamic situation, with a positive modulation of the inflammation, in 30% of patients (grey zone). iii) a shaping of celiac microbiota towards intestinal homeostasis through a bifidogenic effect that Gluten Friendly induced by its digestion on Lactobacilli and other microorganism butyrate-producing species that contribute to the gut “ health ecology” of celiac subjects