Consumption of hypoallergenic flour prevents gluten-induced airway inflammation in Brown Norway rats

Brown Norway rats were immunized with gluten, and then fed a diet containing hypoallergenic flour or an amino acid mixture. The rats were then made to inhale a solubilized gluten to induce gluten-specific bronchial asthma. The antibody levels in the serum of rats were measured by ELISA, and cell counts were done on cytospin preparations of bronchoalveolar lavage fluid. Body weight was decreased after allergen challenge in rats fed the amino acid mixture but not in rats fed the hypoallergenic flour. Antibody levels in the serum were significantly lower in rats fed hypoallergenic flour than in those fed the amino acid mixture. Differential cell counts in the bronchoalveolar lavage fluid showed that the numbers of eosinophils, lymphocytes, and neutrophils were significantly lower in rats fed the hypoallergenic flour than in those fed the amino acid mixture. These results suggest that hypoallergenic flour actively suppresses the allergic reactions, probably by inducing oral tolerance.     

Novel Method for Producing Hypoallergenic Wheat Flour by Enzymatic Fragmentation of the Constituent Allergens and Its Application to Food Processing

A novel method is proposed to produce hypoallergenic wheat flour suitable for patients allergic to wheat. Wheat flour was mixed with a cellulase solution, and the mixture was incubated at 50°C for 1 h to hydrolyze the carbohydrate allergens. The hydrolysate was further incubated with actinase at 40°C for 1 h while gently stirring to decompose the proteinaceous allergens. The product was evaluated for its allergenicity by an enzyme-linked immunosorbent assay, the results of which suggested negative allergenicity in most cases. The product changed to a batter state that was difficult to process by the usual methods. Gelatinization of the starch in the product and the addition of a surfactant were beneficial for food processing.     

Novel method for producing hypoallergenic wheat flour by enzymatic fragmentation of the constituent allergens and its application to food processing

A novel method is proposed to produce hypoallergenic wheat flour suitable for patients allergic to wheat. Wheat flour was mixed with a cellulase solution, and the mixture was incubated at 50 degrees C for 1 h to hydrolyze the carbohydrate allergens. The hydrolysate was further incubated with actinase at 40 degrees C for 1 h while gently stirring to decompose the proteinaceous allergens. The product was evaluated for its allergenicity by an enzyme-linked immunosorbent assay, the results of which suggested negative allergenicity in most cases. The product changed to a batter state that was difficult to process by the usual methods. Gelatinization of the starch in the product and the addition of a surfactant were beneficial for food processing.     

Analysis of food allergen structures and development of foods for allergic patients

Food allergy is one of the important health problems, and countermeasures are socially required. We have been undertaking studies on wheat allergens and their epitopes, and have developed a method for producing hypoallergenic wheat flour by enzymatic modification. The hypoallergenic products are now provided to patients. More noteworthy, by taking hypoallergenic cupcakes over a long period, more than half of patients are hyposensitized and become able to eat normal wheat products. This suggests that the hypoallergenic wheat flour can act as anti-allergenic via allergen-specific immunotolerance. This series of studies was followed by expansive research on food allergy: analysis of epitopes of bovine serum albumin (the major beef allergen), isolation and identification of inhibitory peptides for allergen absorption at the intestine, evaluation of hesperetin as an inhibitor of degranulation of mast cells, and the development of PCR detection methods for verifying allergen labeling and for identifying hidden allergic ingredients in processed foods.     

IgE-reactive 60 kDa Glycoprotein Occurring in Wheat Flour

A new IgE-reactive glycoprotein with a molecular size of 60 kDa was isolated from wheat flour. The N-terminal amino acid sequence of the protein was LDPDESEXVTRYFRIR. The 8th amino acid residue would have been Asn to which the peroxidase-type giycochain was attached. The IgE-binding activity of the glycoprotein was rendered negligible by the enzymatic treatment applied for hypoallergenic flour production.     

IgE-reactive 60 kDa glycoprotein occurring in wheat flour.

A new IgE-reactive glycoprotein with a molecular size of 60 kDa was isolated from wheat flour. The N-terminal amino acid sequence of the protein was LDPDESEXVTRYFRIR. The 8th amino acid residue would have been Asn to which the peroxidase-type glycochain was attached. The IgE-binding activity of the glycoprotein was rendered negligible by the enzymatic treatment applied for hypoallergenic flour production.     

Production Potency of Folate,Vitamin B12, and Thiamine by Lactic Acid Bacteria Isolated from Japanese Pickles

Hypoallergenic wheat products in the form of pasta-like noodles and bread were fabricated. Wheat flour was added with a 0.6-fold weight of water dissolving collagenase to obtain hypoallergenic flour batter. Methods for producing hypoallergenic pasta-like noodles and bread from the batter were designed. In the noodle making, the batter was mixed with an oligosaccharide with a mild sweetness, a surfactant, and salt, and the mixture was extruded under heating. Both retorting and refrigerating processes were applied to the noodles. In the bread making a mixture of the batter, glucose, citric acid, sodium hydrogen carbonate and salt was baked at 180°C. The addition of glucose contributed to generation of faborable flavor and color.     

Isolation and characterization of a novel polysaccharide as a possible allergen occurring in wheat flour

A new polysaccharide with a molecular weight of 5.0 x 10(4) was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with beta-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.     

Chemical and nutritional properties of hypoallergenic wheat flour

The chemical and nutritional properties were investigated of hypoallergenic wheat flour (HWF) prepared by the cellulase-actinase treatment. HWF was composed mainly of oligopeptides and free amino acids, and its average molecular weight was lower than 1,000. Feeding tests on rats showed that, with respect to the PER, GOT and GPT activities and other nutritional indices, the HWF diet was almost equivalent to the control diet which had been prepared from normal wheat flour (NWF). No abnormality was apparent in the main organs after the HWF diet had been fed for 3 weeks. The small intestinal absorption of the HWF diet was found normal by measuring the free amino acid concentration in the intestinal tract and in the portal vein plasma. These data suggest that the absorption of amino acids from the HWF diet was comparable with or more efficient than that from a simulated free amino acid diet.     

Chemical and Nutritional Properties of Hypoallergenic Wheat Flour

The chemical and nutritional properties were investigated of hypoallergenic wheat flour (HWF) prepared by the cellulase-actinase treatment. HWF was composed mainly of oligopeptides and free amino acids, and its average molecular weight was lower than 1,000. Feeding tests on rats showed that, with respect to the PER, GOT and GPT activities and other nutritional indices, the HWF diet was almost equivalent to the control diet which had been prepared from normal wheat flour (NWF). No abnormality was apparent in the main organs after the HWF diet had been fed for 3 weeks. The small intestinal absorption of the HWF diet was found normal by measuring the free amino acid concentration in the intestinal tract and in the portal vein plasma. These data suggest that te absorption of amino acids from the HWF diet was comparable with or more efficient than that from a simulated free amino acid diet.     

Improved Synthesis of 3-14C-3-Hydroxy-3-methylglutaryl Coenzyme A

A new polysaccharide with a molecular weight of 5.0×10⁴ was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with β-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.     

Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis

Background

Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS) and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition.

Results

Plants of Triticum aestivum 'Butte 86' were grown with or without post-anthesis fertilization (PAF) and quantitative 2-dimensional gel electrophoresis (2-DE) was used to analyze protein composition of the resulting flour. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, high molecular weight glutenin subunits (HMW-GS) and serpins as well as some alpha-gliadins increased in proportion with PAF. In contrast, alpha-amylase/protease inhibitors, farinins, purinins and puroindolines decreased in proportion. Decreases were also observed in several low molecular weight glutenin subunits (LMW-GS), globulins, defense proteins and enzymes. The ratio of HMW-GS to LMW-GS in the flour increased from 0.61 to 0.95 and the ratio of gliadins to glutenins increased from 1.02 to 1.30 with PAF. Because flour protein content doubled with PAF from 7 to 14%, most protein types actually increased in absolute amount (μg/mg flour protein). Data further suggest that flour proteins change with PAF according to their content of sulfur-containing amino acids Cys + Met.

Conclusions

A 2-DE approach revealed changes in the wheat flour proteome due to PAF that are important for flour quality and immunogenic potential. The work forms a baseline for further studies of the effects of environmental variables on flour protein composition and provides clues about the regulation of specific flour protein genes. The study also is important for identifying targets for breeding programs and biotechnology efforts aimed at improving flour quality.     

DNA microarray analyses of the effects of dietary proteins

Dietary proteins and amino acids serve not only as a building block for body components but also as regulators of a variety of body functions. A great many of the functions of ingested proteins, their peptide fragments, and amino acids have been characterized and some have been brought to practical application. Using a GeneChip DNA microarray system, we first compared the gene expression profiles among rats fed on 12% casein, 12% gluten, and protein-free diets for one week. The results revealed that a few hundred genes in the liver and muscle were up- or down-regulated by more than two-fold after feeding of the gluten or the protein-free diet. Interesting findings included the induction of genes for synthesis and catabolism of cholesterol by gluten feeding. In addition, we performed a study to examine the effect of the consumption of an enzymatically produced, hypoallergenic wheat flour on gene expression profiles in rats. The results confirmed the safety of this novel food product.     

Hydrolysis of wheat flour with amylase preparations and individual enzymes

Rheological properties of wheat flour were studied in the course of its processing (cooking and saccharification). The effects of commercial alpha-amylase preparations were compared during flour preparation. Test preparations were equally potent in decreasing the viscosity of an all-grain batch. Homogenous glucoamylases isolated from Aspergillus differed in the presence or absence of the starch-binding domain. The starch-binding domain provided for the high activity of glucoamylase on insoluble starch, but gave no advantages in saccharification of pretreated wheat flour.     

Hydrolysis of wheat flour with amylase preparations and individual enzymes

Rheological properties of wheat flour were studied in the course of its processing (cooking and saccharification). The effects of commercial α-amylase preparations were compared during flour preparation. Test preparations were equally potent in decreasing the viscosity of an all-grain batch. Homogenous glucoamylases isolated from Aspergillus differed in the presence or absence of the starch-binding domain. The starch-binding domain provided for the high activity of glucoamylase on insoluble starch, but gave no advantages in saccharification of pretreated wheat flour.     

Expression and epitope analysis of the major allergenic protein Fag e 1 from buckwheat

Seeds of common buckwheat (Fagopyrum esculentum) contain valuable nutritive substances but also allergenic proteins that cause hypersensitive reactions. Thus, the development of hypoallergenic buckwheat would make this important pseudo-cereal available to allergic people. A major allergenic protein of buckwheat is Fag e 1. We isolated the respective cDNA, coding for a 22 kDa protein, from a recently developed autogamous strain of common buckwheat and confirmed its immunoglobulin E (IgE)-binding activity using recombinant Fag e 1 and sera of allergic patients. The derived amino acid sequence from Fag e 1 cDNA was used to synthesize an overlapping peptide library on nitrocellulose membranes for the determination of the Fag e 1 epitopes. We identified eight epitopes and the critical amino acids for IgE-binding within the epitopes. This epitope analysis of a major allergenic protein of buckwheat should help therapeutic efforts and aid in the development of hypoallergenic buckwheat.     

Effectiveness of enzyme preparations of fodder in the degradation of nonstarch polysaccharides from grain substrates

The effects of commercial and laboratory preparations were compared in the course of treatment of components of compound fodder. The most potent preparations were selected for the treatment of soybean flower, sunflower meal, and wheat and barley flour. Preparation 181–1008, which had a high proteinase activity, provided the highest yield of protein from soybean flour and sunflower meal. Preparations aGA, AG20X, and VR, characterized by high activities of pectinase and α-galactosidase, as well as laboratory preparation B2000Mix with a high activity of α-galactosidase, provided the highest yield of sugars from soybean flour. Preparations with high α-galactosidase activity were the most potent in hydrolyzing soluble carbohydrates from soybean flour. The highest yield of reducing sugars was observed after treatment of wheat and barley flour with preparations B2000Mix and aGA. Xylanase activity of these preparations was lower than that of preparations 3.130.2 and TG20X. Preparations 3.130.2 and TG20X were the most potent in hydrolyzing wheat middlings.     

The future of antigen-specific immunotherapy of allergy

More than 25% of the population in industrialized countries suffers from immunoglobulin-E-mediated allergies. The antigen-specific immunotherapy that is in use at present involves the administration of allergen extracts to patients with the aim to cure allergic symptoms. However, the risk of therapy-induced side effects limits its broad application. Recent work indicates that the epitope complexity of natural allergen extracts can be recreated using recombinant allergens, and hypoallergenic derivatives of these can be engineered to increase treatment safety. It is proposed that these modified molecules will improve the current practice of specific immunotherapy and form a basis for prophylactic vaccination.