Wheat flour prepared from novel wheat

Abstract

The purpose of the present invention is to provide a food product having improved stickiness and meltability in the mouth as compared with food products produced from GA-SX wheat flour.　The problem is solved by altering the allele at a high-molecular-weight glutenin gene Glu-D1 of GA-SX wheat (a wheat in which the enzymatic activity of GBSSI-A1 is not deficient, the enzymatic activities of GBSSI-B1 and GBSSI-D1 are deficient, and the enzymatic activities of any two of SSIIa-A1, SSIIa-B1 and SSIIa-D1 are deficient) to a d-type allele.

Description

Flour prepared from new wheat

[0001] The present invention relates to a novel wheat strain that can be used as a raw material for wheat flour compositions used in food production, and more specifically to wheat flour prepared from wheat strains that lack the enzyme activities of two granule-bound starch synthase I (GBSSI) enzymes and two starch synthase type IIa (SSIIa) enzymes and that contain a d-type allele of the high-molecular-weight glutenin gene Glu-D1.

[0002] Wheat flour is widely used as an ingredient in various processed foods that are consumed daily as meals or snacks. Many of these processed foods are produced through a heating process, but changes in quality occur immediately after the heating process is completed. For example, bread has a very soft and moist texture immediately after baking. However, after a few hours or days, it becomes hard and dry. This phenomenon is generally called food aging, but in this invention it is referred to as deterioration. Since the progression of deterioration is directly related to the taste, reducing this progression is an important issue for the food industry.

[0003] To address this issue, a wheat flour (GA-SX wheat flour) was developed by milling wheat that lacked the enzyme activity of granule-bound starch synthase I (GBSSI)-A1, which is responsible for amylose synthesis, but lacked the enzyme activity of GBSSI-B1 and D1, and lacked the enzyme activity of any two of the enzymes starch synthase IIa (SSIIa)-A1, B1, and D1, which are involved in the side chain elongation of amylopectin (Patent Document 1: Japanese Patent No. 6226165). This wheat starch has a low amylose content and a structure in which the amylopectin side chains are shortened, resulting in a slow rate of starch retrogradation. As a result, the texture is soft, deterioration after production is slow, and the deliciousness can be maintained for a long time; these effects are particularly excellent in GA-SA wheat flour (wheat flour obtained by milling wheat that is not deficient in GBSSI-A1, but is deficient in the enzyme activities of GBSSI-B1 and D1, is not deficient in SSIIa-A1, and is deficient in SSIIa-B1 and D1).

[0004] However, foods made with GA-SX wheat flour suffer from the drawbacks of chewing and poor melt-in-the-mouth texture, particularly in bakery foods such as white bread. These chewy textures and poor melt-in-the-mouth texture are thought to be due to the starch composition described above. Wheat varieties with all three GBSSIs functioning contain approximately 25% amylose, whereas mutants lacking one contain 23-24%, a 1-2% reduction in amylose. Mutants lacking two GBSSIs produce approximately 20% amylose, while those lacking all three produce waxy starch containing no amylose. This difference in amylose content affects the gelatinization and retrogradation properties of starch. It is known that amylose retrogrades faster than amylopectin in gelatinized starch in the short term. Therefore, starches with a high amylose content tend to harden due to retrogradation, while waxy starches lacking amylose maintain a soft state. For example, in the case of bread, the texture is affected by the properties of starch, so the lower the amylose content, the softer the bread becomes, but on the other hand, as the proportion of amylopectin increases, the bread becomes chewy and melts in the mouth. GA-SX wheat has an amylose content of 20% or less (Non-Patent Documents 1 and 2), which is thought to be why it has a chewy texture and poor melt-in-the-mouth feel.

[0005] On the other hand, glutenin and gliadin are the major proteins in wheat, and when they coexist and come into contact with water, they form viscoelastic gluten. Glutenin forms large polymers by forming disulfide bonds between its molecules, contributing to the elasticity (strength) of wheat flour dough. Gliadin exists in the form of weakly bonded monomers via hydrogen bonds and other mechanisms, contributing to the extensibility of wheat flour dough. Glutenin is broadly classified into high-molecular-weight glutenin and low-molecular-weight glutenin. High-molecular-weight glutenin is encoded by the Glu-A1, B1, and D1 loci located on the long arms of wheat chromosomes 1A, 1B, and 1D, while low-molecular-weight glutenin is encoded by the Glu-A3, B3, and D3 loci located on the short arms of chromosomes 1A, 1B, and 1D. Numerous alleles (alleles) are known at each of these six loci, and the molecular weight and expression level of the encoded subunits vary depending on the type of allele, which is known to affect the secondary processability of wheat flour. Glu-D1 alleles include a (having a high-molecular-weight glutenin subunit pair "2+12"), c (4+12), d (5+10), and f (2.2+12) types (Non-Patent Document 3), and among them, Glu-D1d is known to be more effective in strengthening dough during breadmaking and increasing the volume of bread than other Glu-D1 alleles (Non-Patent Documents 4 and 5). Similarly, Glu-B3 alleles such as Glu-B3b, B3h, and B3i have been shown to be more effective in strengthening dough during breadmaking than other Glu-B3 alleles (c, j, ae) (Non-Patent Documents 4 and 6).

[0006] The present inventors have conducted extensive research to obtain wheat flour of even higher quality, and have unexpectedly found that the drawbacks of GA-SX wheat flour can be eliminated by converting GA-SX wheat to Glu-D1d allele, leading to the completion of the present invention. Furthermore, the present inventors have found that the drawbacks of GA-SX wheat flour can be further eliminated by converting GA-SX wheat to Glu-B3b or Glu-B3h allele, leading to the completion of the present invention.

[0007] Patent No. 6226165

[0008] Inokuma et al. , J. Agric. Food Chem. 2016, 64, 4, 941-947Inokuma et al. , J. Agric. Food Chem. 2021, 69, 7, 2271-2278 Payne and Lawrence, Cereal Research Communications, (1983) 11, 1, 29-35 Tatsuya Ikeda Journal of the Japanese Society of Food Chemical Engineering (2017) 64, 3, 171-176 Takata et al. , Breeding Science (2000) 50, 303-308 Zhang et al. , 2012, BMC Plant Biology, 12:243 Vrinten et al. , Mol. Gen. Genet. (1999), 261: 463-471 Saito et al. , Mol. Breeding, 2009, 23, 209-217 Shimbata et al. , 2005, Theor. Appl. Genet. , 111, 6, 1072-1079 Osakabe et al. , Proc. Natl. Acad. Sci. USA (2010) 107(26): 12034-12039 Ishikawa et al. (2005) Tohoku National Agricultural Research Center Research Report, 27-37 R. B. Gupta and K. W. Shepherd, Theor Appl. Genet. (1990) 80:65-74 Kojima et al. (2017) NARO Report Crop Development Center 1, 1-13 Wang et al. , Theor. Appl. Genet. 2009, 118: 525-539 Fukuda et al. (2010) Breeding Research 12, 87-95 Inagaki, M. (2001) Journal of Agriculture, Forestry and Fisheries Technology Research, Vol. 24, No. 12, 44-49 Nakamura et al. Genome, 2002, 45, 1150-1156

[0009] An object of the present invention is to provide a food product that has improved chewiness and melt-in-the-mouth properties compared to foods made from GA-SX wheat flour.

[0010] As a result of extensive research to solve the above problems, the present inventors have found that the defects of GA-SX wheat flour can be eliminated by converting the high-molecular-weight glutenin gene Glu-D1 of GA-SX wheat (wheat that does not lack the enzyme activity of GBSSI-A1, but lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, and lacks the enzyme activity of any two of SSIIa-A1, SSIIa-B1, and SSIIa-D1) to a d-type allele, thereby completing the present invention. Furthermore, the present inventors have found that the defects of GA-SX wheat flour can be further eliminated by converting the low-molecular-weight glutenin gene Glu-B3 to a b-type allele or an h-type allele, thereby completing the present invention.

[0011] That is, the present invention encompasses the following aspects: [1] Wheat flour (GA-SX / GD1d wheat flour) obtained by milling harvested wheat that does not lack the enzyme activity of GBSSI-A1, but lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, and lacks the enzyme activity of any two of SSIIa-A1, SSIIa-B1, and SSIIa-D1, and that has a d allele for the high-molecular-weight glutenin gene Glu-D1. [2] Wheat flour according to [1], which further has a b allele for the low-molecular-weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3b wheat flour). [3] Wheat flour according to [1], which further has an h allele for the low-molecular-weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3h wheat flour). [4] A flour composition comprising the wheat flour according to any one of [1] to [3]. [5] A method for producing a food product using the wheat flour according to any one of [1] to [3]. [6] The method according to [5], wherein the food product is a bakery food product.

[0012] Foods made using GA-SX wheat flour show reduced hardening (deterioration) during storage for several days after production. However, these products can have undesirable textures, such as poor melt-in-the-mouth texture and a chewy texture. By using wheat flour prepared from the wheat of the present invention, it is possible to obtain products with improved texture while maintaining the same level of deterioration reduction as conventional GA-SX wheat flour.

[0013] The wheat used in the present invention is wheat that does not lack the enzyme activity of GBSSI-A1, but lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, and lacks the enzyme activity of any two of SSIIa-A1, SSIIa-B1, and SSIIa-D1, and has a d-type allele for the high-molecular-weight glutenin gene Glu-D1 (GA-SX / GD1d wheat).Preferably, the wheat also has a b-type allele for the low-molecular-weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3b wheat) or has an h-type allele for the low-molecular-weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3h wheat).

[0014] Common wheat is an allohexaploid with three homoeologous genomes, A, B, and D, numbered 1 through 7 (1A-7A, 1B-7B, and 1D-7D). GBSSI, also known as Waxy (Wx), is a granule-bound starch synthase involved in the synthesis of amylose in wheat endosperm starch. GBSSI(Wx)-A1, GBSSI(Wx)-B1, and GBSSI(Wx)-D1 are encoded by genes located on chromosomes 7A, 4A, and 7D, respectively. Mutants lacking each enzyme function are known, and the amylose content varies depending on the combination of mutants. SSIIa is an enzyme involved in the elongation of the side chains (branch chains) of amylopectin in wheat endosperm starch. Like GBSSI, SSIIa-A1, B1, and D1 are functional in common wheat, and defective mutants for each are also known. SSIIa-A1, SSIIa-B1, and SSIIa-D1 are encoded by genes located on chromosomes 7A, 7B, and 7D, respectively. When one of the three enzymes is deficient, the amylopectin side chains are slightly shortened. When two enzymes are deficient, the degree of shortening increases, and when all three enzymes are deficient, the side chains are shortened to the greatest extent, resulting in a high-amylose wheat with an amylose content of over 30%.

[0015] "Lack of enzymatic activity" means that a protein having normal enzymatic activity is not functioning in the wheat plant, preferably that a protein having normal enzymatic activity is not expressed. Specific examples include mutations in gene sequence (such as substitution, deletion, insertion, inversion, or translocation of one or more bases, including deletion of the entire gene region), deficiency in mRNA transcription, deficiency in protein translation, and inhibition of enzymatic activity in the wheat plant. Any of these may be present as long as the enzymatic activity is reduced or eliminated to less than 10%, preferably less than 5%, and more preferably less than 1% of the wild-type enzymatic activity.

[0016] In this specification, "wheat that does not lack the enzyme activity of GBSSI-A1, but lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, and lacks the enzyme activity of any two of SSIIa-A1, SSIIa-B1, and SSIIa-D1" is referred to as "GA-SX wheat." GA-SX wheat includes the following wheats, which are combinations of two types of SSIIa deficient in enzyme activity: GA-SA wheat: wheat not deficient in GBSSI-A1 enzyme activity, deficient in GBSSI-B1 and GBSSI-D1 enzyme activities, not deficient in SSIIa-A1 enzyme activity, and deficient in SSIIa-B1 and SSIIa-D1 enzyme activities; GA-SB wheat: wheat not deficient in GBSSI-A1 enzyme activity, deficient in GBSSI-B1 and GBSSI-D1 enzyme activities, not deficient in SSIIa-B1 enzyme activity, and deficient in SSIIa-A1 and SSIIa-D1 enzyme activities; GA-SC wheat: wheat that does not lack the enzyme activity of GBSSI-A1, but lacks the enzyme activities of GBSSI-B1 and GBSSI-D1, and does not lack the enzyme activity of SSIIa-D1, but lacks the enzyme activities of SSIIa-A1 and SSIIa-B1. GA-SX wheat is wheat that can be produced by known methods, for example, the method described in JP 2013-188206 A.

[0017] As examples of so-called wild-type genes that are not deficient in enzyme activity, the sequences (genomic DNA and protein) of GBSSI-A1, B1, D1 and SSIIa-A1, B1, D1 are known, and are registered in GenBank under the following accession numbers. Each of these sequences is shown in the sequence listing as shown in Table 1 below.

[0018]

[0019] These sequences are examples of wild-type sequences, and naturally occurring wheat (including improved wheat varieties) may contain enzyme proteins with equivalent activity but with partial differences in their nucleotide or amino acid sequences. In the present invention, the terms "GBSSI-A1 gene" and "GBSSI-A1 protein" encompass not only those with a sequence completely identical to the nucleotide or amino acid sequence shown in the Sequence Listing, but also those with sequences containing natural mutations that do not impair enzymatic activity. The same applies to other enzymes. Such naturally occurring mutant sequences typically have 90% or more, for example, 95% or more, or 98% or more identity to the respective nucleotide or amino acid sequences shown in the Sequence Listing. In this specification, these GBSSI-A1, B1, D1, SSIIa-A1, B1, and D1 genotypes are referred to as GBSSI-A1a, B1a, D1a, SSIIa-A1a, B1a, and D1 alleles. As an example of a mutant lacking enzyme activity compared to these wild-type forms, a GBSSI-A1 mutant is known in which the expression of the encoded GBSSI-A1 protein is lost due to a genetic mutation in which 23 base pairs are deleted at the junction between the first exon and the following intron in the gene sequence of wild-type GBSSI-A1 (Wx-A1) and a different sequence of four bases is inserted (Non-Patent Document 7). This mutant is referred to herein as the GBSSI-A1b allele. A GBSSI-B1 mutant is known in which the entire gene region from the start codon to the stop codon of wild-type GBSSI-B1 (Wx-B1) is deleted (Non-Patent Document 8), and this mutant is referred to herein as the GBSSI-B1b allele. A known GBSSI-D1 mutant is characterized by a genetic mutation in which 588 bases around the stop codon are deleted and a 12-base different sequence is inserted in the wild-type GBSSI-D1 (Wx-D1) gene sequence, resulting in loss of GBSSI-D1 expression (Non-Patent Document 7). This mutant is referred to herein as the GBSSI-D1b allele. A known SSIIa-A1 mutant is characterized by a genetic mutation in which a 289-base region including the start codon is deleted and a 8-base different sequence is inserted in the wild-type SSIIa-A1 gene sequence, resulting in loss of SSIIa-A1 expression (Non-Patent Document 9). This mutant is referred to herein as the SSIIa-A1b allele.In SSIIa-B1, a mutant is known in which a 175-base insertion occurs within exon 8 in the wild-type SSIIa-B1 gene sequence, resulting in the creation of a stop codon, resulting in the loss of normal expression of the SSIIa-B enzyme protein (Non-Patent Document 9). This mutant is referred to herein as the SSIIa-B1b allele. In SSIIa-D1, a mutant is known in which a genetic mutation occurs in which 63 bases are deleted from the wild-type SSIIa-D1 gene sequence around the junction region of exon 5 and the following intron, resulting in the loss of normal expression of the SSIIa-D enzyme protein (Non-Patent Document 9). In the present invention, this mutant is referred to as the SSIIa-D1b allele.

[0020] "Glutenin" is the main protein in wheat, and when it comes into contact with water in the presence of gliadin, it forms viscoelastic gluten. Glutenin forms huge polymers by forming disulfide bonds between its molecules, and is involved in the elasticity (strength) of wheat flour dough. Gliadin exists in the form of a monomer weakly bonded by hydrogen bonds, etc., and is involved in the extensibility of wheat flour dough. Glutenin is broadly divided into high molecular weight glutenin and low molecular weight glutenin.

[0021] "High molecular weight glutenin" is encoded by the Glu-A1, B1, and D1 loci located on the long arms of wheat chromosomes 1A, 1B, and 1D, while "low molecular weight glutenin" is encoded by the Glu-A3, B3, and D3 loci located on the short arms of wheat chromosomes 1A, 1B, and 1D. Numerous alleles (alleles) are known at each of these six loci, and the molecular weight and expression level of the encoded subunits vary depending on the type of allele, which is known to affect the secondary processability of wheat flour. The wheat used in the present invention is GA-SX wheat, and the high molecular weight glutenin gene Glu-D1 is a d-type allele (GA-SX / GD1d). Preferably, the wheat further contains a b-type allele for the low-molecular-weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3b wheat), or a wheat further contains an h-type allele for the low-molecular-weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3h wheat). In another embodiment, the wheat is GA-SX wheat, the high-molecular-weight glutenin gene Glu-D1 is a d-type allele, and preferably the wheat further contains an i-type allele for the low-molecular-weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3i wheat).

[0022] The wheat used in the present invention, which is a GA-SX wheat, can be produced by crossing known wheat varieties lacking any combination of six enzymes in order to obtain genetic traits related to the deficiency of GBSSI and SSIIa enzyme activities. Mutagenesis may be performed using radiation treatment (gamma rays, beta rays, X-rays, neutrons, etc.), chemical treatment (ethyl methanesulfonate, etc.), or other mutagenesis treatments, followed by selection of the desired enzyme-deficient mutants for use in crossing. Various methods for producing monocotyledonous plant transformants are known, including genetic engineering techniques for disrupting the function of a target gene. For example, methods for inhibiting the expression of a target gene using RNAi or antisense techniques are available. Furthermore, gene disruption methods for disrupting only the target gene in plants are also known (Non-Patent Document 10). Therefore, these transformants can also be produced using genetic engineering techniques. In combination with the deficiency of two GBSSI and two SSIIa enzyme activities, the high-molecular-weight glutenin gene Glu-D1 allele type is the d type. Preferably, the allele type of the low-molecular-weight glutenin gene Glu-B3 is type b or type h. This may be achieved by first producing wheat having two GBSSI and two SSIIa enzyme activity deficiencies and then introducing the desired allele types of the high-molecular-weight glutenin gene Glu-D1, and preferably also the glutenin gene Glu-B3, or by selecting a wheat variety that has the desired allele types of the high-molecular-weight glutenin gene Glu-D1, and preferably also the glutenin gene Glu-B3, during the process of producing wheat having two GBSSI and two SSIIa enzyme activity deficiencies.

[0023] The Glu-D1d allele is based on the classification and nomenclature proposed in Non-Patent Document 3. The presence or absence of the Glu-D1d allele can be identified by detecting a unique base sequence in the Dx5 gene (GenBank accession number X12928: SEQ ID NO: 13), which encodes subunit 5 of the Glu-D1d allele. This identification can be achieved by PCR using the Dx_F, Dx5_F, and Dx_R primers (Dx_F: SEQ ID NO: 20, Dx5_F: SEQ ID NO: 21, Dx_R: SEQ ID NO: 22) described in Non-Patent Document 11. Alternatively, the region containing this unique mutation can be amplified using appropriately designed primers, and the gene sequence can be analyzed to confirm that it matches this sequence. Alternatively, a fraction containing high-molecular-weight glutenin protein can be extracted from wheat, separated by SDS-PAGE, and compared with known wheat samples to identify a band specific to the Glu-D1d allele (Non-Patent Document 3).

[0024] The Glu-B3b, B3h, and B3i alleles are based on the classification and nomenclature proposed in Non-Patent Document 12. These alleles can be identified using the following method. Whether or not a gene has a Glu-B3b allele can be determined by examining whether or not it has a sequence specific to Glu-B3b among the gene sequences described in Non-Patent Document 14 (GenBank Accession No. EU369719: SEQ ID NO: 14). This can be achieved by performing PCR using the SB2F and SB2R primers (SB2F: SEQ ID NO: 23, SB2R: SEQ ID NO: 24) described in Non-Patent Document 14 and examining whether an amplified fragment of the desired length is obtained. Alternatively, PCR can be performed using the LB1F and LB1R or LB4F and LB4R primers (LB1F: SEQ ID NO: 25, LB1R: SEQ ID NO: 26, LB4F: SEQ ID NO: 27, LB4R: SEQ ID NO: 28) described in Non-Patent Document 14, and the gene sequence of the resulting amplified fragment is analyzed to determine whether it matches the Glu-B3b gene sequence (GenBank accession numbers EU369700: SEQ ID NO: 15 or EU369719: SEQ ID NO: 14). Alternatively, a fraction containing low-molecular-weight glutenin protein can be extracted from wheat, separated by SDS-PAGE, and compared with known wheat samples to identify a band specific to the Glu-D1d allele (Non-Patent Document 12). Known wheat varieties that carry the Glu-B3b allele include "Takunekomugi" and "Nanbukomugi" (Non-Patent Document 13).

[0025] Whether or not a mutant has the Glu-B3h allele can be identified by examining whether or not it has a sequence specific to Glu-B3h among the gene sequences described in Non-Patent Document 14 (GenBank Accession No. EU369717: SEQ ID NO: 16). This can be achieved by performing PCR using the SB8F and SB8R primers (SB8F: SEQ ID NO: 29, SB8R: SEQ ID NO: 30) described in Non-Patent Document 14 and examining whether an amplified fragment of the desired length is obtained. Alternatively, PCR can be performed using the LB3F and LB3R primers (LB3F: SEQ ID NO: 31, LB3R: SEQ ID NO: 32) described in Non-Patent Document 14, and analyzing the gene sequence of the resulting amplified fragment to determine whether it matches the Glu-B3h gene sequence (GenBank Accession No. 369717: SEQ ID NO: 18). Alternatively, similar to the Glu-B3b allele, this can be confirmed by SDS-PAGE. Known wheat varieties that have the Glu-B3h allele include "Horoshiri Komugi" and "Haruyutaka" (Non-Patent Document 13).

[0026] Whether or not a gene has a Glu-B3i allele can be identified by examining whether or not it has a sequence specific to Glu-B3i among the gene sequences (GenBank accession number EU369720: SEQ ID NO: 19) described in Non-Patent Document 14. This can be achieved by performing PCR using the SB9F and SB9R primers (SB9F: SEQ ID NO: 33, SB9R: SEQ ID NO: 34) described in Non-Patent Document 14, and examining whether an amplified fragment of the desired length is obtained. Alternatively, PCR can be performed using the LB3F and LB3R or LB4F and LB4R primers (LB3F: SEQ ID NO: 35, LB3R: SEQ ID NO: 36, LB4F: SEQ ID NO: 37, LB4R: SEQ ID NO: 38) described in Non-Patent Document 14, and the gene sequence of the resulting amplified fragment is analyzed to determine whether it matches the Glu-B3i gene sequence (GenBank accession numbers EU369718: SEQ ID NO: 18, EU369720: SEQ ID NO: 17, or EU369714: SEQ ID NO: 19). Alternatively, similar to the Glu-B3b allele, it can be confirmed by SDS-PAGE. Known wheat varieties that carry the Glu-B3i allele include "Norin 61," "Minaminokaori," and "Iwainodaichi" (Non-Patent Document 15).

[0027] The gene sequences and primer sequences that can be used to identify each allele are shown in the sequence listings in Tables 2 and 3.

[0028]

[0029]

[0030] The wheat flour of the present invention is wheat flour (GA-SX / GD1d wheat flour) obtained by milling the harvest of the aforementioned GA-SX / GD1d wheat. Preferably, the wheat flour is wheat flour (GA-SX / GD1d / GB3b wheat flour) obtained by milling the harvest of GA-SX / GD1d / GB3b wheat, and more preferably, wheat flour (GA-SX / GD1d / GB3h wheat flour) obtained by milling the harvest of GA-SX / GD1d / GB3h wheat. The milling method is not particularly limited, and a typical milling method used to produce wheat flour from the harvest (caryopsis or seeds) of conventional wheat varieties can be used. The form of the wheat flour is not particularly limited; for example, it may be wheat flour from which components such as "bran" have been removed through a conventional milling process, or unfractionated whole wheat flour.

[0031] The wheat flour of the present invention can also be provided as a flour composition in which it is mixed with other wheat flours or flours other than wheat flour. Examples of other wheat flours include wheat flours such as strong flour, all-purpose flour, and soft flour, as well as wheat-derived flours not classified as any of these. Examples of flours other than wheat flour include, but are not limited to, flours derived from grains such as rice, rye, barley, corn, buckwheat, soybean, barnyard millet, foxtail millet, and amaranth. In the flour composition of the present invention, the content of the wheat flour of the present invention is preferably 5 to 100% by mass, more preferably 25 to 100% by mass, and even more preferably 50 to 100% by mass, based on the total amount of the flour composition. Most preferably, it is 100% by mass.

[0032] The wheat flour and flour compositions of the present invention have a d-type allele for the high-molecular-weight glutenin gene Glu-D1 (GA-SX / GD1d), which reduces deterioration to the same extent as conventional GA-SX wheat flour, while improving the chewiness and melt-in-the-mouth characteristics that have been problems with foods, particularly bakery foods, produced from conventional GA-SX wheat flour and flour compositions containing GA-SX wheat flour, thereby producing products with improved texture.Furthermore, the low-molecular-weight glutenin gene Glu-B3 has a b-type allele or an h-type allele, which makes it possible to produce products with an even improved texture.

[0033] The flour or flour composition of the present invention can be used to produce various foods that use flour or flour-containing flour compositions. Examples of such foods include bakery foods such as breads, cakes, baked goods, and pizzas; noodles such as udon and Chinese noodles; fried foods such as tempura and fries; baked foods such as gyoza, spring rolls, and shumai (wheat-flour-containing skins); and fish paste foods such as kamaboko and chikuwa. Preferably, the food is a bakery food. The food can be produced by a commonly used production method, except for using the flour or flour composition of the present invention.

[0034] Examples of bakery foods include breads such as white bread, French bread, rolls, and sweet rolls; fried breads such as yeast donuts; steamed breads; pizzas such as pizza pies; cakes such as sponge cakes; and baked goods such as cookies and biscuits. The bakery foods of the present invention can be produced, for example, by kneading the wheat flour or grain flour composition of the present invention with various auxiliary ingredients commonly used in the production of bakery foods, such as chemical leavening agents such as baking soda, yeast, yeast food, salt, sugars, oils and fats, eggs, dairy products, and water, to form a dough, which is then left to rise by fermentation or baked or deep-fried as is. Additives such as vitamins and minerals can be added as needed. Conventional production methods may be used to produce the bakery foods of the present invention.

[0035] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0036] <1. Development of Wheat> To develop the wheat of the present invention, wheat having the alleles in the table below was used.

[0037] (1) Development of SK wheat. SK wheat was developed by introducing the GBSSI-D1b, SSIIa-B1b, and D1b alleles from wheat (ii) into wheat (i) through sequential backcrossing. Backcrossing is a breeding method in which one parent variety (the recurrent parent) is crossed again with the progeny obtained from the cross of two varieties. Repeated crosses with the recurrent parent are called sequential backcrossing. This method is often used to efficiently introduce genes (e.g., disease resistance) that a particular superior variety lacks while maintaining many of its characteristics. The parent used in the recurrent cross is called the recurrent parent, and the parent that donates the desired gene is called the nociceptive parent. To develop SK wheat, sequential backcrossing was performed using wheat (i) as the recurrent parent and wheat (ii) as the nociceptive parent. The individuals obtained in each successive backcross cycle were identified for alleles at the GBSSI-D1, SSIIa-B1, and D1 loci, and individuals heterozygous for the a and b alleles at all three loci were selected for the next backcross. After backcrossing, the selected individuals were selfed to obtain progeny, and individuals homozygous for each of the eight alleles listed in Table 1 were selected.

[0038] (2) Production of HK wheat: Successive backcrossing was performed using wheat (iii) as the recurrent parent and wheat (iv) as the nodal parent, and wheat (iii)-A, in which the GBSSI-A1b and GBSSI-D1b alleles were introduced into wheat (iii), was selected. Similarly, successive backcrossing was performed with wheat (iii) using wheat (v) as the nodal parent, and wheat (iii)-B, in which the SSIIa-A1b, B1b, and D1b alleles were introduced into wheat (iii), was selected. Wheat (iii)-A and (iii)-B were crossed, and genetic fixation was performed using the haploid breeding method (Non-Patent Document 16). From the resulting population (DH population), HK wheats homozygous for each allele of the eight loci listed in Table 1 were selected.

[0039] (3) Creation of N1-1 wheat Wheat (vii) was crossed with wheat (viii), and the resulting F1 generation individuals were backcrossed with wheat (vi) as the recurrent parent. From this progeny, several individuals were selected that were homozygous for each allele of GBSSI-A1a, B1b, and SSIIa-A1a, and heterozygous for GBSSI-D1, SSIIa-B1, and D1. A next-generation population was obtained from these individuals, and then genetic fixation was performed using the haploid breeding method to obtain a DH population. From this DH population, N1-1 wheat was obtained that was homozygous for each allele of the eight loci listed in Table 1.

[0040] In the above crossbreeding and selection processes, the alleles at each locus were determined according to the following methods. Allele determination at the GBSSI-A1 and D1 loci was performed according to the method in Non-Patent Document 17. Allele determination at the GBSSI-B1 locus was performed according to the method in Non-Patent Document 8. Allele determination at the SSIIa-A1, B1, and D1 loci was performed according to the method in Non-Patent Document 9. Allele determination at the Glu-D1d allele was performed according to the method in Non-Patent Document 11. Allele determination at the Glu-B3b and B3h alleles was performed according to the method in Non-Patent Document 14. The Glu-B3i allele was identified by sequencing the gene sequence of a fragment amplified using the LB3F and LB3R primers described in Non-Patent Document 14 under the PCR conditions described in the same document, and examining the homology with the sequence of the Glu-B3i allele (GenBank accession number EU369718, SEQ ID NO: 18). DFBI wheat (MK 5-5 NIL wheat described in Non-Patent Document 2), a conventional GA-SX wheat, was used as a comparison example. The genotypes of each wheat are shown in Table 1 below.

[0041] Table 1. Genotypes (alleles) of each wheat variety

[0042] 2. Preparation of Wheat Flour The harvested wheat raw material was ground using a test mill manufactured by Buehler Co. Water was added to the wheat raw material to adjust the moisture content to 14%, and the wheat raw material was left to stand overnight. The wheat raw material was then placed in the test mill and ground to prepare flour with a milling yield of 60%.

[0043] <3. Bread-making test> Using the above wheat flour, two loaves of bread were made using the 70% standard dough method. 70 parts by weight of 100 parts by weight of wheat flour were used, and 2.3 parts by weight of yeast, 0.1 parts by weight of yeast food, and 40 parts by weight of water were added. Using a commercially available bread mixer (SK Mixer Co., Ltd., product name: SK200), the dough was mixed at low speed for 2 minutes and medium speed for 2 minutes to obtain a dough. This dough was fermented for 4 hours at a temperature of 27°C and a relative humidity of 75%. To this dough, 30 parts by weight of wheat flour, 5 parts by weight of sugar, 2 parts by weight of salt, 5 parts by weight of shortening, 2 parts by weight of skim milk powder, and an appropriate amount of water were added. The amount of water was determined in advance for each test group so that the dough properties (elasticity and stickiness) were equivalent. After mixing at low speed for 2 minutes, medium speed for 3 minutes, and high speed for 1 minute, the dough was scraped off, and further mixing was performed at low speed for 1 minute, medium speed for 3 minutes, and high speed for 7 minutes. The dough was fermented for 20 minutes, divided into 460-gram portions, molded, and placed in baking molds. The dough was then proofed at 38°C and 85% humidity. When the fermented dough reached 1 cm above the mold, it was baked (reel oven, 210°C, 25 minutes). After baking, the loaves were removed from the molds and allowed to cool at room temperature for 1 hour. They were then sealed in polyethylene bags, and one loaf was left at room temperature until the next day, while the other loaf was left until the third day. Each loaf was sliced ​​to a thickness of 12.5 mm before tasting, and 10 panelists evaluated the loaves according to the criteria shown in the evaluation criteria table (Table 2). The next-day evaluation was based on a score of 3.0 for the DFBI slices. For the third-day evaluation, the DFBI slices frozen the day after baking and thawed at room temperature for 4 hours on the day of evaluation were used as the standard (3.0 points).

[0044] Table 2 Evaluation criteria table

[0045] <Production Example> Bread was produced using wheat flour prepared from SK, HK, N1-1 and DFBI, and a sensory evaluation was carried out on the day after baking and on the third day. The results are shown in Table 3.

[0046] Table 3. Sensory evaluation results

[0047] In terms of softness, SK, HK, and N1-1 were equivalent to DFBI. On the other hand, in terms of crunchiness and melt-in-the-mouth feel, all three samples (SK, HK, and N1-1) scored higher than DFBI on both the day after baking and the third day. Among the three samples, HK was rated higher than N1-1 on both the day after baking and the third day, with SK receiving an even higher rating. From the above, it can be seen that the presence of Glu-D1d in GA-SX wheat significantly improved the crunchiness and melt-in-the-mouth feel of bread. Furthermore, this effect was greatest when Glu-B3h was also present, followed by Glu-B3b and B3i, in that order.

Claims

1. Wheat flour (GA-SX / GD1d wheat flour) obtained by milling a harvested wheat which is not deficient in the enzyme activity of GBSSI-A1, is deficient in the enzyme activities of GBSSI-B1 and GBSSI-D1, is deficient in the enzyme activity of any two of SSIIa-A1, SSIIa-B1 and SSIIa-D1, and has a d-type allele in the high molecular weight glutenin gene Glu-D1.

2. The wheat flour according to claim 1, further comprising a b-type allele in the low molecular weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3b wheat flour).

3. The wheat flour according to claim 1, further comprising an h-type allele in the low molecular weight glutenin gene Glu-B3 (GA-SX / GD1d / GB3h wheat flour).

4. A flour composition comprising the wheat flour according to any one of claims 1 to 3.

5. A method for producing food, which uses the wheat flour according to any one of claims 1 to 3.

6. The method of claim 5, wherein the food product is a bakery food product.

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