FIELD OF THE INVENTION

The invention relates to a method for reduc ^¬ ing the content of gluten proteins in a cereal starch fraction, to a cereal starch fraction obtained by the method, and to the uses thereof. BACKGROUND OF THE INVENTION

Proline is an amino acid frequently occurring in the sequence of cereal storage proteins. Proline makes a polypeptide chain to turn in a tight helical structure packing it effectively in the storage pro- tein bodies of the seed. Prolamins are gluten proteins rich in proline, and in addition to their high proline content they are also rich in glutamine. Prolamins are characterized by their water-insolubility, but instead they are typically soluble in aqueous alcohol. The prolamins of three cereals, i.e. wheat, rye and bar ^¬ ley, which are also called Triticeae cereals, are con ^¬ sidered to contain sequences harmful to those having gluten intolerance or celiac disease.

Celiac disease, also referred to as gluten enteropathy or non-tropical sprue, is an autoimmune disorder of the small intestine that occurs in genet ^¬ ically predisposed people of all ages. The character ^¬ istic symptom of this disease is the damage of the ep ^¬ ithelium of small bowel and intolerance to wheat glu- ten. The products containing wheat proteins as well as barley and rye should be eliminated from the diet of people suffering from this disease.

The triggering peptide causing celiac disease still remains somewhat unclear. However, it has been known that the toxic sequence is present in prolamins of some cereals, and that these proteins, which are generally called as gluten proteins, are characterised by a high content of proline amino acid.

By degrading the harmful prolamins it is pos ^¬ sible to decrease or even eliminate their toxicity and to produce products or ingredients, which can safely be used by persons being gluten intolerants. Prior art recognizes the use of enzymes for the hydrolysis or degradation of prolamins or gluten proteins. However, the use of enzymes has the drawback of being expensive to use in this kind of process.

The inventors have recognized a need for an effective method for reducing the content of gluten proteins in a cereal starch fraction in a non- enzymatic manner.

PURPOSE OF THE INVENTION

The purpose of the invention is to provide a new type of method for reducing the content of gluten proteins in a cereal starch fraction, and to provide a cereal starch fraction with reduced gluten content for different applications.

SUMMARY

The method according to the present invention is characterized by what is presented in claim 1.

The cereal starch fraction according to the present invention is characterized by what is present ^¬ ed in claim 10.

The food ingredient according to the present invention is characterized by what is presented in claim 11.

The food product according to the present in ^¬ vention is characterized by what is presented in claim The use according to the present invention is characterized by what is presented in claim 13.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing, which is included to provide a further understanding of the invention and constitutes a part of this specification, illus ^¬ trates one embodiment of the invention and together with the description helps to explain the principles of the invention. In the drawing:

Fig. 1 shows pasting properties of barley starch oxidized by Cu ²⁺ /H ₂ 0 ₂ at pH 4.5 and 7.4 as ana ^¬ lyzed by rapid visco analyser (RVA) . The control was native barley starch.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for reducing the content of gluten proteins in a cereal fraction, wherein gluten protein comprises proline, and wherein the method comprises the step of: a) sub ^¬ jecting the cereal fraction to an oxidative treatment for degrading the gluten proteins into peptides.

The present invention relates to a method for reducing the content of gluten proteins in a cereal starch fraction, wherein gluten protein comprises proline, and wherein the method comprises the step of: a) subjecting the cereal starch fraction to an oxidative treatment for degrading the gluten proteins into pep ^¬ tides .

The expression "gluten proteins" or any corresponding expression should be understood in this specification, unless otherwise stated, as proteins found in cereal that are considered to be harmful to a person with celiac disease and/or an intolerance to gluten. In one embodiment of the present invention the gluten proteins are cereal prolamins and/or glutelins found in the Triticeae cereals, i.e. in wheat, rye and barley. The prolamins of these cereals in particular are considered to contain protein sequences harmful to those having gluten intolerance or celiac disease. These prolamins include gliadin, which can be found in wheat, hordein which can be found in barley, and se- calin, which can be found in rye.

The expression "cereal fraction" or any corresponding expression should be understood in this specification, unless otherwise stated, as any suita ^¬ ble fraction of cereal that can be used in the method according to the present invention.

In one embodiment of the present invention step a) is carried out in an aqueous suspension.

The cereal fraction used in the present in ^¬ vention is a cereal starch fraction. In one embodiment of the present invention the cereal starch frac ^¬ tion comprises wheat, rye, barley, oat, or any combi ^¬ nation thereof. In one embodiment of the present in- vention the cereal starch is selected from a group consisting of wheat starch, rye starch, barley starch, and any combination thereof. In one embodiment of the present invention the cereal starch fraction comprises wheat, rye, barley, or any combination thereof.

In one embodiment of the present invention step a) of subjecting the cereal starch fraction to an oxidative treatment comprises subjecting the cereal starch fraction to at least one oxidizing agent. In one embodiment of the present invention step a) of subjecting the cereal starch fraction to an oxidative treatment comprises subjecting the cereal starch frac ^¬ tion to the presence of at least one oxidizing agent. In one embodiment of the present invention the at least one oxidizing agent is selected from a group consisting of hydrogen peroxide, ascorbic acid, tita ^¬ nium oxide, and any combination thereof. In one embodiment of the present invention step a) of subjecting the cereal starch fraction to an oxidative treatment comprises subjecting the cereal starch fraction to heat, shear forces, homogenisation, sonication, or to any combination thereof. Such treatments can also be used to control structural proper ^¬ ties, e.g. viscosity and sliminess of polysaccharides, of the cereal starch fraction while at the same time promoting gelling properties of polysaccharide frag- ments.

In one embodiment of the present invention step a) of subjecting the cereal starch fraction to an oxidative treatment comprises subjecting the cereal starch fraction to heat, shear forces, homogenisation, sonication, or to any combination thereof before, simultaneously and/or after subjecting the cereal starch fraction to at least one oxidizing agent.

The inventors surprisingly found out that the content of gluten proteins could be reduced in a cere- al starch fraction by using an oxidizing treatment. Without limiting the invention to any specific theory about why the method of the present invention results in this advantage, it is to be considered that the ox ^¬ idizing treatment affects proline present in the amino acid sequence of gluten proteins. The bond between proline and an adjacent amino acid in the amino acid sequence is cleaved resulting in the gluten protein being degraded. The degradation of the gluten proteins results in smaller peptides being formed. The formed peptides are found to be bioactive and potentially health promoting and/or flavouring agents, and are not harmful to persons suffering from celiac disease or to individuals being gluten intolerants. The advantage of the method according to the present invention is that the cereal starch fraction can be treated such that it can be consumed by persons suffering from celiac dis ^¬ ease or being gluten intolerants. The degradation of gluten proteins can result in the release of bioactive peptides that can have health promoting effects such as controlling blood pressure. The method according to the present invention can be applied for modifying ce- real gluten proteins, also called as cereal prolamin proteins, like wheat gliadin and barley hordein.

In one embodiment of the present invention the molar mass of the resulting peptide is less than 4000 Da, preferably less than 2000 Da, and more pref- erably less than 1000 Da.

In one embodiment of the present invention step a) comprises subjecting the cereal starch frac ^¬ tion to an oxidative treatment in the presence of at least one catalyzing agent, wherein the catalyzing agent comprises metal and/or transition metal. In one embodiment of the present invention the catalyzing agent comprises copper and/or iron. In one embodiment of the present invention the catalyzing agent compris ^¬ es Cu(II) and/or Fe(II).

When using at least one catalyzing agent in step a) together with at least one oxidizing agent, e.g. hydrogen peroxide, a so-called Fenton-type metal catalyzed oxidation reaction can take place, e.g. in the following manner:

Cu ⁺ /Fe ²⁺ + H ₂ 0 ₂ → ·ΟΗ + OH ^" + Cu ²⁺ /Fe ³⁺

AH ₂ + 2Cu ²⁺ /Fe ³⁺ → A + 2H ⁺ + 2Cu ⁺ /Fe ²⁺

AH ₂ + 0 ₂ → A + H ₂ 0 ₂ The use of the at least one catalysing agent will speed up the reactions taking place in step a) .

In one embodiment of the present invention the method comprises adjusting the pH in step a) to a value of 3 - 7, preferably 4 - 5, and more preferably to about 4,5. In one embodiment of the present inven ^¬ tion the method comprises adjusting the pH in step a) for modifying the properties of the cereal starch fraction. The pH can be adjusted in step a) such that e.g. cereal starch being treated, is simultaneously gelatinized. The degradation or cleavage of gluten proteins or prolamins was found to be efficient at an acidic pH, and especially at a pH of 3 - 6. Further, it was surprisingly found out that when using acidic pH conditions in step a) , the oxidative treatment has no effect on cereal starch gelatinization . When using a neutral pH, the oxidation step affects the gelatini- zation properties of cereal starch modifying its past ^¬ ing properties. By the use of the method according to the present invention it is possible to modify the properties of cereal starch simultaneously with de ^¬ grading the gluten proteins present therein. Without limiting the invention to any specific theory about why the method of the present invention results in the aforementioned advantage, it is to be considered that the acidic pH affects the radical attack, resulting from the oxidative treatment, on the cereal starch fraction.

In one embodiment of the present invention the peptides formed in step a) are flavor enhancing and/or bioactive.

In one embodiment of the present invention step a) is performed at a temperature of 5 - 50 °C .

In one embodiment of the present invention step a) is allowed to continue until the content of gluten proteins in the cereal starch fraction is at most 100 ppm, preferably at most 20 ppm. In one embod- iment of the present invention step a) is allowed to continue for up to 50 hours. In one embodiment of the present invention step a) is allowed to continue for 1 - 50 hours, preferably 1 - 4 hours.

In one embodiment of the present invention step a) is stopped by adding at least one chelating agent. In one embodiment of the present invention step a) is stopped by adding ethylenediaminetetraacetic ac ^¬ id (EDTA) .

The inventors surprisingly found out that by the method according to the present invention it is possible to reduce the gluten proteins content of a cereal composition. Especially it was found out that the content of gluten proteins in cereal starch could be reduced by the oxidative treatment according to the present invention such that the cereal starch is suit- able to be used by persons suffering from celiac dis ^¬ ease or being gluten intolerants. It was surprisingly found out that by subjecting a cereal starch fraction, such as cereal starch fraction, to e.g. an oxidizing agent in accordance with the present invention the gluten proteins present therein were degraded while maintaining the desired properties of the cereal starch fraction. Degradation of the gluten proteins results in the toxic effects of gluten being removed from the cereal starch fraction. It was found out that the method according to the present invention affected the properties of the treated cereal starch fraction in a desired manner.

The present invention further relates to a cereal starch fraction obtainable by the method ac- cording to the present invention.

The present invention further relates to a food ingredient comprising the cereal starch fraction according to the present invention.

The present invention further relates to a food product produced using the cereal starch fraction according to the present invention.

The present invention further relates to the use of the method according to the present invention for providing a gluten-free cereal starch fraction.

The present invention further relates to the use of the method according to the present invention for providing flavour enhancing and/or bioactive peptides .

The method according to the present invention can be used for removing gluten proteins from a cereal starch fraction. The removal of the gluten proteins can be carried out by degrading the gluten proteins into peptides. Thus, the method according to the pre ^¬ sent invention can result in gluten proteins being degraded into peptides which can be flavour enhancing and/or bioactive. I.e. simultaneously with reducing the gluten content level of the cereal starch fraction beneficial, e.g. bioactive, peptides are being formed.

The method according to the present invention can be used in order to produce food that meets the requirements for gluten-free standards. The cereal starch fraction obtained by the present invention can be consumed by persons having gluten sensitivity.

Fibre products, baked products, and beer can be mentioned as examples of such food products.

The embodiments of the invention described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined to ^¬ gether to form a further embodiment of the invention. A method, a cereal composition, a food ingredient, a food or a use, to which the invention is related, may comprise at least one of the embodiments of the inven ^¬ tion described hereinbefore.

An advantage of the method according to the present invention is that gluten proteins in a cereal starch fraction, i.e. plant storage proteins, can be degraded such that the cereal starch fraction treated is suitable to be used by e.g. a person suffering from celiac disease. An advantage of the obtained cereal starch fraction is that it is tolerated by a person being gluten intolerant or gluten sensitive.

An advantage of the present invention is that it provides an effective, low cost, and gentle method to be used for degrading gluten proteins in a cereal starch fraction without any need for e.g. thermal in- activation as the reaction can end when the compounds, e.g. oxidizing agent and catalyzing agent, for the re- action are consumed.

An advantage of the present invention is that it is possible to produce e.g. cereal starch, having reduced or eliminated content of gluten proteins.

An advantage of the present invention is that when gluten proteins present in a cereal starch frac ^¬ tion are degraded by the method according to the pre ^¬ sent invention small peptides that can act as flavor precursors or health beneficial bioactive peptides are produced .

The present invention has an advantage of providing a new method for the production of gluten- free foods .

EXAMPLES

Reference will now be made in detail to the embodiments of the present invention, an example of which is illustrated in the accompanying drawings.

The description below discloses some embodi ^¬ ments of the invention in such a detail that a person skilled in the art is able to utilize the invention based on the disclosure. Not all steps of the embodi ^¬ ments are discussed in detail, as many of the steps will be obvious for the person skilled in the art based on this specification.

EXAMPLE 1 - Oxidation of a wheat starch fraction with hydrogen peroxide catalyzed by copper

In this example 2 mg/ml of wheat starch was incubated in the presence of 0.05 mM CuS0 ₄ and 5 mM hydrogen peroxide at pH 4.5. The incubation tempera ^¬ ture was 37 ^° C. The reaction was stopped by 1 mM EDTA. The gliadin content of the wheat starch frac ^¬ tion was determined by using a SDS-PAGE gel. From the results it could be seen that the treatment caused breakdown of gliadin present in the wheat starch frac- tion. With a longer incubation time the degradation of gliadin went further. The gliadin was almost totally degraded after eight hours of incubation, and totally degraded after 24 hours of incubation. EXAMPLE 2 - Oxidation of a barley starch fraction by a copper catalyzed hydrogen peroxide induced reaction

In this example 3 g of barley starch was in ^¬ cubated in 25 ml of 0.1 M phosphate buffer with the presence of 0.05 mM CuS0 ₄ and 5 mM hydrogen peroxide at pH values of 4.5 and 7.4. The incubation tempera ^¬ ture was 37 ^° C. After 4 hours the reaction was stopped by adding catalase.

The pasting property of the oxidized barley starch was analyzed by Rapid Visco Analyser (Newport Scientific RVA-4) . The reaction mixture was stirred and heated, causing the barley starch to gelatinize and to form a paste.

Oxidation of the barley starch at pH 7.4 re- suited in a decrease of its final viscosity, while at pH 4.5 the final viscosity was comparable to native starch. The results are presented in Fig. 1.

The treatment caused breakdown of residual gluten proteins present in the barley starch. The deg- radation of the gluten proteins, or prolamins, of the treated barley starch was confirmed by Western blot analysis. The analysis of the barley starch with the R5 ELISA test indicated that the content of gluten proteins in the treated barley starch was below 3 ppm. In the example above the oxidative treatment is carried out by using an oxidizing agent, but the oxidation treatment could also had been initiated or boosted by other means such as by using e.g. homogeni- zation, heating or sonication, as will be obvious to a skilled person in light of this specification. Thus, the invention is not limited to using the aforementioned oxidative treatment in particular and the ad ^¬ vantages of the invention can be readily obtained by the skilled person in light of this specification also with the oxidative treatments mentioned above.

It is obvious to a person skilled in the art that with the advancement of technology, the basic idea of the invention may be implemented in various ways. The invention and its embodiments are thus not limited to the examples described above; instead they may vary within the scope of the claims.