in the present invention , a method that provides for the detoxification of gluten proteins , in particular from wheat grain , is described . the method for making said flour detoxified by toxic epitopes of gluten comprises the following phases : a ) hydration of the grains , in particular that of wheat , with mains water for about 1 hour ; for 100 grams of mature grains 500 ml of water is added . this phase is necessary to trigger the chemical reactions functional to the detoxification of gluten proteins . b ) removal of the water . this phase is carried out by draining with a sieve to retain the grains and remove the water . c ) microwave treatment of hydrated grains for two minutes using a power of 1000 watt . at this stage what is important , is not the temperature within the oven , but the power of the electromagnetic waves that trigger through the water contained in the grains , the reactions of detoxification . d ) cooling of the cereal grains treated with microwaves at room temperature ( 20 °- 22 ° c .). all the phases described above are mandatory : the hydration of the grains for an hour allowing the seed to accumulate the amount of water required to trigger , in the presence of electromagnetic waves , the detoxification reactions of gluten . the power of 1000 watt , applied according to the previously described procedure , is sufficient for two minutes in order that the energy accumulated by the water favours the production of singlet oxygen radicals , hydroxyl radical or hydrogen by the cellular metabolism ( peroxidases , lipoxygenase , etc .). these highly reactive compounds inside the seed of wheat involve polymerization reactions of gluten proteins localized in sectors different from the caryopsis ( protein bodies present in the aleurone layer and protein bodies present in the endosperm ) by intermolecular bonds and / or intramolecular bonds with conformational change . the slowly cooling to a temperature of about 20 ° c . allows the completion of the chemical reactions triggered by the action of the electromagnetic waves and the water . the method illustrated in the present invention 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 . in particular , albumins and globulins are not incorporated in the polymers of high molecular weight but coagulate and interact with gliadins forming an aggregate of molecular weight intermediate to that of gliadins and albumins and globulins revealed as a new peak called “ intermediate protein ” ( ip ) peak . the participation of ω - gliadins to these changes suggests that the interaction between the proteins takes place not only through the formation of disulfide bonds but also through the formation of covalent bonds involving tyrosine residues . 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 grains , hydrated and subjected to treatment with microwaves , generates water radicals that trigger the polymerization of gluten proteins . this polymerization appears to be favoured when gluten proteins are segregated in the protein bodies due to the fact that within these protein bodies are very close to each other allowing an easy interaction between the various classes depending on the type of body protein in which they are ; in the case of the vacuolar protein bodies ( aleuronic layer ) we can have interactions between gliadins , lmw and albumins / globulins , in the case of protein bodies of endoplasmic type ( endosperm layer ) we can only have interactions between hmw . the polymerization induced by the water and by the electromagnetic waves does not determine the loss of free sulfhydryl groups , necessary for the formation of gluten , resulting in a protein network although different in conformation ( fig5 a ) ( due to the interaction of the different aggregates mentioned above ) from the gluten network which is formed naturally ( fig5 b ), but which ensures the adequate technological properties of transformation of wheat flour in a final product . the polymerization of gluten proteins by water radicals favoured by electromagnetic waves produces the formation of covalent bonds between these gluten proteins . the formation of these bonds between gluten proteins inside the protein bodies allows a sort of masking of the toxic epitope , as shown in fig6 , such as to make the site of attack of gliadins not accessible and / or not recognizable at the intestinal level by the transglutaminase , which in celiac disease conditions induces the immune response . the structural changes of gluten proteins of detoxified flours are highlighted through the test that can determine the gluten index , evaluating the strength of the same . the gluten of detoxified flours , passing completely through the grid of the device , undergoes a leaching of the components of the grain , evidencing the presence of structural changes in the gluten protein . these structural changes of the gluten of detoxified flours are better analyzed in fig2 and 3 ; fig2 shows the change of the protein pattern of gliadins extracted from different samples of grains treated with the method of detoxification object of the present invention ( a and b — durum wheat semolina ), compared with other untreated samples ( c , d , e — durum wheat semolina , f , g , h — soft wheat flour ), the gliadins are gluten proteins which are soluble in alcohol that contain toxic epitopes recognized in the intestine . the decrease in the intensity of the electrophoresis bands indicates a qualitative and quantitative change of gliadins . quantitative because most of them , due to the processes of polymerization induced by the microwave treatment , have been rendered insoluble in an ethanol solution to 70 % and therefore not detectable on the electrophoresis gel . qualitative because the few visible bands are however aggregates of gliadins ( which undergo a polymerization by microwaves ) to the lowest molecular weight and therefore extractable in ethanol solution and detectable on the electrophoresis gel , but however non - toxic in accordance with the immunological findings performed in vitro ( fig4 ). fig3 a and 3 b , on the other side , show two graphs that indicate the protein profile of respectively soluble and insoluble proteins , extracted from detoxified grains ( the curves with shades of gray dark ) and untreated grians ( the curve with shades of lighter gray ) by se - hplc ; fig3 a indicates two main peaks , one on the right that represents the polymers of high molecular weight ( hmw and lmw ); the one on the left represents the oligomers and monomers of gliadin ; the graph shows the decrease of the solubility of gluten proteins after the detoxification treatment of the present invention . fig3 b indicates an increase of the two peaks in the detoxified flour , indicating an increase in insoluble proteins after the treatment of detoxification . these structural changes of gluten proteins would be likely to deprive gluten from its toxicity , thanks to a masking of the attack site of the transglutaminase ( fig6 ) and allowing the maintenance of the technical properties of the dough due to the maintenance of the free sulfhydryl groups , necessary for the formation of the gluten network ( fig5 a and 5 b ). regarding the gluten &# 39 ; s loss of toxicity present in treated flours , fig4 shows a graph of a test of immunological cells lymphocytes ( t cells ) of celiac patients ; in this chart the production of interferon - γ in lines of t lymphocytes taken from intestinal mucosa of celiac patients is represented ; the cellular lines have been proved to be highly responsive to gliadin from hexaploid wheat ( tryptic digest pepto - pt ) after deamination with transglutaminase tissue , while any immunological reactivity to gliadin extracted from flours a and b treated at concentrations of 50 and 100 mg / ml has been observed . lastly , regarding the maintenance of technological properties of the detoxified flour , we know that in the kneading with water , the flour absorbs water causing the binding of gliadins and glutenins and , therefore , the formation of gluten network that influence the formation of the viscoelastic mass for entraining gas . flour detoxified by this method retains the ability to kneading the dough as it does not loose the ability to form bonds between free cysteine disulfide groups ( fig5 a and 5 b ) necessary for the formation of the network ; the dough obtained with the detoxified flours maintains the characteristic of extensibility , which , as is known , is mainly due to gliadin , loosing partially elasticity and viscosity due to the formation of aggregates of protein bodies through covalent bonds . in particular , the loss of a part of elasticity is due to the interaction between the sub units of hmw , through covalent bonds that involve amino acids of the central domain of the protein subunit of high molecular weight , which is known , from studies in the literature , determining the elasticity of the dough . the loss of viscosity of the dough is instead due to the interaction of the gliadins between them , as they are responsible for this rheological characteristic . a first advantage of the method is that from those grains and flour it will be possible to produce non - toxic food for people with celiac desease , with organoleptic characteristics equivalent in taste and appearance to those commonly used in the mediterranean alimentation . the second advantage is an economic advantage , due to the raw material used , wheat ( italy is one of the largest producers of wheat in the world ), instead of corn and all structuring substances ( tires , agar , gelatine , etc .) which are expensive , but also for the use during the experimentation of only mains water and electromagnetic waves for a short time ; consequently , the gluten - free products will no longer be expensive as they are now . the third advantage is of health type , as the wheat flours are less starchy than those of corn ( used until know for the production of gluten - free products ) and therefore the resulting products are characterized by a more low glycemic index and therefore such products would be ideal for the feeding of patients who , in addition to celiac disease , also suffer from diabetes mellitus type 1 , an association frequently observed because of the likely common genetic substrate shared by the two diseases . the fourth advantage is the simplicity the procedure , easily applicable also to other grains including , for example , the barley to produce beer , free from toxic ordeine ( proteins similar to the gliadins of wheat ) or oats for make products for breakfast , also free from toxic substances ( proteins similar to gliadins of wheat ). the fifth advantage is the production of foods that determine , through their use in time and by large numbers of the population , not only people with celiac disease , a reduction in the incidence of celiac disease in the population due to the smaller immunogenic effect of the detoxified product the present invention has been described for illustrative purpose , without limitation , but it is to be understood that variations and / or modifications may be made by the experts in the art without departing from the scope of protection , as defined by enclosed claims .
the method allows to obtain gluten detoxified flours suitable for the preparation of bakery products and pasta made from wheat . with the use of the method gluten proteins undergo structural changes that do not activate in patients suffering from celiac disease , the cascade of inflammatory cytokines . such structural changes , in addition , do not affect the technical properties of the flours that form the dough , therefore allowing the preparation of detoxified products , similar in taste and appearance to those commonly used in mediterranean diet and which are intended not only for the people who suffer from intolerance to gluten , but for the whole population . the widespread use of such detoxified products in large part of the population has the purpose , in a totally innovative way , to provoke the reduction of gluten &# 39 ; s effects in people &# 39 ; s health and therefore to the decrease in the incidence of celiac disease .