WO2012111327A1 - Produit alimentaire contenant un gel d'amidon de riz - Google Patents

Produit alimentaire contenant un gel d'amidon de riz Download PDF

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Publication number
WO2012111327A1
WO2012111327A1 PCT/JP2012/000995 JP2012000995W WO2012111327A1 WO 2012111327 A1 WO2012111327 A1 WO 2012111327A1 JP 2012000995 W JP2012000995 W JP 2012000995W WO 2012111327 A1 WO2012111327 A1 WO 2012111327A1
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WIPO (PCT)
Prior art keywords
starch
enzyme
derived
food
treated
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PCT/JP2012/000995
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English (en)
Japanese (ja)
Inventor
敬司 市原
純矢 福田
中村　充
賢一 栗田
Original Assignee
グリコ栄養食品株式会社
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Application filed by グリコ栄養食品株式会社 filed Critical グリコ栄養食品株式会社
Priority to JP2012557839A priority Critical patent/JPWO2012111327A1/ja
Publication of WO2012111327A1 publication Critical patent/WO2012111327A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
    • A23G3/42—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds characterised by the carbohydrates used, e.g. polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/346—Finished or semi-finished products in the form of powders, paste or liquids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/212—Starch; Modified starch; Starch derivatives, e.g. esters or ethers
    • A23L29/219—Chemically modified starch; Reaction or complexation products of starch with other chemicals

Definitions

  • the present invention relates to a rice starch gel-containing food, a high-viscosity and starch-forming rice starch granule, a food prepared from the rice starch granule, and a method for producing the same.
  • the present invention relates to a method for producing a rice starch gel-containing food using an enzyme capable of improving the gel-forming ability of starch.
  • gelling agents When designing processed foods, the use of gelling agents is important for improving texture and physical properties, and various product developments are possible depending on how they are used.
  • natural polymers such as agar, gelatin, gellan gum, xanthan gum, locust bean gum, carrageenan, pectin, sodium alginate, tamarind seed gum, psyllium seed gum, microcrystalline cellulose, curdlan, starch, or carboxymethylcellulose Synthetic polymers such as (CMC) or methylcellulose are used as gelling agents.
  • CMC carboxymethylcellulose Synthetic polymers
  • gelling agents When these gelling agents are used, they may be used alone, but in order to form a gel having more diverse properties, for example, two or more kinds of gelling agents such as native gellan gum and guar gum are used in combination. Has been studied and used (Patent Document 1).
  • gelatin is weak against acids and alkalis
  • agar is also weak against acids.
  • Starch is not only raw starch, but also has a variety of physical properties by being added to food ingredients using processed starch (also called modified starch), which is a modified starch, such as acetate starch and phosphorylated starch, as a gelling agent.
  • modified starch also called modified starch
  • a modified starch such as acetate starch and phosphorylated starch
  • Patent Documents 2, 3 and 4 show examples in which crosslinked starch is used for bread, confectionery or noodles.
  • cross-linked starch having a high degree of cross-linking is added to foods, the hardness and viscosity of the gel can be increased, but the final product has the disadvantage of having a powdery texture and inferior flavor.
  • Starch is a material used for various purposes, and its most important functions are its thickening function and gel forming function. Particularly in the food industry, the thickening function and the gel-forming function of starch are widely used for the formation of food shapes, physical properties and textures.
  • the starch structure differs slightly depending on the raw material plant (for example, corn, potato, wheat, cassava, etc.), and as a result, the thickening function and the gel forming function also differ depending on the raw material plant. For this reason, those skilled in the art have long selected natural starch to be used according to the purpose. For example, wheat starch has been used extensively for fishery products. This is because wheat starch has an excellent gel forming function.
  • tapioca starch is generally used for foods that are highly transparent and require a sticky feeling.
  • the characteristics required in the recent food industry have become more sophisticated, and it has become impossible to cope with the problem simply by changing the natural starch used. Therefore, it is necessary to modify the thickening function or gel forming function of starch.
  • the most widely used means for modifying the thickening function or gel forming function of starch is chemical modification of starch.
  • the method of introducing a new cross-linking point between starch molecules using an appropriate chemical cross-linking agent and the method of applying a chemical treatment such as a method of introducing an appropriate functional group have a remarkable thickening function or gel forming function.
  • a chemical treatment such as a method of introducing an appropriate functional group
  • it has become widely used.
  • starches that have been subjected to such chemical treatment have been designated as food additives and are subject to legal regulations. For this reason, a technique for modifying the thickening function or the gel forming function of starch without chemical treatment has been expected.
  • starch enzyme treatment technology As a technology for modifying starch without chemical treatment, there is a starch enzyme treatment technology. Since an enzyme generally acts on a substrate dissolved in water, the usual enzyme treatment is performed after the starch is completely dissolved in water. Lower molecular weight molecules such as dextrin, starch syrup, maltooligosaccharide, maltose, glucose and the like are produced by cleaving the starch by dissolving hydrolase or glycosyltransferase in starch dissolved in water. However, in the enzyme treatment with these hydrolases or glycosyltransferases, starch molecules are cleaved to become low molecular weight molecules. Therefore, in general, the thickening function and gel forming function of the obtained molecules are the thickening function of starch. And it was thought to be lower or disappear than the gel-forming function.
  • Patent Document 5 discloses a technique in which an enzyme is allowed to act in the form of starch granules in water without dissolving the starch in water.
  • an enzyme when starch is enzymatically treated, it is generally dissolved in water before the enzyme treatment. However, it is not always necessary to dissolve the starch in water before the enzyme treatment.
  • a hydrolase such as ⁇ -amylase or glucoamylase is not dissolved in water and can act on starch particles suspended in water to produce reducing sugar. .
  • Patent Document 5 also discloses that, as a result, the viscosity of starch that has undergone enzyme treatment is lower than the viscosity of starch that has not undergone enzyme treatment.
  • Patent Document 5 suggests that a starch having an improved thickening function or a gel-forming function can be obtained by allowing a hydrolase or a glycosyltransferase to act on starch particles, compared to starch that has not been treated with an enzyme. Neither disclosed nor disclosed.
  • Patent Documents 6 to 10 also disclose a technique for causing a hydrolase to act on insoluble starch granules.
  • these inventions by making hydrolase act on starch granules, holes are made in the surface of the starch granules to create porous starch granules, and the porous starch granules are used as a powder substrate or a porous carrier.
  • the technology is disclosed.
  • Patent Documents 6 to 10 do not suggest or disclose that starch having improved thickening function and gel-forming function can be obtained by allowing a hydrolase or glycosyltransferase to act on starch granules.
  • the object of the present invention is not to make a hole in the surface of the enzyme-treated starch granule, but has nothing to do with the improvement of the thickening function and the gel-forming function and whether the surface of the enzyme-treated starch granule has a hole.
  • the enzyme-treated starch forms a hard gel in the heated food.
  • the enzyme-treated starch granules of the present invention can be used for heated foods.
  • the hardness of the gel formed using the enzyme-treated starch granules can be adjusted by adjusting the degree of enzyme treatment.
  • the hardness of the gel affects the food texture and texture. Therefore, the texture of food can be affected by using the method of the present invention. In this way, the enzyme-treated starch granules of the prior art and the enzyme-treated starch granules used in the present application are completely different in use and usage.
  • the present invention is intended to solve the above-described problems, and an object thereof is to provide a food containing rice starch gel having a desired degree of hardness and a method for producing the same.
  • a specific embodiment of the present invention without using chemical modification of rice starch, providing rice flour or rice starch excellent in thickening function or gel forming function, and providing food containing the rice flour or rice starch And a method for producing the rice flour or rice starch and food.
  • the present inventors have developed a specific hydrolase or glycosyltransferase having the property of improving the gel-forming ability of starch under the condition that starch does not dissolve. It was found that a starch having an excellent thickening function and a gel forming function can be obtained by acting on the grains, and the present invention was completed based on this.
  • hydrolase or glycosyltransferase is allowed to act on starch, the starch is cleaved to become lower molecules, and the viscosity and gel-forming ability of the resulting molecule are higher than the viscosity and gel-forming ability of starch before enzyme treatment. It is thought that it decreases or disappears.
  • the same hydrolase or glycosyltransferase as the hydrolase or glycosyltransferase that produces such excellent starch when it is allowed to act on starch granules under conditions where the starch does not dissolve can be obtained by dissolving the starch in water.
  • the starch viscosity is lowered, and a starch excellent in the thickening function or the gel forming function cannot be obtained.
  • the present invention cannot be predicted by conventional general knowledge and common general knowledge possessed by those skilled in the art.
  • the present inventors first filed an application for wheat starch, tapioca starch, and corn starch, when the above-mentioned hydrolase or glycosyltransferase is used, and starch having excellent thickening function and gel-forming function can be obtained. It was. The present inventors further found that starch having excellent thickening function and gel-forming function can be obtained by using the above-mentioned hydrolase or glycosyltransferase for rice flour or rice starch as well. Went.
  • the enzyme treatment conditions for starch granules may vary depending on the specificity of the enzyme and the origin of the starch granules.
  • a starch suspension is first prepared by suspending starch granules in ion exchange water or a buffer solution. When the pH of the starch suspension needs to be adjusted, the pH is adjusted to an appropriate pH of the enzyme. While the starch suspension is heated to a temperature at which the starch granules do not disintegrate (preferably about 10 ° C. to about 70 ° C.), the enzyme is added, for example, within about 24 hours (preferably about 1 hour to about 70 ° C.). (20 hours) The reaction can be carried out.
  • the enzyme and the saccharide eluted by enzymatic degradation are removed by a washing and dehydration process, which is a conventional method for preparing starch, and the target enzyme-treated starch granules can be obtained through a drying process.
  • the present invention is, for example, as follows: (Item 1) A method for producing rice starch gel-containing food, Mixing rice flour or rice starch with an enzyme; Treating the rice flour or starch granules in the rice starch with the enzyme at a temperature of about 10 ° C. or higher and about 70 ° C.
  • the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase and cyclodextrin glucanotransferase having the property of improving the gel-forming ability of starch.
  • (Item 2) The method according to Item 1, wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from the genus Aspergillus, and cyclodextrin glucanotransferase.
  • the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from the genus Aspergillus, and cyclodextrin glucanotransferase.
  • the above enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from Aspergillus oryzae, ⁇ -amylase derived from Aspergillus niger, and cyclodextrin glucanotransferase. 2. The method according to 2.
  • the enzyme is an amyloglucosidase derived from Aspergillus niger commercially available as AMG from Novozyme, an amyloglucosidase derived from Aspergillus niger commercially available as OPTIDEX L-400 from Genencor, and an asper derived from DAZISO as DIAZYME X4NPil Amyloglucosidase, Aspergillus niger-derived amyloglucosidase commercially available as glucoamylase "Amano" SD from Amano Enzyme, Rhizopus niveus-derived amyloglucosidase commercially available as Amuroenzyme from Amano Enzyme, and marketed as a smear from Shin Nippon Chemical Industry Rhizopus Ryzae-derived amyloglucosidase, Aspergillus niger-derived ⁇ -glucosidase commercially available as transglucosidase L “A
  • the enzyme is (1) a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11. Encoded by a nucleic acid molecule that is encoded and has starch hydrolyzing activity, or (2) a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule consisting of a base sequence complementary to the base sequence of SEQ ID NO: 13;
  • the stringent conditions include 50% formamide, 5 ⁇ SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt solution (0 .2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyrrolidone), 10% dextran sulfate Hybridization at 65 ° C.
  • the enzyme has (1) an amino acid sequence having at least 95% homology with the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12, and has starch hydrolysis activity.
  • (Item 7) The method according to any one of Items 1 to 6, wherein the starch granule is a starch granule of untreated starch, physical-treated starch or chemically modified starch.
  • the starch granule is a starch granule of untreated starch, and the starch granule is not subjected to chemical modification or physical treatment at any stage until the starch granule obtains a starch gel-containing food by the method, Items 1 to 7 The method of any one of these.
  • the starch granule is a starch granule of untreated starch or physical treated starch, and further includes a step of chemically modifying the enzyme-treated starch granule, and the chemically modified enzyme-treated starch granule is converted into the food material and water.
  • the starch granule is a starch granule of untreated starch or chemically modified starch, and further includes a step of physically treating the enzyme-treated starch granule, and the enzyme-treated starch granule is physically treated with the food material and water. 8. The method according to any one of items 1 to 7, wherein the method is mixed with.
  • (Item 16) The enzyme according to any one of items 11 to 15, wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from Aspergillus genus, and cyclodextrin glucanotransferase. Food.
  • the above-mentioned enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from Aspergillus oryzae, ⁇ -amylase derived from Aspergillus niger, and cyclodextrin glucanotransferase.
  • the present invention is also, for example, as follows: (Item 1A) A cooked rice starch-containing food made from enzyme-treated starch granules having high viscosity and gel-forming ability,
  • the rice starch-containing food is a food produced by a method comprising heating after mixing the food material and the enzyme-treated starch granules,
  • the enzyme-treated starch granule is a starch granule obtained by treating rice flour or an untreated starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve,
  • the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified
  • the enzyme-treated starch granules can form a gel having a Young's modulus higher than the Young's modulus of the untreated starch granules or a rupture stress higher than the rupture stress of the untreated starch granules when measured with a rhe
  • a cooked starch-containing food made from enzyme-treated rice starch granules having high viscosity and gel-forming ability is a food produced by a method comprising heating after mixing the food material and the enzyme-treated rice starch granules,
  • the enzyme-treated rice starch granule is a starch obtained by treating rice flour or an untreated starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve,
  • the enzyme-treated rice starch granules are not modified with hydroxyl groups at the 2nd, 3rd and 6th positions of the glucose residue,
  • the enzyme-treated rice starch granules are gels having a Young's modulus of 6.0 ⁇ 10 5 dyn / cm 2 or more and 2.2 ⁇ 10 6 dyn / cm 2 or less or a breaking stress of 40 g or more and 250 g or less as measured with a rheometer. Can form a food.
  • (Item 5A) The food according to any one of Items 1A to 4A, wherein the food is a high-moisture food, and the amount of water in the food is higher than 40 g and lower than 95 g per 100 g of edible portion.
  • (Item 6A) The item according to any one of Items 1A to 5A, wherein the food is selected from the group consisting of Japanese confectionery, oil-and-fat-containing food, gel food, processed fish and livestock meat, sauce and sauces, and noodles. Food.
  • (Item 8A) The food according to any one of Items 1A to 4A and 7A, wherein the food is selected from the group consisting of bakery products, pastry products, and fried foods.
  • Items 1A to 8A wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, cyclodextrin glucanotransferase and ⁇ -amylase having the property of improving the gel forming ability of starch. Foodstuff of any one of.
  • (Item 10A) The food according to Item 9A, wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, cyclodextrin glucanotransferase, and ⁇ -amylase derived from the genus Aspergillus.
  • the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, cyclodextrin glucanotransferase, and ⁇ -amylase derived from the genus Aspergillus.
  • the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from Aspergillus oryzae, cyclodextrin glucanotransferase, and ⁇ -amylase derived from Aspergillus niger. Food listed.
  • the enzyme-treated starch granule is a starch obtained by treating rice flour or untreated starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve, In the enzyme-treated starch granules, the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified,
  • the enzyme-treated starch granules can form a gel having a Young's modulus higher than the Young's modulus of the untreated starch granules or a rupture stress higher than the rupture stress of the untreated starch granules when measured with a rheometer.
  • the enzyme-treated starch granule is a starch granule obtained by treating rice flour or an untreated starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve, In the enzyme-treated starch granules, the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified, The enzyme-treated starch granules can form a gel having a Young's modulus higher than the Young's modulus of the untreated starch granules or a rupture stress higher than the rupture stress of the untreated starch granules when measured with a rheometer. Starch granules.
  • the enzyme-treated starch granule has a Young's modulus of 110% or more and 850% or less (110% or more and 330% or less in one embodiment) of the Young's modulus of the untreated starch granule, as measured with a rheometer, or The starch granules according to item 17A, which can form a gel having a breaking stress of 110% or more and 600% or less of the breaking stress of the untreated starch granules.
  • the enzyme-treated starch granule is a starch obtained by treating rice flour or untreated starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve, In the enzyme-treated starch granules, the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified,
  • the enzyme-treated starch granules are gels having a Young's modulus of 6.0 ⁇ 10 5 dyn / cm 2 or more and 2.2 ⁇ 10 6 dyn / cm 2 or less or a breaking stress of 40 g or more and 250 g or less as measured by a rheometer.
  • Items 14A to 14A wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, cyclodextrin glucanotransferase, and ⁇ -amylase having the property of improving the gel forming ability of starch.
  • the starch of any one of these.
  • the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from Aspergillus oryzae, cyclodextrin glucanotransferase, and ⁇ -amylase derived from Aspergillus niger.
  • (Item 19A) A method for producing enzyme-treated rice starch granules having high viscosity and gel-forming ability, Including a step of treating rice flour or untreated starch granules of rice starch with an enzyme at a temperature of 10 ° C. or higher and 70 ° C. or lower, The method wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, cyclodextrin glucanotransferase and ⁇ -amylase having the property of improving the gel-forming ability of starch.
  • (Item 20A) The method according to Item 19A, wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, cyclodextrin glucanotransferase, and ⁇ -amylase derived from the genus Aspergillus.
  • the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, cyclodextrin glucanotransferase, and ⁇ -amylase derived from the genus Aspergillus.
  • Item 21A Item 19A wherein the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from Aspergillus oryzae, cyclodextrin glucanotransferase and ⁇ -amylase derived from Aspergillus niger.
  • the method according to 20A is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase derived from Aspergillus oryzae, cyclodextrin glucanotransferase and ⁇ -amylase derived from Aspergillus niger.
  • the enzyme is an amyloglucosidase derived from Aspergillus niger commercially available as AMG from Novozyme, an amyloglucosidase derived from Aspergillus niger commercially available as OPTIDEX L-400 from Genencor, and an asper derived from DANASCO as DIAZYME X4NPil Amyloglucosidase, Ameroglucosidase derived from Aspergillus niger commercially available as Amano SD from Amano Enzyme, Amiloglucosidase derived from Rhizopus niveus commercially available as Gluczyme AF6 from Amano Enzyme, and marketed as a smear from Shin Nippon Chemical Industry Rhizopus oryza Amyloglucosidase derived from Aspergillus niger ⁇ -glucosidase derived from Aspergillus niger commercially available as Transglucosidase
  • the enzyme is encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having a complementary sequence of the base sequence of (1) SEQ ID NO: 1, 3, 5, 7, 9, or 11; And (2) is encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule consisting of a base sequence complementary to the base sequence of SEQ ID NO: 13, and has transglycosylation activity.
  • the stringent conditions are 50% formamide, 5 ⁇ SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyrrolidone), 10% dextrose sulfate Run, and hybridization at 65 ° C.
  • the enzyme has (1) an amino acid sequence having at least 95% homology with the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12, and has starch hydrolysis activity.
  • the enzyme-treated starch granule is a starch obtained by treating rice flour or untreated starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve, In the enzyme-treated starch granules, the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified,
  • the enzyme is an amyloglucosidase derived from Aspergillus niger commercially available as AMG from Novozyme, an amyloglucosidase derived from Aspergillus niger commercially available as OPTIDEX L-400 from Genencor, an Aspergillus derived from Aspergillus niger commercially available as DIAZYME X4NP from DANISCO, An amyloglucosidase derived from Aspergillus niger commercially available as glucoamylase “
  • the enzyme-treated starch granule is a starch granule obtained by treating rice flour or an untreated starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve, In the enzyme-treated starch granules, the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified,
  • the enzyme is (1) encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having a complementary sequence of the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11, and starch hydrolysis Or (2) encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule consisting of a base sequence complementary to the base sequence of SEQ ID NO: 13, and has transglycosylation activity,
  • the stringent conditions were: 50% formamide, 5 ⁇
  • the enzyme-treated starch granule is a starch granule obtained by treating an untreated rice starch or a starch granule of rice starch with an enzyme under conditions where the starch granule does not dissolve, In the enzyme-treated starch granules, the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified,
  • the enzyme has (1) an amino acid sequence having at least 95% homology to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12, and has starch hydrolysis activity, or (2) A starch granule having an amino acid sequence having at least 95% homology with the amino acid sequence of SEQ ID NO: 14 and having transglycosylation activity.
  • starch that has been chemically treated for bracken is often used, but requires combined treatment such as acetylation treatment and phosphoric acid crosslinking treatment.
  • starch granules developed this time are starches that have improved these disadvantages.
  • raw starch, physical-treated starch or bleached starch is used as a raw material and it is produced under conditions that are not chemically modified at any stage of the production process, Addition to ordinary foods, or foods mainly composed of starch, can be used for all foods as “food handling” without being limited in use.
  • raw starch, physically-treated starch or bleached starch is used as a raw material and it is manufactured under conditions that are not chemically modified at any stage of the manufacturing process, it is prepared using starch hydrolase or glycosyltransferase.
  • Enzyme-treated starch granules do not correspond to chemically modified processed starch of food additives. Therefore, if the enzyme-treated starch granules of the present invention prepared using starch hydrolase or glycosyltransferase are used, it is possible to prepare food without adding food additives.
  • the enzyme-treated starch granules of the present invention are more effective than untreated starch. Since it has a high gel-forming ability but is not forcedly bonded, it can be sufficiently gelatinized even at a normal heating temperature to develop a viscosity. Furthermore, although the obtained paste liquid is sufficiently gelatinized, it has little stringiness.
  • the gel obtained by using the starch granules of the present invention at a high concentration is very elastic.
  • the food of the present invention Even when a processed food or a physically processed starch is used as a raw material, or a food is produced under conditions where chemical modification or physical treatment is performed at any stage of the food production process, the food of the present invention
  • the gel is stiffer and has a different texture as compared to the case of using the corresponding starch produced without enzyme treatment. Therefore, according to the present invention, a food having a texture different from the conventional one can be provided.
  • starch granules refers to crystalline starch molecules.
  • the starch granule may be an untreated starch granule, or may be a starch granule obtained by chemically modifying or physically treating the untreated starch granule. If it is preferred to use enzyme-treated starch classified as food, the starch granules used are untreated starch granules obtained from plants. Plants store starch molecules as granules (ie, as large crystals) within amyloplasts. This granule is called a starch granule.
  • starch molecules are bonded together by hydrogen bonds or the like. Therefore, the starch granules are hardly dissolved in water as they are and are not easily digested. When starch granules are heated with water, they swell and loosen molecules into a colloidal form. This change is called “gelatinization”.
  • the size and form of starch granules vary depending on the plant from which the starch granules were obtained.
  • Rice has a double grain structure in which a large number of angular starch granules having a diameter of several ⁇ m are accumulated in amyloplasts. In the present invention, various commercially available starch granules can be used.
  • Starch granules may be prepared by a method such as purification of starch granules from plants or the like and used in the present invention.
  • the starch molecules are strongly bound to each other, so that the enzyme does not act easily.
  • the starch granules used in the present invention are isolated or purified from plants, but have been subjected to acid treatment, chemical modification treatment and heat treatment. There is nothing.
  • the term “untreated” starch granule is a naturally occurring starch granule that is derived from other components (for example, proteins, lipids, etc.) coexisting in the natural state. It refers to starch granules that have not been subjected to treatments other than those necessary for separation.
  • each step in the method for preparing starch granules such as a process of removing impurities from plants and the like to purify starch, is not included in the processing of starch granules in this specification.
  • starch granule any starch granule can be used as long as it is a commercially available starch granule.
  • the starch granule used in the present invention may be a starch granule that has been treated by subjecting untreated starch granule to chemical modification or physical treatment.
  • chemically modified starch granules include acetylated adipic acid crosslinked starch, acetylated oxidized starch, acetylated phosphate crosslinked starch, sodium octenyl succinate starch, acetate starch, oxidized starch, bleached starch, hydroxypropylated phosphate crosslinked Starch, hydroxypropyl starch, phosphoric acid crosslinked starch, phosphorylated starch and phosphorylated monoesterified phosphoric acid crosslinked starch.
  • “Acetylated adipic acid-crosslinked starch” refers to a product obtained by esterifying starch with acetic anhydride and adipic anhydride.
  • “Acetylated oxidized starch” refers to a product obtained by treating starch with sodium hypochlorite and then esterifying with acetic anhydride.
  • “Acetylated phosphate cross-linked starch” refers to a product obtained by esterifying starch with sodium trimetaphosphate or phosphorus oxychloride and acetic anhydride or vinyl acetate.
  • Starch sodium octenyl succinate refers to a product obtained by esterifying starch with octenyl succinic anhydride.
  • Starch acetate refers to a product obtained by esterifying starch with acetic anhydride or vinyl acetate.
  • Oxidized starch is obtained by treating starch with sodium hypochlorite, and in accordance with the purity test method described in Ministry of Health, Labor and Welfare Notification No. 485, carboxy group (also referred to as carboxyl group) in sample starch. ) When the carboxy group is 1.1% or less. However, even if the amount of carboxy group is within this range, “bleached starch” is not included in the definition of “oxidized starch”.
  • the “bleached starch” was obtained by treating starch with sodium hypochlorite, and analyzed the carboxy group in the sample starch according to the purity test method described in Ministry of Health, Labor and Welfare Notification No. 485. In some cases, the carboxy group is 0.1% or less, the test result by the “confirmation test (3)” of the oxidized starch described in the Ministry of Health, Labor and Welfare Notification No. 485 is negative, and the starch properties such as viscosity are generated. This can reasonably explain that the change is not due to oxidation. Even if the amount of carboxy group is 0.1% or less, those whose starch properties such as viscosity are changed from natural starch are classified as oxidized starches and are not handled as food in Japan but as food additives .
  • “Hydroxypropylated phosphate cross-linked starch” refers to a product obtained by esterifying starch with sodium trimetaphosphate or phosphorus oxychloride and etherifying with propylene oxide. “Hydroxypropyl starch” refers to a product obtained by etherifying starch with propylene oxide. “Phosphate cross-linked starch” refers to a product obtained by esterifying starch with sodium trimetaphosphate or phosphorus oxychloride. “Phosphorylated starch” refers to a product obtained by esterifying starch with orthophosphoric acid, potassium salt or sodium salt thereof, or sodium tripolyphosphate.
  • Phosphoric acid monoesterified phosphoric acid crosslinked starch means a product obtained by esterifying starch with orthophosphoric acid, potassium salt or sodium salt thereof or sodium tripolyphosphate, and esterifying with sodium trimetaphosphate or phosphorus oxychloride. .
  • starch particles examples include wet heat-treated starch and heat-suppressed starch.
  • the starch granules used in the present invention are provided as rice flour (for example, glutinous rice flour or glutinous rice flour) or rice starch (for example, glutinous rice starch or glutinous rice starch).
  • rice flour for example, glutinous rice flour or glutinous rice flour
  • rice starch for example, glutinous rice starch or glutinous rice starch
  • the term “rice flour” refers to pulverized polished rice grains.
  • “Scouring” refers to removing rice cake by polishing rice grains excluding rice husk. There is a whitening ratio as an index of the degree of whitening.
  • the milling rate is calculated by ⁇ (weight of milled rice after milling) / (weight of raw rice grains excluding rice husk) ⁇ ⁇ 100.
  • the polishing rate of 10% means that the weight of the koji is 10%, and the weight of the resulting polished rice grains is 90%.
  • the raw material of the rice flour used in the present invention may be japonica rice or indica rice. Japonica rice is preferred.
  • the raw material of rice flour may be sticky rice or sticky rice.
  • the raw material for the rice flour may be broken rice.
  • the milling rate of the rice flour used as the raw material for the rice flour is preferably about 10% or more, more preferably about 15% or more, and most preferably about 20% or more.
  • the polishing rate is preferably about 95% or less, and more preferably about 90% or less.
  • the protein content of rice flour used in the present invention is usually about 1% by weight or more, and may be, for example, about 5% by weight or about 6% by weight or more.
  • the protein content of the rice flour used in the present invention is preferably about 10% by weight or less, more preferably about 9% by weight or less, still more preferably about 8% by weight or less, about 7% by weight. It is particularly preferred that it is no more than wt%, and most preferred is no more than about 6 wt%.
  • the rice flour must be one that has not been subjected to a treatment (for example, heat treatment) that disrupts the structure of the starch granules in the production process.
  • Rice flour is generally used for rice flour noodles (rice noodles, rice noodles, etc.), Japanese confectionery (sheep, buns, rice crackers, etc.), Western confectionery (cookies, cakes, etc.), bread, dumpling skins, and grilled skins.
  • rice starch means starch refined from rice grains.
  • Rice starch can be produced, for example, by removing protein from the raw rice and purifying it.
  • a general method for producing rice starch will be described in more detail. For example, about 50% of the rice protein is removed and the rice grains are softened by immersing with an alkaline solution to separate the protein from the raw rice and soften the rice grains.
  • crude starch milk is obtained by grinding, adding an alkali liquid further.
  • the crude starch milk is washed with water (for example, 4 to 5 times) using a combination of a classification type or a nozzle type centrifuge to further remove proteins, thereby obtaining a purified starch milk.
  • the purified starch milk thus obtained is neutralized with hydrochloric acid, washed with water, dehydrated, dried and then purified to obtain purified rice starch having a protein content of about 0.3% or less.
  • Rice starch needs to have not been subjected to a treatment (for example, heat treatment) that disrupts the structure of starch granules in the production process.
  • Rice starch is a multi-faceted double grain, generally having an average particle size of about 2 to about 5 micrometers, the smallest among commercially available starches. For this reason, rice starch is often expensive because production is difficult and yield is low.
  • rice starch Since rice starch is a fine particle, it can be changed to a smooth surface by adhering to the uneven surface to give a smooth feel. Therefore, rice starch is often used for industrial materials such as photographic paper and cosmetics, and for lubricants such as hand flour, dusting powder and sprinkle powder for foods.
  • untreated starch When using untreated starch as starch granules, it is preferable to use untreated rice starch granules.
  • modified starch When using modified starch as starch granules, acetylated adipic acid cross-linked starch, acetylated oxidized starch, acetylated phosphoric acid cross-linked starch, sodium octenyl succinate starch, starch acetate, oxidized starch, bleached starch It is preferable to use hydroxypropylated phosphoric acid crosslinked starch, hydroxypropyl starch, phosphoric acid crosslinked starch, phosphorylated starch, or phosphoric acid monoesterified phosphoric acid crosslinked starch. In the case of using a physically treated starch, it is preferably a wet heat-treated starch or a heat-inhibited starch of rice flour or rice starch.
  • Chemical modification alters the physical properties of untreated starch granules.
  • cross-linking such as phosphate cross-linking and adipic acid cross-linking generally makes the gel formed using the resulting starch granules harder and less turbid than the gel formed using untreated starch granules.
  • Hydroxypropylation, acetylation, and oxidation treatment generally improves the gel formed using the resulting starch granules to be more transparent and softer than the gel formed using untreated starch granules.
  • Octenyl succinic acid treatment can generally allow the gel formed using the resulting starch granules to contain oil.
  • Physical treatment also modifies the physical properties of untreated starch granules.
  • moist heat treatment generally makes gels formed using the resulting starch granules harder than gels formed using untreated starch granules, and reduces paste viscosity.
  • heat suppression treatments generally make the gel formed using the resulting starch granules harder than the gel formed using untreated starch granules.
  • those having a long dry heat treatment time often exhibit a low paste viscosity, such as highly crosslinked starch.
  • the starch granules used in the present invention contain as little impurities as possible.
  • the content of impurities in the starch granules is preferably about 10% by weight or less, more preferably about 5% by weight or less, and still more preferably about 1% by weight or less.
  • Enzymes that can be used in the present invention are starch hydrolase or glycosyltransferase.
  • Starch hydrolases are roughly classified into ⁇ -amylase, ⁇ -amylase, amyloglucosidase, isoamylase, pullulanase and ⁇ -glucosidase.
  • enzymes classified into the same enzyme for example, ⁇ -amylase
  • enzymes classified into the same enzyme are considered to have different characteristics such as enzyme reaction specificity and substrate specificity when the producing bacteria are different. Since these starch hydrolases and glycosyltransferases are very widely distributed in animals, microorganisms, and plants, it can be said that there are an infinite variety of starch hydrolases and glycosyltransferases.
  • the starch hydrolyzing enzyme that can be used for producing the starch granules of the present invention is starch hydrolysate selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, and ⁇ -amylase having the property of improving the gel-forming ability of starch. It is a degrading enzyme.
  • ⁇ -amylase having the property of improving the gel-forming ability of starch means that the Young's modulus or breaking stress of starch granules after enzyme treatment is measured by the determination method described below.
  • the ⁇ -amylase is 10% or more higher than the Young's modulus or breaking stress of starch granules before enzyme treatment.
  • the starch hydrolase used in the present invention is preferably an enzyme classified as ⁇ -amylase, amyloglucosidase, isoamylase, or ⁇ -glucosidase. Enzymes classified as ⁇ -amylase or pullulanase are not preferred. An enzyme classified as amyloglucosidase, isoamylase or ⁇ -glucosidase is considered to be able to produce enzyme-treated starch granules having high viscosity and gel-forming ability when applied to starch granules.
  • Determination of whether an enzyme classified as ⁇ -amylase is ⁇ -amylase having the property of improving the gel-forming ability of starch can be determined by the following determination method.
  • glycosyltransferase that can be used for producing the starch granules of the present invention is cyclodextrin glucanotransferase.
  • An ⁇ -amylase having the property of improving the gel forming ability of starch can be identified by the following method. 900 g of ion-exchanged water is added to 400 g of wheat starch and suspended, and each enzyme is added thereto. The amount of reducing sugar released into the suspension by the reaction is measured to determine the decomposition rate. When the decomposition rate reaches 15%, the starch granules are collected by filtration, washed with water, and dried. Using the enzyme-treated starch granules thus obtained, the Young's modulus and breaking stress are determined by rheometer analysis.
  • the enzyme When the Young's modulus or breaking stress of the starch granules after the enzyme treatment is higher by 10% or more than the Young's modulus or breaking stress of the starch granules before the enzyme treatment, the enzyme has the property of improving the gel forming ability of the starch. It is determined that it has ⁇ -amylase. As an example, Table 1A below shows the determination results of various starch hydrolases.
  • the starch granules used in the method of the present invention are provided as rice flour or rice starch. Whether or not ⁇ -amylase has the property of improving the gel-forming ability of starch is determined by using wheat starch. Used as one indicator.
  • a starch paste is prepared so that the concentration of rice flour is 30% by weight in terms of dry matter, and that of rice starch or wheat starch is 20% by weight in terms of dry matter, and filled into a crehalon casing with a folding width of 45 mm. To do. This is heated up to 90 ° C. at 1 ° C./min and held at 90 ° C. for 30 minutes. Thereafter, the mixture was allowed to cool for 30 minutes in a constant temperature water bath at 20 ° C., and then cooled to 5 ° C. in a refrigerator. After cooling, the sample is stored refrigerated at 5 ° C.
  • rheometer RT-2010J-CW manufactured by Rheotech.
  • the measurement conditions of the rheometer were a break test as a test item, a sample height of 25 mm, an adapter with a viscosity sphere ⁇ 5 (diameter 5 mm, area 19.635 mm 2 ), and a sample moving speed (breaking speed) of 6 cm / Measure in min.
  • breaking stress g
  • Young's modulus (dyn / cm 2 ).
  • starch granules of the present invention is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase having the property of improving the gel forming ability of starch, and cyclodextrin glucanotransferase. Enzymes are used.
  • the enzyme is selected from the group consisting of amyloglucosidase; isoamylase; ⁇ -glucosidase, ⁇ -amylase from the genus Aspergillus and cyclodextrin glucanotransferase.
  • the enzyme is selected from the group consisting of amyloglucosidase, isoamylase, ⁇ -glucosidase, ⁇ -amylase from Aspergillus oryzae, ⁇ -amylase from Aspergillus niger and cyclodextrin glucanotransferase.
  • the enzyme is an Aspergillus niger-derived amyloglucosidase commercially available as AMG from Novozyme, an Aspergillus niger-derived amyloglucosidase commercially available as OPTIDEX L-400 from Genencor, and an Asperglu as Derzyme X4NPil commercially available as DIAZYME X4NPil.
  • Amyloglucosidase Aspergillus niger-derived amyloglucosidase commercially available as glucoamylase "Amano" SD from Amano Enzyme, Rhizopus niveus-derived amyloglucosidase commercially available as Amuroenzyme from Amano Enzyme, and marketed as a smear from Shin Nippon Chemical Industry Rhizop Aspergillus niger-derived ⁇ -glucosidase from Aspergillus niger, marketed as transglucosidase L “Amano” from Amylozyme derived from S.
  • Aspergillus niger-enzyme enzyme derived from Aspergillus niger, from Aspergillus niger, commercially available as Transglucosidase L-500 Aspergillus oryzae-derived ⁇ -amylase commercially available as A, Aspergillus oryzae-derived ⁇ -amylase available from Shin Nippon Chemical Industry, Aspergillus niger-derived ⁇ -amylase available from Danisco as AMYLEX A3, New Japan Commercially available from the chemical industry as Sumiteam AS Aspergillus niger-derived ⁇ -amylase, Pseudomonas amyloderamosa-derived isoamylase commercially available from Sigma as an isoamylase, Novozyme as Bacillus licheniformis-derived cyclodextrin glucanoenzyme as a cyclodextrin glucanoenzyme conserved as a cyclodextrin
  • the enzyme is starch hydrolase, which is stringent with a nucleic acid molecule having a sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11. It is encoded by a nucleic acid molecule that hybridizes under conditions and has starch hydrolyzing activity.
  • the stringent conditions are 50% formamide, 5 ⁇ SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate.
  • the starch hydrolase has an amino acid sequence having at least 95% homology to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 and has starch hydrolyzing activity. Have.
  • ⁇ -Amylase is present in many microorganisms, animals and plants.
  • microorganisms producing the ⁇ - amylase Aspergillus spp (e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus flavus, Aspergillus kawachii, such as Aspergillus sclerotiorum) ,; Bacillus genus (e.g., Bacillus subtilis, Bacillus acidocaldarius, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus cereus, Bacillus licheniformis, etc .; Geobacillus genus (eg, Geobac) llus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus thermodenitrificans, etc.); Lactobacillus gen
  • Lactobacillus manihotivorans Lactobacillus manihotivorans); otherwise, Pseudomonas sp. Pyrococcus furiosus, Rhizopus microsporus, Thermotoga maritima, Vibrio sp. Etc.
  • animal-derived ⁇ -amylase is human pancreas, human saliva, human urine, porcine pancreas, bovine pancreas, carp intestine, etc.
  • plant-derived ⁇ -amylase is barley, rice, wheat, oats, rye, It has been confirmed that it exists in soybeans and broad beans.
  • the organism producing ⁇ -amylase is not limited to these.
  • the ⁇ -amylase may be commercially available, prepared from these organisms by methods known in the art, or genetic recombination based on the amino acid sequence or base sequence of ⁇ -amylase of these organisms. It may be prepared by a method or may be chemically synthesized. Any ⁇ -amylase known in the art can be used as long as it has the property of degrading the ⁇ -1,4-glucoside bond in the endo form.
  • the ⁇ -amylase used in the present invention is preferably an ⁇ -amylase belonging to the genus Aspergillus, and most preferably an ⁇ -amylase derived from Aspergillus oryzae or Aspergillus niger.
  • a nucleotide sequence encoding a typical ⁇ -amylase derived from Aspergillus oryzae is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.
  • a nucleotide sequence encoding a representative ⁇ -amylase derived from Aspergillus niger is shown in SEQ ID NO: 3, and its amino acid sequence is shown in SEQ ID NO: 4.
  • ⁇ -Amylases between related species are considered to have very high homology and show similar enzymatic activity. Therefore, it is considered that the ⁇ -amylase derived from Aspergillus oryzae has an amino acid sequence having a very high homology with SEQ ID NO: 2 and exhibits the same enzyme activity.
  • ⁇ -amylase derived from Aspergillus oryzae has been shown to have the property of improving the gel-forming ability of starch, the ⁇ -amylase having the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence having high homology thereto It is considered that ⁇ -amylase having a property of improving the gel-forming ability of starch.
  • ⁇ -amylase having the amino acid sequence of SEQ ID NO: 2 and homology thereto It is considered that ⁇ -amylase having a high amino acid sequence also has the property of improving the gel forming ability of starch.
  • the ⁇ -amylase used in the present invention is not an amylase derived from Bacillus amyloliquefaciens. This is because the amylase derived from Bacillus amyloliquefaciens cannot produce starch having high viscosity and gel forming ability.
  • ⁇ -amylases are commercially available. Examples of commercially available ⁇ -amylases are described below: Biozyme F1OSD (Origin Aspergillus oryzae; Amano Enzyme Co., Ltd.), Biozyme A (Origin Aspergillus oryzae; Amano Enzyme Inc.), Cochase (Origin Aspergillus oryzae Chemicals; Sumiteam L (origin Aspergillus oryzae; Shinnihon Chemical Industry Co., Ltd.), AMYLEX A3 (origin Aspergillus niger; Danisco Japan Ltd.), Green Amil A (origin Aspergillus oryzae; Danisco Japan Ltd.), VERON AX (Origin Aspergillus oryzae; Higuchi Shokai), VERON GX (Origin Aspergillus o yzae; Higuchi Shokai Co., Ltd.), VERON M4
  • Such a commercially available ⁇ -amylase is analyzed for amino acids to determine the amino acid sequence, a DNA sequence is designed based on the amino acid sequence, and the DNA sequence is introduced into Escherichia coli or the like to introduce the same amino acid as the commercially available ⁇ -amylase. It is possible to produce an ⁇ -amylase having the sequence.
  • amyloglucosidase refers to an enzyme that hydrolyzes the 1,4- ⁇ bond at the non-reducing end of a sugar chain such as starch to produce ⁇ -D-glucose. Amiloglucosidase hydrolyzes the ⁇ -1,4-glucoside chain from the non-reducing end, and the ⁇ -1,6-glucoside chain also degrades at a slow rate.
  • the amyloglucosidase is called glucan 1,4- ⁇ -glucosidase.
  • amyloglucosidase is also referred to as exo-1,4- ⁇ -D-glucosidase, 1,4- ⁇ -D-glucan glucohydrolase, glucoamylase, ⁇ -amylase, lysosomal ⁇ -glucosidase or acid maltase.
  • Amiloglucosidase is classified as EC 3.2.1.3.
  • Amyloglucosidase is present in many microorganisms, animals and plants.
  • microorganisms producing amyloglucosidase Aspergillus spp (e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus candidus, Aspergillus terreus, Aspergillus awamori, Aspergillus phoenicis, etc.
  • Aspergillus saitoi e.g., Candida antarctica, Candida tsukubaensis etc.
  • Rhizopus genus for example, Rhizopus delemar, Rhizopus delmar, Rhizopus javanicus, Rhizopus niveus, Rhizopus ni eus, Rhizopus oligosporus, Rhizopus oryzae etc.
  • Saccharomyces genus e.g.
  • Saccharomyces cerevisiae Saccharomyces diastaticus, Saccharomyces diastaticus, Saccharomyces fibuligera
  • Other Clostridium thermoamylolyticum Cladosporium resinae, Lentinus edodes, Mucor rouxianus, Magnaporthe grisea, Monascus kaoliang, Paecilomyces varioti , Penicillium oxalicum, T ermomyces lanuginosus, like Trichoderma reesei.
  • animal-derived amyloglucosidase is present in the small intestinal mucosa of humans, rats, and mice, and plant-derived amyloglucosidase is present in beets and the like.
  • the organism that produces amyloglucosidase is not limited to these.
  • amyloglucosidase may be commercially available, prepared from these organisms by methods known in the art, or by genetic recombination methods based on the amino acid sequence or base sequence of the amyloglucosidase of these organisms. It may be prepared or chemically synthesized. As long as it has the property of decomposing ⁇ -1,4-glucoside bond and ⁇ -1,6-glucoside bond into glucose units from the non-reducing terminal side in the exo form to produce ⁇ -glucose, any known in the art Amiloglucosidase can be used.
  • amyloglucosidase used in the present invention is preferably an amyloglucosidase belonging to the genus Aspergillus or an amyloglucosidase belonging to the genus Risopus, and most preferably an amyloglucosidase derived from Aspergillus niger, or an amyloglucosidase derived from Risopus nieveus.
  • a nucleotide sequence encoding a typical amyloglucosidase derived from Aspergillus niger is shown in SEQ ID NO: 5, and its amino acid sequence is shown in SEQ ID NO: 6.
  • Amyloglucosidase between related species is considered to have very high homology and show similar enzymatic activity. Therefore, amyloglucosidase derived from Aspergillus niger has an amino acid sequence that is very homologous to SEQ ID NO: 6, and is considered to exhibit the same enzyme activity.
  • amyloglucosidase derived from commercially available Aspergillus niger has been shown to have starch hydrolyzing activity
  • starch is also used for amyloglucosidase having the amino acid sequence of SEQ ID NO: 6 and amyloglucosidase having an amino acid sequence highly homologous thereto. It is thought to have hydrolytic activity.
  • amyloglucosidase used in the present invention is not amyloglucosidase derived from Candida tsukubaensis. This is because amyloglucosidase derived from Candida tsukubaensis cannot produce starch having high viscosity and gel forming ability.
  • amyloglucosidases are commercially available. Examples of commercially available amyloglucosidases are described below: Gluc SG (Origin Rhizopus niveus; Amano Enzyme Co., Ltd.), Gluczyme AF6 (Origin Rhizopus niveus; Amano Enzyme Co., Ltd.), Gluczyme NL4.2 (Origin Aspergillus Tenno; Enzyme), glucoamylase “Amano” SD (origin Aspergillus niger; Amano Enzyme Ltd.), GODO-ANGH (origin Aspergillus niger; Joint Alcohol Co., Ltd.), OPTIDEX L-400 (Origin Aspergillus; Genencor Kyowa Co., Ltd.), OPTIDEX L (Origin Aspergillus niger; Genencor Kyowa Co., Ltd.), Sumi Team (Origin R) izpus oryzae
  • amyloglucosidase having.
  • Isoamylase refers to an enzyme that decomposes ⁇ -1,6-glucoside bonds at branch points such as amylopectin and glycogen to produce amylose-like linear polysaccharides. Isoamylase is also called glycogen 6-glucanohydrolase. Isoamylases are classified as EC 3.2.1.68. The isoamylase can be from any organism that produces isoamylase.
  • Isoamylase is present in many microorganisms, animals and plants.
  • microorganisms that produce isoamylase include Flavobacterium sp. Bacillus sp. Other examples include Pseudomonas amyloderamosa, Sulfolobus solfatricus and the like.
  • animal-derived isoamylase exists in human pancreas, etc.
  • plant-derived isoamylase exists in rice (Oryza sativa), potato (Solanum tuberosum) tuber, Arabidopsis thaliana, etc. .
  • the organism that produces isoamylase is not limited to these.
  • Isoamylases may be commercially available, prepared from these organisms by methods known in the art, or by genetic recombination methods based on the amino acid sequence or base sequence of the isoamylase of these organisms. It may be prepared or chemically synthesized. Any isoamylase known in the art can be used as long as it has the property of degrading the ⁇ -1,6-glucoside bond of amylopectin in an endo form.
  • the isoamylase used in the present invention is preferably an amylase belonging to the genus Flavobacterium or Pseudomonas, more preferably Flavobacterium sp.
  • Flavobacterium sp A nucleotide sequence encoding a representative isoamylase derived from is shown in SEQ ID NO: 7, and its amino acid sequence is shown in SEQ ID NO: 8.
  • a nucleotide sequence encoding a typical isoamylase derived from Pseudomonas amyloderamosa is shown in SEQ ID NO: 9, and its amino acid sequence is shown in SEQ ID NO: 10.
  • Isoamylases have very high homology among closely related species and are thought to exhibit similar enzyme activity. Therefore, Flavobacterium sp.
  • the derived isoamylase has an amino acid sequence having a very high homology with SEQ ID NO: 8, and is considered to exhibit the same enzyme activity.
  • Flavobacterium sp Since the derived isoamylase has been shown to have starch hydrolyzing activity, isoamylase having the amino acid sequence of SEQ ID NO: 8 and isoamylase having an amino acid sequence having high homology thereto also have starch hydrolyzing activity. It is thought to have. Similarly, since commercially available isoamylase derived from Pseudomonas amyloderamosa has been shown to have starch hydrolyzing activity, isoamylase having the amino acid sequence of SEQ ID NO: 10 and isoamylase having an amino acid sequence highly homologous thereto Is also considered to have starch hydrolysis activity.
  • isoamylases are commercially available. Examples of commercially available isoamylases are described below: GODO-FIA (Origin Flavobacterium odoratum; Joint Alcohol Co., Ltd.), Isoamylase (Origin Pseudomonas sp .; Sigma Aldrich).
  • Such a commercially available isoamylase is analyzed for amino acid to determine its amino acid sequence, a DNA sequence is designed based on the amino acid sequence, and the DNA sequence is introduced into Escherichia coli or the like to obtain the same amino acid sequence as that of the commercially available isoamylase. It is possible to produce isoamylase having the same.
  • ⁇ -Glucosidase refers to an enzyme that hydrolyzes the ⁇ -1,4-glucoside bond at the non-reducing end to produce ⁇ -glucose.
  • ⁇ -Glucosidase is referred to as ⁇ -D-glucoside glucohydrolase.
  • ⁇ -Glucosidase is also called maltase, glucoinvertase or glucoside sucrase.
  • ⁇ -D-Glucosidase is classified as EC 3.2.1.20.
  • ⁇ -Glucosidase is present in many microorganisms, animals and plants.
  • microorganisms producing ⁇ - glucosidase Aspergillus spp (e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus fumigatus, Aspergillus nidulans, etc.); Bacillus genus (e.g., Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus caldovelox, Bacillus cereus, Bacillus licheniformis, Bacillus thermoglucosidius, Bacillus sp., Bacillus subtilis, Bacillus revis, Bacillus stearothermophilus; Lactobacillus genus (Lactobacillus acidophilus, etc.
  • Lactobacillus brevis Lactobacillus brevis
  • Penicillium genus Penicillium brevicompactum, Penicillium citrinum, Penicillium oxalicum, Penicillium purpurogenum
  • Pyrococcus sp Pyrococcus furiosus, such as Pyrococcus woesei
  • Saccharomyces genus Saccharomyces genus (Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Sacc haromyces fibuligera, Saccharomyces oviformis, Saccharomyces carlsbergensis, Saccharomyces logos, etc.);.
  • Escherichia coli can be mentioned animal origin ⁇ - glucosidase, molluscs, crustaceans , Invertebrates such as insects, and vertebrates such as fish, amphibians, reptiles, birds and mammals.
  • Plant-derived ⁇ -glucosidase is present in beans, rice, buckwheat, corn, sugar beet seeds, etc. To do And have been confirmed. The organism that produces ⁇ -glucosidase is not limited to these.
  • ⁇ -Glucosidase may be commercially available, prepared from these organisms by methods known in the art, or genetic recombination based on the amino acid sequence or base sequence of ⁇ -glucosidase of these organisms. It may be prepared by a method or may be chemically synthesized. Any ⁇ -glucosidase known in the art as long as it has the property of degrading ⁇ -1,4- and ⁇ -1,6-glucoside bonds in glucose units from the non-reducing end side to produce ⁇ -glucose. Can be used.
  • the ⁇ -glucosidase used in the present invention is preferably an Aspergillus genus ⁇ -glucosidase, and more preferably an Aspergillus niger-derived ⁇ -glucosidase.
  • a nucleotide sequence encoding a representative ⁇ -glucosidase derived from Aspergillus niger is shown in SEQ ID NO: 11, and its amino acid sequence is shown in SEQ ID NO: 12.
  • ⁇ -Glucosidase between related species is considered to have very high homology and show similar enzymatic activity. Therefore, it is considered that the ⁇ -glucosidase derived from Aspergillus niger has an amino acid sequence having a very high homology with SEQ ID NO: 12, and exhibits the same enzyme activity.
  • ⁇ -glucosidase having the amino acid sequence of SEQ ID NO: 12 and ⁇ -glucosidase having an amino acid sequence highly homologous thereto Is also considered to have starch hydrolysis activity.
  • transglucosidase L 500 oil Aspergillus; Genencor Kyowa Co., Ltd.
  • transglucosidase L “Amano” oil Aspergillus niger; Amano Enzyme Ltd.
  • ⁇ -Glucosidase Origin Bacillus stearothermophilus (Sigma Aldrich)
  • ⁇ -Glucosidase origin rice; Sigma Aldrich
  • ⁇ -Glucosidase origin Saccharomyces cerevisiae Algrich
  • ⁇ -Glucosidase Sigma Aldrich
  • ⁇ -Glucosidase Spinning stock Company
  • Amino acid analysis of such a commercially available ⁇ -glucosidase is performed to determine the amino acid sequence, a DNA sequence is designed based on the amino acid sequence, and the DNA sequence is introduced into Escherichia coli and the like to introduce the same amino acid as the commercially available ⁇ -glucosidase. It is possible to produce ⁇ -glucosidase having the sequence.
  • Cyclodextrin glucanotransferase is also called CGTase and is classified as EC 2.4.1.19.
  • CGTase is an enzyme that can catalyze the transglycosylation reaction (ie, heterogenization reaction) of maltooligosaccharide.
  • CGTase recognizes 6-8 glucose chains at the non-reducing end of the donor molecule and carries out a transfer reaction so that this part is cyclized.
  • CGTase is preferably a cyclodextrin glucanotransferase from Bacillus licheniformis commercially available as Torzyme from Novozyme and Paenibacillus macerans commercially available as a contigzyme from Amano Enzyme (also classified as a transdextrin from Bacillus macerans). It is selected from the group consisting of optimum pH 6.0).
  • CGTase may be commercially available, may be prepared from a CGTase-producing organism by methods known in the art, or may be prepared by a genetic recombination method based on the amino acid sequence or base sequence of CGTase of the CGTase-producing organism. Or may be chemically synthesized. Any CGTase known in the art can be used as long as it has transglycosylation activity and activity to improve the gel-forming ability of starch.
  • starch hydrolases or glycosyltransferases When producing the starch granules of the present invention, a plurality of types of starch hydrolases or glycosyltransferases may be used in combination.
  • ⁇ -glucosidase is preferably used in combination with ⁇ -amylase because it alone hardly reacts with starch granules.
  • derived from an organism with an enzyme does not only mean that the enzyme is directly isolated from the organism, but also the amino acid sequence of the enzyme or the base encoding the enzyme. It also means making an enzyme with the same amino acid sequence based on the sequence in another organism. For example, when a gene encoding the enzyme obtained from the organism is introduced into E. coli and the enzyme is isolated from the E. coli, the enzyme is said to be “derived” from the organism.
  • the enzyme is added in a large excess to the starch granules. Therefore, the amount of enzyme is expressed in wt%. It need not be expressed in units (U).
  • allelic variants can exist in nature.
  • allelic variants in addition to the enzymes exemplified above, as long as they have a desired activity, such naturally occurring variants and variants in which mutations are artificially introduced into the natural enzymes are also included.
  • the modified enzyme preferably has an activity equal to or higher than that of the enzyme before the modification is introduced.
  • the amino acid sequence of starch hydrolase used in the present invention is the amino acid sequence of starch hydrolase used in the examples of the present application or the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 (ie, Control amino acid sequence), i.e., 100% identical, and in another embodiment, the amino acid sequence may vary by a certain number of amino acids compared to the control amino acid sequence. There is at least one such change (preferably one or several; there is no particular upper limit, for example, about 50 or less, about 40 or less, about 30 or less, about 20 or less, about 10 or less, etc. ) Amino acid deletions, substitutions (including conservative and non-conservative substitutions) or insertions.
  • This change may occur at the amino terminal or carboxy terminal position of the control amino acid sequence, or may occur at any position other than these terminals. Changes in amino acid residues may be interspersed one by one or several residues may be continuous.
  • One skilled in the art can easily select a target enzyme having desired properties.
  • a gene encoding the target enzyme may be directly chemically synthesized. Such chemical synthesis methods are well known in the art.
  • Enzyme modification can be performed using methods well known in the art, such as site-directed mutagenesis, mutagenesis using mutagens (treating the gene of interest with a mutagen such as nitrite, ultraviolet treatment, etc. Performing), performing error-prone PCR, and the like.
  • Site-directed mutagenesis is preferably used from the viewpoint of easily obtaining the target mutation. This is because if site-directed mutagenesis is used, a target modification can be introduced at a target site. Alternatively, a nucleic acid molecule having a target sequence may be directly synthesized. Such chemical synthesis methods are well known in the art. Site-directed mutagenesis techniques are described, for example, in Nucl. Acid Research, Vol. 10, pp. 6487-6500 (1982).
  • hydrophobicity index of amino acids can be taken into account.
  • the importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982).
  • the hydrophobic nature of amino acids contributes to the secondary structure of the protein produced, and then the protein and other molecules (eg starch hydrolase or glycosyltransferase, substrate, receptor, DNA, antibody, antigen, etc.) Define the interaction.
  • Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties.
  • One amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a substantially similar biological function (eg, a protein that is substantially equivalent in enzymatic activity)
  • the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.
  • the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.
  • an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • “conservative substitution” refers to substitution in which the hydrophilicity index and / or hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above.
  • conservative substitutions are well known to those of skill in the art and include, but are not limited to, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; And valine, leucine, and isoleucine.
  • the enzyme used in the method of the present invention may be isolated from a natural microorganism that produces the target enzyme.
  • a microorganism that produces the target enzyme can be prepared using an appropriate medium (for example, L broth (1% Bactto-Tryptone (Difco Laboratories, Detroit, Mich., USA), 0.5% Bacto-Yeast Extract (Difco) , 0.5% NaCl, pH 7.3)) and cultured overnight at a suitable temperature (eg, about 30 ° C. to about 40 ° C.) with shaking.
  • a suitable temperature eg, about 30 ° C. to about 40 ° C.
  • the culture solution is centrifuged to precipitate the cells, and a culture supernatant is obtained.
  • the obtained culture supernatant is concentrated with a UF membrane to obtain a target enzyme solution.
  • a nucleic acid molecule containing a base sequence encoding the target enzyme is introduced into an appropriate host cell to express the enzyme, and the expressed enzyme is used in the host cell or its culture. It can be obtained by purifying from the liquid.
  • a nucleic acid molecule (also referred to as a gene) containing a base sequence encoding a natural enzyme is trypsinized with the purified enzyme obtained as described above, and the resulting trypsin-treated fragment is separated by HPLC.
  • a nucleic acid molecule also referred to as a gene
  • the resulting trypsin-treated fragment is separated by HPLC.
  • identifying the N-terminal amino acid sequence of the peptide fragment by a peptide sequencer, and then screening an appropriate genomic library or cDNA library using a synthetic oligonucleotide probe created based on the identified amino acid sequence Can be obtained.
  • the basic strategies for preparing oligonucleotide probes and DNA libraries and for screening them by nucleic acid hybridization are well known to those skilled in the art.
  • screening may be performed by hybridization using a nucleic acid probe containing at least a part of this base sequence to obtain a nucleic acid molecule containing another type of enzyme gene. it can.
  • a nucleic acid probe containing at least a part of this base sequence to obtain a nucleic acid molecule containing another type of enzyme gene. It can.
  • Such methods are known in the art.
  • degenerate primers corresponding to regions conserved in the amino acid sequences of various enzymes can be prepared, and the base sequences of the enzymes can be obtained by PCR. Such methods are known in the art.
  • the obtained nucleic acid molecules can be subcloned using methods well known to those skilled in the art.
  • a plasmid containing the target gene can be easily obtained by mixing ⁇ phage containing the target gene, appropriate E. coli, and appropriate helper phage.
  • the gene of interest can be subcloned by transforming appropriate E. coli with a solution containing the plasmid.
  • the obtained transformant is cultured to obtain plasmid DNA by, for example, alkaline SDS method, and the base sequence of the target gene can be determined. Methods for determining the base sequence are well known to those skilled in the art.
  • primers synthesized based on the base sequence of the DNA fragment Aquifex aeolicus, Rhodothermus obamesis, Bacillus stearothermophilus, Bacillus caldellox, Bacillus therogenus, etc. It can also be used to directly amplify enzyme genes.
  • the base sequence encoding the amino acid sequence of the enzyme used in the method of the present invention may be changed to a certain number as compared with the nucleotide sequence encoding the control amino acid sequence (that is, the control base sequence). .
  • Such changes may be selected from the group consisting of at least one nucleotide deletion, substitutions including transitions and transversions, or insertions. This change may occur at the position of the 5 'end or 3' end of the control base sequence, or may occur at any position other than these ends.
  • the change in base may be interspersed with one base at a time, or may be continuous with several bases.
  • the change in the base can cause nonsense, missense or frameshift mutation in the coding sequence, and can change the enzyme encoded by the base sequence after such a change.
  • this enzyme is starch hydrolase
  • this enzyme is, for example, the amino acid sequence of starch hydrolase used in the Examples or SEQ ID NOs: 2, 4, 6, 8, 10 Or at least about 20%, preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, particularly preferably at least about 60%, at least about 70%, at least Has an identity of about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, and starch hydrolysis activity (in certain cases) Preferably has the property of improving the gel-forming ability of starch.
  • this enzyme is, for example, at least about 20% relative to the amino acid sequence of the glycosyltransferase used in the Examples or the amino acid sequence of SEQ ID NO: 14.
  • enzymes or nucleic acid molecules those having a sequence that is not identical but homologous to the amino acid sequence of the enzyme or the base sequence encoding the enzyme can also be used.
  • Such enzymes or nucleic acid molecules having homology to natural enzymes or nucleic acid molecules include, for example, GENETYX-WIN Ver. In 4.0 maximum matching, when compared under the above conditions, for nucleic acids, at least about 30%, at least about 35%, at least about 40%, at least about 45% relative to the sequence to be compared.
  • nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having a complementary sequence of a nucleotide sequence encoding a naturally known starch hydrolase (eg, SEQ ID NO: 1, 3, 5, 7, 9, or 11)
  • the encoded starch hydrolase can be used in the method of the present invention as long as it has starch hydrolyzing activity (a property that improves the gel-forming ability of starch in certain cases).
  • starch hydrolase Coded by a nucleic acid molecule containing a modified base sequence obtained by modifying a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having a complementary sequence of a base sequence encoding a naturally known starch hydrolyzing enzyme
  • the starch hydrolase that is produced can also be used in the method of the present invention as long as it has the ability to produce starch with high viscosity and gel-forming ability.
  • One skilled in the art can easily select the desired starch hydrolase gene.
  • a glycosyltransferase encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having a complementary sequence to a nucleotide sequence encoding a glycosyltransferase known in nature (eg, SEQ ID NO: 13) has a glycosyltransferase activity. As long as it has the property of improving the gel-forming ability of starch in certain cases, it can be used in the method of the present invention.
  • nucleic acid molecule containing a modified base sequence obtained by modifying a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having a complementary sequence of a base sequence encoding a glycosyltransferase known in nature.
  • Glycosyltransferases can also be used in the methods of the invention as long as they have the ability to produce starch with high viscosity and gel-forming ability. Those skilled in the art can easily select a desired glycosyltransferase gene.
  • stringent conditions refers to conditions that hybridize to specific sequences but not to non-specific sequences.
  • the setting of stringent conditions is well known to those skilled in the art and is described, for example, in Molecular Cloning (Sambrook et al., Supra).
  • “Stringent conditions” are, for example, 50% formamide, 5 ⁇ SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution (0.2% BSA, 0 .2% Ficoll 400 and 0.2% polyvinylpyrrolidone), hybridization at 65 ° C.
  • a polynucleotide that hybridizes under stringent conditions specifically includes, for example, 50% formamide, 5 ⁇ SSC (750 mM NaCl, 75 mM quencher) using a filter on which colony or plaque-derived DNA is immobilized.
  • Trisodium acid 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyrrolidone), 10% dextran sulfate, and 20 ⁇ g / ml
  • a 0.1- to 2-fold concentrated SSC (saline-sodium citrate) solution the composition of the 1-fold concentrated SSC solution is 150 mM chloride
  • Sodium, 15 mM Que Using a sodium it refers to a polynucleotide which can be identified by using a condition that the filter washed with 65 ° C. conditions.
  • the nucleic acid molecule used for producing the enzyme used in the method of the present invention may be a nucleic acid molecule modified conservatively with respect to a nucleic acid molecule containing a base sequence encoding a natural enzyme.
  • “Nucleic acid molecule conservatively modified with respect to a nucleic acid molecule containing a base sequence encoding a natural enzyme” means a base sequence encoding an amino acid sequence that is the same as or essentially the same as the amino acid sequence of a natural enzyme.
  • An “amino acid sequence essentially identical to the amino acid sequence of a natural enzyme” refers to an amino acid sequence having essentially the same enzymatic activity as the natural enzyme.
  • the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • the codon can be changed to GCC, GCG or GCU without changing the encoded alanine.
  • the codon is any arbitrary encoding that amino acid without altering the specific amino acid encoded. It can be changed to another codon.
  • Such nucleotide sequence variations are “silent mutations,” which are one species of conservatively modified mutations. All base sequences herein that encode a polypeptide also include all possible silent variations of the nucleic acid. Silent mutation includes “silent substitution” in which the encoded amino acid does not change and the case where the nucleic acid does not encode an amino acid (for example, mutation in an intron, mutation in other untranslated region, etc.). When a nucleic acid encodes an amino acid, silent mutation is synonymous with silent substitution. As used herein, “silent substitution” refers to substituting a base sequence encoding a certain amino acid with another base sequence encoding the same amino acid in the base sequence.
  • a polypeptide having an amino acid sequence encoded by a base sequence generated by silent substitution has the same amino acid sequence as the original polypeptide.
  • each codon in a nucleic acid except AUG, which is usually the only codon that encodes methionine, and TGG, which is usually the only codon that encodes tryptophan, produces a functionally identical molecule. It is understood that it can be modified.
  • each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.
  • the base sequence encoding the enzyme used in the present invention can be changed according to the frequency of codon usage in the organism introduced for expression. Codon usage reflects the frequency of use of genes that are highly expressed in the organism. For example, if it is intended to be expressed in E. coli, optimize for expression in E. coli according to published codon usage frequency tables (eg, Sharp et al., Nucleic Acids Research 17, No. 17, pages 8207 (1988)). be able to.
  • An expression vector can be prepared using a nucleic acid molecule containing a base sequence modified as described above. Methods for producing expression vectors using specific nucleic acid sequences are well known to those skilled in the art.
  • a “vector” refers to a nucleic acid molecule capable of transferring a target base sequence to a target cell. Such a vector can be autonomously replicated in the target cell or can be integrated into the chromosome of the target cell and contains a promoter at a position suitable for transcription of the modified nucleotide sequence.
  • a vector can be a plasmid.
  • an “expression vector” refers to a vector capable of expressing a modified base sequence (that is, a base sequence encoding a modified enzyme) in a target cell.
  • the expression vector includes various regulatory elements such as a promoter that regulates its expression, and, if necessary, factors necessary for replication in the target cell and selection of recombinants. (For example, an origin of replication (ori) and a selectable marker such as a drug resistance gene).
  • the modified nucleotide sequence is operably linked so as to be transcribed and translated. Regulatory elements include promoters, terminators and enhancers.
  • the base sequence encoding the secretory signal peptide is bound upstream of the modified base sequence in the correct reading frame.
  • the type of expression vector used for introduction into a particular organism eg, bacterium
  • the types of regulatory elements and other factors used in the expression vector can vary depending on the cell of interest, This is a matter well known to those skilled in the art.
  • a “terminator” is a sequence that is located downstream of a protein coding region and is involved in termination of transcription when a base sequence is transcribed into mRNA, and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.
  • promoter refers to a region on DNA that determines the transcription start site of a gene and directly regulates the transcription frequency. It is. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, the promoter The region can be estimated.
  • the putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.
  • enhancer can be used to increase the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers may be used, but one may be used or may not be used.
  • operably linked refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or translational regulation in which the desired base sequence results in expression (ie, activation). It is placed under the control of the array. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.
  • ⁇ Enzymatic genes may be processed to operably link the modified nucleic acid sequence to the regulatory element. For example, when the distance between the promoter and the coding region is too long and a decrease in transcription efficiency is expected, or the interval between the ribosome binding site and the translation initiation codon is not appropriate.
  • processing means include digestion with restriction enzymes, digestion with exonucleases such as Bal31 and ExoIII, or introduction of site-specific mutations using single-stranded DNA such as M13 or PCR.
  • the target enzyme is expressed by introducing the expression vector prepared as described above into cells.
  • expression of an enzyme means that a base sequence encoding the enzyme is transcribed and translated in vivo or in vitro to produce the encoded enzyme.
  • Examples of cells (also referred to as hosts) into which expression vectors are introduced include prokaryotes and eukaryotes.
  • a cell into which an expression vector is introduced can be easily selected in consideration of various conditions such as ease of expression of the target enzyme, ease of culture, speed of growth, and safety.
  • Examples of such cells include microorganisms such as bacteria and fungi. More preferable examples of the cells include mesophilic microorganisms (for example, yeast, mold, Escherichia coli, Bacillus subtilis).
  • the cell may be a microbial cell, but may be a plant, animal cell or the like.
  • the starch hydrolase may have been post-translationally processed.
  • the technique for introducing an expression vector into a cell can be any technique known in the art. Examples of such techniques include transformation, transduction, transfection and the like. Such a technique for introducing a nucleic acid molecule is well known in the art and frequently used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experiment Method” Yodosha, 1997, and the like.
  • any material usually used in enzyme treatment can be used as long as it does not interfere with the action of the enzyme.
  • examples of such other materials include salts and buffering agents.
  • the rate of enzyme reaction is dramatically improved by adding an appropriate specific salt to each enzyme. Therefore, it is preferable to add such a specific salt.
  • the treatment time can be shortened by adding an appropriate salt to each enzyme.
  • Examples of such a combination of an enzyme and a salt include a combination of amyloglucosidase and a metal ion (for example, sodium ion, potassium ion, calcium ion or magnesium ion).
  • amyloglucosidase eg, derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor
  • sodium chloride or sodium sulfate eg, sodium sulfate
  • the starch degradation rate was 1.5 to 2 times faster than the system in which potassium chloride was not added.
  • Enzyme-treated starch granules are produced by treating starch granules with starch hydrolase or glycosyltransferase. Details of each step will be described below.
  • starch grains provided as rice flour or rice starch, starch hydrolase or glycosyltransferase, a buffering agent, and a solvent dissolving the same are used as main materials. All of these materials are usually added at the start of the reaction, but any of these materials may be added during the reaction.
  • the solvent used in the production method of the present invention can be any solvent as long as it does not impair the enzyme activity of the enzyme used.
  • a typical solvent is water (for example, ion exchange water, purified water, tap water, etc.).
  • the solvent may be water in the cell lysate obtained accompanying the enzyme when preparing the enzyme.
  • a reaction solution is prepared.
  • the reaction solution can be prepared, for example, by adding starch granules provided as rice flour or rice starch and starch hydrolase or glycosyltransferase to a suitable solvent.
  • the enzyme may be added after preparing starch suspension by suspending starch granules in a solvent (for example, water or buffer).
  • the reaction solution may be prepared by mixing a suspension containing starch granules and a solution containing an enzyme. Any buffer may be added to this reaction solution for the purpose of adjusting the pH, if necessary, as long as the enzyme reaction is not inhibited.
  • the starch granules are suspended and not dissolved in the reaction solution, other components such as enzymes are dissolved, so that the starch solution is called a reaction solution.
  • the pH of the reaction solution can be arbitrarily set as long as the enzyme used can exert its activity.
  • the pH of the reaction solution is preferably around the optimum pH of the enzyme used.
  • the pH of the reaction solution is typically about 2 or more, preferably about 3 or more, more preferably about 4 or more, particularly preferably about 5 or more, particularly preferably about 6 or more. Most preferably, it is about 7 or more.
  • the pH of the reaction solution is typically about 13 or less, preferably about 12 or less, more preferably about 11 or less, particularly preferably about 10 or less, particularly preferably about 9 or less. Most preferably, it is about 8 or less.
  • the pH of the reaction solution is typically within ⁇ 3 of the optimum pH of the enzyme used, preferably within ⁇ 2 of the optimum pH, and more preferably at the optimum pH. Within ⁇ 1, most preferably within ⁇ 0.5 of the optimum pH.
  • the amount of starch granules in the reaction solution can be arbitrarily set as long as it is an amount capable of enzymatic reaction.
  • the amount of starch granules in the reaction solution is preferably about 5% by weight or more, more preferably about 10% by weight or more, further preferably about 20% by weight or more, and most preferably about 30% by weight or more. It is.
  • the amount of starch granules in the reaction solution is preferably about 60% by weight or less, more preferably about 50% by weight or less, still more preferably about 40% by weight or less, and most preferably about 35% by weight or less. It is.
  • the amount of the enzyme in the reaction solution can be arbitrarily set as long as the enzyme reaction is possible.
  • the amount of the enzyme is preferably an amount sufficient to carry out the reaction within a reasonable time. The larger the amount of enzyme, the shorter the time required for the reaction, and the smaller the amount of enzyme, the longer the time required for the reaction. If the amount of the enzyme is too large, the cost becomes very high, and further, the enzyme may be aggregated to form a precipitate, so it is preferable to set appropriately.
  • the amount of the enzyme in the reaction solution is preferably about 0.01% by weight or more, more preferably about 0.05% by weight or more, and further preferably about 0.1% by weight based on the dry weight of the starch granules. % Or more.
  • the amount of the enzyme in the reaction solution is preferably about 10% by weight or less, more preferably about 5% by weight or less, and still more preferably about 1% by weight or less based on the dry weight of the starch granules. Since the amount of the enzyme in the reaction solution may be a sufficient amount for the enzymatic reaction to proceed, it is not necessary to examine the enzyme activity (number of units) in detail.
  • the reaction solution is then reacted by heating as necessary by methods known in the art.
  • the solution temperature in the reaction step can be any temperature as long as the starch granules do not substantially collapse.
  • the reaction temperature is preferably a temperature at which the enzyme to be used can exhibit its activity sufficiently and retains its activity sufficiently (that is, it is difficult to deactivate).
  • the temperature of the solution in this reaction step is preferably a temperature at which activity of about 50% or more, more preferably about 80% or more of the activity of the enzyme contained in this solution before the reaction remains after a predetermined reaction time. For example, this temperature can be the optimum temperature ⁇ 10 ° C.
  • the reaction temperature is preferably about 10 ° C. or higher, more preferably about 10 ° C. or higher, further preferably about 15 ° C. or higher, even more preferably about 20 ° C. or higher, particularly preferably about 30 ° C. And most preferably above 40 ° C.
  • the reaction temperature is preferably about 70 ° C. or less, more preferably about 65 ° C. or less, particularly preferably about 60 ° C. or less, and most preferably 55 ° C. or less.
  • the reaction time can be arbitrarily set in consideration of the reaction temperature, the amount of enzyme for starch granules, and the like.
  • the reaction time is preferably about 1 hour or longer, and can be, for example, about 2 hours or longer, about 3 hours or longer, about 6 hours or longer, about 12 hours or longer.
  • the reaction time is not particularly limited, but is preferably about 72 hours or less, more preferably about 48 hours or less, even more preferably about 36 hours or less, particularly preferably about 24 hours or less, and most preferably about 20 hours or less. .
  • the enzyme-treated starch granules can be used as they are, depending on the application, but the enzyme-treated starch granules are washed and dehydrated to remove the used enzyme and carbohydrates eluted by enzymatic degradation. It is preferable to do.
  • the enzyme-treated starch granules can be washed and dehydrated by any method known in the art. Washing and dehydration of starch granules is a common method for preparing starch and is generally performed. Furthermore, it is preferable to dry the starch after dehydration to obtain the target enzyme-treated starch granules. Drying of the starch after dehydration can be performed by any method known in the art.
  • the starch granules subjected to the enzyme treatment can be chemically modified as desired. Not only when the starch granules used in the enzyme treatment are untreated starch grains or starch grains that have been physically treated, but also when any modified starch starch grains are used, the chemical modification of the kind applied to the modified starch Can be subjected to different types of chemical modifications. Examples of chemical modifications include acetylation, adipic acid crosslinking, oxidation, bleaching, phosphoric acid crosslinking, octenyl succinic acid treatment, hydroxypropylation, phosphorylation and phosphoric acid monoesterification. These chemical modification methods are well known in the art.
  • the starch granules subjected to the enzyme treatment can be physically treated as desired. Not only when the starch granules used for enzyme treatment are untreated starch granules or modified starch granules, but also when starch granules that have undergone some physical treatment are used, physical treatment of a different type from the physical treatment may be applied. it can. Examples of physical treatment include wet heat treatment and heat suppression treatment.
  • “Humid heat treatment” refers to heating to about 95 to about 125 ° C. in a closed container under a relative humidity of about 100% in a low moisture state that does not gelatinize starch.
  • the “low moisture state that does not gelatinize starch” indicates, for example, a moisture content of about 50% or less.
  • the low moisture state that does not gelatinize starch may be, for example, about 35% or less, about 30% or less, about 25% or less, or about 20% or less.
  • the heating time of the wet heat treatment can vary depending on the method of the wet heat treatment.
  • the pressure is first reduced to about 0 to about 500 Torr (about 0 to 66.661 kPa), and then pressurized steam is introduced to about 100 ° C.
  • Heat treatment is performed by holding at about 150 ° C. for about 2 minutes to about 120 minutes.
  • the wet heat treatment is described in various documents and can be performed according to any wet heat treatment method known in the art.
  • the wet heat treatment is described in, for example, JP-A-6-145203, JP-A-4-130102, and monthly food chemical 2010-2 (P.37-42).
  • the temperature and time of the wet heat treatment can be appropriately set depending on the target starch and its physical properties.
  • Heat suppression treatment refers to strengthening the crystal structure of starch granules by subjecting the starch granules dried to extremely low moisture to dry heat treatment.
  • Starch granules dried to very low moisture refers to starch granules having a moisture content of less than about 1%.
  • the water content of the starch granules to be heat-suppressed is preferably about 0%.
  • a method for drying starch granules to extremely low moisture is described in, for example, Japanese Patent Application Laid-Open No. 2008-2223032. For example, after adjusting the pH of starch granules to a pH of 7.0 or more, the water content is about 1%. It may be a method of dehydrating until less than.
  • the pH when drying to low moisture is preferably pH 7 or more, more preferably greater than pH 8, preferably pH 7.5 to 10.5, and more preferably pH 8 to 9.5.
  • the dehydration may be thermal dehydration or non-thermal dehydration.
  • heat treatment is performed at a sufficient temperature for a sufficient time to suppress starch.
  • the starch is heat treated at a sufficient temperature for a sufficient time to render the starch non-agglomerated.
  • the preferred heating temperature for the heat suppression treatment is higher than about 100 ° C.
  • the heat treatment temperature is preferably about 200 ° C. or less.
  • the heating temperature for the heat suppression treatment is more preferably about 120 ° C. to about 180 ° C., particularly preferably about 140 ° C.
  • the level of inhibition depends on pH, heating temperature and heating time.
  • the higher the pH the more highly controlled starch is obtained.
  • the higher the heat treatment temperature the more highly controlled starch is obtained.
  • the longer the heat treatment time the more highly controlled starch is obtained.
  • the heat treatment time for the heat suppression treatment can be, for example, about 3 hours or more, and preferably about 20 hours or less.
  • the heat suppression treatment is described in various documents, and can be performed according to any heat suppression treatment method known in the art.
  • the heat suppression treatment is described in, for example, US Pat. No. 6,221,420, International Publication No. 95/04082, and Japanese Patent Application Laid-Open No. 2008-2223032.
  • the temperature, time, etc. of the heat suppression treatment can be appropriately set depending on the target starch and its physical properties. Physical processing can be performed according to methods well known in the art.
  • wet heat-treated starch examples include “Delica Star Series”, “Natura Star Series”, “Amygel” manufactured by Sanwa Starch Co., Ltd., and “Road Star” manufactured by Nippon Shokuhin Kako Co., Ltd.
  • heat-suppressed starch examples include “Novation Series” manufactured by National Starch.
  • the enzyme-treated starch granules of the present invention are enzyme-treated starch granules having high viscosity and gel-forming ability, and the enzyme-treated starch granules are a step of mixing rice flour or rice starch and an enzyme.
  • high viscosity for enzyme-treated starch granules means that the suspension obtained by dispersing the enzyme-treated starch granules in water and heating them has a high viscosity.
  • it refers to enzyme-treated starch granules that exhibit a high maximum viscosity when the viscosity characteristics are analyzed by amylograph.
  • the enzyme-treated starch granule of the present invention is an enzyme-treated starch granule having a high viscosity and a gel-forming ability
  • the enzyme-treated starch granule is a rice flour under a condition in which the starch granule does not dissolve.
  • starch granules obtained by treating untreated starch granules of rice starch with starch hydrolyzing enzyme, and the enzyme-treated starch granules are modified with hydroxyl groups at the 2nd, 3rd and 6th positions of glucose residues.
  • the enzyme-treated starch granules form a gel having a Young's modulus higher than the untreated starch granules or a rupture stress higher than that of the untreated starch granules as measured by a rheometer. be able to.
  • the amylograph measurement is performed, for example, as follows. 450 ml of water so that a predetermined amount of enzyme-treated starch granules (for example, wheat starch granules is 8.5% by weight, rice starch granules is 9.0% by weight, and rice flour is 9% by weight in terms of dry matter) Then, a starch granule suspension is prepared, put into a sample container, and heated to 50 ° C. while rotating them. Thereafter, the temperature is raised to 95 ° C. at 1.5 ° C./min and held at 95 ° C. for 15 minutes. Subsequently, it is cooled at 1.5 ° C./min.
  • a predetermined amount of enzyme-treated starch granules for example, wheat starch granules is 8.5% by weight, rice starch granules is 9.0% by weight, and rice flour is 9% by weight in terms of dry matter
  • the amylograph is measured by VISCOGRAPH-E manufactured by Brabender, the rotation speed of the sample container is 75 rpm, and the measurement cartridge is 700 cmg.
  • the viscosity when the viscosity reaches the peak is defined as the maximum viscosity
  • the difference between the maximum viscosity and the viscosity when held at 95 ° C. for 15 minutes is defined as breakdown. This difference is also called breakdown viscosity. If the difference between the maximum viscosity and the viscosity when held at 95 ° C. for 15 minutes is less than 100 BU, the starch granules are said to have “no breakdown”.
  • the enzyme-treated starch granules of the present invention are prepared from untreated starch and are not chemically modified or physically treated
  • the enzyme-treated starch granules of the present invention are measured by amylograph under the conditions described in 3.1 above.
  • the enzyme-treated starch granules obtained by performing the enzyme treatment of starch granules (which may be any of untreated starch, chemically modified starch, or physically-treated starch) by the method of the present invention are the conditions described in 3.1 above.
  • about 50% or more (more preferably about 60% or more, particularly preferably about 70% or more, most preferably about 80% or more) of the maximum viscosity of the starch granules used for the enzyme treatment Preferably, it has a maximum viscosity of about 90% or more or about 100% or more.
  • the maximum viscosity of the enzyme-treated starch granules obtained by performing the enzyme treatment of starch granules (which may be any of untreated starch, chemically modified starch, or physically treated starch) by the method of the present invention,
  • starch granules which may be any of untreated starch, chemically modified starch, or physically treated starch
  • the maximum viscosity of the starch granules used for enzyme treatment about 110% or less, about 100% or less, and the like.
  • the maximum viscosity of natural rice flour when measured by amylograph under the above conditions is about 1000 BU to about 1200 BU.
  • the enzyme-treated starch granules of the present invention when measured by amylography under the conditions described in 3.1 above.
  • the maximum viscosity of the treated starch granules is preferably about 900 BU or more, more preferably about 920 BU or more, particularly preferably about 940 BU or more, most preferably about 960 BU or more, for example, about 1000 BU or more, about It may be 1050 BU or more, about 1100 BU or more, or about 1150 BU or more.
  • the maximum viscosity of the enzyme-treated starch granules of the present invention when measured by amylograph under the conditions described in 3.1 above may be, for example, about 1350 BU or less, about 1300 BU or less, about 1250 BU or less, or about 1200 BU or less.
  • the maximum viscosity of natural rice starch when measured by amylograph under the conditions described in 3.1 above is about 1000 BU to about 1200 BU.
  • the enzyme-treated starch granules of the present invention when measured by amylograph under the conditions described in 3.1 above is used.
  • the maximum viscosity of the enzyme-treated rice starch granules is preferably about 860 BU or more, more preferably about 900 BU or more, even more preferably about 920 BU or more, particularly preferably about 940 BU or more, and most preferably about For example, it may be about 1000 BU or more, about 1050 BU or more, about 1100 BU or more, or about 1150 BU or more.
  • the maximum viscosity of the enzyme-treated rice starch granules of the present invention as measured by amylograph under the above conditions can be, for example, about 1350 BU or less, about 1300 BU or less, about 1250 BU or less, or about 1200 BU or less.
  • the enzyme-treated starch granules of the present invention are prepared from untreated starch granules and neither chemically modified nor physically treated, the enzyme-treated starch granules of the present invention are amylographed under the conditions described in 3.1 above. It has a breakdown viscosity when measured. While some conventional starches do not have a breakdown viscosity when measured by amylograph, the enzyme-treated starch granules of the present invention are broken when measured by amylograph under the conditions described in 3.1 above. Has down viscosity.
  • the enzyme-treated starch granules of the present invention are enzyme-treated starch granules obtained by enzymatic treatment of starch granules that have been chemically or physically modified, the starch granules used for the enzyme treatment are listed in 3.1 above. If it has a breakdown viscosity when measured by the amylograph under the described conditions, the enzyme-treated starch granules of the present invention have a breakdown viscosity when measured by the amylograph.
  • the resulting enzyme-treated starch granules have a breakdown viscosity of about 100 BU or more.
  • the breakdown viscosity of the enzyme-treated starch granules obtained when measured with an amylograph under the conditions described in 3.1 above is preferably Is about 600 BU or more, more preferably about 650 BU or more, still more preferably about 700 BU or more, and most preferably about 800 BU or more.
  • untreated starch granules are provided as rice flour and are not chemically modified or physically treated
  • there is a particular upper limit on the breakdown viscosity of enzyme-treated starch granules obtained when measured by amylography under the conditions described in 3.1 above For example, about 1000 BU or less, about 950 BU or less, about 900 BU or less, about 850 BU or less, or about 800 BU or less.
  • the breakdown viscosity of the enzyme-treated starch granules obtained when measured with an amylograph under the conditions described in 3.1 above is: Preferably, it is about 600 BU or more, more preferably about 650 BU or more, still more preferably about 700 BU or more, and most preferably about 800 BU or more.
  • the breakdown viscosity of the enzyme-treated starch granules obtained when measured by amylograph under the conditions described in 3.1 above is particularly there is no upper limit, but it may be, for example, about 1000 BU or less, about 950 BU or less, about 900 BU or less, about 850 BU or less, about 800 BU or less, about 700 BU or less, about 600 BU or less, about 500 BU or less, about 400 BU or less, or about 300 BU or less.
  • starch paste forms a starch gel by cooling when the concentration exceeds a predetermined level.
  • the physical properties of the starch gel, as well as the viscosity vary depending on the origin of the starch and the production method, and are used in various foods in consideration of the characteristics of the gelled physical properties.
  • Several methods for measuring the physical properties of the gel have been put into practical use. One of them is a method using a rheometer.
  • the gel-forming ability is measured with a rheometer, for example, by filling a starch paste solution into a casing, and after heating, for example, for 16 hours or 21 days (for example, at about 5 ° C.) and refrigerated, and at room temperature (for example, about 25 ° C.). After returning, it can be performed by measuring the gel physical properties with a rheometer.
  • the specific measurement method using a rheometer is as described in 1.2.2 above.
  • the enzyme-treated starch granule of the present invention is prepared from untreated rice flour and is not chemically modified or physically treated, the enzyme-treated starch granule produces, for example, a starch gel under the conditions described in 1.2.2 above.
  • it when measured with a rheometer, it has a breaking stress of 110% or more and 850% or less of the breaking stress of the untreated starch granules (that is, untreated rice flour) or 110% or more of the Young's modulus of the untreated starch grains 600 % Or less (in one embodiment, 110% or more and 330% or less).
  • the enzyme-treated starch granule of the present invention is prepared from untreated rice starch and is not chemically modified or physically treated
  • the enzyme-treated starch granule is obtained, for example, by subjecting a starch gel under the above-mentioned conditions of 1.2.2.
  • a starch gel under the above-mentioned conditions of 1.2.2.
  • it has a breaking stress of 110% or more and 850% or less of the breaking stress of untreated starch granules (ie, untreated rice starch) or 110% of the Young's modulus of the untreated starch granules.
  • it has a Young's modulus of 600% or less (in one embodiment, 330% or less).
  • Enzyme-treated starch granules obtained by performing the enzyme treatment of starch granules (which may be any of untreated starch, chemically modified starch, or physically treated starch) in both cases of rice starch and rice flour.
  • starch granules which may be any of untreated starch, chemically modified starch, or physically treated starch
  • starch granules obtained by performing the enzyme treatment of starch granules (which may be any of untreated starch, chemically modified starch or physical-treated starch) by the method of the present invention
  • starch granules which may be any of untreated starch, chemically modified starch or physical-treated starch
  • breaking stress of the enzyme-treated starch granules obtained by performing the enzyme treatment of starch granules (which may be any of untreated starch, chemically modified starch or physical-treated starch) by the method of the present invention.
  • starch gel is prepared under the conditions of 1.2.2 and measured with a rheometer, it is about 600% or less, about 550% or less, or about 500% of the breaking stress of the starch granules used for the enzyme treatment.
  • it may be about 450% or less, about 400% or less, about 300% or less, about 200% or less.
  • the enzyme-treated starch granules obtained by carrying out the enzyme treatment of starch granules (which may be any of untreated starch, chemically modified starch, or physically treated starch) by the method of the present invention are, for example, the above 1.2.2.
  • a starch gel is prepared under the conditions of the above and measured with a rheometer, it is about 110% or more (more preferably about 120% or more, particularly preferably about 130% or more) of the Young's modulus of the starch granules used for the enzyme treatment, Most preferably, it has a Young's modulus of about 140% or more.
  • the Young's modulus of the enzyme-treated starch granules obtained by performing the enzyme treatment of starch granules (which may be any of untreated starch, chemically modified starch, or physically treated starch) by the method of the present invention,
  • starch granules which may be any of untreated starch, chemically modified starch, or physically treated starch
  • the Young's modulus of the starch granules used for the enzyme treatment is about 850% or less, about 800% or less, about 750%.
  • the breaking stress of the obtained enzyme-treated starch granule is, for example, under the condition of 1.2.2 above.
  • the starch gel is prepared and measured with a rheometer, it is preferably about 45 g or more, more preferably about 50 g or more, still more preferably about 60 g or more, particularly preferably about 65 g or more. Most preferably, it is about 70 g or more.
  • a starch gel When a starch gel is prepared under the condition 2 and measured with a rheometer, it may be, for example, about 150 g or less, about 140 g or less, about 130 g or less, about 120 g or less, about 110 g or less, or about 100 g or less.
  • the enzyme-treated starch granules of the present invention are prepared from untreated rice starch and are not chemically modified or physically treated, for example, a starch gel is prepared under the conditions of 1.2.2 and measured with a rheometer.
  • the breaking stress of the obtained enzyme-treated starch granules is preferably about 15 g or more, more preferably about 20 g or more, further preferably about 30 g or more, and most preferably about 40 g or more.
  • the breaking stress of the enzyme-treated starch granules in this case is also, for example, about 45 g or more, about 50 g or more, about 55 g or more, about 60 g or more, about 65 g or more, about 70 g or more, about 80 g or more, about 90 g or more, about 100 g or more. , About 110 g or more, or about 120 g or more.
  • the untreated starch granules are rice starch and are not chemically modified or physically treated, there is no particular upper limit to the breaking stress of the resulting enzyme-treated starch granules.
  • a starch gel When a starch gel is prepared and measured with a rheometer, it may be, for example, about 170 g or less, about 160 g or less, about 150 g or less, about 140 g or less, about 130 g or less, about 120 g or less, about 100 g or less, or about 100 g or less. .
  • the Young's modulus of the starch granules is preferably about 7.0 ⁇ 10 5 dyn / cm 2 or more, more preferably about 7.1 ⁇ 10 5 dyn / cm 2 or more, and further preferably about 7.2 ⁇ . 10 5 dyn / cm 2 or more, and most preferably about 7.3 ⁇ 10 5 dyn / cm 2 or more.
  • the untreated starch granules are provided as rice flour and not subjected to chemical modification or physical treatment, there is no particular upper limit on the Young's modulus of the enzyme-treated starch granules obtained.
  • a gel is prepared and measured with a rheometer, for example, about 2.5 ⁇ 10 6 dyn / cm 2 or less, about 2.4 ⁇ 10 6 dyn / cm 2 or less, about 2.3 ⁇ 10 6 dyn / cm 2 or less. 2 or less, about 2.2 ⁇ 10 6 dyn / cm 2 or less, and the like about 2.1 ⁇ 10 6 dyn / cm 2 or less, or about 2.0 ⁇ 10 6 dyn / cm 2 or less.
  • untreated starch granules are provided as rice starch and are not chemically modified or physically treated, for example, an enzyme obtained when a starch gel is prepared and measured with a rheometer under the conditions of 1.2.2 above
  • the Young's modulus of the treated starch granules is preferably about 2.5 ⁇ 10 5 dyn / cm 2 or more, more preferably about 2.6 ⁇ 10 5 dyn / cm 2 or more, and further preferably about 2.7.
  • the Young's modulus of the enzyme-treated starch granules in this case is also, for example, about 3.0 ⁇ 10 5 dyn / cm 2 or more, about 3.2 ⁇ 10 5 dyn / cm 2 or more, about 3.4 ⁇ 10 5 dyn / cm 2 or more, about 3.6 ⁇ 10 5 dyn / cm 2 or more, about 3.8 ⁇ 10 5 dyn / cm 2 or more, about 4.0 ⁇ 10 5 dyn / cm 2 or more, about 4.2 ⁇ 10 5 dyn / cm 2 or more, about 4.4 ⁇ 10 5 dyn / cm 2 or more, about 4.6 ⁇ 10 5 dyn / cm 2 or more, about 4.8 ⁇ 10 5 dyn / cm 2 or more, about 5.0 ⁇ 10 5 dyn / cm 2 or more, about 5.2 ⁇ 10 5 dyn / cm 2 or more, about 5.4 ⁇ 10 5 dyn
  • a starch gel is prepared and measured with a rheometer, for example, about 4.0 ⁇ 10 6 dyn / cm 2 or less, about 3.8 ⁇ 10 6 dyn / cm 2 or less, about 3.5 ⁇ 10 6 dyn / cm 2 or less, about 2.5 ⁇ 10 6 dyn / cm 2 or less, about 2.4 ⁇ 10 6 dyn / cm 2 or less, about 2.3 ⁇ 10 6 dyn / cm 2 or less, about 2.2 ⁇ 10 6 dyn / cm 2 or less, about 2.1 ⁇ 10 6 dyn / cm 2 or less, or about 2.0 ⁇ 10 6 dyn / cm 2 or less.
  • the resulting enzyme treated starch granules are broken down when measured, for example, under the conditions of 3.1 above.
  • the breaking stress is about 55 to about 100 (g) or the Young's modulus is about 7 having .0 ⁇ 10 5 ⁇ about 2.0 ⁇ 10 6 (dyn / cm 2).
  • the resulting enzyme treated starch granules are broken when measured, for example, under the conditions of 3.1 above.
  • the breaking stress is about 15 to about 100 (g) or the Young's modulus is about 2.5 ⁇ 10 5 to about 2.0 ⁇ 10 6 (dyn / cm 2) either with or rupture stress from about 120 to about 170, (g) or Young's modulus of about 2.5 ⁇ 10 6 to about 4.0 ⁇ 10 6 (dyn / cm 2 )
  • the gel forming ability can be improved in the same manner as described above.
  • adipic acid group content, acetyl group content, carboxyl group content, vinyl acetate content, octenyl succinic acid group content, hydroxypropyl group content and propylene chlorohydrin content And it can be judged that the sample starch is not the starch which received the chemical modification by confirming that these content is not increasing from these content about raw material raw starch. It is preferable to use the adipic acid group content, the acetyl group content, the carboxyl group content, the octenyl succinic acid group content, the hydroxypropyl group content, and the propylene chlorohydrin content as judgment criteria.
  • bleached starch that has been bleached using sodium hypochlorite is permitted to be distributed as food.
  • This bleached starch can also be determined by measuring the carboxyl group content by the same purity test method as that for the oxidized starch.
  • Chemically modified processed starches other than the above 11 processed starches are not approved by the Food Sanitation Law and cannot be used for food. Therefore, chemically modified starches other than the above 11 items are basically not used in Japan and are not distributed. Therefore, in practice, when the starch of the present invention is confirmed whether or not the hydroxyl groups at the 2-position, 3-position and 6-position of the glucose residue are not modified, chemical modification other than the above chemical modification is not performed. There is no need to check.
  • the hydroxyl groups at the 2nd, 3rd and 6th positions of the glucose residue are not modified
  • all the hydroxyl groups at the 2nd, 3rd and 6th positions of the glucose residue are not modified. It is preferable, however, some modification may be included in the case where some modification is performed in a natural state.
  • the total number of hydroxyl groups at the 2nd, 3rd and 6th positions of the glucose residue it is preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably.
  • the food of the present invention comprises a step of mixing rice flour or rice starch and an enzyme; the rice flour or starch granules in the rice starch are treated with the enzyme at a temperature of about 10 ° C. or more and about 70 ° C. or less.
  • the food of the present invention is a cooked rice starch-containing food made from enzyme-treated starch granules having high viscosity and gel-forming ability.
  • the starch-containing food of the present invention is a food produced by a method comprising mixing the food material and the enzyme-treated starch granules and then heating.
  • rice starch gel-containing food refers to food containing rice starch gel. If it contains rice starch gel, the food as a whole need not be in the form of a gel.
  • gel foods such as custard pudding and gel-like Japanese confectionery such as crumbs and eels form a gel as a whole.
  • Oils and fats-containing foods such as whipped cream and ice cream, and sauces such as meat sauce are not gel-like as a whole food, but are contained in the starch gel-containing food of the present invention because they contain fine starch gels.
  • bakery products, pastry products, etc. are also included in the starch gel-containing food of the present invention because they contain a starch gel that once formed a gel during the production process and reduced in moisture by baking or the like.
  • the food product of the present invention may be prepared using enzyme-treated rice starch granules.
  • the starch granules produced by the method of the present invention can be used for the same applications as conventional starches.
  • the enzyme-treated rice starch granules of the present invention for food, the physical properties and texture of the food are modified.
  • the enzyme-treated rice starch granules of the present invention can be used for almost all food-drinking compositions or food additive compositions that have been conventionally prepared using starch.
  • any material usually used in the intended composition and food can be used as long as it does not interfere with the excellent effect obtained by the enzyme-treated rice starch granules.
  • the starch of the present invention forms a gel in the food of the present invention.
  • the enzyme-treated rice starch granules of the present invention impart a body, a natural elasticity due to a strong gel-forming ability, and a moderate mouth-melting feeling when used in high moisture foods. Furthermore, the enzyme-treated rice starch granules of the present invention can give a mochi or moist feeling superior to conventional rice flour and rice starch in high moisture foods.
  • a high moisture type food means a food having a water content of more than 40 g per 100 g of the edible portion in a fed state. Examples of high moisture foods include Japanese confectionery, oil and fat-containing foods, gel foods, fish and livestock meat processed foods, sauces and sauces, and noodles.
  • the enzyme-treated rice starch granules of the present invention can impart a smooth texture that is easy to break when used in low-moisture foods. Furthermore, the enzyme-treated rice starch granules of the present invention can impart a crispy feeling or fragrance superior to conventional rice flour and rice starch in low moisture foods.
  • a low moisture food means a food having a water content of 40 g or less per 100 g of the edible portion in the state of eating. Examples of low moisture foods include bakery products, pastry products, fried food products, jelly candy products, and the like.
  • high moisture foods and low moisture foods are classified according to whether the moisture content per 100 g of edible portion is higher than 40 g or 40 g or less.
  • foods with a moisture content of about 40 g (35-50 g) per 100 g of edible portion may exhibit contradictory physical properties depending on the form even if the moisture content is the same.
  • the determination is based on the amount of moisture in the clothing part, excluding ingredients.
  • the amount of water per 100 g of edible portion of various foods is illustrated below (from the 5th edition supplemented Japanese food standard ingredient table; the amount of water in parentheses): (1) Bakery: white bread (38.0 g), hard biscuits (2.6 g), pie dough (32.0 g), sanitary bolo (4.5 g); (2) Japanese confectionery: Uiro (54.5 g), Kuzumanju (45.0 g), Daifuku-an (41.5 g); (3) Western confectionery: sponge cake (32.0 g), castella (25.6 g), hot cake (40.0 g); (4) Oil and fat-containing foods: whipped cream (milk fat type, 42.1 g), whipped cream (vegetable fat type, 41.2 g), ice cream (ice milk: 65.6 g, lacto ice: 60.4 g); (5) Gel food: Custard pudding (74.1 g); (6) Fish meat, processed meat products: Sumaki kamaboko (75.8 g), grilled kamaboko (72.8 g), winner (53.0 g); (7)
  • the enzyme-treated rice starch granules of the present invention for such foods, for example, the following physical properties are improved as compared with the case where conventional starch is used: (1) In bakery products, it is soft and gives a mouthfeel that melts well. Examples of bakery products include bread, cookies, biscuits, pizza dough, puff pastry, ice cream cone cup, monaca skin, cream puff skin, and the like.
  • Japanese confectionery In Japanese confectionery, it has moderate hardness and brittleness and imparts moderate viscoelasticity and a crisp texture. Examples of Japanese confectionery include crumbs, owls, and buns.
  • fat and oil-containing foods it has a moderate body feeling and shape retention, and has a good mouth melt and a smooth texture.
  • examples of the fat and oil-containing food include custard cream, flower paste, filling, whipped cream, ice cream (for example, ice milk, lacto ice) and the like.
  • a gel-like food it has a smooth and smooth texture while having a good elasticity and a good mouth melt.
  • gel foods include jelly, pudding, mousse, yogurt, sesame tofu and the like.
  • Sauces and sauces have a good body feeling and shape retention, are well applied to foods and are difficult to drip, and have a low stickiness and stringiness, giving a smooth texture.
  • Examples of sauces and sauces include salmon grilled sauce, mitarashi dumpling sauce, fruit sauce, white sauce, dressing and the like.
  • fried foods include tempura and fried shrimp.
  • noodles it gives a chewy and chewy texture.
  • Examples of noodles include udon, somen, cold wheat, Chinese noodles, buckwheat, macaroni, spaghetti and the like.
  • jelly candy In jelly candy, it has a moderate elasticity and also has a good mouth melt and a smooth texture.
  • Examples of jelly candy include jelly candy and jelly beans.
  • the enzyme-treated rice starch granules of the present invention can be used in the same amount as that of starch conventionally used in the food.
  • a part of the conventional starch may be used, and the rest may be replaced with the enzyme-treated rice starch granules of the present invention.
  • the enzyme-treated rice starch granules of the present invention are preferably about 50% by weight or more, more preferably about 60% by weight or more, and still more preferably about 70% by weight or more of the usual amount of starch used.
  • the amount is preferably about 80% by weight or more, particularly preferably about 90% by weight or more, and most preferably 100% by weight. That is, it is most preferable to replace the total amount of conventional starch with the enzyme-treated rice starch granules of the present invention.
  • the method for producing a starch gel-containing food of the present invention comprises the step of mixing rice flour or rice starch and an enzyme; the temperature of the starch granules in the rice flour or rice starch is about 10 ° C or higher and about 70 ° C or lower.
  • a step of obtaining an enzyme-treated rice starch granule by treating with the enzyme a step of mixing a food material, the enzyme-treated rice starch granule, and water to obtain a mixture; and heating the mixture to treat the enzyme in the mixture
  • a step of gelatinizing the rice starch granules and a step of cooling and gelling the mixture containing the gelatinized enzyme-treated rice starch granules to obtain a starch gel-containing food.
  • starch granules are not used after being subjected to enzyme treatment in the food production process.
  • the steps of mixing rice flour or rice starch and enzyme, and treating the starch granules in the rice flour or rice starch with the enzyme at a temperature of about 10 ° C. or more and about 70 ° C. or less to obtain enzyme-treated rice starch granules are as described above. It can be carried out as detailed in “2.2 Enzymatic reaction”.
  • the starch granules can be starch granules of untreated starch, physically treated starch or chemically modified starch.
  • the starch granules are starch granules of untreated starch, physical-treated starch or bleached starch, and starch starch-containing food is obtained using this starch granule.
  • the starch granules are not chemically modified.
  • the starch granule is a starch granule of untreated starch or physical treated starch, further comprising chemically modifying the enzyme-treated rice starch granule, wherein the chemically modified enzyme-treated rice starch granule is used as a food material and Mix with water.
  • the starch granule is a starch granule of untreated starch or chemically modified starch, further comprising a step of physically treating the enzyme-treated rice starch granule, wherein the physically treated enzyme-treated rice starch granule is a food product. Mix with ingredients and water.
  • the food material, the enzyme-treated rice starch granules and water are mixed to obtain a mixture.
  • the mixing method and the mixing ratio of the food material, the enzyme-treated rice starch granules and the water can be performed according to the mixing method and mixing ratio in the normal production method of the target food.
  • the enzyme-treated rice starch granules in the mixture are gelatinized by heating the mixture.
  • This heating can be cooking. Heating can be performed under the same conditions as cooking in the usual production method of the target food.
  • the gelatinized enzyme-treated rice starch granules are cooled and gelled to obtain a starch gel-containing food. Cooling may be performed by leaving the heated mixture at room temperature, or may be performed by cooling in a refrigerator or the like.
  • the food of the present invention can be produced in the same manner as in the case of using ordinary starch, except that the enzyme-treated rice starch granules are used.
  • the method for producing a starch-containing food of the present invention includes a step of adding and mixing enzyme-treated rice starch granules to a food material; and a step of cooking the mixture.
  • the enzyme-treated rice starch granules of the present invention have superior viscosity and gel-forming ability compared to conventional untreated starch. Therefore, the enzyme-treated rice starch granules of the present invention are added to a food material, mixed, and the mixture is cooked to gelatinize the enzyme-treated rice starch granules, and then cooled to form a gel. Therefore, the resulting cooked food has better physical properties than the cooked food using conventional unprocessed starch (for example, excellent body feeling, natural elasticity, good melting in the mouth, smooth texture, crisp texture) , Soft texture, etc.).
  • the food may be a beverage.
  • “cooking” refers to heating a mixture of food material and starch.
  • the cooking may be heating at a temperature higher than the collapse temperature of the starch granules.
  • the mixture of food material and starch can be heated at about 70 ° C or higher, about 80 ° C or higher, about 90 ° C or higher, or about 95 ° C or higher.
  • the cooking is performed at a temperature that does not cause excessive denaturation of the food material and starch.
  • the mixture of food material and starch can be heated at about 200 ° C. or less, about 150 ° C. or less, about 130 ° C. or less, or about 110 ° C. or less.
  • the cooking time can be the normal cooking time of the target food.
  • Heat cooking is preferably performed in the presence of some moisture.
  • the starch granules normally swell when heated in the presence of a predetermined amount or more of water, increasing the transparency and increasing the viscosity. If the food material contains more moisture than necessary, it is not necessary to add water to the mixture of the food material and starch, but if the food material is low in moisture, water may be added to the mixture of the food material and starch. preferable. In the case of foods that do not contain food materials other than water and starch, such as sugar-free suzuyu, water is regarded as a food material.
  • Heat cooking can be part of the method for producing the intended food.
  • a gel food such as jelly
  • it can be cooled at a temperature of, for example, about 5 to 10 ° C. after cooking.
  • SEQ ID NO: 1 is a nucleotide sequence encoding an ⁇ -amylase from Aspergillus oryzae
  • SEQ ID NO: 2 is the amino acid sequence of an ⁇ -amylase from Aspergillus oryzae
  • SEQ ID NO: 3 is a nucleotide sequence encoding an ⁇ -amylase from Aspergillus niger
  • SEQ ID NO: 4 is the amino acid sequence of an ⁇ -amylase from Aspergillus niger
  • SEQ ID NO: 5 is a nucleotide sequence encoding amyloglucosidase from Aspergillus niger
  • SEQ ID NO: 6 is the amino acid sequence of amyloglucosidase from Aspergillus niger
  • SEQ ID NO: 7 is Flavobacterium sp.
  • the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
  • the viscosity was measured with a Brabender amylograph, and the gel properties were measured with a Rheotech rheometer.
  • the physical properties of each starch and the physical properties of the gel prepared from each starch were measured according to the following analysis and evaluation methods.
  • rheometer rheometer measurement conditions are as follows: a break test as a test item, a sample height of 25 mm, an adapter with a viscosity ball ⁇ 5 (diameter 5 mm, area 19.635 mm 2 ), and the sample moving speed (break speed). Measurement was performed at 6 cm / min. At this time, the hardness of the starch gel was evaluated by breaking stress (g) and Young's modulus (dyn / cm 2 ).
  • the decomposition rate of starch granules was measured by the following method.
  • the amount (g) of free reducing sugar contained in the supernatant obtained by centrifuging (3000 rpm, 5 minutes) the starch-decomposed suspension after the enzyme reaction was measured by the phenol-sulfuric acid method.
  • the percentage of the amount of reducing sugar released was determined relative to the total amount of starch (g) before the enzyme reaction.
  • Test example 1 Comparison between liquid reaction and solid reaction
  • (1) Liquid reaction After adding 250 g of ion-exchanged water to 90 g (dry weight) of untreated rice flour, adjusting to pH 5.0, heating in a boiling water bath to prepare a paste solution in which the starch in the rice flour is completely dissolved did.
  • ⁇ -amylase from Aspergillus oryzae was added in an amount of 0.1 wt% (dry weight of rice flour), adjusted to a total weight of 300 g, and stirred at 50 ° C. to carry out the enzyme reaction. After 30 minutes, the sample was left in a boiling water bath for 10 minutes to inactivate the enzyme, thereby preparing Sample 1.
  • gel properties were measured and evaluated by breaking stress and Young's modulus. The results are shown in Table 1B.
  • the obtained sample 1 When the enzyme was allowed to act after gelatinizing the starch in the rice flour, the obtained sample 1 was confirmed to have a marked decrease in viscosity, and no longer had a viscosity property of starch and formed a gel. On the other hand, when the enzyme was reacted with the starch grains in the rice flour retained, it was confirmed that the obtained sample 2 retained the viscosity physical properties of the starch and formed a hard gel. Therefore, it was confirmed that it is important to react the enzyme with the starch granules as they are without gelatinizing the starch grains in the rice flour.
  • the ⁇ -amylase used was as follows: Reference Example 2: Aspergillus oryzae-derived “Biozyme A” manufactured by Amano Enzyme Co., Ltd .; optimum pH 5.0; Reference Example 3: “AMYLEX A3” from Aspergillus niger, manufactured by DANISCO; optimum pH 5.0; Reference Example 4: Derived from Bacillus subtilis, “Novamil” manufactured by Novo; optimum pH 5.0; Reference Example 5: " ⁇ -amylase” derived from Bacillus amyloliquefaciens and manufactured by Sigma-Aldrich; optimum pH 6.0; Reference Example 6: Bacillus sp. Origin, Novo “Maltogenase L”; optimum pH 5.0; or Reference Example 7: Bacillus richeniformis origin, Novo “Termamyl Mill 120L”; optimum pH 6.0.
  • ⁇ -amylase derived from Aspergillus oryzae and ⁇ -amylase derived from Aspergillus niger have the property of improving the gel-forming ability of starch.
  • Examples and comparative examples with rice flour (Comparative Example 1) Japanese polished white rice (Japonica rice, whitening ratio 20%) was used as the raw material rice, and after sufficient washing, it was immersed in water overnight. After soaking, the water was removed, and water was added in the same volume as the apparent volume of the rice after soaking, followed by grinding and sieving according to conventional techniques. The starch milk thus obtained was dehydrated and dried according to the conventional technique to obtain rice flour having a protein content of 5.5% by weight. The untreated rice flour obtained in this way was analyzed for viscosity characteristics and gel physical properties using an amylograph and a rheometer according to the method described in “1. Analysis and Evaluation Method” above without performing enzyme treatment. The results are shown in Table 3.
  • Examples 1-5 and Comparative Examples 2-3 Treatment with enzyme reaction alone (Example 1) 900 g of ion-exchanged water was added to 400 g of untreated rice flour of the same lot used in Comparative Example 1 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, ⁇ -amylase (derived from Aspergillus oryzae, “Biozyme A” manufactured by Amano Enzyme Co., Ltd .; optimum pH 5.0) was added at 1% by weight (vs. starch dry weight). Then, the enzyme reaction was carried out by stirring at 50 ° C. for 18 hours. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • ⁇ -amylase derived from Aspergillus oryzae, “Biozyme A” manufactured by Amano Enzyme Co., Ltd .; optimum pH 5.0
  • Example 2 900 g of ion-exchanged water was added to 400 g of untreated rice flour of the same lot used in Comparative Example 1 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight of ⁇ -amylase (derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0) (based on dry weight of starch) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • ⁇ -amylase derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0
  • Example 3 900 g of ion-exchanged water was added to 400 g of untreated rice flour of the same lot used in Comparative Example 1 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Example 4 900 g of ion-exchanged water was added to 400 g of untreated rice flour of the same lot used in Comparative Example 1 to prepare a starch granule suspension.
  • ⁇ -glucosidase derived from Aspergillus niger, “Transglucosidase L-500” manufactured by Genencor; optimum pH 5.0
  • the enzyme reaction was performed by stirring at 50 ° C. for 18 hours. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • Example 5 900 g of ion-exchanged water was added to 400 g of untreated rice flour of the same lot used in Comparative Example 1 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 0.1% by weight (vs. starch dry weight) of isoamylase (derived from Flavobacterium sp., “GODO-FIA” manufactured by Godo Sake; optimum pH 5.5) was added. Then, the enzyme reaction was carried out by stirring at 50 ° C. for 18 hours. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • isoamylase derived from Flavobacterium sp., “GODO-FIA” manufactured by Godo Sake; optimum pH 5.5
  • Comparative Examples 4 to 5 and Examples 6 to 7 Combination of chemical treatment and enzyme reaction (Comparative Example 4) To 500 g of untreated rice flour of the same lot used in Comparative Example 1, 750 g of a 6.7% (w / w) aqueous sodium sulfate solution was added to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 8.5, 22.5 g of vinyl acetate monomer was added, and the reaction was performed by stirring at 30 ° C. for 40 minutes. After 40 minutes, the pH of the suspension was adjusted to 6.0 to stop the reaction. After completion of the reaction, centrifugal filtration and air drying were performed to recover starch acetate granules. The viscosity characteristics and gel physical properties of the obtained starch acetate granules were analyzed with an amylograph and a rheometer according to the method described in “1. Analysis and Evaluation Method” above. The results are shown in Table 4.
  • Example 6 900 g of ion-exchanged water was added to 400 g of the starch acetate granules prepared in Comparative Example 4 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Example 7 900 g of ion-exchanged water was added to 400 g of phosphoric acid crosslinked starch granules prepared in Comparative Example 5 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Comparative Example 6 and Example 8 Combination of physical treatment and chemical treatment (Comparative Example 6) Ion-exchanged water is added to 2 kg of untreated rice flour of the same lot used in Comparative Example 1, adjusted to a moisture content of 32%, packed in a 3 L glass beaker with as little space as possible, and the upper part made of aluminum. After covering with foil, wet heat treatment was performed by heating at 120 ° C. for 15 minutes. After completion of the wet heat treatment, the product was dried by air blowing to collect the wet heat treated starch granules. The viscosity characteristics and gel physical properties of the obtained heat-treated starch granules were analyzed with an amylograph and a rheometer according to the method described in “1. Analysis and Evaluation Method” above. The results are shown in Table 4.
  • Example 8 900 g of ion-exchanged water was added to 400 g of the wet heat-treated starch granules prepared in Comparative Example 6 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and blast drying were performed to recover the wet heat enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Viscosity characteristics and gel physical properties of the obtained wet heat enzyme-treated starch granules were analyzed with an amylograph and a rheometer according to the method described in “1. Analysis and Evaluation Method” above. Further, after the reaction was completed, the decomposition rate was determined using a part of the reaction solution according to the method described in “1. Analysis and Evaluation Method” above. The results are shown in Table 4.
  • Examples 9-12 Production of genetically modified enzymes (Example 9) (Method for preparing ⁇ -amylase derived from Aspergillus oryzae) An EcoRI recognition site (GAATTC) was added to both ends of the base sequence of SEQ ID NO: 1 in the sequence listing to chemically synthesize double-stranded DNA. This synthetic DNA was completely decomposed with the restriction enzyme EcoRI, and then mixed with pYCDE1 (Method in Enzymology, 101, pp. 192-201 (1983)) that had been completely decomposed with EcoRI in advance and ligated. Escherichia coli TG1 was transformed with the ligation reaction solution, and a transformant into which the synthetic gene was correctly introduced was selected. Plasmid pYAMY1 carried by this transformant was prepared.
  • GATTC EcoRI recognition site
  • pYAMY1 is introduced into the yeast host DBY746 according to the method of Ito et al. (J. bacteriol., Vol. 153, 163 to 168 (1983)), and can be grown in a medium not containing tryptophan by complementation of the tryptophan requirement.
  • a transformant was obtained. This transformant was inoculated into 100 ml of a synthetic medium consisting of 2% glucose, 0.67% yeast nitrogen base, 24 mg / l L-uracil, 24 mg / l L-histidine, 36 mg / l L-leucine, pH 5.7. And cultured with shaking at 30 ° C. for 120 hours.
  • the supernatant obtained by centrifuging the culture solution (5000 rpm, 10 minutes) was concentrated using a hollow fiber type UF membrane module having a molecular weight of 10,000 cut to prepare ⁇ -amylase derived from Aspergillus oryzae.
  • This ⁇ -amylase has the amino acid sequence of SEQ ID NO: 2.
  • Example 10 (Method for preparing ⁇ -amylase derived from Aspergillus niger) An EcoRI recognition site (GAATTC) was added to both ends of the base sequence of SEQ ID NO: 3 in the sequence listing to chemically synthesize double-stranded DNA.
  • This synthetic DNA was completely decomposed with the restriction enzyme EcoRI, and then mixed with pYCDE1 (Method in Enzymology, 101, pp. 192-201 (1983)) that had been completely decomposed with EcoRI in advance and ligated.
  • Escherichia coli TG1 was transformed with the ligation reaction solution, and a transformant into which the synthetic gene was correctly introduced was selected. Plasmid pYAMY2 carried by this transformant was prepared.
  • pYAMY2 was introduced into the yeast host DBY746 according to the method of Ito et al. (J. bacteriol., Vol. 153, 163 to 168 (1983)) and complemented with the tryptophan requirement, so that it can grow in a medium containing no tryptophan.
  • a transformant was obtained. This transformant was inoculated into 100 ml of a synthetic medium consisting of 2% glucose, 0.67% yeast nitrogen base, 24 mg / l L-uracil, 24 mg / l L-histidine, 36 mg / l L-leucine, pH 5.7. And cultured with shaking at 30 ° C. for 120 hours.
  • the supernatant obtained by centrifuging the culture solution (5000 rpm, 10 minutes) was concentrated using a hollow fiber type UF membrane module having a molecular weight of 10,000 cut to prepare Aspergillus niger-derived ⁇ -amylase.
  • This ⁇ -amylase has the amino acid sequence of SEQ ID NO: 4.
  • Example 11 (Method for preparing amyloglucosidase from Aspergillus niger)
  • An EcoRI recognition site (GAATTC) was added to both ends of the base sequence of SEQ ID NO: 5 in the sequence listing to chemically synthesize double-stranded DNA.
  • This synthetic DNA was completely decomposed with the restriction enzyme EcoRI, and then mixed with pYCDE1 (Method in Enzymology, 101, pp. 192-201 (1983)) that had been completely decomposed with EcoRI in advance and ligated.
  • Escherichia coli TG1 was transformed with the ligation reaction solution, and a transformant into which the synthetic gene was correctly introduced was selected. Plasmid pYGLU1 carried by this transformant was prepared.
  • pYGLU1 was introduced into the yeast host DBY746 according to the method of Ito et al. (J. bacteriol., Vol. 153, 163 to 168 (1983)) and complemented with tryptophan requirement, so that it can grow in a medium containing no tryptophan.
  • a transformant was obtained. This transformant was inoculated into 100 ml of a synthetic medium consisting of 2% glucose, 0.67% yeast nitrogen base, 24 mg / l L-uracil, 24 mg / l L-histidine, 36 mg / l L-leucine, pH 5.7. And cultured with shaking at 30 ° C. for 120 hours.
  • the supernatant obtained by centrifuging the culture solution (5000 rpm, 10 minutes) was concentrated using a hollow fiber UF membrane module with a molecular weight of 10,000 cut to prepare Aspergillus niger-derived amyloglucosidase.
  • This amyloglucosidase has the amino acid sequence of SEQ ID NO: 6.
  • Example 12 (Method for preparing isoamylase derived from Flavobacterium sp.) An EcoRI recognition site (GAATTC) was added to both ends of the base sequence of SEQ ID NO: 7 in the sequence listing to chemically synthesize double-stranded DNA.
  • This synthetic DNA was completely decomposed with the restriction enzyme EcoRI, and then mixed with pYCDE1 (Method in Enzymology, 101, pp. 192-201 (1983)) that had been completely decomposed with EcoRI in advance and ligated.
  • Escherichia coli TG1 was transformed with the ligation reaction solution, and a transformant into which the synthetic gene was correctly introduced was selected. Plasmid pYISO1 carried by this transformant was prepared.
  • pYISO1 was introduced into the yeast host DBY746 according to the method of Ito et al. (J. bacteriol., Vol. 153, 163 to 168 (1983)) and complemented with tryptophan requirement, so that it can grow in a medium containing no tryptophan.
  • a transformant was obtained. This transformant was inoculated into 100 ml of a synthetic medium consisting of 2% glucose, 0.67% yeast nitrogen base, 24 mg / l L-uracil, 24 mg / l L-histidine, 36 mg / l L-leucine, pH 5.7. And cultured with shaking at 30 ° C. for 120 hours.
  • the supernatant obtained by centrifuging the culture solution (5000 rpm, 10 minutes) was concentrated using a hollow fiber type UF membrane module having a molecular weight of 10,000, and Flavobacterium sp. Origin isoamylase was prepared.
  • This isoamylase has the amino acid sequence of SEQ ID NO: 8.
  • Example 9A to Example 12A A starch granule suspension was prepared by adding 900 g of ion-exchanged water to 400 g of untreated natural rice flour of the same lot used in Comparative Example 1. After adjusting the pH of the suspension to pH 5.0, 1% by weight of ⁇ -amylase (vs. dry weight of starch) was added, and the mixture was stirred at 50 ° C. for 18 hours to carry out the enzyme reaction. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules. The viscosity characteristics and gel properties of the obtained enzyme-treated starch granules were analyzed with an amylograph and a rheometer according to the method described in “1. Analysis and Evaluation Method” above. Further, after the reaction was completed, the decomposition rate was determined using a part of the reaction solution according to the method described in “1. Analysis and Evaluation Method” above. The results are shown in Table 4 below.
  • Example 9A ⁇ -amylase prepared in Example 9
  • Example 10A ⁇ -amylase prepared in Example 10
  • Example 11A ⁇ -amylase prepared in Example 11
  • Example 12A ⁇ -amylase prepared in Example 12.
  • the gel-forming ability can be improved by acting ⁇ -amylase, amyloglucosidase or isoamylase on the rice starch grains in the rice flour.
  • Prototype example Next, the present invention will be described in detail with reference to prototype examples, but the present invention is not limited to these prototype examples. Further, unless otherwise specified, “part” means “part by mass”.
  • the texture of the cookie of the prototype 1 was better than the texture of the cookie of the prototype 1 as compared to the hard and crunchy texture. And the cookie of Prototype Example 1 had a very light texture and was easy to eat.
  • the dough at the time of molding of the cookie of Prototype Example 1 is very dry and smooth compared to the dough at the time of molding of the cookie of Prototype Comparative Example 1, and there is no adhesion to hands or rolling pins, and workability is also good It was very good.
  • the sponge cake of Prototype Example 2 has a good bulge after firing and a large volume compared to the sponge cake of Prototype Comparative Example 2, and has a soft texture that is soft and fluffy. Had.
  • the obtained roll cake was refrigerated at 4 ° C., and the texture of the next day was evaluated. As a result of the next day, the roll cake of prototype comparative example 3-1 felt a sticky texture.
  • the roll cake of trial comparison example 3-2 had a soft texture compared to the roll cake of trial comparison example 3-1, but had a fluffy texture.
  • the roll cake of the prototype example 3-1 has a sticky texture
  • the roll cake of the prototype example 3-2 has a sticky texture.
  • the texture was soft and soft, and the mouth melted well.
  • the flour paste of Prototype Example 4-1 had a smooth texture with good mouth melting while retaining shape retention.
  • the flour paste of Prototype Example 4-2 was not as smooth as Prototype Example 4-1, but was given a body feeling and had a mouthfeel and good texture.
  • the flour paste of the prototype comparative example 4 had a mouthfeel that was poor in melting in the mouth and was not rough and smooth.
  • the fried food cooked with oil after immersing ingredients such as shrimp, baked bread, and cooked with oil had a crispy and light texture.
  • the fried food obtained by using the fried food batter of the prototype comparative example 5 had a weak crispy texture and was difficult to feel lightness.
  • the resulting raw udon was boiled in boiling water for 10 minutes and then put in hot soup to evaluate the texture.
  • the boiled udon of Prototype Comparative Example 6-1 had a feeling of stickiness but was poor in elasticity and was soft.
  • the boiled udon of the trial comparative example 6-2 was given a sticky feeling and lost its soft texture, but was poor in elasticity.
  • the boiled udon of Prototype 6-1 is given elasticity in addition to the moist texture, and the texture improvement effect is recognized.
  • the boiled udon of Prototype 6-2 is also elastic in addition to the moist feeling. In addition, it was given a slickness and a good texture.
  • the texture of the next day was evaluated for the obtained bread.
  • the bread of prototype comparative example 7 had a sticky texture, but at the same time had a sticky feeling.
  • the bread of Prototype Example 7 was soft and light, and had a resilient texture.
  • the sacrificial dumpling sacrificial dumpling of Prototype Example 8 had a good body feeling and shape retention, and the glue to the dumpling was hard to fall off.
  • the sacrificial dumpling sauce of Prototype Example 8 had a smooth and meltable mouthfeel.
  • the sacrificial dumpling sacrificial dumpling had a weak body feeling and shape retention.
  • the sacrificial dumpling sacrificial dumpling had a feeling of roughness and a very poor melting of the mouth.
  • the dumpling of the prototype comparative example 9 had a texture that was weak in body feeling and shape retention and poor in melting in the mouth.
  • the dumpling of Prototype Example 9 had a good body feeling and shape retention, and had a very good mouthfeel.
  • the sausage of Prototype Comparative Example 10-1 had a texture that was poor in elasticity and chewy.
  • the sausages of the prototype comparison example 10-2 and the prototype comparison example 10-3 were more elastic and crunchy than the sausage of the prototype comparison example 10-1, but were soft and had a poor texture.
  • the sausages of Prototype Example 10-1 and Prototype Example 10-2 were very good in texture and elasticity.
  • the sausage of Prototype Example 10-2 had a good texture with sufficient texture in addition to elasticity and body feeling.
  • the rice starch granules used in the following Comparative Examples and Examples are rice starch made by Sigma-Aldrich or based on a modification thereof. Rice starch manufactured by Sigma-Aldrich is derived from rice. (Comparative Example 7) Viscosity characteristics were analyzed with an amylograph and a rheometer in accordance with the method described in “1. Analysis and Evaluation Method” without applying enzyme treatment to untreated rice starch granules (rice starch; manufactured by Sigma Aldrich). The results are shown in Table 16.
  • Example 13 900 g of ion-exchanged water was added to 400 g of untreated rice starch granules (rice starch; Sigma Aldrich) of the same lot used in Comparative Example 7 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Example 14 900 g of ion-exchanged water was added to 400 g of untreated rice starch granules (rice starch; Sigma Aldrich) of the same lot used in Comparative Example 7 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight of ⁇ -amylase (derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0) (based on dry weight of starch) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • ⁇ -amylase derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0
  • Comparative Example 8 900 g of ion-exchanged water was added to 400 g of untreated rice starch granules (rice starch; Sigma Aldrich) of the same lot used in Comparative Example 7 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight of ⁇ -amylase (from Bacillus subtilis, Novo's “Novamil”; optimum pH 5.0) was added at 50 ° C. The enzyme reaction was carried out by stirring for 18 hours. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • ⁇ -amylase from Bacillus subtilis, Novo's “Novamil”; optimum pH 5.0
  • Example 15 900 g of ion-exchanged water was added to 400 g of untreated rice starch granules (rice starch; Sigma Aldrich) of the same lot used in Comparative Example 7 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 0.1% by weight (based on dry weight of starch) of isoamylase (derived from Flavobacterium odoratum, “GODO-FIA” manufactured by Godo Sake; optimum pH 5.5) was added. The enzyme reaction was carried out by stirring at 50 ° C. for 18 hours. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • isoamylase derived from Flavobacterium odoratum, “GODO-FIA” manufactured by Godo Sake; optimum pH 5.5
  • Example 16 900 g of ion-exchanged water was added to 400 g of phosphoric acid crosslinked starch granules prepared in Comparative Example 9 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Example 17 900 g of ion exchange water was added to 400 g of the starch acetate granules prepared in Comparative Example 10 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Example 18 900 g of ion exchange water was added to 400 g of the starch acetate granules prepared in Comparative Example 10 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight of ⁇ -amylase (derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0) (based on dry weight of starch) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and air drying were performed to collect enzyme-treated starch granules.
  • ⁇ -amylase derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0
  • Example 19 900 g of ion-exchanged water was added to 400 g of the wet heat-treated starch granules prepared in Comparative Example 11 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight (based on dry weight of starch) of amyloglucosidase (derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and blast drying were performed to recover the wet heat enzyme-treated starch granules.
  • amyloglucosidase derived from Aspergillus niger, “OPTIDEX L-400” manufactured by Genencor; optimum pH 4.4
  • Example 20 900 g of ion-exchanged water was added to 400 g of the wet heat-treated starch granules prepared in Comparative Example 11 to prepare a starch granule suspension. After adjusting the pH of the suspension to pH 5.0, 1% by weight of ⁇ -amylase (derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0) (based on dry weight of starch) was added. The enzyme reaction was carried out by stirring for 18 hours at ° C. After completion of the reaction, centrifugal filtration and blast drying were performed to recover the wet heat enzyme-treated starch granules.
  • ⁇ -amylase derived from Aspergillus niger, “AMYLEX A3” manufactured by DANISCO; optimum pH 5.0
  • enzyme-treated starch granules having strong gel forming ability and high viscosity are provided.
  • a highly elastic food with a body and natural elasticity can be obtained.
  • a low-moisture food that melts well in the mouth is obtained.

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Abstract

La présente invention concerne un procédé pour la production d'un produit alimentaire contenant un gel d'amidon de riz et qui comprend: une étape pour le mélange de poudre de riz ou d'amidon de riz et d'une enzyme ; une étape pour le traitement des granules d'amidon dans l'amidon de riz ou la poudre de riz au moyen d'une enzyme à une température comprise entre environ 10°C et environ 70°C inclusivement, permettant d'obtenir des granules d'amidon soumis à un traitement enzymatique ; une étape pour l'obtention d'un mélange par le mélange d'un ingrédient alimentaire, des granules soumis à un traitement enzymatique, et de l'eau ; une étape pour la gélatinisation des granules d'amidon soumis à un traitement enzymatique par le chauffage du mélange ; et une étape pour l'obtention d'un produit alimentaire contenant un gel d'amidon par le refroidissement et la gélification du mélange contenant les granules d'amidon soumis à un traitement enzymatique gélatinisés.
PCT/JP2012/000995 2011-02-16 2012-02-15 Produit alimentaire contenant un gel d'amidon de riz WO2012111327A1 (fr)

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