Foods containing buckwheat with a mutation in the SBE1 gene

Abstract

To obtain a buckwheat plant with modified starch properties and provide a buckwheat-containing food product with improved physical properties. [Solution] According to the present invention, a buckwheat-containing food product is provided which contains the seeds or pulverized product of a buckwheat plant whose starch gelatinization onset temperature is 90% or less compared to the wild type, and / or whose starch gelatinization peak temperature is 85% or less compared to the wild type.

Description

Technical Field

[0001] The present invention relates to a buckwheat-containing food containing the fruit or a pulverized product thereof of a buckwheat plant whose gelatinization start temperature is 90% or less compared to the wild type and / or whose gelatinization peak temperature is 85% or less compared to the wild type. The present invention relates to a method for producing a buckwheat-containing food, which includes using the fruit or a pulverized product thereof of a buckwheat plant whose gelatinization start temperature is 90% or less compared to the wild type and / or whose gelatinization peak temperature is 85% or less compared to the wild type as a raw material.

Background Art

[0002] Cultivated species of buckwheat plants classified in the Polygonaceae family include buckwheat (Fagopyrum esculentum) (hereinafter referred to as common buckwheat), tartary buckwheat (F. tataricum), and perennial buckwheat (F. cymosum). In Japan, common buckwheat and tartary buckwheat are mainly used as foods.

[0003] Generally, when using grains as foods, ensuring softness after food production is one of the important factors, and starch has a great influence on them. Starch is roughly classified into amylose in which glucose is polymerized linearly and amylopectin with branched structures. In grains, when the chain length of amylopectin becomes shorter, the food remains soft even when cooled, the gelatinization temperature decreases, and the water separation during freezing and thawing decreases.

[0004] Amylopectin chain length is elongated by starch synthase (SSIIa). Rice amylopectin is mainly classified into S-type and L-type depending on the presence or absence of SSIIa activity. S-type amylopectin has a higher proportion of short amylopectin side chains and a lower starch gelatinization initiation temperature than L-type amylopectin. For example, Japonica rice lacks SSIIa and therefore has S-type amylopectin, which does not harden easily when cooled. Indica rice has L-type amylopectin and tends to become crumbly when cooled. Furthermore, it has been reported that buckwheat plants lacking SSIIa activity have urea disintegration rates that are 1.5 times higher than the wild type, and their gelatinization peak temperature is 95% or less than that of the wild type (Patent Document 1).

[0005] Starch branching enzyme 1 (SBE1) is an enzyme involved in the formation of amylopectin side chains. It is thought that SSIIa extends these side chains. It has been reported that rice varieties into which a functionally deficient rice SBE1 gene has been introduced have a higher proportion of short amylopectin side chains and reduced gluten hardening ability (Non-Patent Literature 1). Furthermore, it has been reported that rice varieties lacking SBE1 activity (SSIIa activity deficient and SBE1 activity deficient) have a starch gelatinization peak temperature measured by differential scanning calorimeter approximately 4°C lower than that of Koshihikari (SSIIa activity deficient) (Non-Patent Literature 2). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2022-140409 [Non-patent literature]

[0007] [Non-Patent Document 1] Suzuki, Taro. 2019, "Development of 'Aichi Mochi 126,' a glutinous rice variety lacking the activity of starch branching enzyme 1 and exhibiting low mochi hardening properties," Journal of Plant Breeding Science, Vol. 21, 28-34. doi: 10.1270 / jsbbr.18J13 [Non-Patent Document 2] Noriaki Aoki, University of Tsukuba, Doctoral dissertation, 2015, "Breeding and physiological studies on suitability for rice flour bread" https: / / cir.nii.ac.jp / crid / 1910020910756854400 [Overview of the project] [Problems that the invention aims to solve]

[0008] In rice, there are reports that gelatinization temperature decreases in varieties lacking both SSIIa activity and SBE1 activity (Non-Patent Literature 2), but there are no reports for rice varieties lacking only SBE1 activity. To date, there is no knowledge as to whether the starch gelatinization temperature actually changes due to the suppression or deficiency of SBE1 activity in buckwheat.

[0009] To expand the demand for buckwheat, it is crucial to develop buckwheat-containing foods with improved physical properties. To meet the diverse tastes and cooking methods of consumers, it is necessary to improve the starch properties of buckwheat seeds and flour. In particular, there is a growing need for buckwheat seeds and flour with a lower starch gelatinization temperature. Therefore, the objective of this invention is to obtain a buckwheat plant with modified starch properties and to provide buckwheat-containing foods with improved physical properties. [Means for solving the problem]

[0010] The inventors obtained a buckwheat plant in which a mutation in the SBE1 gene had occurred, resulting in a lower starch gelatinization temperature compared to the wild type, from among buckwheat plants that had undergone mutation treatment. They found that food products made from this buckwheat plant showed suppressed deterioration of physical properties, thus completing the present invention.

[0011] The present invention provides the following inventions. [1] Buckwheat-containing food products containing the seeds or pulverized product of a buckwheat plant whose gelatinization onset temperature is 90% or less of that of the wild type, and / or whose gelatinization peak temperature is 85% or less of that of the wild type. [2] The buckwheat-containing food described in [1], wherein deterioration of physical properties due to the passage of time after manufacturing is suppressed. [3] Buckwheat-containing food as described in [1] or [2], wherein the water separation rate after 7 days of refrigerated storage is less than 20%. [4] A buckwheat-containing food product as described in any one of [1] to [3], wherein the water separation rate after 7 days of frozen storage is less than 20%. [5] A buckwheat-containing food according to any one of [1] to [4], which contains the seeds or crushed product of a buckwheat plant having reduced or deficient starch branching enzyme 1 (SBE1) activity. [6] A method for suppressing deterioration of the physical properties of buckwheat-containing foods, characterized by using the seeds or pulverized material of buckwheat plants having reduced or deficient starch branching enzyme 1 (SBE1) activity as raw materials. [7] A method for producing a buckwheat-containing food product, comprising using the seeds or pulverized material of a buckwheat plant whose gelatinization onset temperature is 90% or less of that of the wild type, and / or whose gelatinization peak temperature is 85% or less of that of the wild type, as a raw material. [Brief explanation of the drawing]

[0012] [Figure 1A] Figure 1A shows the estimated cDNA sequence of the wild-type buckwheat SBE1b gene. In the mutant, the cytosine (C) at position 74 of sequence number 2 is deleted. [Figure 1B] Figure 1B shows the putative cDNA sequence in alternative splicing of the wild-type buckwheat SBE1b gene. In the mutant, the adenine (A) at position 712 of sequence number 10 is changed to cytosine (C). [Figure 1C] Figure 1C shows the amino acid sequences of the SBE1b putative translation product (wild type) and the SBE1b putative translation product (mutant = buckwheat UD1). In the mutant, a frameshift occurs due to the deletion of cytosine (C) at position 74 of the putative cDNA sequence of the buckwheat SBE1b gene (SEQ ID NO: 2). As a result, the amino acid residue at position 26 of the amino acid sequence of the buckwheat SBE1b putative translation product is threonine (T) in the wild type (SEQ ID NO: 4), but proline (P) in the mutant (SEQ ID NO: 5). In the mutant (SEQ ID NO: 5), an early stop codon occurs at position 46. [Figure 1D]Figure 1C shows the amino acid sequence of the SBE1b putative translation product (wild type) when alternative splicing occurs (SEQ ID NO: 11). In the buckwheat SBE1b putative translation product amino acid sequence (SEQ ID NO: 11), the amino acid residue at position 238 is lysine (K) in the wild type, but glutamine (Q) in the mutant. [Figure 1E] Figure 1E shows the genome sequence of the wild-type buckwheat SBE1a gene (SEQ ID NO: 6). The bases of the exon region are shown in bold and underlined. [Figure 1F] Figure 1F shows the genome sequence of the wild-type buckwheat SBE1b gene (SEQ ID NO: 7). Bases in the exon region are shown in bold and underlined. In alternative splicing, it is presumed that the ATG located at position 124 in the genome sequence shown in SEQ ID NO: 7 is used as the translation start codon, and the region from positions 4373 to 5168 (shown in italics) is spliced ​​out as an intron, resulting in the splicing product shown in SEQ ID NO: 10. On the other hand, in the cDNA base sequence shown in SEQ ID NO: 2 (which can be considered another splicing product), the ATG located at position 4373 becomes the translation start codon. [Figure 2] Figure 2 shows the urea disintegration properties of buckwheat seeds from the wild type "IH3" and the SBE1b mutant "Sugae UD1 strain". [Figure 3] Figure 3 is a graph showing the gene expression levels of SSIIa1, SSIIa2, SBE1a, and SBE1b genes in maturing seeds. [Figure 4] Figure 4 is a graph showing the change over time in the breaking load of refrigerated noodle sheets produced from buckwheat flour of wild-type buckwheat "IH3", low-amylose buckwheat "Kyushu No. 12" (lacking only GBSSa), and SBE1b mutant buckwheat "Sukei UD1". [Figure 5] Figure 5 is a graph comparing the amount of water released from mochi (rice cakes filled with sweet bean paste) after refrigeration (left) and after freezing (right) storage, for mochi made using wild-type or Su-type UD1 buckwheat flour. [Figure 6] Figure 6 is a graph comparing the amount of water released from steamed bread made using wild-type or Su-type UD1 buckwheat flour after refrigeration (left) and after freezing (right).

BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The description of the present invention given below may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, the positions in a base sequence are counted starting from the base at the 5'-end of the base sequence as the 1st base. The positions in an amino acid sequence are counted starting from the amino acid at the N-terminus of the sequence as the 1st amino acid.

[0014] [Buckwheat-containing food] The present invention provides a buckwheat-containing food containing the fruit or a pulverized product thereof of a buckwheat genus plant in which the starch gelatinization start temperature is 90% or less compared to the wild type and / or the starch gelatinization peak temperature is 85% or less compared to the wild type.

[0015] The present invention also provides a buckwheat-containing food containing the fruit or a pulverized product thereof of a buckwheat genus plant that satisfies at least one of the following. · The starch urea breakdown property is increased compared to the wild type. · The alkali breakdown property of buckwheat grains is increased compared to the wild type. · The starch gelatinization start temperature measured by a differential scanning calorimeter is 59.5°C or lower. · The starch gelatinization peak temperature measured by a differential scanning calorimeter is 65°C or lower.

[0016] In this specification, a "buckwheat genus plant in which the starch gelatinization start temperature is 90% or less compared to the wild type and / or the starch gelatinization peak temperature is 85% or less compared to the wild type" may be referred to as a "buckwheat genus plant with reduced gelatinization temperature".

[0017] The present invention provides a buckwheat-containing food in which deterioration of physical properties over time after production is suppressed, using the fruit or a pulverized product thereof of a buckwheat genus plant with reduced gelatinization temperature as a raw material.

[0018] In this specification, with respect to buckwheat-containing foods, "physical properties" refers to physical properties related to texture (hardness, elasticity, chewiness, dryness, freshness, etc.), quality (shelf life, hardening due to refrigeration or freezing, syneresis due to refrigeration or freezing, etc.), and appearance (gloss, color, etc.). In certain embodiments, the deterioration of the physical properties of buckwheat-containing foods is due to hardening over time after manufacturing. In certain embodiments, the deterioration of the physical properties of buckwheat-containing foods is due to hardening after refrigeration.

[0019] The physical properties of buckwheat-containing foods, particularly their hardness, can be measured as the breaking load when the food's thickness (2.0 mm) is compressed over 2 seconds (0.5 mm / sec). The breaking load can be measured using, for example, a physical property testing system (Yamaden Co., Ltd. RE2-3305C-1). The measurement temperature can be set to 25°C.

[0020] The inventors compared the breaking loads of noodle dough made from buckwheat flour of wild-type buckwheat "IH3", low-amylose buckwheat "Kyushu No. 12" (lacking only GBSSa), and SBE1b mutant buckwheat "Sukei UD1 series," as shown in the examples described below. SBE1b mutant buckwheat "Sukei UD1 series" is a buckwheat plant with a reduced gelatinization temperature. The breaking load of wild-type buckwheat "IH3" and low-amylose buckwheat "Kyushu No. 12" increased with the passage of storage time, but the breaking load of SBE1b mutant buckwheat "Sukei UD1 series" did not increase even after 9 days. Breaking load is an indicator of hardness. In SBE1b mutant buckwheat "Sukei UD1 series," deterioration of physical properties due to the passage of time after manufacturing was suppressed.

[0021] In certain embodiments, when the buckwheat-containing food of the present invention is processed into a 2 mm thick noodle sheet, the breaking load is 1.5 N or less, 1.4 N or less, 1.3 N or less, 1.2 N or less, 1.1 N or less, or 1.0 N or less. In certain embodiments, the breaking load of the buckwheat-containing food of the present invention does not increase after storage at 4°C. The breaking load was measured by holding the noodle sheet at 25°C and compressing it in the thickness direction at a speed of 0.5 mm / sec. For the buckwheat-containing food of the present invention, the breaking load was measured on the day of manufacture (immediately after manufacture without refrigeration), as well as on the 3rd, 6th, and 9th days of storage at 4°C. Although the largest change compared to the day of manufacture was observed on the 6th day of storage (-16.3% compared to the day of manufacture), the breaking load did not increase compared to the day of manufacture, confirming that the softness of the noodles is well maintained even during refrigerated storage.

[0022] In a specific embodiment, the buckwheat-containing food of the present invention has a syneresis rate of less than 20% after 7 days of refrigerated storage, for example, less than 19%, less than 18%, or 17% or less. Refrigerated storage was performed by covering the food with a kitchen towel, placing it in an airtight container, and storing it at 4°C for 7 days. After storage, the food was left at room temperature (25°C) for 60 minutes before its weight was measured. The water separation rate (%) was calculated using the following formula. Water separation rate (%) = (Weight at the start of storage - Weight at the time of measurement) ÷ Weight at the start of storage × 100

[0023] In a specific embodiment, the buckwheat-containing food of the present invention has a water separation rate of less than 20% after 7 days of frozen storage. The buckwheat-containing food of the present invention has a water separation rate of less than 20% after 7 days of frozen storage, for example, less than 19%, less than 18%, or 17% or less. Frozen storage was performed by covering the food with a kitchen towel, placing it in a sealed container, and storing it at -20°C for 7 days. After storage, the weight of the food was measured after leaving it at room temperature (25°C) for 60 minutes. The water separation rate (%) was calculated using the above formula.

[0024] In certain embodiments, the buckwheat-containing food of the present invention exhibits a lower water separation rate after 7 days of refrigerated or frozen storage compared to food produced using wild buckwheat as a raw material under the same conditions. The water separation rate is 70% or less, 60% or less, or 55% or less of the water separation rate of the comparative food made from wild buckwheat. The water separation rate serves as an indicator of softness and freshness.

[0025] Using buckwheat seeds or flour with a low starch gelatinization temperature in food production can shorten cooking times and provide a smoother, softer texture. A lower starch gelatinization temperature may also improve workability during food production (dough cohesiveness, stretchability, etc.) and shorten the boiling time for buckwheat. Buckwheat seeds and flour with a low starch gelatinization temperature also contribute to suppressing the deterioration of physical properties over time after production, such as hardening and starch retrogradation during refrigerated storage, and suppressing the decrease in moisture retention. Starch retrogradation is a major cause of hardening and deterioration of texture in buckwheat-containing foods during refrigerated storage, but starch with a low gelatinization temperature is less likely to recrystallize, thus maintaining a good texture in buckwheat-containing foods even during storage. Such improvements are expected to enhance consumer convenience and satisfaction, and expand the market for buckwheat-containing foods.

[0026] In relation to the present invention, "seeds" refer to the seeds of a plant of the genus Buckwheat, and include buckwheat grains. Buckwheat grains are unmilled buckwheat seeds, particularly those with the hull removed, known as hulled grains. Buckwheat grains may include the bran, endosperm, and germ. In some embodiments, buckwheat grains may not include the bran.

[0027] In this invention, the pulverized material is obtained by roller milling buckwheat (grains) and contains buckwheat flour. The pulverized material is separated using a sieve to obtain buckwheat flour. In roller milling, the buckwheat is turned into flour from the center outwards, and the flours are called first-grade flour, second-grade flour, and third-grade flour in the order they emerge.

[0028] The present invention relates to buckwheat-containing foods such as noodles (fresh noodles, boiled noodles, etc.), bread (e.g., sliced ​​bread, sweet bread, bagels, steamed bread, and butter rolls), pizza, mixed flours (e.g., fried chicken flour, tempura flour, bread mix, pancake mix, okonomiyaki mix, and takoyaki mix), confectionery (cookies, biscuits, crackers, bolo, snacks, sponge cake, manju, dango, senbei, arare, okaki, etc.), and mochi. Buckwheat-containing foods further include boiled buckwheat grains, buckwheat porridge (e.g., kasha), and hulled buckwheat grains for mixed grain rice. Buckwheat is used as grains (kasha) in Russia, for example, but because of its high amylose content, the starch tends to retrograde over time after cooking, resulting in a poor texture (it becomes hard and brittle). However, in this invention, by using buckwheat seeds from a plant of the genus Buckwheat, which has a low starch gelatinization temperature, the texture does not deteriorate easily even after time has passed since cooking.

[0029] In relation to the present invention, when referring to starch gelatinization temperature or gelatinization temperature, unless otherwise specified, it refers to the value (°C) measured for buckwheat flour (endosperm) obtained by milling buckwheat seeds obtained from buckwheat plants using a conventional milling machine. The gelatinization temperature can be measured by differential scanning calorimetry (DSC).

[0030] More specifically, for example, after removing the hulls from buckwheat seeds, approximately 10 mg of buckwheat flour is weighed out and placed in a silver pan. Distilled water is added to achieve a starch concentration of 30% (based on dry weight, weight / weight). Using this sealed pan with the added distilled water as a reference, the scan is performed from 25°C to 130°C at a heating rate of 2°C / minute.

[0031] Specifically, for the buckwheat plants that are the raw materials for the buckwheat-containing food of the present invention, the degree of decrease in the gelatinization peak temperature (gelatinization peak temperature for the target buckwheat plant (°C) / gelatinization peak temperature for the wild type (°C) × 100) is 90% or less, preferably 89% or less, more preferably 88% or less, even more preferably 87% or less, 86% or less, and 85% or less. According to the inventors' studies, the gelatinization peak temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 85% when compared to wild-type buckwheat.

[0032] Alternatively, for the buckwheat plant used as a raw material for the buckwheat-containing food of the present invention, the gelatinization peak temperature is 65°C or lower, preferably 64°C or lower, and more preferably 63°C or lower. According to the inventors' studies, the gelatinization peak temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 62.7°C.

[0033] Specifically, for the buckwheat plants that are the raw materials for the buckwheat-containing food of the present invention, the degree of decrease in the gelatinization onset temperature (gelatinization onset temperature for the target buckwheat plant (°C) / gelatinization onset temperature for the wild type (°C) × 100) is 85% or less, preferably 84% or less, more preferably 83% or less, and even more preferably 82% or less. According to the inventors' studies, the gelatinization onset temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 81.2% when compared to wild-type buckwheat.

[0034] Alternatively, for the buckwheat plant used as a raw material for the buckwheat-containing food of the present invention, the gelatinization start temperature is 59.5°C or lower, preferably 59°C or lower, more preferably 58°C or lower, 57°C or lower, 56°C or lower, or 55°C or lower. According to the inventors' studies, the peak gelatinization temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 54.5°C.

[0035] The buckwheat plant used as a raw material for the buckwheat-containing food of the present invention has improved starch properties. Specifically, compared to conventional buckwheat plants, it has a higher proportion of short-chain amylopectin side chains, resulting in increased urea-disintegration and alkali-disintegration properties, as well as a lower gelatinization temperature. Measuring amylopectin chain length requires special equipment and is relatively complicated to perform. However, evaluating urea-disintegration and alkali-disintegration properties does not require special equipment, and if the difference is large, it can be observed with the naked eye, and the procedure is simple. By evaluating the urea-disintegration and alkali-disintegration properties of the buckwheat plant used as a raw material for the buckwheat-containing food of the present invention, it is possible to estimate the inhibitory effect on the deterioration of physical properties over time after manufacturing.

[0036] In relation to the present invention, when referring to urea disintegration, unless otherwise specified, it refers to the urea disintegration properties of buckwheat flour (endosperm) obtained by milling buckwheat seeds obtained from the target buckwheat plant using a conventional milling machine. Urea disintegration properties may sometimes be expressed as the degree of urea disintegration in the starch of whole grain flour, but those skilled in the art can perform appropriate calculations for buckwheat flour milled using a milling machine and determine the difference in the degree of urea disintegration by comparing it with buckwheat flour distributed on the market. Generally, buckwheat flour is produced by rolling buckwheat (grains) and separating the crushed material with a sieve.

[0037] The urea-disintegrating properties of starch can be quantified by colorimetrically measuring the iodine-starch reaction with starch solubilized in 1-4 M urea from buckwheat flour. More specifically, for example, after removing the hulls from buckwheat seeds, approximately 20 mg of buckwheat flour, ground in a mortar, is weighed into a 0.5 ml tube, 1 ml of 3 M urea is added, and the mixture is stirred at 25°C for 16 hours (200 rpm). 180 μL of iodine solution (0.002% iodine-0.02% potassium iodide solution) is added to 20 μL of the supernatant, and the absorbance at 690 nm is measured using a microplate reader (ThermoScientific: MultiskanFC). At this time, the absolute value of the measurement obtained (absorbance divided by the weight of the buckwheat flour) may be used for evaluation, but it may also be evaluated using a relative value compared to a control buckwheat flour (buckwheat flour obtained from wild-type buckwheat, or commercially available buckwheat flour).

[0038] A high proportion of short-chain amylopectin side chains in starch can be detected as a difference in urea disintegration, that is, a difference in the degree to which amylopectin is solubilized in the presence of urea. In other words, for example, when starch is disintegrated (solubilized) in the presence of 3M urea, short amylopectin is more easily solubilized than long amylopectin. Therefore, the amylopectin chain length can be measured by comparing the soluble fractions after color development using the iodine-starch reaction (Hirokazu Sato, Shinichi Saito, Tomohiko Yoshida. Selection method of glutinous rice varieties based on mochi hardening properties, gelatinization characteristics and urea disintegration properties. Journal of the Crop Science Society of Japan, 74(3), 310-315 (2005) doi:10.1626 / jcs.74.310).

[0039] The alkali-disintegrating properties of buckwheat grains can be determined by observing whether or not starch dissolves after immersing the grains in an alkaline solution for several hours. For example, if hulled buckwheat grains are immersed in a 1.5% potassium hydroxide solution and left to stand at room temperature, the grains are considered highly alkali-disintegrating if starch dissolution is observed, and less alkali-disintegrating if no starch dissolution is observed.

[0040] If the urea-disintegrating properties of buckwheat or the alkali-disintegrating properties of buckwheat grains can be increased, it is possible to keep cooked foods soft, and by modifying their physical properties, it is thought that their use can be expanded to food applications other than noodles.

[0041] (SBE1 activity-deficient buckwheat plants) In one embodiment, the present invention provides a buckwheat-containing food product containing the seeds or pulverized product of a buckwheat plant having reduced or deficient starch branching enzyme 1 (SBE1) activity. Starch branching enzyme 1 (SBE1) is an enzyme that synthesizes amylopectin side chains and is a type of enzyme that catalyzes the formation of α-1,6-glucosidic bonds by glycosyltransferase reactions.

[0042] In this invention, reduced or deficient starch branching enzyme 1 (SBE1) activity means that the function of the SBE1 protein in the plant is reduced or not functioning normally compared to the wild type. In this invention, reduced or not functioning normally of the SBE1 protein is not limited to cases where SBE1 activity is reduced by a specific mechanism. Reduced or deficient activity includes not only the suppression of SBE1 enzyme activity but also the suppression of SBE1 gene expression. More specifically, suppression of activity includes not only the inhibition of SBE1 activity but also mutations in the SBE1 gene that prevent the production of active SBE1, reduced SBE1 activity, and mutations in the expression regulatory regions of the SBE1 gene (promoter region, transcription factor binding region, splicing-related region, etc.) that suppress SBE1 production.

[0043] Whether SBE1 activity is reduced or absent can be evaluated by the proportion of short amylopectin side chains (approximately 6-11 degrees of polymerization) compared to the wild type. Furthermore, a higher proportion of short amylopectin side chains compared to the wild type can be evaluated by increased urea disintegration, increased alkali disintegration, a lower gelatinization initiation temperature, and a lower gelatinization peak temperature, as will be described later. The proportion of short amylopectin side chains can also be measured by freeing the side chains through enzymatic debranching (e.g., isoamylase treatment), followed by separation and quantification using HPAEC-PAD (High Performance Anion Exchange Chromatography / Pulsed Amylopectin Peripheral Detection). This method allows for highly accurate quantification of the short chain content.

[0044] As shown in the examples described below, in buckwheat plants, the SBE1a and SBE1b genes are expressed, and the SBE1a and SBE1b proteins are considered to function normally. Buckwheat plants in which these proteins function normally can be said to be wild-type plants. In this specification, when referring to buckwheat plants, SBE1 refers to SBE1a and SBE1b unless otherwise specified. In a particular embodiment, "wild-type buckwheat plant" refers to variety IH3 (developed by the Hokkaido Agricultural Research Center). This variety is available from the National Agriculture and Food Research Organization (Plant Production Science, 20(4), 384-388).

[0045] In buckwheat plants exhibiting reduced or deficient starch branching enzyme 1 (SBE1) activity, the reduction or deficiency of SBE1 activity is attributed to mutations in the SBE1 gene. Mutations in the SBE1 gene according to this invention can be obtained through spontaneous mutation or natural selection during breeding, as well as through chemical mutagenesis treatment or introduction using artificial genome editing technology. Chemical mutagenesis treatment is not particularly limited and includes methods such as inducing mutations by base substitution using alkylating agents such as ethyl methanesulfonate (EMS). On the other hand, artificial genome editing technology is not particularly limited and can be achieved by introducing deletions, substitutions, or insertions into target base sequences using CRISPR / Cas systems, TALENs, ZFNs, etc.

[0046] Buckwheat plants with reduced or absent SBE1 activity have mutations in at least one of the SBE1a and SBE1b genes, which suppress the function of the gene.

[0047] In buckwheat plants with reduced or absent SBE1 activity, the wild-type SBE1 gene having the aforementioned mutation may be one of the following: (1) The SBE1a gene as defined in (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 6, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 6, and which encodes a protein having SBE1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 3, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence. (2) The SBE1b gene as defined in (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 7, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 7, and which encodes a protein having starch branching enzyme 1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 4 or 11, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence.

[0048] With regard to the present invention, considering polymorphisms of the SBE1 gene among species, varieties, and strains of buckwheat plants, these variations are also included within the scope of the present invention. Therefore, the base identity to SEQ ID NO: 6 or SEQ ID NO: 7 is 90% or higher, and may be 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99%. Furthermore, with regard to the present invention, considering polymorphisms of the SBE1 protein sequence among species, varieties, and strains of buckwheat plants, these variations are also included within the scope of the present invention. Therefore, the amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 11 is 90% or higher, and may be 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99%.

[0049] The sequence listing of this application shows the sequence of the buckwheat SBE1a gene (wild type) as sequence number 6 and the sequence of the buckwheat SBE1b gene (wild type) as sequence number 7. In addition, the putative cDNA sequence of the buckwheat SBE1a gene (wild type) is shown as sequence number 1, the putative cDNA sequence of the buckwheat SBE1b gene (wild type) is shown as sequence number 2, and the putative cDNA sequence in alternative splicing of the buckwheat SBE1b gene (wild type) is shown as sequence number 10.

[0050] Furthermore, the sequence listing of this application includes the amino acid sequence of buckwheat SBE1a (wild type) as SEQ ID NO: 3, the amino acid sequence of buckwheat SBE1b (wild type) as SEQ ID NO: 4, and the amino acid sequence of buckwheat SBE1b (wild type, alternative splicing product (protein isoform produced by alternative splicing)) as SEQ ID NO: 11. Alternative splicing is a fundamental gene expression regulatory mechanism inherent in buckwheat plants, and this alternative splicing product is considered to be the normal wild-type isoform of the SBE1b gene.

[0051] In one embodiment, mutations in the SBE1 gene are, for example, nucleotide changes due to the substitution, deletion, or insertion of one or more nucleotides compared to the wild-type sequence. Mutations that the buckwheat plants of the present invention may have may include, for example, nonsensical mutations (such as nonsense and missense mutations), frameshift mutations, in-frame insertions or deletions, splice site mutations, and / or mutations in expression regulatory regions such as promoters or enhancers. Nonsense mutations (which result in stop codons) and missense mutations generally produce incomplete and nonfunctional proteins. Frameshift mutations cause a shift in the codon reading frame, resulting in the translation of a protein with a completely different amino acid sequence from the original, often leading to the appearance of early stop codons and the production of incomplete proteins. Splice site mutations generally result in abnormal splicing, the generation of abnormal mRNA, and the production of nonfunctional proteins. Mutations in expression regulatory regions may lead to a decrease in mRNA transcripts compared to the wild-type sequence, thereby reducing protein synthesis. These phenomena are also true for multiple mRNAs resulting from alternative splicing. The mutations in the buckwheat plants of the present invention are not limited to the above mutations, but may be any mutations that lead to a decrease or deficiency of SBE1 activity.

[0052] In one embodiment, the mutation in the above-mentioned buckwheat plant is a frameshift mutation, which results in the appearance of an early stop codon and prevents the synthesis of normal SBE1 protein, thus reducing or eliminating SBE1 activity. In another embodiment, the mutation in the above-mentioned buckwheat plant is a missense mutation, which results in amino acid substitution and prevents the synthesis of normal SBE1 protein, thus reducing or eliminating SBE1 activity. In another embodiment, the alternative splicing shown in the present invention is an inherent characteristic of buckwheat plants, but even in that case, the above-mentioned missense mutation in the buckwheat plant of the present invention leads to the synthesis of abnormal SBE1 protein, resulting in reduced or eliminating SBE1 activity. As shown in the examples described later, the buckwheat plant "Sugaya UD1 strain" having a mutation in the SBE1 gene has a frameshift mutation in the SBE1b gene, and it is considered that the function of the SBE1b gene is suppressed. Specifically, a frameshift occurs due to the deletion of cytosine (C) at position 74 of the predicted cDNA sequence (SEQ ID NO: 2) of the buckwheat SBE1b gene (position 4446 in the genome sequence (SEQ ID NO: 7)). As a result, the amino acid residue at position 26 of the amino acid sequence of the predicted buckwheat SBE1b translation product is threonine (T) in the wild type (SEQ ID NO: 4), but proline (P) in the mutant (SEQ ID NO: 5), and the subsequent amino acid sequence also differs from the wild type. Furthermore, in the mutant (SEQ ID NO: 5), a stop codon occurs at position 46, and the amino acid length is significantly shorter compared to the wild type. Therefore, it is thought that the activity of the buckwheat SBE1b protein is deficient in the "Su-type UD1 strain." In addition, the "Su-type UD1 strain," a buckwheat plant with a mutation in the SBE1 gene, has a substitution from a basic amino acid residue to a neutral amino acid residue in the amino acid sequence of a key region that is highly conserved in SBE1 orthologs, etc., in the alternative splicing product. It is thought that this substitution significantly reduces or eliminates the activity of the buckwheat SBE1b protein. Specifically, in the mutant, the adenine (A) at position 712 of the putative cDNA (SEQ ID NO: 10) in the alternative splicing of the wild-type buckwheat SBE1b gene (position 8194 in the genome sequence (SEQ ID NO: 7)) is changed to cytosine (C).This mutation causes the amino acid residue at position 238 of the amino acid sequence (SEQ ID NO: 11) of the buckwheat SBE1b predicted translation product to be glutamine (Q) in the mutant, whereas in the wild type it is lysine (K).

[0053] Therefore, the present invention is (i) A polynucleotide containing a sequence in which the base at position 4446 in the sequence shown in SEQ ID NO: 7 is deleted, or a polynucleotide in which the base at position 8194 in the sequence shown in SEQ ID NO: 7 is changed from adenine to cytosine, (ii) A polynucleotide containing a sequence in which the base at position 4446 is deleted in a sequence that has 90% or more sequence identity with the sequence shown in Sequence ID No. 7, or a polynucleotide in which the base at position 8194 in the sequence shown in Sequence ID No. 7 is changed from adenine to cytosine. The present invention provides a buckwheat-containing food product containing the seeds or crushed product of a buckwheat plant having [specific characteristic].

[0054] Sequence ID 7 is the sequence of the buckwheat SBE1b gene (wild type). The cytosine at position 4446 in the sequence shown in Sequence ID 7 corresponds to the cytosine at position 74 in the putative cDNA sequence of the buckwheat SBE1b gene (Sequence ID 2). Also, the adenine at position 8194 in the sequence shown in Sequence ID 7 corresponds to the adenine at position 712 in the putative cDNA sequence of the buckwheat SBE1b gene in alternative splicing (Sequence ID 10).

[0055] The mutations described above are not the only ones that may produce similar activity deficiency effects. For example, mutations that delete or replace cysteine ​​residues related to the three-dimensional structure of a protein that greatly affects its activity, mutations that delete or replace amino acids in the active site with amino acids of a different polarity, or mutations that delete or replace one or more amino acids with other amino acids of a different polarity in an amino acid region that is highly conserved among plants are thought to reduce or eliminate SBE1 activity, and similar effects can be expected from such mutations.

[0056] The presence of a gene mutation can be confirmed by methods such as those listed below: decoding the mutated base sequence itself using base sequence analysis methods such as the Sanger assay or next-generation DNA analysis technology; detecting differences in the cleavage status of genomic DNA or PCR amplification fragments using restriction enzymes that recognize the mutated base sequence; detecting differences in the mutated base sequence using technologies such as LGC Genomics' KASP (Kompetitive Allele Specific PCR) genotyping assay or high-resolution melting curve (HRM) analysis; or PCR-SSCP (Single Nucleotide Conformation Polymorphism). Detection may also be performed using a DNA marker linked to the mutated base region.

[0057] Mutations in the SBE1 gene or its expression regulatory region may be carried out by various means. Examples of methods for obtaining buckwheat plants with mutations in the SBE1 gene or its expression regulatory region include selection from mutant strains, genetic engineering, RNA interference, genome editing, artificial genome synthesis, and genome methylation. In a preferred embodiment, selection from mutant strains is used because it allows for more reliable production of the plant.

[0058] Buckwheat plants with reduced or deficient SBE1 activity may be bred from existing varieties or lines. Buckwheat plants obtained in this way can be distinguished from existing varieties or lines by at least one of the following characteristics: a high proportion of short amylopectin side chains, high urea-disintegration and alkali-disintegration properties, and a low gelatinization peak temperature.

[0059] There are no particular limitations on the varieties and lines used as starting materials when acquiring mutant strains, or the varieties and lines used in breeding. Examples of buckwheat varieties include Kitawase Soba, Botan Soba, Kitayuki, Hashikami Wase, Iwate Wase, Mogami Wase, Hashikami Wase, Hitachi Aki Soba, Shinano No. 1, Shinshu Oosoba, Shinano Natsu Soba, Fukui Zairaishu, Kochi Zairaishu, Miyazaki Ootsubu, AOI, KOMA, NARO-FE-1, Akiakane, Gamma no Irodori, Kitano Mashu, Kitamitsuki, Great Ruby, Cobalt no Chikara, Sachiizumi, Sanrutin, and Soba Intermediate Parent No. 1. Examples of buckwheat varieties include Tachiakane, Dewakaori, Toyomusume, Natsumi, Nijiyutaka, Horominori, Miyazaki Ootsubu, Ruchiking, Reranokaori, Natsuyoshi, Aizu no Kaori, Kaida Wase, Miyazaki Wase Kaori, Takamine Ruby, Takamine Ruby 2011, Yamagata BW5, Izumo no Mai, Haru no Ibuki, Hitachi Akisoba, Shin'ei Red, Shinshu Oosoba, Nagano S11, Nagano S8, Shimada Scarlet, Hida No. 1, and Hokkai No. 3. Examples of buckwheat varieties include Shin'ei Yellow, Hokkai T8, Hokkai T9, Hokkai T10, Hokuriku No. 4, Ki no Chikara, Ki no Takara, Ki no Yutaka, Daizen, Shinano Kurotsubu, Daruma Dattan, Aeon no Kisai, Manten Kirari, Nishi no Haruka, and Aeon no Kisai. Many of these can be obtained from seed companies or related organizations.

[0060] An example of a buckwheat plant with reduced or absent SBE1 activity is the "Su-kei UD1 line." The "Su-kei UD1 line" is self-pollinating and is a line obtained by the method described in the examples herein, in which the mutant SBE1 gene is fixed to homozygosity. The Su-kei UD1 line has increased urea disintegration compared to the wild type, and this increase in urea disintegration is a stable trait observed over multiple growing seasons.

[0061] The inventors of this invention were the first to demonstrate that a deficiency in SBE1 activity in buckwheat plants alters the properties of the starch they contain. Starch branching enzyme 1 (SBE1) is an enzyme involved in the formation of amylopectin side chains, and it is thought that SSIIa extends these side chains. In rice, there are reports on varieties lacking both SSIIa and SBE1 activity (e.g., Non-Patent Document 1), but there are no reports on the properties of starch in varieties lacking only SBE1 activity. Therefore, it was impossible to predict what effect a lack of SBE1 activity in buckwheat would have on the properties of starch.

[0062] Furthermore, rice has one SBE1 gene, while buckwheat has two. The two isozymes in buckwheat may have different effects. In addition, rice is a monocotyledonous plant, while buckwheat is a dicotyledonous plant, and they are phylogenetically distant. Therefore, it was impossible to predict how effective the remaining isozymes would be without actually inducing mutations in the isozymes in buckwheat.

[0063] The present invention provides a buckwheat-containing food product containing the seeds or ground product of a buckwheat plant whose starch properties have been modified by reducing or eliminating the activity of at least one of the following proteins. (A) Proteins consisting of the amino acid sequences shown in Sequence ID No. 3, 4, or 11; (B) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 3, 4, or 11, and that has starch branching enzyme 1 activity in buckwheat plants; (C) A protein having the activity of starch branching enzyme 1 of buckwheat plants, consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 3, 4, or 11.

[0064] Furthermore, with respect to the present invention, considering the polymorphism of SBEI due to differences in species, varieties, and strains, these mutations are also included within the scope of the present invention. Therefore, the amino acid sequence identity to SEQ ID NOs: 3, 4, or 11 is 90% or higher, preferably 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99%. Similarly, the number of deletions, substitutions, or additions in the amino acid sequence is 1 to 60, but may be 1 to 30, 1 to 9, or 1 to 4. If the substitutions are to amino acids with similar properties, there may be even more substitutions, etc. Means for creating polynucleotides or proteins relating to such amino acid sequences are well known to those skilled in the art. With respect to the present invention, the fact that a protein has the starch branching enzyme 1 activity of buckwheat plants can be confirmed, for example, by measuring the amylopectin chain length and observing a high ratio of short amylopectin side chains. The ratio of short-chain amylopectin side chains can be measured by freeing the side chains through enzymatic debranching and then separating and quantifying them using HPAEC-PAD. Furthermore, it can also be confirmed by measuring urea disintegration, alkali disintegration, and starch gelatinization temperature.

[0065] In this invention, when referring to a base sequence (sometimes called a nucleotide sequence) or an amino acid sequence, unless otherwise specified, it means the percentage of matching nucleotides or amino acids shared between two sequences when the two sequences are aligned in the most optimal manner. That is, identity can be calculated as (number of matching positions / total number of positions) × 100, and can be calculated using commercially available algorithms. Such algorithms are incorporated into the NBLAST and XBLAST programs described in Altschuletal., J.Mol.Biol.215(1990)403-410. More specifically, the search and analysis of the identity of base sequences or amino acid sequences can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, the parameters can be appropriately set by those skilled in the art, or the default parameters of each program may be used. The specific methods of these analysis methods are also well known to those skilled in the art.

[0066] In this specification, when referring to a high degree of identity with respect to a base sequence or amino acid sequence, unless otherwise specified, it means a sequence identity of at least 70%, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97.5% or more, and even more preferably 99% or more.

[0067] [Methods to suppress physical property degradation] The present invention provides a method for suppressing the deterioration of the physical properties of buckwheat-containing foods, characterized by using buckwheat seeds or their pulverized product as raw materials, in which the activity of starch branching enzyme 1 (SBE1) is reduced or deficient. The deterioration of the physical properties of buckwheat-containing foods occurs over time after manufacturing.

[0068] The present invention provides a method for suppressing the deterioration of the physical properties of buckwheat-containing foods, which can suppress the deterioration of physical properties of refrigerated or frozen foods. According to the present invention, it is possible to provide buckwheat-containing foods that retain their softness and freshness even after refrigeration or freezing. In a particular embodiment, the present invention provides a method for improving the physical properties of foods stored at chilled temperatures, such as boiled noodles.

[0069] In certain embodiments, the present invention further provides a method for preventing deterioration of the texture of boiled noodles, characterized by using the seeds or pulverized product of buckwheat plants having reduced or deficient starch branching enzyme 1 (SBE1) activity as a raw material. The texture of boiled noodles can be evaluated by sensory evaluation by a panel of experts.

[0070] By using buckwheat flour obtained from buckwheat plants with reduced or deficient starch branching enzyme 1 (SBE1) activity as a raw material, the noodles become softer and chewier immediately after production compared to those made with buckwheat flour from wild-type buckwheat. Furthermore, even after refrigeration, the noodles remain soft, chewier, less crumbly, and more delicious. Using buckwheat flour obtained from buckwheat plants with reduced or deficient starch branching enzyme 1 (SBE1) activity as a raw material can prevent deterioration of texture after refrigeration.

[0071] In certain embodiments, the present invention further provides a method for preventing deterioration of the texture of buckwheat puffed food, characterized by using buckwheat seeds or their pulverized product as a raw material, wherein the buckwheat seeds have reduced or deficient starch branching enzyme 1 (SBE1) activity. In this specification, "buckwheat puffed food" refers to food made using buckwheat flour, and means food made by puffing up dough containing buckwheat flour through heating such as baking, steaming, or frying. Specifically, examples include Western-style confectionery, Japanese-style confectionery, bread, etc. According to the present invention, it is possible to provide buckwheat puffed food that retains its softness and freshness even after refrigeration or freezing.

[0072] [Method for producing buckwheat-containing food products] The present invention provides a method for producing buckwheat-containing food products, comprising using the seeds or pulverized product of a buckwheat plant whose gelatinization onset temperature is 90% or less of that of the wild type, and / or whose gelatinization peak temperature is 85% or less of that of the wild type, as a raw material. The method may include mixing the seeds or pulverized product with other raw materials, and heating the mixture of the seeds or pulverized product and the other raw materials.

[0073] Buckwheat flour dough is difficult to handle, and to make foods with a high buckwheat content, it is necessary to add binders or other auxiliary ingredients. For example, there is a technique to make buckwheat noodles by gelatinizing buckwheat flour to increase its adhesiveness and using it as a binder (e.g., Patent No. 4403144), but this requires high-temperature processing using a twin-screw extruder, which may result in the loss of buckwheat flavor. In addition, there is the problem of the added step of gelatinizing the buckwheat flour after milling. According to the method for producing buckwheat-containing foods of the present invention, by using the seeds or pulverized products of buckwheat plants with a reduced gelatinization temperature as raw materials, foods with a high buckwheat content (for example, 100% buckwheat noodles) can be easily produced.

[0074] In one embodiment, the present invention provides a method for producing a buckwheat-containing food product, which includes kneading a raw material containing pulverized seeds of a buckwheat plant having a gelatinization onset temperature of 90% or less compared to the wild type, and / or a gelatinization peak temperature of 85% or less compared to the wild type, and water, to produce a dough, and shaping the dough.

[0075] In certain embodiments, the present invention provides a method for producing chilled noodles, comprising kneading a raw material containing pulverized seeds of a buckwheat plant having a gelatinization onset temperature of 90% or less compared to the wild type, and / or a gelatinization peak temperature of 85% or less compared to the wild type, and water, to produce a dough, and shaping the dough. Chilled noodles are noodles that are distributed or stored in the chilled temperature range, and may be fresh noodles, semi-fresh noodles, or boiled noodles. In certain embodiments, it is preferable that the chilled noodles are boiled noodles that are distributed and stored in the chilled temperature range. The "chilled temperature range" refers to temperature conditions in which the average temperature is within the range of 0°C to 10°C.

[0076] Chilled boiled noodles are in high demand because they can be easily eaten by pouring hot or cold noodle soup over them, or by loosening them with water and dipping them in hot or cold noodle soup. However, there was a problem with soba noodles becoming hard and crumbly when stored at chilled temperatures. By using buckwheat flour obtained from buckwheat plants with a lower gelatinization temperature as a raw material, compared to using buckwheat flour from wild buckwheat, the noodles remain soft, chewy, and less crumbly even after refrigeration, resulting in delicious noodles.

[0077] The method for producing buckwheat-containing food products of the present invention may include materials other than the seeds or pulverized products of buckwheat plants, as long as they do not hinder the property deterioration suppression effect of the present invention. For example, other powder materials besides buckwheat flour include wheat flour, rice flour, and starch flour. Auxiliary materials include water, whole eggs, egg whites, egg yolks, seaweed binders, yam binders, oils and fats, milk, and dairy products. Depending on the type of buckwheat-containing food product produced by the method of the present invention, the type and amount of auxiliary materials can be selected as long as they do not hinder the property deterioration suppression effect of the present invention. The final water content relative to the total powder raw material can be in the range of approximately 35-98%, approximately 40-98%, approximately 50-98%, or approximately 60-98%, with the total moisture content of the powder raw material being 0%. [Examples]

[0078] Example 1: Acquisition of a buckwheat plant with an SBE1 gene mutation method Mutation treatment 100g of self-pollinating buckwheat (Fagopyrum esculentum, strain name IH3) seeds were placed in 200mL of ethyl methanesulfonate (0.1%~2.5%) and stirred at 25°C or similar conditions at 150 revolutions per minute. After 2 to 16 hours, the seeds were removed and sown in pots or fields (NARO Kyushu Okinawa Agricultural Research Center (2421 Suya, Koshi City, Kumamoto Prefecture 861-1192) or NARO Hokkaido Agricultural Research Center (4-banchi, Shinsei Minami 9-sen, Memuro-cho, Kasai-gun, Hokkaido 082-0081)). Seeds were obtained from each plant after fruiting. The harvested seeds were sown again in pots or fields, and leaves and seeds were collected for subsequent analysis.

[0079] Urea disintegration test The hulls of fully ripened buckwheat seeds were removed, immersed in 3M urea for 3 days, and then iodine solution was added to observe the color change. Specifically, after removing the hulls from the buckwheat seeds, approximately 20 mg of buckwheat flour, ground in a mortar, was weighed into a 0.5 mL tube, 1 mL of 3M urea was added, and after standing at 25°C, 180 μL of iodine solution (0.002% iodine - 0.02% potassium iodide solution) was added to 20 μL of the supernatant. A bluish-black color was determined to indicate high urea disintegration.

[0080] Gelatinization onset temperature, gelatinization peak temperature, enthalpy The hulls were removed from fully ripened buckwheat seeds, which were then crushed into beads in a plastic tube. The results were then measured using a differential scanning calorimetry (DSC, Seiko Instruments Inc., DSC6100). 10 mg (dry weight) of crushed buckwheat seeds was weighed into a 70 μL silver sealed sample container (Seiko Instruments Inc. for DSC), and distilled water was added to a concentration of 30% (w / w) (dry weight). After sealing the sample container, a sealed sample container containing the same weight of distilled water was used as a blank, and the gelatinization temperature and heat quantity were measured at a heating rate of 2 °C / min from 25 °C to 130 °C. The obtained data was analyzed using the software attached to the differential scanning calorimeter, and the gelatinization temperature and enthalpy change were calculated.

[0081] Enthalpy is the "heat of gelatinization," and a larger value indicates that more heat is required for gelatinization (it is harder to gelatinize). The gelatinization properties of amylopectin molecules are influenced by the length of their side chains (degree of polymerization). As the side chains become longer, the number of hydrogen bonds between them increases, which increases the crystallinity of the amylopectin molecules and requires more thermal energy to gelatinize (break down) the molecules. Therefore, the higher the amylopectin content in starch granules and the longer the side chain length, the more heat of gelatinization is required.

[0082] Genotype determination Genomic DNA was extracted from leaves, and the sequences obtained using a next-generation DNA sequencer (Illumina HiSeq2000) were mapped to a reference genome sequence to investigate mutations. The reference genome was obtained based on information from the Buckwheat Genome Database (BGDB) https: / / buckwheat.kazusa.or.jp / .

[0083] result 32,000 seeds were treated with ethyl methanesulfonate, and 12,000 plants were collected. These were then selected using urea disintegration testing, followed by genotyping, and two lines with mutations in the SBE1 gene were obtained. One of these two lineages is the "Su-type UD1 lineage." The "Su-type UD1 lineage" has a single nucleotide deletion in the exon of the SBE1b gene, resulting in an early stop codon, so its translation product was expected to be shorter than that of the wild type (Figure 1C). Therefore, SBE1b activity may be reduced or absent. Furthermore, RNA sequencing data analysis suggested that at least two translation products of SBE1b may be generated from the same genome through alternative splicing. Due to alternative splicing, in the wild type, it is thought that in addition to the SBE1b putative translation product with the amino acid sequence shown in SEQ ID NO: 4, an SBE1b putative translation product with the amino acid sequence shown in SEQ ID NO: 11 is produced. In the "Su-type UD1 lineage," it is thought that the lysine at position 238 of the amino acid sequence shown in SEQ ID NO: 11 is changed to glutamine. This mutation may be causing reduced or absent SBE1b activity. To fix the mutation in a homozygous state, seeds were sown again, and the process of self-pollination and confirmation of the presence or absence of the gene mutation was repeated. Once the gene mutation was fixed in a homozygous state, the plant was designated as the SBE1 gene mutant buckwheat plant "Sugae UD1 line". The SSIIa1, SSIIa2, and SBE1a genes in the "Sugae UD1 strain" were wild-type. The SSIIa1, SSIIa2, SBE1a, and SBE1b genes in the self-pollinating buckwheat strain "IH3" were confirmed to be wild-type. As shown in Figure 2, the "Sugaya UD1 strain" showed increased urea disintegration compared to the wild-type "IH3". As shown in Table 1, compared to the wild-type "IH3," the "Sugae UD1" strain had a starch gelatinization onset temperature approximately 12°C lower, a gelatinization peak temperature approximately 11°C lower, and an enthalpy change of approximately 3 J / g less. A t-test showed a statistically significant difference at the 0.1% level from the mean (N=3). [Table 1]

[0084] In the "Su-type UD1 strain," a mutation in the SBE1b gene leads to a functional defect in the gene's translation product, which is thought to shorten the amylopectin side chains and lower the starch gelatinization initiation temperature. The "Su-type UD1 strain" is a mutation in which only one of the two SBE1 genes is deleted, but the gelatinization peak temperature is reduced by approximately 11°C, which is comparable to the 10°C reduction in gelatinization peak temperature obtained when both SSIIa and SBE1 are deleted in rice. The basis for claiming that the gelatinization peak temperature of "rice lacking SSIIa and SBE1" is 10°C lower compared to "rice with wild-type SSIIa and SBE1" is that there is a report that the gelatinization peak temperature of "rice lacking SSIIa and SBE1" is 4°C lower compared to "rice lacking SSIIa and wild-type SBE1" (Non-Patent Literature 2), and furthermore, there is a report that both the gelatinization onset temperature and peak temperature are about 5°C lower for general japonica rice (Nipponbare; gelatinization onset temperature 60.0°C, gelatinization peak temperature 66.0°C) compared to indica rice (representatively BMBirinari rice; gelatinization onset temperature 65.5°C, gelatinization peak temperature 70.5°C) (Kyoko Tanaka et al., "Comparison of Cooking Characteristics of Myanmar Indica Rice and Japanese Japonica Rice," Journal of Food Science and Technology of Japan, Vol.41, No.L11~17 (2008)). In buckwheat, it is thought that a sufficient gelatinization temperature effect was obtained by simply deleting the SBE1b gene. In cereal starch synthesis, it is thought that SBE1 transfers a short glucose chain to amylose, generating a short amylopectin chain (side chain formation), and that SSIIa then extends this side chain (Reference: Yasunori Nakamura, "Rice as a material for starch synthesis research," Journal of the Japan Society for Biotechnology, 2017, Vol. 95, No. 4). However, since amylopectin is present even in rice lacking SBE1, it is thought that there are other side chain formation mechanisms besides SBE1.

[0085] Example 2: Comparison of gene expression levels method Total RNA was extracted from the endosperm of buckwheat seeds during the maturation process of wild-type buckwheat ("IH3"), converted to cDNA, and then the base sequence was obtained using a next-generation DNA sequencer. The gene transcript quantity was then investigated as TPM (transcripts per million) by mapping it to a reference sequence.

[0086] result In wild-type buckwheat ("IH3"), we confirmed the expression of the SSIIa1, SSIIa2, SBE1a, and SBE1b genes. These four genes are thought to be expressed as functional proteins. We confirmed that in buckwheat, two genes exist at each of four different loci.

[0087] Example 3: Comparison of breaking loads during refrigerated storage of noodle dough method After hulling the fully matured seeds of wild buckwheat "IH3", low-amylose buckwheat "Kyushu No. 12" (lacking only GBSSa), and SBE1b mutant buckwheat "Sukei UD1", the seeds were milled in a mortar and pestle, and water was added to achieve a final moisture content of 83% to produce 2mm thick noodle sheets. The noodle sheets were boiled in boiling water for 10 minutes, cooled in cold water, and then stored in a sealed plastic bag at 4°C for up to 9 days to prevent drying. For the above-mentioned refrigerated noodle sheets, the breaking strength was measured under the following conditions using a physical property testing system (Yamaden Co., Ltd. RE2-3305C-1) on the day of manufacture (no refrigeration, immediately after manufacture), the 3rd day, the 6th day, and the 9th day. • The temperature during measurement was 25℃ • Compressing the noodle sheet (2.0 mm thick) at 0.5 mm / sec

[0088] result The breaking load of the wild-type buckwheat "IH3" and the low-amylose buckwheat "Kyushu No. 12" increased with the passage of storage time, but it did not increase for the SBE1b mutant buckwheat "Sukei UD1". Since fracture load is an indicator of hardness, it is thought that the SBE1b mutant buckwheat "Sukei UD1" has the characteristic of retaining its softness (not hardening easily) when stored in the refrigerator.

[0089] Example 4: Evaluation of water release In this example, the amount of water released from steamed bread and mochi after refrigeration and after freezing was compared between the wild type and the Su-type UD1 strain. Generally, it is known that when water inside food is released to the outside during refrigeration or freezing (so-called syneresis), the food hardens, and its texture and moisture retention deteriorate.

[0090] method: About Daifuku Wild-type and Su-type UD1 daifuku were milled after molting, and 15% of the flour weight in sugar and 200% in water were added. After mixing well, the mixture was steamed for 10 minutes to produce daifuku. After being left at room temperature (25°C) for 60 minutes, the daifuku were wrapped in two layers of kitchen towels, placed in polyethylene packs, and stored at 5°C (refrigerated) or -20°C (frozen). During refrigerated storage, the weight of the daifuku was measured over time, and the percentage decrease from the weight at the start of refrigerated storage was calculated as the water separation rate. The weight was measured after leaving the daifuku at room temperature (25°C) for 60 minutes to return to room temperature. After measuring the weight, the kitchen towels were replaced with new ones. The experiment was conducted in sets of three. About steamed buns Wild-type and cultivated UD1 wheat were milled after molting, and 0.6% salt, 3% baking powder, 50% sugar, and 200% water were added to the flour weight. After mixing well, the mixture was steamed for 10 minutes to produce steamed bread. After being left at room temperature (25°C) for 60 minutes, the bread was wrapped in two layers of kitchen towels, placed in polyethylene packs, and stored at 5°C (refrigerated) or -20°C (frozen). During refrigerated storage, the weight of the steamed bread was measured over time, and the percentage decrease from the weight at the start of refrigeration was calculated as the water loss rate. The weight was measured after leaving the steamed bread at room temperature (25°C) for 60 minutes to return to room temperature. After measuring the weight, the kitchen towels were replaced with new ones. The experiment was conducted in sets of three.

[0091] Results: The results are shown in Figures 5 and 6. About Daifuku In a comparison after 7 days of refrigerated storage, the water separation rate for the wild type was 20.3%, compared to 11.5% for the Su-type UD1 strain, which was below 56.7% of the wild type's rate. In a comparison after 7 days of frozen storage, the water separation rate for the wild type was 25.1%, compared to 13.4% for the Su-type UD1 strain, which was below 53.4% ​​of the wild type's rate. About steamed buns In a comparison after 7 days of refrigerated storage, the water separation rate for the wild type was 41.6%, compared to 17.0% for the Su-type UD1 strain, which was below 40.9% for the wild type. In a comparison after 7 days of frozen storage, the water separation rate for the wild type was 34.7%, compared to 17.6% for the Su-type UD1 strain, which was below 50.8% for the wild type. Under both refrigerated and frozen storage conditions, the water separation rate in the UD1 strain was lower than that of the wild type, confirming that the mutant strain retained moisture well even after storage. Furthermore, when we tasted and evaluated daifuku and steamed buns after 7 days of refrigeration and 7 days of freezing, in both cases, the Su-type UD1 strain was superior in softness and moisture compared to the wild type. [Industrial applicability]

[0092] Buckwheat plants in which the starch-forming enzyme of the present invention has been mutated exhibit a lower starch gelatinization temperature compared to the wild type. Therefore, it is believed that by using the technology of the present invention, it will be possible to cultivate buckwheat plants with altered softness in cooked food, thereby meeting consumer needs.

[0093] (Sequence Listing) Sequence ID 1: Buckwheat SBE1a gene, estimated cDNA, wild type Sequence ID No. 2: Buckwheat SBE1b gene, estimated cDNA, wild type Sequence ID 3: Buckwheat SBE1a protein amino acid sequence (wild type) Sequence ID No. 4: Buckwheat SBE1b protein amino acid sequence (wild type) Sequence ID 5: Buckwheat SBE1b protein amino acid sequence variant Sequence ID 6: Buckwheat SBE1a gene, genome, wild type Sequence ID 7: Buckwheat SBE1b gene genome, wild type Sequence ID No. 8: Buckwheat SSIIa1 gene genome, wild type Sequence ID 9: Buckwheat SSIIa2 gene, genome, wild type Sequence ID No. 10: Buckwheat SBE1b gene, presumed cDNA, wild type, alternative splicing product. Sequence ID No. 11: Buckwheat SBE1b protein amino acid sequence, wild type, alternative splicing product.

Claims

1. Buckwheat-containing food products containing the seeds or crushed product of a buckwheat plant whose starch gelatinization onset temperature is 90% or less compared to the wild type, and / or whose starch gelatinization peak temperature is 85% or less compared to the wild type.

2. The buckwheat-containing food according to claim 1, wherein deterioration of physical properties due to the passage of time after manufacturing is suppressed.

3. The buckwheat-containing food according to claim 1, wherein the water separation rate after 7 days of refrigerated storage is less than 20%.

4. The buckwheat-containing food according to claim 1, wherein the water separation rate after 7 days of frozen storage is less than 20%.

5. The buckwheat-containing food according to claim 1, comprising seeds or a pulverized product of a buckwheat plant having reduced or deficient starch branching enzyme 1 (SBE1) activity.

6. A method for suppressing the deterioration of the physical properties of buckwheat-containing foods, characterized by using the seeds or crushed material of buckwheat plants having reduced or deficient starch branching enzyme 1 (SBE1) activity as raw materials.

7. A method for producing a buckwheat-containing food product, comprising using the seeds or pulverized product of a buckwheat plant whose gelatinization onset temperature is 90% or less of that of the wild type, and / or whose gelatinization peak temperature is 85% or less of that of the wild type, as a raw material.

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