JP2013172710A - Production method for low temperature gelatinizing mutant rice, rice finished good, and food - Google Patents

Abstract

Kind Code: A1 The present invention provides a processed rice product and food that maintain softness, and a method for producing low-temperature gelatinized mutant rice that is easy to gelatinize and difficult to age.
A method for producing a low-temperature gelatinized mutant rice includes a step of mutating rice bran D and a step of selecting low-temperature gelatinized mutant rice using a urea solution. Following the step of selecting the low-temperature gelatinized mutant rice, the step of mating the low-temperature gelatinized mutant rice and fertile rice, the step of selecting fertile individuals from the F ₂ generation individuals of the mating progeny, and fertility Selecting a gelatinized easily individual by detecting a mutation in the core promoter region of the starch branching enzyme I gene of chromosome 6 contained in the sample derived from the individual. Gelatinization is easy using at least one of HvSSR11-27 marker and HvSSR11-28 marker and at least one of HvSSR11-48 marker and HvSSR11-50 marker using DNA in a sample derived from a fertile individual as a template. The method further includes the step of selecting a sex individual.
[Selection] Figure 4

Description

  The present invention relates to a method for producing low-temperature gelatinized mutant rice, processed rice products, and foods.

  Rice, which is a regular diet by the Japanese, is one of the few grains in Japan with a very high domestic self-sufficiency rate. From the viewpoint of improving the food self-sufficiency rate in Japan, rice consumption is being promoted as a national measure.

  Due to changes in consumer lifestyles, foods purchased at convenience stores, supermarkets, etc. are brought home and eaten at home dining tables, so-called “meal” is becoming established. Examples of common meals include lunch boxes, rice balls, sushi and the like, and cooked rice is used for them.

  In recent years, the problem of wheat allergy has become obvious, and in order to avoid it, bread using rice flour instead of wheat (hereinafter referred to as rice flour bread) is becoming widespread. Rice flour bread is attracting attention because rice has high nutritional value and low calories compared to wheat.

  Lunch boxes using cooked rice such as those mentioned above are delivered in a refrigerated state and displayed in the store, so the cooked rice tends to harden during delivery and display, and the rice flour bread tends to harden after baking. There was a problem. In addition, a rice bran product using glutinous rice also has a problem that the texture is impaired since it becomes harder over time after being sown and molded. This is because starch contained in rice is gelatinized by rice cooking and heat processing, and then ages when cooled. Several methods for solving such problems have been found and reported.

  Additives mainly composed of sugars with high water retention, such as trehalose (Patent Document 1), enzymes (amylase and papain) (Patent Document 2), diglycerin fatty acid ester (Patent Document 3), or edible oils and fats when cooking rice The method of suppressing that cooked rice becomes hard by adding (patent document 4) is reported. Moreover, the method of manufacturing the rice flour bread | pan which is hard to become hard by adding amylase is reported (patent document 5). Furthermore, a method of obtaining cooked rice with less texture deterioration during low-temperature storage by using low-amylose rice having a strong amylose content of 15% or less has been reported (Patent Document 6). In addition, rice bran D is known as a rice variety having a low gelatinization start temperature (Non-patent Document 1). In addition, it has been reported that, for cocoon products, the natural polysaccharide pullulan, disaccharide, and emulsifier are used as anti-curing agents for moss (Patent Document 7).

Japanese Patent Laid-Open No. 7-147916 Japanese Patent Laid-Open No. 7-31396 JP-A-7-39325 Japanese Patent Laid-Open No. 4-141052 JP 2010-187559 A JP-A-9-322725 JP-A-5-84046

Kazuyuki Okamoto et al., Breeding Studies, 6 (Another 2), p316, 2004

  However, in the methods described in Patent Documents 1 to 5 and 7, since polysaccharides, enzymes and the like are added, not only does the production cost and work procedure increase, but consumers who want to reduce food additives Going back to needs. Moreover, in the method of patent document 6, since the sticky low amylose rice is used, in the packing line to the container for cooking of cooked rice, the problem that manufacturing efficiency falls because cooked rice adheres to a machine. I left it. Furthermore, the rice bran D described in Non-Patent Document 1 has left a problem regarding gelatinization characteristics in terms of cooked rice and rice flour bread that meet the needs of consumers.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a processed rice product and food product that can maintain softness, and a method for producing low-temperature gelatinized mutant rice that is easily gelatinized and hardly aged. .

In order to achieve the above object, a method for producing low-temperature gelatinized mutant rice according to the first aspect of the present invention,
Mutating 旱 ignorant D;
A process of selecting low-temperature gelatinized mutant rice using a urea solution;
including,
It is characterized by that.

The method for producing the low-temperature gelatinized mutant rice is as follows:
Following the step of selecting the low-temperature gelatinized mutant rice,
Crossing the low-temperature gelatinized mutant rice with fertile rice;
Selecting fertile individuals from the F ₂ generation of progenies of the mating;
Selecting a gelatine-friendly individual by detecting a mutation in the core promoter region of the starch branching enzyme I gene of chromosome 6 contained in the sample derived from the fertile individual;
May be included.

The method for producing the low-temperature gelatinized mutant rice is as follows:
HvSSR11-27 marker (Forward Primer: SEQ ID NO: 3, Reverse Primer: SEQ ID NO: 4) and HvSSR11-28 marker (Forward Primer: SEQ ID NO: 5, Reverse Primer: Sequence) using DNA in the sample derived from the fertile individual as a template And at least one of HvSSR11-48 marker (Forward Primer: SEQ ID NO: 7, Reverse Primer: SEQ ID NO: 8) and HvSSR11-50 marker (Forward Primer: SEQ ID NO: 9, Reverse Primer: SEQ ID NO: 10). A step of selecting an easily gelatinizable individual using at least one of them may be further included.

  The processed rice product according to the second aspect of the present invention comprises the low-temperature gelatinized mutant rice obtained by the low-temperature gelatinized mutant rice production method according to the first aspect of the present invention.

  The food according to the third aspect of the present invention includes the processed rice product according to the second aspect of the present invention.

  The processed rice product according to the fourth aspect of the present invention comprises low-temperature gelatinized mutant rice obtained by mutating rice bran D.

  The amylopectin short chain index value of the low-temperature gelatinized mutant rice may be 110% or more of the amylopectin short chain index value of Aoi Dignity D.

  The gelatinization temperature of the low-temperature gelatinized mutant rice may be 2 ° C. or more lower than the gelatinization temperature of the rice bran D.

  The low-temperature gelatinized mutant rice may be fertile low-temperature gelatinized mutant rice obtained by crossing low-temperature gelatinized mutant rice and fertile rice.

  The food according to the fifth aspect of the present invention includes the processed rice product according to the fourth aspect of the present invention.

  ADVANTAGE OF THE INVENTION According to this invention, the production method of the low-temperature gelatinization variation | mutation rice which is easy to gelatinize and cannot be aged easily can be provided.

It is a figure which shows one of the microplates used at the process of selecting low temperature gelatinization mutant rice using a urea solution. It is a figure which shows amylopectin chain length distribution and DP9 / DP17 of each rice variety. It is a figure which shows the hardness of cooked rice of each rice varieties. It is a figure which shows the hardness after baking of the gluten addition rice flour bread | bread of each rice varieties. It is a figure which shows the mapping of a novel gelatinization easy gene. It is a figure which shows the hardness of the koji by the fermented low temperature gelatinization mutant rice by a present Example. (A) is a figure of the result of Sapporo material, (b) is a figure of the result of Fukuyama material.

  Hereinafter, embodiments of the present invention will be described in detail.

(1. Low temperature gelatinized rice)
The low-temperature gelatinized mutant rice used in the processed rice product according to the present invention will be described in detail below.

  The low-temperature gelatinized mutant rice according to the present invention can be obtained by mutating the rice cultivar D which is an upland rice variety. In the present specification, the low-temperature gelatinized mutant rice refers to a mutant rice having a characteristic that it is more gelatinized and less susceptible to aging than the rice bran D. The method of mutation treatment will be described in detail later.

  The amylopectin short-chain index value of the low-temperature gelatinized mutant rice according to the present invention is, for example, 110% or more of that of strawberry ignorance D. In the present specification, the amylopectin short chain index value is calculated as follows. Calculate the total number of moles of glucose detected from the degree of glucose polymerization (hereinafter referred to as DP) 5 to DP35 of the amylopectin side chain of rice starch, the mole ratio (A) occupied by the side chain of DP9 with respect to the total number of moles detected, and the total number of moles detected The molar ratio (B) occupied by the side chain of DP17 with respect to the number is determined. A value obtained by dividing A by B is defined as an amylopectin short chain index value (hereinafter referred to as DP9 / DP17). The higher the DP9 / DP17 value, the higher the amylopectin short chain content in rice.

  The sample of rice used for the above-mentioned DP9 / DP17 measurement is, for example, preparing purified starch from rice by referring to the cold alkali soaking method (Yamamoto et al., Starch Science, 20, p99-104, 1973). It is preliminarily prepared by a method of gelatinizing, limitedly decomposing amylopectin with isoamylase, and using side chains as linear components. The sample is, for example, fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid (8-aminopyrene-1,3,6-trisulfonic acid: ATPS), and a capillary electrophoresis apparatus (model name: P / ACE system MDQ, Beckman Coulter)).

  As described above, DP9 / DP17 of the low-temperature gelatinized mutant rice according to the present invention is 110% or more, and preferably 111% or more, of that of the rice cake D. Therefore, the amylopectin short chain content of the low-temperature gelatinized mutant rice according to the present invention is higher than that of the potato dignity D. DP9 / DP17 of the low-temperature gelatinized mutant rice according to the present invention is, for example, 1.45 or more, preferably 1.50 or more, more preferably 1.55 or more. In addition, in so-called low amylose rice, texture deterioration during low-temperature storage is suppressed by reducing the amylose content, while low-temperature gelatinized mutant rice according to the present invention has a high amylopectin short chain content and is easily gelatinized. Has the property of being difficult to age.

  The gelatinization temperature of the low-temperature gelatinized mutant rice according to the present invention is lower than that of the rice bran D, for example, 2 ° C. or more. In this specification, the gelatinization temperature is a value obtained by measuring a temperature increase rate of 2 ° C./min from 25 ° C. to 130 ° C. using a differential scanning calorimeter (for example, model name: DSC6100 (manufactured by Seiko Instruments Inc.)). It is calculated from. Specifically, the gelatinization endothermic peak is measured with a differential scanning calorimeter, and the temperature at the time when the gelatinization endothermic peak shows the apex is defined as the gelatinization temperature.

  As the rice sample used for the above gelatinization temperature measurement, for example, purified starch prepared from rice with reference to the same cold alkali immersion method as described above can be used.

  As described above, the gelatinization temperature of the low-temperature gelatinized mutant rice according to the present invention is lower than that of the rice bran D, for example, 2 ° C. or more, preferably 2.2 ° C. or more. Therefore, the low-temperature gelatinized mutant rice according to the present invention has a characteristic that it is easily gelatinized and not easily aged as compared with the rice bran D. The gelatinization temperature of the low-temperature gelatinized mutant rice according to the present invention is, for example, 66 ° C. or less, preferably 65 ° C. or less, more preferably 64.5 ° C. or less.

(2. Production method of low temperature gelatinized mutant rice)
The method for producing a low-temperature gelatinized mutant rice according to the present invention includes a step of mutating the rice bran D and a step of selecting low-temperature gelatinized mutant rice using a urea solution.

  In the step of mutating cocoon ignorant D, for example, the mutation treatment is carried out by converting seeds of moth ignorant D into sodium azide, methylnitrosourea (MNU), N-bis (2-hydroxypropyl) nitrosamine (DHPN) and the like. It is performed by immersing in the solution containing. For example, a mutation treatment method using radiation such as cobalt 60 may be used. Any mutation treatment method that exhibits the effects of the present invention can be selected as appropriate.

  The process of selecting low-temperature gelatinized mutant rice using a urea solution will be described below. Starch, which is easily gelatinized at low temperatures, has the property of dissolving in a low-concentration urea solution. In order to obtain low-temperature gelatinized mutant rice that is more easily gelatinized at a low temperature, first, brown rice of 旱 Unknown D is immersed in urea solutions of various concentrations, and a urea limit concentration at which the brown rice of 旱 Unknown D is not dissolved is set. For example, when the 2.5M urea solution does not dissolve, the 2.6M urea solution does not dissolve, and the 2.7M urea solution dissolves, the urea limit concentration becomes “2.6M”. Next, the brown rice of the mutant rice obtained by mutating the rice bran D is immersed in a urea solution having a set urea limit concentration (for example, soaked at 20 ° C. for 18 hours). Mutant rice that dissolves in the urea solution of this urea limit concentration can be a candidate for low-temperature gelatinized mutant rice that is easier to gelatinize than 旱 Unknown D. In addition, it can confirm that the mutant rice melt | dissolved in the urea solution by adding an iodine solution to the urea solution which soaked the mutant rice, and visually observing dyeing reddish brown or bluish purple by the iodine starch reaction. By repeating such a process for each generation, individuals inherited and fixed in low temperature gelatinization property can be selected as low temperature gelatinized mutant rice.

  A specific example of the process of selecting low-temperature gelatinized mutant rice using a urea solution will be described below. As described above, after nurturing the seeds that have undergone mutation treatment, they are cultivated in paddy fields, and the seeds are harvested for each individual rice. Using the single-edged razor, the brown rice of the obtained seed is divided into a half grain having an embryo and a half grain not having an embryo. A half grain having no germ is immersed in a 2.6 M (urea limit concentration) urea aqueous solution, and the half seed dissolved in the 2.6 M urea aqueous solution is selected as a candidate for low temperature gelatinized mutant rice. Half seeds with brown rice germ of the selected seeds are sown and cultivated to harvest the next generation seeds. Similarly to the above, selection of candidates for low-temperature gelatinization mutant rice using a urea solution is repeated for each generation, and individuals with inherited and fixed low-temperature gelatinization properties can be made into low-temperature gelatinization mutant rice.

  In the process of selecting low-temperature gelatinized mutant rice using a urea solution, it can be carried out easily without using special reagents, devices and techniques, and low-temperature gelatinized mutant rice can be reliably selected.

(3. Processed rice products and foods made of low-temperature gelatinized mutant rice)
Examples of processed rice products made of low-temperature gelatinized mutant rice according to the present invention include rice flour, cooked rice, and processed rice.

  The rice flour according to the present invention can be obtained, for example, by pulverizing the aforementioned low-temperature gelatinized mutant rice. For example, milling devices such as air mills, rice bran, stamp mills, roll mills, stone mills (dry milling or wet milling), hammer mills (dry milling or wet milling) etc. For example, it is obtained by grinding to a particle size of about 200 μm or less, about 100 μm or less, or about 50 μm or less. You may mix the rice flour obtained by a different milling apparatus. The rice may be washed before pulverization by the milling apparatus, and when the rice is washed, the rice may be dried before or after pulverization by the milling apparatus. Moreover, you may process rice with the enzyme liquid containing pectinase etc. before the grinding | pulverization by a flour mill. Further, the rice flour obtained by the milling device may be sieved. If it is the preparation method of the rice flour which shows the effect of this invention, it can select suitably.

  The rice flour according to the present invention includes those obtained by further converting the rice flour obtained by the milling device into purified starch. When the purified starch is prepared by, for example, the cold alkali soaking method (Yamamoto et al., Starch Science, 20, p99-104, 1973), the low-temperature gelatinized mutant rice is ground to a particle size of about 0.5 mm or less, for example. Then, purification may be performed. Any method for preparing purified starch that exhibits the effects of the present invention can be selected as appropriate.

  The rice flour according to the present invention may appropriately contain preservatives, excipients and the like that are usually used in rice flour products.

  Since the low-temperature gelatinized mutant rice according to the present invention has a characteristic that it is easily gelatinized and difficult to age, foods such as bread using rice flour containing this low-temperature gelatinized mutant rice are not easily hardened even after heat processing. Maintained.

  The cooked rice according to the present invention can be obtained, for example, by cooking the aforementioned low-temperature gelatinized mutant rice with a known rice cooker. As described above, the low-temperature gelatinized mutant rice according to the present invention has the characteristics of being easily gelatinized and not easily aged, so that the rice cooked with this low-temperature gelatinized mutant rice is not easily hardened even after cooking and refrigeration, and the softness is maintained. The

  In this specification, the hardness of cooked rice can be measured using a hardness / viscosity meter (for example, product name: RHS1A (made by Satake)). The increase rate of the hardness after refrigeration at 4 ° C. for 24 hours with respect to the hardness of the cooked rice according to the present invention after heat dissipation is smaller than that of 旱 ignorant D, for example, less than 50%, preferably less than 45%. Preferably it is 43% or less.

  The cooked rice according to the present invention maintains its softness even after cooking and refrigeration, so it can be suitably used for lunch for lunch, rice balls and the like. Moreover, since softness is maintained even after rice cooking and refrigeration without using an additive, manufacturing costs and work procedures such as lunch boxes and rice balls can be reduced. Furthermore, since softness is maintained even after cooking and refrigeration without using low amylose rice, the adhesion of cooked rice to machines is reduced in the packing line to containers such as lunch boxes and rice balls, and production efficiency is improved. be able to.

  Processed cooked rice according to the present invention is, for example, aseptically filled with cooked rice according to the present invention in a retort pouch, tray or the like. As described above, the cooked rice according to the present invention can be suitably used for processed cooked rice because the softness is maintained even after cooking and refrigeration. Moreover, the manufacturing cost and work procedure of processed cooked rice can be reduced similarly to the above-mentioned, and manufacturing efficiency can be improved.

  In addition, if it is the processed rice product which has the effect of this invention, it can select suitably besides rice flour, cooked rice, and processed rice.

  Next, the food according to the present invention includes the aforementioned processed rice product. Examples of the food include gluten-added rice flour bread and rice flour blend bread.

  The gluten-added rice flour bread according to the present invention is made, for example, by adding wheat active gluten to the rice flour according to the present invention. The blending ratio is, for example, 80% rice flour: 20% wheat active gluten. The aforementioned low-temperature gelatinized mutant rice has a characteristic that it is easily gelatinized and difficult to age. Therefore, the gluten-added rice flour bread using rice flour produced from this low-temperature gelatinized mutant rice is hard to be hardened even after baking, and the softness is maintained. .

  In this specification, the hardness of gluten-added rice flour bread (mixing ratio = rice flour 80%: wheat active gluten 20%) is obtained by thickening a bread sliced to a thickness of 1.4 cm with a plunger having a diameter of 3.6 cm. The stress at that time is expressed by a value (g) measured using a texture analyzer (for example, model name: TA.XTplus (manufactured by Stable Micro System)). The hardness value on the third day after baking of the gluten-added rice flour bread according to the present invention is significantly lower than, for example, the same gluten-added rice flour bread using the rice flour of Kaki Achi D, for example, 200 g or less. The increase rate of the gluten-added rice flour bread according to the present invention on the third day after baking to the hardness on the first day after baking is, for example, 30% or less, preferably 26% or less. Thus, the gluten-added rice flour bread according to the present invention maintains its softness even after baking without using other additives.

  The rice flour blend bread according to the present invention is made, for example, by blending the flour of the super strong wheat variety “Yumechikara” with the rice flour according to the present invention. The blending ratio is, for example, 20% rice flour: 80% from Yumechi. Since the aforementioned low-temperature gelatinized mutant rice has the characteristics of being easily gelatinized and difficult to age, the rice flour blend bread using the rice flour produced by this low-temperature gelatinized mutant rice is not easily hardened even after baking, and the softness is maintained.

In this specification, the hardness of the rice flour blend bread (mixing ratio = 20% rice flour: 80% from yumechi) is sealed in a polyethylene bag after the bread cooled to room temperature is stored at room temperature for 3 days. Thereafter, the bread is sliced to a thickness of 2 cm, a 3 cm × 3 cm square sample is cut out from the center of each slice, and the sample is subjected to a rheometer (for example, model name: model RE33005 (manufactured by Yamadensha)). It can be expressed as stress (N / m ² ) when compressed to 1 cm. Hardness of rice flour blends pan according to the invention the firing 3 days after, lower than that of Hideri unknown D, for example, 4600N / ^{m 2} or less, preferably 4500N / ^{m 2} or less. Thus, the rice flour blend pan according to the present invention maintains its softness even after baking without using additives.

  Examples of the food according to the present invention include confectionery, noodles, dumpling skin, rice paper, baby food (retort) and the like in addition to the above-mentioned gluten-added rice flour bread and rice flour blend bread. Since the aforementioned low-temperature gelatinized mutant rice has the characteristics of being easily gelatinized and difficult to age, the confectionery, noodles and the like according to the present invention are not easily hardened even after processing, and the softness is maintained. In addition, if it is a food with the effect of this invention, it can select suitably.

(4. Fermented low temperature gelatinized mutant rice)
The low-temperature gelatinized mutant rice according to the present invention may be fertile low-temperature gelatinized mutant rice obtained by crossing low-temperature gelatinized mutant rice (fertile) and fertile rice. The fermented low temperature gelatinized rice according to the present invention will be described in detail below.

Fertile low-temperature gelatinized mutant rice according to the present invention is obtained by crossing the aforementioned low-temperature gelatinized mutant rice (fertile) and fertile rice, and selecting fertile and easily gelatinized individuals from the F ₂ generation individuals of the mating progeny. It is obtained by selecting. The method will be described in detail later.

  When the fermented low temperature gelatinized mutant rice according to the present invention is made into a koji, the koji has a property of slow curing (that is, softness is maintained). Specifically, in the case where the fermented low temperature gelatinized mutant rice according to the present invention is brewed into rice cake, the rice derived from the fertile rice to be mated is made into rice cake, or the aforementioned low temperature gelatinized mutated rice and rice cake are used. Compared to the case of rice rice that had not inherited the starch characteristics of low temperature gelatinized mutant rice (described later) by crossing with rice, it was hardened (that is, softness was maintained). ) The hardness of the wrinkles can be measured with a texture analyzer, for example, after 24 hours of standing at 5 ° C.

(5. Production method of fermented low temperature gelatinized mutant rice)
The method for producing fertile low-temperature gelatinized mutant rice according to the present invention comprises a step of mutating strawberry dignity D, a step of selecting low-temperature gelatinized mutant rice using a urea solution, and a subsequent low-temperature gelatinized mutant rice ( The step of mating fertile) and fertile rice, the step of selecting fertile individuals from the F ₂ generation of the progeny, and the starch branching enzyme I of chromosome 6 contained in the samples derived from fertile individuals And selecting a gelatinized easily individual by detecting a mutation in the core promoter region of the gene. The step of mutating the potato dignity D and the step of selecting low-temperature gelatinized mutant rice using a urea solution are the same as described above.

  In the process of crossing low temperature gelatinized mutant rice (fertile) and fertile rice, as fertile rice, for example, Kitayukimochi, Koganemochi, Hiyokumochi, Hakuchomochi, Himenomochi, Mangetsumochi, Shinba double rice cake, Kaguramochi Varieties such as Kiju-don, Mochi-minori, Kurenai-mochi, Hidekomochi, Mochihikari, Mine-no-mochi. From the viewpoint of good cocoon quality, Kitayukimochi can be preferably used. Any fertile rice that exhibits the effects of the present invention can be selected as appropriate.

In the step of selecting a waxiness individuals from F ₂ generation individuals progeny, as a method of selecting a glutinous rice, for example, may be selected those which can be determined that the waxiness from the appearance of brown rice, rice DNA is extracted from leaves and fertile individuals are selected by PCR using a known DNA marker (for example, the marker described in Wanchana et al., Plant Science, 165, p1193-1199, 2003). May be. Any method for selecting fertile individuals that exhibit the effects of the present invention can be selected as appropriate.

  In the step of selecting a gelatinizable easily individual by detecting a mutation in the core promoter region of the starch branching enzyme I gene (Sbe1 gene) of chromosome 6 contained in a sample derived from a fertile individual, The “sample” means, for example, a DNA sample extracted from rice (eg, rice leaves) determined to be fertile. As a method for detecting this mutation, for example, using DNA in a sample derived from a dwarf individual as a template, a marker based on insertion / deletion in the core promoter region of the Sbe1 gene (Forward Primer: SEQ ID NO: 1, Reverse Primer). : SEQ ID NO: 2) (hereinafter referred to as “Sbe1 marker” in the present specification), a method of amplifying by PCR method, separating PCR products by agarose gel electrophoresis, and detecting mutation by band position It is done. It is judged that the starch characteristic that the amylopectin short chain ratio of the low-temperature gelatinized mutant rice varieties D has a high short chain ratio is determined by one gene, and the locus chromosome region is the Sbe1 gene of chromosome 6. It is reported that it is near the locus (Umemoto et al., Japanese Society of Crop Science, Vol. 77, No. 1, 2008, p284-285). Therefore, by detecting mutations (insertion / deletion) in the core promoter region of the Sbe1 gene using the Sbe1 marker, rice individuals that inherited starch characteristics (high amylopectin short-chain ratio) that are easily gelatinized from 旱 Unknown D And a rice individual that has not been inherited can be clearly distinguished from each other, and a rice individual that has inherited the easily gelatinized starch characteristics (an easily gelatinized individual) can be reliably selected.

  In the method for producing fertile low-temperature gelatinized mutant rice according to the present invention, HvSSR11-27 marker (Forward Primer: SEQ ID NO: 3, Reverse Primer: SEQ ID NO: 4) using DNA in a sample derived from fertile individuals as a template (Table 4) 1) and at least one of HvSSR11-28 marker (Forward Primer: SEQ ID NO: 5, Reverse Primer: SEQ ID NO: 6) (Table 1), and HvSSR11-48 marker (Forward Primer: SEQ ID NO: 7, Reverse Primer: SEQ ID NO: 8) (Table 1) and HvSSR11-50 marker (Forward Primer: SEQ ID NO: 9, Reverse Primer: SEQ ID NO: 10) (Table 1) A step of selecting a body may be further included. Specifically, the DNA in a sample derived from a fertile individual is used as a template to amplify by PCR using the above marker, and the PCR product is separated by electrophoresis using, for example, an agarose gel. Detect polymorphism. As a result of intensive studies, the present inventors have found that a novel gelatinization gene is located between a region amplified by the HvSSR11-27 marker and a region amplified by the HvSSR11-50 marker ( Hereinafter, this novel gelatinization susceptibility locus is referred to as “Lgt (t) locus”). As a result, it was found that by using such a marker in addition to the Sbe1 marker, it is possible to select rice individuals (individuable gelatinized individuals) that have inherited starch characteristics that are more easily gelatinized.

The specific example of the production method of the fermented low temperature gelatinization mutant rice by this invention is demonstrated below. It was mated "Kita Yukimochi" a variety of previously described resulting cold gelatinized mutant rice (japonica type property) and waxiness rice, to obtain a subsequent generations F ₂ seeds. Using F ₂ seeds as brown rice, select individuals that can be judged fertile from the appearance. This individual is bred and DNA is extracted from the leaves. Using this DNA as a template, at least one of the Sbe1 marker, HvSSR11-27 marker and HvSSR11-28 marker, and at least one of HvSSR11-48 marker and HvSSR11-50 marker (for example, Sbe1 marker) And an HvSSR11-28 marker and an HvSSR11-48 marker), an individual whose Sbe1 locus is the same genotype (sbe1) as a cold-gelatinized mutant rice (mutant strain), and an Lgt (t) locus is a cold paste An individual having the same genotype (lgt) as the mutated rice (mutant strain), and an individual having the same genotype (sbe1 / lgt) as the low temperature gelatinized mutant rice (mutant strain) in both the Sbe1 locus and the Lgt (t) locus Sort out. The fertile rice (fermented low-temperature gelatinized mutant rice) obtained in this way has the property of easy gelatinization, and the soft rice is maintained soft. In addition, in the individuals having the same genotype (sbe1 / lgt) as the low-temperature gelatinized mutant rice (mutant strain) in both the Sbe1 locus and the Lgt (t) locus, either the Sbe1 locus or the Lgt (t) locus has a low-temperature gelatinization mutation. Compared to individuals with the same genotype as rice (mutant strains), the soot is softer.

(6. Processed rice products and foods made of fermented low temperature gelatinized mutant rice)
Examples of processed rice products made of fermented low-temperature gelatinized mutant rice according to the present invention include, for example, glutinous rice, glutinous rice flour (eg, white flour), and cocoon products (eg, brewed rice cake, chopped rice cake). For example, sticky rice is obtained by milling fertile low temperature gelatinized mutant rice according to the present invention, and rice bran powder is obtained by grinding (for example after milling) fermented low temperature gelatinized mutant rice according to the present invention, The cocoon product is obtained by steaming the above-mentioned glutinous rice and then molding it with a rice grinder. Since the koji product obtained in this way uses fermented low-temperature gelatinized mutant rice that has a slow curing property, it does not become hard even after a certain period of time since it has been sown, and additives such as sugars (prevention of curing) Softness is maintained without adding the agent.

  Examples of the food containing the above processed rice products include raw confectionery such as Daifuku rice cake, white ball dumpling, rice flour blend bread and the like. Such food uses fermented low-temperature gelatinized mutant rice having a slow-curing property, so that it does not become hard even after a certain time has elapsed after processing, and the softness is maintained. For this reason, soft confectionery such as Daifuku-dono maintains softness without the addition of sugars or other additives (anti-curing agents), white ball dumplings are hard to harden even when cooled, and rice flour blend breads are soft even after baking. Can be maintained.

  These processed rice products may appropriately contain preservatives, excipients and the like that are usually used. In addition, if it is the processed rice product and foodstuff which show the effect of this invention, it can select suitably.

  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

[Example 1]
(Selection of mutagenized and low temperature gelatinized rice)
40 g of seeds of the upland rice variety “Kaki-chiri D” (distributed from Biotechnology Research Institute, Ibaraki Prefectural Agricultural Research Center) in an aqueous solution of 1 mM sodium azide solution (sodium azide (product name: sodium azide, Wako Pure Chemical Industries, Ltd.)) (100 mM) was diluted with 100 mM phosphate buffer (pH 3.0)) and immersed in 400 mL at 25 ° C. for 6 hours for mutation treatment. The seed after the mutation treatment was washed twice with 400 mL of distilled water and then dried at room temperature (for details of the mutation treatment, see Hasegawa and Inoue, Japan Journal of Breeding 30, p301-308, 1980). ).

  As described above, seeds mutated were nurtured, cultivated in paddy fields, and seeds were harvested for each rice plant. The obtained seeds were used as brown rice by removing rice husks. Using a single-edged razor, 16 grains of brown rice per individual rice were divided into two halves, one with the germ and the other without the germ. Half grains with germs and half grains without germs were placed in holes at corresponding locations on different 96-well microplates. 2.6M (urea limit concentration) urea aqueous solution (pH 7.0) (urea (product name: urea, manufactured by Wako Pure Chemical Industries, Ltd.)) was used as an aqueous solution in each hole of a microplate containing half grains without embryos. 1) 200 μL was added and allowed to stand at 20 ° C. for 18 hours. Then, iodine solution (0.1% iodine (product name: iodine, manufactured by Wako Pure Chemical Industries, Ltd.) / 1% potassium iodide (product name: potassium iodide, Nacalai Tesque) was added to each hole to which urea aqueous solution was added. 20 μL of an aqueous solution) was added dropwise to confirm the solubility of brown rice in an aqueous urea solution. Brown rice dyed reddish brown or bluish purple by the iodine solution (low-temperature gelatinized mutant rice candidate column in FIG. 1) was selected as a candidate for low-temperature gelatinized mutant rice.

  As described above, brown rice selected as a candidate for low-temperature gelatinized mutant rice was sown and cultivated, and the next-generation seeds were harvested. Selection of candidates for low-temperature gelatinized mutant rice was repeated for each generation by the above-described method using an aqueous urea solution, and finally, M5 generation seeds were obtained as low-temperature gelatinized mutant rice. The obtained low-temperature gelatinized mutant rice was named “mutant strain 202”.

[Example 2]
(Measurement of DP9 / DP17)
In order to evaluate the amylopectin short chain ratio of the mutant line 202 obtained as described above, DP9 / DP17 was measured. As a comparison, DP9 / DP17 of Kashihikari D, which is the original variety, and Koshihikari, which is the standard variety, were also measured.

  Mutant strain 202, rice bran D, and Koshihikari were polished to give white rice. The white rice was pulverized to a particle size of 0.5 mm or less using a sample mill (CYCLOTEC 1093, manufactured by FOSS TECATOR) to obtain white rice powder. Using this white rice flour, purified starch was prepared by the following method with reference to the cold alkali soaking method (Yamamoto et al., Starch Science, 20, p99-104, 1973). 1,200 mL of 0.1% sodium hydroxide aqueous solution (product made from sodium hydroxide (product name: sodium hydroxide, manufactured by Nacalai Tesque)) cooled to 4 ° C. was added to 200 g of the white rice flour. The mixture was stirred at 3 ° C. for 3 hours and then allowed to stand at 4 ° C. for 15 hours. After discarding the supernatant sodium hydroxide aqueous solution, the precipitate was suspended in distilled water and washed. The suspension was passed through a 150 μm sieve. The sieve passing material was centrifuged at 2,000 rpm and 4 ° C. for 3 minutes. The supernatant is discarded, and the precipitate is suspended by adding 1,600 mL of a 0.1% aqueous sodium hydroxide solution (same as above) cooled to 4 ° C., and stirring and standing at 4 ° C. again as described above. went. Thereafter, washing of the precipitate with distilled water was repeated until the supernatant reached pH 7. The suspension was passed through a sieve of 100 μm and subsequently 75 μm, and the sieved product was dried by ventilation at 25 ° C. The sieved product after drying was pulverized using a mortar and pestle, and this was used as purified starch.

  The chain length distribution of the side chain of amylopectin in the purified starch of mutant line 202, 旱 不知 D, and Koshihikari prepared as described above was measured. The measurement was carried out by the following method with reference to previously reported reports (O'Shea and Morell; Carbohydrate Research, 307, p1-12, 1998, and Fujita et al; Plant Science, 160, p595-602, 2001).

  1 mg of each purified starch was weighed into a microtube, 285 μL of deionized distilled water was added and stirred, and then 15 μL of 5N sodium hydroxide was added and stirred again. This microtube was placed in boiling water for 5 minutes to gelatinize the starch. After allowing to cool, 9.6 μL of 100% acetic acid was added to neutralize, 50 μL of 600 mM sodium acetate buffer (pH 4.6), 10 μL of 2% sodium azide, and 690.4 μL of deionized distilled water were added and mixed. Thereafter, 5 μL (about 700 units) of isoamylase (isoamylase obtained from the culture solution of Pseudomonas amyloderamosa, Hayashibara Biochemical Research Institute) was reacted to limit the amylopectin, and the side chain was used as a linear component. This was reacted with 8-aminopyrene-1,3,6-trisulfonic acid (8-aminopyrene-1,3,6-trisulfonic acid: ATPS) (product name: Labeling Dye (ATPS), manufactured by Beckman Coulter, Inc.). Fluorescently labeled. Detection of the labeled linear chain was performed using a capillary electrophoresis apparatus (model name: P / ACE system MDQ, Beckman Coulter, Inc.). The total number of moles detected from DP5 to DP35 of each purified starch was calculated, the mole ratio (A) occupied by the side chain of DP9 relative to the total moles detected, and the mole ratio occupied by the side chain of DP17 relative to the total moles detected (B ) Was calculated and a numerical value (DP9 / DP17) obtained by dividing A by B was calculated.

  The results are shown in FIG. In the figure, the difference in molar ratio represents a value calculated by subtracting the molar ratio of each amylopectin side chain of the corresponding Koshihikari from the molar ratio of each amylopectin side chain of the mutant strain 202 or 旱 ignorant D. The amylopectin short-chain molar ratio was higher in the acupuncture D and mutant line 202 than in Koshihikari. In the mutant strain 202, DP9 / DP17 was 143% for Koshihikari, and 111% for the wandering ignorant D.

  From the above, it was shown that the amylopectin short chain ratio of the mutant strain 202 is higher than the amylopectin short chain ratio of Satsukeni D.

Example 3
(Measurement of gelatinization temperature)
The gelatinization temperature of the mutant line 202 obtained as described above was measured. As a comparison, the gelatinization temperature of the cultivar Dakichi D and the standard cultivar Koshihikari was also measured.

  The gelatinization temperature was measured using a differential scanning calorimeter (model name: DSC6100, manufactured by Seiko Instruments Inc.). Purified starch was prepared in the same manner as in Example 2 from the mutant line 202, 旱 ignorant D, and Koshihikari. 10 mg of each purified starch was weighed into a sealed sample container (silver) (product code: 560-003, for DSC100 system, Epollide Service) having a capacity of 70 μL, distilled water was added, and the starch concentration was 30% (w / w w) (in terms of dry weight). After sealing the sealed sample container, the gelatinization temperature was measured at a temperature increase rate of 2 ° C./min from 25 ° C. to 130 ° C. using a sealed sample container containing only distilled water as a blank. The obtained data was analyzed with the software attached to the differential scanning calorimeter, and the gelatinization temperature was calculated. Specifically, the gelatinization endothermic peak was measured with the above differential scanning calorimeter, and the temperature at the time when the gelatinization endothermic peak showed the apex was defined as the gelatinization temperature.

  The results are shown in Table 2. The gelatinization temperature of the mutant line 202 was 6.1 ° C. lower than that of Koshihikari, and 2.2 ° C. lower than that of Kakuhichi D.

  From the above, it was shown that the gelatinization temperature of the mutant strain 202 is lower than the gelatinization temperature of the moths Dignity D and Koshihikari. Therefore, it was clarified that the mutant strain 202 is more gelatinized and less aged than the silkworm ignorant D.

Example 4
(Evaluation of cooked rice)
The hardness of the cooked rice of the mutant strain 202, the rice bran D, and Koshihikari was measured as follows.

  Mutant strain 202, rice bran D, and Koshihikari were polished into white rice and cooked with an electric rice cooker (product number: SR-W180, manufactured by National Corporation). The hardness of cooked rice is measured using a hardness / viscose meter (model name: RHS1A, manufactured by Satake Co., Ltd.) according to the method already reported (Ochi et al., Taste Technology Research Group, 6, p16-19, 2005). I went. 8.0 g of cooked rice was weighed, packed in the sample ring attached to the hardness / viscosity meter and shaped, and then subjected to hardness measurement. The hardness was analyzed using the program attached to the hardness / viscosity meter. The measurement of the hardness of cooked rice was performed after heat dissipation from cooked rice and after refrigeration at 4 ° C. for 24 hours.

  The results are shown in FIG. After the rice cooking heat dissipation, the rice cooked rice of the mutant line 202 had the same degree of hardness as the rice bran D and tended to be harder than Koshihikari. However, after 24 hours of refrigeration at 4 ° C., the cooked rice of the mutant line 202 was softer than the rice bran D and remarkably softer than Koshihikari. The rate of increase in hardness after refrigeration at 4 ° C. for 24 hours is 42% in the mutant line 202 as compared with 137% Koshihikari and 50% D. wisdom, and the rate of increase in hardness of the mutant line 202 is Koshihikari and It was shown to be small compared to the 旱 Dignity D.

  From the above, it was shown that the cooked rice of the mutant strain 202 is harder to be harder than that of the rice bran D even after refrigeration, and the softness is maintained.

Example 5
(Evaluation of gluten-added rice flour bread)
A gluten-added rice flour bread was made using the mutant strain 202, rice bran D, and Koshihikari processed into rice flour, and the hardness and taste after baking were evaluated.

  First, rice flour was prepared as follows from the mutant strain 202, strawberry ignorance D, and Koshihikari. Rice flour was prepared as follows by a wet air-flow pulverization method with enzyme treatment. Rice of various varieties was polished into white rice samples. 1700 g of each white rice sample was washed four times with tap water, and then washed once with hot water at around 40 ° C. in order to improve the action of an enzyme (described later) added by raising the temperature of the rice. After draining water, trisodium citrate dihydrate (product name: trisodium citrate dihydrate, manufactured by Nacalai Tesque) (0.3%) and pectinase (product name: pectinase G “Amano”), A white rice sample was dipped in 5 L of an enzyme solution (about 40 ° C.) containing Amano Enzyme Co., Ltd. (0.05%) and allowed to stand in a 45 ° C. incubator for 1 hour. After the treatment, the white rice sample was washed with tap water, dehydrated, and ground using a jet mill (model name: SPM-R290, manufactured by Nishimura Seisakusho). The rotation speed of the pulverizer was set to 50 Hz.

  Next, using the rice flour of each of the rice varieties obtained above, bread making is performed as follows according to the no-time method (Yamauchi et al, Food Sci. Technol. Res. 10, p247-253, 2004). (Mixing ratio = rice flour 80%: wheat active gluten 20%). Formulation is a weight ratio with a moisture content of 14%, each rice flour 80%, active gluten (product name: Emmasoft EX-100, manufactured by Riken Vitamin Co.) 15%, and gluten formulation (product name: Dowmaster FR, RIKEN) Vitamin Co., Ltd.) 5%. The above was made into 100%, and it mixed with the ratio of sugar 7%, skim milk powder 3%, salt 1.5%, shortening 8%, and dry yeast 1.5%. The amount of water added was 75% with respect to the weight of rice flour, active gluten, and gluten preparation, and the amount of water was also adjusted for 14% moisture correction. Based on the above ratio, the amount of each material used was adjusted and mixed so that the weight after addition was 1250 g. Bread dough is kneaded using a can-to-mixer (model name: HPi-20M, manufactured by Kanto Blender Kogyo Co., Ltd.), 400 g of bread dough is a fluororesin processed bread type (sulton bread type, for 1 kg, length 190 mm, width 90 mm, And was fermented at 38 ° C. and 80% humidity until the dough height reached the top of the mold (about 1 hour). The mold containing bread dough was then placed in a gas oven preheated to 185 ° C. and baked for 28 minutes. The baked bread was removed from the mold and cooled to room temperature.

  The bread after baking was sliced to a thickness of 1.4 cm, and the center of the bread was pushed by a plunger having a diameter of 3.6 cm with a thickness of 25%, and the stress at that time was measured with a texture analyzer (model name: TA.XTplus, Stable Micro System) was used, and the measured value was determined as the bread hardness. The bread hardness was measured on the first, second, and third days after baking.

  The results are shown in FIG. 4 (in the figure, error bars are SD (n = 3)). The bread hardness of the mutant line 202 was not more than 200 g even on the third day, and was significantly softer than Koshihikari and Kaki-Unknown D. Moreover, the increase rate of the 3rd day after baking with respect to the bread hardness of the 1st day after baking was 26% in the mutant line 202 and 21% in the strawberry ignorance D with respect to 119% Koshihikari. Thus, the rate of increase in hardness of the mutant line 202 after firing was significantly lower than that of Koshihikari, which was comparable to that of the strawberry ignorant D.

  The taste evaluation of the gluten-added rice flour bread was performed by the following method. Based on bread using commercially available rice flour (product name: low-strength rice flour, manufactured by Nurisha), mutant strain 202, rice bran D and Koshihikari bread were evaluated. The evaluation items were five items of “appearance”, “fragrance”, “softness”, “moist feeling”, and “overall evaluation”. “3”, “2”, “1”, “0”, “0”, “−1”, “−2”, or very good It was evaluated in 7 grades of “-3” which is inferior to. The bread taste was evaluated after about 24 hours of baking. The number of evaluators was 20.

  The results are shown in Table 3. The values shown in Table 3 are average values obtained by dividing the sum of products of the respective scores and the number of evaluators by the total number of evaluators. The breads of the mutant line 202 were superior to the breads of Kaki-chichi D and Koshihikari in “softness”, “moistness” and “overall evaluation”.

  From the above, it was shown that the gluten-added rice flour bread using the mutant line 202 was maintained soft even after one day of baking and excellent in taste as compared with using the rice bran D and Koshihikari.

Example 6
(Evaluation of rice flour blend bread)
A rice flour blend pan mixed with ultra-strong wheat flour was made using the mutant line 202, rice bran D and Koshihikari processed into rice flour, and the swell and hardness after baking and the taste were evaluated.

  First, purified starch was prepared in the same manner as in Example 2 from the mutant line 202, 旱 ignorant D, and Koshihikari. 160g flour of super strong wheat cultivar "Yumechikara" (obtained from Ebetsu Flour Mills (2010)), 40g of refined starch of each rice cultivar obtained above, 10g of granulated sugar, shortening 10 g, 4 g of salt, 4 g of dry yeast, and 1 mL of 100 ppm ascorbic acid were mixed and breaded as follows (blending ratio = 20% rice flour: 80% from Yumechi). The amount of water added was 133 mL. The dough was kneaded while monitoring the load on the motor of the mixer (200 g pin mixer, manufactured by National MFG), and the mixer was stopped about 10 seconds after the load peaked. The dough was divided into 100 g portions, placed in a fermenter at 30 ° C. for 20 minutes, and then fermented at 38 ° C. and 85% humidity for 70 minutes. Then, it was baked in an oven preheated to 200 ° C. for 25 minutes using a pan mold (length: 123 mm, width: 70 mm, height: 50 mm). Moreover, the bread | baking and baking similarly to the above using 200g of wheat flour of "Yumechikara" without using rice flour was made into the bread for a comparison (compounding ratio = 0% of rice flour: 100% from Yumechi).

  Bread bulge was evaluated by specific volume. The specific volume was calculated by cooling the baked bread for 1 hour at room temperature, measuring the volume and weight of the bread, and dividing the volume by the weight.

  The hardness of the bread was measured according to the method described previously (Yamauchi et al., Journal of Japanese Society of Food Science and Technology; 46, p212-219, 1999). The bread that had been allowed to cool to room temperature was sealed in a polyethylene bag and stored at room temperature for 3 days. Thereafter, the bread was sliced to a thickness of 2 cm, and a 3 cm × 3 cm square sample was cut from the center of each slice. Using a rheometer (model name: model RE33005, manufactured by Yamaden Co., Ltd.), the cut sample was used as the hardness of the bread when the sample was compressed to 1 cm. The bread hardness was measured 3 days after baking.

  The results are shown in Table 4. In the table, there is no significant difference between the numerical values of the same alphabet by the Turkey test (p <0.05). The parentheses indicate the ratio when the comparative bread (composition ratio = rice flour 0: yumechi to 100) is 100. There was no difference in bread bulge (specific volume) between 旱 ignorant D and Koshihikari, but the mutant strain 202 was higher than 旱 ignorant D and Koshihikari. In particular, the specific volume in the mutant line 202 was significantly higher than that in Koshihikari, and it was confirmed visually that the bread of the mutant line 202 had a good bulge. In addition, the bread hardness of the mutant line 202 bread was softer than that of the potato ignorant D and Koshihikari, and in particular, significantly softer than that of Koshihikari.

  The taste evaluation of the rice flour blend bread was performed by the following method. Based on the comparative bread (combination ratio = rice flour 0%: 100% from yumechi), the rice flour blend pans of the mutant line 202, Aoi Shichi D, and Koshihikari were evaluated. The evaluation items are “baked color” (score: 1 to 10), “uniformity” (score: 1 to 5), “cortex (whether there is no brittleness or cracks in the colored part) Etc.) ”(score: 1-5),“ sudachi ”(score: 1-10),“ hue ”(score: 1-5),“ tactile sensation ”(score: 1-5),“ fragrance ”( It was 9 items of "score: 1-15", "taste" (score: 1-15), and "overall rating" (highest score: 70). In each item, the score of the comparative bread was 80% of the perfect score, and each rice flour blend bread was evaluated based on that. Taste evaluation was carried out 1 day and 3 days after baking. The number of evaluators was 2 after 1 day of firing and 2 after 3 days of firing.

  The results are shown in Table 5. The values shown in Table 5 are average values obtained by dividing the sum of products of the respective scores and the number of evaluators by the total number of evaluators. On the other hand, there were evaluation items that were inferior to Koshihikari in Kashiwa Dignity D, whereas there was no difference from Koshihikari or superior to Koshihikari except for “fragrance” 3 days after firing of mutant strain 202. In the mutant line 202, “cortex”, “tactile sensation”, “fragrance” (after 1 day of baking), “taste” (after 3 days of baking), and “overall evaluation” were superior to those of the wandering ignorant D. In particular, in the “tactile sensation”, the wisdom D decreased to “3.5” after 3 days from “3.75” in 1 day after firing, but “4.25” after 1 day from firing in the mutant strain 202, Even after 3 days, it was maintained at “4”. Furthermore, in the “taste”, it was “11.75” one day after baking in both the potato ignorant D and the mutant strain 202, whereas in the mutant strain 202, it decreased to “9” after three days. Even after 3 days, it was maintained at “9.25”.

  From the above, the rice flour blend bread using the mutant line 202 has a good swell at the time of baking, is soft after baking, and is excellent in taste as compared with that using the rice bran D. Indicated.

Example 7
(Mating between mutant line 202 and fertile varieties)
In order to obtain easy-to-gelatin fertile rice (fertile low-temperature gelatinized mutant rice), mutant line 202 and sticky rice varieties “Kitayukimochi” (distributed from Agricultural Research Headquarters, Hokkaido Research Institute, Kamikawa Agricultural Experiment Station) It was mated, to ensure the F ₂ generation individuals of the seed of the progeny.

(Determination of new low temperature gelatinization mutation locus)
Among the aforementioned F ₂ generations individuals, in order to screen individuals gelatinization easily soluble, first, as follows, to determine the new low temperature gelatinisation mutation locus.

In order to determine a new low-temperature gelatinization mutant locus, 200 progeny F ₂ generations in which a mutant strain 202 and Koshihikari were crossed as materials were used. This F ₂ generation 200 individuals, those mutant lines 202 have investigated the genotype of a known gelatinization easy genes (starch branching with enzyme 1, SBE1), fixed to the mutant lines 202 homozygous or Koshihikari homozygote I chose. The method is as follows.

First, the F ₂ generation individuals progeny were crossed and mutant lines 202 and Koshihikari cultivated by practice method, 1-2 sheets mature leaf was dried overnight at 50 ° C.. The dried leaves (about 5 cm) were cut with scissors for dissection, placed in a 2 mL microtube with glass beads, and dried overnight at 50 ° C. with the lid open. The dried sample was pulverized into a powder by applying it to a pulverizer (Shake Master, Biomedical Science) for 2 minutes.

Then, DNA was extracted from the _{F 2} generation of individuals. For extraction of DNA, a CTAB (Cetyl trimethyl ammonium bromide) method scaled down was used. The aforementioned powdery dry sample was put into a microtube, and a 1.5-fold CTAB solution (1.5% CTAB, 75 mM Tris-HCl (pH 8.0), 15 mM EDTA (pH 8.0), 1.05 M NaCl). ) After adding 800 μL and stirring well, it was placed in a constant temperature water bath at 56 ° C. for 30 minutes. Thereafter, 500 μL of chloroform / isoamyl alcohol (24: 1, v / v) was added, and the microtube was laid and shaken for 20 minutes at a speed of 75 strokes / minute. Thereafter, the mixture was centrifuged at 10,000 g for 5 minutes, 700 μL of the supernatant was transferred to another 2 mL microtube, 500 μL of chloroform / isoamyl alcohol was added, and the mixture was shaken for 20 minutes at a speed of 75 strokes / minute. After centrifugation (10,000 g × 5 minutes), 650 μL of the supernatant was transferred to another 2 mL microtube, and 650 μL of CTAB precipitation buffer (1% CTAB, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0)). Was added and mixed to precipitate DNA. The precipitated DNA was pelleted by centrifugation (10,000 g × 5 minutes), and the supernatant was discarded. The pellet was washed once with 99.5% ice-cold ethanol and then with 70% ice-cold ethanol, and then the DNA was dried at 60 ° C. for about 20 minutes. Thereafter, 100 μL of 1/10 concentration TE solution was added to the DNA and gently stirred to dissolve the DNA. The concentration of DNA was measured and diluted to 20 ng / μL using 1/10 concentration TE solution.

  Next, the Sbe1 locus genotype was determined by PCR using a DNA marker. The DNA marker includes a primer pair that is a marker (insertion / deletion marker) based on an insertion / deletion mutation in the core promoter region of the Sbe1 gene, Sbe1 — 5end — 12I / D_U (SEQ ID NO: 1) and Sbe1 — 5end — 12I / D_L (SEQ ID NO: 2) (Umemoto et al. (2008)).

The composition of the PCR reaction solution (10 μL) is as follows.
GoTaq mix (Promega) 5.0μL
Sterile water 4.0μL
Template DNA (20 ng / μL) 0.8 μL
Primer mixture (20 μM) 0.2 μL

The conditions for PCR are as follows.
1. DNA denaturation: 95 ° C., 2 minutes 2. DNA denaturation: 94 ° C., 30 seconds Annealing: 60 ° C., 30 seconds 4. Elongation reaction: 72 ° C., 30 seconds (35 cycles from 2 to 4 above)
5. Extension reaction: 72 ° C, 5 minutes

The amplified PCR products were separated by electrophoresis using 3% agarose gel, the position of the band, the progeny _{F 2} generation 200 individuals with mutant lines 202 and Koshihikari, SBE1 locus genotypes (mutant lines 202 homozygous , Koshihikari homotype, or heterotype). This F ₂ generations of individuals, in order to facilitate genetic analysis of the novel gelatinization easy of having the mutant lines 202, dismissed for heterozygous, were selected only the mutant strain 202 homozygous and Koshihikari homozygote.

Using 24 F ₃ seeds that were born to each F ₂ individual selected as described above, the genotype of the new gelatinization susceptibility of the F ₂ individual was examined by the same method as in Example 1 using the urea aqueous solution. It was. Individuals with ambiguous gelatinization results by a method using an aqueous urea solution were not included in the genetic analysis. On top of that, SBE1 seat Koshihikari homozygous 5 individuals, mutant lines 202 homozygous 12 individuals, and the _{F 2} individuals in total 33 individuals heterozygous 16 individuals were subjected to genetic analysis.

Preliminary mapping of a novel gelatinization susceptibility gene was performed for the 33 F ₂ individuals described above using a DNA marker. Among these HvSSR markers (Singh et al., Molecular Breeding, 25, p359-364, 2010) as this DNA marker, a clear polymorphism was confirmed between the mutant line 202 and Koshihikari, which are parents of the genetic analysis population. 46 markers (Table 6) were used. The composition of the PCR reaction solution, PCR conditions, and analysis of amplification products are the same as described above. The amplified PCR products were separated by electrophoresis using 3% agarose gel, and the genotype was determined by the position of the band.

The mapping data F ₂ generations 33 individual genotypes used in the analysis shown in FIG. In the locus chromosome region of the novel gelatinization gene, the coincidence rate between the gelatinization genotype and the DNA marker genotype is high. As a result of the analysis, it was suggested that a novel gelatinizable gene exists between the region amplified by HvSSR11-27 and the region amplified by HvSSR11-50 of chromosome 11. Therefore, SSR markers 202-9 (Forward Primer: SEQ ID NO: 99, Reverse Primer: SEQ ID NO: 100) and 202-16 (Forward Primer: SEQ ID NO: 101, Reverse Primer: SEQ ID NO: 102) located between the two markers ( As a result of performing analysis by adding Table 7), the matching rate was further increased. From this, it was almost determined that the novel gelatinization gene is located between the region amplified with HvSSR11-27 and the region amplified with HvSSR11-50. Therefore, the selection of a new easily gelatinizable DNA marker possessed by the mutant strain 202 is based on a DNA marker based on a polymorphism of a base sequence located between a region amplified by HvSSR11-27 and a region amplified by HvSSR11-50. It was found that it can be implemented by using.

(Selection of fertile rice individuals that are easily gelatinized)
As mentioned above, the F ₂ generation individuals obtained by crossing the mutant line 202 and the rice cultivar “Kitayukimochi” are divided into two groups, and the subsequent cultivation is performed separately in Sapporo City, Hokkaido and Fukuyama City, Hiroshima Prefecture. (Hereinafter referred to as “Sapporo material” or “Fukuyama material”, respectively).

(Culture and selection of Sapporo materials)
The above-mentioned cross progeny F ₂ generation 1109 individuals (seed) were used as brown rice, and 240 individuals that could be judged fertile from the appearance were selected. These were raised by a customary method, planted in a paddy field of Hokkaido Agricultural Research Center (Sapporo City, Hokkaido), and DNA was simply adjusted from the leaves. A simple DNA preparation method is as follows. One to two mature rice leaves were dried at 50 ° C. overnight. The dried leaves (about 2 cm) were cut with scissors for dissection, put into 2 mL microtubes with glass beads, and dried overnight at 50 ° C. with the lid open. The dried sample was pulverized into a powder by applying it to a pulverizer (shaking master, biomedical science) for 2 minutes. Add 400 μL of extraction buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1 M NaCl) to the microtube containing the sample powder, vigorously stir, centrifuge at 10,000 g for 10 minutes, and 350 μL of supernatant. Was transferred to a new 1.5 mL microtube. To this, 350 μL of isopropanol was added and mixed well by inversion, followed by centrifugation at 10,000 g for 10 minutes to precipitate DNA. After discarding the supernatant, the pellet was dried at room temperature for about 1 hour with the microtube lid kept open. Thereafter, the pellet was dissolved in 30 μL of 1/10 TE solution. 0.8 μL of this solution was used for the PCR reaction.

  Using this prepared DNA as a template, the genotypes of the Sbe1 locus, which is a known gelatinization locus, and the aforementioned novel gelatinization locus were examined. As a DNA marker, a primer pair that is a marker (insertion / deletion marker) based on an insertion / deletion mutation in the core promoter region of the Sbe1 gene, Sbe1 — 5end — 12I / D_U (SEQ ID NO: 1) and Sbe1 — 5end — 12I / D_L (SEQ ID NO: 2), HvSSR11-28 marker (Forward Primer: SEQ ID NO: 5, Reverse Primer: SEQ ID NO: 6) and HvSSR11-48 marker (Forward Primer: SEQ ID NO: 7, Reverse Primer: SEQ ID NO: 8) were used. The analysis of the PCR reaction solution composition, PCR conditions, and amplification product for DNA marker analysis is the same as described above. The amplified PCR products were separated by electrophoresis using 3% agarose gel, and the genotype was determined by the position of the band.

By selecting this DNA marker, both the Sbe1 locus and the new gelatinization susceptibility locus (Lgt (t) locus) of the F ₂ population are homozygous individuals (genotypes) of the fertile parent variety “Kitayukimochi”. : SBE1 / LGT), the Sbe1 locus is the mutant strain 202 homotype, the Lgt (t) locus is the “Kitayukimochi” homotype (genotype: sbe1 / LGT), and the Sbe1 locus is the “Kitayukimochi” homotype, Lgt ( t) Mutant strain 202 homotype (genotype: SBE1 / lgt) was selected for the locus, and mutant strain 202 homotype (genotype: sbe1 / lgt) was selected for both loci. These genotypes are summarized in the table below.

  When these selected individuals were harvested and white rice was adjusted, 8 g of white rice necessary for the hardenability test of koji was obtained. SBE1 / LGT was 6 individuals, sbe1 / LGT was 4 individuals, and SBE1 / lgt was 5 There were 5 individuals, sbe1 / lgt.

(Cultivation and selection of Fukuyama materials)
To practice cultivation above the progeny of the F ₂ generation 780 individuals (seeds) in the water rice paddy field of the National Agricultural Research Center for Western Region (Fukuyama City, Hiroshima Prefecture), the simple adjusted (Simple adjustment method of DNA to DNA from the leaves above the same as). Selection of fertile individuals was performed by a method using a DNA marker described in Wanchana et al., Plant Science, 165, p1193-1199, 2003, rather than determination based on the appearance of brown rice. The selection of the Sbe1 locus and the Lgt (t) locus was performed in the case of the above Sapporo material except that the HvSSR11-50 marker (Forward Primer: SEQ ID NO: 9 and Reverse Primer: SEQ ID NO: 10) was used instead of the HvSSR11-48 marker. The same was done.

  When these selected individuals were harvested and white rice was adjusted, the individuals that were able to adjust 8 g of white rice necessary for koji hardening test were 2 SBE1 / LGT, 3 sbe1 / LGT, and 2 SBE1 / lgt , Sbe1 / lgt was 3 individuals.

(Measurement of curability of rice cake)
8g of white rice from each sample of Sapporo and Fukuyama materials is absorbed and steamed, then it is sprinkled into a bowl with a small rice bowl for testing "Mini Usagi" (Applied Nutrition Food Research Laboratories), and the dough is made 7mm thick Molded. After being placed at 5 ° C. for 24 hours, a cylindrical probe having a diameter of 2 mm was attached to a texture analyzer (Stable Micro System, TA-XT2i), and the maximum positive load when 3 mm was penetrated at a speed of 2 mm / second ( g) was measured as hardness. In addition, sclerosis | hardenability measurement was performed by the same method with Sapporo material and Fukuyama material. In addition, the analysis of Sapporo materials was conducted after adding a sample of the fertile parent cultivar “Kitayukimochi” that was also cultivated in Sapporo (5 repeated measurements). We did not cultivate “Kitayukimochi” because it was extremely fast.

(Results of Sapporo materials)
The result of Sapporo material is shown in FIG. As a result of the hardness measurement, there is a significant difference between the hardness of the kite of “Kitayukimochi” and the hardness of the kite of SBE1 / LGT (both Sbe1 and Lgt (t) loci are “Kitayukimochi” genotypes). There was no difference. On the other hand, SBE1 / lgt and sbe1 / LGT (Sbe1 locus or Lgt (t) locus is mutant strain 202 genotype) tend to be softer than “Kitayukimochi”. In addition, sbe1 / lgt (both Sbe1 and Lgt (t) loci are mutated strain 202 genotypes) tends to be softer than SBE1 / lgt and sbe1 / LGT cocoons, and moreover, “Kitayukimochi” cocoons and The result was significantly softer than the SBE1 / LGT sputum.

(Results of Fukuyama materials)
The result of the Fukuyama material is shown in FIG. Similar to the results for the Sapporo material, the SBE1 / lgt and sbe1 / LGT kites tend to be softer than the SBE1 / LGT kites, and the sbe1 / lgt kites tend to be softer.

  From these facts, in addition to the Sbe1 locus, by selecting individuals whose novel gelatinization locus (Lgt (t) locus) was a mutant strain 202 genotype, it was possible to reliably select fertile individuals that were difficult to cure. It has been clarified that the koji made from the fermented low-temperature gelatinized rice obtained in this example is not hard and remains soft even after a certain period of time has passed since it was sown. It was done.

  As described above, according to the present invention, a processed rice product and food that maintain softness and taste, and a method for producing low-temperature gelatinized mutant rice that is easy to gelatinize and difficult to age are provided.

  Using the low-temperature gelatinized mutant rice according to the present invention, for example, after cooking, cooked rice, processed rice, gluten-added rice flour bread, rice flour blended bread, confectionery, noodles, dumpling skin, rice paper, baby food (retort), etc. It is possible to produce a food that is hard to be hardened and maintains its softness. In addition, using the fermented low-temperature gelatinized mutant rice according to the present invention, for example, raw confectionery such as rice cake products, Daifuku rice cake, white ball dumpling, rice flour blend bread, etc. A maintained food product can be produced. According to the present invention, since the taste and quality of these foods can be improved, there are advantages not only for consumers, but also for manufacturers and distributors, such as an increase in shelf life of foods.

Claims (10)

Mutating 旱 ignorant D;
A process of selecting low-temperature gelatinized mutant rice using a urea solution;
including,
A method for producing low-temperature gelatinized mutant rice characterized by the above. Following the step of selecting the low-temperature gelatinized mutant rice,
Crossing the low-temperature gelatinized mutant rice with fertile rice;
Selecting fertile individuals from the F ₂ generation of progenies of the mating;
Selecting a gelatine-friendly individual by detecting a mutation in the core promoter region of the starch branching enzyme I gene of chromosome 6 contained in the sample derived from the fertile individual;
including,
The method for producing low-temperature gelatinized mutant rice according to claim 1. HvSSR11-27 marker (Forward Primer: SEQ ID NO: 3, Reverse Primer: SEQ ID NO: 4) and HvSSR11-28 marker (Forward Primer: SEQ ID NO: 5, Reverse Primer: Sequence) using DNA in the sample derived from the fertile individual as a template And at least one of HvSSR11-48 marker (Forward Primer: SEQ ID NO: 7, Reverse Primer: SEQ ID NO: 8) and HvSSR11-50 marker (Forward Primer: SEQ ID NO: 9, Reverse Primer: SEQ ID NO: 10). Further comprising the step of selecting an easily gelatinizable individual using at least one of them,
The method for producing low-temperature gelatinized mutant rice according to claim 2.   The processed rice product which consists of low-temperature gelatinization variation rice obtained by the production method of any one of Claims 1 thru | or 3.   A food comprising the processed rice product according to claim 4.   A processed rice product made from low-temperature gelatinized mutant rice obtained by mutating 旱 Unknown D. The amylopectin short chain index value of the low-temperature gelatinized mutant rice is 110% or more of the amylopectin short chain index value of 旱 ignorant D,
The processed rice product according to claim 6. The gelatinization temperature of the low-temperature gelatinized mutant rice is 2 ° C. or more lower than the gelatinization temperature of Kashiwa Dignity D,
Processed rice products according to claim 6 or 7, characterized in that. The low-temperature gelatinized mutant rice is a fertile low-temperature gelatinized mutant rice obtained by crossing low-temperature gelatinized mutant rice and fertile rice,
The processed rice product according to any one of claims 6 to 8, wherein   A food comprising the processed rice product according to any one of claims 6 to 9.