WO2018011714A1 - Preparation of cereal products suitable for celiac patients by the creation of supramolecular structures between proteins and chitosan - Google Patents

Abstract

The present invention relates to a method for detoxifying wheat flour (Triticum spp.) and gluten by forming supramolecular structures with chitosan. The invention also relates to the corresponding detoxified flour and gluten having toxic epitope values reduced by 65 and 88%, respectively, for patients with celiac disease, when compared with non-modified or original flour and gluten. In addition, the invention relates to the use of the detoxifying method for other cereals and their respective products, in particular other Gramineae (Poaceae), subfamily Pooideae, especially the species of the Triticeae tribe, such as barley (Hordeum spp.), rye (Secale spp.), Triticale (x Triticosecale), and of the Aveneae tribe, such as oats (Avena spp.), and their germination products.

Description

 DESCRIPTION

 "DETOXIFICATION PROCESS OF WHEAT AND GLUTEN FLOUR THROUGH FORMATION OF SUPRAMOLECULAR STRUCTURES WITH CHITOSANA AND RESPECTIVE FLOUR OF WHEAT AND GLUTEN DETOXIFIED FOR CONSUMPTION BY CELL DISEASES

Technical Domain

 The present invention relates to a detoxification process of wheat flour (Triticum spp.) And gluten through the formation of supramolecular structures with chitosan and their detoxified flour and gluten in which patient-toxic epitope reduction values are obtained. 65 and 88% when compared with unmodified or original flour and gluten, respectively. It also concerns the use of the detoxification process in other cereals and their products, in particular in other grasses (Poaceae), subfamily Pooideae, mainly of Triticeae species such as barley (Hordeum spp.), Rye (Secale spp.), triticale (x Triticosecale) and the Aveneae tribe as oats (Avena spp.), as well as their germinated products.

Background

Celiac disease is an autoimmune disease that translates into an enteropathy that occurs after contact with gluten in genetically susceptible individuals (1, 2). The disease causes villous atrophy of the small intestine mucosa, causing impaired nutrient absorption, the most common symptoms being diarrhea, growth retardation, anemia and fatigue (1). Additionally, other symptoms such as neurological complications, hormonal dysregulation and primary biliary cirrhosis have been described (3-5). This disease is more common in western populations than previously thought, having a prevalence in these populations of approximately 1% (2) and being underdiagnosed in Portugal (1). The only effective treatment is a strictly gluten-free diet, with no drugs to prevent damage or the body's autoimmune response (6). Given the relevance of this problem, research on the allergenicity of wheat and other cereal proteins is particularly important (7).

Wheat (Triticum spp.) Is one of the main cereals grown by man and its grain is the major source of protein in the human diet. The annual world production of this cereal is around 715 million tons, providing approximately 20% of calories. Wheat has the largest global area of production (-220 million ha) and the largest international transaction volume of a simple agronomic crop (-150 million tonnes), meaning about $ 50 billion (8). It contains a protein complex, gluten, which is primarily responsible for the unique rheological behavior of its flours (9) and makes it a cereal of choice in its diverse uses: traditional bakery and confectionery products such as bread , cookies, biscuits, pizza bases, pasta and pasta, cakes of various kinds, among others. The main protein components of gluten are gliadin and glutenins which confer mass extensibility and elasticity, respectively (10).

A key step in the pathogenesis of celiac disease occurs when certain gluten peptides, namely Gliadin derivatives (rich in glutamine residues) are deactivated by tissue transglutaminase (TG2), increasing their affinity for human leukocyte antigen (HLA) -DQ2 or -DQ8, thereby enhancing the autoimmune reaction (11, 12). Alphagliadin epitopes are considered to be of greater clinical relevance for both the adaptive and innate responses that lead to celiac disease (13-18). Some amino acid sequences have been reported to have strong immunogenic potential. All peptides that were active in celiac disease contained the Gln-Gln-Gln-Pro and Pro-Ser-Gln-Gln sequences. In contrast, inactive peptides contained none of these sequences. Therefore, these sequences are considered of high importance for the development of the disease (19, 20). The immunological activity of these amino acid sequences in celiac disease has been confirmed by other investigators (21, 22). In addition, a 33-residue peptide resistant to intestinal proteases and containing three of the most immunogenic epitopes was identified as a major stimulant of the inflammatory gluten response (15, 23).

To date, there is no pharmacological treatment for gluten intolerant patients and the only effective route is the strictly gluten-free continuous diet, which sometimes results in social problems (6). Given the incomplete response of many patients to the gluten-free diet as well as the difficulty of long-term dietary adherence, the development of new effective therapies to control the symptoms and reversal of inflammation and organ damage becomes extremely important. . In this sense, some promising strategies have been conducted, both therapeutically and with a view to gluten modification to reverse this scenario. For example, gluten and its peptides may be hydrolyzed by prolylendopeptidases and / or other glutenases allowing the production of safe products for celiac patients (24). Enzymes can also be ingested as a prophylactic measure, in which dietary gluten is hydrolyzed by co-ingested peptidases, thus avoiding the specific immunological reactions of celiac disease in the small intestine (25). The use of these enzymes to detoxify gluten or even its co-administration in the diet has led to several patent applications (26-30).

However, the use of peptidases for gluten modification has some limitations, namely the loss of technological quality of gluten and pasta because they are hydrolysed products without the protein structure necessary to maintain elasticity and extensibility properties. Regarding the co-ingestion of peptidases, the main limitations are that they are rapidly hydrolyzed by gastric pepsin (31, 32), making oral administration difficult and also inactivated by trypsin and chymotrypsin in the presence of salts. bile ducts (33).

Other pharmacological approaches are highlighted, namely the use of polymeric binders (random hydroxyethyl methacrylate (HEMA) and sodium 4-styrene sulfonate (SS) copolymers) which through non-covalent binding to gliadins prevent their digestion and thus suppress gliadin-induced toxicity in the intestinal epithelium (34), paracellular permeability modulators (35), transglutaminase-2 inhibitors (36-41), peptide vaccination (42-44), intestinal regeneration enhancers (45) and natural killer NKG2D receptor blockers and MICA ligand protein (46, 47). In addition, the Glycine-type 2 polypeptide (GLP-2) demonstrated significant repair activity of the small and large intestine mucosal epithelium, increasing the intestine's ability to digest and absorb nutrients. Its use for the treatment of gastrointestinal disorders and disorders has been described (48, 49). Also, the use of antibodies with specific activity against gluten or gluten-derived peptides that may inhibit gluten deactivation or its peptides derived by tissue tranglutaminase is a potential strategy for reducing gluten toxicity (50).

In any case, the treatment of celiac disease or its control by a pharmacological approach will have the counterpart of the treatment targeting the endogenous effectors of the disease and in this sense, the modification of gluten (exogenous effector) aiming at its detoxification becomes a very attractive strategy compared to pharmacological treatment. Prior evaluation of the overall toxicity of the products is a crucial advantage as the heterogeneity of celiac disease is well known (18). It is now known that not only gliadin, but all gluten proteins can be toxic to at least one celiac patient. (51).

A method of reducing food allergenicity using supercritical carbon dioxide or liquid nitrogen has been described (52). Also Ribonucleic Acid Interference (RNAi) technology was used to reduce gliadin expression in wheat lines which were subsequently evaluated as having a low level of toxicity for celiac patients (53). Recent research has shown that altering gamma-gliadin expression in different genetic backgrounds has no direct effect on the technological quality of wheat masses.

(54). With regard to RNAi technology, the heterogeneity of the disease, the genetic stability of the lines over the generations and the fact that the end product is a genetically modified organism (GMO) and all the various or potential issues arising therefrom are pointed out. as the biggest limitations of this approach

(55).

Other approaches have been conducted to modify glutamine residues such as the treatment of wheat flour with microbial transglutaminase for the production of hypoallergenic flours (56) or the modification of wheat flour to interfere with the deamidation of wheat residues. glutamine by prior chemical or enzymatic deamidation followed by enzymatic derivatization of the formed carboxylic group leading to a patent application (57). In addition, the ability of peptide conjugation by transglutaminase has been described (58, 59) and its use for product development and enhancement of certain characteristics such as texture through protein cross-linking, bridging the lysine deficiency of certain foods, among others. others led to several patent applications (60-69). The use of microbial transglutaminase in combination with an amine donor to detoxify gluten for celiac patients has recently been described (70, 71). Through modification Selective screening of glutamine residues present on toxic epitopes by microbial transglutaminase (transamidation) using lysine or methyl or ethyl esters of lysine prevents the process of deamidation driven by tissue transglutaminase in the human body, which is a key step in the development of immune response in celiac patients (72).

Several studies were subsequently conducted using the action of microbial transglutaminase and lysine methyl ester as substrate in intact wheat flour (73), lysine ethyl ester as substrate in wheat flour previously devoid of albumin and globulin (endosperm proteins). (74, 75), or L-lysine as a gluten substrate previously hydrolyzed by pepsin followed by trypsin (76). In all of these studies a marked decrease in celiac patients' toxicity of modified products was found. The major disadvantage of industrial application is the high cost of the substrate used. In addition, all work cited above involving the use of lysine or lysine methyl or ethyl esters as amino group donors for transglutaminase requires an assessment of the final rheological / technological quality of the modified products. More recently, a study using the action of microbial transglutaminase and n-butylamine as a substrate in wheat flour and its gluten has been published, in which the extent of the transamidation reaction is greatly increased through the use of reducing conditions. In addition, the increased hydrophobicity of the treated proteins resulted in better baking properties of the treated flour compared to the original flour (77). The present invention describes a novel process of detoxification of intact wheat flour and its gluten through the formation of supramolecular structures between proteins and chitosan natural polysaccharide, resulting in products intended for consumption by celiac patients and having a number of advantages. and differences with respect to the processes described above, namely:

 • Chitosan is a non-toxic polysaccharide, is hypoallergenic, biocompatible and biodegradable;

 • Chitosan is a cheaper product than products previously used for detoxification of flour and gluten such as L-lysine, methyl and ethyl esters of lysine;

 • Derived products are compatible with celiac disease;

 • Ease of production, ie fast production without high temperatures and no toxic side products;

 • Obtaining products sensorially, viscoelastically and mechanically similar to classic products.

In addition, the present invention further relates to the use of the process in other cereals and their products, namely barley (Hordeum spp.), Rye (Secale spp.), Triticale (x Triticosecale Wittmack) and oats (Avena spp. ), as well as their germinated products, as some proteins of these cereals have similar immunological reactivity to wheat gluten for celiac patients (2). summary

 The present invention relates to a detoxification process of wheat flour (Triticum spp.) And gluten by forming supramolecular structures with chitosan and their flour and gluten in which toxic epitope reduction values are obtained for celiac patients. about 65 and 88% compared to unmodified or original flour and gluten. In addition, it concerns the use of the process in other cereals and their products, namely barley (Hordeum spp.), Rye (Secale spp.), Triticale (x Triticosecale) and oats (Avena spp.) And sprouted products.

In a preferred embodiment, chitosan may originate from crustaceans or fungi.

In a preferred embodiment, the detoxification process involves the use of chitosans with different degrees of polymerization (SD = 2 to SD> 1,000,000) and acetylation degrees (0 to 60%).

In a preferred embodiment, the detoxification process involves the use of amino sugars. In yet another preferred embodiment the protein: chitosan ratio ranges from 0.5: 1 to 10: 1 (w / w), preferably 1.9: 1.

In a preferred embodiment, the detoxification process involves the use of sugars with different degrees of polymerization (SD = 2 to SD> 1,000,000). In another preferred embodiment, the detoxification process involves the reaction at a pH value from 1 to 9, preferably at pH 6.

In a preferred embodiment glutathione is used as reducing agent in a concentration between 0.1 mM to 500 mM, preferably 20 mM.

In another preferred embodiment, the flour and proteins may originate from oats, rye, barley and triticale or their germinated products, such as malt.

The present invention further describes the process for obtaining wheat, oat, barley or triticale flour and their detoxified proteins for consumption by celiac patients described above, comprising the following steps:

- dispersion of chitosan in acetic acid (0.05 mol / L) in a protein: chitosan ratio ranging from 0.5: 1 to 10: 1 (w / w), preferably 1.9: 1 followed by pH adjustment between 4 to 9 using a buffer system, preferably 6;

 dispersion of the flour in the buffered solution containing chitosan in a protein: chitosan ratio ranging from 0.5: 1 to 10: 1 (w / w), preferably 1.9: 1;

adding a reducing agent at a final concentration of 0.1 to 500 mM, preferably 20 mM. The reducing agent used may be glutathione or dithioerythritol (DTE) or dithiothitrol (DTT) or tris 2-carboxyethyl phosphine (TCEP), preferably glutathione; incubating the mixture at a temperature between 4 to 80 ° C, preferably 40 ° C;

 - incubation lasting 30 minutes to 72 hours, preferably 24 hours;

 - separating the flour from the solution by centrifugation or filtration with water washes;

 - flour recovery and freeze drying, oven drying, vacuum oven drying, atomization.

The present invention also describes the process for obtaining detoxified wheat gluten for consumption by celiac patients described above comprising the following steps:

 - dispersion of chitosan in acetic acid (0.05 mol / L) in a protein: chitosan ratio ranging from 0.5: 1 to 10: 1 (w / w), preferably 1.9: 1 followed by pH adjustment between 4 to 9 using a buffer system, preferably 6;

 - dispersion of gluten obtained from the aqueous washing of the produced dough of wheat flour with the addition of 1% NaCl in the buffered solution containing chitosan in a protein: chitosan ratio ranging from 0,5: 1 to 10: 1 ( w / w), preferably 1.9: 1;

 adding a reducing agent at a final concentration of 0.1 to 500 mM, preferably 20 mM. The reducing agent used may be glutathione or dithioerythritol (DTE) or dithiothitrol (DTT) or tris 2-carboxyethyl phosphine (TCEP), preferably glutathione;

 incubating the mixture at a temperature between 4 to 80 ° C, preferably 40 ° C;

- incubation lasting 30 minutes to 72 hours, preferably 24 hours; - separating gluten from solution by centrifugation or filtration with water washes;

 - Gluten recovery and subsequent freeze drying, oven drying, vacuum oven drying, atomization.

Brief Description of the Figures

 For an easier understanding of the present application, attached are figures which represent preferred embodiments which, however, are not intended to limit the art disclosed herein.

Figure 1. Nature of the association between gliadin and chitosan in a 1: 1 (w / w) ratio at different pHs and under reducing and non-reducing conditions.

Figure 2. Gluten x-ray spectrum containing supramolecular structures with chitosan (¾¾¾¾) and chitosan treated under similar conditions ().

Figure 3. Characterization of the effect of the presence of supramolecular structures on flour and gluten in relation to the release of toxic epitopes measured by antibody R5 (A), digestibility (B), and tissue transglutaminase (C) deamidation activity. * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001; n = 3.

Figure 4. Kieffer microextension extension curves of dough produced by flour containing supramolecular structures with chitosan (* »» ») and control flour, ie treated under similar conditions Figure 5. Process for obtaining gluten-chitosan supramolecular structures in flour of various origins and gluten isolated from wheat flour.

Description of embodiments

 Referring to the figures, some embodiments are now described in more detail, which are not, however, intended to limit the scope of the present application.

Detoxified wheat flour and gluten for consumption by celiac patients, objects of the present invention, are different from products made for the same purpose in that they contain supramolecular structures produced between gluten proteins and chitosan, which is a safe polysaccharide. for human consumption. These supramolecular structures result from the non-covalent interaction by hydrogen bridges between gluten proteins, namely gliadin, and chitosan, under reducing conditions (Figure 1). In addition, the re-oxidation of disulfide bridges results in mechanical entrapment and consequent stabilization of these structures. Analysis of the formation of high molecular weight complexes and the nature of the protein-chitosan association was conducted by molecular exclusion chromatography. For this, a column (30cm x 7.8mm, particles 8μπι) was used TSK-Gel G4000 SW _XL (TOSOH Bioscience, Japan) at 50 ° C and a solution of 0.2mol / L acetic acid / 0.15 mol / L ammonium acetate (pH 4.5) as eluent. A flow rate of 0.5 mL / min and a photodiode detector (Dionex PDA-100) were used. Detection was performed at 280 nm. The sample volume used was about 50 µl. The analytical column was previously calibrated with blue dextran (-2000 kDa), urease (-545 kDa), bovine serum albumin (-66 kDa), cytochrome c (-12 kDa), insulin (-6 KDa) and tyrosine (-181 Da).

X-ray diffraction (Figure 2) and infrared spectroscopy (Table 1) show the profound change that the association between proteins and chitosans, and the formation of supramolecular structures, have on gluten. X-ray diffraction analysis was performed using the PANalytical X'Pert Pro X-ray diffractometer equipped with the X'Celerator detector. Measurements were conducted using Cu-Κ radiation (40kV; 30mA) in Bragg-Bentane geometry (7-60 ° angular range 2Θ). Infrared spectroscopy was performed on a Bruker Alpha with Platinum ATR module in the range 4000 to 600 cr ¹ at a resolution of 4 cm ^-1 . Spectra resulted from the addition of 32 scans. The deconvolution of the second derivative of the amide I band was performed using the PeakFIT software (Systat).

Table 1. Infrared spectroscopy and assignment of secondary protein structures of control gluten containing supramolecular structures.

1681 Round β6.03 ± 0.02 5.76 ± 0.08 Ρ = 0.0048

1689 Sheet β3.37 ± 0.02 2.73 ± 0.05 Ρ <0.0001

1696 Sheet β1.89 ± 0.07 1.49 ± 0.06 Ρ = 0.0017

∑ Sheet β 52, 48 51, 92 (-1.07%)

 ∑ Propeller 25, 08 26.28 (+ 4.78%)

 ∑ Round β 22, 44 21.80 (-2.85%)

The result in terms of application to the production of tolerable products for celiac patients is a clear decrease in the digestibility of gluten proteins, namely gliadins, resulting in a product in which more than 65% of celiac patients' toxic epitopes are eliminated. damsidation process conducted by tissue transglutaminase, a key step in the pathogenesis of celiac disease (11, 12), inhibited (Figure 3).

For the quantification of toxic epitopes in flour and gluten, controls and associated with chitosan, the competitive RIDASCREEN® Gliadin commercial product was used. This product is based on the competitive Enzyme-Linked Immunosorbent Assay (ELISA) and monoclonal antibody R5, which is recommended by Codex Alimentarius and recognizes among others the potentially toxic sequence QQPFP, which occurs repeatedly in flour proteins. and gluten. The competitive format of this assay has the advantage of detecting individual peptide fragments compared to the sandwich ELISA format. The limit of detection is 1.36 ppm gliadin and the limit of quantitation is 5 ppm gliadin. All RIDASCREEN® Gliadin competitive product instructions were strictly followed and material preparation was done as described by Gessendorfer et al. (78), also a recommendation of the test used. Briefly, the original and detoxified flour and gluten were dispersed in 5% distilled water and the pH adjusted to 1.8 using 1 M HCl solution (79). Then 2.5 mg pepsin was added and reacted for four hours while stirring at 37 ° C. Thereafter the pH was adjusted to 7.8 with 1 M NaOH solution and 2.5 mg trypsin was added to the reaction mixture for an additional four hours while stirring at 37 ° C. Finally, the pH was adjusted to 4.5 with a 1 M HCl solution, and the tryptic digest were centrifuged at 4000 xg for twenty minutes at room temperature. The supernatant was decanted, frozen and lyophilized. The dried residues obtained correspond to the tryptic-peptide (PT) digestions of flour and gluten, controls and associated with chitosan. This protocol for digestion of flour and gluten with proteases similar to those present in the human digestive system allows to mimic the digestion process.

Digestibility was determined by quantifying peptides resulting from peptide-tryptic hydrolysis by molecular exclusion chromatography as described above. Tissue transglutaminase deamidation analysis was conducted according to van de Wal et al. (80) and using the commercial Ammonia assay kit (Megazyme, Bray, Ireland).

Chitosan used for the detoxification of the mentioned cereal flours, including wheat (Triticum spp.) And gluten, may have a molecular weight ranging from 2 glucosamine units to over 1 million glucosamine units. Other amino sugars may also be used for this process. The chitosan used for the detoxification of the mentioned cereal flours, including wheat (Triticum spp.) And gluten may still be naturally partially acetylated, with an acetylation degree of up to 60%.

Also the chitosan used for detoxification of the mentioned cereal flours, including wheat (Triticum spp.) And gluten can be obtained from the exoskeleton of crustaceans such as crab or shrimp, or obtained from the cell walls of fungi such as Agaricus bisporus or Aspergillus niger.

Chitosan is previously dissolved in an acid solution to promote its solubility, for example using dilute acetic acid solutions, typically with a concentration of 1%.

The detoxification reaction of flour or gluten with chitosan occurs at a temperature ranging from 4 ° C to 80 ° C, preferably at 40 ° C, for thirty minutes to seventy-two hours, preferably twenty-four hours.

After this detoxification period the flour or gluten is separated from the reaction mixture by centrifugation or filtration, with successive washes with water to remove the reducing agent and the unreacted chitosan.

Detoxified flour or gluten and after its recovery are dried by lyophilization or oven drying or vacuum drying or spray drying. Flour or gluten containing the supramolecular structures retains or improves the recognized organoleptic, viscoelastic and mechanical characteristics of similarly treated wheat and gluten flours (Figure 4) and can be transformed into traditional bakery and pastry products, such as bread, cookies, biscuits, pizza bases, pasta and pasta, cakes of a different nature, among others.

For the determination of the extensibility and extension resistance of flour containing supramolecular structures and its comparison with control flour, the Kieffer micro-extensograph (81) was used. In short, the masses were obtained by mixing water calculated according to AACC procedure 54-40.02 (82), and allowed to stand 20 min at 30 ° C under water saturated atmosphere conditions. After this time the masses were pressed in a preheated Teflon mold at 30 ° C and allowed to stand for a further 40 min at 30 ° C in a water saturated atmosphere. The teflon mold and mass measuring system were the SMS / Kieffer Dough and Gluten Extensibility Rig with the Stable Micro Systems TA-XT2 texturometer.

In summary, by using chitosan in the presence of a reducing agent (Figure 5), it is possible to alter the allergenic properties of gluten proteins, eliminating toxicity in more than 65% of reactive epitopes in celiac disease without loss of organoleptic characteristics, viscoelastic and mechanical components of wheat and gluten flours and can be made into bakery traditional pastries such as bread, crackers, biscuits, pizza crusts, pasta and pasta, cakes of a different nature, among others, the detoxified flour and gluten being a unique and different product from all products described or existing in market .

Application Examples

Example 1

 For the detoxification of wheat flour (Triticum spp.) 437 mg of chitosan obtained from the crab exoskeleton with a 66% deacetylation degree and a molecular weight of 580 kDa were initially dissolved in a 0.05 mol / L acetic acid solution. . After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (6.14 g / l). The mixture is kept at 40 ° C for 24 hours. After this detoxification period the flour is dialyzed and dried by lyophilization.

Example 2

For detoxification of gluten isolated from wheat flour (Triticum spp.), 437 mg of chitosan obtained from the crab exoskeleton with a 66% deacetylation degree and a molecular weight of 580 kDa were initially dissolved in an acetic acid solution. at 0.05 mol / l. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. THE Gluten (20 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (6.14 g / l). The mixture is kept at 40 ° C for 24 hours. After this detoxification period the gluten is dialyzed and freeze dried.

Example 3

 For detoxification of wheat flour (Triticum spp.) 1g of chitosan obtained from the cell wall of Agaricus bisporus was initially dissolved with a deacetylation degree of 78% and a molecular weight of 60-120 kDa in a 1% acetic acid solution. %. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 5.0 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (3.07 g / l). The mixture is kept at 30 ° C for forty eight hours. After this detoxification period the flour is separated from the reaction mixture by filtration, washed successively with water and dried in a vacuum oven.

Example 4

For detoxification of gluten isolated from wheat flour (Triticum spp.), 1 g of chitosan obtained from the cell wall of Agaricus bisporus was initially dissolved with a degree of deacetylation of 78% and a molecular weight of 60-120 kDa. 1% acetic acid solution. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 7.5 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. Gluten (20 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (6.14 g / l). THE The mixture is kept at 50 ° C for twelve hours. After this detoxification period the gluten is separated from the reaction mixture by centrifugation, washed successively with water and dried in a vacuum oven.

Example 5

 For detoxification of wheat flour (Triticum spp.), 1 g of chitosan obtained from the Aspergillus niger cell wall was initially dissolved with a deacetylation degree of 83% and a molecular weight of 60-120 kDa in a 1% acetic acid solution. %. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6.5 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (6.14 g / l). The mixture is kept at 40 ° C for 24 hours. After this detoxification period the flour is separated from the reaction mixture by centrifugation, washed successively with water and dried by lyophilization.

Example 6

For detoxification of gluten isolated from wheat flour (Triticum spp.), 1 g of chitosan obtained from the Aspergillus niger cell wall was initially dissolved with a deacetylation degree of 83% and a molecular weight of 60-120 kDa in a 1% acetic acid solution. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6.5 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. Gluten (20 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (6.14 g / l). The mixture is kept at 40 ° C for 24 hours. After this detoxification period the gluten is separated from the reaction mixture by centrifugation, washed successively with water and dried by lyophilization.

Example 7

 For detoxification of wheat flour (Triticum spp.) Initially 1g of chitosan obtained from the crab exoskeleton with a degree of 80% deacetylation and a molecular weight of 900 kDa was dissolved in a 1% acetic acid solution. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6.5 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (10 g / l) and ten biocatalyst units per gram of protein. The mixture is kept at 40 ° C for 24 hours. After this detoxification period the flour is separated from the reaction mixture by centrifugation, washed successively with water and dried by lyophilization.

Example 8

For detoxification of gluten isolated from wheat flour (Triticum spp.), 1 g of chitosan obtained from the crab exoskeleton with a degree of deacetylation of 80% and a molecular weight of 900 kDa was initially dissolved. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6.5 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. Gluten (20 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (10 g / l). The mixture is kept at 40 ° C for 24 hours. After this detoxification period gluten is separated the reaction mixture by centrifugation, washed successively with water and dried by lyophilization.

Example 9

 For detoxification of barley flour (Hordeum spp.), 1 g of chitosan obtained from the crab exoskeleton was initially dissolved with a 66% deacetylation degree and a molecular weight of 580 kDa in a 0.05 mol / acetic acid solution. L. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (3.07 g / l). The mixture is kept at 30 ° C for forty eight hours. After this detoxification period the flour is separated from the reaction mixture by filtration, washed successively with water and dried in a vacuum oven.

Example 10

For detoxification of rye flour (Secale spp.) 1g of chitosan obtained from the cell walls of Agaricus bisporus was initially dissolved with a deacetylation degree of 78% and a molecular weight of 60-120 kDa in a 1% acetic acid solution. %. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6.0 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (6.14 g / l). The mixture is kept at 40 ° C for forty eight hours. After this detoxification period the flour is separated from the mixture. reaction mixture, washed successively with water and dried in a vacuum oven.

Example 11

 For detoxification of triticale flour (x Triticosecale) 1g of chitosan obtained from the cell walls of Agaricus bisporus was initially dissolved with a deacetylation degree of 78% and a molecular weight of 60-120 kDa in a 1% acetic acid solution. . After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 5.0 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (3.07 g / l). The mixture is kept at 30 ° C for forty eight hours. After this detoxification period the flour is separated from the reaction mixture by filtration, washed successively with water and dried in a vacuum oven.

Example 12

For detoxification of oatmeal (Avena spp.) Initially 1g of chitosan obtained from the crab exoskeleton with a 98% deacetylation degree and a molecular weight of 220-300 kDa was dissolved in a 1% acetic acid solution. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6.5 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (3.07 g / l). The mixture is kept at 35 ° C for 24 hours. After this detoxification period the flour is separated from the reaction mixture. by filtration, washed successively with water and dried in a vacuum oven.

Example 13

 For detoxification of malt produced from barley, 1g of chitosan obtained from the cell walls of Agaricus bisporus was initially dissolved with a 78% deacetylation degree and a molecular weight of 60-120 kDa in a 1% acetic acid solution. After stirring and complete dissolution of the chitosan, the pH of the solution is adjusted and buffered to pH 6.0 with a 0.5 mol / L sodium phosphate buffer solution and the volume adjusted to 50 mL. The flour (256.8 g / l) is dispersed in the above solution by stirring, followed by the addition of glutathione (3.07 g / l). The mixture is kept at 30 ° C for forty eight hours. After this detoxification period the flour is separated from the reaction mixture by filtration, washed successively with water and dried in a vacuum oven.

The present description is, of course, by no means restricted to the embodiments set forth herein and a person of ordinary skill in the art may foresee many possibilities for modification thereof without departing from the general idea as defined in the claims. The preferred embodiments described above are obviously combinable with each other. The following claims further define preferred embodiments.

Bibliography Antunes H, et al. (2006) First determination of celiac disease prevalence in a Portuguese population. Minutes Port. 19: 115-120.

van Heel DA & West J (2006) Recent advances in coeliac disease. Gut 55 (7): 1037-1046.

Pengiran Tengah DSNA, AJ Wills, & Holmes GKT (2002) Neurological complications of celiac disease. Postgraduate Medical Journal 78 (921): 393-398.

Collin P, Kaukinen K, Vanlimki M, & Salmi J (2002) Endocrinological Disorders and Celiac Disease. Endocrine Reviews 23 (4): 464-483.

Kingham JG & Parker DR (1998) The association between primary biliary cirrhosis and celiac disease: a study of relative prevalences. Gut 42 (1): 120-122.

Kupper C (2005) Dietary guidelines and implementation for celiac disease. Gastroenterology 128 (4 Suppl 1): S121-127.

Ribeiro M, Poet P, & Churches G (2014) The Genetic Variability of Wheat Can Ensure Safe Products for Celiac Disease Patients? International Journal of Celiac Disease 2 (1): 24-26.

FAOSTAT (2015) (http://faostat.fao.org/).

Payne PI, Holt LM, Jackson EA, Law CN, & Damania AB (1984) Wheat storage proteins: their genetics and their potential for manipulation by plant breeding. Phil Trans R. Soc. B 304 (1120): 359-371.

MacRitchie F (1992) Physicochemical properties of wheat proteins in relation to functionality. Adv. Food Nutr. Res. , ed John EK (Academic Press), Vol 36, pp 1- 87.

Di Sabatino A, et al. (2012) The function of tissue transglutaminase in celiac disease. Autoimmune Rev. 11 (10): 746-753. Schuppan D, Junker Y, & Barisani D (2009) Celiac Disease: From Pathogenesis to Novel Therapies. Gastroenterology 137 (6) .1912-1933.

Arentz-Hansen EH, McAdam SN, Molberg O, Kristiansen C, & Sollid LM (2000) Production of a panel of recombinant gliadins for the characterization of T cell reactivity in celiac disease. Gut 46 (1): 46-51. Arentz-Hansen H, et al. (2000) The Intestinal T Cell Response to -Gliadin in Adult Celiac Disease is Focused on a Single Deamidated Glutamine Targeted by Tissue Transglutaminase. J. Exp. Med. 191 (4): 603-612. Arentz-Hansen H, et al. (2002) Celiac lesion T cells recognize epitopes that cluster in regions of gliadin rich in proline residues. Gastroenterology 123 (3): 803-809.

Anderson RP, Degano P, AJ Godkin, Jewell DP, & AVS Hill (2000) In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med 6 (3): 337-342.

Maiuri L, et al. (2003) Association between innate response to gliadin and activation of pathogenic T cells in celiac disease. Lancet 362 (9377): 30-37.

Camarca A, et al. (2009) Intestinal T cell responses to gluten peptides are largely heterogeneous: implications for a peptide-based therapy in celiac disease. J Immunol 182 (7): 4158-4166.

from Ritis G, et al. (1988) In vitro (organ culture) studies of the toxicity of specific A-gliadin peptides in celiac disease. Gastroenterology 94 (1): 41-49.

Marsh MN (1992) Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity ('celiac sprue'). Gastroenterology 102 (1): 330-354.

Wieser H (1996) Relationship between gliadin structure and coeliac toxicity. Acta Paediatr 85 (s412): 3-9.

Maruyama N, et al. (1998) Identification of major wheat allergens by means of the Escherichia coli expression system. European journal of biochemistry / FEBS 255 (3): 739-745.

Shan L, et al. (2005) Identification and analysis of multivalent proteolytically resistant peptides from gluten: implications for celiac sprue. J. Proteome Res. 4 (5): 1732-1741.

Pyle GG, et al. (2005) Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue. Clin Gastroenterol Hepatol 3 (7): 687-694.

Bethune MT & Khosla C (2012) Oral enzyme therapy for celiac sprue. Methods in enzymology 502: 241-271.

Shan L, Bethune M, Khosla C, & Gass J (2013).

Kumar P (2013).

Hausch F, Gray G, Shan L, & Khosla C (2007).

Shan L, et al. (2008).

Marti T, Oyvind M, & Sollid LM (2009).

Shan L, Marti T, Sollid LM, Gray GM, & Khosla C (2004) Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue. Biochem J 383 (Pt 2): 311-318.

Stepniak D, et al. (2006) Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease. American journal of physiology. Gastrointestinal and liver physiology 291 (4): G621-629. Gass J, Vora H, Hofmann AF, Gray GM, & Khosla C (2007) Enhancement of dietary protein digestion by conjugated bile acids. Gastroenterology 133 (1): 16-23.

Pinier M, et al. (2009) Polymeric binders suppress gliadin-induced toxicity in the intestinal epithelium. Gastroenterology 136 (1): 288-298.

Kelly CP, et al. (2009) Safety, Tolerability and Effects On Intestinal Permeability of Larazotide Acetate in Celiac Disease: Results of a Phase IIB 6- Week Gluten-Challenge Clinical Trial. Gastroenterology 136 (5): A-474.

Siegel M & Khosla C (2007) Transglutaminase 2 inhibitors and their therapeutic role in disease states. Pharmacology & therapeutics 115 (2): 232-245. Siegel M, Xia J, & Khosla C (2007) Structure-based design of alpha-starch aldehyde containing gluten peptide analogues to modulators of HLA-DQ2 and transglutaminase 2. Bioorganic & medicinal chemistry 15 (18) .6253-6261.

Xia J, Siegel M, Bergseng E, Sollid LM, & Khosla C (2006) Inhibition of HLA-DQ2-mediated antigen presentation by analogues of a high affinity 33-residue peptide from alpha2-gliadin. Journal of the American Chemical Society 128 (6): 1859-1867.

Ozaki S, et al. (2010) Potent transglutaminase inhibitors, aryl beta-aminoethyl ketones. Bioorganic & medicinal chemistry letters 20 (3): 1141-1144.

Ozaki S, et al. (2011) Potent transglutaminase inhibitors, dithio beta-aminoethyl ketones. Bioorganic & medicinal chemistry letters 21 (1): 377-379.

Khosla C & Choi K (2007).

Keech CL, Dromey J, Chen Z, Anderson RP, & McCluskey J (2009) Immune Tolerance Induced By Peptide Immunotherapy in An HLA Dq2-Dependent Mouse Model of Gluten Immunity. Gastroenterology 136 (5): A-57.

Anderson RP, Stewart JA, Dromey JA, & Tye-Din JA (2011).

Anderson RP, Hill AVS, & Jewell DP (2006).

Zhao J, et al. (2007) R-spondinl, novel novel intestinotrophic mitogen, ameliorates experimental colitis in mice. Gastroenterology 132 (4): 1331-1343. Tang F, et al. (2009) Cytosolic PLA2 is required for CTL-mediated immunopathology of celiac disease via NKG2D and IL-15. J Exp Med 206 (3): 707-719.

Kjellev S, et al. (2007) Inhibition of NKG2D receptor function by antibody therapy attenuates transfer-induced colitis in SCID mice. European journal of immunology 37 (5): 1397-1406.

Bridon DP, et al. (2006).

Drucker DJ (1998).

Fox BS (2007).

Tye-Din JA, et al. (2010) Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease. Know . Transi Med. 2 (41): 41ra51.

Girsh LS (2001).

Gil-Humans J, Piston F, Tollefsen S, Sollid LM, & Clay F (2010) Effective shutdown in the expression of celiac disease-related wheat gliadin T-cell epitopes by RNA interference. Proceedings of the National Academy of Sciences of the United States of America 107 (39): 17023-17028.

Gil-Humanes J, Piston F, Gimenez MJ, Martin A, & Barro F (2012) The introgression of RNAi silencing of gamma gliadins in commercial lines of bread changes in the mixing and technological properties of the dough. PloS one 7 (9): e45937. 55. Laursen L (2016) Will Europe toast GM wheat for gluten sufferers? Nature biotechnology 34 (4): 369-371.

 56. Watanabe M, Suzuki T, Ikezawa Z, & Arai S (1994) Controlled Enzymatic Treatment of Wheat Proteins for Production of Hypoallergenic Flour. Biosci Biotech Bioch. 58 (2): 388-390.

 57. Sjoestroem H & Noren 0 (1999).

 58. Johansen NL, Zundel M, & Dorwald FZ (2007).

 59. Johansen NL, Zundel M, & Dorwald FZ (2009).

 60. Costes C & Frouin A (1991).

 61. Yamazaki K & Soeda T (2002).

 62. Kuraishi C, Sato K, & Tanaka H (1999).

 63. Schuhmann F (2002).

 64. Mallee L, Franciscus M, & Bronts M (2001).

 65. Boumans L & Wijngaards G (2001).

 66. Yajima M & Katahira R (1995).

 67. Gang D (2005).

 68. Piedmont W (2004).

 69. Yamazaki K & Nishimura Υ (2002).

 70. Gianfrani C, et al. (2007) Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease. Gastroenterology 133 (3): 780-789.

 71. Rossi M, Gianfrani C, & Sicilian RA (2008).

 72. Green PHR & Cellier C (2007) Celiac Disease. New England Journal of Medicine 357 (17): 1731-1743.

 73. Mazzarella G, et al. (2012) Reintroduction of gluten following flour transamidation in adult celiac patients: a randomized, controlled clinical study. Clin. Dev. Immunol. 2012: 329150.

74. Mazzeo MF, et al. (2013) Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour. Biochim. Biophys. Minutes 1830 (11): 5166-5174.

 75. Lombardi E, et al. (2013) Selective inhibition of gliadin-specific, cell-mediated immune response by transamidation with microbial transglutaminase. J. Leukoc. Biol. 93 (4): 479-488.

 76. Elli L, et al. (2012) Immunological effects of transglutaminase-treated gluten in coeliac disease. Hmm. Immunol. 73 (10): 992-997.

 77. Ribeiro M, et al. (2015) Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties. Scientific reports 5: 18041.

 78. Gessendorfer B, Koehler P, & Wieser H (2009) Preparation and characterization of enzymatically hydrolyzed prolamins from wheat, rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten. Anal. Bioanal. Chem. 395 (6): 1721-1728.

 79. Kasarda DD, Woodard KM, & Adalsteins AE (1998) Resolution of High Molecular Weight Glutenin Subunits by a New SDS-PAGE System Incorporating a Neutral pH Buffer. Cereal Chemistry Journal 75 (1): 70-71.

 80. van de Wal Y, et al. (1998) Cutting Edge: Selective Deamidation by Tissue Transglutaminase Strongly Enhances Gliadin-Specific T Celi Reactivity. The Journal of Immunology 161 (4): 1585-1588.

 81. Kieffer R, Wieser H, Henderson MH, & Graveland A (1998) Correlations of the Breadmaking Performance of Wheat Flour with Rheological Measurements on a Microscale. J. Cereal I know. 27 (1): 53-60.

82. AACC (Final approval November 8, 1995; Reapproval November 3, 1999.) Approved Methods of Analysis, 11th Ed Method 54-40.02. Mixograph Method. (St. Paul, MN U.S.A.).

 AACC (1983) AACCI Method 10-10.03. Optimized Straight Dough Bread-Making Method.

Claims

1. A method for efficiently detoxifying cereal products, germinated products derived therefrom, and protein fractions derived therefrom, for celiac patients through the supramolecular association between potentially toxic proteins and amino polysaccharides.

A method according to claim 1 wherein the cereal product is wheat flour (Triticum spp.).

A method according to claim 1 wherein the cereal product is barley flour (Hordeum spp.).

A method according to claim 1 wherein the cereal product is rye flour (Secale spp.).

A method according to claim 1 wherein the cereal product is triticale flour (x Triticosecale).

A method according to claim 1 wherein the cereal product is the wheat germinated product (Triticum spp.).

A method according to claim 1 wherein the cereal product is the germinated barley product (Hordeum spp.).

A method according to claim 1 wherein the cereal product is the germinated rye product (Secale spp.).

A method according to claim 1 wherein the cereal product is the germinated triticale (x Triticosecale) product.

A method according to claim 1 wherein the cereal product is gluten isolated from wheat (Triticum spp.).

Cereal flour, germinated cereal and gluten products which according to the preceding claims have a lower concentration of toxic epitopes for celiac patients as compared to unmodified or original flour, germinated products and gluten.

Process for obtaining cereal flour, germinated products and gluten as described in claims 1-11, characterized in that it comprises the following steps:

 - dispersion of amino polysaccharides in acetic acid (0.05 mol / L) in a protein: polysaccharide ratio which may range from 0.5: 1 to 10: 1 (w / w), preferably 1.9: 1 followed by pH adjustment between 4 to 9 using a buffer system, preferably 6;

- Dispersing cereal flour, germinated products or gluten in the buffered solution containing the amino polysaccharides in a protein: polysaccharide ratio ranging from 0.5: 1 to 10: 1 (g / g), preferably 1.9: 1; adding a reducing agent at a final concentration of 0.1 to 500 mM, preferably 20 mM;

 incubating the mixture at a temperature between 4 to 80 ° C, preferably 40 ° C;

 - incubation lasting 30 minutes to 72 hours, preferably 24 hours;

 - separating the flour from the solution by centrifugation or filtration with water washes;

 - flour recovery and freeze drying, oven drying, vacuum oven drying, atomization.

A process according to the preceding claim, characterized in that the detoxifying amino polysaccharide is chitosan with a degree of polymerization between 2 monosaccharide units and up to one million units of monosaccharides and degree of acetylation of the amino groups between 0% and 60%.

Process according to Claim 13, characterized in that the chitosan originates from the crustacean exoskeleton or fungal cell walls.

Cereal and gluten flours according to the preceding claims, characterized in that they are viscoelastically superior to unmodified or original products.

Cereal and gluten flours according to the preceding claims, characterized in that they are sensory products similar to those of classical products.

A process according to any preceding claim wherein a reducing agent is used prior to modification of cereal flour, germinated products and gluten.

Process according to Claims 12-14, characterized in that the reducing agent may be glutathione, dithioerythritol (DTE), dithiothitrol (DTT) and tris 2-carboxyethyl phosphine (TCEP).

Food or dietary product prepared with cereal flour, germinated cereal products and gluten detoxified by the amino polysaccharides according to the preceding claims.