Combination product suitable for celiac patients, comprising a gluten containing flour and an additive

Abstract

The present application for the first time discloses a combination product of a gluten containing flour and an additive, wherein the additive comprises ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, such as of approximately 2.2-3% of the flour weight, and Zinc at a concentration of 0.02-0.05%, such as of approximately 0.034% of the flour weight, characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten. The application further discloses a baking process for producing baked products comprising the herein described combination product and the baked products themselves, characterized in that the consumption of the baked products does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient.

Description

[0001] COMBINATION PRODUCT SUITABLE FOR CELIAC PATIENTS, COMPRISING A GLUTEN CONTAINING FLOUR AND AN ADDITIVE

[0002] Field of invention

[0003] The present invention relates to a combination product of a gluten containing flour and an additive, characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten in a gluten intolerant patient.

[0004] Background of the invention

[0005] Celiac disease

[0006] Celiac disease (a gluten intolerance) is an HLA-linked inflammatory autoimmune condition that primarily affects the small intestine. The classical symptoms are pain, diarrhea, and nutrient malabsorption. In genetically predisposed individuals, celiac disease may develop in response to a gluten-containing diet. Celiac disease develops in genetically predisposed individuals who possess specific proteins (HLADQ8 / DQ2) on immune cells that bind gluten-derived deamidated peptides in the gastrointestinal tract and then starts an autoimmune immune response.

[0007] About 98% of individuals diagnosed with celiac disease possess the combination, or either of, the specific genes, HLADQ8 and HLADQ2. About 20-30 % of the world population is genetically predisposed to celiac disease, however “only” 1-2% develop the disease, indicating that gluten exposure is not the only trigger. The HLADQ8 and HLADQ2 genes are coding for antigen presenting proteins (HLA antigens) on immune cells, and this particular type presents fragments of gluten-derived deamidated peptides to T-cells (CD4+), which triggers the proliferation of gluten-specific T helper (Thl )-cells, increasing the sensitivity to gluten-derived deamidated peptides and therefore become drivers of the celiac immune response.

[0008] The disease can develop in individuals with said particular genetic predisposition when they eat gluten proteins from wheat, rye or barley. In the intestinal wall, transglutaminase, is a key mediator in the development of celiac disease. When eating gluten, the enzyme forms so-called deamidated peptides that are responsible for activating the immune system in these individuals.

[0009] Initiation of celiac disease involves intestinal tissue transglutaminase 2 (TG2) In recent years, it has been uncovered that the auto-immune response to gluten involves transglutaminase 2 (TG2 or tTG), harbored in the intestinal wall. TG2’s function is to crosslink proteins / peptides in the presence of a primary amine. However, if a primary amine is unavailable, TG2 will use water instead to commence its enzymatic activity, thereby producing deamidated peptides. The deamidation reaction renders the peptides negatively charged. The deamidated peptides will be further broken down in the intestinal wall, however, this intermediary is relatively long-lived and therefore the tissue levels are relatively high. Unfortunately, the HLADQ8 / DQ2 proteins on antigen-presenting immune cells of celiac-predisposed individuals have a high affinity for the deamidated peptides and if the levels get high enough, an immune response is triggered.

[0010] The key for initiation of an (auto-) immune response towards gluten is the production of the deamidated peptides, which is catalyzed by an intestinal enzyme, TG2 (E.C. 2.3.2.13). TG2 binds specifically to a binding motive (gln-X-pro) on gluten peptides and catalyzes transamidation of the peptides in the presence of a primary amine, and deamidation of the peptides in the presence of water. The deamidation reaction produces negatively charged peptides, converting glutamine (gin) to glutamic acid (glu) residues. The activity of TG2 is regulated by the trace metal ions calcium (Ca2+) and Zinc (Zn2+), whereas Ca2+promotes, and Zn2+inhibits its activity. Therefore, the dietary levels of calcium and Zinc may also affect the intestinal level of deamidated peptides after a gluten-containing meal.

[0011] In Northern Europe, this causes problems since the intake of dairy (calcium-rich) products is relatively high and wheat, rye, and barley are staple foods. If the diet is also poor in Zinc, the development of celiac disease is further promoted. If celiac disease is already manifest, there are very small chances to cure the condition, since the immune system “remembers” the antigen (deamidated peptides) and autoantigen (TG2) by circulating antibodies for a certain length of time. The only option is to avoid stimulating the immune system.

[0012] Celiac disease is an auto-immune disease in which the immune system attacks TG2 by producing autoantibodies towards the enzyme. The immune system-assisted removal of TG2 (TG2 auto-antibodies) is associated with upregulation of TG2 in the small intestine in active celiac diseased ], further escalating the disease. Antibodies towards transglutaminases (TG) are thus sensitive diagnostic markers for celiac disease. There is also data suggesting that a diagnosis of celiac disease could likely be made on serology alone without confirmatory biopsy if the TG titre is high. That TG2 is key in celiac disease is evident since about 98% of incident celiac patients present with antibodies directed towards transglutaminases, which is therefore used as the primary diagnostic marker for incident celiac disease. On a strict gluten-free diet, the serum level of TG antibodies (Anti-tTG) decreases and after time is suppressed to a level of a non-celiac individual. This can be used to estimate compliance to a gluten-free diet in the long-term perspective.

[0013] The present inventors have prior identified the food additive ascorbyl palmitate (E304i), to be able to reduce transamidation of gliadin (by 80% at 15 pM). The degree of deamidation of gliadin was measured by an antibody towards deamidated gliadin (dGDP), which confirmed that there was no significant deamidation of gliadin in the presence of ascorbyl palmitate or ascorbyl palmitate with Zinc chloride.

[0014] Ascorbyl palmitate (E304i) is generally recognized as safe (GRAS) and is approved by the U.S. Food and Drug Administration (USFDA) as a food additive. It is frequently used as an antioxidant or as a fat-soluble form of vitamin C. The European Food safety authority (EFSA) has, in line with the American authorities, also concluded that there is no safety concern regarding E304i as a food additive. According to EFSA, ascorbyl palmitate is considered to have the same biological effects as ascorbic acid, and from human data it was concluded that supplementary doses of about 1 g / person per day in addition to normal dietary intakes are not associated with adverse gastrointestinal effects, but that gastrointestinal acute effects may occur at doses exceeding 3-4 g / day.

[0015] Zinc sulphate is the compound normally used for Zinc fortification of foods. The recommended daily intake is 11 mg / day for men and 8 mg / day for women in the U.S (13). However, the intestinal absorption of Zinc from the diet has been reported to be 15-60%. Dietary Zinc supplements come in daily doses in the range of 10-50 mg and a common supplemental dose is about 30 mg / day. An upper tolerable level for Zinc has been estimated to 40 mg / day for adults and 12 mg / day for children from 4-8 years in the U.S. Symptoms of mildly overdosing Zinc is mainly related to gastrointestinal discomfort while yet higher doses negatively affect body copper balance. Zinc salts such as Zinc chloride, Zinc acetate, and Zinc sulphate are approved within the E.U. as dietary Zinc supplements.

[0016] It is well known that even minor amounts of gluten residues are detrimental to celiac patients and can set into spin an escalating auto-immune reaction with severe consequences for the patient. Since the immune system “remembers” the antigen (deamidated peptides) and autoantigen (TG2) by circulating antibodies for a certain length of time. The only option is to avoid stimulating the immune system. Thus, when producing baked goods for consumption by celiac patients, the additive needs to be fully effective at the time point of digestion, i.e., after the baking process and the remaining and active amount of ascorbyl palmitate needs to be constant, so as to guarantee that no auto-immune response in which the immune system attacks TG2 by producing autoantibodies towards the enzyme is initiated at all.

[0017] Therefore, it is paramount to be able to provide a food product with a guaranteed effectivity that in each consumed product reliably, fully and completely inhibits and / or at least sufficiently reduces the gluten driven deamidation of gliadin in the gut of the celiac patient.

[0018] Summary of the present invention

[0019] The present invention for the first time provides gluten-intolerant individuals, such as celiac patients, with sustainable, cheap, wheat- and rye-based products of high nutritional quality compared to the alternatives that are common today (rice and corn-based consumer products) and thus greatly increases the quality of life for these individuals.

[0020] The approach taken herein is to prevent intestinal TG2 from binding to gluten peptides by adding the food additive E304i , which by the inventors has previously found to interact strongly with TG2 binding motives on gliadin. In this way, the catalysis of both transamidation and deamidation reactions are prevented. Therefore, either the transamidation or deamidation reactions could be acessd to prove if the binding of TG2 was hindered by E304L Although, it is the deamidation reaction that initiates the celiac immune response. In studies by Italian researchers, an approach based on the same starting point was used, in which they used a microbial transglutaminase to crosslink a primary amine to gliadin extracted from wheat flour, which was observed to block the production of the cytokine interferon y (IFN- y) in gluten-restricted T-cells (4.). In the same study, results suggested that transamidated gliadin still could bind DQ2 surface protein on antigen-presenting cells. Some years later, the research group conducted a human intervention trial with celiac patients on the concept and found that as many as 37% (on day 15) in the group challenged with transamidated flours experienced clinical relapse whilst the relapse in the control group challenged with non-deamidated flours was 75% (5.), raising the question if not transamidated gliadin peptides could be immunogenic as well, but to a much lesser extent than deamdiated intermediaries.

[0021] In the present study, the highest concentration of the TG2 binding motive blocker E304i was limited to 30 mg E304i / g flour to be suitable for human ingestion. At this level (30 mg / g flour), two bread rolls can be ingested daily, without exceeding the recommended daily allowance (RDA). Even higher levels have been reported in the literature and the RDA differs in the EU and in the US, suggesting that there seem to be no real toxicity risk. About 80% of E304i seems to be retained after heating and the breakdown products are unharmful (ascorbic acid and palmitic acid) as reported by EFSA.

[0022] The present application for the first time discloses a combination product of a gluten containing flour and an additive, wherein the additive comprises ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, such as of approximately 2.2-3% of the flour weight, and Zinc at a concentration of 0.02-0.05%, such as of approximately 0.034% of the flour weight, characterized in that the consumption of the combination product does not elicit an celiac immune response towards gluten in the human intestine of a celiac patient.

[0023] In one embodiment, a combination product of a gluten containing flour and an additive comprises flour which contains at the most 10-15 g protein / 100 g flour, such as at the most 10 g protein / 100 g flour. Typically, the flour is wheat flour and / or rye flour.

[0024] In one embodiment, a combination product of a gluten containing flour and an additive comprises ascorbyl palmitate (E304i) at a concentration of between 15-30 mg / g flour, such as between 22-30 mg / g flour, such as at the most 30 mg / g.

[0025] In one embodiment, a combination product of a gluten containing flour and an additive comprises Zinc at a concentration of 0.02-0.05%, such as of approximately 0.034% of the flour weight.

[0026] In one embodiment, a combination product of a gluten containing flour and an additive comprises Zinc sulphate with a molecular weight of 179.47 g / mol at a concentration of between 0.05-0.2% of the flour weight, such as of 0.1 -1.5% of the flour weight, such as of approximately 0.149% of the flour weight. In one embodiment, the current invention relates to a combination product of a gluten containing flour and an additive, wherein the additive prevents TG2 antibody production with at least 51 %±10% after consumption of the combination product in a celiac-intolerant subject.

[0027] In one embodiment, the current invention relates to a combination product of a gluten containing flour and an additive, wherein the additive fully prevents TG2 antibody production after consumption of the combination product in a celiac-intolerant subject.

[0028] Consumption of a combination product of a gluten containing flour and an additive according to the present invention does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient. This can e.g., be measured as a standardised immunological determination of one or more celiac specific biomarker(s) in a sample from a celiac patient, such as, as a standardized immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient. Typically, the consumption of the combination product is determined not to elicit a celiac immune response towards gluten in the human intestine of a celiac patient when the level of tTG is below 10 U / mL, measured as a standardized immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient. In one embodiment, the consumption of the combination product is determined not to elicit a celiac immune response towards gluten in the human intestine of a celiac patient when the level of tTG IgA is below 10 U / mL, such as below 9 U / mL, such as at the most 7 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient. In one embodiment, the consumption of the combination product is determined not to elicit a celiac immune response towards gluten in the human intestine of a celiac patient when the level of tTG IgG is below 10 U / mL, such as below 9 U / mL, such as at the most 7 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient.

[0029] The present invention further relates to a baked product comprising a combination product of a gluten containing flour and an additive according to any of the preceding claims, characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

[0030] A baked product according to the current invention can be a bakery product. A bakery product can be selected from the group consisting of bread, portion bread, roll, toast bread, baguette, hamburger bun, Danish pastry, French pastry, puff pastry, muffin and cake.

[0031] A baked product according to the current invention is intended for use in a daily serving of Zn, which does not exceed 1 .25 mg / kg of body massperday as well as for use in a daily serving of ascorbyl palmitate (E304i) of no more than 3-4 g / day. In embodiments, the baked product is intended for use in a daily serving of ascorbyl palmitate (E304i) of no more than 1 .6 g / day, or 2.4 g / day, dependent on the weight of the baked product.

[0032] The current invention in one aspect relates to a process for producing a baked product according to the current invention, the process comprising the steps of: a) mixing a. the additive comprising i) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc at a concentration of 0.02- 0.05% of the flour weight, b. water, c. a lipid and / or fat, d. yeast, and e. gluten-containing flour, b) proofing the dough at room temperature, and c) baking the product.

[0033] During a process for producing a baked product according to the present invention, the temperature inside the baked product during the baking step c) does not exceed 100°C. Typically, the heat used during a process for producing a baked product according to the present invention, in the baking step c) does not exceed 225°C, 220°C, 210°C, 200°C, 195°C or 190°C.

[0034] In a currently preferred process for producing a baked product according to the current invention, the heat in the baking step c) does not exceed 200°C, such as does not exceed 195°C.

[0035] In one embodiment, the process for producing a baked product according to the current invention comprises solving the additive in water, oil and water, in butter and water, or in milk before mixing it with the yeast and gluten containing flour in step a). In a process for producing a baked product according to the current invention, no further gluten is added throughout the baking process than the gluten comprised in the gluten- containing flour.

[0036] In a process for producing a baked product according to the current invention, no further gluten containing flour is added in the baking process than is mixed with the other ingredients in mixing step a) of the baking process.

[0037] A process for producing a baked product according to the current invention can be used to produce a baked product selected from the group consisting of bread, portion bread, roll, toast bread, baguette, hamburger bun, Danish pastry, French pastry, puff pastry, muffin and cake.

[0038] In one aspect, the current invention also relates to the use of a combination product of a gluten containing flour and an additive according to the current invention in the preparation of a baked product comprising a gluten containing flour, wherein the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient.

[0039] Also envisioned is a premix comprising a gluten-containing flour and an additive comprising i) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05% of the flour weight, as well as the use of said premix in the preparation of a baked product comprising a gluten containing flour, wherein the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient.

[0040] Further, the invention relates to a kit of parts comprising a gluten-containing flour and an additive comprising i) ascorbyl palmitate (E304i) at a concentration of between 1 .1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05% of the flour weight and the use of said kit of parts in the preparation of a baked product comprising a gluten- containing flour, wherein the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient.

[0041] The baked product can again be selected from the group consisting of bread, portion bread, roll, toast bread, baguette, hamburger bun, Danish pastry, French pastry, puff pastry, muffin and cake. Figure legends

[0042] Figure 1 : Overview of the study design. The upper group (BA) started with the wheat rolls containing the E304i / Zinc additive and the lower group (AB) stared with the reference wheat rolls.

[0043] Figure 2: Inclusion scheme.

[0044] Figure 3: PPM Gluten per gram flour. Below 20 PPM is regarded as "gluten free”.

[0045] Figure 4: Dose-dependent decrease in human TG2 transamidation of wheat and rye flours containing the food additive ascorbyl palmitate exposed to simulated gastrointestinal digestion. Data are presented as percentage of control (0 mg E304i / g of flour) and are means ± std dev, n=3 experiments.

[0046] Figure 5: Participant (n=9) blood levels of dGDP after intake of reference wheat bread rolls (blue dots) and after intake of E304i / Zinc supplemented wheat rolls. Non-compliant individuals were excluded from the study. Non-compliance was counted as, 1 ) Reported no intake of intervention bread rolls and 2) Reported intake of gluten-containing foods.

[0047] Detailed description of the invention

[0048] A flour additive that can make wheat products celiac-safe, will increase life quality tremendously for individuals with celiac disease. It can also aid celiac parents in keeping their children on a gluten-free diet, since children with a celiac parent are often predisposed to develop celiac disease. In addition, other groups with related genetic traits, such as certain diabetics and rheumatics are also at higher risk of developing celiac disease and would thus also greatly benefit from a food product with a guaranteed effective solution that in each consumed product reliably, fully, and completely inhibits and / or at least sufficiently reduces the gluten driven deamidation of gliadin in the gut of the consumer.

[0049] Today, the celiac patients have to rely on special-made, expensive, and often inferior products available on the market. In addition, new celiac-safe wheat or rye products according to the current invention can be marketed at a relatively low cost compared to the present special products. The use of two known and approved additives in such consumption products has a great potential since it has a solid scientific base as a safe additive, and with the herein for the first time described baking process, the effectivity of the additives can be guaranteed.

[0050] The herein presented combined additive of ascorbyl palmitate and Zinc can be a lifechanger for many people. The experiments disclosed in the experimental section are limited to testing done on only confirmed celiac disease with intestinal involvement and only two types of flour from wheat and rye. Still, since the effect of the additives is on the gluten component in the gut of the consumer, there is no reason to believe that the effect will not be that same for any gluten-comprising flour, as long as the content of gluten can be defined and controlled and the herein described novel baking process is followed.

[0051] The scientific question underlying the current invention was whether gluten-containing bread with ascorbyl palmitate in combination with Zinc gives rise to the production of celiac-specific antibodies and inflammatory mediators in tissue samples from celiac disease. The immune response is in the experimental section compared with the immune response from control bread without the addition of ascorbyl palmitate and Zinc, respectively untreated control. The supplement is shown to prevent the production of celiac-specific antibodies and inflammatory mediators by inhibiting the transglutaminase- mediated immune response.

[0052] Firstly, TG2 binding to gluten peptides in wheat and rye flours were estimated after a simulated gastrointestinal digestion step by running human TG2 transamidation assays, to determine the amount (mg) of ascorbyl palmitate per g of flour needed to prevent TG2 binding. This information was used to make breakfast wheat rolls used in a blind pilot study (n=2 participants) to evaluate if the participants could taste the E304i / Zinc additive in the wheat rolls. Lastly, a randomized double-blind cross-over intervention in healthy participants was conducted in which the endpoint was dGDP measured in participant blood, to determine if ingestion of wheat rolls containing the E304i / Zinc additive would lead to less or no TG2 processing of gliadin in comparison to reference wheat rolls (no additives).

[0053] The present invention relates to a combination product of a gluten containing flour and an additive characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

[0054] As is shown in the experimental section, by in vitro data in a human intervention study in healthy participants, the current invention for the first time discloses the ratio of E304i and Zinc additives to flour for full prevention of TG2 binding in baked products which still are palatable for human consumption and do not exceed the recommended daily intake of the additives. The experiments disclosed herein investigated if E304i could prevent TG2 processing of gluten in flours and if the effect was evident after simulated gastrointestinal digestion. The outcome in vivo in a human cross-over intervention study in healthy nonceliac participants further confirmed these findings. TG2 transamidation experiments (in vitro) of digested wheat and rye flours supplemented with E304i at 30 mg / g indicated full prevention of TG2 processing. In the intervention study, participant serum levels of deamidated gliadin peptides (dGDP) increased after intake of reference wheat rolls (80 g a day for a week; 41% ± 4% compared to washout), whilst intake of the intervention E304i / zinc sulphate wheat rolls generated a modest response (80 g a day for a week; 8% ±10 % of control). The difference between the groups (32.8% ±15.6%) was significant (p=0.00003, n=9), confirming that E304i / zinc addition to wheat rolls prevented TG2 deamidation of gluten. In conclusion, the herein disclosed study shows that E304i / zinc addition to wheat rolls prevents TG2 deamidation of gluten in non-celiac participants.

[0055] A combination product of a gluten containing flour and an additive

[0056] The current invention relates to a combination product of a gluten containing flour and an additive, wherein the additive comprises: i) ascorbyl palmitate (E304i) at a concentration of between 1 .1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05% of the flour weight, such as of approximately 0.034% of the flour weight, characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

[0057] In one embodiment, the combination product of a gluten containing flour and an additive comprises: i) ascorbyl palmitate (E304i) at a concentration of approximately 2.2-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05% of the flour weight, such as of approximately 0.034% of the flour weight.

[0058] Ascorbyl palmitate (E304i)

[0059] Flour treatment agents improve the baking properties of the flour. Ascorbic acid (vitamin C) is an example and is used in almost all types of flour. The traditional quantity is very small, approx. 2-4 g per 100 kg of flour, but it is an important additive that is traditionally used to strengthen the gluten of the flour and to improve the stability of the dough.

[0060] Ascorbyl palmitate is actually a vitamin C ester (i.e. , vitamin C that has been esterified to a fatty acid). In this case, vitamin C is esterified to the saturated fatty acid, palmitic acid, resulting in a fat-soluble form of vitamin C. Ascorbyl palmitate has been added to a number of skin creams due to interest in its antioxidant properties as well as its importance in collagen synthesis. Although ascorbyl palmitate is also available as an oral supplement, it is likely that most of it is hydrolyzed (broken apart) to ascorbic acid and palmitic acid in the digestive tract before it is absorbed.

[0061] In addition to its use as a source of vitamin C, it is also used as an antioxidant food additive (E number E304). Ascorbyl palmitate is further known as a fat-soluble antioxidant used to increase the shelf life of vegetable oils and potato chips. It is approved for use as a food additive in the EU, the U.S., Canada, Australia, and New Zealand. Ascorbyl palmitate is also marketed as "vitamin C ester". It is synthesized by acylation vitamin C using different acyl donors.

[0062] Ascorbyl palmitate is an amphipathic molecule, meaning one end is water-soluble and the other end is fat-soluble. This dual solubility allows it to be incorporated into cell membranes. When incorporated into the cell membranes of human red blood cells, ascorbyl palmitate has been found to protect them from oxidative damage and to protect alpha-tocopherol (a fat-soluble antioxidant) from oxidation by free radicals. However, the protective effects of ascorbyl palmitate on cell membranes have only been demonstrated in the test tube. Taking ascorbyl palmitate orally probably is thought not to result in any significant incorporation into cell membranes because most of it appears to be hydrolyzed (broken apart into palmitate and ascorbic acid) in the human digestive tract before it is absorbed. The ascorbic acid released by the hydrolysis of ascorbyl palmitate appears to be as bioavailable as ascorbic acid alone. The presence of ascorbyl palmitate in oral supplements contributes to the ascorbic acid content of the supplement and probably helps protect fat-soluble antioxidants in the supplement.

[0063] Ascorbic acid is synthetically produced and is only added to wheat flour. Small amounts are added, 3-5 g per ton. It strengthens the gluten, which results in a fluffier bread.

[0064] Ascorbic acid is known to break down when heated at 45 °C. Sulfur in proteins links the gluten together and makes it stronger. Ascorbic acid is traditionally added when the flour is milled, in an amount that should give the flour the same baking properties. In small mills, ascorbic acid is not added and this results in a flour with varying baking properties.

[0065] A number of possible problems with very large doses of vitamin C have been suggested, mainly based on in vitro experiments or isolated case reports, including genetic mutations, birth defects, cancer, atherosclerosis, kidney stones, "rebound scurvy", increased oxidative stress, excess iron absorption, vitamin B12 deficiency, and erosion of dental enamel. However, none of these alleged adverse health effects have been confirmed, and there is no reliable scientific evidence that large amounts of vitamin C (up to 10 grams / day in adults) are toxic or detrimental to health.

[0066] Ascorbyl palmitate is generally recognized as safe, GRAS- approved by the FDA (U.S. Food and Drug Administration) as a food additive. It is frequently used as an antioxidant or as a fat-soluble form of vitamin C. The European Food safety authority (EFSA) has in line with the American authorities also concluded that there is no safety concern regarding ascorbyl palmitate (E304i) as a food additive. According to EFSA, ascorbyl palmitate is considered to have the same biological effects as ascorbic acid, and from human data it was concluded that supplementary doses of about 1 g / person / day in addition to normal dietary intakes are not associated with adverse gastrointestinal effects, but gastrointestinal acute effects may occur at doses exceeding 3-4 g / day (EFSA Journal 2013; 11 (2):3104). The intervention meal described in the experimental section does not exceed 1 g ascorbyl palmitate / participant / day.

[0067] In the current invention, the additive comprises ascorbyl palmitate (E304i) at a concentration of between 15-30 mg / g flour, such as between 20-30 mg / g flour, such as 22-30 mg / g flour, such as between 25-30 mg / g flour such as between 20-25mg / g flour such as between 28-30 mg / g flour such as at the most 15, 20, 25 or 30 mg / g flour. In embodiments, the additive comprises ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, such as of approximately 1.1-2% of the flour weight such as of approximately 2-3% of the flour weight such as of approximately 2.2-2.5% of the flour weight such as of approximately 2.5-3% of the flour weight, or such as of approximately 2.2-3% of the flour weight.

[0068] In embodiments, the additive comprises ascorbyl palmitate (E304i) at a concentration of no more than 3% of the flour weight, such as of no more than 1.1 % of the flour weight, such as of no more than 1.5% of the flour weight, such as of no more than 2% of the flour weight, such as of no more than 2.2% of the flour weight, such as of no more than 2.3% of the flour weight, such as of no more than 2.5% of the flour weight , such as of no more than 2.75% of the flour weight.

[0069] In general, the amount of ascorbyl palmitate (E304i) used is adapted to the content of gluten in the flour used and calculated so as to be efficient to neutralize the gluten content completely, or at least sufficiently to reduce the effect of the gluten in the flour after baking and consumption of the baked product, so as not to elicit any immunological reaction to the gluten in the gut of the gluten-intolerant individual who consumes the baked product.

[0070] Zinc

[0071] Zinc sulphate is the compound normally used for Zinc fortification of foods. The recommended daily intake is 11 mg / day for men and 8 mg / day for women (US figures). However, the intestinal absorption of Zinc from the diet has been reported to be 15-60%.

[0072] In the current context, the term “Zinc” is used interchangably with Zn2+.

[0073] Dietary Zinc supplements come in daily doses in the range of 10-50 mg and a common supplemental dose is about 30 mg / day. An upper tolerable level for Zinc has been estimated to 50 mg / day for adults and 10 mg / day for children from 4 years of age (E.U.). Symptoms of mildly overdosing Zinc is mainly related to gastrointestinal discomfort while yet higher doses negatively affect copper balance. Zinc salts such as ZnCh, Zn acetate, and Zn sulphate are approved within the EU as dietary Zinc supplements. The intervention bread used in the experimental section does not exceed the recommended daily intake of Zinc sulphate.

[0074] The current invention relates to a combination product of a gluten containing flour and an additive, wherein the additive comprises: i) ascorbyl palmitate (E304i), and ii) Zinc (Zn2+) at a concentration of 0.02-0.05% of the flour weight, such as of approximately 0.034% of the flour weight, such as of approximately 0.035% of the flour weight, such as of approximately 0.036% of the flour weight, such as of approximately 0.037% of the flour weight, such as of approximately 0.038% of the flour weight, such as of approximately 0.039% of the flour weight, such as of approximately 0.033% of the flour weight, such as of approximately 0.032% of the flour weight, such as of approximately 0.031 % of the flour weight, such as of approximately 0.03% of the flour weight, such as of approximately 0.02% of the flour weight, such as of approximately 0.04% of the flour weight, such as of approximately 0.05% of the flour weight, such as of no more than 0.05% of the flour weight, such as of no more than 0.034% of the flour weight, such as of no more than 0.03% of the flour weight, such as of no more than 0.02% of the flour weight, such as at a concentration of between 0.02-0.04% of the flour weight, such as at a concentration of between 0.03-0.04% of the flour weight, such as at a concentration of between 0.03- 0.035% of the flour weight, such as at a concentration of between 0.02-0.025% of the flour weight, such as at a concentration of between 0.03-0.034% of the flour weight, such as at a concentration of between 0.025-0.035% of the flour weight, or such as at a concentration of between 0.04-0.05% of the flour weight.

[0075] In embodiments, the current invention relates to a combination product of a gluten containing flour and an additive, wherein the additive comprises: i) ascorbyl palmitate ( E304 i ), and ii) Zinc sulphate with a molecular weight of 179.47g / mol which should be comprised at a concentration of approximately 0.149% of the flour weight, such as at a concentration of between 0.1- 0.15% of the flour weight, such as at a concentration of between 0.1- 0.14% of the flour weight, such as at a concentration of between 0.1- 0.13% of the flour weight, such as at a concentration of between 0.1- 0.12% of the flour weight, such as at a concentration of between 0.1- 0.11 % of the flour weight, such as at a concentration of no more than 0.15% of the flour weight, such as at a concentration of no more than 0.149% of the flour weight, such as at a concentration of no more than 0.14% of the flour weight, such as at a concentration of no more than 0.13% of the flour weight , such as at a concentration of no more than 0.12% of the flour weight, such as at a concentration of no more than 0.11 % of the flour weight, or such as at a concentration of no more than 0.1 % of the flour weight.

[0076] It will be apparent to the person skilled in the art that although the embodiments shown in the experimental section relate to the use of Zinc (Zn2+) in the form of Zinc sulphate, any sort of Zinc salts such as ZnCI2, Zn acetate, and Zn sulphate can be used interchangeably, as long as the amount of Zinc salt is correctly calibrated to be equivalent to the amount of (Zn2+), or Zinc sulphate with a molecular weight of 179.47 g / mol as disclosed above.

[0077] The flour

[0078] Gluten is a protein group found in wheat, and gluten-like proteins are also found in rye and barley. In the case of gluten intolerance, the intestinal mucosa is damaged by very small amounts of gluten and therefore all food containing gluten must be avoided. Nowadays, gluten-intolerant adults can eat products that contain guaranteed pure oats (pure = no contamination with the other types of grain by using the same mills). The production of gluten-free bread must be done completely separately from the regular bakery operation, as very small amounts of gluten can cause great harm to those with gluten intolerance.

[0079] Gluten content in flour can be measured by Agraquant® gluten G12 ELISA (range 2-200 ppm). This assay measures a stretch of amino acids (one out of seven TG2-binding motives) most common in the immunogenic peptide alpha-2-gliadin, also known as the 33- mere, which is translated into gluten content in ppm.

[0080] To be considered gluten-free, a product must contain below 20 ppm (20 mg / kg) gluten. There is also a label “very low gluten content” which refers to a gluten content below 100 ppm.

[0081] Flour means fine-grained substance and flour can be made from almost any kind of grain. There are flours made from many different types of grain. The five most common types of grain are:

[0082] Wheat

[0083] Wheat flour is our most common flour and is made from the kernel of wheat. When wheat flour and water are processed or kneaded, a gluten network is formed, which retains the gas produced by the yeast. The result is a bread with good volume and fine porosity. Wheat flour is divided into different types according to how porous it is and airy breads they can provide - the higher the gluten quality, the better the volume. Wheat flour is made from part of the wheat kernel while whole wheat flour is made from the whole wheat kernel.

[0084] Many people want nice, porous breads, and therefore wheat flour is used in almost all breads, even the really coarse ones. Whole wheat bread often contains some wheat flour or a small amount of wheat gluten to improve volume.

[0085] The grain kernel consists of three parts

[0086] The shell part is rich in dietary fibre, B vitamins, minerals and biologically active substances. Bran is a concentrate of the shell parts of the kernel. Wheat bran is thus ground husk parts of wheat.

[0087] The germ is the basis for the growing plant and is rich in minerals, B vitamins, vitamin E and bioactive substances.

[0088] The flour body contains starch and some proteins.

[0089] When regular wheat flour is milled, approximately 70% of the original wheat kernel is used. But in whole wheat flour, 100% of the wheat kernel is used. Graham flour is whole grain flour from wheat. Regular rye flour always contains 100% whole grain, while sifted rye flour does not. In bread, whole grains can be included as flour, semolina, flakes, crushed, cut or whole kernels.

[0090] Rye

[0091] Rye flour is often used to bake bread that is dark in colour. The rye kernel contains less protein (gluten) than the wheat kernel, so the rye bread is not as porous as the wheat bread. Most bread contains rye flour because it makes the bread last longer and makes it extra filling.

[0092] Rye wheat is, just as it sounds, a cross between rye and wheat. Rye is the richest in fiber of the four grains, and also contains more fiber than almost all fruits and vegetables.

[0093] Barley

[0094] Today, barley is rarely included in bread. Barley has a grayish color, distinct taste, and does not add volume to leavened bread. A soft bread that is baked solely on barley will therefore be compact and crumbly. Barley is included as the main ingredient in bread where the volume does not play a major role, as in traditional Norrland thin bread. Sometimes grains are added in small amounts to give flavour variety and useful dietary fibers to the bread.

[0095] The current invention relates to a combination product of a gluten containing flour and an additive as described herein, wherein the flour contains at the most 10-15g protein / 100g flour, such as, wherein the flour contains at the most 10g protein / 100g flour.

[0096] In embodiments, a gluten containing flour as described herein, relates to flour which contains at the most 10-15g protein / 100g flour, such as, at the most 10g protein / 100g flour, such as, at the most 11g protein / 100g flour, such as, at the most 12g protein / 100g flour, such as, at the most 13g protein / 100g flour, such as, at the most 14g protein / 100g flour, or such as, at the most 15g protein / 100g flour.

[0097] Typically, a combination product of a gluten containing flour and an additive according to the current invention comprises flour which is selected from the group consisting of wheat flour, barley, and rye flour. In a currently preferred embodiment, a combination product of a gluten containing flour and an additive according to the current invention comprises flour which is wheat flour and / or rye flour. The current invention in consequence relates to a combination product of a gluten containing flour and an additive according to any of the preceding claims, wherein the additive comprises ascorbyl palmitate (E304i) at a concentration of between 15-30 mg / g flour, such as 22-30 mg / g flour, such as at the most 30 mg / g, and wherein the flour contains at the most 10-15 g protein / 100 g flour, such as at the most 10 g protein / 100 g flour and wherein the flour is wheat flour and / or rye flour.

[0098] Fat and / or Lipids

[0099] Bakers use fat in the bread to make it taste better, have a smoother texture, crumble less and stay fresh longer. Typically, bread contains no more than 2-6 grams of fat per 100 grams of bread.

[0100] The fat also acts as a carrier of flavours and aromas.

[0101] Bread that contains a lot of seeds is often a bit fattier than other breads but, on the other hand, is rich in fiber and other benefits.

[0102] Different types of fat

[0103] Fat is needed as a source of energy and for the body to absorb fat-soluble vitamins (vitamins A, D, E and K). Fat also contains the vital fatty acids linoleic acid and linolenic acid.

[0104] There are different types of fat that have different health effects in the body. Le., saturated fat, monounsaturated fat, polyunsaturated fat, hydrogenated fat, hardened fat and trans fats.

[0105] Vegetable fats and oils and vegetable margarine and margarine are what are most often used for bread. Rapeseed oil, which has a very healthy composition, is the main fat raw material when large bakeries bake bread. Sunflower oil, olive oil and palm oil are also used, although to a lesser extent. Animalic fats, especially those that are found in dairyproducts are often added to pasties or buns.

[0106] In the current context, any of the herein mentioned fats, as well as other fats known to be used in the field of the art, can be added to the dough or the ready baked product without changing the inherent characteristics of the consumer product, i.e., that the consumption of the combination product does not elicit a celiac immune response towards gluten. The choice of fat will be obvious to the person skilled in the art depending on the desired end baked product to be achieved.

[0107] The immune response

[0108] A combination product of a gluten containing flour and an additive according to the current invention does not elicit a celiac immune response towards gluten in the human intestine of a gluten intolerant individual. Typically, the immune response is measured as a standardised immunological determination of one or more celiac specific biomarker(s) in a sample from a celiac patient.

[0109] A celiac patient

[0110] The combination product of a gluten containing flour and an additive, according to the current invention is characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten in the human intestine of a gluten intolerant individual.

[0111] In the current context, a gluten intolerant individual is a patient suffering from a gluten- related disorder, selected from the group consisting of a celiac patient having a IgG or IgA response to gluten or gluten-like proteins, such as, but not limited to, secalins and hordeins.

[0112] Celiac disease is also known as coeliac disease, celiac sprue, non-tropical sprue, and gluten sensitive enteropathy. In the current context, these terms are used interchangeably.

[0113] In the current context, the term “Celiaci” is used to describe Celiac disease (Gluten intolerance or Gluten-induced enteropathy), which is classified as an autoimmune disease, where reaction against the enzyme transglutaminase (tTG) is triggered by gluten in wheat and / or the corresponding prolamins in rye and barley, which clinically results in an inflammation of the small intestinal mucosa, which in turn leads to villus atrophy.

[0114] Inflammation and mucosal atrophy can lead to gastrointestinal symptoms as well as deficiency states due to malabsorption.

[0115] The terms “celiac”, ’’celiacs” and “celiac patient”, “celiac patients” are herein used to describe one or more individual(s) who suffer from celiac disease. In a current preferred embodiment, the combination product of a gluten containing flour and an additive, according to the current invention is characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

[0116] A standardised immunological determination of one or more celiac specific biomarker(s)

[0117] Celiac disease is an auto-immune disease in which the immune system attacks TG2 by producing autoantibodies towards the enzyme. The immune system-assisted removal of TG2 (TG2 auto-antibodies) is associated with upregulation of TG2 in the small intestine in active celiac diseased ], further escalating the disease. Antibodies towards transglutaminases (TG) are thus sensitive diagnostic markers for celiac disease. There is also data suggesting that a diagnosis of celiac disease could likely be made on serology alone without confirmatory biopsy if the TG titre is high.

[0118] Antibodies towards deamidated gliadin peptides (anti-dGDP) is a feature of celiac disease, which is directly connected to TG2 because of its responsibility for the deamidation of GDP (dGDP). This is an earlier marker for gluten intake (compared to anti- tTG) in celiac patients, as well as a marker for celiac disease. Endomysial antibodies (anti-EMA) are also connected to TG2. About 90-95% of celiac patients produce these antibodies which make them a further choice as a diagnostic marker. Endomysial antibodies are partly produced locally in the celiac intestine, which make them suitable markers in ex vivo studies of celiac tissue cultures (biopsy studies) and, which is also true for anti-dGDP, which can be measured already in the culture medium after 24 hours.

[0119] In the current context, the immune response is measured as a standardised immunological determination of one or more celiac specific biomarker(s) in a sample from a celiac patient.

[0120] As is demonstrated in experiment 2, the herein for the first time disclosed combination product of a gluten containing flour and an additive prevents production of TG2 antibodies with at least 51 %±10% after exposure of the combination product to a biopsy of a celiac- intolerant subject, compared to TG2 antibody production after exposure of the same amount of gluten containing flour without an additive to a biopsy of said celiac-intolerant subject, suggesting that consumption of the combination product will not lead to an increase of celiac associated immunological markers in a celiac-intolerant subject, or will at least not lead to a clinically relevant increase of celiac associated immunological markers in a celiac-intolerant subject.

[0121] In one aspect, the current invention consequently relates to a combination product of a gluten containing flour and an additive, wherein the additive essentially fully, and / or at least to a clinically relevant degree, prevents production of TG2 antibodies after consumption of the combination product in a celiac-intolerant subject, as measured as at least preventing TG2 antibody production with at least 51 %±10% after exposure of the combination product to a biopsy of a celiac-intolerant subject, compared to TG2 antibody production after exposure of the same amount of gluten containing flour without an additive to a biopsy of said celiac-intolerant subject. This suggests that consumption of the combination product will not lead to an increase of celiac associated immunological markers in a celiac-intolerant subject, or will at least not lead to a clinically relevant increase of celiac associated immunological markers in a celiac-intolerant subject, such as measured as at least preventing TG2 antibody production with at least 61 , 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 , 50, 49, 48, 47, 46, 45, 44, 43, 42 or 41%.

[0122] A sample from a celiac patient

[0123] In one embodiment, the immune response of a combination product of a gluten containing flour and an additive according to the current invention can be measured as a standardised immunological determination of one or more celiac specific biomarker(s) in a sample from a celiac patient.

[0124] A sample from a celiac patient can e.g., be a tissue, a blood (serum or plasma) or another biological sample.

[0125] In one embodiment, the immune response of a combination product of a gluten containing flour and an additive according to the current invention can be determined, wherein the immune response is measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient.

[0126] The consumption of a combination product of a gluten containing flour and an additive, according to the current context, is determined not to elicit a celiac immune response towards gluten in the human intestine of a celiac patient when the level of tTG is below 10 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient. The consumption of a combination product of a gluten containing flour and an additive, according to the current context, is determined not to elicit a celiac immune response towards gluten in the human intestine of a celiac patient when the level of tTG IgA is below 10 U / mL, such as below 9 U / mL, such as at the most 7 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient.

[0127] The consumption of a combination product of a gluten containing flour and an additive, according to the current context, is determined not to elicit a celiac immune response towards gluten in the human intestine of a celiac patient when the level of tTG IgG is below 10 U / mL, such as below 9 U / mL, such as at the most 7 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient.

[0128] A baked product

[0129] The current invention relates to a baked product comprising a combination product of a gluten containing flour and an additive according to the current invention, characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

[0130] In one embodiment, such a baked product is called “the intervention bread” (40 g buns with poppy seeds) as described in the experimental section. The intervention breads are; one endosperm wheat bread (high gluten content) and one endosperm rye bread (low gluten content). The experiment describes reference buns without the additive of the current invention (APZN) and the APZN-containing buns. Bread samples are analyzed for gluten content with the standard method Agraquant® gluten G12 ELISA (range 2-200 ppm).

[0131] In embodiments, a baked product according to the current invention is a bakery product. Typically, a baked product according to the current invention is selected from the group consisting of bread, portion bread, roll, toast bread, baguette, hamburger bun, Danish pastry, French pastry, puff pastry, muffin and cake. A daily serving

[0132] A baked product according to the current context is intended for use in a daily serving of Zn, which does not exceed the recommended daily dose, which is at the most 1 .25 mg / kg bw / day.

[0133] A baked product according to the current context is intended for use in a daily serving of ascorbyl palmitate (E304i) which does not exceed the recommended daily dose, which is at the most 3-4 g / day.

[0134] In embodiments, a baked product according to the current context is intended for use in a daily serving of ascorbyl palmitate (E304i) of at the most 1.6 g / day, such as at the most 2.4 g / day, dependent on the weight of the baked product.

[0135] The baking process

[0136] In one aspect, the current invention relates to a process for producing a baked product according to the current invention, comprising a combination product of a gluten containing flour and an additive, wherein the baking process does not destroy the effectivity of the ascorbyl palmitate (E304i) comprised in the additive, or at least leaves enough ascorbyl palmitate (E304i) in the baked product, so that the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient.

[0137] During the experiments leading up to the current invention, it was surprisingly found that the ascorbyl palmitate (E304i) was especially sensitive to temperatures over RT in the proofing step b) and would lose sufficient effectivity in the final baked product if the proofing step b) was carried out at temperatures between 30-37°C. Thus, the currently disclosed baking process comprises only proofing in the proofing step b) in RT or below, such as at 0-30°C, such as at no more than 2, 4, 6, 8, 10, 12, 14, 16 or 18°C, such as at no more than 19°C, such as at no more than 20°C, such as at no more than 21 °C, such as at no more than 22°C, such as at no more than 23°C, such as at no more than 24°C, such as at no more than 25°C, such as at no more than 26°C, such as at no more than 27°C, such as at no more than 28°C, such as at no more than 29°C or such as at no more than 30°C.

[0138] Furthermore, the ascorbyl palmitate (E304i) was also sensitive to temperatures over 200°C in the baking step c) and would lose sufficient effectivity in the final baked product if the baking step c) was carried out at too high temperatures, such as above 190, 195, 199, 200, 205, 210, 215, 220 or 225°C.

[0139] Irrespective of the exact heat used in the baking step c), the inner temperature of each baked product should not exceed 80-100°C, such as 90-100°C, such as 100°C.

[0140] In consequence, the baking process of the current invention comprises the steps of: a) mixing

[0141] 1 ) the additive comprising:

[0142] 1) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05%, such as of approximately 0.034% of the flour weight,

[0143] 2) water,

[0144] 3) optionally a lipid and / or fat,

[0145] 4) yeast, and

[0146] 5) gluten-containing flour, b) proofing the dough at room temperature, and c) baking the product.

[0147] In embodiments, the baking process of the current invention comprises the steps of: a) mixing

[0148] 1 ) the additive comprising:

[0149] 1) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05%, such as of approximately 0.034%% of the flour weight,

[0150] 2) water,

[0151] 3) optionally a lipid and / or fat,

[0152] 4) yeast, and

[0153] 5) gluten-containing flour, b) proofing the dough at room temperature, and c) baking the product at no more than 195°C.

[0154] The experimental section encloses embodiments wherein all ingredients are mixed in a bowl, whereupon the raw dough weighs 50 g, is admixed with Meltec-water mixture (0.6 g) + poppy seeds (1 .7 g), then left to proofing in room Temperature (RT) for 65 min and is finally baked for 12 min at 195°C. The baked products are ready buns of approximately 42 g each.

[0155] The experimental section encloses additional embodiments wherein all ingredients are mixed in a bowl, whereupon the raw dough weighs 50 g, is admixed with Meltec-water mixture (0.6 g) + poppy seeds (1.7 g), then left to proofing in room Temperature (RT) for 75 min and is finally baked for 12 min at 195°C. The baked products are ready buns of approximately 42 g each.

[0156] The baked products can be cooled in RT and then freezed.

[0157] The current invention thus in one embodiment relates to a process for producing a baked product according to the current invention, wherein the temperature inside the baked product during the baking step c) does not exceed 100°C.

[0158] The current invention in one embodiment relates to a process for producing a baked product according to the current invention, wherein the heat in the baking step c) does not exceed 225°C, 220°C, 210°C, 200°C, 195°C or 190°C.

[0159] The current invention in one embodiment relates to a process for producing a baked product according to the current invention, wherein the heat in the baking step c) does not exceed 195°C.

[0160] In a baking process according to the current invention, the additive can be solved in water, oil and water, in butter and water, or in milk before mixing it with the yeast and gluten containing flour in step a). The current invention thus in one embodiment relates to a process for producing a baked product according to the current invention, wherein the additive is solved in oil and water, and wherein the heat in the baking step c) does not exceed 200°C.

[0161] Since it is imperative to control the gluten / additive ration in the combination product described herein, the current invention relates to a process for producing a baked product according to the current invention, wherein no further gluten is added throughout the baking process than the gluten comprised in the gluten containing flour. Typically, no further gluten containing flour is added in the baking process than is mixed with the other ingredients in mixing step a) of the baking process. The process for producing a baked product according to the current invention produces a baked product, which is selected from the group consisting of bread, portion bread, roll, toast bread, baguette, hamburger bun, Danish pastry, French pastry, puff pastry, muffin and cake. In consequence, in one embodiment, the current invention relates to a baked product produced by the herein for the first time disclosed baking process, characterized in that the proofing step b) is carried out RT.

[0162] In one embodiment, the current invention relates to a baked product produced by the herein for the first time disclosed baking process, characterized in that at heat in the baking step c) does not exceed 195°C.

[0163] In one embodiment, the current invention relates to a baked product produced by the herein for the first time disclosed baking process, characterized in that the temperature inside the baked product during the baking step c) does not exceed 100°C.

[0164] A baked product produced by the herein for the first time disclosed baking process is characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

[0165] Below are selected embodiments of 1 ) a wheat bun dough without additive, 2) a wheat bun dough with additive and 3) a rye bun dough with additive.

[0166] Table 1: A wheat bun dough without additive

[0167] Table 2: A wheat bun dough with additive

[0168] Table 3: A rye bun dough with additive

[0169] A Premix

[0170] The current invention further relates to a premix comprising a gluten containing flour and an additive comprising: i) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05%, such as of approximately 0.034% of the flour weight.

[0171] The invention also relates to a premix comprising a gluten containing flour and an additive according to the current invention in the preparation of a baked product comprising a gluten containing flour, wherein the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient.

[0172] A kit of parts

[0173] A kit of parts is also envisioned, comprising a gluten containing flour and an additive comprising i) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05%, such as of approximately 0.034% of the flour weight.

[0174] In one embodiment, the invention relates to the use of a kit of parts comprising a gluten containing flour and an additive according to the current invention in the preparation of a baked product comprising a gluten containing flour, wherein the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient. Uses

[0175] A combination product of a gluten containing flour and an additive according to the current invention can be used in the preparation of a baked product comprising a gluten containing flour, wherein the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient. Said baked product can be a baked product selected from the group consisting of bread, portion bread, roll, toast bread, baguette, hamburger bun, Danish pastry, French pastry, puff pastry, muffin and cake.

[0176] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0177] As used herein, the term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Similarly, the term “consisting essentially of” is intended to include embodiments encompassed by the term “consisting Of”.

[0178] As used herein, the term “about” modifying the quantity of an ingredient or reactant employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities.

[0179] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the invention.

[0180] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0181] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated to disclose and described the methods and / or materials in connection with which the publications are cited.

[0182] Other embodiments

[0183] It is to be understood that while the present invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims.

[0184] Other aspects, advantages, and modifications are within the scope of the following claims.

[0185] Experimental section

[0186] The present invention is further illustrated by the following non-limiting experiments.

[0187] Example 1

[0188] In the present study, the aims were to 1 ) estimate the ratio of E304i to flour for full prevention of TG2 binding, 2) to conduct pilot experiments to evaluate taste of wheat rolls containing those amounts of E304i , 3) to evaluate the in vitro data in a human intervention study in healthy participants.

[0189] First, TG2 binding to gluten peptides in wheat and rye flours after a simulated gastrointestinal digestion step by running human TG2 transamidation assays was estimated, to determine the amount (mg) of E304i per g flour needed to prevent TG2 binding. This information was used to make breakfast wheat rolls used in a blind pilot study (n=2 participants) to evaluate if the participants could taste the E304i / zinc additive in the wheat rolls. Lastly, a randomized double-blind cross-over intervention in healthy participants was conducted in which the endpoint was dGDP measured in participant blood, to determine if ingestion of wheat rolls containing the E304i / zinc additive would lead to less or no TG2 processing of gliadin in comparison to reference wheat rolls (no additives).

[0190] Background

[0191] In celiac disease, intestinal transglutaminase 2 (TG2) produces immunogenic peptides by deamidation of gluten and gluten-like proteins from wheat, barley and rye. These products drive the celiac immune response. The aim was to investigate if E304i can prevent TG2 processing of gluten in full flours and if the effect was evident after simulated gastrointestinal digestion. The aim of the study was also to confirm the outcome in vivo in a human cross-over intervention study in healthy participants.

[0192] Experimental setup

[0193] Materials and methods

[0194] Table 4: Flours

[0195] Determination of gluten content (TG2 binding motives) in wheat and rye flours

[0196] A commercially available assay to estimate gluten content (AgraQuant® ELISA gluten G12; Romer labs) was chosen since the assay antibody (G12) is specific to the TG2 binding motive QLP, which the studies focus on. The assay was performed according to the manufacturers’ instructions. After data analysis the theoretical content of E304i (per gram of flours) was calculated that was estimated to be needed to fully cover all TG2 binding sites in the wheat and rye flours.

[0197] Simulated gastrointestinal digestion of flour samples

[0198] Wheat and rye flour samples (provided by Lantmannen, Finland) went through a simulated gastrointestinal digestion (in vitro) according to a simplified standard protocol with modifications. Briefly, flour samples (1 g) were suspended in water (10 ml) and digested with pepsin solution containing 0.16 mg / L of HCI (0.1 mM) at pH 2 for 1 hour at 37°C. Then the pH was adjusted to 7 by the addition of NaHCC (1 M). The samples were further digested with pancreatin solution (1 .7 ml / sample containing pancreatin; 4 g / l) for 1 hour at 37°C. After the digestion, the digests were cooled down to inhibit further digestion. TG2 transamidation assay To estimate TG2 binding to gluten peptides in full flours, a transamidation assay based on the method of Skovbjerg et. alhas been developed.

[0199] Briefly, 96-well plates were coated overnight at 4°C on a rotary shaker at 20 rpm, with freshly in digested flour samples, with and without E304i at 5,10, and 30 mg / g flour. The wells were washed three times with TBS (5 mM) + Tween (0.05%) and blocked for 1 h in room temperature using Sigma’s Coating stabilizer and blocking buffer (# C9483, SigmaAldrich, St. Louis, MO, US). Biotin solution, containing 5-(biotinamido) pentylamine (0.165 mg / ml) mixed with Tris-HCI (5 mM), CaCI2 (5 mM) and dithiothreitol (10 mM), mixed with TG2 (0.5 U / ml, human recombinant; #T022, Zedira, Darmstadt, Germany) was made fresh and added to each well to incubate for 1 h in 37°C and 30 rpm. The plate was then washed and europium-streptavidin (1 pg / ml, DELFIA; PerkinElmer, Waltham, MA, US) in DELFIA assay buffer (PerkinElmer) was added and the plate incubated for 1 h in room temperature. After another washing, Delfia enhancement buffer (PerkinElmer) was added and the plate incubated for 5 minutes, after which the fluorescence (345 nm excitation, 617 nm emission) was measured. The reduction in transamidation by E304i was compared to untreated flour controls and presented as % of control flours.

[0200] A randomized double-blind intervention study in healthy volunteers

[0201] Study design

[0202] The study was a randomized, double blind, 4-week human dietary intervention with a cross-over design in healthy participants. Celiac disease and other diagnosed enteropathies were among the exclusion criteria. It was also a requirement that the participants were eating gluten products as a part of their habitual diet (to avoid asymptomatic celiacs and to promote compliance). The endpoint was dGDP in the participants' blood as an indicator of TG2 deamidation of gluten peptides in the intestinal wall. The 4-week intervention started with a wash-out week (gluten-free diet), to reduce participant blood levels of dGDP. The participants were instructed to not eat gluten containing foods, except for the intervention bread, throughout the whole study. The participants met the research staff at the screening and once a week during the intervention period. At the weekly meetings, the participants were provided with gluten- free staples such as pasta, rice, and bread, in addition to the intervention bread rolls. The nutritionist / dietician followed up on the previous week food diary and the participants well- being / health status. The study design is outlined in figure 1.

[0203] Participants

[0204] 56 individuals of both genders between the age of 18-50 years (BMI 18.5-32) were prescreened in the first round (on the phone), out of those, 34 individuals went through the screening process (a visit + blood analysis) and then 24 were invited to participate in the study and block-randomized (starting with A or B) into each intervention arm (AB or BA). Out of these, 4 participants were excluded during the intervention. Reasons for the late exclusion were the common cold / possible Corona virus infection. 20 participants went through the full intervention study. Figure 2 shows the inclusion scheme.

[0205] The intervention meal

[0206] The intervention meal (breakfast) consisted of 2 wheat rolls (a 42 g) with poppy seeds. The purpose of the poppy seeds was to cover differences in appearance of the crust to ensure the blinding of the study. Standard recipe wheat rolls were baked at Lantmannen Unibake’s production site in Finland. One reference dough and one dough supplemented with ascorbyl palmitate (3% of flour weight) and Zinc sulphate heptahydrate (0.149% of the flour weight) was produced. The wheat rolls were packaged in color-coded bags and transported frozen to Sweden. The identity of the color codes was not known for the personnel in contact with the participants. The cold chain was not broken until the weekly ration had reached the participants. The instructions were that the breakfast rolls could be prepared with any gluten-free spread of the participant’s own choice.

[0207] Table 5: Reference and additive containing (AP) baked product

[0208] Table 6: recipe for the test baking

[0209] MELTEC® HIFOOD Code Codice: 1000182 5Kg / 1000202 20 kg / 1000203 IBC

[0210] One intervention wheat roll (42 g baked roll) contained 0.84 g E304i and the intake of 2 of these rolls per day contained 1.6 g of ascorbyl palmitate / participant / day. The intervention wheat rolls (per 42 g of baked roll) contained 6.2 mg of zinc per roll, leading to an intake of 12.4 mg of zinc per day during the intervention study. Zinc chloride was used as the Zinc salt in the preclinical studies and Zinc sulphate was the choice for the intervention wheat rolls in the human study.

[0211] Biological samples

[0212] Blood samples were collected (heparin and serum tubes) at the study visits by nurses at Gothia clinical trial center (CTC) at Sahlgrenska University hospital, Gothenburg, Sweden. Samples were kept on ice and immediately processed and aliquoted. All samples were stored at -20 °C during the day of collection and then transferred to -80 °C freezers at Chalmers University of Technology. Samples were analysed for dGDP with ELISA (primary antibody ab36729; Abeam).

[0213] Statistical analysis

[0214] Basic statistical analyses were made with Microsoft® Excel for Mac (version 16.67). Two- tailed T-tests were done based on unequal (2,3) or equal variances (2,2). The Sdev.P function was used to estimate the standard deviation on population level and sdev for population samples. To our knowledge, there are no previous published data on the serum levels of deamidated gliadin peptides since the measure is normally antibodies to deamidated gliadin in celiac patients or healthy individuals. Therefore, the assumptions for the sample size calculations were made based on preliminary measurements in our lab. We used an online tool to estimate sample size for a cross-over study where the outcome is a measurement (hedwig.mgh.harvard.edu / sample_size / js / js_crossover_quant.html). 40 participants were to enter the study assuming a power of 80% and a two-sided significance level of 5% and a potential drop-out of 18%. After the study, the power was recalculated using the achieved results and it was estimated that the power based on the compliant participants, n=9, was 90% (5% two-sided significance). Thus, the original calculations greatly underestimated the difference between the groups. The randomization was done using an online tool (sealedenvelope.com / simple-randomiser / v1 / lists).

[0215] Conclusions

[0216] TG2 transamidation of digested wheat and rye flours supplemented with E304i at 30 mg / g was not significantly different from baseline, indicating full prevention of TG2 processing. In the human intervention study, wheat rolls supplemented with E304i and Zinc (sulphate), an inhibitor of TG2 activity, was compared to reference wheat rolls. The participants who reported compliance to the study (n=9; 45%) showed a significant increase in serum- deamidated gliadin peptides after the reference wheat rolls (141 % ± 4% of control) (41 % ± 4% compared to washout), but only a slight increase from the E304i / Zinc (sulphate) wheat rolls (108% ±10 % of control) (80 g a day for a week; 8% ±10 % of control). The intervention study confirmed that E304i / Zinc sulphate addition to wheat rolls prevents TG2 deamidation of gluten in healthy non-celiac participants. The difference between the groups (32.8% ± 15.6%) was significant (p=0.00003, n=9), confirming that E304i / zinc addition to wheat rolls prevented TG2 deamidation of gluten. In conclusion, this study shows that E304i / zinc addition to wheat rolls prevents TG2 deamidation of gluten in non- celiac participants.

[0217] Results Gluten content as estimated by the G12 antibody-based commercial gluten test

[0218] The AgraQuant assay was developed by Romer labs to measure gluten content. The method builds on an antibody (G12) that binds one immunogenic amino acid sequence in wheat (QPQLPY) in which QLP is one of the binding motives of TG2. The theoretical content of TG2 binding sites was calculated to roughly estimate the amount of E304i needed to cover all TG2 binding sites in the flours (Table 7)

[0219] Table 7: Theoretical amounts of E304i to cover the QXP binding sites (mg / g flour).

[0220] The relative levels of experimentally estimated gluten (with the G12 assay) correlated with the number of QXP motives and calculated amount of E304i to cover them (Figure 3. vs table 7.) Figure 3 shows PPM Gluten per gram flour (or PPM QPQLPY, in which QLP is one (out of “seven) TG2-binding motives). The results indicate: Wheat >Rye. Below 20 PPM is regarded as "gluten free”.

[0221] Transglutaminase processing of wheat and rye flours

[0222] Dose-response experiments with three different concentrations of E304i (5, 10 and 30 mg E304i / g flour; n=3) were conducted. 30 mg E304i / g flour was required to fully prevent TG2 processing of wheat and rye flours as anticipated by the theoretical estimations. At this level (30 mg / g flour), two bread rolls can be ingested daily, without exceeding the recommended daily allowance (RDA). About 80% of ascorbyl palmitate has been reported to be retained after heating and the breakdown products are unharmful (ascorbic acid and palmitic acid).

[0223] The prevention of the transamidation is predicted to decrease further with the addition of Zinc (chloride) to inhibit the intrinsic activity of TG2 as was observed with pure gliadin in previous studies. The biscuit flour, which showed the highest content of TG2 processing of the untreated refined wheat flours (except for the sifted spelt flour) showed a marked decrease of TG2 processing already at low E304i levels (10 mg / g flour, 71 % decrease, with no addition of Zinc). The TG2 activity in both wheat and rye flours were not sufficiently lowered at 10 mg E304i Zg flour. All the flours were not significantly different from baseline regarding TG2 processing at 30 mg E304i Zg flour, which was the ideal case and therefore sufficient. This was the rationale for choosing to start with 30 mg E304i Zg flour in the human intervention study.

[0224] Figure 4 shows the dose-dependent decrease in human TG2 transamidation of wheat and rye flours containing the food additive ascorbyl palmitate exposed to simulated gastrointestinal digestion. Data are presented as percentage of control (0 mg E304i / g flours) and are means ± stddev, n=3 experiments.

[0225] Sensory study of bread rolls with added E304i / Zinc sulphate- a pilot study

[0226] A pilot study (n=2) to evaluate if the E304i addition to a wheat roll would cause sensory issues was undertaken. The results indicated that there was no difference in taste of wheat rolls containing E304i or E304i / Zinc sulphate compared to the control wheat roll with no additives. The blindfolded participants could not identify which roll contained E304i / Zn. However, the E304i additive was observed to be sensitive to high heat (200°C oven temperature) since the crust appeared with tiny dark spots. These spots were only visible on the crust. Therefore, we added poppy seeds to the intervention bread rolls to facilitate the blinding of the study.

[0227] The E304i / Zinc sulphate bread roll intervention study

[0228] Serum levels of dGDP

[0229] Participant blood levels of dGDP was measured in serum samples taken at baseline and then after each washout and intervention week (a total of 5 sampling events, see fig. 1 ). It was seen that serum levels of dGDP were easily affected by ingestion of gluten, with the consequence that non-compliance or intake of trace amounts of gluten-containing foods gave large effects on the data. It also gave the tool to presumably identify non-intervention bread eaters (n=3). Non-compliance was counted as, 1) Reported no intake of intervention bread rolls and 2) Reported intake of gluten-containing foods (in addition to the intervention wheat rolls). Base levels of dGDP in participants after washout differed by a factor of approximately 2.5 (compliant participants, n=9) and in average were 422 ng dGDP / ml serum among the reported compliant participants. In a pilot pre-study before the intervention, dGDP levels were estimated in serum from an individual on a habitual gluten- free diet (non-celiac) to 28.2 ng dGDP / ml serum and after an arbitrary gluten intake for 2 weeks, the serum concentration of dGDP was 106.9 ng / ml, indicating that dGDP levels are sensitive to gluten exposure and may be a candidate (compliance) biomarker for gluten intake (here used as a marker for deamidation of gluten by TG2). These results also indicate that the difference between the intervention groups would be greater if the washout periods were considerably longer.

[0230] The compliance of the study participants was 45% (n=9 individuals) out from the 20 individuals who finalized the study. Among the compliant individuals, it was sometimes evident that there was an increased basal level of TG2 processing, suggesting that the participants were still exposed to unreported gluten. This was not considered as non- compliance since the participants were not instructed to keep a strict gluten-free diet, only to avoid gluten-containing foods such as wheat pasta. The outcome of the study was based on the individuals with reported compliance (n=9). These were n=6 from the AB group and n=3 from the BA group (where bread A was the reference roll and B the E304i / Zinc-added wheat roll). The serum levels of dGDP in response to the E304i / Zinc- added wheat roll and the reference roll respectively, were normalized to each participant dGDP status before the intervention week (washout-levels). The percentage change within each participant was then compared on group level.

[0231] The percentage increase in participant serum dGDP after one week of having two reference wheat rolls a day (gluten exposure) in comparison to having the E304i / Zinc- added wheat rolls was 32.8% ± 15.7% on group level (statistical power 90%), see Fig. 5. The increase in participant serum-dGDP after the E304i / Zinc-added wheat rolls vs. washout was in average (108 ±10 %) 8 ± 10 % and after intake of the reference wheat rolls, 41 % ± 4% vs. washout.

[0232] Figure 5 shows Participant (n=9) blood levels of dGDP after intake of reference wheat bread rolls (blue dots) and after intake of E304i / Zinc supplemented wheat rolls. Data were corrected for reported non-compliance. Non-compliance was counted as, 1) Reported no intake of intervention bread rolls and 2) Reported intake of gluten-containing foods.

[0233] The study in particular researched the dGDP levels after having the E304i / Zinc sulphate bread rolls in comparison to the washout with no bread, with the optimal outcome at 100% a.k.a. no difference. The data showed individual differences with an average of 108%. This small, but significant increase on group level after having the E304i / Zinc wheat rolls was likely caused by small additions of pure wheat flour after the mixing process at the bakery. This was done since the dough was too soft. This source of error was not known by the project team until after the intervention study was finalized. In hindsight, the outcome (through lower baseline dGDP) would have benefited from the participants being on a strict gluten free diet (except for the intervention wheat rolls) during the 4-week intervention.

[0234] Health reports during the study period

[0235] The participants self-reported health status was in general good comparing before and after the study. Four participants reported adverse effects during the study. Three of these reported skin rashes and one reported nausea. Two of the skin rashes were improved during the intervention with the E304i / Zinc bread rolls and one of the rashes appeared during the week with the reference bread. It was concluded that the E304i / Zinc sulphate additive was unlikely the problem. In one of the cases, an allergic reaction towards poppy seeds could have been the cause. The participant with nausea did not improve during the washout week and developed additional symptoms seemingly not associated with either E304i / Zinc sulphate or poppy seeds.

[0236] Conclusions

[0237] The study investigated if E304i could prevent TG2 processing of gluten in flours and if the effect was evident after simulated gastrointestinal digestion. It also confirmed the outcome in vivo in a human cross-over intervention study in healthy nonceliac participants. TG2 transamidation experiments (in vitro) of digested wheat and rye flours supplemented with E304i at 30 mg / g indicated full prevention of TG2 processing. In the intervention study, participant serum levels of deamidated gliadin peptides (dGDP) increased after intake of reference wheat rolls (80 g a day for a week; 41 % ± 4% compared to washout), whilst intake of the intervention E304i / zinc sulphate wheat rolls generated a modest response (80 g a day for a week; 8% ± 10 % of control). The difference between the groups (32.8% ± 15.6%) was significant (p=0.00003, n=9), confirming that E304i / zinc addition to wheat rolls prevented TG2 deamidation of gluten. In conclusion, this study shows that E304i / zinc addition to wheat rolls prevents TG2 deamidation of gluten in non-celiac participants.

[0238] The prevention of transamidation was predicted to decrease further with the addition of a zinc salt to inhibit the intrinsic activity of TG2, as was observed with pure gliadin in previous studies. The compliance of the study participants was 45% (n=9 individuals) out of the 20 individuals who finalized the study. Among the compliant individuals, it was sometimes evident that there was an increased basal level of dGDP, suggesting that the participants were still exposed to unreported gluten. This was not considered as non- compliance since the participants were not instructed to keep a strict gluten-free diet, only to avoid gluten-containing foods such as wheat-based pasta and bread etc. The outcome (through lower baseline dGDP) would have benefited from the participants being on a strict gluten free diet (except for the intervention wheat rolls) and this was a weakness of the study. The study also indicated that the difference between the intervention groups would be greater if the washout periods were considerably longer. The short washout periods were therefore another weakness of the study. A long-term study with participants on gluten free diets with the E304i / zinc added wheat rolls as the only gluten exposure would have been ideal. However, in conclusion, this study shows that E304i / zinc addition to wheat rolls prevents TG2 deamidation of gluten in non- celiac participan

[0239] The study outcome was successful from two perspectives, 1 ) The addition of E304i / Zinc sulphate to wheat rolls prevented deamidation of gluten-derived peptides as indicated by serum dGDP levels, 2) dGDP indirectly showed to be a sensitive marker for gluten intake.

[0240] Example 2

[0241] Tissue studies outside the body on patient material from celiac disease center

[0242] There is now a major aspect that needs to be investigated before a celiac intervention can take place; the demonstration that E304i / Zinc sulphate addition to wheat products does not provoke a celiac immune response (investigated in vitro or ex vivo).

[0243] In the above food intervention study in healthy subjects it was observed that the compound, APZn (i.e., ascorbyl palmitate in combination with Zinc) in bread at a certain concentration in relation to the amount of gluten-containing flour used and after a baking process with proofing at RT and a low baking temeperature), inhibited the deamidation of gluten peptides in vivo, an important step in the immune system's recognition process in gluten intolerant people. The current experiment is conducetd to show that APZn containing bread does not cause an immune response in individuals with celiac disease either. Frst a tissue study outside the body and then a smaller intervention study in celiac patients is conducted to ensure that the APZn's inhibition of the deamidation also implies a lack of celiac disease-specific immune response.

[0244] The scientific question is whether gluten-containing bread with ascorbyl palmitate in combination with Zinc gives rise to the production of celiac-specific antibodies and inflammatory mediators in tissue samples from celiac disease. The immune response is compared with the immune response from control bread without the addition of ascorbyl palmitate and Zinc, respectively untreated control. It is forseen that the supplement prevents the production of celiac-specific antibodies and inflammatory mediators by inhibiting the transglutaminase-mediated immune response.

[0245] Experimental setup

[0246] Wheat and rye buns, with and without the additive APZn, are exposed to a simulated gastrointestinal digestion procedure.

[0247] Three duodenal biopsies per individual are collected from celiac and non-celiac patients. The procedure is done during routine gastroendoscopy in collaboration at an endoscopy unit. The specific number of patients is determined by availability, but aims at 15 individuals, treated celiacs are included as they have non-detectable / very low levels of celiac antibodies at baseline. After collection of the tissue samples, the tissues will be placed in pre-heated cell medium (DMEM, HEPES 15 mM, L-glutamine 2 mM, penicillin 100 u / ml, and 0,1 mg / ml streptomycin) containing 1 ) APZn bread digest (1 mg / ml), 2) reference bread digest (1 mg / ml) or 3) only cell medium, negative control. Each person serves as her / his own control. The incubation lasts for 24 h after which the medium is collected and analyzed for celiac-associated antibodies (Anti-EMA IgA, Anti- TG IgA, and / or anti dGDP). In addition, the intercellular adhesion molecule ICAM-1 , associated with celiac tissue and serum is also measured. This method has successfully been used in studies investigating gliadin toxicity in celiac disease and is therefore a plausible approach to estimate celiac safety of the APZn wheat product, without the risk of causing harm.

[0248] Materials and methods

[0249] The study is a biopsy study, where intestinal biopsies are collected from celiac disease subjects and from controls without celiac disease. The scoping takes place during regular remission control (celiac disease) and scoping for dyspepsia symptoms (controls), and is performed by medical staff.

[0250] 3 pcs. Intestinal biopsies (from the duodenum, standard size 2-3 mm) are collected per individual and scoping case. In total, 10 celiac patients and 5 controls without celiac disease are scoped. A total of 15 individuals and a total of 45 intestinal biopsies.

[0251] Reliability, in the cases of the patient's health status in relation to the celiac disease diagnosis, is ensured by the samples initially receiving the patient's social security number in addition to the study code and the project manager receiving an extract from the patient's medical record with information on diagnosis and relevant data on markers for celiac disease and inflammation. The study also needs to know if there are immune deficiencies that can affect the inflammatory response in the ex vivo experimental part.

[0252] The samples are immediately placed in 6-well plates with prewarmed medium (DMEM, HEPES 15 mM, L-glutamine 2 mM, penicillin 100 u / ml, and 0.1 mg / ml streptomycin). The samples are only marked with the study code. To each respective wells will then be added, 1 ) e APZn bread (wheat or rye) and 2) control bread and 3) no gluten-containing treatment, empty digested. The bread has first undergone a simulated gastrointestinal digestion and the digestate is in the duodenal (small intestine) phase. Each patient is thus its own control. The incubation is 24 h, where medium and tissue is collected and analyzed for celiac disease-associated antibodies and inflammatory mediators (such as An-EMA IgA, An-TG IgA, an dGDP and ICAM- 1). Bread-specific components and transglutaminase products can also be analyzed. The method has been successfully used in several published works, where the toxicity of gliadin in celiac disease has been studied.

[0253] Suitable patients are entered during referral processing. Patients called to the endoscopy unit for remission control (celiac disease) and patients called for biopsy for dyspeptic symptoms 5 controls without celiac disease are recruited from the patients called for scoping due to dyspeptic symptoms. These patient samples are formally included in the study if they have no pathological findings on endoscopy. Pathological findings such as inflammation or bleeding can affect the study results and therefore leads to exclusion.

[0254] The study is laid out so that there is a positive and a negative control (baseline) in addition to the study product itself, which reduces the number of patients needed to obtain reliable results. The purpose of the study is to determine whether the APZn bread produces an immune response rather than to measure the size of the response. Strictly speaking, n=3 is therefore required as the smallest possible number to be able to determine whether the APZn bread is significantly different from the positive control or non-significantly different from the patient's baseline. To ensure that n=3 is achieved, we take in n=5 patients for the APZn bread with wheat and rye and control patients respectively. The control patients receive the APZn bread with wheat (highest gluten content).

[0255] The intestinal tissue samples are cultured and exposed to the intervention bread and reference bread (pre-processed to mimic gastrointestinal digestion). The endpoints are anti-TG IgA, anti-EMA IgA, anti-dGDP IgA, ICAM-1. The choice of endpoints and experimental parameters are done according to published literature on celiac biopsy studieslf the data turns out to be inconclusive, other, in the field well-kniwnceliac markers of inflammation can be estimated. In addition, metabolites or components from the breads in the tissue samples can be chosen to be analysed

[0256] Results

[0257] Results from patients with a positive screening test for celiac disease (Transglutaminase Ig A >7 U / ml) are reported in this report (n=3). Duodenal biopsies have been taken from the patients during routine endoscopy examinations at Sahlgrenska University Hospital in Gothenburg or Kungalv Hospital in Kungalv. The biopsies (3 per patient) have been immediately transported to the laboratory at Chalmers University of Technology for cultivation and supplementation of sample bread. Samples (blank digest, reference bread or bread containing E304i / zinc sulfate according to experiment 1 (APZn bread produces from dough supplemented with ascorbyl palmitate (3% of flour weight) and Zinc sulphate heptahydrate (0.149% of the flour weight) was produced) were then added to each biopsy and incubated for 24 hours. After incubation, nutrient medium and biopsies were collected and frozen at -150 °C. Antibodies against TG2 (TG IgA) in medium were then analyzed with a commercial ELISA (Euro Hospital).

[0258] The wheat bread containing E304i / zinc sulfate (1 g E304i / 10 mg zinc per bun) led to an average of 51 %±10% (n=3 Patients) lower levels of TG2 antibodies in medium from the biopsies compared to the wheat bread without the additive (the reference bread). In addition, lower levels of TG2 antibodies were measured in the medium after exposure with bread containing E304i / zinc sulfate, compared to medium from biopsies without bread exposure (n=2 patients), which suggests a potential anti-inflammatory effect of the additive. The higher TG2 activity in non-bread-exposed biopsies may mean that the patients were exposed to gluten, knowingly or unconsciously, with subclinical representation (low clinical values of TG IgA). One patient had low TG2 background activity from the biopsy that increased with exposure to the unsupplemented bread, but no decrease was seen after exposure to bread containing E304i / zinc sulfate.

[0259] References

[0260] 1 . Molberg, O., et aL, Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med, 1998. 4(6): p. 713-7.

[0261] 2. Skovbjerg, H., et aL, Gliadin is a good substrate of several transglutaminases: possible implication in the pathogenesis of coeliac disease. Scand J Gastroenterol, 2002. 37(7): p. 812-7.

[0262] 3. Engstrom, N., et aL, Towards Celiac-safe foods: Decreasing the affinity of transglutaminase 2 for gliadin by addition ofascorbyl palmitate and ZnCI2 as detoxifiers. Sci Rep, 2017. 7(1): p. 77.

[0263] 4. Gianfrani C, Siciliano RA, Facchiano AM, Camarca A, Mazzeo MF, Costantini S, et aLT ransamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease. Gastroenterology. 2007;13(3):780-9

[0264] 5. Mazzarella G, Salvati VM, laquinto G, Stefanile R, Capobianco F, Luongo D, et aL Reintroduction of gluten following flour transamidation in adult celiac patients: a randomized, controlled clinical study. Clin Dev Immunol. 2012;2012:329150

Claims

Claims1 . A combination product of a gluten containing flour and an additive, wherein the additive comprises: i) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc at a concentration of 0.02-0.05% of the flour weight, characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

2. A combination product of a gluten containing flour and an additive according to claim 1 , wherein the additive comprises: i) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc sulphate with a molecular weight of 179.47 g / mol at a concentration of between 0.1-1 .5% of the flour weight, characterized in that the consumption of the combination product does not elicit a celiac immune response towards gluten.

3. A combination product of a gluten containing flour and an additive according to claim 1 or 2, wherein the additive comprises: i) ascorbyl palmitate (E304i) at a concentration of approximately 2.2-3% of the flour weightthat the consumption of the combination product does not elicit a celiac immune response towards gluten.

4. A combination product of a gluten containing flour and an additive according to any one of claims 1 or 3, wherein the additive comprises: i) Zinc at a concentration of 0.034% of the flour weight.

5. A combination product of a gluten containing flour and an additive according to any one of claims 1 - 4, wherein the flour contains at the most 10-15 g protein / 100 g flour, such as at the most 10 g protein / 100 g flour.

6. A combination product of a gluten containing flour and an additive according to any one of claims 1-5, wherein the flour is wheat flour and / or rye flour.

7. A combination product of a gluten containing flour and an additive according to any of the preceding claims, wherein the additive comprises ascorbyl palmitate (E304i) at a concentration of between 15-30 mg / g flour, such as 22-30 mg / g flour, such as at the most 30mg / g.

8. A combination product of a gluten containing flour and an additive according to any one of claims 1 - 7, wherein the additive prevents production of TG2 antibodies with at least 51 %±10% after exposure of the combination product to a biopsy of a celiac-intolerant subject.

9. A combination product of a gluten containing flour and an additive according to claim 8, wherein the additive fully prevents production of TG2 antibodies after consumption of the combination product in a celiac-intolerant subject.

10. A combination product of a gluten containing flour and an additive according to any of the preceding claims, wherein the immune response is measured as a standardised immunological determination of one or more celiac specific biomarker(s) in a sample from a celiac patient.

11. A combination product of a gluten containing flour and an additive according to any of the preceding claims, wherein the immune response is measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient, wherein the consumption of the combination product is determined not to elicit a celiac immune response towards gluten in the human intestine of a celiac patient when the level of tTG is below 10 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient, and / or when the level of tTG IgA is below 10 U / mL, such as below 9 U / mL, such as at the most 7 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient, and / or when the level of tTG IgG is below 10 U / mL, such as below 9 U / mL, such as at the most 7 U / mL, measured as a standardised immunological determination of anti-tTG antibodies in an intestinal tissue culture from a celiac patient.

12. A baked product comprising a combination product of a gluten containing flour and an additive according to any of the preceding claims, characterized in that theconsumption of the combination product does not elicit a celiac immune response towards gluten, which is a bakery product, such as selected from the group consisting of bread, portion bread, roll, toast bread, baguette, hamburger bun, Danish pastry, French pastry, puff pastry, muffin and cake.

13. A baked product according to claim 12, for use in a daily serving of Zn, which does not exceed 1 .25 mg / kg bw / day and / or for use in a daily serving of ascorbyl palmitate (E304i) of no more than 3-4 g / day, such as for use in a daily serving of ascorbyl palmitate (E304i) of no more than 1 ,6 g / day.

14. A process for producing a baked product according to any one of claims 12-13, comprising a combination product of a gluten containing flour and an additive according to any one of claims 1-11 , the process comprising the steps of: a) mixing1 ) the additive comprising : i) ascorbyl palmitate (E304i) at a concentration of between 1.1-3% of the flour weight, and ii) Zinc sulphate with a molecular weight of 179.47 g / mol at a concentration of between 0.005-0.0125% of the flour weight,2) water,3) a lipid and / or fat,4) yeast, and5) the gluten containing flour, b) proofing the dough at room temperature, and c) baking the product, wherein the heat in the baking step c) does not exceed 225°C, 220°C, 210°C, 200°C, 195°C or 190°C.

15. The use of a combination product of a gluten containing flour and an additive according to any one of claims 1-11 in the preparation of a baked product comprising a gluten containing flour, wherein the consumption of the baked product does not elicit a celiac immune response towards gluten in the human intestine of a celiac patient.

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