US 2013 0022711A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0022711 A1 Ichihara et al. (43) Pub. Date: Jan. 24, 2013

(54) FOOD PRODUCT CONTAINING STARCH Publication Classification GEL STARCH GRANULE, PRODUCTION METHOD AND USE THEREOF (51) Int. Cl. A2.3L. I./0522 (2006.01) (75) Inventors: Takashi Ichihara, Osaka (JP); Junya (52) U.S. Cl...... 426/48; 426/578; 426/549 Fukuda, Osaka (JP); Masakazu Kimura, Osaka (JP); Kenichi Kurita, Osaka (JP) (57) ABSTRACT (73) Assignee: GLICO FOODS CO.,LTD., Osaka (JP) Here is provided a method of producing a starch gel-contain ing food, the method comprising the steps of treating starch (21) Appl. No.: 13/383,060 granules with an enzyme at a temperature of about 10°C. or higher and about 70° C. or lower to obtain an enzyme-treated (22) PCT Filed: Aug. 11, 2010 starch; mixing a food material, the enzyme-treated Starch and water to obtain a mixture; heating the mixture thereby gela (86) PCT NO.: PCT/UP2O1 O/OOSO46 tinizing the enzyme-treated Starch in the mixture; and cooling S371 (c)(1), the mixture containing the gelatinized enzyme-treated Starch (2), (4) Date: Sep. 24, 2012 thereby gelling the starch to obtain a starch gel-containing food, wherein the enzyme is selected from the group consist (30) Foreign Application Priority Data ing of amyloglucosidase, isoamylase, C-glucosidase, C.-amy lase having a characteristic capable of improving a gel form Aug. 18, 2009 (JP) ...... 2009-189567 ing ability of a starch, and cyclodextrin glucanotransferase. US 2013/0022711 A1 Jan. 24, 2013

FOOD PRODUCT CONTAINING STARCH gel can be enhanced, but there is such a drawback that a final GEL STARCH GRANULE, PRODUCTION product has powdery texture and also is inferior in flavor. METHOD AND USE THEREOF Also, in the case where a starch having a low crosslinking degree is added to a food, since a large amount of the starch TECHNICAL FIELD needs to be used so as to obtain the desired hardness, the obtained food has increased powdery texture, thus causing 0001. The present invention relates to a starch gel-contain deterioration of quality of a final product. Therefore, there is ing food, a starch having a high viscosity and a gel forming a limit on the use amount of the starch having a low crosslink ability, a food containing the starch, and a method of produc ing degree. In addition, processing of the starch utilizing a ing thereof. More particularly, the present invention relates to chemical reaction also has such problems that there is a strict a method of producing a starch gel-containing food using an legal restriction on a processing method and a processing enzyme capable of improving a gel forming ability of a degree so as to secure safety, and that it is not necessarily starch. Suited to needs of consumers who require security and safety. 0010 For the purpose of deigning these processed foods, it BACKGROUND ART is urgently necessary to develop a processing technique to 0002 With diversification of foods, foods having various obtain a processed Starch which exhibits various physical shapes, physical properties and textures have been required. properties and has high safety. Particularly, intense interest has recently been shown towards 0011. As a result of intensive studies, we have found that a melt in mouth and texture as important physical properties for food with rich elasticity, crispy sensation and the like can be the purpose of designing foods. Also in the fields related to prepared by adopting the steps of treating starch granules deglutition and care toward which intense interest has with a starch hydrolase or a glycosyltransferase in advance; recently been shown, texture has been studied as important then mixing the resultant with a food material and water, and physical properties. heating the mixture. 0003. In the case of designing processed foods, utilization 0012. A starch is a material utilized for various purposes of a gelling agent is important so as to improve texture and and the most important function thereof is the thickening physical properties, and it is possible to develop various prod function and the gel forming function. Particularly in the food ucts according to how to use. industry, the thickening function and the gel forming function 0004 For the purpose of altering physical properties of of the starch are widely utilized for forming the shape, physi foods, various gelling agents have hitherto been added to food cal properties and texture of a food. The structure of a starch materials in the case of preparing foods. delicately varies depending on plant from which the starch 0005. In food processing, natural macromolecules such as derived (for example, corn, potato, wheat, and cassava). As a agar, gelatin, gellan gum, Xanthan gum, locust bean gum, result, the thickening function and gel forming function also carrageenan, pectin, Sodium alginate, Tamarind seed gum, vary depending on the plant from which the starch derived. psyllium seed gum, microcrystalline cellulose, curdlan, and Therefore, those skilled in the art have been selected a native starch; or synthetic macromolecules such as carboxymethyl starch to be used for a long time depending on the purpose. cellulose (CMC) or methyl cellulose are commonly used as For example, a wheat starch has often been used in a fish paste gelling agents. product for a long time. The reason is that the wheat Starch is 0006. In the case of using these gelling agents, gelling excellent in gel forming function. For example, a cassava agents may be sometimes used alone, however, in order to starch is commonly utilized in a food which has high trans form gels having more various characteristics, for example, parency and requires Sticky texture. However, with the use of two or more kinds of gelling agents such as native advancement of characteristics required in the current food gellan gum and guar gum in combination is studied and industry, it becomes impossible to cope with the advancement utilized (Patent Document 1). only by changing a native starch to be used. Therefore, there 0007. However, there are few combinations which can arises the need to alter the thickening function or the gel synergistically change the gel strength of foods. Even if it is forming function of a starch. possible to synergistically change the gel strength, the gel 0013 Means which are used most commonly to alter the obtained thereby does not have nice physical properties. Mix thickening function or the gel forming function of a starch is ing of two or more kinds of gelling agents is a defect due to a chemical modification of a starch. Above all, techniques of being complicated and that many materials are very expen applying a chemical treatment, Such as a technique of intro S1V. ducing a new crosslinking point between starch molecules 0008 Furthermore, there is such a restriction on use in using a Suitable chemical crosslinking agent and a technique food processing that, for example, a gelatin is inferior in of introducing a suitable functional group have widely been resistance to an acid and an alkali, and also an agar is inferior utilized so as to remarkably alter the thickening function or in resistance to an acid. the gel forming function. However, a starch Subjected to Such 0009 Starches have successfully produced various physi a chemical treatment has been specified as a food additive cal properties by adding not only raw starches but also a from October, 2008 in Japan, and thus restricted by law. processed starch obtained by chemically modifying starches Therefore, there has been required a technique in which the (also referred to as a chemically modified Starch) such as thickening function or the gel forming function of a starch is starch acetate and monostarch phosphate as agelling agent to altered without a chemical treatment. food materials. For example, Patent Documents 2, 3 and 4 0014. The technique of altering a starch without a chemi indicate examples in which a crosslinked Starch is utilized in cal treatment includes a technique of an enzymatic treatment a white table bread, confectioneries or noodles. However, in of a starch. Since an enzyme commonly acts on a substrate the case where a crosslinked starch having a high crosslinking dissolved in water, an enzymatic treatment is usually carried degree is added to a food, the hardness and the Viscosity of a out after completely dissolving a starch in water. A hydrolytic US 2013/0022711 A1 Jan. 24, 2013

enzyme or a glycosyltransferase is allowed to act on a starch food. Therefore, use of the method of the present invention dissolved in water to cleave the starch, thereby producing can exert an influence on texture of the food. As described molecules having a lower molecular weight such as dextrin, above, the enzyme-treated Starch granules of the prior art and starch syrup, maltooligosaccharide, maltose, and glucose. the enzyme-treated Starch granules used in the present appli However, in the enzymatic treatment with a hydrolytic cation quite differ in application and usage. enzyme or a glycosyltransferase, a starch molecule is cleaved 0017. As described above, it was conventionally impos to form low-molecular weight molecules. Therefore, it has sible to provide a starch excellent in thickening function or been commonly considered that the thickening function and gel forming function without utilizing a chemical modifica the gel forming function of the obtained molecule deteriorate tion of a starch. as compared with the thickening function and the gel forming 0018. Also, in the prior art, no attention was paid at all function of the starch, or are lost. whether or not an enzyme has characteristics capable of 0.015. Also, Patent Document 5 discloses, as a method of improving a gel forming ability of a starch. It was not also altering physical properties of a starch, a technique in which found at all whether or not industrial advantages are exerted an enzyme is allowed to act on a starch in the form of starch by characteristics of an enzyme capable of improving a gel granules in water without dissolving them in water. Patent forming ability of a starch. Document 5 discloses that although a starch has convention ally been dissolved in water before an enzymatic treatment in PRIOR ART DOCUMENTS the case of subjecting the starch to the enzymatic treatment, it is not necessarily required to dissolve the starch in water Patent Documents before the enzymatic treatment, and it is possible to subject 0019 Patent Document 1: Japanese Laid-open Patent starch granules, which are not dissolved in water but Sus Publication No. 10-215795 pended in water, to the enzymatic treatment. Specifically, it is 0020 Patent Document 2: Japanese Patent Gazette No. disclosed that a hydrolytic enzyme such as C.-amylase or 3,723,860 glucoamylase can act on starch granules, which are not dis 0021. Patent Document 3: Japanese Patent Gazette No. Solved in water but Suspended in water, and thus a reducing 3.312,225 Sugar can be produced. Patent Document 5 also discloses as a 0022. Patent Document 4: Japanese Patent Publication for result of this that the viscosity of the starch subjected to the Opposition No. 7-63324 enzymatic treatment is lower than that of the starch which is 0023 Patent Document 5: Japanese Laid-open Patent not subjected to the enzymatic treatment. However, Patent Publication No. 6-269291 Document 5 neither suggests nor discloses that a starch hav 0024 Patent Document 6: Japanese Laid-open Patent ing improved thickening function or gel forming function as Publication No. 2003-2198.13 compared with the starch, which is not subjected to the enzy (0025 Patent Document 7: Japanese Patent Gazette No. matic treatment, is obtained by allowing a hydrolytic enzyme 4,170,062 or a glycosyltransferase to act on starch granules. 0026 Patent Document 8: Japanese Laid-open Patent 0016 Patent Documents 6 to 10 also disclose a technique Publication No. 1-159047 of allowing a hydrolytic enzyme to act on insoluble starch 0027 Patent Document 9: Japanese Laid-open Patent granules. These inventions disclose a technique in which the Publication No. 5-112469 action of a hydrolytic enzyme on starch granules opens pores 0028 Patent Document 10: Japanese Laid-open Patent on the Surfaces of starch granules to make porous starch Publication No. 8-277230 granules, and the porous starch granules are utilized as a powdered base material or a porous carrier. However, Patent SUMMARY OF THE INVENTION Documents 6 to 10 neither suggests nor discloses that a starch having improved thickening function and gel forming func Problems to be Solved by the Invention tion is obtained by allowing a hydrolytic enzyme or a glyco Syltransferase to act on starch granules. An object of the 0029. The present invention is intended to solve the above present invention is not to open pores on the Surfaces of problems, and it is an object of the invention to provide a food enzyme-treated Starch granules, and there is not any relation containing a starch gel having the desired degree of hardness ship between an improvement in thickening function and gel and a method of producing the same. In a specific embodi forming function, and whether or not pores are opened on the ment of the present invention, objects are to provide a starch Surfaces of enzyme-treated Starch granules. If a heated food is excellent in thickening function or gel forming function with produced using the enzyme-treated Starch of the present out utilizing a chemical modification of a starch; a food con invention, the enzyme-treated Starch forms a hard gel in the taining the starch; and a method of producing the starch and heated food. The enzyme-treated starch of the present inven food. tion is usable in the heated food. On the other hand, in the prior art, it is important that pores are present on the Surfaces Means for Solving the Problems of starch granules. If starch granules after Subjected to the 0030 The present inventors have intensively studied so as enzymatic treatment and water are mixed and then heated, to solve the above problems and have found that a starch starch granules are collapsed and pore-opened states thereof excellent in thickening function and gel forming function is are lost. Therefore, those skilled in the art did not consider to obtained by allowing a specific hydrolytic enzyme or glyco use a pore-opened starch of the prior art in the heated food. In Syltransferase having characteristics capable of improving a the present invention, it is possible to adjust the hardness of a gel forming ability of a starch to act on starch granules under gel to be formed using an enzyme-treated Starch by adjusting the condition where a starch is not dissolved, and thus have the degree of the enzymatic treatment. The hardness of the gel completed the present invention based on this finding. It is exerts an influence on texture, chewiness, and the like of the commonly considered that when a hydrolytic enzyme or a US 2013/0022711 A1 Jan. 24, 2013 glycosyltransferase is allowed to act on a starch, the starch is derived from Aspergillus niger commercially available from cleaved to form smaller molecules, and therefore the viscos Genencor as OPTIDEX L-400, amyloglucosidase derived ity and the gel forming ability of the obtained molecules from Aspergillus niger commercially available from deteriorate as compared with the viscosity and gel forming DANISCO as DIAZYME X4NP. amyloglucosidase derived ability of the starch before being subjected to the enzymatic from Aspergillus niger commercially available from Amano treatment, or are lost. Actually, when the same hydrolytic Enzyme as glucoamylase "Amano' SD, amyloglucosidase enzyme or glycosyltransferase as the hydrolytic enzyme or derived from Rhizopus niveus commercially available from glycosyltransferase capable of producing such an excellent Amano Enzyme as Gluczyme AF6, amyloglucosidase starch when being allowed to act on starch granules under the derived from Rhizopus Oryzae commercially available from condition where a starch is not dissolved in water is allowed SHIN NIHON CHEMICALS Corporation as Sumizyme, to act on a starchafter dissolving the starch in water, the starch C-glucosidase derived from Aspergillus niger commercially Viscosity decreases, and thus a starch excellent in thickening available from Amano Enzyme as transglucosidase L function or gel forming function cannot be obtained. As "Amano', C.-glucosidase derived from Aspergillus niger described above, the present invention cannot be conceived commercially available from Genencor as Transglucosidase from the conventionally general knowledge and technical L-500, C-amylase derived from Aspergillus Oryzae commer common sense possessed by those skilled in the art. cially available from Amano Enzyme as Biozyme A, C.-amy 0031. The conditions of an enzymatic treatment of starch lase derived from Aspergillus Oryzae commercially available granules can vary depending on the specificity of the enzyme from SHIN NIHON CHEMICALS Corporation as Sum and the origin of starch granules. For example, first, starch izyme L. C.-amylase derived from Aspergillus niger commer granules are suspended in ion-exchange water or a buffer cially available from Danisco as AMYLEX A3, C.-amylase Solution to prepare a starch Suspension. In the case where the derived from Aspergillus niger commercially available from pH adjustment of the starch Suspension is required, the pH is SHIN NIHONCHEMICALS Corporation as Sumizyme AS, adjusted to the optimum pH of the enzyme. While warming isoamylase derived from Pseudomonas amyloderamosa this starch Suspension at the temperature at which starch commercially available from Sigma as isoamylase, cyclodex granules are not degraded (preferably from about 10° C. to trin glucanotransferase derived from Bacillus licheniformis about 70° C.), the enzyme is added and the reaction can be commercially available from Novozyme as Toruzyme, and carried out, for example, within about 24 hours (preferably cyclodextrin glucanotransferase derived from Paenibacillus from about for 1 hour to about 20 hours). Then, the enzyme macerans (Bacillus macerans) commercially available from and a carbohydrate eluted by enzymatic hydrolysis are Amano Enzyme as Cyclodextrin glucanotransferase removed by the washing and dehydration steps which are a Amano’. conventional method of preparing a starch, followed by the 0042 (Item 5) The method according to Item 1, wherein: drying step, and thus the objective enzyme-treated Starch (1) the enzyme is encoded by a nucleic acid molecule which granules can be obtained. is capable of hybridizing under stringent conditions with a 0032. The present invention is, for example, as follows: nucleic acid molecule consisting of a base sequence comple 0033 (Item 1) A method of producing a starch gel-con mentary to a base sequence of SEQID NO: 1, 3, 5, 7, 9 or 11, taining food, the method comprising the steps of and has a starch hydrolysis activity; or 0034 treating starch granules with an enzyme at a tem (2) the enzyme is encoded by a nucleic acid molecule which perature of about 10°C. or higher and about 70° C. or lower is capable of hybridizing under stringent conditions with a to obtain an enzyme-treated Starch; nucleic acid molecule consisting of a base sequence comple 0035 mixing a food material, the enzyme-treated starch mentary to a base sequence of SEQ ID NO: 13, and has a and water to obtain a mixture; transglycosylation activity; wherein the stringent conditions 0036 heating the mixture thereby gelatinizing the are hybridization in a solution containing 50% formamide, enzyme-treated Starch in the mixture; and 5xSSC (750 mM. NaCl, 75 mM trisodium citrate), 50 mM 0037 cooling the mixture containing the gelatinized sodium phosphate (pH 7.6), 5xDenhardt’s solution (0.2% enzyme-treated Starch thereby gelling the starch to obtain a BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyrrolidone), 10% starch gel-containing food, wherein dextran Sulfate and 201g/ml denatured sheared salmon sperm 0038 the enzyme is selected from the group consisting of DNA at 65°C., and subsequent washing under the condition amyloglucosidase, isoamylase, C-glucosidase, C.-amylase of 65° C. using an SSC solution having a 0.1 to 2-fold con having a characteristic capable of improving a gel forming centration (a composition of an SSC solution having a 1-fold ability of a starch, and cyclodextrin glucanotransferase. concentration is 150 mM sodium chloride and 15 mM sodium 0039 (Item 2) The method according to Item 1, wherein citrate). the enzyme is selected from the group consisting of amylo 0043 (Item 6) The method according to Item 1, wherein: glucosidase, isoamylase, C-glucosidase, C.-amylase derived (1) the enzyme has an amino acid sequence having at least from the genus Aspergillus, and cyclodextrin glucanotrans 95% or more of homology with an amino acid sequence of ferase. SEQID NO: 2, 4, 6, 8, 10 or 12, and has a starch hydrolysis 0040 (Item 3) The method according to Item 1, wherein activity; or the enzyme is selected from the group consisting of amylo (2) the enzyme has an amino acid sequence having at least glucosidase, isoamylase, C-glucosidase, C.-amylase derived 95% or more of homology with an amino acid sequence of from Aspergillus Oryzae, C.-amylase derived from Aspergillus SEQID NO: 14, and has a transglycosylation activity. niger, and cyclodextrin glucanotransferase. 0044 (Item 7) The method according to Item 1, wherein 0041) (Item 4) The method according to Item 1, wherein the starch granules are starch granules of an untreated Starch, the enzyme is selected from the group consisting of amylo a physically treated Starch or a chemically modified Starch. glucosidase derived from Aspergillus niger commercially 0045 (Item 8) The method according to Item 1, wherein available from Novozyme as AMG. amyloglucosidase the starch granules are starch granules of an untreated Starch, US 2013/0022711 A1 Jan. 24, 2013

and the starch granules have been neither chemically modi 0062 (Item 2A) The food according to Item 1A, wherein fied nor physically treated in any stage until the starch gel the untreated Starch is a untreated wheat starch, the enzyme containing food is obtained by the method. treated Starch is a enzyme-treated wheat starch, and 0046 (Item 9) The method according to Item 1, wherein 0063 the enzyme-treated wheat starch is capable of form the starch granules are starch granules of an untreated Starch ing a gel having a Young's modulus which accounts for 110% or a physically treated Starch, the method further comprises or more and 500% or less (110% or more and 330% or less in the step of chemically modifying the enzyme-treated Starch, an embodiment) of the Young's modulus of the untreated and the chemically modified enzyme-treated Starch is mixed wheat starch, or a rupture stress which accounts for 110% or with the food material and water. more and 300% or less of the rupture stress of the untreated 0047 (Item 10) The method according to Item 1, wherein wheat starch, when measured by a rheometer. the starch granules are starch granules of an untreated Starch 0064 (Item 3A) The food according to Item 1A, wherein or a chemically modified starch, the method further com the untreated Starch is a untreated cassava starch, the enzyme prises the step of physically treating the enzyme-treated treated Starch is a enzyme-treated cassava starch, and starch, and the physically treated enzyme-treated Starch is 0065 the enzyme-treated cassava starch is capable of mixed with the food material and water. forming a gel having a Young's modulus which accounts for 0048 (Item 11) A starch gel-containing food produced by 110% or more and 500% or less (110% or more and 330% or the method according to Item 1. less in an embodiment) of the Young's modulus of the 0049 (Item 12) The food according to Item 11, wherein untreated cassava starch, or a rupture stress which accounts the food is a high moisture content type food and the amount for 110% or more and 300% or less of the rupture stress of the of moisture of the food is more than 40g and less than 95g per untreated cassava starch, when measured by a rheometer. 100 g of the edible portion. 0.066 (Item 4A) The food according to Item 1A, wherein 0050 (Item 13) The food according to Item 11, wherein the untreated Starch is a untreated corn starch, the enzyme the food is selected from the group consisting of traditional treated Starch is a enzyme-treated corn starch, and Japanese-style confectioneries, fat- or oil-containing foods, 0067 the enzyme-treated corn starch is capable of form gelatinous foods, fish meat and animal meat processed foods, ing a gel having a Young's modulus which accounts for 110% Salsa and sauces, and noodles. or more and 500% or less (110% or more and 330% or less in 0051 (Item 14) The food according to Item 11, wherein an embodiment) of theYoung's modulus of the untreated corn the food is a low moisture content type food and the amount starch, or a rupture stress which accounts for 110% or more of moisture of the food is 1 g or more and 40g or less per 100 and 300% or less of the rupture stress of the untreated corn g of the edible portion. starch, when measured by a rheometer. 0052 (Item 15) The food according to Item 11, wherein 0068 (Item 5A) A heat-cooked starch-containing food the food is selected from the group consisting of bakeries, containing an enzyme-treated wheat starch having high vis Western-style confectioneries, and fried foods. cosity and gel-forming ability, 0053 (Item 16) The food according to Item 11, wherein the enzyme is selected from the group consisting of amylo 0069 the starch-containing food is a food produced by a glucosidase, isoamylase, C-glucosidase, C.-amylase derived method comprising the steps of mixing a food material and from the genus Aspergillus, and cyclodextrin glucanotrans the enzyme-treated wheat starch and then heating them, ferase. 0070 the enzyme-treated wheat starch is a starch obtained 0054 (Item 17) The food according to Item 11, wherein by treating starch granules of untreated wheat starch with a the enzyme is selected from the group consisting of amylo starch hydrolase under the condition where the starch gran glucosidase, isoamylase, C-glucosidase, C.-amylase derived ules are not dissolved, from Aspergillus Oryzae, C.-amylase derived from Aspergillus 0071 the enzyme-treated wheat starch is not modified on niger, and cyclodextrin glucanotransferase. hydroxyl groups at the positions 2, 3 and 6 of the glucose 0055 (Item 18) The food according to Item 11, wherein residues, the starch is derived from cassava, corn or wheat. 0072 the enzyme-treated wheat starch can form a gel hav 0056. In a specific embodiment, the present invention is, ing a Young's modulus of 5.0x10° dyn/cm or more and 8.0x for example, as follows: 10° dyn/cm or less, or a rupture stress of 150 g or more and 0057 (Item 1A) A heat-cooked starch-containing food 450 g or less, when measured by a rheometer. containing an enzyme-treated Starch having high viscosity 0073 (Item 6A) A heat-cooked starch-containing food and gel-forming ability, containing an enzyme-treated cassava starch having high vis 0058 the starch-containing food is a food produced by a cosity and gel-forming ability, method comprising the steps of mixing a food material and 0074 the starch-containing food is a food produced by a the enzyme-treated Starch and then heating them, method comprising the steps of mixing a food material and 0059 the enzyme-treated starch is a starch obtained by the enzyme-treated cassava starch and then heating them, treating starch granules of untreated Starch with a starch 0075 the enzyme-treated cassava starch is a starch hydrolase under the condition where the starch granules are obtained by treating starch granules of untreated cassava not dissolved, starch with an starch hydrolase under the condition where the 0060 the enzyme-treated starch is not modified on starch granules are not dissolved, hydroxyl groups at the positions 2, 3 and 6 of the glucose 0076 the enzyme-treated cassava starch is not modified on residues, hydroxyl groups at the positions 2, 3 and 6 of the glucose 0061 the enzyme-treated starch can form a gel having a residues, Young's modulus higher than that of the untreated starch or a 0077 the enzyme-treated cassava starch can form a gel rupture stress higher than that of the untreated starch, when having a Young's modulus of 5.2x10 dyn/cm or more and measured by a rheometer. 2.7x10° dyn/cm or less (5.2x10 dyn/cm or more and 1.6x US 2013/0022711 A1 Jan. 24, 2013

10° dyn/cm or less in one embodiment), or a rupture stress of 0.095 the enzyme-treated starch is not modified on 55 g or more and 150 g or less, when measured by a rheom hydroxyl groups at the positions 2, 3 and 6 of the glucose eter. residues, 0078 (Item 7A). A heat-cooked starch-containing food 0096 the enzyme-treated starch can form a gel having a containing an enzyme-treated corn starch having high viscos Young's modulus higher than that of the untreated starch or a ity and gel-forming ability, rupture stress higher than that of the untreated starch, when 0079 the starch-containing food is a food produced by a measured by a rheometer. method comprising the steps of mixing a food material and 0097 (Item 17A) An enzyme-treated starch having high the enzyme-treated corn Starch and then heating them, Viscosity and gel-forming ability, 0080 the enzyme-treated corn starch is a starch obtained 0.098 the enzyme-treated starch is a starch obtained by by treating starch granules of untreated corn starch with an treating starch granules of untreated Starch with an starch starch hydrolase under the condition where the starch gran hydrolase under the condition where the starch granules are ules are not dissolved, not dissolved, 0081 the enzyme-treated corn starch is not modified on 0099 the enzyme-treated starch is not modified on hydroxyl groups at the positions 2, 3 and 6 of the glucose hydroxyl groups at the positions 2, 3 and 6 of the glucose residues, residues, 0082 the enzyme-treated corn starch can form a gel hav 0100 the enzyme-treated starch can form a gel having a ing a Young's modulus of 6.0x10° dyn/cm or more and 9.0x Young's modulus higher than that of the untreated starch or a 10° dyn/cm or less, or a rupture stress of 210 g or more and rupture stress higher than that of the untreated starch, when 450 g or less (220g or more and 450 g or less in one embodi measured by a rheometer. ment), when measured by a rheometer. 0101 (Item 18A) The starch according to Item 17A, 0083 (Item 8A) The food according to any one of Items wherein the untreated starch is a untreated wheat starch, the 1A to 7A, wherein the starch is forming a gel in the food. enzyme-treated Starch is a enzyme-treated wheat Starch, and 0084 (Item 9A) The food according to any one of Items 0102 the enzyme-treated wheat starch is capable of form 1A to 8A, wherein the food is a high moisture content type ing a gel having a Young's modulus which accounts for 110% food and the amount of moisture of the food is more than 40 or more and 500% or less (110% or more and 330% or less in g and less than 95g per 100 g of the edible portion. an embodiment) of the Young's modulus of the untreated 0085 (Item 10A) The food according to any one of Items wheat starch, or a rupture stress which accounts for 110% or 1A to 9A, wherein the food is selected from the group con more and 300% or less of the rupture stress of the untreated sisting of traditional Japanese-style confectioneries, fat- or wheat starch, when measured by a rheometer. oil-containing foods, gelatinous foods, fish meat and animal 0103 (Item 19A) The starch according to Item 17A, meat processed foods, Salsa and sauces, and noodles. wherein the untreated Starch is a untreated cassava starch, the I0086 (Item 11A) The food according to any one of Items enzyme-treated Starch is a enzyme-treated cassava starch, and 1A to 8A, wherein the food is a low moisture content type 0104 the enzyme-treated cassava starch is capable of food and the amount of moisture of the food is 1 g or more and forming a gel having a Young's modulus which accounts for 40 g or less per 100 g of the edible portion. 110% or more and 500% or less (110% or more and 330% or 0087 (Item 12A) The food according to any one of Items less in an embodiment) of the Young's modulus of the 1A to 8A and 11A, wherein the food is selected from the untreated cassava starch, or a rupture stress which accounts group consisting of bakeries, Western-style confectioneries, for 110% or more and 300% or less of the rupture stress of the and fried foods. untreated cassava starch, when measured by a rheometer. 0088 (Item 13A) The food according to any one of Items 0105 (Item 20A) The starch according to Item 17A, 1A to 12A, wherein the starch hydrolase is selected from the wherein the untreated Starch is a untreated corn starch, the group consisting of amyloglucosidase, isoamylase, C-glu enzyme-treated Starch is a enzyme-treated corn starch, and cosidase, and C-amylase having characteristics capable of 0106 the enzyme-treated corn starch is capable of form improving a gel-forming ability of a starch. ing a gel having a Young's modulus which accounts for 110% I0089 (Item 14A) The food according to Item 13A, or more and 500% or less (110% or more and 330% or less in wherein the starch hydrolase is selected from the group con an embodiment) of theYoung's modulus of the untreated corn sisting of amyloglucosidase, isoamylase, C-glucosidase, and starch, or a rupture stress which accounts for 110% or more C.-amylase derived from the genus Aspergillus. and 300% or less of the rupture stress of the untreated corn 0090 (Item 15A) The food according to Item 13A, starch, when measured by a rheometer. wherein the starch hydrolase is selected from the group con 0107 (Item 21A) An enzyme-treated wheat starch having sisting of amyloglucosidase, isoamylase, C-glucosidase, high viscosity and gel-forming ability, C.-amylase derived from Aspergillus Oryzae, and C-amylase 0.108 the enzyme-treated wheat starch is a starch obtained derived from Aspergillus niger: by treating starch granules of untreated wheat starch with an 0091 (Item 16A) A method of producing a starch-contain starch hydrolase under the condition where the starch gran ing food, the method comprising the steps of ules are not dissolved, 0092 adding and mixing an enzyme-treated starch to a 0109 the enzyme-treated wheat starch is not modified on food material; and hydroxyl groups at the positions 2, 3 and 6 of the glucose 0093 heat-cooking the mixture; residues, 0094 the enzyme-treated starch is a starch obtained by 0110 the enzyme-treated wheat starch can form a gel hav treating starch granules of untreated Starch with a starch ing a Young's modulus of 5.0x10° dyn/cm or more and 8.0x hydrolase under the condition where the starch granules are 10° dyn/cm or less, or a rupture stress of 150 g or more and not dissolved; 450 g or less, when measured by a rheometer. US 2013/0022711 A1 Jan. 24, 2013

0111 (Item 22A) An enzyme-treated cassava starch hav from the group consisting of amyloglucosidase derived from ing high viscosity and gel-forming ability, Aspergillus niger commercially available from Novozyme as 0112 the enzyme-treated cassava starch is a starch AMG, amyloglucosidase derived from Aspergillus niger obtained by treating starch granules of untreated cassava commercially available from Genencor as OPTIDEXL-400, starch with an starch hydrolase under the condition where the amyloglucosidase derived from Aspergillus niger commer starch granules are not dissolved, cially available from DANISCO as DIAZYMEX4NP amy 0113 the enzyme-treated cassava starch is not modified on loglucosidase derived from Aspergillus niger commercially hydroxyl groups at the positions 2, 3 and 6 of the glucose available from Amano Enzyme as glucoamylase "Amano' residues, SD, amyloglucosidase derived from Rhizopus niveus com 0114 the enzyme-treated cassava starch can form a gel mercially available from Amano Enzyme as Gluczyme AF6, having a Young's modulus of 5.2x10 dyn/cm or more and amyloglucosidase derived from Rhizopus Oryzae commer 2.7x10° dyn/cm or less (5.2x10 dyn/cm or more and 1.6x cially available from SHIN NIHON CHEMICALS Corpora 10° dyn/cm or less in one embodiment), or a rupture stress of tion as Sumizyme, C-glucosidase derived from Aspergillus 55 g or more and 150 g or less, when measured by a rheom niger commercially available from Amano Enzyme as trans eter. glucosidase L Amano'. C-glucosidase derived from 0115 (Item 23A) An enzyme-treated corn starch having Aspergillus niger commercially available from Genencor as high viscosity and gel-forming ability, Transglucosidase L-50, C.-amylase derived from Aspergillus 0116 the enzyme-treated corn starch is a starch obtained Oryzae commercially available from Amano Enzyme as by treating starch granules of untreated corn starch with an Biozyme A, C.-amylase derived from Aspergillus Oryzae com starch hydrolase under the condition where the starch gran mercially available from SHIN NIHON CHEMICALS Cor ules are not dissolved, poration as Sumizyme L, C.-amylase derived from Aspergil 0117 the enzyme-treated corn starch is not modified on lus niger commercially available from Danisco as AMYLEX hydroxyl groups at the positions 2, 3 and 6 of the glucose A3, C.-amylase derived from Aspergillus niger commercially residues, available from SHIN NIHON CHEMICALS Corporation as 0118 the enzyme-treated corn starch can form a gel hav Sumizyme AS, and isoamylase derived from Pseudomonas ing a Young's modulus of 6.0x10° dyn/cm or more and 9.0x amyloderamosa commercially available from Sigma as 10° dyn/cm or less, or a rupture stress of 210 g or more and isoamylase. 450 g or less (220g or more and 450 g or less in one embodi I0128 (Item 31A) The method according to any one of ment), when measured by a rheometer. Items 27A to 30A, wherein the starch hydrolase is encoded by 0119 (Item 24A) The starch according to any one of Items a nucleic acid molecule which is capable of hybridizing under 18A to 23A, wherein the starch hydrolase is selected from the stringent conditions with a nucleic acid molecule having a group consisting of amyloglucosidase, isoamylase, C-glu complementary sequence of the base sequence of SEQ ID cosidase, and C-amylase having characteristics capable of NO: 1, 3, 5, 7, 9 or 11, and has a starch hydrolysis activity; improving a gel-forming ability of a starch. wherein the stringent conditions are hybridization in a solu 0120 (Item 25A). The starch according to Item 24A, tion containing 50% formamide, 5xSSC (750 mM NaCl, 75 wherein the starch hydrolase is selected from the group con mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), sisting of amyloglucosidase, isoamylase, C-glucosidase, and 5xDenhardt’s solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% C.-amylase derived from the genus Aspergillus. polyvinylpyrrolidone), 10% dextran sulfate and 20 ug/ml 0121 (Item 26A) The starch according to Item 24A, denatured sheared salmon sperm DNA at 65° C., and subse wherein the starch hydrolase is selected from the group con quent washing under the condition of 65° C. using an SSC sisting of amyloglucosidase, isoamylase, C-glucosidase, Solution having a 0.1 to 2-fold concentration (a composition C.-amylase derived from Aspergillus Oryzae, and C-amylase of an SSC solution having a 1-fold concentration is 150 mM derived from Aspergillus niger: sodium chloride and 15 mM sodium citrate). 0122 (Item 27A) A method of producing an enzyme I0129 (Item 32A) The method according to any one of treated Starch having high viscosity and gel-forming ability, Items 27A to 30A, wherein the starch hydrolase has an amino the method comprising the step of acid sequence having at least 95% or more of homology with 0123 treating starch granules of untreated starch with a an amino acid sequence of SEQID NO: 2, 4, 6, 8, 10 or 12, starch hydrolase at a temperature of 10°C. or higher and 70° and has a starch hydrolysis activity. C. or lower; 0.130 (Item 33A) An enzyme-treated starch having high 0.124 the starch hydrolase is selected from the group con Viscosity and gel-forming ability, sisting of amyloglucosidase, isoamylase, C-glucosidase, and I0131 the enzyme-treated starch is a starch obtained by C.-amylase having a characteristic capable of improving a gel treating starch granules of untreated Starch with a starch forming ability of a starch. hydrolase under the condition where the starch granules are 0.125 (Item 28A) The method according to Item 27A, not dissolved; wherein the starch hydrolase is selected from the group con I0132 the enzyme-treated starch is not modified on sisting of amyloglucosidase, isoamylase, C-glucosidase, and hydroxyl groups at the positions 2, 3 and 6 of the glucose C.-amylase derived from the genus Aspergillus. residues, 0126 (Item 29A) The method according to Item 27A or 0.133 the starch hydrolase is selected from the group con 28A, wherein the starch hydrolase is selected from the group sisting of amyloglucosidase derived from Aspergillus niger consisting of amyloglucosidase, isoamylase, C-glucosidase, commercially available from Novozyme as AMG, amyloglu C.-amylase derived from Aspergillus Oryzae, and C-amylase cosidase derived from Aspergillus niger commercially avail derived from Aspergillus niger: able from Genencor as OPTIDEXL-400, amyloglucosidase 0127 (Item 30A). The method according to any one of derived from Aspergillus niger commercially available from Items 27A to 29A, wherein the starch hydrolase is selected DANISCO as DIAZYME X4NP. amyloglucosidase derived US 2013/0022711 A1 Jan. 24, 2013 from Aspergillus niger commercially available from Amano 0.143 Since a conventional starch having a strong gel Enzyme as glucoamylase "Amano' SD, amyloglucosidase forming ability cannot sufficiently undergo Swelling and derived from Rhizopus niveus commercially available from gelatinization in a usual heating temperature Zone, powderi Amano Enzyme as Gluczyme AF6, amyloglucosidase ness is likely to be left when added to a food. In order to derived from Rhizopus Oryzae commercially available from Sufficiently Swell and gelatinize the conventional starch hav SHIN NIHON CHEMICALS Corporation as Sumizyme, ing a strong gel forming ability, heating at higher temperature C-glucosidase derived from Aspergillus niger commercially than usual heating temperature Zone of a food is required. As available from Amano Enzyme as transglucosidase L for a starch Subjected to an acid treatment and a starch having "Amano', C.-glucosidase derived from Aspergillus niger enriched in amylose fraction, they are excellent in gel form commercially available from Genencor as Transglucosidase ing ability, however they do not exhibit viscosity or hardly L-50, C-amylase derived from Aspergillus Oryzae commer exhibit viscosity, and thus the application of them has been cially available from Amano Enzyme as Biozyme A, C.-amy limited. Even in the case of such a starch Subjected to an acid lase derived from Aspergillus Oryzae commercially available treatment, the gel forming ability can be improved as com from SHIN NIHON CHEMICALS Corporation as Sum pared to the prior art by the enzymatic treatment according to izyme L. C.-amylase derived from Aspergillus niger commer the method of the present invention while maintaining a cer cially available from Danisco as AMYLEX A3, C-amylase tain degree of viscosity. derived from Aspergillus niger commercially available from 0144. Furthermore, although a chemically treated starch is SHIN NIHON CHEMICALS Corporation as Sumizyme AS, often used for a bracken-starch dumpling (Warabimochi), it is and isoamylase derived from Pseudomonas amyloderamosa necessary to use an acetylation treatment and a phosphate commercially available from Sigma as isoamylase. crosslinking treatment in combination. 0134 (Item 34A) An enzyme-treated starch having high 0145 The starch developed in this time is a starch in which Viscosity and gel-forming ability, these defects have been improved. In the case where an 0135 the enzyme-treated starch is a starch obtained by untreated Starch, a physically treated Starch, or a bleached treating starch granules of untreated Starch with a starch starch is used as a raw material, and the starch developed in hydrolase under the condition where the starch granules are this time is produced under the condition where a chemical not dissolved; treatment is not applied in any stage of the production pro 0.136 the enzyme-treated starch is not modified on cess, the addition to a usual food, or the application in a food hydroxyl groups at the positions 2, 3 and 6 of the glucose containing a starch as a main raw material is not limited, and residues, the starch can be used in all foods “dealt as a food'. 0.137 the starch hydrolase is encoded by a nucleic acid 0146 In the case where an untreated starch, a physically molecule which is capable of hybridizing under stringent treated Starch or a bleached Starch is used as a raw material conditions with a nucleic acid molecule having a complemen and the enzyme-treated Starch of the present invention is tary sequence of the base sequence of SEQID NO: 1, 3, 5, 7, produced under the condition where a chemical treatment is 9 or 11, and has a starch hydrolysis activity; wherein the not applied in any stage of the production process, the stringent conditions are hybridization in a solution containing enzyme-treated Starch of the present invention prepared by 50% formamide, 5xSSC (750 mM. NaCl, 75 mM trisodium using a starch hydrolase or a glycosyltransferase does not citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt's correspond to a processed Starch obtained by a chemical solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyr modification in a food additive. Therefore, it is possible to rolidone), 10% dextran sulfate and 20 g/ml denatured prepare a food without the addition of a food additive if the sheared salmon sperm DNA at 65°C., and subsequent wash enzyme-treated Starch of the present invention prepared by ing under the condition of 65° C. using an SSC solution using a starch hydrolase or a glycosyltransferase is used. having a 0.1 to 2-fold concentration (a composition of an SSC 0.147. In the case where an untreated starch is used as a raw solution having a 1-fold concentration is 150 mM sodium material and an enzyme-treated Starch is produced under the chloride and 15 mM sodium citrate). condition where neither a chemical treatment nor a physical 0138 (Item 35A) An enzyme-treated starch having high treatment is applied in any stage of the production process, Viscosity and gel-forming ability, since the enzyme-treated Starch used in the present invention 0139 the enzyme-treated starch is a starch obtained by has a higher gel forming ability than that of the untreated treating starch granules of untreated Starch with a starch starch and is free from a forcible bond, the starch can suffi hydrolase under the condition where the starch granules are ciently undergo gelatinization even at usual heating tempera not dissolved; ture and can exhibit viscosity. Furthermore, the obtained 0140 the enzyme-treated starch is not modified on starch paste has less spinnability regardless of being suffi hydroxyl groups at the positions 2, 3 and 6 of the glucose ciently gelatinized. The gel obtained by using a high concen residues, tration of the starch of the present invention is very rich in 0141 the starch hydrolase has an amino acid sequence elasticity. That is, in the case where the starch of the present having at least 95% or more of homology with an amino acid invention is added to a high moisture content type food, a sequence of SEQID NO: 2, 4, 6, 8, 10 or 12, and has a starch body can be imparted and also natural elasticity can be hydrolysis activity. imparted by a strong gel forming ability. On the other hand, in the case where the starch of the present invention is added to Effects of the Invention a low moisture content type food, texture with nice melt in 0142. According to the present invention, a starch “having mouth can be imparted to a food. Furthermore, there is less a strong gel forming ability and a high viscosity’ which have restriction even in the operation step due from the viewpoint never been achieved by a conventional starch has been Suc of gelatinization characteristics of them. cessfully developed by using an enzyme having characteris 0.148. Even in the case where a processed starch or a physi tics capable of improving a gel forming ability of a starch. cally treated Starch is used as a raw material or a food is US 2013/0022711 A1 Jan. 24, 2013

produced under the condition where a chemical modification plants or the like to purify a starch is not encompassed in a or a physical treatment is applied in any stage of the produc treatment of Starch granules in the present description. It is tion process of a food, a food of the present invention has a possible to use, as starch granules, any starch granules as long harder geland has different texture as compared with the case as they are usually commercially available starch granules. where a food is produced using a corresponding starch pro duced without being Subjected to an enzymatic treatment. 0153. In another specific embodiment, the starch granules Therefore, according to the present invention, it is possible to used in the present invention may be starch granules treated provide a food having texture which is different from that of by Subjecting untreated Starch granules to a chemical modi the prior art. fication or a physical treatment. Examples of the chemically modified Starch granules include an acetylated distarch adi pate, an acetylated oxidized starch, an acetylated distarch MODE FOR CARRYING OUT THE INVENTION phosphate, a starch sodium octenyl Succinate, a starch 0149 Hereinafter, the present invention will be described acetate, an oxidized Starch, a bleached starch, a hydroxypro in detail. pyl distarch phosphate, a hydroxypropyl Starch, a distarch 0150 (1. Materials) phosphate, a monostarch phosphate, and a phosphated dis tarch phosphate. The “acetylated distarch adipate” refers to (1.1 Starch Granules) those obtained by esterifying a starch with acetic anhydride and adipic anhydride. The “acetylated oxidized starch” refers 0151. In the present description, the term “starch gran to those obtained by treating a starch with sodium hypochlo ules' refers to starch molecules in a crystalline state. The rite and then esterifying it with acetic anhydride. The “acety starch granules may be untreated Starch granules, or may be lated distarch phosphate” refers to those obtained by esteri starch granules obtained by a chemical modification or a fying a starch with sodium trimetaphosphate or phosphorus physical treatment of untreated Starch granules. In the case oxychloride and acetic anhydride or vinyl acetate. The where an enzyme-treated Starch classified as a food is pref “starch sodium octenyl succinate” refers to those obtained by erably used, starch granules to be used are untreated Starch esterifying a starch with octenyl Succinic anhydride. The granules obtained from plants. Plants store starch molecules “starch acetate' refers to those obtained by esterifying a as granules (i.e., as a large crystal) in amyloplasts. The gran starch with acetic anhydride or vinyl acetate. The “oxidized ules are called Starch granules. In the starch granules, starch starch” refers to those obtained by treating a starch with molecules are mutually bonded through a hydrogen bond or Sodium hypochlorite, wherein the content of carboxyl groups the like. Therefore, starch granules are not easily dissolved in is 1.1% or less when carboxyl groups (also referred to as water as they are, and are not also easily digested. When the carboxyl groups) in a sample starch are analyzed in accor starch granules are heated together with water, they are Swol dance with the method for the purity test described in Minis len and molecules are disentangled to form a colloid. This try of Health and Welfare Notification No. 485. Provided that, change is called 'gelatinization'. The size and shape of the even when the amount of a carboxyl group is within the above starch granules vary depending on plants from which the range, the "bleached starch' is not included in the definition starch granules are obtained. For example, an average granule of the “oxidized starch'. The "bleached starch” refers to those size of corn starch granules (corn Starch) is from about 12 um obtained by treating a starch with sodium hypochlorite, to about 15um and is slightly smaller, and the size is relatively wherein the content of carboxyl groups is 0.1% or less when uniform, than that of other starch granules. Starch granules of carboxyl groups in a sample starch are analyzed in accor wheat and barley are classified into two kinds in size: large dance with the method for the purity test described in Minis sized starch granules having a granule size of about 20 um to try of Health and Welfare Notification No. 485, and wherein about 40 um, and Small-sized starch granules having a gran the test results of “Confirmation test (3) of the oxidized ule size of several um. Rice has a compound starch granule starch described in Ministry of Health and Welfare Notifica structure in which many Small angular starch granules having tion No. 485 are negative and wherein it can be reasonably a diameter of several um are accumulated in amyloplast. The explained that a change in properties, such as Viscosity, of the average granule size of potato starch granules is about 40 um starch is not caused by oxidation. Those in which, even if the and is the largest among those which are commonly used as a amount of carboxyl groups is 0.1% or less, properties such as starch raw material. In the present invention, commercially viscosity of the starch change from those of the native starch available various starch granules can be used. Starch granules are classified as the oxidized starch, and are not dealt as a food may be prepared by the method of, for example, purifying in Japan but dealt as food additives. The “hydroxypropyl starch granules from plants and used in the present invention. distarch phosphate” refers to those obtained by esterifying a 0152 Ina state of starch granules, the enzyme hardly acts starch with sodium trimetaphosphate orphosphorus oxychlo on starch granules since starch molecules are strongly bonded ride and etherifying it with propylene oxide. The “hydrox to each other. In a specific embodiment for obtaining an ypropyl starch” refers to those obtained by etherifying a enzyme-treated starch to be treated as a food, the starch starch with propylene oxide. The “distarch phosphate” refers granules used in the present invention are isolated or purified to those obtained by esterifying a starch with sodium trimeta from plants, but are not subjected to an acid treatment, a phosphate or phosphorus oxychloride. The "monostarch chemical modification treatment and a heat treatment. In the phosphate” refers to those obtained by esterifying a starch present description, the term “untreated starch granules refer with orthophosphoric acid, a potassium salt or a sodium salt to starch granules which are naturally produced and are not thereof, or sodium tripolyphosphate. The “phosphated dis Subjected to a treatment other than treatments required to tarch phosphate” refers to those obtained by esterifying a separate starch granules from other components (for starch with orthophosphoric acid, a potassium salt or a example, protein and lipid) coexisting in a natural state. Sodium salt thereof, or sodium tripolyphosphate, and esteri Accordingly, the respective steps in the method of preparing fying it with sodium trimetaphosphate or phosphorus oxy starch granules, such as the step of removing impurities from chloride. US 2013/0022711 A1 Jan. 24, 2013

0154 Examples of the types of the physically treated the starch paste lower than those of the gel formed by using starch granules include a heat-moisture-treated Starch and a the untreated Starch granules. For example, commonly, the thermally inhibited starch. thermal inhibition treatment often makes the gel formed by 0155 The starch granules used in the present invention using the obtained Starch granules harder than the gel formed may be eithera aboveground starch or a underground starch. by using the untreated Starch granules. Also, when the time of Examples of the underground starch include a cassava starch, the dry heat treatment is long, the obtained starch often exhib a potato starch, a Sweet potato starch, and a kudzu Starch. its low viscosity of the starch paste like a highly crosslinked Examples of the aboveground starch include a wheat starch, a starch. corn Starch (for example, a high amylose corn starch, a usual 0159. It is preferred that the starch granules used in the corn starch, and a waxy corn starch), a rice starch (for present invention contain impurities as low as possible. The example, a glutinous rice starch and a nonglutinous rice content of impurities in the starch granules is preferably about starch), a bean starch (for example, a green gram starch, a pea 10% by weight or less, more preferably about 5% by weight starch, an adzuki bean starch, and a fava bean starch), and an or less, and still more preferably about 1% by weight or less. Amaranthus starch. The starch granules used in the present invention are preferably starches derived from cassava, corn, (1.2 Enzyme) or wheat. In the case where the untreated starch is used as the 0160 The enzyme usable in the present invention is a starch granules, an untreated cassava starch, an untreated corn starch hydrolase or a glycosyltransferase. The starch hydro starch or an untreated wheat starch is preferably used. In the lase is roughly classified into C.-amylase, B-amylase, amylo case where the chemically modified starch is used as the glucosidase, isoamylase, pullulanase, and C-glucosidase. starch granules, it is preferred to use an acetylated distarch However, even in the enzymes classified as the same enzyme adipate, an acetylated oxidized Starch, an acetylated distarch (for example, C.-amylase), if the microorganisms producing phosphate, a starch sodium octenyl Succinate, a starch the enzyme are different, it is considered that features such as acetate, an oxidized Starch, a bleached starch, a hydroxypro reaction specificity and Substrate specificity of the enzymes pyl distarch phosphate, a hydroxypropyl Starch, a distarch are different. Since these starch hydrolases and glycosyltrans phosphate, a monostarch phosphate or a phosphated distarch ferase are very widely distributed in animals, microorganisms phosphate of a cassava starch, a corn starch or a wheat starch. and plants, it can be said that there are infinite kinds of starch In the case where the physically treated starch is used, it is hydrolases and glycosyltransferases. preferred to use a heat-moisture-treated starch or a thermally 0.161 The starch hydrolase usable in the production of the inhibited Starch of a cassava starch, a corn Starch or a wheat starch of the present invention is a starch hydrolase selected starch. from the group consisting of amyloglucosidase, isoamylase, 0156 Since the structure of the starch delicately varies C-glucosidase, and C.-amylase having characteristics capable depending on the origin, features of physical properties vary of improving a gel forming ability of a starch. In the present depending on the origin. For example, although the untreated description, the “C.-amylase having characteristics capable of wheat starch has a high gel forming ability, the starch paste improving a gel forming ability of a starch' is C.-amylase thereof has a low viscosity and the starch paste is opaque. wherein the Young's modulus or rupture stress of the starch Although the untreated cassava starch has a low gel forming after the treatment with the enzyme is 10% or more higher ability, the starch paste thereof has a high viscosity and the than the Young's modulus or rupture stress of the starch starch paste has high transparency and the degree of retrogra before the treatment with the enzyme, when measured by the dation is a middle degree. Particularly, although the untreated judgment method described below. The starch hydrolase used cassava starch is inexpensive, the starch paste thereof is trans in the present invention is preferably an enzyme classified as parent, and it therefore has a merit of being easily added, the C.-amylase, amyloglucosidase, isoamylase, or C-glucosidase. application thereof is limited because of their low gel forming The enzyme classified as B-amylase or pullulanase is not ability. Furthermore, the untreated native wheat starch could preferable. It is considered that the enzyme classified as amy not be used in the application where viscosity is required loglucosidase, isoamylase or C-glucosidase can produce an because of a low viscosity of the starch paste. Although the enzyme-treated Starch having a high viscosity and a gel form untreated corn starch has a high gel forming ability, the starch ing ability if these enzymes are allowed to act on starch paste thereofhas slightly low viscosity, and the starch paste is granules. However, in the case of the enzyme classified as opaque and has high retrogradation property. C.-amylase, not all enzymes can be suitably utilized and 0157. The chemical modification alters physical proper C.-amylase having characteristics capable of improving a gel ties of the untreated Starch granules. Commonly, crosslinking forming ability of a starch needs to be selected, and the starch Such as phosphate crosslinking or adipate crosslinking often of the present invention cannot be produced evenifan C.-amy makes the gel formed by using the obtained starch granules lase not having this activity is used. harder and higher turbidity than the gel formed by using the 0162. It is possible to judge whether or not the enzyme untreated Starch granules. Generally, hydroxypropylation, classified as C.-amylase is C.-amylase having characteristics acetylation and oxidation treatments often improve transpar capable of improving a gel forming ability of a starch, by the ency of and make softer the gel formed by using the obtained following judgment method. starch granules as compared with the gel formed by using the 0163 Examples of the glycosyltransferase usable in the untreated Starch granules. Commonly, the treatment with production of the starch of the present invention include octenyl Succinic acid can make it possible for the gel formed cyclodextrin glucanotransferase. using the obtained starch granules to contain oil. 0164 (1.2.1 Method of Judging C.-Amylase Having Char 0158. The physical treatment also alters physical proper acteristics Capable of Improving Gel Forming Ability of ties of the untreated Starch granules. For example, commonly, Starch) the heat-moisture treatment often makes the gel formed by 0.165. The C.-amylase having characteristics capable of using the obtained starch granule harder and the Viscosity of improving a gel forming ability of a starch can be judged by US 2013/0022711 A1 Jan. 24, 2013

the following method. To 400 g of a wheat starch, 900 g of measurement, the hardness of the starch gel is evaluated by a ion-exchange water is added thereby Suspending the wheat rupture stress (g) and a Young's modulus (dyn/cm). starch, and each enzyme is added thereto. The amount of a reducing Sugar released in the Suspension by the reaction is (0169 (1.2.3: Preferred Example Used in Present Applica measured to determine a degradation ratio. When the degra tion) dation ratio reaches 15%, starch granules are recovered by 0170 In order to produce the starch of the invention, an filtration, washed with water and then dried. Using the enzyme selected from the group consisting of amyloglucosi enzyme-treated Starch thus obtained, a Young's modulus and dase, isoamylase, C-glucosidase, C.-amylase having charac a rupture stress are determined by rheometer analysis. In the teristics capable of improving a gel forming ability of a case where theYoung's modulus or rupture stress of the starch starch, and cyclodextrin glucanotransferase is used. after treatment with the enzyme increases by 10% or more as compared with the Young's modulus or rupture stress of the 0171 In a specific embodiment, the enzyme is selected starch before treatment with the enzyme, the enzyme is from the group consisting of amyloglucosidase, isoamylase, judged as C.-amylase having characteristics capable of C-glucosidase, C.-amylase derived from the genus Aspergil improving a gel forming ability of the starch. As an example, lus, and cyclodextrin glucanotransferase. the judgment results of various starch hydrolases are shown in 0172. In a specific embodiment, the enzyme is selected Table 1A below. from the group consisting of amyloglucosidase, isoamylase, TABLE 1A Rupture stress Young's modulus Name of Measured Measured enzyme Product name value Relative value Relative group Origin (Selling agency) (g) 0: 1 (dyn/cm) %** Judgment Before 141 100 4,601,665 100 Reference treatinent with enzyme C-amylase Aspergilius oryzae Biozyme A 197 140 5,518,329 120 Usable (Amano Enzyme) C-amylase Aspergilius niger AMYLEXA3 211 150 5,465,779 119 Usable (DANISCO) C-amylase Bacilius subtilis Novamyl (Novo) Not measurable since gel is not formed Not usable because of being too soft C-amylase Bacilius C-amylase Not measurable since gel is not formed Not usable amyloiquefaciens (Reagent) (Sigma) because of being too soft C-amylase Bacilius sp. Maltogenase L. Not measurable since gel is not formed Not usable (Novo) because of being too soft C-amylase Bacilius Termamyl 120L Not measurable since gel is not formed Not usable licheniformis (Novo) because of being too soft *Relative rupture stress = {(rupture stress for after treatment with enzyme)/(rupture stress for before treatment with enzyme)} x 100 *Relative Young's modulus = {(Young's modulus for after treatment with enzyme)/(Young's modulus for before treatment with enzyme)) x 100

0166 As described above, it is possible to easily decide C-glucosidase, C.-amylase derived from Aspergillus Oryzae, whether or not various C.-amylases have characteristics C.-amylase derived from Aspergillus niger, and cyclodextrin capable of improving a gel forming ability of a starch. It is glucanotransferase. noted that a specific method of rheometer analysis is as 0173. In a preferred embodiment, the enzyme is selected described in 1.2.2 below. from the group consisting of amyloglucosidase derived from Aspergillus niger commercially available from Novozyme as (0167 (1.2.2 Specific Method of Rheometer Analysis) AMG, amyloglucosidase derived from Aspergillus niger 0168 A starch paste is prepared so that the concentration commercially available from Genencor as OPTIDEXL-400, of the starch is 20% by weight on the dry matter basis, and amyloglucosidase derived from Aspergillus niger commer then filled in a Krehalon casing having a folding width of 45 cially available from DANISCO as DIAZYMEX4NP amy mm. This starch paste filled in the casing is heated to 90° C. at loglucosidase derived from Aspergillus niger commercially 1° C./min and maintained at 90° C. for 30 minutes. Then the available from Amano Enzyme as glucoamylase "Amano' starch paste is left to cool in a constant-temperature water SD, amyloglucosidase derived from Rhizopus niveus com bath at 20° C. for 30 minutes and then cooled to 5° C. in a mercially available from Amano Enzyme as Gluczyme AF6, refrigerator. After cooling, it is refrigeration stored at 5°C. for amyloglucosidase derived from Rhizopus Oryzae commer 16 hours, then it is left at room temperature (about 25°C.) for cially available from SHIN NIHON CHEMICALS Corpora 4 hours to return the temperature of it to room temperature, tion as Sumizyme, C-glucosidase derived from Aspergillus and then measurements by a rheometer (RT-2010J-CW) niger commercially available from Amano Enzyme as trans manufactured by Rheotech Inc. is performed. The measure glucosidase L Amano'. C-glucosidase derived from ment is carried out under the measurement conditions of the Aspergillus niger commercially available from Genencor as rheometer: a test item: a rupture test; a height of a sample: 25 Transglucosidase L-50, C.-amylase derived from Aspergillus mm, and a movement rate (rupture rate) of a sample: 6 Oryzae commercially available from Amano Enzyme as cm/min, using an adapter of a spherical jig for measurement Biozyme A, C.-amylase derived from Aspergillus Oryzae com viscosity (25 (diameter: 5 mm, area: 19.635 mm). At the mercially available from SHIN NIHON CHEMICALS Cor US 2013/0022711 A1 Jan. 24, 2013 poration as Sumizyme L, C.-amylase derived from Aspergil 0179 An O-amylase used in the present invention is pref lus niger commercially available from Danisco as AMYLEX erably an O-amylase from genus Aspergillus, and most pref A3, C.-amylase derived from Aspergillus niger commercially erably an O-amylase derived from Aspergillus Oryzae or available from SHIN NIHON CHEMICALS Corporation as Aspergillus niger. Sumizyme AS, isoamylase derived from Pseudomonas amy 0180 A nucleotide sequence encoding typical C.-amylase loderamosa commercially available from Sigma as isoamy derived from Aspergillus Oryzae is shown in SEQID NO: 1. lase, cyclodextrin glucanotransferase derived from Bacillus and its amino acid sequence is shown in SEQ ID NO: 2. A licheniformis commercially available from Novozyme as nucleotide sequence encoding typical C.-amylase derived ToruZyme, and cyclodextrin glucanotransferase derived from from Aspergillus niger is shown in SEQ ID NO: 3, and its Paenibacillus macerans (Bacillus macerans) commercially amino acid sequence is shown in SEQID NO: 4. It is consid available from Amano Enzyme as Cyclodextrin glucan ered that C.-amylases of closely related species have a very otransferase “Amano'. high homology and exhibit the similar enzyme activities. 0.174. In a specific preferred embodiment, the enzyme is a Therefore, it is considered that C.-amylases derived from starch hydrolase, and the starch hydrolase is encoded by a Aspergillus Oryzae have amino acid sequences having a very nucleic acid molecule which is capable of hybridizing under high homology to SEQ ID NO: 2 and exhibit the similar stringent conditions with a nucleic acid molecule having a enzyme activities. Since it is shown that a commercially complementary sequence to the base sequence of SEQ ID available C-amylase derived from Aspergillus Oryzae has NO: 1, 3, 5, 7, 9 or 11 and has a starch hydrolysis activity; characteristics capable of improving a gel forming ability of wherein the stringent conditions are hybridization in a solu a starch, it is considered that C.-amylase having an amino acid tion containing 50% formamide, 5xSSC (750 mM NaCl, 75 sequence of SEQID NO: 2 and C.-amylase having an amino mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), acid sequence which has a high homology thereto also have 5xDenhardt’s solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% characteristics capable of improving a gel forming ability of polyvinylpyrrolidone), 10% dextran sulfate and 20 ug/ml a starch. Similarly, since it is shown that a commercially denatured sheared salmon sperm DNA at 65° C., and subse available C.-amylase derived from Aspergillus niger has char quent washing under the condition of 65° C. using an SSC acteristics capable of improving a gel forming ability of a Solution having a 0.1 to 2-fold concentration (a composition starch, it is considered that C.-amylase having an amino acid of an SSC solution having a 1-fold concentration is 150 mM sequence of SEQID NO: 2 and C.-amylase having an amino sodium chloride and 15 mM sodium citrate). acid sequence which have a high homology thereto also have characteristics capable of improving a gel forming ability of (0175. In a preferred embodiment, the starch hydrolase has a starch. an amino acid sequence having at least 95% or more of 0181. The C-amylase used in the present invention is not homology with an amino acid sequence of SEQID NO: 2, 4, an amylase derived from Bacillus amyloliquefaciens. The 6, 8, 10 or 12, and has a starch hydrolysis activity reason is that the amylase derived from Bacillus amylolique 0176 (1.2.4. C.-Amylase) faciens cannot produce a starch having a high viscosity and a 0177 C-Amylases existin many microorganisms, animals gel forming ability. and plants. Examples of microorganisms which produce an 0182. A lot of C.-amylase is commercially available. C.-amylase include those of genus Aspergillus (for example, Examples of the commercially available O.-amylase are Aspergillus Oryzae, Aspergillus niger, Aspergillus awamori, described below: Biozyme F1OSD (origin: Aspergillus Aspergillus flavus, Aspergillus kawachii, Aspergillus sclero Oryzae, Amano Enzyme Inc.), Biozyme A (origin: Aspergil tiorum and the like); those of genus Bacillus (for example, lus Oryzae, Amano Enzyme Inc.), Kokulase (origin: Aspergil Bacillus subtilis, Bacillus acidocaldarius, Bacillus amy lus Oryzae; Mitsubishi-Kagaku Foods Corporation), Sum loliquefaciens, Bacillus Stearothermophilus, Bacillus cereus, izyme L (origin: Aspergillus Oryzae; SHIN NIHON Bacillus licheniformis and the like); those of genus Geoba CHEMICALS Corporation), AMYLEX A3 (origin: cillus (for example, Geobacillus Stearothermophilus, Geoba Aspergillus niger; Danisco Japan Ltd.), GRINDAMYLA cillus thermodenitrificans, Geobacillus thermodenitrificans (origin: Aspergillus Oryzae; Danisco Japan Ltd.), VERON and the like); those of genus Lactobacillus (for example, AX (origin: Aspergillus oryzae: HIGUCHI INC.), VERON Lactobacillus amylovorus, Lactobacillus cellobioses, Lacto GX (origin: Aspergillus oryzae: HIGUCHI INC.), VERON bacillus manihotivorans and the like); further more, M4 (origin: Aspergillus oryzae: HIGUCHI INC.), VERON Pseudomonas sp., Pyrococcus furiosus, Rhizopus ELS (origin: Aspergillus Oryzae; HIGUCHI INC.), Sum microsporus, Thermotoga maritima, Vibrio sp. and the like. izyme AS (origin: Aspergillus niger; SHIN NIHONCHEMI Furthermore, it is confirmed that the C.-amylase derived from CALS Corporation), Bakezyme P500 (origin: Aspergillus animals exist in human pancreas, human saliva, human urine, Oryzae; Nihon Siber Hegner K.K.), and C.-Amylase (origin: porcine pancreas, bovine pancreas, carp intestinal tract and Aspergillus Oryzae, Sigma-Aldrich Corporation). the like, and that the C-amylase derived from plants exist in 0183 Such commercially available C.-amylase is sub barley, rice, wheat, oat, rye, soybean, and fava bean. The jected to amino acid analysis to determine the amino acid organisms that produce an O-amylase are not limited to them. sequence thereof, and a DNA sequence is designed based on 0.178 C.-Amylase may be commercially available one or the amino acid sequence, and then the DNA sequence is may be prepared from these organisms by a method known in introduced into E. coli or the like, and thus C.-amylase having the art, or may be prepared by a genetic recombination the same amino acid sequence as that of the commercially method based on an amino acid sequence or a base sequence available O-amylase can be produced. of C-amylase of these organisms, or may be chemically Syn 0.184 (1.2.5 Amyloglucosidase) thesized. Any O-amylase known in the art can be used as long 0185. Amyloglucosidase refers to an enzyme capable of as it has properties of cleaving an O-1,4-glucoside bond in the producing B-D-glucose by hydrolyzing a 1.4-O bond at a end type. non-reducing terminal of a carbohydrate chain of a starch or US 2013/0022711 A1 Jan. 24, 2013 the like. The amyloglucosidase hydrolyzes an O-1,4-gluco Candidatsukubaensis cannot produce a starch having a high side chain from a non-reducing terminal, and also an O-1,6- Viscosity and a gel forming ability. glucoside chain, although the degradation rate is low. A sys 0191) A lot of amyloglucosidase is commercially avail tematic name of the amyloglucosidase is glucan 1,4-O- able. Examples of the commercially available amyloglucosi glucosidase. Another name of the amyloglucosidase is exo dase are described below: GlucS G (origin: Rhizopus niveus; 1,4-O-D-glucosidase, 1,4-O-D-glucan glucohydrolase, Amano Enzyme Inc.), Gluczyme AF6 (origin: Rhizopus glucoamylase, Y-amylase, lysosomal C-glucosidase, or acidic niveus; Amano Enzyme Inc.), Gluczyme NL4.2 (origin: maltase. The amyloglucosidase is classified as EC 3.2.1.3. Aspergillus niger, Amano Enzyme Inc.), Brewing glucoamy 0186 Amyloglucosidases exist in many microorganisms, lase "Amano' SD (origin: Aspergillus niger; Amano Enzyme animals and plants. Examples of microorganisms which pro Inc.), GODO-ANGH (origin: Aspergillus niger; GODO duce an amyloglucosidase include those of genus Aspergillus SHUSEICO., LTD.), OPTIDEXL-400 (origin: Aspergillus (for example, Aspergillus niger; Aspergillus Oryzae, Aspergil niger; Genencor Kyowa), OPTIDEXL (origin: Aspergillus lus candidus, Aspergillus terreus, Aspergillus awamori, niger; Genencor Kyowa), Sumizyme (origin: Rhizopus Aspergillus phoenicis, Aspergillus Saitoi and the like); those oryzae; SHINNIHON CHEMICALS Corporation), Sum of genus Candida (for example, Candida antarctica, Candida izyme SG (origin: Rhizopus sp.: SHIN NIHON CHEMI tsukubaensis and the like); those of genus Rhizopus (for CALS Corporation), Sumizyme HG (origin: Rhizopus example, Rhizopus deleimar; Rhizopus delmar, Rhizopus jav oryzae; SHIN NIHON CHEMICALS Corporation), GLU anicus, Rhizopusniveus, Rhizopusniveus, Rhizopus Oli COZYME #20000 (origin: Rhizopus sp.: Nagase Chemtex gosporus, Rhizopus Oryzae and the like); those of genus Sac Corporation). AMG (origin: Aspergillus niger; Novozymes charomyces (for example, Saccharomyces cerevisiae, Japan Ltd.), GLUTASEAN (origin: Aspergillus niger; HBI Saccharomyces diastaticus, Saccharomyces diastaticus, Sac Enzymes Ltd.), UNIASE K, 2K (origin: Rhizopus sp.: charomyces fibuligera); further more, Clostridium ther YAKULT PHARMACEUTICAL INDUSTRY CO., LTD.), moamylolyticum, Cladosporium resinae, Lentinus edodes, UNIASE 30 (origin: Rhizopus sp.: YAKULT PHARMA Mucor rouxianus, Magnaporthe grisea, Monascus kaoliang, CEUTICAL INDUSTRY CO.,LTD.), UNIASE 60F (origin: Paecilomyces varioti, Penicillium oxalicum, Thermomyces Rhizopus sp.:YAKULT PHARMACEUTICAL INDUSTRY lanuginosus, Trichoderma reesei and the like. Furthermore, it CO., LTD.), MAGNUX JW-201 (origin: Rhizopus sp.: is confirmed that an amyloglucosidase derived from animals Rakuto Kasei Industrial Co., Ltd.), GRINDAMYL AG (ori exists in mucosa membrane of Small intestine of human, rat gin Aspergillus sp.; Danisco Japan Ltd.), DIAZYME X4NP and mice, and that an amyloglucosidase derived from plants (origin: Aspergillus niger, Danisco Japan Ltd.), Bakezyme exists in beet and the like. The organisms that produce an AG800 (origin: Aspergillus niger; Nihon Siber Hegner K.K.), amyloglucosidase are not limited to them. Amyloglucosidase (origin: Aspergillus niger, Sigma-Aldrich 0187 Amyloglucosidase may be commercially available Corporation), Amyloglucosidase (origin: Rhizopus sp.: one or may be prepared from these organisms by a method Sigma-Aldrich Corporation), and Glucoamylase (origin: known in the art, or may be prepared by a genetic recombi Rhizopus sp., Toyobo Co., Ltd.). nation method based on an amino acid sequence or a base 0.192 Such commercially available amyloglucosidase is sequence of amyloglucosidase of these organisms, or may be Subjected to amino acid analysis to determine the amino acid chemically synthesized. Any amyloglucosidase known in the sequence thereof, and a DNA sequence is designed based on art can be used as long as it has properties of cleaving an the amino acid sequence, and then the DNA sequence is C-1,4-glucoside bond and an O-1,6-glucoside bond in an exo introduced into E. coli or the like, and thus amyloglucosidase type from a non-reducing terminal side in a glucose unit to having the same amino acid sequence as that of the commer produce B-glucose. cially available amyloglucosidase can be produced. 0188 An amyloglucosidase used in the present invention (0193 (1.2.6 Isoamylase) is preferably an amyloglucosidase from genus Aspergillus or 0194 Isoamylase refers to an enzyme which cleaves an an amyloglucosidase from genus Rizopus, and most prefer C-1,6-glucoside bond of at a branched point of amylopectin, ably an amyloglucosidase derived from Aspergillus niger or glycogen, or the like to produce amylose-like linear polysac an amyloglucosidase derived from Rizopus niveus. charides. Another name of the isoamylase is glycogen 6-glu 0189 A nucleotide sequence encoding typical amyloglu canohydrolase. The isoamylase is classified as EC3.2.1.68. cosidase derived from Aspergillus niger is shown in SEQID The isoamylase can be derived from any organism capable of NO: 5, and its amino acid sequence is shown in SEQID NO: producing isoamylase. 6. It is considered that amyloglucosidase of closely related 0.195 Isoamylases existin many microorganisms, animals species have a very high homology and exhibit the similar and plants. Examples of microorganisms which produce an enzyme activities. Therefore, it is considered that amyloglu isoamylase include Flavobacterium sp.: Bacillus sp.: further cosidase derived from Aspergillus niger have amino acid more, Pseudomonas amyloderamosa, Sulfolobus solfatari sequences having a very high homology to SEQID NO: 6 and cus and the like. Furthermore, it is confirmed that an isoamy exhibit the similar enzyme activities. Since it is shown that a lase derived from animals exists in human pancreas and the commercially available amyloglucosidase derived from like, and that an isoamylase derived from plants exists in Aspergillus niger has starch hydrolysis activity, it is consid Oryza sativa, potato (Solanum tuberosum) tuber, Arabidopsis ered that amyloglucosidase having an amino acid sequence of thaliana and the like. The organisms that produce an isoamy SEQID NO: 6 and amyloglucosidase having an amino acid lase are not limited to them. sequence which has a high homology thereto also have starch 0196) Isoamylase may be commercially available or may hydrolysis activity. be prepared from these organisms by a method known in the 0190. The amyloglucosidase used in the present invention art, or may be prepared by a genetic recombination method is not an amyloglucosidase derived from Candida tsukubaen based on an amino acid sequence or a base sequence of sis. The reason is that the amyloglucosidase derived from isoamylase of these organisms, or may be chemically synthe US 2013/0022711 A1 Jan. 24, 2013 sized. Any isoamylase known in the art can be used as long as firiosus, Pyrococcus woesei and the like), those of genus it has properties of cleaving an O-1,6-glucoside bond of amy Saccharomyces (Saccharomyces Carlsbergensis, Saccharo lopectin in the end type. myces cerevisiae, Saccharomyces fibuligera, Saccharomyces 0.197 An isoamylase used in the present invention is pref Oviformis, Saccharomyces Carlsbergensis, Saccharomyces erably an isoamylase from genus Flavobacterium or genus logos and the like); furthermore, Candida tropicalis, Pseudomonas, and more preferably an isoamylase derived Schizosaccharomyces pombe, Sulfolobus solfataricus, Ther from Flavobacterium sp. or an isoamylase derived from motoga maritima, Escherichia coli and the like. It is con Pseudomonas amyloderamosa. firmed that the C-glucosidase derived from animals widely 0198 A nucleotide sequence encoding typical isoamylase exist within a range from invertebrate animals such as mol derived from Flavobacterium sp. is shown in SEQID NO: 7. lusks, crustaceans, and insects to vertebrate animals such as and its amino acid sequence is shown in SEQID NO: 8. A fishes, amphibians, reptiles, birds, and mammalians, and the nucleotide sequence encoding typical isoamylase derived C-glucosidase derived from plants exist in beans, rice, buck from Pseudomonas amyloderamosa is shown in SEQID NO: wheat, corn, beet seeds and the like. It is noted that organisms 9, and its amino acid sequence is shown in SEQID NO: 10. It capable of producing O-glucosidase are not limited to them. is considered that isoamylase of closely related species have 0204 C-Glucosidase may be commercially available one a very high homology and exhibit the similar enzyme activi or may be prepared from these organisms by a method known ties. Therefore, it is considered that isoamylase derived from in the art, or may be prepared by a genetic recombination Flavobacterium sp. have amino acid sequences having a very method based on an amino acid sequence or a base sequence high homology to SEQ ID NO: 8 and exhibit the similar of C-glucosidase of these organisms, or may be chemically enzyme activities. Since it is shown that a commercially synthesized. Any O-glucosidase known in the art can be used available isoamylase derived from Flavobacterium sp. has as long as it has properties of cleaving an O-1,4-glucoside starch hydrolysis activity, it is considered that isoamylase bond and an C-1,6-glucoside bond in an exo type from a having an amino acid sequence of SEQID NO: 8 and isoamy non-reducing terminal side in a glucose unit to produce lase having an amino acid sequence which has a high homol C-glucose. ogy thereto also have starch hydrolysis activity. Similarly, 0205 An O-glucosidase used in the present invention is since it is shown that a commercially available isoamylase preferably an O-glucosidase from genus Aspergillus, and derived from Pseudomonas amyloderamosa has starch more preferably an O-glucosidase derived from Aspergillus hydrolysis activity, it is considered that isoamylase having an niger: amino acid sequence of SEQ ID NO: 10 and isoamylase 0206. A nucleotide sequence encoding typical C-glucosi having an amino acid sequence which has a high homology dase derived from Aspergillus niger is shown in SEQID NO: thereto also have starch hydrolysis activity. 11, and its amino acid sequence is shown in SEQID NO: 12. 0199. A lot of isoamylase is commercially available. It is considered that C-glucosidase of closely related species Examples of the commercially available isoamylase are have a very high homology and exhibit the similar enzyme described below: GODO-FIA (origin: Flavobacterium Odo activities. Therefore, it is considered that C-glucosidase ratum: GODO SHUSEICO.,LTD.), and Isoamylase (origin: derived from Aspergillus niger have amino acid sequences Pseudomonas sp.: Sigma-Aldrich Corporation). having a very high homology to SEQID NO: 12 and exhibit 0200. Such commercially available isoamylase is sub the similar enzyme activities. Since it is shown that a com jected to amino acid analysis to determine an amino acid mercially available C-glucosidase derived from Aspergillus sequence thereof, and a DNA sequence is designed based on niger has starch hydrolysis activity, it is considered that the amino acid sequence, and then the DNA sequence is C-glucosidase having anamino acid sequence of SEQID NO: introduced into E. coli or the like, and thus isoamylase having 12 and C-glucosidase having an amino acid sequence which the same amino acid sequence as that of the commercially has a high homology thereto also have starch hydrolysis activ available isoamylase can be produced. ity. 0201 (1.2.7 C.-Glucosidase) 0207. A lot of C-glucosidase is commercially available. 0202 C.-Glucosidase refers to an enzyme which hydro Examples of the commercially available C-glucosidase are lyzes an O-1,4-glucoside bond at a non-reducing terminal to described below: Transglucosidase L 500 (origin: Aspergil produce C-glucose. Systematic name of the C-glucosidase is lus; Genencor Kyowa), Transglucosidase L'Amano' (origin: C-D-glucoside glucohydrolase. Another name of the C-glu Aspergillus niger, Amano Enzyme Inc.), O-Glucosidase (ori cosidase is maltase, glucoinvertase, or glucoside Sucrase. The gin: Bacillus Stearothermophilus; Sigma-Aldrich Corpora C-D-glucosidase is classified as EC 3.2.1.20. tion), O-Glucosidase (origin: rice; Sigma-Aldrich Corpora 0203 O-Glucosidases exist in many microorganisms, ani tion), O-Glucosidase (origin: Saccharomyces cerevisiae: mals and plants. Examples of microorganisms which produce Sigma-Aldrich Corporation), O-Glucosidase (origin: an C-glucosidase include those of genus Aspergillus (for Aspergillus niger, Sigma-Aldrich Corporation), and O-Glu example, Aspergillus Oryzae, Aspergillus niger, Aspergillus cosidase (origin: Microorganism; Toyobo Co., Ltd.). awamori, Aspergillus fumigatus, Aspergillus nidulans and 0208 Such commercially available C-glucosidase is sub the like); those of genus Bacillus (for example, Bacillus amy jected to amino acid analysis to determine the amino acid loliquefaciens, Bacillus amylolyticus, Bacillus caldovelox, sequence thereof, and a DNA sequence is designed based on Bacillus cereus, Bacillus licheniformis, Bacillus thermoglu the amino acid sequence, and then the DNA sequence is cosidius, Bacillus sp., Bacillus subtilis, Bacillus brevis, introduced into E. coli or the like, and thus C-glucosidase Bacillus Stearothermophilus; those of genus Lactobacillus having the same amino acid sequence as that of the commer (Lactobacillus acidophilus, Lactobacillus brevis and the cially available C-glucosidase can be produced. like); those of genus Penicillium (Penicillium brevicompac 0209 (1.2.8 Cyclodextrin Glucanotransferase) tum, Penicilliumcitrinum, PenicilliumOxalicum, Penicillium 0210 Cyclodextrin glucanotransferase is also called purpurogenium); those of genus Pyrococcus (Pyrococcus CGTase and is classified as EC2.4.1.19. CGTase is an enzyme US 2013/0022711 A1 Jan. 24, 2013

capable of catalyzing a transglycosylation reaction (i.e., dis cation, or the amino acid sequence of SEQID NO: 2, SEQID proportionation reaction) of maltooligosaccharide. CGTase NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, or is an enzyme which performs a transfer reaction so as to SEQID NO: 12; or this amino acid sequence may, in another recognize 6 to 8 glucose-chain at a non-reducing terminal of embodiment, be altered in up to a certain number of amino donor molecules thereby cyclizing this portion to produce acids compared with a reference amino acid sequence. Such cyclodextrin having a degree of polymerization of 6 to 8 and alterations can be selected from the group consisting of a noncyclic limit dextrin. As an example of CGTase usable in deletion, a Substitution (including conservative and non-con the present invention, CGTase derived from well-known servative substitution), or an insertion of at least 1 (preferably microorganisms or a commercially available CGTase can be 1 or several; there is no specific upper limit, for example, used. CGTase is preferably selected from the group consist about 50 or less, about 40 or less, about 30 or less, about 20 or ing of cyclodextrin glucanotransferase derived from Bacillus less, about 10 or less, or the like) amino acids. This alteration licheniformis commercially available from Novozyme as may occur at a position of an amino terminus or a carboxyl ToruZyme, and cyclodextrin glucanotransferase (optimum terminus of a reference amino acid sequence, or may occurat pH 6.0) derived from Paenibacillus macerans (also classified any position other than these termini. Alteration of an amino as Bacillus macerans) commercially available from Amano acid residue may be interspersed with one residue, or a few Enzyme as Cyclodextrin glucanotransferase "Amano'. residues may be contiguous. Those skilled in the art can easily 0211 CGTase may be commercially available or may be selecta objective enzyme having a desired property. Alterna prepared from CGTase producing organisms by a method tively, a gene encoding the objective enzyme may be directly known in the art, or may be prepared by a genetic recombi chemically synthesized. Methods for such chemical synthesis nation method based on an amino acid sequence or a base are well-known in the art. sequence of CGTase of CGTase producing organisms, or may 0218 Modification to enzyme can be carried out using a be chemically synthesized. Any CGTase known in the art can method well-known in the art, for example, by carrying out be used as long as it has a transglycosylation activity, and an site-directed mutagenesis, mutagenesis with a mutagen activity capable of improving agel forming ability of a starch. (treatment of a Subject gene with a mutagenic agent such as 0212 (1.2.9 Use of Enzymes in Combination) nitrite, or treatment with UV rays), or error-prone PCR. It is 0213. In the case of producing the starch of the present preferable to use site-directed mutagenesis from the view invention, multiple kinds of Starch hydrolases or glycosyl point that the objective mutation is easily obtained, because transferases may be allowed to act on in combination. Par the objective modification can be introduced at an objective ticularly, since C-glucosidase alone does not easily react with site when site-directed mutagenesis is used. Alternatively, a starch granules, it is preferred to use in combination with nucleic acid molecule having an objective sequence may be C.-amylase. directly synthesized. Such chemical synthesis methods are 0214 (1.2.10 Common Explanation about Enzymes) well-known in the art. Techniques of site-directed mutagen 0215. In the present description, the fact that the enzyme is esis are described in, for example, Nucl. Acid Research, Vol. "derived from certain organisms means not only the fact that 10, pp. 6487-6500 (1982). the enzyme is directly isolated from the organisms, but also 0219 Upon design of the aforementioned modification, the fact that an enzyme having the same amino acid sequence the hydrophobicity index of an amino acid can be considered. is produced from another organisms based on an amino acid Significance of a hydrophobic amino acid index upon impar sequence of the enzyme possessed by the organisms, or a base tation interacting biological function to a protein is generally sequence encoding the amino acid sequence. For example, recognized in the art (Kyte.J and Doolittle, R. F. J. Mol. Biol. also in the case of introducing a gene encoding the enzyme 157 (1): 105-132, 1982). The hydrophobic nature of an amino obtained from the organisms into E. coli and isolating the acid contributes to the secondary structure of a produced enzyme from the E. coli, it is said that the enzyme is "derived protein and, then, defines interaction between the protein and from the organisms. other molecule (e.g. starch hydrolase or glycosyltransferase, 0216. In the present description, a large excess amount of Substrate, receptor, DNA, antibody, antigen and the like). An the enzyme is added to starch granules. Therefore, the amount amino acid is assigned a hydrophobicity index based on of the enzyme is represented by % by weight. It is not neces hydrophobicity and a nature of a charge thereof. They are: sary to represent it by the unit (U). isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylala 0217 Many C.-amylases, amyloglucosidases, isoamy nine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); ala lases, C-glucosidases and cyclodextrin glucanotransferases nine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); are known, and, therefore, many natural base sequences and tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine amino acid sequences of these enzymes are known. It is (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid known that variants (so-called allele variants) having a (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). slightly different sequence from the natural sequences can 0220. It is well-known in the art to substitute a certain occur naturally. Such naturally occurring variants and vari amino acid with another amino acid having a similar hydro ants created by artificially mutating the natural enzymes, in phobicity index, thereby, a protein still having Substantially addition to the enzymes exemplified above, can be used in the similar biological functions (e.g. protein Substantially method of the present invention insofar as they have a desired equivalent in enzyme activity) can be produced. In Such an activity. Variant enzymes preferably have activity equal to, or amino acid substitution, a hydrophobicity index is preferably higher than, that of the enzyme before modification. For within +2, more preferably within +1, further preferably example, the amino acid sequence of a starch hydrolase used within +0.5. It is understood in the art that such the substitu in the present invention, in a certain embodiment, may be tion of an amino acid based on hydrophobicity is efficient. As identical with (that is, 100% identical with) an amino acid described in U.S. Pat. No. 4,554,101, the following hydro sequence (that is, a reference amino acid sequence) of the philicity index is assigned to amino acid residues: arginine starch hydrolase used in the Examples of the present appli (+3.0); lysine (+3.0); aspartic acid (+3.0+1); glutamic acid US 2013/0022711 A1 Jan. 24, 2013

(+3.0t1); serine (+0.3); asparagine (+0.2); glutamine (+0.2): Synthesis (edited by M. J. Gait, 1984); and Nucleic Acid glycine (O); threonine (-0.4); proline (-0.5+1); alanine (-0. Hybridization (edited by B. D. Hames & S.J. Higgins, 1984). 5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); 0225. Alternatively, based on homology to a base valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2. sequence of a certain enzyme gene for which a base sequence 3); phenylalanine (-2.5); and tryptophan (-3.4). It is under encoding enzyme is known, screening can be conducted by stood that an amino acid can be substituted with another hybridization using nucleic acid probes containing at least a amino acid which has a similar hydrophilicity index, and can part of this base sequence, thereby, a nucleic acid molecule still provide a biological equivalent. In Such the amino acid containing another kind of the enzyme gene may be acquired. substitution, the hydrophilicity index is preferably within +2, Such methods are known in the art. more preferably within +1, and further preferably within 0226. Alternatively, degenerate primers corresponding to O.5. a region which is conserved in the amino acid sequence of 0221. In the present invention, “conservative substitution' various enzymes are prepared, and PCR is performed, and the refers to substitution in which a hydrophilicity index or/and a base sequence of the enzyme may be acquired. Such methods hydrophobicity index are similar, as described above, are known in the art. between the original amino acid and an amino acid to be 0227. When a genome library is screened, the resulting Substituted, in amino acid substitution. Examples of conser nucleic acid molecule can be subcloned using methods well vative substitution are well-known to those skilled in the art, known to those skilled in the art. For example, by mixing W and include, but are not limited to Substitution among the phage containing an objective gene, Suitable Escherichia coli following each group, for example: arginine and lysine; and Suitable helper phage, a plasmid containing an objective glutamic acid and aspartic acid; serine and threonine; gene can be easily obtained. Thereafter, by transforming Suit glutamine and asparagines; and Valine, leucine, and isoleu able Escherichia coli using a solution containing the plasmid, C1G. an objective gene can be subcloned. By culturing the resulting 0222. The enzyme used in the method of the present inven transformant, a plasmid DNA may be obtained, for example, tion may be isolated from naturally occurring microorgan by an alkaline SDS method, and the base sequence of the isms producing the above-mentioned enzyme of interest. For objective gene can be determined. A method of determining a example, firstly, a microorganism producing the enzyme of base sequence is well-known to those skilled in the art. Fur interest is inoculated into a Suitable medium (for example, L ther, using primers synthesized based on a base sequence of a broth (1% Bacto-Tryptone (Difco Laboratories, Detroit, DNA fragment, and using a polymerase chain reaction (PCR) Mich., USA), 0.5% Bacto-Yeast Extract (Difico), 0.5% NaCl, employing, for example, the genomic DNA of Aquifex aeoli pH 7.3) and cultured at appropriate temperature (for example, cus, Rhodothermus Obamensis, Bacillus Stearothermophilus, about 30° C. to about 40°C.) overnight with shaking. Then, Bacillus caldovelox, Bacillus thermocatenulatus, Bacillus this culture is centrifuged to precipitate the microbial cells caldolyticus or the like as a template, an enzyme gene may be and then obtained a culture supernatant. The obtained culture directly amplified. Supernatant is concentrated with UF membrane to obtain an 0228. Alternatively, the enzyme gene can be chemically enzyme liquid of interest. When further purification is neces synthesized based on a known base sequence. sary, a solution containing a purified enzyme of interest can 0229. A base sequence encoding an amino acid sequence be obtained by combining fractionation with ion-exchange of the enzyme used in the method of the present invention chromatography on Q-Sepharose or the like, fractionation may be altered in up to certain number of nucleotides as with gel filtration chromatography on Sephacryl S-200HR compared with the nucleotide sequence (that is, the reference (manufactured by Pharmacia) or the like and fractionation nucleotide sequence) encoding the reference amino acid with hydrophobic chromatography on Phenyl-TOYOPEARL sequence described above. Such alterations can be selected 650M (manufactured by Tosoh Corporation) or the like, if from the group consisting of a deletion of at least one nucle necessary. otide, Substitution with at least one nucleotide, including 0223) Alternatively, the enzyme used in the method of the transition and transversion, or an insertion of at least one present invention can be obtained by introducing a nucleic nucleotide. This alteration may occur at a position of the 5' acid molecule containing a base sequence encoding enzyme terminus or the 3' terminus of a reference nucleotide of interest into a suitable host cell, to express the enzyme, and sequence, or may occur at any position other than these ter purifying the expressed enzyme from the host cell or its mini. Alteration of a base may be interspersed with one base, culture liquid. or a few bases may be contiguous. 0224 Purified enzyme obtained resultingly is treated with 0230. A nucleotide alteration can generate a nonsense, trypsin, the resulting trypsin treated fragment is separated by missense or frame shift mutation in a code sequence, and thus HPLC, and the amino acid sequence of the N-terminus of any alteration of the enzyme encoded by such a altered base of the separated peptide fragments is determined using a sequence can be effected. peptide sequencer. Then, using synthetic oligonucleotide 0231. In the case where the enzyme used in the present probes prepared based on the identified amino acid sequence, invention is a starch hydrolase, it is preferred that this enzyme a suitable genome library or a cDNA library is screened, has at least about 20%, preferably at least about 30%, more thereby, a nucleic acid molecule (also referred to as a gene) preferably at least about 40%, still more preferably at least comprising a base sequence encoding natural enzyme can be about 50%, and particularly preferably at least about 60%, obtained. Fundamental strategies for preparing the oligo about 70%, about 80%, about 90%, about 95%, about 96%, nucleotide probes and DNA libraries, and screening them by about 97%, about 98% or about 99% of identity against an hybridization of nucleic acids, are well-known to those amino acid sequence of a starch hydrolase used in Examples, skilled in the art. For example, see Sambrooketal. Molecular oran amino acid sequence of SEQID NO: 2, 4, 6, 8, 10 or 12, Cloning: A Laboratory Manual (1989); DNA Cloning, Vol and has a starch hydrolysis activity (characteristics capable of umes I and II (edited by D. N. Glover, 1985); Oligonucleotide improving a gel forming ability of a starch in a specific case). US 2013/0022711 A1 Jan. 24, 2013

0232. In the case where the enzyme used in the present 0236 A transglycosylase, which is encoded by a nucleic invention is a glycosyltransferase, it is preferred that this acid molecule which is capable of hybridizing understringent enzyme has at least about 20%, preferably at least about 30%, conditions with a nucleic acid molecule having a complemen more preferably at least about 40%, still more preferably at tary sequence of a base sequence (for example, SEQID NO: least about 50%, and particularly preferably at least about 13) encoding a natural known transglycosylase, can be used 60%, about 70%, about 80%, about 90%, about 95%, about in the method of the present invention as long as it has a 96%, about 97%, about 98% or about 99% of identity against transglycosylase activity (characteristics of improving a gel an amino acid sequence of a glycosyltransferase used in forming ability of a starch in a specific case). A transglyco Examples, or an amino acid sequence of SEQID NO: 14, and Sylase, which is encoded by a nucleic acid molecule contain has a transglycosylation activity (characteristics capable of ing an altered base sequence obtained by altering a nucleic improving a gel forming ability of a starch in a specific case). acid molecule which is capable of hybridizing understringent 0233. In the present specification, the identity of conditions with a nucleic acid molecule having a complemen sequences is calculated using maximum matching of GENE tary sequence of a base sequence encoding a natural known TYX-WINVer, 4.0 (Genetics Co., Ltd.). This program aligns transglycosylase can also be used in the method of the present sequence data to be analyzed, and sequence data to be com invention as long as it has an ability to produce a high viscos pared so that amino acid pairs matched between sequences ity starch having a gel forming ability. Those skilled in the art become greatest while Substitution and deletion are consid can easily select a desired transglycosylase gene. ered, and thereupon, gives a score to each of Matches, Mis 0237 As used in the present description, the term “strin matches, and Gaps, calculates a sum, outputs alignment at the gent conditions' refers to conditions under which a sequence Smallest Sum, and calculates identity thereupon (Reference: hybridizes with a specific sequence, but not with a non-spe Takashi, K., and Gotoh, O. 1984. Sequence Relationships cific sequence. Selection of appropriate Stringent conditions among Various 4.5 S RNA Species J. Biochem. 92: 1173 is well-known to those skilled in the art, and is described, for 1177). In the present specification, the percentage identity of example, in Molecular Cloning (Sambrook, et al., Supra). For sequences is calculated using maximum matching of GENE example, “stringent conditions' are hybridization in a solu TYX-WINVer, 4.0 under the condition of Matches=-1; Mis tion containing 50% formamide, 5xSSC (750 mM NaCl, 75 matches=1; Gaps=1; *N+=2. mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 0234. As a natural enzyme or nucleic acid molecule, an 5xDenhardt’s solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% enzyme or nucleic acid molecule having a sequence that is not polyvinylpyrrolidone), 10% dextran sulfate and 20 ug/ml identical with, but is homologous to, the amino acid sequence denatured sheared salmon sperm DNA at 65° C., and subse of the enzyme or the base sequence encoding the amino acid quent washing under the condition of 65° C. using an SSC sequence of the enzyme can also be used. Such an enzyme or (saline-sodium citrate) solution having a 0.1 to 2-fold con nucleic acid molecule having homology with the natural centration (a composition of an SSC solution having a 1-fold enzyme or nucleic acid molecule includes, but are not limited concentration is 150 mM sodium chloride and 15 mM sodium to, in the case of a nucleic acid, nucleic acid molecules con citrate). Therefore, for example, a polynucleotide being taining a base sequence having at least about 30%, about capable to hybridize under Stringent conditions means, spe 35%, about 40%, about 45%, about 50%, about 55%, about cifically, a polynucleotide which can be identified using the 60%, about 65%, about 70%, about 75%, about 80%, about conditions under which hybridization is performed at 65° C. 85%, about 90%, about 95%, or about 99% identity with a in a solution containing 50% formamide, 5xSSC (750 mM comparison Subject sequence, and, in the case of an enzyme, NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate includes, but are not limited to, enzymes having an amino (pH 7.6), 5xDenhardt’s solution (0.2% BSA, 0.2% Ficoll 400 acid sequence having at least about 40%, about 45%, about and 0.2% polyvinyl pyrrolidone), 10% dextran sulfate, and 20 50%, about 55%, about 60%, about 65%, about 70%, about ug/ml denatured sheared salmon sperm DNA using a filter on 75%, about 80%, about 85%, about 90%, about 95%, or about which a DNA derived from a colony or a plaque has been 99% identity with a comparison Subject sequence, when com immobilized, and a filter is washed under the condition of 65° pared in maximum matching in for example GENETYX C. using an SSC (Saline-sodium citrate) solution having a 0.1 WINVer, 4.0 under the conditions described above. to 2-fold concentration (a composition of an SSC solution 0235 A starch hydrolase, which is encoded by a nucleic having a 1-fold concentration is 150 mM sodium chloride, 15 acid molecule which is capable of hybridizing understringent mM sodium citrate). conditions with a nucleic acid molecule having a complemen Specifically, the conditions mean, for example, that tary sequence of a base sequence (for example, SEQID NO: 0238 A nucleic acid molecule used for producing an 1, 3, 5, 7, 9 or 11) encoding a natural known starch hydrolase, enzyme used in the present method may be a nucleic acid can be used in the method of the present invention as long as molecule which was conservatively modified relative to a it has a starch hydrolysis activity (characteristics of improv nucleic acid molecule comprising a base sequence encoding ing agel forming ability of a starchina specific case). A starch a natural enzyme. The “nucleic acid molecule which was hydrolase, which is encoded by a nucleic acid molecule con conservatively modified relative to a nucleic acid molecule taining an altered base sequence obtained by altering a comprising a base sequence encoding natural enzyme” refers nucleic acid molecule which is capable of hybridizing under to a nucleic acid molecule comprising a base sequence encod stringent conditions with a nucleic acid molecule having a ing an amino acid sequence which is the same or essentially complementary sequence of a base sequence encoding a natu the same as an amino acid sequence of the natural enzyme. ral known starch hydrolase can also be used in the method of The "amino acid sequence which is essentially the same as an the present invention as long as it has an ability capable of amino acid sequence of the natural enzyme” refers to an producing a high viscosity starch having agel forming ability. amino acid sequence having essentially the same enzyme Those skilled in the art can easily select a desired starch activity as that of the natural enzyme. Due to the degeneracy hydrolase gene. of the genetic code, many functionally equivalent base US 2013/0022711 A1 Jan. 24, 2013 sequences encode any prescribed amino acid sequence. For 0242. As used in the present description, an “expression example, codons GCA, GCC, GCG and GCU all encode the vector” refers to a vector which can express a modified base amino acid alanine. Therefore, at all positions where alanine sequence (i.e. base sequence encoding modified enzyme) in is specified by a GCA codon, the codon can be changed to an objective cell. An expression vector contains, in addition to GCC, GCG or GCU without changing the encoded alanine. a modified base sequence, various regulation elements such Similarly, regarding an amino acid which can be encoded by as a promoter regulating expression thereof and, if necessary, a plurality of codons, at all positions where the amino acid is factors necessary for replication in an objective cell and selec specified by a codon, the codon can be changed to any another tion of a recombinant (e.g. origin of replication (ori), and a codon encoding the amino acid without changing the particu selectable marker Such as a drug resistant gene). In an expres lar amino acid coded. Such a variation in a base sequence is a sion vector, a modified base sequence is operably linked so “silent mutation” which is one kind of conservatively modi that it is transcribed and translated. Regulation elements fied mutation. All base sequences in the present specification include a promoter, a terminator and an enhancer. In addition, which encode a polypeptide also include all possible silent when secretion of an expressed enzyme outside a cell is mutations of the nucleic acid. Silent mutation includes “silent intended, a base sequence encoding a secretion signal peptide Substitution' in which a coded amino acid is not changed, and is linked upstream of a modified base sequence in the correct the case where a nucleic acid does not originally encode an reading frame. It is well-known to those skilled in the art that amino acid (for example, a mutation at an intron portion, a both the type of an expression vector used for introduction mutation at other untranslated region and the like). When a into a particular organism (e.g. bacterium), and the kind of a certain nucleic acid encodes an amino acid, silent mutation regulation element and other factors used in the expression has the same meaning as that of silent Substitution. In the vector, can vary depending on an objective cell. present specification, “silent substitution” refers to substitu 0243 As used in the present description, a “terminator” is tion of a base sequence encoding a certain amino acid with a sequence which is situated downstream of a protein coding another base sequence encoding the same amino acid, in a region, and is involved in termination of transcription upon base sequence. Based on the phenomenon of degeneracy in transcription of a base sequence into an mRNA, and in the the genetic code, in the case where there area plurality of base addition of a poly A sequence. It is known that a terminator sequences encoding a certain amino acid (for example, gly influences the expression level of a gene by involving the cine and the like), such silent substitution is possible. There stability of an mRNA. fore, a polypeptide having an amino acid sequence encoded 0244 As used in the present description, a “promoter' by a base sequence produced by silent Substitution has the refers to a region on a DNA which determines a transcription same amino acid sequence as that of the original polypeptide. initiation site of a gene, and directly regulates the transcrip In the art, it is understood that each codon in a nucleic acid tion frequency, and is a base sequence to which a RNA poly (except for AUG which is the only codon usually encoding merase binds, thereby, initiating transcription. Since the methionine, and TGG which is the only codon usually encod region of a promoter is usually a region about 2 kbp or less ing tryptophan) can be modified in order to produce function upstream of a first exon of a putative protein coding region in ally the same molecule. Therefore, each silent mutation of a many cases, when a protein coding region in a genome base nucleic acid encoding a polypeptide is implicitly included in sequence is predicted using a DNA analyzing Software, a each described sequence. Preferably, such a modification can promoter region can be putative. A putative promoter region be performed so that substitution of cysteine, which is an varies with every structural gene, and is usually upstream of a amino acid that greatly influences the conformation of a structural gene without limitation, and may be downstream of polypeptide, is avoided. a structural gene. Preferably, a putative promoter region is 0239. A base sequence encoding enzyme used in the present about 2 kbp or less upstream of a first exon translation present invention can be changed in conformity with a codon initiation point. usage in an organism into which the sequence is introduced 0245. As used in the present description, an "enhancer for expression. Codon usage reflects the usage in a gene can be used for enhancing the expression efficiency of an which is highly expressed in the organism. For example, objective gene. Such an enhancer is well-known in the art. A when expression is intended in Escherichia coli, the sequence plurality of enhancers can be used, or only one may be used, can be made to be optimal for expression in Escherichia coli or may not be used at all. according to the published codon usage table (for example, 0246. As used in the present description, “operably Sharp, et al., Nucleic Acids Research 16, No. 17, p. 8207 linked refers to when a desired base sequence is placed under (1988)). the control of a transcription and translation regulating 0240 An expression vector can be made using a nucleic sequence (e.g. promoter, enhancer and the like) or a transla acid molecule comprising the base sequence modified as tion regulating sequence which effect expression (i.e. opera described above. A method for preparing an expression vector tion). In order that a promoter is operably linked to a gene, using a particular nucleic acid sequence is well-known to usually, a promoter is disposed immediately upstream of the those skilled in the art. gene, but it is not necessary that the promoter is disposed 0241 When a nucleic acid molecule is referred to in the adjacent to the gene. present specification, a “vector” refers to a nucleic acid mol 0247. In order to operably link a modified nucleic acid ecule which can transfer an objective base sequence into an sequence to the aforementioned regulation element, an objective cell. Examples of such vectors include a vector enzyme gene should be processed in some cases. Examples which can autonomously replicate in an objective cell, or can include the case where the distance between a promoter and a be incorporated into a chromosome of an objective cell, and coding region is too long, and reduction in a transcription has a promoter at a position Suitable for transcribing a modi efficiency is predicted, the case where the distance between a fied base sequence. In the present specification, the vector ribosome binding site and a translation initiation codon is not may be a plasmid. Suitable, and the like. Examples of processing mean include US 2013/0022711 A1 Jan. 24, 2013 digestion with a restriction enzyme, digestion with an exonu (0255 (2.1 Preparation of Suspension) clease such as Bal31 and ExoIII, or introduction of site 0256 In the production method of the present invention, directed mutation using a single-stranded DNA such as M13 for example, starch granules, a starch hydrolase or a glyco or PCR. Syltransferase, a buffer agent, and a solvent dissolving them 0248. Then, the expression vector prepared as described are used as main materials. Although all of these materials are above is introduced into a cell, thereby, the objective enzyme usually added at the time of initiation of a reaction, any is expressed. material among these materials may be further added during 0249. As used in the present description, “expression' of the reaction. The solvent used in the production method of the an enzyme refers to in vivo or in vitro transcription and present invention can be any solvent as long as it is a solvent translation of a base sequence encoding the enzyme, and which does not impair an enzyme activity of the enzyme to be production of the encoded enzyme. used. The typical solventis water (for example, ion-exchange 0250) A cell into which an expression vector is introduced water, purified water, and tap water). The solvent may be (also referred to as a host) includes prokaryotes and eukary moisture of a crushed cell liquid obtained in association with otes. A cell into which an expression vector is introduced can the enzyme upon preparing the enzyme. be easily selected, taking various conditions such as ease of 0257. In the production method of the present invention, expression of objective enzyme, ease of culturing, growth first, a reaction Solution is prepared. The reaction solution can rate, and safety into consideration. Examples of Such cells be obtained, for example, by adding starch granules and a include microorganisms such as bacteria and fungi. Examples starch hydrolase or a glycosyltransferase to a suitable solvent. of more preferable cells include mesophilic microorganisms For example, the enzyme may be added after preparing a (e.g. yeast, mold, Escherichia coli, Bacillus subtilis). A cell starch Suspension by Suspending starch granules in the Sol may be a microorganism cell, or may be a plant or animal cell. vent (for example, water or buffer solution). Alternatively, the Depending on the cell to be used, a starch hydrolase can be an reaction solution may be prepared by mixing a Suspension enzyme which has undergone post-translational processing. containing starch granules with a solution containing an 0251. In the method of the present invention, the technique enzyme. To this reaction solution, any buffer agent may be of introducing an expression vector into a cell may be any optionally added for the purpose of adjusting the pH as long technique known in the art. Examples of Such techniques as it does not inhibit the enzyme reaction. It is noted that include, for example, transformation, transduction, and trans although starch granules are not dissolved but Suspended in fection. Such techniques of introducing a nucleic acid mol the reaction solution, it is called as reaction solution, since ecule are well-known in the art, and are conventional, and are other components such as an enzyme are dissolved therein. described, for example, in Ausubel F. A., et al. ed. (1988), 0258. The pH of the reaction solution can be arbitrarily set Current Protocols in Molecular Biology, Wiley, New York, as long as it is the pH at which the enzyme to be used can exert N.Y.; Sambrook J, et al. (1987) Molecular Cloning: A Labo an activity. The pH of the reaction solution is preferably ratory Manual, 2nd Ed., Cold Spring Harbor Laboratory around the optimum pH of the enzyme to be used. The pH of Press, Cold Spring Harbor, N.Y., and Bessatsu Jikken-igaku the reaction solution is typically about 2 or more, preferably “Idenshidounyu & Hatsugen kaiseki jikkenhou, Yodosha, about 3 or more, still more preferably about 4 or more, par 1997. ticularly preferably about 5 or more, particularly preferably about 6 or more, and most preferably about 7 or more. The pH (1.3 Other Materials) of the reaction solution is typically about 13 or less, prefer 0252. In the production of enzyme-treated starch granules, ably about 12 or less, still more preferably about 11 or less, any material used usually in an enzymatic treatment can be particularly preferably about 10 or less, particularly prefer used as long as it does not obstruct an action of the enzyme. ably about 9 or less, and most preferably about 8 or less. In an Examples of such other material include salts and buffer embodiment, the pH of the reaction solution is typically agents. Since it is commonly known that a rate of an enzyme within the optimum pH +3, preferably within the optimum pH reaction can be drastically improved by adding a specific salt +2, still more preferably within the optimum pH +1, and most Suitable to each enzyme, it is preferred to add Such a specific preferably within the optimum pH +0.5, of the enzyme to be salt. It is possible to shorten the treatment time by adding Such used. a suitable salt to each enzyme. Examples of the combination 0259. The amount of the starch granules in the reaction of the enzyme and the salt include a combination of amylo Solution can be arbitrarily set as long as it is the amount which glucosidase and a metalion (for example, Sodium ion, potas enables the enzyme reaction. The amount of the starch gran sium ion, calcium ion, or magnesium ion). As a result of a test ules in the reaction solution is preferably about 5% by weight by the present inventors, for example, in the case of treating or more, more preferably about 10% by weight or more, still an untreated native cassava starch with amyloglucosidase (for more preferably about 20% by weight or more, and most example, “OPTIDEXL-400' derived from Aspergillus niger, preferably about 30% by weight or more. The amount of the manufactured by Genencor), a degradation rate of the starch starch granules in the reaction Solution is preferably about in the system, in which 100 ppm (in terms of a metal ion) of 60% by weight or less, more preferably about 50% by weight Sodium chloride, or Sodium Sulfate, or potassium chloride, or or less, still more preferably about 40% by weight or less, and calcium chloride, or magnesium chloride is added, increased most preferably about 35% by weight or less. by 1.5 to 2 times as compared with the system in which no 0260 The amount of the enzyme in the reaction solution metal ion is added. can be arbitrarily set as long as it is the amount which enables 0253 (2. Method for Producing Enzyme-Treated Starch the enzyme reaction. The amount of the enzyme is preferably Granule) the amount enough to carryout the reaction within a reason 0254 Enzyme-treated starch granules are produced by able time. As the amount of the enzyme increases, the time treating starch granules with a starch hydrolase or a glyco required to the reaction becomes shorter. As the amount of the syltransferase. Details of each step will be described below. enzyme decreases, the time required to the reaction becomes US 2013/0022711 A1 Jan. 24, 2013 longer. When the amount of the enzyme is too large, the cost enzyme-treated Starch granules by drying the starch after increases too much and the enzyme may be sometimes aggre dehydration. Drying of the starch after dehydration can be gated to form a precipitate. Therefore, it is preferred to appro carried out by any method known in the art. priately set the amount of the enzyme. 0267 (2.4 Chemical Modification) 0261 The amount of the enzyme in the reaction solution is 0268. The starch granules subjected to the enzymatic preferably about 0.01% by weight or more, more preferably treatment can be subjected to a chemical modification, if about 0.05% by weight or more, and still more preferably desired. Not only in the case where the starch granules used in about 0.1% by weight or more, based on the solid content of the enzymatic treatment are untreated Starch granules or the starch granules. The amount of the enzyme in the reaction starch granules Subjected to a physical treatment, but also in solution is preferably about 10% by weight or less, more the case where starch granules of Some chemically modified preferably about 5% by weight or less, and still more prefer starch are used, it is possible to be subjected to a chemical ably about 1% by weight or less, based on the solid content of modification which is different from various chemical modi the starch granules. The amount of the enzyme in the reaction fications applied to the chemically modified starch. Examples Solution may be the amount enough to enable proceeding of of the chemical modification include acetylation, adipate the enzyme reaction. Therefore, it is not necessary to examine crosslinking, oxidation, bleaching, phosphate crosslinking, in detail about an activity (number of units) of the enzyme. treatment with octenyl Succinic acid, hydroxypropylation, 0262 (2.2 Enzyme Reaction) phosphorylation, and phosphoric acid monoesterification. 0263. Next, the reaction solution is reacted optionally by These chemical modification methods are well known in the heating using a method known in the art. The solution tem art. These chemical modifications can be carried out to any perature in the reaction step can be any temperature as long as degree as long as they are within the scope permitted by the it is the temperature at which the starch granules are not Food Sanitation Law of Japan. In Japan, in order that the substantially collapsed. The reaction temperature is prefer chemically modified Starch is approved as a food additive, it ably the temperature at which an enzyme to be used can is essential that various chemical Substances in a sample sufficiently exert an activity and sufficiently retain an activity starch are analyzed in accordance with a method for a purity (that is, less likely to be inactivated). The temperature of the test described in Ministry of Health and Welfare Notification Solution in this reaction step is preferably the temperature at No. 485 and the obtained analytical results meet the following which about 50% or more, and more preferably about 80% or standards: more of the activity of the enzyme contained in this solution (a) Acetylated distarch adipate: the content of adipic acid before the reaction remains after a predetermined reaction groups shall be 0.135% or less and the content of acetyl time. For example, this temperature can be an optimum tem groups shall be 2.5% or less; perature +10°C., more preferably an optimum temperature (b) Acetylated oxidized starch: the content of acetyl groups +5, still more preferably an optimum temperature +1°C., and shall be 2.5% or less and the content of carboxyl groups shall most preferably an optimum pH +0.5°C., of the enzyme to be be 1.3% or less; used. The reaction temperature is preferably about 10° C. or (c) Acetylated distarch phosphate: the content of acetyl higher, more preferably about 10° C. or higher, still more groups shall be 2.5% or less and the content of phosphorus preferably about 15° C. or higher, further more preferably shall be 0.14% or less in terms of P: about 20°C. or higher, particularly preferably about 30°C. or (d) Starch sodium octenyl Succinate: the content of octenyl higher, and most preferably 40° C. or higher. The reaction succinic acid groups shall be 3.0% or less; temperature is preferably about 70° C. or lower, more pref erably about 65° C. or lower, particularly preferably about (e) Starch acetate: the content of acetyl groups shall be 2.5% 60° C. or lower, and most preferably 55° C. or lower. or less; 0264. The reaction time can be arbitrarily set taking the (f) Oxidized starch: the content of carboxyl groups shall be reaction temperature, the amount of the enzyme to starch 1.1% or less; granules into consideration. The reaction time can be prefer (g) Hydroxypropyl distarch phosphate: the content of ably for about 1 hour or more, for example, about 2 hours or hydroxypropyl groups shall be 7.0% or less and the content of more, about 3 hours or more, about 6 hours or more, and about phosphorus shall be 0.14% or less in terms of P: 12 hours or more. Although there is no particular upper limit (h) Hydroxypropyl starch: the content of hydroxypropyl of the reaction time, the reaction time is preferably about 72 groups shall be 7.0% or less; hours or less, more preferably about 48 hours or less, still (i) Distarch phosphate: the content of phosphorus shall be more preferably about 36 hours or less, particularly prefer 0.5% or less in terms of P: ably about 24 hours or less, and most preferably about 20 (j) Monostarch phosphate: the content of phosphorus shall be hours or less. 0.5% or less in terms of P: 0265 (2.3 Post-Treatment) (k) Phosphated distarch phosphate: the content of phosphorus 0266 The starch granules subjected to the enzymatic shall be 0.5% or less in terms of P: treatment can be used as they are depending on the applica (1) Bleached starch; the content of carboxyl groups shall be tion. However, it is preferred that the enzyme used and glu 0.1% or less, the test results of “Confirmatory test (3) of the cide eluted by enzymatic hydrolysis are removed by washing oxidized starch described in Ministry of Health and Welfare the starch granules Subjected to the enzymatic treatment, and Notification No. 485 shall be negative, and it shall be reason followed by dehydration. Washing and dehydration of the ably explained that a change in properties, such as Viscosity, starch granules Subjected to the enzymatic treatment can be of the starch is not caused by oxidation. Regarding the coun carried out by any method known in the art. Washing and tries other than Japan, any degree of a chemical treatment can dehydration of the starch granules are conventional methods be carried out as long as it is within the scope permitted in that used for preparation of a starch, and are commonly carried country. Some kinds of chemical modifications can be used in out. Furthermore, it is preferred to obtain the objective combination. US 2013/0022711 A1 Jan. 24, 2013 20

0269 (2.5 Physical Treatment) inhibition treatment is more preferably from about 120° C. to 0270. The starch granules subjected to the enzymatic about 180°C., particularly preferably from about 140°C. to treatment can be subjected to a physical treatment, if desired. about 160° C., and most preferably from about 160° C. The Not only in the case where the starch granules used in the level of inhibition depends on the pH, heating temperature enzymatic treatment are untreated Starch granules or a chemi and heating time. As the pH becomes higher, a more highly cally modified starch, but also in the case where the starch inhibited starch is obtained. As the temperature of the heat granules Subjected to some physical treatment are used, it is treatment becomes higher, a more highly inhibited Starch is possible to be subjected to a physical treatment which is obtained. As the time of the heat treatment becomes longer, a different from the physical treatment. Examples of the physi more highly inhibited starch is obtained. The thermal treat cal treatment include a heat-moisture treatment and a thermal ment time for a thermal inhibition treatment can be, for inhibition treatment. example, about 3 hours or more, and preferably about 20 0271 The “heat-moisture treatment” refers to heating to a hours or less. The thermal inhibition treatment is described in temperature of about 95 to about 125° C. in a low moisture various documents and can be carried out in accordance with state where a starch is not gelatinized in a closed container any thermal inhibition treatment method known in the art. under the condition of a relative humidity of about 100%. The The thermal inhibition treatment is described, for example, in “low moisture state where a starch is not gelatinized indi U.S. Pat. No. 6,221,420, Pamphlet of International Publica cates, for example, the moisture content of about 50% or less. tion No. WO95/04082, and Japanese Laid-open Patent Pub The low moisture state where a starch is not gelatinized may lication No. 2008-223,032. The temperature, time, and the like be, for example, the moisture content of about 35% or less, of the thermal inhibition treatment can be appropriately set about 30% or less, about 25% or less, or about 20% or less. depending on the objective starch and physical properties The heating time of the heat-moisture treatment can vary thereof. The physical treatment can be carried out in accor depending on the method of the heat-moisture treatment. For dance with the method well known in the art. example, a heat-moisture treatment is carried out in accor 0273 Examples of the heat-moisture-treated starch dance with the method described in Japanese Laid-open include, for example, “Delicastar series”, “Naturastar series'. Patent Publication No. 6-145203, a heat treatment is carried and “AMYLOGEL manufactured by SANWA CORN out by first decompressing to a pressure of about 0 to 500 torr STARCH CO.,LTD.; and “ROADSTER manufactured by (about 0 to 66.661 kPa) and then introducing pressurized Nihon Shokuhin Kako Co., Ltd. Examples of the thermally steam, followed by retention at about 100° C. to about 150° C. inhibited Starch include “NOVATION Series' manufactured for about 2 minutes to about 120 minutes. The heat-moisture by National Starch Corp. treatment is described in various documents and can be car 0274 (3. Characteristics of Enzyme-Treated Starch Gran ried out in accordance with any heat-moisture treatment ules of the Present Invention) method known in the art. The heat-moisture treatment is 0275. In a specific embodiment, the enzyme-treated starch described, for example, in Japanese Laid-open Patent Publi of the present invention is an enzyme-treated Starch having a cation No. 6-145203, Japanese Laid-open Patent Publication high viscosity and a gel forming ability, and the enzyme No. 4-1301 02, A Technical Journal on Food Chemistry & treated Starch is an enzyme-treated Starch obtained by treating Chemicals 2010-2 (P. 37-42) and the like. The temperature, starch granules with an enzyme at a temperature of about 10 time and the like of the heat-moisture treatment can be appro C. or higher and about 70° C. or lower. priately set depending on the objective starch and physical 0276. In another specific embodiment, the enzyme-treated properties thereof. starch of the present invention is an enzyme-treated Starch 0272. The “thermal inhibition treatment refers to the fact having a high viscosity and a gel forming ability; the enzyme that a crystal structure of starch granules is reinforced by treated Starch is a starch obtained by treating starch granules Subjecting starch granules dried to extremely low water con of an untreated Starch with a starch hydrolase under the con tent to a dry heat treatment. The “starch granules dried to dition where the starch granules are not dissolved; the extremely low water content” refers to starch granules whose enzyme-treated Starch is not modified on hydroxyl groups at moisture content is less than about 1%. The moisture content the positions 2, 3 and 6 of the glucose residues; and the of the starch granules subjected to a thermal inhibition treat enzyme-treated Starch can form agel having a Young's modu ment is preferably about 0%. The method of drying starch lus higher than that of the untreated Starch or a rupture stress granules to extremely low water content is described, for higher than that of the untreated starch, when measured by a example, in JP-A-2008-223,032 and can be, for example, a rheometer. method in which the pH of starch granules is adjusted to the (0277 (3.1 Viscosity) pH of 7.0 or more and then dehydration is carried out until the 0278. It is well known that when a starch is heated together moisture content reaches less than about 1%. In the case of with a predetermined amount or more of water, starch gran drying to low water content, the pH is preferably 7 or more, ules generally cause a gelatinization phenomenon Such as more preferably more than 8, still more preferably from 7.5 to Swelling, an increase in transparency, and an increase in vis 10.5, and further more preferably from 8 to 9.5. The dehy cosity. The starch granules are collapsed by furtherheating. In dration may be either thermal dehydration or nonthermal order to measure a change in Viscosity associated with a series dehydration. In the case of a dry heat treatment, a heat treat of these events, an amylograph manufactured by Brabender ment is carried out at a sufficient temperature for the time Inc. is practical and is widely used, although there are some enough to inhibit a starch. Preferably, a heat treatment is methods. The amylograph is that in which the object is heated carried out at a Sufficient temperature for the time enough to at a predetermined rate and a relationship between the tem make a starch non-aggregative. The heating temperature for a perature and the viscosity of the object is recorded. That is, thermal inhibition treatment is preferably higher than about starch granules undergo Swelling with heating, while mani 100° C. The heat treatment temperature is preferably about festation of viscosity and an increase in Viscosity arise in the 200° C. or lower. The heating temperature for a thermal amylograph. Then, when the Swelling of the starch granules US 2013/0022711 A1 Jan. 24, 2013

becomes to maximum, the Viscosity also reaches a peak. This more, particularly preferably about 450 BU or more, most Viscosity is called maximum viscosity. Furtherheating causes preferably about 500 BU or more, for example, about 550 BU collapse of the starch granules and simultaneously causes a or more, about 570 BU or more, about 600 BU or more, or decrease in Viscosity. This degree of the decrease in Viscosity about 650 BU or more. In a specific embodiment, it is possible is called breakdown. A viscosity curve obtained by this amy to make the maximum viscosity of the enzyme-treated wheat lograph varies depending on the origin and production starch granules of the present invention when measured by an method of the starch, and is a measuring method showing amylograph under the above conditions to about 660 BU or features of the starch. more, about 670 BU or more, or about 700 BU or more. The 0279 For example, the measurement by the amylograph is maximum viscosity of the enzyme-treated wheat starch gran carried out as follows. A starch Suspension is prepared in 450 ules of the present invention when measured by an amylo ml of water so as to obtain a predetermined amount of graph under the above conditions can be, for example, about enzyme-treated Starch granules (for example, the concentra 900 BU or less, about 850 BU or less, about 800 BU or less, tion of a wheat starch is 8.5% by weight, the concentration of or about 750 BU or less. a corn starch is 7.0% by weight, and the concentration of a 0282 For example, regarding the corn starch, a maximum cassava starch is 6.0% by weight, on the dry matter basis), put Viscosity of the native corn starch when measured by an in a sample container, and then warmed to 50° C. while amylograph under the above conditions is from about 400 BU rotating them. Then the suspension is heated to 95°C. at 1.5° to about 500 BU. On the other hand, in the case where the C./min and maintained at 95°C. for 15 minutes, followed by enzyme-treated Starch granules of the present invention are cooling at 1.5°C./min. The measurement is carried out using prepared from an untreated corn starch and have not been an amylograph VISCOGRAPH-E manufactured by Bra Subjected to either a chemical modification or a physical bender Inc. under the conditions of a rotation number of a treatment, a maximum viscosity of the enzyme-treated corn sample container of 75 rpm and a measuring cartridge of 700 starch granules of the present invention when measured by an cmg. Wherein, the viscosity reached to a peak is regarded as amylograph under the above conditions may be preferably a maximum viscosity, and a difference between this maxi about 250 BU or more, more preferably about 270 BU or mum viscosity and a viscosity at the point after maintaining at more, particularly preferably about 300 BU or more, most 95° C. for 15 minutes is regarded as breakdown. This differ preferably about 350 BU or more, for example, about 400 BU ence is also called as a breakdown viscosity. When the differ or more, about 420 BU or more, about 440 BU or more, or ence between the maximum viscosity and the Viscosity at the about 450 BU or more. The maximum viscosity of the point after maintaining at 95°C. for 15 minutes is less than enzyme-treated corn starch granules of the present invention 100 BU, it is said that the starch has no “breakdown'. when measured by an amylograph under the above conditions 0280. In the case where the enzyme-treated starch gran can be, for example, about 600 BU or less, about 550 BU or ules of the present invention are prepared from an untreated less, about 520 BU or less, or about 500 BU or less. starch and have not been subjected to either a chemical modi 0283 For example, regarding the cassava starch, a maxi fication or a physical treatment, it is preferred that the mum viscosity of the native cassava starch when measured by enzyme-treated Starch granules of the present invention have an amylograph under the above conditions is from about 700 a maximum viscosity which accounts for about 50% or more BU to about 800 BU. On the other hand, in the case where the (more preferably about 60% or more, particularly preferably enzyme-treated Starch granules of the present invention are about 70% or more, and most preferably about 80% or more, prepared from an untreated cassava starch and have not been about 90% or more, or about 100% or more) of the maximum Subjected to either a chemical modification or a physical Viscosity of the untreated Starch, when measured by an amy treatment, a maximum viscosity of the enzyme-treated cas lograph under the above conditions. There is no particular sava starch granules of the present invention when measured upper limit of the maximum viscosity of the enzyme-treated by an amylograph under the above conditions may be prefer starch of the present invention. For example, the maximum ably about 500 BU or more, more preferably about 520 BU or viscosity of the enzyme-treated starch of the present inven more, particularly preferably about 530 BU or more, most tion can be about 300% or less, about 250% or less, about preferably about 550 BU or more, for example, about 600 BU 200% or less, about 150% or less, about 110% or less, and or more, about 620 BU or more, about 630 BU or more, or about 100% or less of the maximum viscosity of the untreated about 650 BU or more. The maximum viscosity of the starch, when measured by an amylograph under the above enzyme-treated cassava starch granules of the present inven conditions. For example, it is preferred that the enzyme tion when measured by an amylograph under the above con treated wheat Starch can form a gel having a viscosity which ditions can be, for example, about 900 BU or less, about 850 accounts for 70% or more and 200% or less (more preferably BU or less, about 800 BU or less, or about 770 BU or less. 80% or more and 200% or less) of the viscosity of the 0284. In the case where the enzyme-treated starch gran untreated wheat starch. ules of the present invention are prepared from an untreated 0281 For example, regarding the wheat starch, a maxi starch and have not been subjected to either a chemical modi mum viscosity of the native wheat starch when measured by fication or a physical treatment, the enzyme-treated Starch an amylograph under the above conditions is from about 550 granules of the present invention have breakdown when mea BU to about 650 BU. On the other hand, in the case where the Sured by an amylograph. Some conventional starches have no enzyme-treated Starch granules of the present invention are breakdown, whereas, the enzyme-treated Starch granules of prepared from an untreated wheat starch and have not been the present invention have breakdown. Subjected to either a chemical modification or a physical 0285 For example, in the case where the untreated starch treatment, a maximum viscosity of the enzyme-treated wheat is a wheat starch, a corn starch or a cassava starch and neither starch granules of the present invention when measured by an a chemical modification nor a physical treatment is carried amylograph under the above conditions may be preferably out, the obtained enzyme-treated Starch has a breakdown about 400 BU or more, more preferably about 420 BU or viscosity of about 100 BU or more. US 2013/0022711 A1 Jan. 24, 2013 22

0286. In the case where the untreated starch is a wheat treatment, it is preferred that the enzyme-treated wheat starch starch and neither a chemical modification nor a physical has a rupture stress which accounts for 110% or more and treatment is carried out, the breakdown viscosity of the 300% or less of the rupture stress of the untreated wheat obtained enzyme-treated starch is preferably about 100 BU or starch, or has a Young's modulus which accounts for 110% or more, more preferably about 120 BU or more, still more more and 500% or less (110% or more and 330% or less in an preferably about 130 BU or more, and most preferably about embodiment) of the Young's modulus of the untreated wheat 150 BU or more. In the case where the untreated starch is a starch. wheat starch and neither a chemical modification nor a physi 0292. In the case where the enzyme-treated starch gran cal treatment is carried out, although there is no particular ules of the present invention are prepared from an untreated upper limit of the breakdown viscosity of the obtained corn Starch and have not been Subjected to either a chemical enzyme-treated starch, the breakdown viscosity of the modification or a physical treatment, it is preferred that the obtained enzyme-treated Starch can be, for example, about enzyme-treated corn starch has a rupture stress which 500 BU or less, about 450 BU or less, about 400 BU or less, accounts for 110% or more and 300% or less of the rupture about 350 BU or less, or about 300 BU or less. stress of the untreated corn starch, or has a Young's modulus 0287. In the case where the untreated starch is a corn starch which accounts for 110% or more and 500% or less (330% or and neither a chemical modification nor a physical treatment less in an embodiment) of the Young's modulus of the is carried out, the breakdown viscosity of the obtained untreated corn starch. enzyme-treated starch is preferably about 100 BU or more, 0293. In the case where the enzyme-treated starch gran more preferably about 110 BU or more, still more preferably ules of the present invention are prepared from an untreated about 120 BU or more, and most preferably about 150 BU or cassava starch and have not been Subjected to either a chemi more. In the case where the untreated Starch is a corn starch cal modification or a physical treatment, it is preferred that the and neither a chemical modification nor a physical treatment enzyme-treated cassava starch has a rupture stress which is carried out, although there is no particular upper limit of the accounts for 110% or more and 300% or less of the rupture breakdown viscosity of the obtained enzyme-treated starch, stress of the untreated cassava starch, or has a Young's modu the breakdown viscosity of the obtained enzyme-treated lus which accounts for 110% or more and 500% or less (33.0% starch can be, for example, about 300 BU or less, about 290 or less in an embodiment) of the untreated cassava starch. BU or less, about 280 BU or less, 200 BU or less, about 190 0294. In the case where the enzyme-treated starch gran BU or less, or about 180 BU or less. ules of the present invention are prepared from an untreated 0288. In the case where the untreated starch is a cassava wheat starch and have not been subjected to either a chemical starch and neither a chemical modification nor a physical modification or a physical treatment, and the untreated Starch treatment is carried out, the breakdown viscosity of the is a wheat starch, the rupture stress of the obtained enzyme obtained enzyme-treated starch is preferably about 300 BU or treated starch is preferably about 150 g or more, more pref more, more preferably about 320 BU or more, still more erably about 160 g or more, still more preferably about 170 g preferably about 330 BU or more, and most preferably about or more, particularly preferably about 180 g or more, and 350 BU or more. In the case where the untreated starch is a most preferably about 200 g or more. In the case where the cassava starch and neither a chemical modification nor a untreated Starch is a wheat starch, although there is no par physical treatment is carried out, although there is no particu ticular upper limit of the rupture stress of the obtained lar upper limit of the breakdown viscosity of the obtained enzyme-treated starch, the rupture stress of the obtained enzyme-treated starch, the breakdown viscosity of the enzyme-treated Starch can be, for example, about 450 g or obtained enzyme-treated Starch can be, for example, about less, about 440 g or less, about 430 g or less, about 420 g or 550 BU or less, about 540 BU or less, about 530 BU or less, less, about 410 g or less, or about 400 g or less. about 500 BU or less, about 480 BU or less, or about 470 BU 0295. In the case where the untreated starch is a corn starch or less. and neither a chemical modification nor a physical treatment 0289 (3.2 Gel Forming Ability) is carried out, the rupture stress of the obtained enzyme 0290. It is well known that when the concentration of a treated starch is preferably about 210 g or more, more pref starch of a starch paste reaches a predetermined concentration erably about 220g or more, still more preferably about 230 g or more, a starch gel is formed by cooling it. Similarly to the or more, and most preferably about 240 g or more, and, in one Viscosity, physical properties of this starch gel vary depend embodiment, is 250 g or more. In the case where the untreated ing on the origin and production method of the starch, and the starch is a corn starch and neither a chemical modification nor starch is used in various foods taking features of this gelling a physical treatment is carried out, although there is no par physical properties into consideration. Some methods of ticular upper limit of the rupture stress of the obtained measuring physical properties of the gel are practically used, enzyme-treated starch, the rupture stress of the obtained and one of them is a method of measuring using a rheometer. enzyme-treated Starch can be, for example, about 450 g or The gel forming ability can be measured by the following less, about 440 g or less, about 430 g or less, about 420 g or method using a rheometer. For example, a starch paste is filled less, about 410 g or less, or about 400 g or less. in a casing, heated, and then refrigerated for 16 hours or 21 0296. In the case where the untreated starch is a cassava days (for example, at about 5°C.) and, after returning to room starch and neither a chemical modification nor a physical temperature (for example, at about 25°C.), physical proper treatment is carried out, the rupture stress of the obtained ties of the gel are measured by a rheometer. enzyme-treated starch is preferably about 55g or more, more 0291. The specific measuring method using a rheometer is preferably about 60 g or more, still more preferably about 65 as described in the aforementioned 1.2.2. In the case where gor more, and most preferably about 70g or more. In the case the enzyme-treated Starch granules of the present invention where the untreated Starch is a cassava starch and neither a are prepared from an untreated wheat starch and have not chemical modification nor a physical treatment is carried out, been subjected to eithera chemical modification or a physical although there is no particular upper limit of the rupture stress US 2013/0022711 A1 Jan. 24, 2013

of the obtained enzyme-treated starch, the rupture stress of enzyme-treated starch has breakdown (about 100 BU or the obtained enzyme-treated Starch can be, for example, more), and a rupture stress of about 210 to about 450 (g) about 150 g or less, about 140 g or less, about 130 g or less, (about 220 to about 450 (g) in one embodiment) or aYoung's about 120g or less, about 110 g or less, or about 100g or less. modulus of about 6,000,000 to about 9,000,000 (dyn/cm). 0297. In the case where the untreated starch is a wheat 0302) In a specific embodiment, in the case where the starch and neither a chemical modification nor a physical untreated Starch is a cassava starch and neither a chemical treatment is carried out, the Young's modulus of the obtained modification nor a physical treatment is carried out, the enzyme-treated starchis preferably about 5.0x10° dyn/cm or obtained enzyme-treated starch has breakdown (about 100 more, more preferably about 5.2x10° dyn/cm or more, still BU or more), and a rupture stress of about 55 to about 150 (g) more preferably about 5.4x10° dyn/cm or more, and most or a Young's modulus of about 520,000 to about 2,700,000 preferably about 5.6x10° dyn/cm or more. In the case where (dyn/cm) (about 520,000 to about 1,600,000 (dyn/cm) in the untreated Starch is a wheat starch and neither a chemical one embodiment). modification nor a physical treatment is carried out, although 0303 Also, in the case where a chemically modified starch there is no particular upper limit of the Young's modulus of or a physically treated Starch is used as starch granules, or in the obtained enzyme-treated starch, the Young's modulus of the case where a chemical modification or a physical treat the obtained enzyme-treated Starch can be, for example, ment is carried out after an enzymatic treatment, an improve about 8.0x10° dyn/cm or less, about 7.5x10° dyn/cm or less, ment in gel forming ability can be obtained similarly to the about 7.0x10° dyn/cm or less, about 6.9x10° dyn/cm or less, above. about 6.8x10° dyn/cm or less, or about 6.7x10° dyn/cm or (0304 (3.3 Enzyme-Treated Starch in which Hydroxyl less. Groups at Positions 2, 3 and 6 of Glucose Residues are not 0298. In the case where the untreated starch is a corn starch Modified) and neither a chemical modification nor a physical treatment 0305. In the case where the enzyme-treated starch gran is carried out, the Young's modulus of the obtained enzyme ules of the present invention are prepared from an untreated treated starch is preferably about 6.0x10° dyn/cm or more, starch, a physically treated Starch or a bleached starch and more preferably about 6.2x10' dyn/cm or more, still more have not been Subjected to chemical modification, since the preferably about 6.3x10° dyn/cm or more, and most prefer enzyme-treated Starch of the present invention is not Sub ably about 6.5x10° dyn/cm or more. In the case where the jected to an artificial chemical treatment, hydroxyl groups at untreated Starch is a corn starch and neither a chemical modi the positions 2,3 and 6 of glucose residues are not modified as fication nor a physical treatment is carried out, although there compared with a native starch (i.e., untreated starch). A is no particular upper limit of the Young's modulus of the starch, in which hydroxyl groups at the positions 2, 3 and 6 of obtained enzyme-treated starch, the Young's modulus of the glucose residues are modified, refers to a modified starch obtained enzyme-treated Starch can be, for example, about (also referred to as a chemically modified starch) subjected to 9.0x10° dyn/cm or less, about 8.9x10° dyn/cm or less, about so-called chemical modification by an industrial process. 8.8x10° dyn/cm or less, about 8.7x10° dyn/cm or less, about According to the ministerial ordinance to revise a part of the 8.6x10° dyn/cm or less, or about 8.5x10° dyn/cm or less. Ordinance For Enforcement of the Food Sanitation Act noti 0299. In the case where the untreated starch is a cassava fied in Ministry of Health and Welfare Notification No. 485 starch and neither a chemical modification nor a physical dated Oct. 1, 2008, the following 11 items of modified treatment is carried out, the Young's modulus of the obtained starches will be dealt as an additive: enzyme-treated starchis preferably about 5.2x10 dyn/cm or 0306 acetylated distarch adipate: more, more preferably about 5.4x10 dyn/cm or more, still 0307 acetylated oxidized starch; more preferably about 5.6x10' dyn/cm or more, and most 0308 acetylated distarch phosphate: preferably about 5.8x10 dyn/cm or more. In the case where 0309 starch sodium octenyl succinate: the untreated Starch is a cassava starch and neither a chemical 0310 starch acetate; modification nor a physical treatment is carried out, although 0311 oxidized starch; there is no particular upper limit of the Young's modulus of 0312 hydroxypropyl distarch phosphate: the obtained enzyme-treated starch, the Young's modulus of 0313 hydroxypropyl starch; the obtained enzyme-treated Starch can be, for example, 0314 distarch phosphate: about 2.7x10° dyn/cm or less, about 2.5x10'dyn/cm or less, 0315 monostarch phosphate; and about 2.4x10° dyn/cm or less, about 2.3x10'dyn/cm or less, 0316 phosphated distarch phosphate. In Ministry of about 2.2x10° dyn/cm or less, about 2.0x10° dyn/cm or less, Health and Welfare Notification No. 485, a method for a about 1.8x10" dyn/cm or less, about 1.6x10'dyn/cm or less, purity test of these starches is described. Therefore, it is about 1.5x10° dyn/cm or less, about 1.4x10° dyn/cm or less, possible to judge that a sample starch is not a starch Subjected about 1.3x10° dyn/cm or less, about 1.2x10'dyn/cm or less, to a chemical modification, for example, by analyzing various or about 1.1x10° dyn/cm or less. chemical Substances in the sample starch, such as adipic acid 0300. In a specific embodiment, in the case where the groups, acetyl groups, and carboxyl groups in accordance untreated Starch is a wheat starch and neither a chemical with a method for a purity test of the above various modified modification nor a physical treatment is carried out, the starches described in Ministry of Health and Welfare Notifi obtained enzyme-treated starch has breakdown (about 100 cation No. 485 dated Oct. 1, 2008, comparing with the results BU or more), and a rupture stress of about 150 to about 450 of analysis of a raw material native starch carried out for (g) or a Young's modulus of about 5,000,000 to about 8,000, comparison reference, and confirming there is no increase in 000 (dyn/cm). the content of corresponding various chemical Substances. 0301 In a specific embodiment, in the case where the Particularly, it is possible to judge that a sample starch is not untreated Starch is a corn starch and neither a chemical modi a starch Subjected to a chemical modification, by measuring fication nor a physical treatment is carried out, the obtained the content of adipic acid groups, the content of acetyl groups, US 2013/0022711 A1 Jan. 24, 2013 24 the content of carboxyl groups, the content of vinyl acetate, ing foods such as whipping cream and ice cream; and sauces the content of octenyl Succinic acid groups, the content of Such as meat sauce, foods are not entirely in a gel form but hydroxypropyl groups, and the content of propylene chloro contain a micro starch gel, and are therefore included in the hydrins, and confirming that the contents of them do not starch gel-containing food of the present invention. Also, increase as compared with those of the raw material native bakeries and Western-style confectioneries are included in starch. It is preferred to use the content of adipic acid groups, the starch gel-containing food of the present invention since the content of acetyl groups, the content of carboxyl groups, they contain a starch gel with the decreased water content the content of octenyl Succinic acid groups, the content of which was obtained by once forming a gel during the produc hydroxypropyl groups, and the content of propylene chloro tion process, and baking the gel. hydrins as evaluation criteria. It is recognized that a bleached 0322. In a specific embodiment, the food of the present starch Subjected to a bleaching treatment using sodium invention can be prepared by using enzyme-treated Starch hypochlorite is distributed as a food. It is also possible to granules. The starch produced by the method of the present judge this bleached starch by measuring the content of car invention can be utilized in the same application as in a boxyl groups using a method for a purity test similar to that in conventional starch. By utilizing the enzyme-treated Starch of the above oxidized starch. The chemical modified starch the present invention in a food, physical properties and tex other than the above modified starches of 11 items cannot be ture of the food are altered. The enzyme-treated starch of the used in a food since it is not recognized by the Food Sanitation present invention can be used in almost all of compositions Law of JAPAN. Therefore, the chemically modified starch for eating and drinking or compositions for food additives other than the above 11 items are not basically used in JAPAN prepared by utilizing a conventional starch. and are not distributed. Accordingly, practically, in the case of 0323. In the food of the present invention, any material confirming whether or not hydroxyl groups at the 2-, 3- and used usually in the objective composition and food can be 6-positions of a glucose residue of the starch of the invention used as long as an excellent effect obtained by the enzyme of the present application are modified, it is not necessary to treated Starch granules is not impaired. In a preferred embodi confirm whether or not a chemical modification other than the ment, the starch of the present invention forms a gel in the above chemical modification has been subjected. food of the present invention. 0317. In the present description, in the case where 0324. In the case where the enzyme-treated starch of the "hydroxyl groups at the positions 2, 3 and 6 of glucose resi present invention is utilized in a high moisture content type dues are not modified, it is preferred that all hydroxyl groups food, it imparts a body, imparts natural elasticity by a strong at the positions 2, 3 and 6 of glucose residues are not modi gel forming ability, and also imparts appropriate smooth tex fied. However, in the case where hydroxyl groups are sub ture in mouth. The high moisture content type food refers to jected to Some modification in a natural State, Some modifi a food in which the amount of moisture per 100g of the edible cations may be contained. In this case, based on the total portion is more than 40 g in a state at the time of eating. number of hydroxyl groups at the positions 2, 3 and 6 of Examples of the high moisture content type food include, for glucose residues, preferably about 70% or more, more pref example, traditional Japanese-style confectioneries, fat or erably about 80% or more, still more preferably about 90% or oil-containing foods, gelatinous foods, fish meat and animal more, particularly preferably about 95% or more, about 96% meat processed foods, Salsa and sauces, and noodles. or more, about 97% or more, about 98% or more, about 99% 0325 In the case where the enzyme-treated starch of the or more, or about 99.5%, and most preferably about 100% of present invention is utilized in a low moisture content type hydroxyl groups are not modified. food, it is possible to impart smooth texture with nice melt in 0318 (4. Food of the Present Invention) mouth. The low moisture content type food refers to a food in 0319. In a specific embodiment, the food of the present which the amount of moisture per 100 g of the edible portion invention is a food produced by a method including the steps is 40 g or less in a state at the time of eating. Examples of the of treating starch granules with an enzyme at a temperature of low moisture content type food include, for example, baker about 10°C. or higher and about 70° C. or lower to obtain an ies, Western-style confectioneries, fried foods, and jelly can enzyme-treated Starch; mixing a food material, the enzyme dies. treated Starch and water to obtain a mixture; heating the 0326. As described above, the high moisture content type mixture thereby gelatinizing the enzyme-treated Starch in the food and the low moisture content type food are classified by mixture; and cooling the mixture containing the gelatinized the amount of moisture, per 100g of the edible portion, which enzyme-treated Starch thereby gelling the starch to obtain a is more than 40 g, or 40g or less. Provided that the food in starch gel-containing food. which the amount of moisture per 100 g of the edible portion 0320 In another specific embodiment, the food of the is around 40 g (35 to 50 g) may sometimes exhibit contradict present invention is a heat cooked Starch-containing food ing physical properties depending on the form, even in the containing an enzyme-treated Starch having a high viscosity case of the same amount of moisture. Also, in the case of the and a gel forming ability. In another specific embodiment, the fried food, it is judged by the amount of moisture for the starch-containing food of the present invention is a food pro coating partin which core food materials have been removed. duced by a method including mixing a food material with the 0327. The amounts of water per 100g of the edible portion enzyme-treated Starch, and then heating the mixture. of various foods are exemplified below (extract from Stan 0321. In the present description, the starch gel-containing dard Tables of Food Composition in Japan (Fifth Revised and food refers to a food containing a starch gel. If the food Enlarged Edition); the number in parenthesis denotes the contains the starch gel, it is not necessary for the food to be amount of moisture): entirely in a gel form. For example, in the case of gelatinous (1) Bakeries: white table bread (38.0 g), hard biscuit (2.6 g), foods such as custard pudding; and gel-like traditional Japa pie pastry (32.0 g), Eisei-boro (4.5 g); nese-style confectioneries such as kudzu Starch cake and (2) Traditional Japanese-style confectioneries: Uiro (54.5 g), Uiro, entire foods form a gel. In the case offat or oil-contain Kudzu-manju (45.0 g), Daifuku-mochi (41.5 g); US 2013/0022711 A1 Jan. 24, 2013

(3) Western-style confectioneries: sponge cake (32.0 g). the present invention preferably accounts for about 50% by Kasutera (25.6 g), hot cake (40.0 g); weight or more, more preferably about 60% by weight or (4) Fat- or oil-containing foods: whipping cream (milk fat more, still more preferably about 70% by weight or more, type, 42.1 g), whipping cream (vegetable fattype, 41.2g), ice further preferably about 80% by weight or more, particularly creams (ice milk: 65.6 g., lactic ice: 60.4 g); preferably about 90% by weight or more, and most preferably (5) Gelatinous foods: custard pudding (74.1 g); 100% by weight, of a usual use amount of the starch. In other (6) Fish meat and animal meat processed foods: Sumaki word, most preferably, the entire amount of a conventional kamaboko (75.8 g), Yakinuki-kamaboko (72.8g), Vienna sau starch is replaced by the enzyme-treated starch of the present sage (53.0 g); invention. (7) Salsa and sauces: Worcester sauce (61.7 g), meat sauce 0330 (5. Method for Producing Starch Gel-Containing (78.8 g), Thousand Island dressing (44.1 g); and Food) (8) Jelly candies: jelly candy (16 g), jellybeans (9.5 g). 0331. In a specific embodiment, the method for producing 0328 By using the enzyme-treated starch of the present a starch gel-containing food of the present invention includes invention in these foods, the following physical properties, the steps of treating starch granules with an enzyme at a for example, are improved as compared with the case of using temperature of about 10° C. or higher and about 70° C. or a conventional starch: lower to obtain an enzyme-treated Starch; mixing a food (1) In bakeries, textures with softness and nice melt in mouth material, the enzyme-treated Starch and water to obtain a is imparted. Examples of bakeries include breads, cookies, mixture; heating the mixture thereby gelatinizing the biscuits, pizza crusts, pie pastries, corn cups for ice creams, enzyme-treated Starch in the mixture; and cooling the mixture pastries of Monaka, and puff of cream puff. containing the gelatinized enzyme-treated Starch thereby gel (2) In traditional Japanese-style confectioneries, appropriate ling the starch to obtain a starch gel-containing food. In the hardness, brittleness, and appropriate viscoelasticity and production of a conventional food, starch granules are not Sticky textures are imparted. Examples of traditional Japa Subjected to an enzymatic treatment during the food produc nese-style confectioneries include kudzu Starch cake, Uiro, tion process. and Manju. (3) In Western-style confectioneries, improvement of Vol 0332 The step of treating starch granules with an enzyme umes by nice puffing after baking as well as Soft and nice at a temperature of about 10°C. or higher and about 70° C. or textures are imparted. Examples of Western-style confection lower to obtain an enzyme-treated Starch can be carried out as described in detail in the aforementioned “2.2 Enzyme Reac eries include sponge cake, chiffon cake, Kasutera, Madeleine, tion'. As described above, the starch granules can be starch financier, pound cake, and Swiss roll. granules of an untreated Starch, a physically treated Starch or (4) In fat- or oil-containing foods, while maintaining appro a chemically modified starch. In the case where it is prefer priate body and shape retention, nice melt in mouth and able to obtain an enzyme-treated Starch which is dealt as a Smooth texture is imparted. Examples of the fat- or oil-con food, starch granules are starch granules of an untreated taining food include custard cream, flour paste, filling, whip starch, a physically treated Starch orableached starch, and the ping cream, and ice creams (for example, ice milk, lactic ice). starch granules are not subjected to a chemical modification (5) In gelatinous foods, while maintaining sticky and chewy, in any stage until a starch gel-containing food is obtained nice melt in mouth and Smooth texture is imparted. Examples using the starch granules. In a specific embodiment, the starch of the gelatinous food include jelly, pudding, mousse, yogurt, granule is a starch granule of an untreated Starch or a physi and goma-dofu. cally treated Starch, the step of chemically modifying the (6) In fish meat and animal meat processed foods, while enzyme-treated starch is further included, and the chemically having elasticity with nice chewiness, the effect of small modified enzyme-treated starch is mixed with the food mate change with time is imparted. Examples of fish meat and meat rial and water. In another specific embodiment, the starch processed foods include kamaboko and sausage. granules are starch granules of an untreated Starch or a chemi (7) In Salsa and sauces, while having nice body and shape cally modified Starch, the step of physically treating the retention, properties of being less likely to cause dropping enzyme-treated Starch is further included, and the physically because of nice adhesion onto a food as well as less stickiness treated enzyme-treated starch is mixed with the food material and thread-forming sensation, and Smooth textures are and water. imparted. Examples of Salsa and sauces include Salsa for split and broiled fish, glaze formitarashi dango, fruit sauce, white 0333 Next, a mixture is obtained by mixing a food mate sauce, and dressing. rial, the enzyme-treated Starch and water. A mixing method (8) In fried foods, crispy light texture is imparted. Examples and a mixing ratio of the food material, the enzyme-treated of fried foods include tempura and fried prawn. starch and water can be a mixing method and a mixing ratio in (9) In noodles, Sticky texture rich in chewiness is imparted. accordance with a usual method for producing the objective Examples of noodles include udon, Somen, hiyamugi, Chi food. nese noodles, buckwheat noodles, macaroni, and spaghetti. 0334 Next, the mixture is heated thereby gelatinizing the (10) Injelly candies, while having appropriate elasticity, nice enzyme-treated Starch in the mixture. The heating can be heat melt in mouth and Smooth texture is imparted. Examples of cooking. Heating can be carried out under the same condi jelly candies include jelly candy and jellybeans. tions as those of heat cooking in a usual method for producing 0329. In the food of the present invention, the enzyme the objective food. treated Starch of the present invention can be used in the same 0335) Next, the mixture containing the gelatinized amount as that of the starch which has been conventionally enzyme-treated Starch is cooled, thereby gelling the starch to used in the food. A conventional starch may be used as a part obtain a starch gel-containing food. Cooling may be carried and the remainder may be replaced by the enzyme-treated out by leaving the mixture after heating at room temperature, starch of the present invention. The enzyme-treated starch of or carried out in a refrigerator or the like. US 2013/0022711 A1 Jan. 24, 2013 26

0336. In the embodiment in which the enzyme-treated 0348 SEQ ID NO: 7 is a nucleotide sequence encoding starch of the present invention is used, the food of the present isoamylase derived from Flavobacterium sp.: invention can be produced in the same method as in the case 0349 SEQID NO: 8 is an amino acid sequence of isoamy of a usual starch, except that the enzyme-treated Starch is lase derived from Flavobacterium sp.: used. The method for producing the starch-containing food of 0350 SEQ ID NO: 9 is a nucleotide sequence encoding the present invention includes the steps of adding an enzyme isoamylase derived from Pseudomonas amyloderamosa, treated Starch to a food material and mixing them; and heat 0351 SEQ ID NO: 10 is an amino acid sequence of cooking the mixture. isoamylase derived from Pseudomonas amyloderamosa, 0337 The enzyme-treated starch of the present invention 0352 SEQID NO: 11 is a nucleotide sequence encoding has excellent viscosity and gel forming ability as compared C-glucosidase derived from Aspergillus niger, with a conventional untreated starch. Therefore, by adding 0353 SEQID NO: 12 is an amino acid sequence of O-glu the enzyme-treated starch of the present invention to the food cosidase derived from Aspergillus niger, material, mixing them and heat cooking the mixture, this 0354 SEQID NO: 13 is a nucleotide sequence encoding enzyme-treated Starch is gelatinized and then cooled to form cyclodextrin glucanotransferase derived from Paenibacillus a gel. Accordingly, the obtained heat cooked material is pro macerans (also classified as Bacillus macerans). vided with excellent physical properties (for example, excel 0355 SEQID NO: 14 is an amino acid sequence of cyclo lent body, natural elasticity, nice melt in mouth, Smooth tex dextrin glucanotransferase derived from Paenibacillus mac ture, Sticky texture, and soft texture) as compared with the erans (also classified as Bacillus macerans). heat cooked material in which a conventional untreated Starch is used. In the present description, the food may also be a EXAMPLES beverage. 0356. Next, the present invention will be described in 0338. In the present description, “heat cooking refers to more detail by way of Examples, but the present invention is heating of a mixture of a food material and a starch. Prefer not limited to these Examples. It is noted that in the Examples, ably, heat cooking can be heating at a collapse temperature or a viscosity was measured by an amylograph from Brabender higher of starch granules. For example, the mixture of a food Inc., and physical properties of a gel were measured by a material and a starch can be heated at about 70° C. or higher, rheometer from Rheotech Inc. about 80° C. or higher, about 90° C. or higher or about 95°C. 0357 (1. Method for Measurement of Viscosity) or higher. Preferably, heat cooking is carried out at a tempera 0358) A viscosity was measured by the following method. ture at which excess denaturation of the food material and the A starch suspension was adjusted in 450 ml of water so that starch does not arise. For example, the mixture of a food the concentration of a wheat starch was 8.5% by weight, the material and a starch can be heated at about 200° C. or lower, concentration of a corn starch was 7.0% by weight and the about 150° C. or lower, about 130° C. or lower or about 110° concentration of a cassava starch was 6.0% by weight, on the C. or lower. Heat cooking is carried out for a usual heat dry matter basis and, put in a sample container, and then cooking time of the objective food. warmed to 50° C. while rotating them. Then the suspension 0339 Heat cooking is preferably carried out in the pres was heated to 95°C. at 1.5°C/min, and maintained at 95°C. ence of Some degree of moisture. Usually, when starch gran for 15 minute, followed by cooling at 1.5°C/min. The mea ules are heated in the presence of a predetermined amount or Surement was carried out using an amylograph VISCO more of water, Swelling arises, transparency increases and GRAPH-Emanufactured by Brabender Inc. under the condi viscosity increases. When the food material contains too tions of a rotation number of the sample container of 75 rpm much moisture, it is not necessary to add water to the mixture and a measuring cartridge of 700 cmg. Wherein, the Viscosity of a food material and a starch. However, when the food reached to a peak was regarded as a maximum viscosity, and material contains Small amount of moisture, it is preferred to a difference between this maximum viscosity and a viscosity add water to the mixture of a food material and a starch. It is soon after maintaining at 95°C. for 15 minutes was regarded noted that in the case of a food which does not contain food as breakdown. materials other than water and a starch, like a Sugar-free 0359 (2. Method for Measurement of Physical Properties kuZuyu, water is considered as the food material. of a Gel) 0340 Heat cooking can be a part of the method for pro 0360 Physical properties of a gel were measured by the ducing the objective food. For example, in the case of a following method. A starch paste was prepared so that the gelatinous food such as jelly, it can be heat cooled after concentration of the starch was 20% by weight on the dry cooking at a temperature of, for example, about 5 to 10°C. matter basis, and then filled in a Krehalon casing having a 0341 (6. Explanation of Sequence) folding width of 45 mm. This starch paste filled in the casing 0342 SEQ ID NO: 1 is a nucleotide sequence encoding was heated to 90° C. at 1° C./min and maintained at 90° C. for C.-amylase derived from Aspergillus Oryzae, 30 minutes. Then, the starch paste was left to cool in a con 0343 SEQID NO: 2 is an amino acid sequence of C.-amy stant-temperature water bath at 20°C. for 30 minutes and then lase derived from Aspergillus Oryzae, cooled to 5°C. in a refrigerator. After cooling, it was refrig eration storaged at 5°C. for 16 hours, then it was left at room 0344 SEQ ID NO: 3 is a nucleotide sequence encoding temperature (about 25°C.) for 4 hours to return the tempera C.-amylase derived from Aspergillus niger, ture of it to room temperature, and then physical properties of 0345 SEQID NO: 4 is an amino acid sequence of C.-amy the gel were measured by a rheometer (RT-2010J-CW) manu lase derived from Aspergillus niger, factured by Rheotech Inc. The measurement was carried out 0346 SEQ ID NO: 5 is a nucleotide sequence encoding under the measurement conditions of the rheometer: a test amyloglucosidase derived from Aspergillus niger, item: a rupture test; a height of a sample: 25 mm; and a 0347 SEQID NO: 6 is an amino acid sequence of amy movement rate (rupture rate) of a sample: 6 cm/min, using an loglucosidase derived from Aspergillus niger, adapter of a spherical jig for measurement viscosity (p5 (diam US 2013/0022711 A1 Jan. 24, 2013 27 eter: 5 mm, area: 19.635 mm). At the measurement, the TABLE 1-continued hardness of the starch gel was evaluated by a rupture stress (g) and a Young's modulus (dyn/cm). Sample 1 Sample 2 Physical (those reacted (those reacted Untreated 0361 (3. Method for Measurement of Degradation Ratio properties in a form of in a form of wheat of Starch Granules) of Gel liquid) solid) starch 0362. A degradation ratio of starch granules was measured by the following method. The amount(g) of released reducing modulus because of dyn/cm’ dyn/cm. Sugars contained in the Supernatant obtained by centrifuga being too soft tion (at 3,000 rpm for 5 minutes) of a starch degraded sus pension after Subjecting to an enzyme reaction was measured 0365. When an enzyme was allowed to act on the starch by a phenol-sulfuric acid method. The percentage of the after gelling, a remarkable decrease in Viscosity was con amount of the released reducing Sugars to the total amount of firmed in the obtained sample 1, and the sample did not retain the starch (g) before Subjecting to an enzyme reaction was Viscosity physical properties of the starch anymore and thus a determined. gel was not formed. On the other hand, when the enzyme is Degradation ratio (%) of starch granules={(amount reacted keeping the starch granules as it is, it was confirmed (g) of released reducing Sugars)x100}/{(total that the obtained sample 2 retained viscosity physical prop amount (g) of starch before enzymatic reaction)}. Equation 1 erties of the starch and a hard gel was formed. Comparative Example 1 Test Example 1 0366 Viscosity characteristics were analyzed by the amy Comparison Between Liquid Reaction and Solid lograph and the rheometer without Subjecting an untreated Reaction native wheat starch to an enzymatic treatment. The results are shown in Table 2-2. 1. Liquid Reaction Examples 1-1 and 1-2 0363 To 15 g (dry weight) of an untreated native wheat starch, 250 g of ion-exchange water was added and, after 0367 To 400 g of an untreated native wheat starch, 900 g adjusting the pH of the mixture to 5.0, the mixture was of ion-exchange water was added to prepare a starch Suspen warmed in a boiled water bath to prepare a starch paste in sion. After adjusting the pH of the suspension to 5.0, 0.1% by which a starch was completely dissolved. To this starch paste, weight (based on starch solid content) of C.-amylase 0.1% by weight (based on starch solid content) of C-amylase (“Biozyme A' derived from Aspergillus Oryzae, manufac (origin: Aspergillus Oryzae) was added to make the total tured by Amano Enzyme Inc.; optimum pH of 5.0) was added weight to 300 g, and stirred at 50° C. to carry out an enzyme and stirred at 50° C. for 1 hour to carry out an enzyme reaction reaction. After 30 minutes, this was left in a boiled water bath and resulted in preparation of a sample having a degradation for 10 minutes to deactivate the enzyme and thereby obtained ratio of about 5% (Example 1-1). Using a similar amount of a sample 1. Using the obtained sample 1, physical properties the enzyme, stirring was carried out at 50° C. for 3 hours to of the gel were measured and evaluated by a rupture stress and prepare a sample having a degradation ratio of about 10% a Young's modulus. (Example 1-2). After completion of the reaction, an enzyme treated starch was recovered by centrifugal filtration and blow 2. Solid Reaction drying. Viscosity characteristics of the obtained enzyme treated Starch were analyzed by the amylograph and the rhe 0364 To 400 g of an untreated native wheat starch (dry ometer. After completion of the reaction, a degradation ratio weight), 900 g of ion-exchange water was added to prepare a was determined using a part of the reaction solution. The starch Suspension. After adjusting the pH of the Suspension to results are shown in Table 2-2. 5.0, 1% by weight (based on starch solid content) of C.-amy lase (origin: Aspergillus Oryzae) was added and stirred at 50° Example 1-3 C. for 18 hours to carry out an enzyme reaction. After comple tion of the reaction, an enzyme-treated Starch was recovered 0368 To 400 g of an untreated native wheat starch, 900 g by centrifugal filtration and blow drying. To 15 g (dry weight) of ion-exchange water was added to prepare a starch Suspen of this enzyme-treated Starch, ion-exchange water was added sion. After adjusting the pH of the suspension to 5.0, 1% by to make the total weight to 300g. This was warmed in a boiled weight (based on Starch Solid content) of C.-amylase water bath to prepare a starch paste in which the starch was (“Biozyme A' derived from Aspergillus Oryzae, manufac completely dissolved, as a sample 2. Using the obtained tured by Amano Enzyme Inc.; optimum pH of 5.0) was added sample 2, physical properties of the gel were measured and and stirred at 50° C. for 18 hours to carryout an enzyme evaluated by a rupture stress and a Young's modulus. reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration and blow dry ing. Viscosity characteristics of the obtained enzyme-treated TABLE 1. starch were analyzed by the amylograph and the rheometer. Sample 1 Sample 2 Also, after completion of the reaction, a degradation ratio was Physical (those reacted (those reacted Untreated properties in a form of in a form of wheat determined using a part of the reaction Solution. The results of Gel liquid) solid) starch are shown in Table 2-2. Rupture Not measurable 206 g 141 g Example 2A StreSS since gel is Young's not formed 5,533,540 4,601,665 0369 To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspen US 2013/0022711 A1 Jan. 24, 2013 28 sion. After adjusting the pH of the suspension to 5.0, 1% by similar amount of the enzyme, stirring was carried out at 50° weight (based on Starch Solid content) of C-amylase C. for 1.5 hours to prepare a sample having a degradation ratio (AMYLEX A3’ derived from Aspergillus niger, manufac of about 10%. After completion of the reaction, an enzyme tured by DANISCO; optimum pH of 5.0) was added and treated starch was recovered by centrifugal filtration and blow stirred at 50° C. for 18 hours to carry out an enzyme reaction. drying. Viscosity characteristics of the obtained enzyme After completion of the reaction, an enzyme-treated Starch treated Starch were analyzed by the amylograph and the rhe was recovered by centrifugal filtration and blow drying. Vis ometer. The results are shown in Table 2-2. cosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and the rheometer. After Examples 3A-1 and 3A-2 completion of the reaction, a degradation ratio was deter 0373) To 400 g of an untreated native wheat starch, 900 g mined using a part of the reaction solution. The results are of ion-exchange water was added to prepare a starch Suspen shown in Table 2-2. sion. After adjusting the pH of the suspension to 5.0, 0.1% by weight (based on starch Solid content) of amyloglucosidase Example 2B (AMG' derived from Aspergillus niger, manufactured by 0370. To 400 g of an untreated native wheat starch, 900 g Novozymes; optimum pH of 4.5) was added and stirred at 50° of ion-exchange water was added to prepare a starch Suspen C. for 2 hours to prepare a sample having a degradation ratio sion. After adjusting the pH of the suspension to 5.0, 1% by of about 5% (Example 3A-1). Also, 0.5% by weight (based on weight (based on starch Solid content) of C-amylase (“Sum starch Solid content) of the similar enzyme was added and izyme AS” derived from Aspergillus niger, manufactured by stirred at 50° C. for 3 hours to prepare a sample having a SHIN NIHON CHEMICALS Corporation; optimum pH of degradation ratio of about 10% (Example 3A-2). After 4.5) was added and stirred at 50° C. for 18 hours to carry out completion of the reaction, an enzyme-treated Starch was an enzyme reaction. After completion of the reaction, an recovered by centrifugal filtration and blow drying. Viscosity enzyme-treated starch was recovered by centrifugal filtration characteristics of the obtained enzyme-treated starch were and blow drying. Viscosity characteristics of the obtained analyzed by the amylograph and the rheometer. enzyme-treated Starch were analyzed by the amylograph and the rheometer. After completion of the reaction, a degradation Example 3A-3 ratio was determined using a part of the reaction Solution. The 0374. To 400 g of an untreated native wheat starch, 900 g results are shown in Table 2-2. of ion-exchange water was added to prepare a starch Suspen sion. After adjusting the pH of the suspension to 5.0, 1% by Comparative Examples 2, 3, 4-3, and 5 to 6 weight (based on starch Solid content) of amyloglucosidase 0371 To 400 g of an untreated native wheat starch, 900 g (AMG' derived from Aspergillus niger, manufactured by of ion-exchange water was added to prepare a starch Suspen Novozymes; optimum pH of 4.5) was added and stirred at 50° sion. After adjusting the pH of the suspension to 5.0, 1% by C. for 18 hours to carry out an enzyme reaction. After comple weight (based on starch Solid content) of C-amylase ("C.- tion of the reaction, an enzyme-treated Starch was recovered amylase 3A derived from Bacillus subtilis, manufactured by by centrifugal filtration and blow drying. Viscosity character HBI, Inc.; optimum pH of 5.9; Comparative Example 2), istics of the obtained enzyme-treated starch were analyzed by C.-amylase (“Novamyl derived from Bacillus subtilis, manu the amylograph and the rheometer. After completion of the factured by Novo; optimum pH of 5.0; Comparative Example reaction, a degradation ratio was determined using a part of 3), C.-amylase (“C.-amylase' derived from Bacillus amy the reaction solution. The results are shown in Table 2-2. loliquefaciens, manufactured by Sigma-Aldrich Corporation; optimum pH of 6.0; Comparative Example 4-3), C-amylase Example 3B (“TERMAMYL 120L derived from Bacillus licheniformis, 0375. To 400 g of an untreated native wheat starch, 900 g manufactured by Novo: optimum pH of 6.0; Comparative of ion-exchange water was added to prepare a starch Suspen Example 5), or O-amylase (“Maltogenase L' derived from sion. After adjusting the pH of the suspension to 5.0, 1% by Bacillus sp., manufactured by Novo: optimum pH of 5.0; weight (based on starch Solid content) of amyloglucosidase Comparative Example 6) was added and stirred at 50° C. for (“OPTIDEXL-400' derived from Aspergillus niger, manu 18 hours to carry out an enzyme reaction. After completion of factured by Genencor; optimum pH of 4.4) was added and the reaction, an enzyme-treated Starch was recovered by cen stirred at 50° C. for 18 hours to carry out an enzyme reaction. trifugal filtration and blow drying. Viscosity characteristics of After completion of the reaction, an enzyme-treated Starch the obtained enzyme-treated starch were analyzed by the was recovered by centrifugal filtration and blow drying. Vis amylograph and the rheometer. After completion of the reac cosity characteristics of the obtained enzyme-treated Starch tion, a degradation ratio was determined by a part of the were analyzed by the amylograph and the rheometer. After reaction solution. The results are shown in Table 2-2. completion of the reaction, a degradation ratio was deter mined using a part of the reaction solution. The results are Comparative Examples 4-1 and 4-2 shown in Table 2-2. 0372 To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspen Example 3C sion. After adjusting the pH of the suspension to 5.0, 0.01% 0376 To 400 g of an untreated native wheat starch, 900 g by weight (based on starch Solid content) of C.-amylase ("C.- of ion-exchange water was added to prepare a starch Suspen amylase' derived from Bacillus amyloliquefaciens, manufac sion. After adjusting the pH of the suspension to 5.0, 1% by tured by Sigma-Aldrich Corporation; optimum pH of 6.0) weight (based on starch Solid content) of amyloglucosidase was added and stirred at 50° C. for 30 minutes to prepare a (“DIAZYMEX4NP” derived from Aspergillus niger, manu sample having a degradation ratio of about 5%. Also, using a factured by DANISCO; optimum pH of 4.0) was added and US 2013/0022711 A1 Jan. 24, 2013 29 stirred at 50° C. for 18 hours to carry out an enzyme reaction. tured by Sigma-Aldrich Corporation; optimum pH of 2.5) After completion of the reaction, an enzyme-treated Starch was added and stirred at 50° C. for 18 hours to carry out an was recovered by centrifugal filtration and blow drying. Vis enzyme reaction. After completion of the reaction, an cosity characteristics of the obtained enzyme-treated Starch enzyme-treated starch was recovered by centrifugal filtration were analyzed by the amylograph and the rheometer. After and blow drying. Viscosity characteristics of the obtained completion of the reaction, a degradation ratio was deter enzyme-treated Starch were analyzed by the amylograph and mined using a part of the reaction solution. The results are the rheometer. After completion of the reaction, a degradation shown in Table 2-2. ratio was determined using a part of the reaction solution. The results are shown in Table 2-2. Example 3D Example 4 0377 To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspen 0381 To 400 g of an untreated native wheat starch, 900 g sion. After adjusting the pH of the suspension to 5.0, 1% by of ion-exchange water was added to prepare a starch Suspen weight (based on starch Solid content) of amyloglucosidase sion. After adjusting the pH of the suspension to 5.0, 0.1% by (“glucoamylase Amano SD' derived from Aspergillus weight (based on starch solid content) of isoamylase (“Re niger, manufactured by Amano Enzyme Inc., optimum pH of agent” derived from Pseudomonas amyloderamosa, manu 4.5) was added and stirred at 50° C. for 18 hours to carry out factured by Sigma-Aldrich Corporation; optimum pH of 3.0) an enzyme reaction. After completion of the reaction, an was added and stirred at 50° C. for 18 hours to carry out an enzyme-treated starch was recovered by centrifugal filtration enzyme reaction. After completion of the reaction, an and blow drying. Viscosity characteristics of the obtained enzyme-treated starch was recovered by centrifugal filtration enzyme-treated Starch were analyzed by the amylograph and and blow drying. Viscosity characteristics of the obtained the rheometer. After completion of the reaction, a degradation enzyme-treated Starch were analyzed by the amylograph and ratio was determined using a part of the reaction Solution. The the rheometer. After completion of the reaction, a degradation results are shown in Table 2-2. ratio was determined using a part of the reaction solution. The results are shown in Table 2-2. Example 3E) Example 5A 0378. To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspen 0382 To 400 g of an untreated native wheat starch, 900 g sion. After adjusting the pH of the suspension to 5.0, 1% by of ion-exchange water was added to prepare a starch suspen weight (based on starch Solid content) of amyloglucosidase sion. After adjusting the pH of the suspension to 5.0, 1% by (“Gluczyme AF6” derived from Rhizopus niveus, manufac weight (based on starch Solid content) of C-glucosidase tured by Amano Enzyme Inc.; optimum pH of 4.5) was added (“Transglucosidase L Amano’ derived from Aspergillus and stirred at 50° C. for 18 hours to carry out an enzyme niger, manufactured by Amano Enzyme Inc., optimum pH of reaction. After completion of the reaction, an enzyme-treated 5.0) was added and stirred at 50° C. for 18 hours to carry out starch was recovered by centrifugal filtration and blow dry an enzyme reaction. After completion of the reaction, an ing. Viscosity characteristics of the obtained enzyme-treated enzyme-treated starch was recovered by centrifugal filtration starch were analyzed by the amylograph and the rheometer. and blow drying. Viscosity characteristics of the obtained After completion of the reaction, a degradation ratio was enzyme-treated Starch were analyzed by the amylograph and determined using a part of the reaction Solution. The results the rheometer. After completion of the reaction, a degradation are shown in Table 2-2. ratio was determined using a part of the reaction solution. The results are shown in Table 2-2. Example 3F Example 5B 0379 To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspen 0383 To 400 g of an untreated native wheat starch, 900 g sion. After adjusting the pH of the suspension to 5.0, 1% by of ion-exchange water was added to prepare a starch Suspen weight (based on starch Solid content) of amyloglucosidase sion. After adjusting the pH of the suspension to 5.0, 1% by (“Sumizyme” derived from Rhizopus oryzae, manufactured weight (based on starch Solid content) of C-glucosidase by SHIN NIHON CHEMICALS Corporation; optimum pH (“Transglucosidase L-500 derived from Aspergillus niger, of 5.0) was added and stirred at 50° C. for 18 hours to carry out manufactured by Genencor, optimum pH of 5.0) was added an enzyme reaction. After completion of the reaction, an and stirred at 50° C. for 18 hours to carry out an enzyme enzyme-treated starch was recovered by centrifugal filtration reaction. After completion of the reaction, an enzyme-treated and blow drying. Viscosity characteristics of the obtained starch was recovered by centrifugal filtration and blow dry enzyme-treated Starch were analyzed by the amylograph and ing. Viscosity characteristics of the obtained enzyme-treated the rheometer. After completion of the reaction, a degradation starch were analyzed by the amylograph and the rheometer. ratio was determined using a part of the reaction Solution. The After completion of the reaction, a degradation ratio was results are shown in Table 2-2. determined using a part of the reaction Solution. The results are shown in Table 2-2. Comparative Example 8 0380. To 400 g of an untreated native wheat starch, 900 g Comparative Examples 10 and 11 of ion-exchange water was added to prepare a starch Suspen 0384. To 400 g of an untreated native wheat starch, 900 g sion. After adjusting the pH of the suspension to 5.0, 1% by of ion-exchange water was added to prepare a starch Suspen weight (based on starch Solid content) of amyloglucosidase sion. After adjusting the pH of the suspension to 5.0, 1% by (“Reagent” derived from Candida tsukubaensis, manufac weight (based on starch solid content) of B-amylase (“OPTI US 2013/0022711 A1 Jan. 24, 2013 30

MALT BBA derived from barley, manufactured by Genen enzyme reaction. After completion of the reaction, an cor; optimum pH of 5.0) or pullulanase (“Pullulanase' enzyme-treated starch was recovered by centrifugal filtration derived from Klebsiella pneumoniae, manufactured by and blow drying. Viscosity characteristics of the obtained Amano Enzyme Inc.; optimum pH of 6.0) was added and enzyme-treated Starch were analyzed by the amylograph and stirred at 50° C. for 18 hours to carry out an enzyme reaction. the rheometer. Also, after completion of the reaction, a deg After completion of the reaction, an enzyme-treated Starch radation ratio was determined by a part of the reaction solu was recovered by centrifugal filtration and blow drying. Vis tion. The results are shown in Table 3-2. cosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and the rheometer. After Comparative Example 14 completion of the reaction, a degradation ratio was deter 0389. To 400 g of an untreated native corn starch, 900 g of mined using a part of the reaction solution. The results are ion-exchange water was added to prepare a starch Suspension. shown in Table 2-2. After adjusting the pH of the suspension to 5.0, 1% by weight Comparative Example 12 (based on starch solid content) of B-amylase (“OPTIMALT BBA derived from barley, manufactured by Genencor, opti 0385 Viscosity characteristics were analyzed by the amy mum pH of 5.0) was added and stirred at 50° C. for 18 hours lograph and the rheometer without Subjecting a corn starch to to carry out an enzyme reaction. After completion of the an enzymatic treatment. The results are shown in Table 3-2. reaction, an enzyme-treated Starch was recovered by centrifu gal filtration and blow drying. Viscosity characteristics of the Example 6 obtained enzyme-treated Starch were analyzed by the amylo 0386 To 400 g of an untreated native corn starch, 900 g of graph and the rheometer. After completion of the reaction, a ion-exchange water was added to prepare a starch Suspension. degradation ratio was determined using a part of the reaction After adjusting the pH of the suspension to 5.0, 1% by weight solution. The results are shown in Table 3-2. (based on starch solid content) of C.-amylase (“Biozyme A” derived from Aspergillus Oryzae, manufactured by Amano Example 8A Enzyme Inc.; optimum pH of 5.0) was added and stirred at 0390 To 400 g of an untreated native corn starch, 900 g of 50° C. for 18 hours to carryout an enzyme reaction. After ion-exchange water was added to prepare a starch Suspension. completion of the reaction, an enzyme-treated Starch was After adjusting the pH of the suspension to 5.0, 0.1% by recovered by centrifugal filtration and blow drying. Viscosity weight (based on starch solid content) of isoamylase (“Re characteristics of the obtained enzyme-treated starch were agent” derived from Pseudomonas amyloderamosa, manu analyzed by the amylograph and the rheometer. Also, after factured by Sigma-Aldrich Corporation; optimum pH of 3.0) completion of the reaction, a degradation ratio was deter was added and stirred at 50° C. for 18 hours to carry out an mined using a part of the reaction solution. The results are enzyme reaction. After completion of the reaction, an shown in Table 3-2. enzyme-treated starch was recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained Comparative Examples 13-1 and 13-2 enzyme-treated Starch were analyzed by the amylograph and 0387 To 400 g of an untreated native corn starch, 900 g of the rheometer. After completion of the reaction, a degradation ion-exchange water was added to prepare a starch Suspension. ratio was determined using a part of the reaction solution. The After adjusting the pH of the suspension to 5.0, 0.01% by results are shown in Table 3-2. weight (based on starch Solid content) (Comparative Example 13-1) of C-amylase (“Reagent” derived from Bacillus amy Example 8B loliquefaciens, manufactured by Sigma-Aldrich Corporation; 0391 To 400 g of an untreated native corn starch, 900 g of optimum pH of 6.0) was added and stirred at 50° C. for 30 ion-exchange water was added to prepare a starch Suspension. minutes, or 1% by weight (based on starch solid content) After adjusting the pH of the suspension to 5.0, 1% by weight (Comparative Example 13-2) of the C-amylase was added (based on starch solid content) of C-amylase (AMYLEX and stirred at 50° C. for 18 hours, to carryout an enzyme A3' derived from Aspergillus niger, manufactured by reaction. After completion of the reaction, an enzyme-treated DANISCO: optimum pH of 5.0) was added and stirred at 50° starch was recovered by centrifugal filtration and blow dry C. for 18 hours to carry out an enzyme reaction. After comple ing. Viscosity characteristics of the obtained enzyme-treated tion of the reaction, an enzyme-treated Starch was recovered starch were analyzed by the amylograph and the rheometer. by centrifugal filtration and blow drying. Viscosity character Also, after completion of the reaction, a degradation ratio was istics of the obtained enzyme-treated starch were analyzed by determined by a part of the reaction solution. The results are the amylograph and the rheometer. After completion of the shown in Table 3-2. reaction, a degradation ratio was determined using a part of the reaction solution. The results are shown in Table 3-2. Examples 7-1 and 7-2 Example 8C 0388 To 400 g of an untreated native corn starch, 900 g of ion-exchange water was added to prepare a starch Suspension. 0392 To 400 g of an untreated native corn starch, 900 g of After adjusting the pH of the suspension to 5.0, 0.5% by ion-exchange water was added to prepare a starch Suspension. weight (based on starch solid content) (Example 7-1) of amy After adjusting the pH of the suspension to 5.0, 1% by weight loglucosidase (AMG' derived from Aspergillus niger, (based on starch Solid content) of C-glucosidase (“Transglu manufactured by Novozymes; optimum pH of 4.5) was added cosidase L Amano derived from Aspergillus niger, manu and stirred at 50° C. for 3 hours, or 1% by weight (based on factured by Amano Enzyme Inc.; optimum pH of 5.0) was starch Solid content) (Example 7-2) of the amyloglucosidase added and stirred at 50° C. for 18 hours to carry out an enzyme was added and stirred at 50° C. for 18 hours, to carry out an reaction. After completion of the reaction, an enzyme-treated US 2013/0022711 A1 Jan. 24, 2013

starch was recovered by centrifugal filtration and blow dry radation ratio was determined by a part of the reaction solu ing. Viscosity characteristics of the obtained enzyme-treated tion. The results are shown in Table 4-2. starch were analyzed by the amylograph and the rheometer. After completion of the reaction, a degradation ratio was Comparative Example 17 determined using a part of the reaction Solution. The results 0397 To 400g of an untreated native cassava starch, 900 g are shown in Table 3-2. of ion-exchange water was added to prepare a starch Suspen Comparative Example 15 sion. After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch solid content) of B-amylase (“OPTI 0393 Viscosity characteristics were analyzed by the amy MALT BBA derived from barley, manufactured by Genen lograph and the rheometer without Subjecting an untreated cor; optimum pH of 5.0) was added and stirred at 50° C. for 18 native cassava starch to an enzymatic treatment. The results hours to carry out an enzyme reaction. After completion of the are shown in Table 4-2. reaction, an enzyme-treated Starch was recovered by centrifu gal filtration and blow drying. Viscosity characteristics of the Example 9 obtained enzyme-treated Starch were analyzed by the amylo 0394. To 400g of an untreated native cassava starch, 900 g graph and the rheometer. After completion of the reaction, a of ion-exchange water was added to prepare a starch Suspen degradation ratio was determined using a part of the reaction sion. After adjusting the pH of the suspension to 5.0, 1% by solution. The results are shown in Table 4-2. weight (based on Starch Solid content) of C-amylase (“Biozyme A' derived from Aspergillus Oryzae, manufac Example 1 1A tured by Amano Enzyme Inc.; optimum pH of 5.0) was added 0398. To 400g of an untreated native cassava starch, 900 g and stirred at 50° C. for 18 hours to carry out an enzyme of ion-exchange water was added to prepare a starch Suspen reaction. After completion of the reaction, an enzyme-treated sion. After adjusting the pH of the suspension to 5.0, 0.1% by starch was recovered by centrifugal filtration and blow dry weight (based on starch solid content) of isoamylase (“Re ing. Viscosity characteristics of the obtained enzyme-treated agent” derived from Pseudomonas amyloderamosa, manu starch were analyzed by the amylograph and the rheometer. factured by Sigma-Aldrich Corporation; optimum pH of 3.0) After completion of the reaction, a degradation ratio was was added and stirred at 50° C. for 18 hours to carry out an determined using a part of the reaction Solution. The results enzyme reaction. After completion of the reaction, an are shown in Table 4-2. enzyme-treated starch was recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained Comparative Examples 16-1 and 16-2 enzyme-treated Starch were analyzed by the amylograph and 0395. To 400g of an untreated native cassava starch, 900 g the rheometer. After completion of the reaction, a degradation of ion-exchange water was added to prepare a starch Suspen ratio was determined using a part of the reaction solution. The sion. After adjusting the pH of the suspension to 5.0, 0.01% results are shown in Table 4-2. by weight (based on starch Solid content) (Comparative Example 16-1) of C.-amylase (“Reagent” derived from Bacil Example 11B lus amyloliquefaciens, manufactured by Sigma-Aldrich Cor 0399. To 400g of an untreated native cassava starch, 900 g poration; optimum pH of 6.0) was added and stirred at 50° C. of ion-exchange water was added to prepare a starch Suspen for 30 minutes, or 1.0% by weight (based on starch solid sion. After adjusting the pH of the suspension to 5.0, 1% by content) (Comparative Example 16-2) of the C-amylase was weight (based on Starch Solid content) of C.-amylase added and stirred at 50° C. for 18 hours, to carryout an (AMYLEX A3' derived from Aspergillus niger, manufac enzyme reaction. After completion of the reaction, an tured by DANISCO; optimum pH of 5.0) was added and enzyme-treated starch was recovered by centrifugal filtration stirred at 50° C. for 18 hours to carry out an enzyme reaction. and blow drying. Viscosity characteristics of the obtained After completion of the reaction, an enzyme-treated Starch enzyme-treated Starch were analyzed by the amylograph and was recovered by centrifugal filtration and blow drying. Vis the rheometer. Also, after completion of the reaction, a deg cosity characteristics of the obtained enzyme-treated Starch radation ratio was determined by a part of the reaction solu were analyzed by the amylograph and the rheometer. After tion. The results are shown in Table 4-2. completion of the reaction, a degradation ratio was deter mined using a part of the reaction solution. The results are Examples 10-1 and 10-2 shown in Table 4-2. 0396 To 400g of an untreated native cassava starch, 900 g of ion-exchange water was added to prepare a starch Suspen Example 11C sion. After adjusting the pH of the suspension to 5.0, 0.5% by 0400. To 400g of an untreated native cassava starch, 900 g weight (based on starch solid content) (Example 10-1) of of ion-exchange water was added to prepare a starch Suspen amyloglucosidase (AMG' derived from Aspergillus niger, sion. After adjusting the pH of the suspension to 5.0, 1% by manufactured by Novozymes; optimum pH of 4.5) was added weight (based on starch Solid content) of C-glucosidase and stirred at 50° C. for 3 hours, or 1% by weight (based on (“Transglucosidase L Amano’ derived from Aspergillus starch Solid content) (Example 10-2) of the amyloglucosidase niger, manufactured by Amano Enzyme Inc., optimum pH of was added and stirred at 50° C. for 18 hours, to carry out an 5.0) was added and stirred at 50° C. for 18 hours to carry out enzyme reaction. After completion of the reaction, an an enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration enzyme-treated starch was recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained and blow drying. Viscosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and enzyme-treated Starch were analyzed by the amylograph and the rheometer. Also, after completion of the reaction, a deg the rheometer. After completion of the reaction, a degradation US 2013/0022711 A1 Jan. 24, 2013 32 ratio was determined using a part of the reaction Solution. The into a yeast host DBY746, and obtained a transformant results are shown in Table 4-2. capable of growing in a tryptophan-free culture medium by 04.01. As a result, in Examples 1 to 11C, it was confirmed complementation of the tryptophan requirement. This trans that a novel starch having both a high viscosity and strong gel formant was inoculated in 100 ml of a synthetic culture characteristics can be prepared by Subjecting to an enzymatic medium consisting of 2% glucose, 0.67% yeast nitrogen base, treatment. Further, using C-amylase derived from Bacillus 24 mg/l L-uracil, 24 mg/l L-histidine and 36 mg/l L-leucine at amyloliquefaciens used in Comparative Examples 4-1 and pH 5.7 and then cultured with shaking at 30°C. for 120 hours. 4-2, 13-1, 13-2, 16-1, 16-2 at a degradation ratio of 40% or less, it was impossible to prepare a starch having both a high 04.09. The supernatant obtained by centrifugation (at Viscosity and strong gel characteristics, which is the object of 5,000 rpm for 10 minutes) of the culture was concentrated the present inventors. Therefore, it has been proved that the using a hollow fiber type UF membrane module having a starch developed by the present inventors is a Substance molecular weight cut-off of 10,000 to prepare C.-amylase which is different from the starch prepared by Japanese Patent derived from Aspergillus niger. This C.-amylase has an amino Gazette No. 2,615,398. acid sequence of SEQID NO: 4. Example 12A Example 12C 0402 (Method for Preparation of C.-Amylase derived 0410 (Method for Preparation of Amyloglucosidase from Aspergillus Oryzae) Derived from Aspergillus niger) 0403. A double stranded DNA was chemically synthe sized by adding an EcoRI recognition site (GAATTC) to both 0411. A double stranded DNA was chemically synthe terminals of a base sequence of SEQID NO: 1 in the sequence sized by adding an EcoRI recognition site (GAATTC) to both listing. This synthetic DNA was completely cleaved by a terminals of a base sequence of SEQID NO. 5 in the sequence restriction enzyme EcoRI, mixed with pYCDE1 (Method in listing. This synthetic DNA was completely cleaved by a Enzymology, 101, pp. 192-201 (1983)) which was previously restriction enzyme EcoRI, mixed with pYCDE1 (Method in completely cleaved by EcoRI, and then ligation was carried Enzymology, 101, pp. 192-201 (1983)) which was previously out. E. coli TG1 was transformed with the ligation reaction completely cleaved by EcoRI, and then ligation was carried Solution and a transformant into which a synthetic gene was out. E. coli TG1 was transformed with the ligation reaction properly introduced was selected. Plasmid pYAMY1 held by solution and a transformant into which a synthetic gene was this transformant was prepared. properly introduced was selected. Plasmid pYGLU1 held by 04.04. In accordance with the method of Ito et al. (J. bac this transformant was prepared. terial.., Vol. 153, 163-168 (1983)), pYAMY1 was introduced 0412. In accordance with the method of Ito et al. (J. bac into a yeast host DBY746, and obtained a transformant terial, Vol. 153, 163-168 (1983)), pYGLU1 was introduced capable of growing in a tryptophan-free culture medium by into a yeast host DBY746, and obtained a transformant complementation of the tryptophan requirement. This trans capable of growing in a tryptophan-free culture medium by formant was inoculated in 100 ml of a synthetic culture complementation of the tryptophan requirement. This trans medium consisting of 2% glucose, 0.67% yeast nitrogen base, formant was inoculated in 100 ml of a synthetic culture 24 mg/l L-uracil, 24 mg/l L-histidine and 36 mg/l L-leucine at medium consisting of 2% glucose, 0.67% yeast nitrogen base, pH 5.7 and then cultured with shaking at 30°C. for 120 hours. 24 mg/l L-uracil, 24 mg/l L-histidine and 36 mg/l L-leucine at 04.05 The supernatant obtained by centrifugation (at pH 5.7 and then cultured with shaking at 30°C. for 120 hours. 5,000 rpm for 10 minutes) of the culture was concentrated using a hollow fiber type UF membrane module having a 0413. The supernatant obtained by centrifugation (at molecular weight cut-off of 10,000 to prepare C-amylase 5,000 rpm for 10 minutes) of the culture was concentrated derived from Aspergillus Oryzae. This C.-amylase has an using a hollow fiber type UF membrane module having a amino acid sequence of SEQID NO: 2. molecular weight cut-off of 10,000 to prepare amyloglucosi dase derived from Aspergillus niger. This amyloglucosidase Example 12B has an amino acid sequence of SEQID NO: 6. 0406 (Method for Preparation of C.-Amylase derived Example 12D from Aspergillus niger) 0407. A double stranded DNA was chemically synthe 0414 (Method for Preparation of Isoamylase Derived sized by adding an EcoRI recognition site (GAATTC) to both from Flavobacterium sp.) terminals of a base sequence of SEQID NO:3 in the sequence 0415. A double stranded DNA was chemically synthe listing. This synthetic DNA was completely cleaved by a sized by adding an EcoRI recognition site (GAATTC) to both restriction enzyme EcoRI, mixed with pYCDE1 (Method in terminals of a base sequence of SEQID NO: 7 in the sequence Enzymology, 101, pp. 192-201 (1983)) which was previously listing. This synthetic DNA was completely cleaved by a completely cleaved by EcoRI, and then ligation was carried restriction enzyme EcoRI, mixed with pYCDE1 (Method in out. E. coli TG1 was transformed with the ligation reaction Enzymology, 101, pp. 192-201 (1983)) which was previously Solution and a transformant into which a synthetic gene was completely cleaved by EcoRI, and then ligation was carried properly introduced was selected. Plasmid pYAMY2 held by out. E. coli TG1 was transformed with the ligation reaction this transformant was prepared. Solution and a transformant into which a synthetic gene was 0408. In accordance with the method of Ito et al. (J. bac properly introduced was selected. Plasmid pYISO1 held by terial, Vol. 153, 163-168 (1983)), pYAMY2 was introduced this transformant was prepared. US 2013/0022711 A1 Jan. 24, 2013

0416) In accordance with the method of Ito et al. (J. bac Example 12B-1 terial.., Vol. 153, 163-168 (1983)), pYISO1 was introduced into a yeast host DBY746, and obtained a transformant 0423 To 400 g of an untreated native wheat starch, 900 g capable of growing in a tryptophan-free culture medium by of ion-exchange water was added to prepare a starch Suspen complementation of the tryptophan requirement. This trans sion. After adjusting the pH of the suspension to 5.0, 1% by formant was inoculated in 100 ml of a synthetic culture weight (based on starch Solid content) of C.-amylase (derived medium consisting of 2% glucose, 0.67% yeast nitrogen base, from Aspergillus niger) prepared in Example 12B was added 24 mg/l L-uracil, 24 mg/l L-histidine and 36 mg/l L-leucine at and stirred at 50° C. for 18 hours to carry out an enzyme pH 5.7 and then cultured with shaking at 30°C. for 120 hours. reaction. After completion of the reaction, an enzyme-treated 0417. The supernatant obtained by centrifugation (at starch was recovered by centrifugal filtration and blow dry 5,000 rpm for 10 minutes) of the culture was concentrated ing. Viscosity characteristics of the obtained enzyme-treated using a hollow fiber type UF membrane module having a starch were analyzed by the amylograph and the rheometer. molecular weight cut-off of 10,000 to prepare isoamylase After completion of the reaction, a degradation ratio was derived from Flavobacterium sp. This isoamylase has an determined using a part of the reaction Solution. The results amino acid sequence of SEQID NO: 8. are shown in Table 5-2 below. Example 12E Example 12C-1 0418 (Method for Preparation of Isoamylase Derived 0424. To 400 g of an untreated native wheat starch, 900 g from Pseudomonas Amyloderamosa) of ion-exchange water was added to prepare a starch Suspen 0419. A double stranded DNA was chemically synthe sion. After adjusting the pH of the suspension to 5.0, 1% by sized by adding an EcoRI recognition site (GAATTC) to both weight (based on starch Solid content) of amyloglucosidase terminals of a base sequence of SEQID NO: 9 in the sequence (derived from Aspergillus niger) prepared in Example 12C listing. This synthetic DNA was completely cleaved by a was added and stirred at 50° C. for 18 hours to carry out an restriction enzyme EcoRI, mixed with pYCDE1 (Method in enzyme reaction. After completion of the reaction, an Enzymology, 101, pp. 192-201 (1983)) which was previously enzyme-treated starch was recovered by centrifugal filtration completely cleaved by EcoRI, and then ligation was carried and blow drying. Viscosity characteristics of the obtained out. E. coli TG1 was transformed with the ligation reaction enzyme-treated Starch were analyzed by the amylograph and Solution and a transformant into which a synthetic gene was the rheometer. After completion of the reaction, a degradation properly introduced was selected. Plasmid pYISO held by ratio was determined using a part of the reaction solution. The this transformant was prepared. results are shown in Table 5-2 below. 0420. In accordance with the method of Ito et al. (J. bac terial.., Vol. 153, 163-168 (1983)), pYISO2 was introduced Example 12D-1 into a yeast host DBY746, and obtained a transformant 0425 To 400 g of an untreated native wheat starch, 900 g capable of growing in a tryptophan-free culture medium by of ion-exchange water was added to prepare a starch Suspen complementation of the tryptophan requirement. This trans sion. After adjusting the pH of the suspension to 5.0, 1% by formant was inoculated in 100 ml of a synthetic culture weight (based on starch Solid content) of isoamylase (derived medium consisting of 2% glucose, 0.67% yeast nitrogen base, from Flavobacterium sp.) prepared in Example 12D was 24 mg/l L-uracil, 24 mg/l L-histidine and 36 mg/l L-leucine at added and stirred at 50° C. for 18 hours to carry out an enzyme pH 5.7 and then cultured with shaking at 30°C. for 120 hours. reaction. After completion of the reaction, an enzyme-treated 0421. The supernatant obtained by centrifugation (at starch was recovered by centrifugal filtration and blow dry 5,000 rpm for 10 minutes) of the culture was concentrated ing. Viscosity characteristics of the obtained enzyme-treated using a hollow fiber type UF membrane module having a starch were analyzed by the amylograph and the rheometer. molecular weight cut-off of 10,000 to prepare isoamylase After completion of the reaction, a degradation ratio was derived from Pseudomonas amyloderamosa. This isoamy determined using a part of the reaction Solution. The results lase has an amino acid sequence of SEQID NO: 10. are shown in Table 5-2 below. Example 12A-1 Example 12E-1 0422 To 400 g of an untreated native wheat starch, 900 g 0426 To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspen of ion-exchange water was added to prepare a starch Suspen sion. After adjusting the pH of the suspension to 5.0, 1% by sion. After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch Solid content) of C.-amylase (derived weight (based on starch Solid content) of isoamylase (derived from Aspergillus Oryzae) prepared in Example 12A was from Pseudomonas amyloderamosa) prepared in Example added and stirred at 50° C. for 18 hours to carryout an enzyme 12E was added and stirred at 50° C. for 18 hours to carry out reaction. After completion of the reaction, an enzyme-treated an enzyme reaction. After completion of the reaction, an starch was recovered by centrifugal filtration and blow dry enzyme-treated starch was recovered by centrifugal filtration ing. Viscosity characteristics of the obtained enzyme-treated and blow drying. Viscosity characteristics of the obtained starch were analyzed by the amylograph and the rheometer. enzyme-treated Starch were analyzed by the amylograph and After completion of the reaction, a degradation ratio was the rheometer. After completion of the reaction, a degradation determined using a part of the reaction Solution. The results ratio was determined using a part of the reaction solution. The are shown in Table 5-2 below. results are shown in Table 5-2 below. US 2013/0022711 A1 Jan. 24, 2013 34

TABLE 2-1 Summary of Names. Origins and Product Names of Enzymes used for Wheat Starch Example Name Origins Product name (Manufacturer) Comp. Ex. 1 Untreated wheat starch Example 1-1 C-amylase Aspergilius Oryzae Biozyme A (Amano Enzyme) Example 1-2 C-amylase Aspergilius Oryzae Biozyme A (Amano Enzyme) Example 1-3 C-amylase Aspergilius Oryzae Biozyme A (Amano Enzyme) Example 2A C-amylase Aspergilius niger AMYLEX A3 (DANISCO) Example 2B C-amylase Aspergilius niger Sumizyme AS (SHIN NIHON CHEMICALS Corporation) Comp. Ex. 2 C-amylase Bacilius subtiis C-amylase 3A (HBI) Comp. Ex. 3 C-amylase Bacilius subtiis Novamyl (Novo) Comp. Ex. 4-1 C-amylase Bacilius amyloiiquefaciens Reagent (Sigma-Aldrich Corporation) Comp. Ex. 4-2 C-amylase Bacilius amyloiiquefaciens Reagent (Sigma-Aldrich Corporation) Comp. Ex. 4-3 C-amylase Bacilius amyloiiquefaciens Reagent (Sigma-Aldrich Corporation) Comp. Ex. 5 C-amylase Bacilius licheniformis TERMAMYL 12OL (Novo) Comp. Ex. 6 C-amylase Bacilius sp. Maltogenase L (Novo) Example 3A-1 amyloglucosidase Aspergilius niger AMG (Novo) Example 3A-2 amyloglucosidase Aspergilius niger AMG (Novo) Example 3A-3 amyloglucosidase Aspergilius niger AMG (Novo) Example 3B amyloglucosidase Aspergilius niger OPTIDEX L-400 (Genencor) Example 3C amyloglucosidase Aspergilius niger DIAZYMEX4NP (DANISCO) Example 3D amyloglucosidase Aspergilius niger Glucoamylase Amano' SD (Amano Enzyme) Example 3E amyloglucosidase Rhizopus nivetis Gluczyme AF6 (Amano Enzyme) Example 3F amyloglucosidase Rhizopus oryzae Sumizyme (SHIN NIHON CHEMICALS Corporation) Comp. Ex. 8 amyloglucosidase Candidatsukubaensis Reagent (Sigma-Aldrich Corporation) Example 4 isoamylase Pseudamonas amyloderanosa Reagent (Sigma-Aldrich Corporation) Example 5A C-glucosidase Aspergilius niger Transglucosidase L'Amano (Amano Enzyme) Example SB C-glucosidase Aspergilius niger Transglucosidase L-500 (Genencor) Comp. Ex. 10 B-amylase Barley OPTIMALT BBA (Genencor) Comp. Ex. 11 pullulanase Klebsiella pneumoniae Pullulanase (Amano Enzyme) Example 13-1 CGTase Bacilius licheniformis Toruzyme 3.0L (Novo) Example 13-2 CGTase Paenibacilius macerans Cyclodextrin glucanotransferase Amano (Amano Enzyme) (Bacilius macerans) Comp, Ex. = Comparative Example

TABLE 2-2 Table 2-2. Summary of Results of Wheat Starch (Starch Concentration for Amylograph: 8.5% Maximum viscosity Rupture stress Young's modulus Degradation Measured Breakdown Measured Measured ratio value Relative viscosity value Relative value Relative Evaluation Example (%) (BU) % (%) (BU) (g) % (%) (dyn/cm) % (%) results Comp. Ex. 1 621 100 126 141 100 4,601,665 100 — Example 1-1 5 672 108 188 167 118 5,188,263 113 USable Example 1-2 8 707 114 221 185 131 5,490,949 119 Usable Example 1-3 19 738 119 279 2O6 146 5,533,540 120 USable Example 2A 15 880 142 365 211 150 5,465,779 119 Usable Example 2B 14 839 135 374 16S 117 5,484.457 119 Usable Comp. Ex. 2 33 398 64 241 5 1 637,600 14 Not usable Comp. Ex. 3 24 14 2 8 Not measurable since gel is not formed Not usable because of being too soft Comp. Ex. 4-1 5 148 24 118 Not measurable since gel is not formed Not usable because of being too soft Comp. Ex. 4-2 13 172 28 142 Not measurable since gel is not formed Not usable because of being too soft Comp. Ex. 4-3 46 658 106 3O2 34 24 1260,110 27 Not usable Comp. Ex. 5 29 535 86 271 37 26 1493,271 32 Not usable Comp. Ex. 6 2O 70 11 33 25 8 834,422 18 Not usable Example 3A-1 4 641 103 16S 218 155 5,520,234 120 USable Example 3A-2 15 719 116 229 263 187 5,890,552 128 Usable Example 3A-3 29 727 117 267 311 221 6,356,475 138. Usable Example 3B 2O 858 138 385 307 218 6,731,469 146 Usable Example 3C 26 873 141 394 313 222 6489,069 141 Usable Example 3D 2O 867 140 369 242 172 5,998.440 13.0 Usable Example 3E 42 806 130 407 283 2O1 5,581,328 121 USable Example 3F 43 808 130 403 286 203 5.941241 129 Usable Comp. Ex. 8 5 736 119 263 119 84 4,096,046 89 Not usable Example 4 6 828 133 3OO 297 211 6,987,728 152 Usable Example 5A 4 746 120 291 166 118 5,142,993 112 USable Example 5B 5 554 89 274 18O 128 6.418,528 139 USable US 2013/0022711 A1 Jan. 24, 2013 35

TABLE 2-2-continued Table 2-2. Summary of Results of Wheat Starch (Starch Concentration for Amylograph: 8.5% Maximum viscosity Rupture stress Young's modulus Degradation Measured Breakdown Measured Measured ratio value Relative viscosity value Relative value Relative Evaluation Example (%) (BU) % (%) (BU) (g) % (%) (dyn/cm) % (%) results Comp. Ex. 10 7 757 122 2S6 145 103 4,385,924 95 Not usable Comp. Ex. 11 4 6O1 97 245 140 99 4,534,673 99 Not usable Example 13-1 14 444 71 422 161 114 5,136,339 112 USable Example 13-2 12 475 76 460 159 113 5,291,915 115 Usable Comp, Ex. = Comparative Example

TABLE 3-1 Summary of Names. Origins and Product Names of Enzymes used for Corn Starch Example Name Origins Product name (Manufacturer) Comp. Ex. 12 Untreated corn starch Example 6 C-amylase Aspergilius Oryzae Biozyme A (Amano Enzyme) Comp. Ex. 13-1 C-amylase Bacilius amyloiquefaciens Reagent (Sigma-Aldrich Corporation) Comp. Ex. 13-2 C-amylase Bacilius amyloiquefaciens Reagent (Sigma-Aldrich Corporation) Example 7-1 amyloglucosidase Aspergilius niger AMG (Novo) Example 7-2 amyloglucosidase Aspergilius niger AMG (Novo) Comp. Ex. 14 3-amylase Barley OPTIMALT BBA (Genencor) Example 8A isoamylase Pseudomonas amyloderanosa Reagent (Sigma-Aldrich Corporation) Example 8B C-amylase Aspergilius niger AMYLEX A3 (DANISCO) Example 8C C-glucosidase Aspergilius niger Transglucosidase L'Amano (Amano Enzyme) Example 14 CGTase Bacilius licheniformis Toruzyme 3.0L (Novo) Comp, Ex. = Comparative Example

TABLE 3-2 Table 3-2: Summary of Results of Corn Starch (Starch Concentration for Amylograph: 7.0% Maximum viscosity Rupture stress Young's modulus Degradation Measured Breakdown Measured Measured ratio value Relative viscosity value Relative value Relative Evaluation Example (%) (BU) % (%) (BU) (g) % (%) (dyn/cm) % (%) results Comp. Ex. 12 476 100 182 171 100 5,603,029 100 — Example 6 8 425 89 161 230 135 6,054,798 108 USable Comp. Ex. 13-1 11 414 87 162 56 33 2,434,858 43 Not usable Comp. Ex. 13-2 40 383 8O 141 153 90 4,318,913 77 Not usable Example 7-1 11 455 96 153 381 223 7,805,888 139 USable Example 7-2 33 432 91 170 348 204 6.219,387 111 Usable Comp. Ex. 14 1 477 100 152 183 107 5,582,293 100 Not usable Example 8A 1 445 93 163 216 126 6,304,730 113 USable Example 8B 23 414 87 176 297 174 5,910,873 105 USable Example 8C 1 451 95 163 220 129 6,731.402 120 USable Example 14 7 285 60 273 222 130 6,546,236 117 Usable Comp, Ex. = Comparative Example

TABLE 4-1 Summary of Names. Origins and Product Names of Enzymes used for Cassava Starch Example Name Origins Product name (Manufacturer) Comp. Ex. 15 Untreated cassava starch Example 9 C-amylase Aspergilius Oryzae Biozyme A (Amano Enzyme) Comp. Ex. 16-1 C-amylase Bacilius amyloiquefaciens Reagent (Sigma-Aldrich Corporation) Comp. Ex. 16-2 C-amylase Bacilius amyloiquefaciens Reagent (Sigma-Aldrich Corporation) Example 10-1 amyloglucosidase Aspergilius niger AMG (Novo) Example 10-2 amyloglucosidase Aspergilius niger AMG (Novo) Comp. Ex. 17 3-amylase Barley OPTIMALT BBA (Genencor) Example 11A isoamylase Pseudomonas amyloderanosa Reagent (Sigma-Aldrich Corporation) US 2013/0022711 A1 Jan. 24, 2013 36

TABLE 4-1-continued Summary of Names. Origins and Product Names of Enzymes used for Cassava Starch Example Name Origins Product name (Manufacturer) Example 11B C-amylase Aspergilius niger AMYLEXA3(DANISCO) Example 11C C-glucosidase Aspergilius niger Transglucosidase L'Amano (Amano Enzyme) Example 15 CGTase Bacilius licheniformis Toruzyme 3.0L(Novo) Comp, Ex. = Comparative Example

TABLE 4-2 Table 4-2. Summary of Results of Cassava Starch (Starch Concentration for Amylograph: 6.0% Maximum Viscosity Rupture stress Young's modulus Degradation Measured Breakdown Measured Measured ratio value Relative viscosity value Relative value Relative Evaluation Example (%) (BU) % (%) (BU) (g) % (%) (dyn/cm) % (%) results Comp. Ex. 15 757 100 509 51 100 472,273 100 — Example 9 6 737 97 434 60 118 567,949 120 USable Comp. Ex. 16-1 11 271 36 2S6 21 41 225,310 48 Not usable Comp. Ex. 16-2 34 112 15 109 Not measurable since gel is not formed Not usable because of being too soft Example 10-1 11 704 93 419 71 139 715,243 151. Usable Example 10-2 28 660 87 388 115 225 1,390,964. 295 Usable Comp. Ex. 17 2 755 100 440 52 102 494,672 105 Not usable Example 11A 2 642 85 377 75 147 701,944 149 USable Example 11B 16 S61 74 328 89 175 969,841 205 USable Example 11C 2 638 84 374 70 137 663.407 140 USable Example 15 7 533 70 529 92 180 2,342,930 496 Usable Comp, Ex. = Comparative Example

TABLE 5-1 TABLE 5-1-continued Summary of Names, Origins and Product Names of Summary of Names, Origins and Product Names of Enzymes used when Enzyme prepared by Genetic Enzymes used when Enzyme prepared by Genetic Recombination is reacted with Wheat Starch Recombination is reacted with Wheat Starch Product name Product name Example Name Origins (Manufacturer) Example Name Origins (Manufacturer) Example 12C-1 amylogluco- Aspergilius niger SEQ ID NO: 6 sidase Comparative Untreated Example 12D-1 isoamylase Flavobacterium sp. SEQID NO: 8 Example 1 wheat starch Example 12E-1 isoamylase Pseudomonas SEQ ID NO: 10 Example 12A-1 C-amylase Aspergillus oryzae SEQID NO: 2 amyloderanosa Example 12B-1 C-amylase Aspergilius niger SEQ ID NO:4

TABLE 5-2

Table 5-2: Summary of Results when Enzyme prepared by Genetic Recombination is reacted with Wheat Starch (Starch Concentration for Amylograph: 8.5%) Maximum viscosity Rupture stress Young's modulus

Degradation Measured Breakdown Measured Measured ratio value Relative viscosity value Relative value Relative Evaluation Example (%) (BU) % (%) (BU) (g) % (%) (dyn/cm) % (%) results

Comparative 621 100 81 141 100 4,601,665 100 — Example 1 Example 12A-1 25 785 126 403 252 179 5,785,782 126 Usable Example 12B-1 15 797 128 309 281 199 6,712,299 146 Usable Example 12C-1 26 806 130 4O7 378 268 6,973,739 152 Usable Example 12D-1 6 781 126 306 285 202 6.425,189 140 Usable Example 12E-1 6 775 125 297 263 187 6,483,006 141 Usable US 2013/0022711 A1 Jan. 24, 2013 37

Trial Production Examples Comparative Trial Production Examples 1 to 4, which were hard and crunchy, and also had texture with poor melt in 0427 Next, the present invention will be described in mouth. In particular, the cookies of Trial Production more detail by way of Trial Production Examples, but the Examples 1-1 and 1-2 had very light texture and were readily present invention is not limited to the following Trial Produc edible. Regarding the dough at the time of shaping, both the tion Example. Unless otherwise specified, “parts' means doughs of Trial Production Examples 1-1 and 1-2 were very “parts by mass'. dry and non-sticky as compared with the doughs of Compara tive Trial Production Examples 1-1 to 1-4, and did not stick to Trial Production Example 1 hands, rolling pin and the like, and also showed very nice Preparation of Cookie operability. 0428. Among the formulations shown in Table 10 below, salt-free butter and shortening were put in a mixer and then Trial Production Example 2 well mixed. Furthermore, white soft Sugar and common salt were added, well mixed, and then ammonium hydrogen car Preparation of Sponge Cake bonate previously dissolved in water was added and well mixed. Finally, a powder Sample obtained by previously mix 0430. Among the formulations shown in Table 11 below, ing soft wheat flour, a starch and baking Soda (sodium hydro whole egg and granulated Sugar were warmed to around a gen carbonate) was added, followed by well mixing until a body temperature while mixing using a hand mixer. Further mass of a dough was formed. The mass of the dough was more, the mixture was stirred by a hand mixer until the mix spread thinly using a rolling pin, cut using a mold and then ture become to a mixture that has a viscosity, fine bubbles and baked in an oven (at 200° C. for 15 minutes) to prepare wholly whitish state. To the mixture, a powder sample cookies. obtained by previously mixing soft wheat flour, a starch and TABLE 10 Comparative Comparative Comparative Comparative Trial Trial Trial Trial Trial Trial Production Production Formulation Production Production Production Production Example Example (Parts) Example 1-1 Example 1-2 Example 1-3 Example 1-4 1-1 1-2

Soft wheat flour 150 150 150 150 150 150 Chemically unmodified 150 cassava starche () Chemically modified 150 cassava starch 1 '' () Chemically modified 150 cassava starch 2^*) Chemically modified 150 cassava starch 3 '' () Starch prepared in 150 Example 9 Starch prepared in 150 Example 10-2 White soft Sugar 120 120 120 120 120 120 Salt-free butter 60 60 60 60 60 60 Shortening 60 60 60 60 60 60 Ammonium hydrogen 3 3 3 3 3 3 carbonate Baking soda 3 3 3 3 3 3 Common salt 1 1 1 1 1 1 Water 34 34 34 34 34 34

Note (1) Chemically modified cassava starch 1: “RK-08”, manufactured by GLICO FOODS CO.,LTD. Note (2) Chemically modified cassava starch 2: “CHEMISTAR 280”, manufactured by GLICO FOODS CO.,LTD. “CHEMISTAR” is a registered trademark of GLICO FOODS CO.,LTD. Chemically modified cassava starch 3: “CHEMISTAR 300S, manufactured by GLICO FOODS CO.,LTD. “CHEMISTAR is a registered trademark of GLICO FOODS CO.,LTD. Note (4) Chemically unmodified cassava starch: untreated native cassava starch,

0429. The obtained cookies showed the following results. wheat gluten was added through sieving, followed by mixing That is, both the cookies of Trial Production Examples 1-1 using a spatula. Finally, a mixture of melted butter and milk and 1-2, in which any one of the starches prepared in was added and mixed. The obtained mixture was poured into Examples 9 and 10-2 was added, were soft and had texture a mold and then baked in an oven (at 200° C. for 15 minutes, with nice melt in mouth as compared with the cookies of then at 190° C. for 18 minutes) to prepare a sponge cake. US 2013/0022711 A1 Jan. 24, 2013 38

TABLE 11 Comparative Comparative Trial Trial Trial Trial Trial Trial Production Production Production Production Formulation Production Production Example Example Example Example (Parts) Example 2-1 Example 2-2 2-1 2-2 2-3 2-4 Soft wheat flour 50 50 50 50 50 50 Chemically unmodified 40 wheat starche () Chemically modified 40 wheat starch '' () Starch prepared in 40 Example 1-3 Starch prepared in 40 Example 2A Starch prepared in 40 Example 3A-3 Starch prepared in 40 Example 5A Wheat gluten' (2) 4.5 4.5 4.5 4.5 4.5 4.5 Whole egg 170 170 170 170 170 170 Granulated Sugar 1OO 100 100 100 100 100 Salt-free butter 35 35 35 35 35 35 Milk 25 25 25 25 25 25

Note (1) Chemically modified wheat starch: “MDSOL 1020”, manufactured by GLICO FOODS CO.,LTD. Note (2) Wheat gluten: “FinegluVP", manufactured by GLICO FOODS CO.,LTD. “Fineglu” is a registered trademark of GLICO FOODS CO.,LTD. Note (3) Chemically unmodified wheat starch: untreated native wheat starch.

0431. The obtained sponge cakes showed the following TABLE 12-continued results. That is, all the sponge cakes of Trial Production Example 2-1 to 2-4, in which any one of the starches prepared Comparative Comparative Trial Trial in Examples 1-3, 2A, 3 A-3 and 5A was added, showed nice Trial Trial Production Production Swelling after baking and had a large Volume, and also had Formulation Production Production Example Example Soft and puffy nice texture as compared with the sponge cakes (Parts) Example 3-1 Example 3-2 3-1 3-2 of Comparative Trial Production Example 2-1 and Compara Chemically 10 tive Trial Production Example 2-2. modified wheat starch Note (1) Trial Production Example 3 Starch prepared in 10 Example 1-3 Preparation of Custard Cream Starch prepared in 10 Example 3A-3 0432 Among the formulations shown in Table 12 below, Salt-free butter 15 15 15 15 granulated Sugar was added to egg yolk beaten well by a KUCEHINA COLOR appro- appro- appro- appro beater, followed by mixing by the beater. To the mixture, a 40OLS Note (2) priate priate priate priate powder sample obtained by previously mixing soft wheat amount amount amount amount flour and a starch was added through sieving, followed by Vanilla essence appro- appro- appro- appro mixing. Furthermore, warmed milk was added and mixed priate priate priate priate with them, the mixture was filtered and put in a pan, and then amount amount amount amount heated. The mixture was stirred by a wooden spatula until the Note (1) mixture become to a mixture that has a viscosity and a smooth Chemically modified wheat starch: “MIDSOL 1020”, manufactured by GLICO FOODS CO.,LTD. state. Finally, butter, a food color and vanilla essence were Note (2) added and mixed with them to prepare a custard cream. KUCHINA COLOR 400LS: Gardenia yellow food color. “KUCHINA COLOR is a reg istered trademark of GLICO FOODS CO.,LTD. TABLE 12 Note (3) Chemically unmodified wheat starch: Untreated native wheat starch. Comparative Comparative Trial Trial Trial Trial Production Production Formulation Production Production Example Example 0433. The obtained custard creams showed the following (Parts) Example 3-1 Example 3-2 3-1 3-2 results. That is, both the custard creams of Trial Production Milk 300 3OO 3OO 3OO Example 3-1 and 3-2, in which any one of starches prepared Egg yolk 35 35 35 35 in Examples 1-3 and 3A-3 was added, had appropriate body Granulated Sugar 60 60 60 60 and shape retention, and had nice melt in mouth and Smooth Soft wheat flour 10 10 10 10 Chemically 10 texture. On the other hand, the custard cream of Comparative unmodified wheat Trial Production Example 3-1 had gel-like physical proper starch Note (3) ties and heavy texture, and also had poor melt in mouth and poor Smoothness. Also, the custard cream of Comparative US 2013/0022711 A1 Jan. 24, 2013 39

Trial Production Example 3-2 had poor body and shape reten TABLE 13-continued tion, and had texture with Stickiness and poor melt in mouth. Comparative Comparative Trial Trial Trial Trial Production Production Trial Production Example 4 Formulation Production Production Example Example (Parts) Example 4-1 Example 4-2 4-1 4-2 Preparation of Milk Pudding Starch prepared in 10 Example 3A-3 0434 Among the formulations shown in Table 13 below, Note (1) granulated Sugar was added to milk and mixed well using a Chemically modified wheat starch: “MIDSOL 1020”, manufactured by GLICO FOODS wooden spatula to dissolve the granulated Sugar. To the mix Note.)CO.,LTD. ture, a starch sample was added and mixed well using the Chemically unmodified wheat starch: Untreated native wheat starch. wooden spatula. The mixture was heated while stirring by the 0435 The obtained milk puddings showed the following wooden spatula until the mixture become to a mixture having results. That is, both the milk puddings of Trial Production a viscosity and a smooth state. The mixture was filled inajelly Examples 4-1 and 4-2, in which any one of the starches cup and quenched in an ice bath to prepare a milk pudding. prepared in Examples 1-3 and 3A-3 was added, had sticky and chewy and also had nice melt in mouth and Smooth texture. TABLE 13 On the other hand, the milk pudding of Comparative Trial Production Example 4-1 was sticky but had texture with hard Comparative Comparative Trial Trial yogurt-like hardness and was therefore inferior in both melt in Trial Trial Production Production mouth and smoothness as compared with those of Trial Pro Formulation Production Production Example Example duction Examples. Also, the milk pudding of Comparative (Parts) Example 4-1 Example 4-2 4-1 4-2 Trial Production Example 4-2 was not firmly gelled and had a texture with stickiness, and also had poor melt in mouth. Milk 170 170 170 170 Trial Production Example 5 Granulated Sugar 10 10 10 10 Chemically 10 Preparation of Kudzu Starch Cake unmodified wheat 0436 Among the formulations shown in Table 14 below, a starch Note (2) mixture of a starch sample and white Soft Sugar was added to Chemically 10 water and white soft sugar was dissolved by well mixing Modified wheat using a wooden spatula. The mixture was heated while stir starch Yo'e () ring using the wooden spatula until the mixture become a Starch prepared in 10 pasty mixture with a viscosity and a transparency state. The Example 1-3 mixture was poured into a mold and quenched in an ice bath to prepare a kudzu Starch cake. TABLE 1.4

Comparative Comparative Comparative Trial Trial Trial Trial Trial Trial Trial Production Production Production Production Formulation Production Production Production Example Example Example Example (Parts) Example 5-1 Example 5-2 Example 5-3 S-1 S-2 S-3 5-4

Fermented wheat 56 starch Chemically unmodified 56 wheat starch '' (2) Chemically modified 56 wheat starch '' () Starch prepared in 56 Example 1-3 Starch prepared in 56 Example 2A Starch prepared in 56 Example 3A-3 Starch prepared in 56 Example 5A White soft Sugar 32 32 32 32 32 32 32 Water 28O 28O 28O 28O 28O 28O 28O

Note (1) Chemically modified wheat starch: “MDSOL 1020”, manufactured by GLICO FOODS CO.,LTD. Note (2) Chemically unmodified wheat starch: Untreated native wheat starch. US 2013/0022711 A1 Jan. 24, 2013 40

0437. The obtained kudzu starch cakes showed the follow TABLE 15-continued ing results. That is, all the kudzu starch cakes of Trial Pro Comparative Comparative Trial Trial duction Example 5-1 to 5-4, in which any one of the starches Trial Trial Production Production prepared in Examples 1-3, 2A, 3A-3 and 5A was added, had Formulation Production Production Example Example a appropriate hardness and brittleness, and had appropriate (Parts) Example 6-1 Example 6-2 6-1 6-2 Viscoelasticity and sticky texture. As compared with a kudzu Starch prepared in 30 starch cake so called in the Kanto area in Comparative Trial Example 1-3 Production Example 5-1, in which a fermented wheat starch Starch prepared in 30 obtained by fermenting for a long period, for example, one or Example 3A-3 more years was used, the kudzu Starch cakes having the Water 300 3OO 3OO 300 identical texture could be prepared without requiringfermen Note (1) tation for a long period in Trial Production Examples. Fur Chemically modified wheat starch: “MIDSOL 1020”, manufactured by GLICO FOODS thermore, the obtained kudzu starch cakes had nice flavor C2.TD.die without having peculiar flavor derived from a fermented Chemically unmodified wheat starch: Untreated native wheat starch. wheat starch and fermentation odor. On the other hand, the kudzu starch cake of Comparative Trial Production Example 0439. The obtained goma-dofus showed the following 5-2 was hard and brittle, and had texture with stickiness in the results. That is, both the goma-dofus of Trial Production mouth. Also, the kudzu starch cake of Comparative Trial Examples 6-1 and 6-2, in which anyone of starches prepared Production Example 5-3 had soft and brittle texture and in Examples 1-3 and 3A-3 was added, had appropriately showed texture far different from that of the kudzu starch cake Sticky texture and appropriate crispy sensation in contrast to a texture with rich elasticity like texture obtained by the so called in the Kanto area, together with those of Compara addition of a kudzu powder, and had readily edible texture tive Trial Production Example 5-2 and Comparative Trial with less Stickiness and Sticking in the mouth. Thus, it could Production Example 5-3. be expected for the obtained goma-dofus to be applied to foods for advanced aged persons, for example. On the other Trial Production Example 6 hand, the goma-dofu of Comparative Trial Production Example 6-1 has soft and strong Sticky texture and the goma Preparation of Goma-dofu dofu of Comparative Trial Production Example 6-2 had hard 0438 Among the formulation shown in Table 15 below, a and brittle texture, but had neither elasticity nor sticky texture, starch sample was added to water and the mixture was heated and thus both goma-dofus of Comparative Trial Production while stirring using a wooden spatula until the mixture Example 6-1 and Comparative Trial Production Example 6-2 become a pasty mixture with a viscosity and a transparency were inferior in deliciousness and ease of eating. state. A sesame paste was added to them and mixed well. The Trial Production Example 7 mixture was filled in a container and then cooled to obtain a goma-dofu. Preparation of Kamaboko 0440 Among the formulations shown in Table 16 below, a fish paste, common salt, Sugar, monosodiumglutamate and TABLE 1.5 potassium Sorbate were put in a (silent) mixer and well mixed Comparative Comparative Trial Trial until the mixture had a viscosity. In order to inhibit a tem Trial Trial Production Production perature rise of the fish paste, a half amount of moisture with Formulation Production Production Example Example ice was added to them and mixed. Then, egg white, Mirin and (Parts) Example 6-1 Example 6-2 6-1 6-2 a starch previously suspended in the remaining water with ice Sesame paste 50 50 50 50 were added to them and mixed well until a homogeneous Chemically 30 mixture was obtained. Indication of the temperature of the unmodified wheat starch Note (2) fish paste after mixing was within a range from 10 to 15° C. Chemically 30 The mixed fish paste was deaerated and filled in a cage. After modified wheat filling, the cage filled with a mixed fish paste was subjected to starch Yo'e () a sterilization step (at 90° C. for 40 minutes) and cooled to prepare a kamaboko. TABLE 16 Comparative Comparative Comparative Trial Trial Trial Trial Trial Production Production Formulation Production Production Production Example Example (Parts) Example 7-1 Example 7-2 Example 7-3 7-1 7-2 Fish paste 1OO 100 1OO 1OO 100 Chemically unmodified 15 wheat starche () Chemically modified 15 wheat starch 1 '' () Chemically modified 15 wheat starch 2^* (2) Starch prepared in 15 Example 1-3 Starch prepared in 15 Example 3A-3 US 2013/0022711 A1 Jan. 24, 2013

TABLE 16-continued Comparative Comparative Comparative Trial Trial Trial Trial Trial Production Production Formulation Production Production Production Example Example (Parts) Example 7-1 Example 7-2 Example 7-3 7-1 7-2 Common salt 3 3 3 3 3 Sugar 2.5 2.5 2.5 2.5 2.5 Egg white 5 5 5 5 5 Mirin 4 4 4 4 4 Monosodium glutamate O.S O.S O.S O.S O.S Potassium sorbate O.3 O.3 O.3 O.3 O.3 Water with ice 40 40 40 40 40

Note (1) Chemically modified wheat starch 1: “MDSOL 1020, manufactured by GLICO FOODS CO.,LTD. Note (2) Chemically modified wheat starch 2: “Ginrin', manufactured by GLICOFOODS CO.,LTD. “Ginrin” is a registered trademark of GLICO FOODS CO.,LTD. Note (3) Chemically unmodified wheat starch: Untreated native wheat starch.

0441. On the next day of production and after one week, TABLE 17-continued the obtained kamabokos were subjected to a sensory test. The kamaboko of Comparative Trial Production Example 7-1 had Comparative Comparative Trial Trial texture with slightly poor elasticity and also had no good Trial Trial Production Production chewiness. The kamaboko of Comparative Trial Production Formulation Production Production Example Example Example 7-2 had hardness but had stiff texture, and also (Parts) Example 8-1 Example 8-2 8-1 8-2 retrogradation of the starch arose in a sensory test after refrig eration for one week, and thus the kamaboko showed dry and Chemically 10 tasteless texture with water separation. The kamaboko of modified cassava Comparative Trial Production Example 7-3 was less likely to starch Note (1) cause change with time due to retrogradation because of the Starch prepared in 10 structure of the starch, but showed greasy texture with poor Example 9 elasticity. As compared with these Comparative Trial Produc Starch prepared in 10 tion Examples, both the kamabokos of Trial Production Example 10-2 Examples 7-1 and 7-2, in which any one of the starches Water 70 70 70 70 prepared in Examples 1-3 and 3A-3 was added, had elasticity Note (1) with nice chewiness and also caused less change with time. Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Note (2) Trial Production Example 8 Chemically unmodified cassava starch: Untreated native cassava starch, Preparation of Glaze for Mitarashi Dango 0443) The obtained glaze for mitarashi dango showed the following results. That is, both the glazes formitarashi dango 0442 Among the formulation shown in Table 17 below, a of Trial Production Example 8-1 and 8-2, in which any one of starch sample was previously Suspended in a part of water. the starches prepared in Examples 9 and 10-2 was added, had The total amount of white Soft Sugar, dark soy sauce, Mirin, starch syrup and the remaining water were put in a pan and nice body and shape retention, and were less likely to drop then mixed well by a wooden spatula. Furthermore, the starch because of nice adhesion onto the dango, and also had less sample previously suspended in water was added to them and Stickiness and thread-forming sensation and had smooth tex heated while stirring using the wooden spatula. The mixture ture. On the other hand, the glaze for mitarashi dango of was heated until the mixture becomes a pasty mixture with a Comparative Trial Production Example 8-1 had gel-like Viscosity and a transparency state, to prepare a glaze for physical properties and heavy texture, and also had poor melt mitarashi dango. in mouth and no Smoothness. The glaze for mitarashi dango of Comparative Trial Production Example 8-2 had poor body TABLE 17 and poor shape retention and caused dropping because of poor adhesion onto a dango, and also had texture with Sticki Comparative Comparative Trial Trial Trial Trial Production Production ness and poor melt in mouth. For example, in freezing distri Formulation Production Production Example Example bution of a split and broiled eel, in order to prevent a salsa for (Parts) Example 8-1 Example 8-2 8-1 8-2 the split and broiled eel from dropping at the time of thawing, White soft Sugar 95 95 95 95 a Salsa having a high viscosity and nice body and shape Dark soy sauce 8O 8O 8O 8O retention may be sometimes used in the final step of baking. Mirin 35 35 35 35 However there is a problem that the salsa having a high Starch syrup 8 8 8 8 Chemically 10 Viscosity usually has strong Stickiness or in a gel-like form, unmodified cassava and also has jellied fish-like physical properties and heavy starch Note (2) texture. Use of the enzyme-treated starch in the present inven tion makes it possible to prepare a split and broiled fish which US 2013/0022711 A1 Jan. 24, 2013 42 is less likely to drop because of nice adhesion onto the eel and egg yolk was added to them and mixed well. Using a homo the like, and has less Stickiness and thread-forming sensation, mixer manufactured by Tokushu Kika Kogyo Co., Ltd. (Now and has Smooth texture. in the name of PRIMIX Corporation), salad oil was slowly added dropwise while mixing with stirring at 8,000 rpm. Trial Production Example 9 After dropwise addition of the entire amount of salad oil, the mixture was mixed with stirring at 8,000 rpm further for 5 Preparation of Fruit Sauce minutes to prepare a dressing. 0444 Among the formulations shown in Table 18 below, a starch sample was previously Suspended in a part of water. TABLE 19 Fruit puree, white Soft Sugar, lemon juice and the total amount Comparative Comparative of the remaining water were put in a pan and heated with Trial Trial Trial Trial stirring using the wooden spatula. Furthermore, the starch Production Production Production Production sample previously Suspended in water was added to them. Formulation Example Example Example Example The mixture was heated until the mixture become a pasty (Parts) 1O-1 10-2 1O-1 10-2 mixture with a viscosity and a transparency state to prepare a Salad oil 38 38 38 38 fruit sauce. Brewed vinegar 10 10 10 10 (acidity: 4.2) Egg yolk 5 5 5 5 TABLE 18 White soft Sugar 5 5 5 5 Common Salt 3 3 3 3 Comparative Comparative Trial Trial Lemon juice 2 2 2 2 Trial Trial Production Production Chemically 2.5 Formulation Production Production Example Example unmodified cassava (Parts) Example 9-1 Example 9-2 9-1 9-2 starch Note (2) Fruit puree 100 1OO 1OO 100 Chemically 2.5 White soft Sugar 10 10 10 10 modified cassava Chemically 3 starch Yo'e () unmodified cassava Starch prepared in 2.5 starch Noe (2) Example 9 Chemically 3 Starch prepared in 2.5 modified cassava Example 10-2 starch Note (1) Monosodium 0.2 0.2 0.2 0.2 Starch prepared in 3 glutamate Example 9 Pepper Suitable Suitable Suitable Suitable Starch prepared in 3 amount amount amount amount Example 10-2 Mustard Suitable Suitable Suitable Suitable Lemon juice 2 2 2 2 amount amount amount amount Water 10 10 10 10 Water 33.8 33.8 33.8 33.8 Note (1) Note (1) Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Note (2) Note (2) Chemically unmodified cassava starch: Untreated native cassava starch, Chemically unmodified cassava starch: Untreated native cassava starch, 0445. The obtained fruit sauces showed the following 0447 The obtained dressings showed the following results. That is, both the fruit sauces of Trial Production results. That is, both the dressings of Trial Production Example 9-1 and 9-2, in which any one of the starches pre Examples 10-1 and 10-2, in which each of the starches pre pared in Examples 9 and 10-2 was added, had nice body and pared in Examples 9 and 10-2 was added, had nice body and shape retention, and had nice adhesion onto a food Such as nice shape retention, and were less likely to drop because of dessert, and had less Stickiness and thread-forming sensation, and smooth texture. On the other hand, the fruit sauce of nice adhesion onto vegetables and the like, and caused less Comparative Trial Production Example 9-1 had gel-like Stickiness and thread-forming sensation, and had Smooth tex physical properties and heavy texture, and also had poor melt ture. On the other hand, the dressing of Comparative Trial in mouth and no Smoothness. The fruit sauce of Comparative Production Example 10-1 had gel-like physical properties Trial Production Example 9-2 had poor body and poor shape and heavy texture, and also had poor melt in mouth and no retention, and caused dropping because of poor adhesion onto smoothness. Also, the dressing of Comparative Trial Produc a food Such as dessert, and also had texture with Stickiness, tion Example 10-2 had poor body and poor shape retention and poor melt in mouth. and caused dropping because of poor adhesion onto Veg etables and the like, and also had texture with Stickiness, and Trial Production Example 10 poor melt in mouth. Preparation of Dressing Trial Production Example 11 0446 Among the formulations shown in Table 19 below, white soft Sugar and a starch sample which are previously Preparation of Batter for Deep-Fried Food mixed in powder state was added to water, and heated with stirring at 90° C. for 10 minutes. Brewed vinegar, common 0448. Among the formulations shown in Table 20 below, salt, lemon juice, and seasonings including monosodium Soft wheat flour and a starch sample previously mixed in a glutamate and the like were added and further heated with powder state were suspended in cold water and mixed well to stirring for 5 minutes. After cooling it to room temperature, prepare a batter for deep-fried food. US 2013/0022711 A1 Jan. 24, 2013

TABLE 20 TABLE 21-continued Comparative Comparative Comparative Comparative Trial Trial Trial Trial Trial Trial Trial Trial Production Production Production Production Formulation Example Example Example Example Production Production Production Production (Parts) 11-1 11-2 11-1 11-2 Formulation Example Example Example Example (Parts) 12-1 12-2 12-1 12-2 Soft wheat flour 150 95 95 95 Chemically 30 Chemically 3 unmodified cassava unmodified cassava starch Noe (2) starch Yee (3) Chemically 30 Chemically 3 modified cassava starch Note (1) modified cassava Starch prepared in 30 starch Note (1) Example 9 Starch prepared in 3 Starch prepared in 30 Example 9 Example 10-2 Starch prepared in 3 Cold water 230 230 230 230 Example 10-2 Water with ice 25 25 25 25 Note (1) Common salt 1.4 1.4 1.4 1.4 Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Note (2) White soft Sugar 1 1 1 1 Chemically unmodified cassava starch: Untreated native cassava starch, Seasoning O.3 O.3 O.3 O.3 pickle solution O.S O.S O.S O.S Pork powder 1 1 1 1 0449 The obtained batters for deep-fried food showed the Spice O.S O.S O.S O.S following results. That is, both the tempuras obtained by Potassium sorbate O.15 O.15 O.15 O.15 dipping ingredients such as prawn in the batters for deep-fried pH adjusting agent O.15 O.15 O.15 O.15 food of Trial Production Examples 11-1 and 11-2, in which Creation Color appro- appro- appro- appro the starch prepared in Example 9 or 10-2 was added, thereby RCNote (2) priate priate priate priate coating the ingredients with the batters, and frying in oil, or amount amount amount amount the fried foods obtained by dipping ingredients such as prawn in the batters for deep-fried food of Trial Production Note (1) Examples 11-1 and 11-2, in which the starch prepared in Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Example 9 or 10-2 was added, thereby coating the ingredients Note (2) Creation Color RC: Cochineal food color, “Creation” is a registered trademark of GLICO with the batters, further coating this with a bread crumbs and FOODS CO.,LTD. frying in oil, had a crispy and light texture. On the other hand, Note (3) tempuras and fried foods, in which the batters for deep-fried Chemically unmodified cassava starch: Untreated native cassava starch, food of Comparative Trial Production Example 11-1 and Comparative Trial Production Example 1 1-2 were used, had 0451. On the next day of production and after one week, poor crispy texture, and hardly made users feel lightness. the obtained sausages were Subjected to a sensory test. The sausage of Comparative Trial Production Example 12-1 had Trial Production Example 12 texture with slightly poor elasticity and had no good chewi ness. The sausage of Comparative Trial Production Example Preparation of Sausage 12-2 had hardness but had stiff texture, and retrogradation of the starch arose in a sensory test after refrigeration for one 0450 Among the formulations shown in Table 21 below, pork arm meat was put in a silent cutter, while cutting the pork week, and thus the sausage showed dry and tasteless texture arm meat at a high speed, casein sodium, common salt, white with water separation. As compared with those of these Com Soft Sugar, a seasoning, a pickle solution, a pork powder, parative Trial Production Examples, both the sausages of spice, potassium Sorbate, a pH adjusting agent and a food Trial Production Examples 12-1 and 12-2, in which the starch color were added and mixed well. When the mixture was prepared in Example 9 or 10-2 was added, had elasticity with formed into a paste, water with ice and lard were added and nice chewiness and also caused less change with time. cutting was continued. Finally, a starch sample was added to them and mixed well to give a homogeneous paste. The paste Trial Production Example 13 was filled in a casing and then sterilized at 80° C. for 40 minutes to prepare a sausage by cooling with running water. Preparation of Raw Udon TABLE 21 0452. To a powder mixture obtained by mixing a starch, Comparative Comparative medium wheat flour and a powdered gluten in the following Trial Trial Trial Trial ratio in accordance with the formulation shown in Table 22 Production Production Production Production Formulation Example Example Example Example below, water for kneading obtained by dissolving 2 parts of (Parts) 12-1 12-2 12-1 12-2 common salt in 40 parts of water was added, followed by Pork arm meat 60 60 60 60 kneading in a vacuum mixer for 12 minutes. Using a noodle Lard 10 10 10 10 making machine, the obtained kneaded mixture was Sub Casein sodium 1 1 1 1 jected to compound and rolling to obtaina noodle strip, which was cut using a cutting-tooth No. 10 to obtain a raw udon. US 2013/0022711 A1 Jan. 24, 2013 44

TABLE 22 TABLE 23-continued Comparative Comparative Comparative Comparative Comparative Trial Trial Trial Trial Trial Trial Trial Trial Production Production Production Production Production Production Production Production Formulation Example Example Example Example Formulation Example Example Example Example (Parts) 13-1 13-2 13-3 13-1 (Parts) 14-1 14-2 14-1 14-2 Wheat flour 8O 8O 8O 8O Chemically 2O Chemically 21 unmodified modified cassava C3SS8W8 starch Yo'e () starch Yee (3) Starch prepared in 21 Chemically 2O Example 9 modified Starch prepared in 21 C3SS8W8 starch 1 Noe () Example 10-2 Chemically 2O Water 15 15 15 15 modified C3SS8W8 Note (1) starch 2 Noe (2) Chemically modified cassava starch: "CHEMISTAR 300S” manufactured by GLICO Starch prepared in 2O FOODS CO.,LTD. “CHEMISTAR is a registered trademark of GLICO FOODS CO.,LTD. Example 10-2 Note (2) Powdered gluten 2 2 2 2 Chemically unmodified cassava starch: Untreated native cassava starch, Salt 2 2 2 2 Water 40 40 40 40 0455 The obtained jelly candies showed the following Note (1) results. That is, both the jelly candies of Trial Production Chemically modified cassava starch 1: “CHEMISTAR 280, manufactured by GLICO F9RS CO.,LTD. “CHEMISTAR” is a registered trademark of GLICO FOODS CO.,LTD. Example 14-1 and 14-2 had appropriate viscoelasticity and die nice melt texture in mouth. On the other hand, the jelly candy Chemically modified cassava starch 2: “RK-08”, manufactured by GLICO FOODS CO., LTD. of Comparative Trial Production Example 14-1 had strong Note (3) elastic sensation, and the jelly candy of Comparative Trial Chemically unmodified cassava starch: Untreated native cassava starch, Production Example 14-2 had strong sticky textures, but both 0453 The obtained raw udon was boiled in boiling water the jelly candies had strong pasty sensation and poor melt in for 10 minutes and dipped in a hot Soup, and then texture was mouth. evaluated. The udon of Comparative Trial Production Example 13-1 and Comparative Trial Production Example Trial Production Example 15 13-2 was poor in elasticity and texture was hardly improved. Regarding the udon of Comparative Trial Production Example 13-3, a slight effect of imparting elasticity was Preparation of Frozen Dessert recognized. However, rigid hardness was merely imparted and this effect may have a bad effect on noodles. On the other 0456. In accordance with the formulation shown in Table hand, regarding the udon of Trial Production Example 13-1, 24 below, while raw materials and water were mixed with the effect of imparting sticky texture with excellent chewiness stirring in the following ratio, the mixture was dissolved by was recognized. heating up to Bx (Brix) 40. The obtained solution was put in an ice cream maker and cooled with stirring for 35 minutes. Trial Production Example 14 The obtained materials was transferred to a container and then frozen to obtain a frozen dessert. Preparation of Jelly Candy TABLE 24 0454. In accordance with the formulations shown in Table 23 below, white Sugar, starch syrup, a starch and water were Comparative Comparative Trial Trial Trial Trial mixed with stirring in the following ratio, the mixture was Production Production Production Production dissolved by heating up to Bx (Brix) 75. The obtained solu Formulation Example Example Example Example tion was filled in a mold, and left at normal temperature for 24 (Parts) 15-1 15-2 15-1 15-2 hours. After confirming that the solution has been solidified, Starch syrup 18 18 18 18 it was removed from the mold to obtain a jelly candy. Granulated Sugar 12 12 12 12 Fresh cream 12 12 12 12 TABLE 23 Vegetable oil 6 6 6 6 and fat Comparative Comparative Chemically 2.4 Trial Trial Trial Trial unmodified cassava Production Production Production Production starch Note (2) Formulation Example Example Example Example Chemically 2.4 (Parts) 14-1 14-2 14-1 14-2 modified cassava starch Yo'e () Sugar 34 34 34 34 Starch prepared in 2.4 Starch syrup 30 30 30 30 Example 9 Chemically 21 Starch prepared in 2.4 unmodified cassava Example 10-2 starch Note (2) Guar gum 0.4 0.4 0.4 0.4 US 2013/0022711 A1 Jan. 24, 2013

TABLE 24-continued mis, manufactured by Novo: optimum pH of 6.0) was added and stirred at 50° C. for 18 hours to carry out an enzyme Comparative Comparative reaction. After completion of the reaction, an enzyme-treated Trial Trial Trial Trial Production Production Production Production starch was recovered by centrifugal filtration and blow dry Formulation Example Example Example Example ing. Viscosity characteristics of the obtained enzyme-treated (Parts) 15-1 15-2 15-1 15-2 starch were analyzed by the amylograph and the rheometer. Emulsifier O.2 O.2 O.2 O.2 Also, after completion of the reaction, a degradation ratio was Water 49 49 49 49 determined by a part of a reaction solution. The results are shown in Table 3-2. As a result, the setback viscosity of it was Note (1) 0 (BU). Chemically modified cassava starch: "CHEMISTAR 300S”, manufactured by GLICO 5.99ps CO.,LTD. “CHEMISTAR” is a registered trademark of GLICO FOODS CO.,LTD. die Example 15 Chemically unmodified cassava starch: Untreated native cassava starch, 0461) To 400g of an untreated native cassava starch, 900 g 0457. The obtained frozen desserts showed the following of ion-exchange water was added to prepare a starch Suspen results. That is, both the frozen desserts of Trial Production sion. After adjusting the pH of the suspension to 5.0, 1% by Examples 15-1 and 15-2 had appropriate viscoelasticity and weight (based on starch Solid content) of cyclodextrin glu sticky texture, and had nice melt texture in mouth. On the canotransferase (“Toruzyme 3.0 L derived from Bacillus other hand, the frozen dessert of Comparative Trial Produc licheniformis, manufactured by Novo: optimum pH of 6.0) tion Example 15-1 had sticky texture and the frozen dessert of was added and stirred at 50° C. for 18 hours to carry out an Comparative Trial Production Example 15-2 also had sticky enzyme reaction. After completion of the reaction, an texture and spinnability. However, both the frozen desserts enzyme-treated starch was recovered by centrifugal filtration had strong pasty sensation and poor melt in mouth. and blow drying. Viscosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and Example 13-1 the rheometer. Also, after completion of the reaction, a deg 0458. To 400 g of an untreated native wheat starch, 900 g radation ratio was determined by a part of a reaction solution. of ion-exchange water was added to prepare a starch Suspen The results are shown in Table 4-2. As a result, the setback sion. After adjusting the pH of the suspension to 5.0, 1% by viscosity of it was 2 (BU). weight (based on starch Solid content) of cyclodextrin glu canotransferase (“Toruzyme 3.0 L derived from Bacillus Comparative Example 18 licheniformis, manufactured by Novo: optimum pH of 6.0) 0462. To 500g of an untreated native cassava starch, 750g was added and stirred at 50° C. for 18 hours to carry out an of an aqueous 6.7% (w/w) sodium sulfate solution was added enzyme reaction. After completion of the reaction, an to prepare a starch Suspension. After adjusting the pH of the enzyme-treated starch was recovered by centrifugal filtration Suspension to 8.5, 7.36 g of a vinyl acetate monomer was and blow drying. Viscosity characteristics of the obtained added and stirred at 30° C. for 40 minutes to allow a reaction enzyme-treated Starch were analyzed by the amylograph and proceed. After 40 minutes, the pH of the suspension was the rheometer. Also, after completion of the reaction, a deg adjusted to 6.0 and the reaction was terminated. After comple radation ratio was determined by a part of a reaction solution. tion of the reaction, starch acetate was recovered by centrifu The results are shown in Table 2-2. As a result, the setback gal filtration and blow drying. Viscosity characteristics of the viscosity of it was 7.0 (BU). obtained starch acetate were analyzed by the amylograph and the rheometer. Example 13-2 0459. To 400 g of an untreated native wheat starch, 900 g Comparative Example 19 of ion-exchange water was added to prepare a starch Suspen 0463) To 500g of an untreated native cassava starch, 785g sion. After adjusting the pH of the suspension to 6.0, 1% by of an aqueous 11% (W/w) sodium sulfate solution was added weight (based on starch Solid content) of cyclodextrin glu to prepare a starch Suspension. After adjusting the pH of the canotransferase (Cyclodextrin Cycrodextrin glucanotrans Suspension to 11.0, 24 g of propylene oxide was added and ferase “Amano” derived from Paenibacillus macerans (Ba stirred at 42°C. for 16 hours to allow a reaction proceed. After cillus macerans), manufactured by Amano Enzyme) was 16 hours, the pH of the suspension was adjusted to 6.0 and the added and stirred at 50° C. for 18 hours to carry out an enzyme reaction was terminated. After completion of the reaction, a reaction. After completion of the reaction, an enzyme-treated hydroxypropyl starch was recovered by centrifugal filtration starch was recovered by centrifugal filtration and blow dry and blow drying. Viscosity characteristics of the obtained ing. Viscosity characteristics of the obtained enzyme-treated hydroxypropyl Starch were analyzed by the amylograph and starch were analyzed by the amylograph and the rheometer. the rheometer. Also, after completion of the reaction, a degradation ratio was determined by a part of a reaction solution. The results are Comparative Example 20 shown in Table 2-2. 0464) To 500g of an untreated native cassava starch, 750g of an aqueous 6.7% (w/w) sodium sulfate solution was added Example 14 to prepare a starch Suspension. After adjusting the pH of the 0460. To 400 g of an untreated native corn starch, 900 g of Suspension to 11.0, 10 ul of phosphorus oxychloride was ion-exchange water was added to prepare a starch Suspension. added and stirred at 30° C. for 1 hour to allow a reaction After adjusting the pH of the suspension to 5.0, 1% by weight proceed. After 1 hour, the pH of the suspension was adjusted (based on starch Solid content) of cyclodextrin glucanotrans to 6.0 and the reaction was terminated. After completion of ferase (“Toruzyme 3.0 L derived from Bacillus lichenifor the reaction, a distarch phosphate was recovered by centrifu US 2013/0022711 A1 Jan. 24, 2013 46 gal filtration and blow drying. Viscosity characteristics of the Example 19 obtained distarch phosphate were analyzed by the amylo 0469 To 400 g of the hydroxypropyl starch prepared in graph and the rheometer. Comparative Example 19,900 g of ion-exchange water was Comparative Example 21 added to prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch solid 0465. To 500g of an untreated native cassava starch, 910 g content) of amyloglucosidase (“OPTIDEX L-400' derived of an aqueous 10% (w/w) sodium sulfate solution was added from Aspergillus niger, manufactured by Genencor, optimum to prepare a starch Suspension. After adjusting the pH of the pH of 4.4) was added and stirred at 50° C. for 18 hours to carry Suspension to 11.0, 16 g of propylene oxide was added and out an enzyme reaction. After completion of the reaction, an stirred at 42°C. for 16 hours to allow an etherification reac enzyme-treated starch was recovered by centrifugal filtration tion proceed. After 16 hours, the temperature of the starch and blow drying. Viscosity characteristics of the obtained Suspension was adjusted to 30°C., 5 Jul of phosphorus oxy enzyme-treated Starch were analyzed by the amylograph and chloride was added and stirred at 30°C. for 1 hour to carry out the rheometer. Also, after completion of the reaction, a deg a crosslinking reaction proceed. After 1 hour, the pH of the radation ratio was determined using a part of the reaction Suspension was adjusted to 6.0 and the entire reaction was Solution. terminated. After completion of the reaction, a hydroxypro pyl distarch phosphate was recovered by centrifugal filtration Example 20 and blow drying. Viscosity characteristics of the obtained hydroxypropyl distarch phosphate were analyzed by the amy 0470 To 400 g of the hydroxypropyl starch prepared in lograph and the rheometer. Comparative Example 19,900 g of ion-exchange water was added to prepare a starch Suspension. After adjusting the pH Example 16 of the suspension to 5.0, 1% by weight (based on starch solid content) of C-amylase (AMYLEX A3” derived from 0466. To 4 Kg of an untreated native cassava starch, 9 Kg Aspergillus niger, manufactured by DANISCO: optimum pH of ion-exchange water was added to prepare a starch Suspen of 5.0) was added and stirred at 50° C. for 18 hours to carry out sion. After adjusting the pH of the suspension to 5.0, 1% by an enzyme reaction. After completion of the reaction, an weight (based on starch Solid content) of amyloglucosidase enzyme-treated starch was recovered by centrifugal filtration (“OPTIDEXL-400' derived from Aspergillus niger, manu and blow drying. Viscosity characteristics of the obtained factured by Genencor; optimum pH of 4.4) was added and enzyme-treated starch were analyzed by the amylograph and stirred at 50° C. for 18 hours to carry out an enzyme reaction. the rheometer. Also, after completion of the reaction, a deg After completion of the reaction, an enzyme-treated Starch radation ratio was determined using a part of the reaction was recovered by centrifugal filtration and blow drying. It is Solution. noted that the degradation ratio of the obtained sample was 21%. Example 21 Example 17 0471 To 400g of the distarch phosphate prepared in Com 0467 To 400 g of the starch acetate prepared in Compara parative Example 20,900 g of ion-exchange water was added tive Example 18, 900 g of ion-exchange water was added to to prepare a starch Suspension. After adjusting the pH of the prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch solid con suspension to 5.0, 1% by weight (based on starch solid con tent) of amyloglucosidase (“OPTIDEXL-400' derived from tent) of amyloglucosidase (“OPTIDEXL-400' derived from Aspergillus niger, manufactured by Genencor, optimum pH Aspergillus niger, manufactured by Genencor, optimum pH of 4.4) was added and stirred at 50° C. for 18 hours to carry out of 4.4) was added and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an an enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration enzyme-treated starch was recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained and blow drying. Viscosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and enzyme-treated Starch were analyzed by the amylograph and the rheometer. Also, after completion of the reaction, a deg the rheometer. After completion of the reaction, a degradation radation ratio was determined using a part of the reaction ratio was determined using a part of the reaction solution. Solution. Example 18 Example 22 0468. To 400 g of the starch acetate prepared in Compara 0472. To 400g of the distarch phosphate prepared in Com tive Example 18, 900 g of ion-exchange water was added to parative Example 20,900 g of ion-exchange water was added prepare a starch Suspension. After adjusting the pH of the to prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch solid con suspension to 5.0, 1% by weight (based on starch solid con tent) of C-amylase (AMYLEXA3’ derived from Aspergillus tent) of C.-amylase (AMYLEXA3’ derived from Aspergillus niger, manufactured by DANISCO; optimum pH of 5.0) was niger, manufactured by DANISCO; optimum pH of 5.0) was added and stirred at 50° C. for 18 hours to carryout an enzyme added and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an enzyme-treated reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration and blow dry starch was recovered by centrifugal filtration and blow dry ing. Viscosity characteristics of the obtained enzyme-treated ing. Viscosity characteristics of the obtained enzyme-treated starch were analyzed by the amylograph and the rheometer. starch were analyzed by the amylograph and the rheometer. Also, after completion of the reaction, a degradation ratio was Also, after completion of the reaction, a degradation ratio was determined using a part of the reaction solution. determined using a part of the reaction solution. US 2013/0022711 A1 Jan. 24, 2013 47

Example 23 Example 27 0473. To 400 g of the hydroxypropyl distarch phosphate 0477 To 500 g of the enzyme-treated starch prepared in prepared in Comparative Example 21, 900 g of ion-exchange Example 16, 750 g of an aqueous 6.7% (w/w) sodium sulfate water was added to prepare a starch Suspension. After adjust Solution was added to prepare a starch Suspension. After ing the pH of the suspension to 5.0, 1% by weight (based on adjusting the pH of the suspension to 11.0, 10ul of phospho starch solid content) of amyloglucosidase (“OPTIDEX rus oxychloride was added and stirred at 30° C. for 1 hour to L-400' derived from Aspergillus niger, manufactured by carry out a reaction. After 1 hour, the pH of the Suspension Genencor, optimum pH of 4.4) was added and stirred at 50° was adjusted to 6.0 and the reaction was terminated. After C. for 18 hours to carry out an enzyme reaction. After comple completion of the reaction, an enzyme-treated distarch phos tion of the reaction, an enzyme-treated Starch was recovered phate was recovered by centrifugal filtration and blow drying. by centrifugal filtration and blow drying. Viscosity character Viscosity characteristics of the obtained enzyme-treated dis istics of the obtained enzyme-treated starch were analyzed by tarch phosphate were analyzed by the amylograph and the the amylograph and the rheometer. Also, after completion of rheometer. Also, after completion of the reaction, a degrada the reaction, a degradation ratio was determined using a part tion ratio was determined by a part of the reaction solution. of the reaction solution. Example 28 Example 24 0478. To 500 g of the enzyme-treated starch prepared in 0474. To 400 g of the hydroxypropyl distarch phosphate Example 16,910 g of an aqueous 10% (w/w) sodium sulfate prepared in Comparative Example 21, 900 g of ion-exchange Solution was added to prepare a starch Suspension. After water was added to prepare a starch Suspension. After adjust adjusting the pH of the Suspension to 11.0, 16 g of propylene ing the pH of the suspension to 5.0, 1% by weight (based on oxide was added and stirred at 42°C. for 16 hours to carry out starch solid content) of C.-amylase (AMYLEX A3' derived an etherification reaction. After 16 hours, the temperature of from Aspergillus niger, manufactured by DANISCO; opti the starch suspension was adjusted to 30° C. and 5 ul of mum pH of 5.0) was added and stirred at 50° C. for 18 hours phosphorus oxychloride was added and stirred at 30°C. for 1 to carry out an enzyme reaction. After completion of the hour to carry out a crosslinking reaction. After 1 hour, the pH reaction, an enzyme-treated Starch was recovered by centrifu of the Suspension was adjusted to 6.0 and the entire reaction gal filtration and blow drying. Viscosity characteristics of the was terminated. After completion of the reaction, an enzyme obtained enzyme-treated Starch were analyzed by the amylo treated hydroxypropyl distarch phosphate was recovered by graph and the rheometer. Also, after completion of the reac centrifugal filtration and blow drying. Viscosity characteris tion, a degradation ratio was determined using a part of the tics of the obtained enzyme-treated hydroxypropyl distarch reaction solution. phosphate were analyzed by the amylograph and the rheom eter. Also, after completion of the reaction, a degradation ratio Example 25 was determined by a part of the reaction Solution. 0475 To 500 g of the enzyme-treated starch prepared in 0479. The measurement results of Comparative Examples Example 16, 750 g of an aqueous 6.7% (w/w) sodium sulfate 18 to 21 and Examples 17 to 28 are shown in Table 25-2. It is Solution was added to prepare a starch Suspension. After noted that in an analysis by the rheometer of the present starch adjusting the pH of the suspension to 8.5, 7.36 g of a vinyl which used the chemical modification and the enzymatic acetate monomer was added and stirred at 30° C. for 40 treatment in combination, after refrigeration storage at 5°C. minutes to carry out a reaction. After 40 minutes, the pH of the for 16 hours the gel did not have the hardness sufficient for the Suspension was adjusted to 6.0 and the reaction was termi measurement. Therefore, it was difficult to compare physical nated. After completion of the reaction, an acetic acid properties of the gels. Therefore, confirmation was carried out enzyme-treated starch was recovered by centrifugal filtration after refrigeration storage at 5°C. for 21 days. The details are and blow drying. Viscosity characteristics of the obtained as follows. acetic acid enzyme-treated Starch were analyzed by the amy 0480. A starch paste was prepared so that the concentra lograph and the rheometer. Also, after completion of the tion of the starch was 20% by weight on the dry matter basis, reaction, a degradation ratio was determined by apart of the and then filled in a Krehalon casing having a folding width of reaction solution. 45 mm. This starch paste filled in the casing was heated to 90° C. at 1°C/minand maintained at 90° C. for 30 minutes. Then, Example 26 the starch paste was left to cool in a constant-temperature water bath at 20° C. for 30 minutes, and then it was cooled to 0476. To 500 g of the enzyme-treated starch prepared in 5°C. in a refrigerator. After cooling, it was refrigerated at 5° Example 16, 785 g of an aqueous 11% (w/w) sodium sulfate C. for 21 days, then it was left at room temperature (about 25° Solution was added to prepare a starch Suspension. After C.) for 4 hours to return the temperature of it to room tem adjusting the pH of the Suspension to 11.0, 24 g of propylene perature, and then the measurement was carried out with a oxide was added and stirred at 42°C. for 16 hours to carry out rheometer (RT-2010J-CW) manufactured by Rheotech Inc. a reaction. After 16 hours, the pH of the suspension was The measurement was carried out under the measurement adjusted to 6.0 and the reaction was terminated. After comple conditions of the rheometer: a test item: a rupture test; a tion of the reaction, a hydroxypropyl enzyme-treated Starch height of a sample: 25 mm; and a movement rate (rupture rate) was recovered by centrifugal filtration and blow drying. Vis of a sample: 6 cm/min, using an adapter of a spherical jig for cosity characteristics of the obtained hydroxypropylenzyme measurement viscosity (p5 (diameter: 5 mm, area: 19.635 treated Starch were analyzed by the amylograph and the rhe mm). At the measurement, the hardness of the starch gel was ometer. Also, after completion of the reaction, a degradation evaluated by a rupture stress (g) and a Young's modulus ratio was determined by a part of the reaction Solution. (dyn/cm). US 2013/0022711 A1 Jan. 24, 2013 48

TABLE 25-1

Summary of Names, Origins and Product Names of Enzymes used for Chemical Modification and Enzymatic Treatment in combination

Example Name of enzyme Origin Kind of chemical modification

Comp. Ex. 18 Acetylation Example 17 amyloglucosidase Aspergilius niger Acetylation Example 18 C-amylase Aspergilius niger Acetylation Example 25 amyloglucosidase Aspergilius niger Acetylation Comp. Ex. 19 Hydroxypropylation Example 19 amyloglucosidase Aspergilius niger Hydroxypropylation Example 20 C-amylase Aspergilius niger Hydroxypropylation Example 26 amyloglucosidase Aspergilius niger Hydroxypropylation Comp. Ex. 20 Phosphate crosslinking Example 21 amyloglucosidase Aspergilius niger Phosphate crosslinking Example 22 C-amylase Aspergilius niger Phosphate crosslinking Example 27 amyloglucosidase Aspergilius niger Phosphate crosslinking Comp. Ex. 21 Hydroxypropylation phosphate crosslinking Example 23 amyloglucosidase Aspergilius niger Hydroxypropylation phosphate crosslinking Example 24 C-amylase Aspergilius niger Hydroxypropylation phosphate crosslinking Example 28 amyloglucosidase Aspergilius niger Hydroxypropylation phosphate crosslinking Comp. Ex. 22 Oxidized starch Example 29 amyloglucosidase Aspergilius niger Oxidized starch Example 30 C-amylase Aspergilius niger Oxidized starch Comp. Ex. 23 (Bleached starch) Example 31 amyloglucosidase Aspergilius niger (Bleached starch) Example 32 C-amylase Aspergilius niger (Bleached starch)

Comp, Ex. = Comparative Example

TABLE 25-2 Table 25-2: Summary of combined use of chemical modification and enzymatic treatment (Starch Concentration for Amylograph: 6.0% Maximum viscosity Rupture stress Young's modulus Degradation Measured Breakdown Measured Measured ratio value Relative viscosity value Relative value Relative Example (%) (BU) % (%) (BU) (g) % (%) (dyn/cm) % (%) Comp. Ex. 18 895 OO 606 121 2,499,026 OO Example17 7 727 81 433 145 2,923,860 17 Example18 9 693 77 429 142 3,023,821 21 Example25 21 797 89 472 215 4,169,554 67 Comp. Ex. 19 1004 OO 670 33 373,715 OO Example19 2O 845 84 550 47 483,149 29 Example20 21 661 66 470 43 669,986 79 Example26 21 824 82 525 71 573,625 53 Comp. Ex. 20 903 OO O 170 3,943,691 OO Example21 21 908 O1 O 283 5,319,388 35 Example22 15 871 96 O 337 7,529,620 91 Example27 21 898 99 O 554 10,027,924 2S4 Comp. Ex. 21 769 OO 403 31 250,328 Example23 19 740 96 413 45 391,117 Example24 21 524 68 356 64 1,218,285 Example28 21 784 O2 2O7 125 1497,293 Comp. Ex. 22 317 OO 227 91 4,468,130 Example29 7 419 32 324 119 5,953,997 Example30 9 411 30 315 101 5,048,987 Comp. Ex. 23 715 OO 181 137 4494,603 Example31 22 673 94 138 224 5,293,378 Example32 31 630 88 143 237 6,764,052 * Degradation ratios in Examples 25 to 28 each refer to a degradation ratio of an enzyme-treated starch used as a base material, US 2013/0022711 A1 Jan. 24, 2013 49

0481. It was confirmed that when the chemical modifica and blow drying. Viscosity characteristics of the obtained tion and the enzymatic treatment are used in combination, enzyme-treated Starch were analyzed by the amylograph and particularly when the distarch phosphate is subjected to the the rheometer. Also, after completion of the reaction, a deg enzymatic treatment, gel forming ability can be enhanced radation ratio was determined using a part of the reaction while maintaining a maximum viscosity. This is an extremely Solution. excellent advantage as compared with the fact that when phosphate crosslinking is increased in a conventional chemi Example 30 cal modification, the gel becomes harder but the maximum Viscosity drastically decreases, thus leading to the cause of In the Case where an Oxidized Starch was Treated powderiness. It was also confirmed for not only the distarch with an O-Amylase phosphate but also other chemically modified starches that, 0485 To 400 g of the oxidized starch prepared in Com by carrying out an enzymatic treatment, it is possible to parative Example 22,900 g of ion-exchange water was added enhance gel forming ability while relatively maintaining the to prepare a starch Suspension. After adjusting the pH of the Viscosity as compared with a conventional chemically modi suspension to 5.0, 1% by weight (based on starch solid con fied starch. tent) of C.-amylase (AMYLEXA3’ derived from Aspergillus niger, manufactured by DANISCO; optimum pH of 5.0) was Comparative Example 22 added and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an enzyme-treated Oxidized Starch starch was recovered by centrifugal filtration and blow dry 0482 To 500g of an untreated native cassava starch, 750 g ing. Viscosity characteristics of the obtained enzyme-treated of ion-exchange water was added to prepare a starch Suspen starch were analyzed by the amylograph and the rheometer. sion. After adjusting the pH of the suspension to 10.0, 2.5g of Also, after completion of the reaction, a degradation ratio was sodium hypochlorite whose effective chlorine amount is 10% determined using a part of the reaction solution. was added and stirred at 30° C. for 2 hours to carry out a reaction, while maintaining the pH of the Suspension at 10.0. Example 31 After 2 hours, the pH of the suspension was adjusted to 6.0 and then 2 g of sodium hydrogen Sulfite was added. Immedi In the Case where a Bleached Starch was Treated ately after stirring, the pH of the Suspension was adjusted to with an Amyloglucosidase 6.0 and the reaction was terminated. After completion of the 0486 To 400 g of the bleached starch prepared in Com reaction, the oxidized starch was recovered by centrifugal parative filtration and blow drying. Viscosity characteristics of the 0487. Example 23, 900 g of ion-exchange water was obtained oxidized starch were analyzed by the amylograph added to prepare a starch Suspension. After adjusting the pH and the rheometer. of the suspension to 5.0, 1% by weight (based on starch solid Comparative Example 23 content) of amyloglucosidase (“OPTIDEX L-400' derived from Aspergillus niger, manufactured by Genencor, optimum Bleached Starch pH of 4.4) was added and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an 0483 To 500g of an untreated native cassava starch, 700 g enzyme-treated starch was recovered by centrifugal filtration of ion-exchange water was added to prepare a starch Suspen and blow drying. Viscosity characteristics of the obtained Sion. After adjusting the pH of the Suspension to 11.0, 2.5g of enzyme-treated Starch were analyzed by the amylograph and sodium hypochlorite whose effective chlorine amount is 10% the rheometer. Also, after completion of the reaction, a deg was added and stirred at 30°C. for 5 minutes while maintain radation ratio was determined using a part of the reaction ing the pH of the suspension at 11. Then, 0.25 g of sodium Solution. metabisulfite was added and stirred for 10 minutes, the pH of the Suspension was adjusted to 6.0 and the reaction was ter Example 32 minated. After completion of the reaction, the bleached starch was recovered by centrifugal filtration and blow drying. Vis In the Case where a Bleached Starch was Treated cosity characteristics of the obtained bleached starch were with an O-Amylase analyzed by the amylograph and the rheometer. 0488 To 400 g of the bleached starch prepared in Com Example 29 parative Example 23,900 g of ion-exchange water was added to prepare a starch Suspension. After adjusting the pH of the In the Case where an Oxidized Starch was Treated suspension to 5.0, 1% by weight (based on starch solid con with an Amyloglucosidase tent) of C.-amylase (AMYLEXA3’ derived from Aspergillus niger, manufactured by DANISCO; optimum pH of 5.0) was 0484. To 400 g of the oxidized starch prepared in Com added and stirred at 50° C. for 18 hours to carry out an enzyme parative Example 22,900 g of ion-exchange water was added reaction. After completion of the reaction, an enzyme-treated to prepare a starch Suspension. After adjusting the pH of the starch was recovered by centrifugal filtration and blow dry suspension to 5.0, 1% by weight (based on starch solid con ing. Viscosity characteristics of the obtained enzyme-treated tent) of amyloglucosidase (“OPTIDEXL-400' derived from starch were analyzed by the amylograph and the rheometer. Aspergillus niger, manufactured by Genencor, optimum pH Also, after completion of the reaction, a degradation ratio was of 4.4) was added and stirred at 50° C. for 18 hours to carry out determined using a part of the reaction Solution. The mea an enzyme reaction. After completion of the reaction, an surement results of Comparative Examples 22 to 23 and enzyme-treated starch was recovered by centrifugal filtration Examples 29 to 32 are shown in Table 25-2. US 2013/0022711 A1 Jan. 24, 2013 50

Comparative Example 24 acteristics of the obtained enzyme-heat-moisture-treated 0489. To 2 kg of an untreated native corn starch, ion starch were analyzed by the amylograph and the rheometer. exchange water was added thereby adjusting the water con Also, after completion of the reaction, a degradation ratio was tent to 21%. The resultant was filled in a 3 L. glass beaker in a determined using a part of the reaction solution. state where blank space was as Small as possible, and the upper portion was covered with an aluminum foil, and then Example 37 heated at 120° C. for 15 minutes to carry out a heat-moisture treatment. After completion of the heat-moisture treatment, 0494. To 400 g of the enzyme-treated starch prepared in the heat-moisture-treated starch was recovered by blow dry Example 33, ion-exchange water was added thereby adjust 1ng. ing the water content to 21%. The resultant was filled in a 1 L glass beaker in a state where blank space was as Small as Example 33 possible, and the upper portion was covered with an alumi 0490. To 4 Kg of an untreated native corn starch, 9 Kg of num foil, and then heated at 120° C. for 15 minutes to carry ion-exchange water was added to prepare a starch Suspension. out a heat-moisture treatment. After completion of the heat After adjusting the pH of the suspension to 5.0, 1% by weight moisture treatment, the heat-moisture-enzyme-treated Starch (based on starch solid content) of amyloglucosidase (“OPTI was recovered by blow drying. Viscosity characteristics of the DEXL-400' derived from Aspergillus niger, manufactured obtained heat-moisture-enzyme-treated Starch were analyzed by Genencor; optimum pH of 4.4) was added and stirred at by the amylograph and the rheometer. Also, after completion 50° C. for 18 hours to carry out an enzyme reaction. After of the reaction, a degradation ratio was determined by a part completion of the reaction, an enzyme-treated Starch was of the reaction solution. recovered by centrifugal filtration and blow drying. It is noted that the degradation ratio of the obtained sample was 34%. Example 38 Example 34 0495 To 400 g of the enzyme-treated starch prepared in 0491. To 4 Kg of an untreated native corn starch, 9 Kg of Example 34, ion-exchange water was added thereby adjust ion-exchange water was added to prepare a starch Suspension. ing the water content to 20%. The resultant was filled in a 1 L After adjusting the pH of the suspension to 5.0, 1% by weight glass beaker in a state where blank space was as Small as (based on starch solid content) of O-amylase (AMYLEX A3' derived from Aspergillus niger, manufactured by possible, and the upper portion was covered with an alumi DANISCO: optimum pH of 5.0) was added and stirred at 50° num foil, and then heated at 120° C. for 15 minutes to carry C. for 18 hours to carry out an enzyme reaction. After comple out a heat-moisture treatment. After completion of the heat tion of the reaction, an enzyme-treated Starch was recovered moisture treatment, the heat-moisture-enzyme-treated Starch by centrifugal filtration and blow drying. It is noted that the was recovered by blow drying. Viscosity characteristics of the degradation ratio of the obtained sample was 28%. obtained heat-moisture-enzyme-treated Starch were analyzed by the amylograph and the rheometer. Also, after completion Example 35 of the reaction, a degradation ratio was determined by a part of the reaction solution. The measurement results of Com 0492 To 400 g of the heat-moisture-treated starch pre pared in Comparative Example 24, 900 g of ion-exchange parative Example 24 and Examples 35 to 38 are shown in water was added to prepare a starch Suspension. After adjust Table 26-2. ing the pH of the suspension to 5.0, 1% by weight (based on starch solid content) of amyloglucosidase (“OPTIDEX TABLE 26-1 L-400' derived from Aspergillus niger, manufactured by Genencor, optimum pH of 4.4) was added and stirred at 50° Summary of Names, Origins and Product Names of Enzymes C. for 18 hours to carry out an enzyme reaction. After comple Used for a Treatment of a Heat-moisture-treated Starch tion of the reaction, an enzyme-heat-moisture-treated Starch was recovered by centrifugal filtration and blow drying. Vis Product name cosity characteristics of the obtained enzyme-heat-moisture Name of of enzyme treated Starch were analyzed by the amylograph and the rhe Example enzyme Origin (manufacture) ometer. Also, after completion of the reaction, a degradation ratio was determined using a part of the reaction solution. Comparative Untreated corn Example 36 Example 24 starch Example 35 amyloglucosidase Aspergillus niger OPTIDEXL-400 0493 To 400 g of the heat-moisture-treated starch pre (Genencor) pared in Comparative Example 24, 900 g of ion-exchange Example 36 C-amylase Aspergillus niger AMYLEXA3 water was added to prepare a starch Suspension. After adjust (DANISCO) ing the pH of the suspension to 5.0, 1% by weight (based on Example 37 amyloglucosidase Aspergillus niger OPTIDEXL-400 starch solid content) of C.-amylase (AMYLEX A3' derived from Aspergillus niger, manufactured by DANISCO; opti (Genencor) mum pH of 5.0) was added and stirred at 50° C. for 18 hours Example 38 C-amylase Aspergillus niger AMYLEXA3 to carry out an enzyme reaction. After completion of the (DANISCO) reaction, an enzyme-heat-moisture-treated Starch was recov ered by centrifugal filtration and blow drying. Viscosity char US 2013/0022711 A1 Jan. 24, 2013 51

TABLE 26-2 Next Day Maximum viscosity Rupture stress Young's modulus Degradation Measured Breakdown Measured Measured ratio value Relative viscosity value Relative value Relative Example (%) (BU) 9% (%) (BU) (g) % (%) (dyn/cm) 9% (%) Comparative 401 100 108 230 1OO 6,091460 1OO Example 24 Example 35 39 351 88 96 439 191 6,566.419 108 Example 36 33 351 88 91 436 190 6,788,705 111 Example 37 34 327 82 74 420 182 6,690,934 110 Example 38 28 349 87 79 428 186 7,132,581 117 * Degradation ratios in Examples 37 and 38 each refer to a degradation ratio of enzyme-treated starch used as a base material.

0496 As described above, the present invention has been it possible to prepare a cookie which is very light and soft and exemplified using a preferred embodiment of the present has texture with nice melt in mouth, and to provide a cookie invention, but the present invention should not be construed to having readily edible texture which is also suited for persons be limited to this embodiment. It is understood that the of advanced age and infants. In addition, since the dough at present invention should be construed for its scope only by the the time of shaping a cookie is very dry and not sticky, the claims. It is understood that those skilled in the art can prac water addition amount at the time of preparing the dough can tice an equivalent range based on the description of the inven be further increased, thus making it possible to increase a tion and the technical common knowledge, from the descrip yield factor. Furthermore, for a food such as kudzu starch tion of the specific preferable embodiment of the present cake so-called in the Kanto area, which requires longtime and invention. It is understood that patents, patent applications much labor heretofore because the preparation of a wheat and publications cited in the present specification should be starch as a raw material requires a fermentation process for a herein incorporated by reference for the content thereofas if long period Such as one or more years, use of an enzyme the contents themselves were specifically described in the treated wheat starch of the present invention makes it possible present specification. to easily prepare a kudzu Starch cake so-called in the Kanto area which does not have fermentation odor derived from a INDUSTRIAL APPLICABILITY fermented wheat starch and has nice flavor, without requiring Such long time and much labor. 0497 As described above, the present invention provides 0499 Furthermore, when the enzyme-treated cassava various industrial advantages by using an enzyme having starch of the present invention is used in noodles, for example, characteristics capable of increasing a maximum viscosity of raw udon, the texture improving effect of impairing Sticky a starch. texture with rich chewiness is recognized, and no adverse 0498. According to the present invention, it becomes pos influence was exerted on factors of the quality of noodles, sible to provide a food having new textures which could not Such as “slippery and Smooth” and 'sogginess'. Thus, it has be obtained by a conventional chemically unmodified starch been found that the addition of this enzyme-treated starch and a chemically modified Starch. For example, use of the easily improve the texture of the noodle to those favored by enzyme-treated cassava starch in the present invention makes Japanese.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 14

<21 Os SEQ ID NO 1 &211s LENGTH: 1848 &212s. TYPE: DNA <213> ORGANISM: Aspergillus oryzae 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1848)

<4 OOs SEQUENCE: 1

atg cct tcc aag gt caca cag tat citg acc ggit gtt coa cac acc gac 48 Met Pro Ser Llys Val Thr Glin Tyr Lieu. Thr Gly Val Pro His Thr Asp 1. 5 1O 15

tgc titg ggt acc gala gca gtt gat acg tct att gala gta gala C9g C9C 96 Cys Lieu. Gly Thr Glu Ala Val Asp Thir Ser Ile Glu Val Glu Arg Arg 2O 25 3 O

atg atg acg cat cog to c tat att togg cta tac ct g to a gala aat cag 144

US 2013/0022711 A1 Jan. 24, 2013 54

- Continued

Met Pro Ser Llys Val Thr Glin Tyr Lieu. Thr Gly Val Pro His Thr Asp

Cys Lieu. Gly. Thr Glu Ala Val Asp Thir Ser Ile Glu Val Glu Arg Arg 2O 25 3O Met Met Thr His Pro Ser Tyr Ile Trp Leu Tyr Lieu Ser Glu Asin Glin 35 4 O 45 Lieu. Phe Leu Val Gly Tyr Phe His Glin Phe His Phe Val Ala Ser His SO 55 6 O Ser Gly Phe Leu Ser Ile Glin Ala Ile Asin Val Thr His Ser Ile Leu 65 70 7s 8O Val Ser Lieu. Thir Ser Lieu. Phe Thr Ser Gly Arg Ser Val Asp Thr Lys 85 90 95 Tyr Val Val Lys Ile Glu Glu Ala Glin His Lieu. Thir Lys Lieu Pro Ser 1OO 105 11 O Trp Asp Thr Pro Asp Asn Ser Leu Met Leu Gln Gly Phe Glu Trp His 115 12 O 125 Val Pro Asp Asp Glin Gly His Trp Lys Arg Lieu. Glin Arg Ser Lieu Val 13 O 135 14 O Ser Leu Lys Ser Ile Gly Val Asp Ser Ile Trp Ile Pro Pro Gly Cys 145 150 155 160 Lys Ala Met Asn. Pro Ser Gly Asin Gly Tyr Asp Ile Tyr Asp Lieu. Tyr 1.65 17O 17s Asp Lieu. Gly Glu Phe Asp Glin Lys Gly Ser Arg Ser Thr Lys Trp Gly 18O 185 19 O Ser Lys Thr Glu Lieu. Glin Ser Lieu Ala Cys Ser Ala Arg Asn Lieu. Gly 195 2OO 2O5 Ile Gly Ile Cys Trp Asp Ala Val Lieu. Asn His Lys Ala Gly Ala Asp 21 O 215 22O Tyr Thr Glu Arg Phe Ser Ala Wall Lys Val Asp Pro Lys Asp Arg Ser 225 23 O 235 24 O Val Glu Ile Phe Ala Ala Arg Glu Ile Glu Gly Trp Val Gly Phe Ser 245 250 255 Phe Pro Gly Arg Gly Gly Ile Tyr Ser Ser Met Lys Tyr Ser Trp His 26 O 265 27 O His Phe Ser Gly Val Asp Trp Asp Glu Ala Arg Llys Lys Asn Ala Ile 27s 28O 285 Tyr Arg Val Ala Ser Lys Arg Trip Ser Asp Asp Val Ala His Glu Lys 29 O 295 3 OO Gly Asn Tyr Asp Tyr Lieu Met Phe Ala Asp Lieu. Asp Tyr Ser Asn Lieu. 3. OS 310 315 32O Glu Val Glin Lys Asp Val Lieu. Arg Trp Gly Glu Trp Ile Gly Ser Glin 3.25 330 335 Lieu Pro Lieu. Trp Gly Met Arg Lieu. Asp Ala Ser Llys His Tyr Ser Ala 34 O 345 35. O Asp Phe Glin Llys Llys Phe Val Asn His Val Arg Ala Thr Val Gly Pro 355 360 365 Glin Ile Phe Phe Val Ala Glu Tyr Trp Ser Gly Asp Val Arg Val Lieu. 37 O 375 38O Met His Tyr Lieu. Glin Llys Met Asp Tyr Glin Lieu. Ser Lieu. Phe Asp Ala 385 390 395 4 OO

Pro Lieu Val Gly Arg Phe Ser Arg Ile Ser Arg Thr Gly Glu Asp Lieu. US 2013/0022711 A1 Jan. 24, 2013 55

- Continued

4 OS 41O 415 Arg Glu Ile Phe Asp Asp Thir Lieu Val Gly Asn Llys Pro Ala His Ala 42O 425 43 O Ile Thr Lieu Val Met Asn His Asp Thr Val Arg Glu Arg Glin Ser Lieu. 435 44 O 445 Glu Ala Pro Ile Ala Ser Phe Phe Llys Pro Lieu Ala Tyr Ala Lieu. Ile 450 45.5 460 Lieu. Lieu. Arg Asp Llys Gly Glin Pro Cys Ile Phe Tyr Gly Asp Lieu. Tyr 465 470 47s 48O Gly Ile Arg Arg Gly Val Lys Asn Pro Met Thr Pro Ser Cys Gly Gly 485 490 495 Llys Lieu Pro Val Lieu Ala Arg Ala Arg Llys Lieu. Tyr Ala Tyr Gly Glu SOO 505 51O Glin Cys Asp Tyr Phe Asp Glin Ala Asn. Cys Ile Gly Phe Val Arg Tyr 515 52O 525 Gly Asn Lieu. His His Pro Ser Gly Lieu Ala Cys Ile Met Ser Asn Gly 53 O 535 54 O Gly Ala Ser Glin Lys Arg Met Tyr Val Gly Arg Ser His Ala Lys Glu 5.45 550 555 560 Arg Trp Thir Asp Ile Lieu. Gly Trp His Pro Llys Thr Val Ile Ile Asp 565 st O sts Lys Lys Gly Tyr Gly Ile Phe Pro Val Ser Ala Met Glin Val Ser Val 58O 585 59 O Trp Val Asn. Ser Ala Ala Glu Ala Arg Glu Ser Lieu. Glin Glu Pro Phe 595 6OO 605 Glu Glu Lys Ile Tyr Glu Asn 610 615

<210s, SEQ ID NO 3 &211s LENGTH: 1497 &212s. TYPE: DNA <213> ORGANISM: Aspergillus niger 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1497

<4 OOs, SEQUENCE: 3 atg gt C gcg tdg td tot Cta titt Ctg tac ggc Ctt cag gt C gog gca 48 Met Val Ala Trp Trp Ser Lieu. Phe Lieu. Tyr Gly Lieu. Glin Val Ala Ala 1. 5 1O 15

Cct gct ttg gCt gca acg cct gcg gac tog catcg caa toc att tat 96 Pro Ala Lieu Ala Ala Thr Pro Ala Asp Trp Arg Ser Glin Ser Ile Tyr 2O 25 3O ttic Ctt Ct c acg gat cattt gca agg acg gat ggg tog acg act gcg 144 Phe Lieu. Lieu. Thir Asp Arg Phe Ala Arg Thr Asp Gly Ser Thir Thr Ala 35 4 O 45 act tt aat act gcg gat cag aaa tac tdt ggit gga aca tig cag ggc 192 Thr Cys Asn Thr Ala Asp Gln Lys Tyr Cys Gly Gly. Thir Trp Glin Gly SO 55 6 O atc at C gac aag titg gac tat at C cag gga atg ggc titc aca gcc at C 24 O Ile Ile Asp Llys Lieu. Asp Tyr Ile Glin Gly Met Gly Phe Thir Ala Ile 65 70 7s 8O tgg atc acc ccc gtt aca gcc cag citg ccc cag acc acc gca tat gga 288 Trp Ile Thr Pro Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly 85 90 95 gat gcc tac cat ggc tac tig cag cag gat at a tact ct ctgaac gala 336

US 2013/0022711 A1 Jan. 24, 2013 57

- Continued

4 OS 41O 415 cgc aag ggc aca gat ggg tog Cag at C gtg act atc ttg to C aac aag 296 Arg Lys Gly Thr Asp Gly Ser Glin Ile Val Thir Ile Lieu. Ser Asn Lys 42O 425 43 O ggit gct tcg ggit gat t c tat acc Ct c to C titg agt ggit gcg ggit tac 344 Gly Ala Ser Gly Asp Ser Tyr Thr Lieu. Ser Lieu. Ser Gly Ala Gly Tyr 435 44 O 445 aca gcc ggc cag caa ttg acg gag gtc att ggc tigc acg acc gtg acg 392 Thr Ala Gly Glin Gln Lieu. Thr Glu Val Ile Gly Cys Thr Thr Val Thr 450 45.5 460 gtt ggit tog gat gga aat gtg cct gtt CCt atg gca ggt ggg ct a cct 44 O Val Gly Ser Asp Gly Asn Val Pro Val Pro Met Ala Gly Gly Lieu Pro 465 470 47s 48O agg gta ttg tat ccg act gag aag ttg gca ggit agc aag at C tit agt 488 Arg Val Lieu. Tyr Pro Thr Glu Lys Lieu Ala Gly Ser Lys Ile Cys Ser 485 490 495 agc ticg tda 497 Ser Ser

<210s, SEQ ID NO 4 &211s LENGTH: 498 212. TYPE: PRT <213> ORGANISM: Aspergillus niger

<4 OOs, SEQUENCE: 4 Met Val Ala Trp Trp Ser Lieu. Phe Lieu. Tyr Gly Lieu. Glin Val Ala Ala 1. 5 1O 15 Pro Ala Lieu Ala Ala Thr Pro Ala Asp Trp Arg Ser Glin Ser Ile Tyr 2O 25 3O Phe Lieu. Lieu. Thir Asp Arg Phe Ala Arg Thr Asp Gly Ser Thir Thr Ala 35 4 O 45 Thr Cys Asn Thr Ala Asp Gln Lys Tyr Cys Gly Gly. Thir Trp Glin Gly SO 55 6 O Ile Ile Asp Llys Lieu. Asp Tyr Ile Glin Gly Met Gly Phe Thir Ala Ile 65 70 7s 8O Trp Ile Thr Pro Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly 85 90 95 Asp Ala Tyr His Gly Tyr Trp Glin Glin Asp Ile Tyr Ser Lieu. Asn. Glu 1OO 105 11 O Asn Tyr Gly Thr Ala Asp Asp Lieu Lys Ala Lieu. Ser Ser Ala Lieu. His 115 12 O 125 Glu Arg Gly Met Tyr Lieu Met Val Asp Val Val Ala Asn His Met Gly 13 O 135 14 O Tyr Asp Gly Ala Gly Ser Ser Val Asp Tyr Ser Val Phe Llys Pro Phe 145 150 155 160 Ser Ser Glin Asp Tyr Phe His Pro Phe Cys Phe Ile Glin Asn Tyr Glu 1.65 17O 17s Asp Glin Thr Glin Val Glu Asp Cys Trp Lieu. Gly Asp Asn. Thr Val Ser 18O 185 19 O Lieu Pro Asp Lieu. Asp Thir Thr Lys Asp Val Val Lys Asn. Glu Trp Tyr 195 2OO 2O5 Asp Trp Val Gly Ser Lieu Val Ser Asn Tyr Ser Ile Asp Gly Lieu. Arg 21 O 215 22O Ile Asp Thr Val Lys His Val Glin Lys Asp Phe Trp Pro Gly Tyr Asn 225 23 O 235 24 O US 2013/0022711 A1 Jan. 24, 2013 58

- Continued

Lys Ala Ala Gly Val Tyr Cys Ile Gly Glu Val Lieu. Asp Gly Asp Pro 245 250 255 Ala Tyr Thr Cys Pro Tyr Glin Asn Val Met Asp Gly Val Lieu. Asn Tyr 26 O 265 27 O Pro Ile Tyr Tyr Pro Leu Lleu. Asn Ala Phe Llys Ser Thr Ser Gly Ser 27s 28O 285 Met Asp Asp Lieu. Tyr Asn Met Ile Asn Thr Val Llys Ser Asp Cys Pro 29 O 295 3 OO Asp Ser Thir Lieu. Lieu. Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg 3. OS 310 315 32O Phe Ala Ser Tyr Thr Asn Asp Ile Ala Lieu Ala Lys Asn Val Ala Ala 3.25 330 335 Phe Ile Ile Lieu. Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Glin Glu 34 O 345 35. O Glin His Tyr Ala Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thir Trp 355 360 365 Lieu. Ser Gly Tyr Pro Thr Asp Ser Glu Lieu. Tyr Llys Lieu. Ile Ala Ser 37 O 375 38O Ala Asn Ala Ile Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val 385 390 395 4 OO Thr Tyr Lys Asn Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met 4 OS 41O 415 Arg Lys Gly Thr Asp Gly Ser Glin Ile Val Thir Ile Lieu. Ser Asn Lys 42O 425 43 O Gly Ala Ser Gly Asp Ser Tyr Thr Lieu. Ser Lieu. Ser Gly Ala Gly Tyr 435 44 O 445 Thr Ala Gly Glin Gln Lieu. Thr Glu Val Ile Gly Cys Thr Thr Val Thr 450 45.5 460 Val Gly Ser Asp Gly Asn Val Pro Val Pro Met Ala Gly Gly Lieu Pro 465 470 47s 48O Arg Val Lieu. Tyr Pro Thr Glu Lys Lieu Ala Gly Ser Lys Ile Cys Ser 485 490 495

Ser Ser

<210s, SEQ ID NO 5 &211s LENGTH: 1923 &212s. TYPE: DNA <213> ORGANISM: Aspergillus niger 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1923)

<4 OOs, SEQUENCE: 5 atgtcg tt C catct cta ctic goc ctg agc ggc ctic gtc. tcc aca ggg 48 Met Ser Phe Arg Ser Lieu. Lieu Ala Lieu. Ser Gly Lieu Val Cys Thr Gly 1. 5 1O 15 ttg gca aat gtg att t cc aag cc gcg acc titg gat tca tig titg agc 96 Lieu Ala Asn Val Ile Ser Lys Arg Ala Thir Lieu. Asp Ser Trp Lieu. Ser 2O 25 3O aac gala gcg acc gtg gct cqt act gcc at C ctgaat aac at C ggg gcg 144 Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Lieu. Asn. Asn. Ile Gly Ala 35 4 O 45 gac ggit gct tog gtg tcg ggc gcg gac tot ggc att gtc gtt gct agt 192 Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser SO 55 6 O

US 2013/0022711 A1 Jan. 24, 2013 61

- Continued

Met Ser Phe Arg Ser Lieu. Lieu Ala Lieu. Ser Gly Lieu Val Cys Thr Gly

Lieu Ala Asn Val Ile Ser Lys Arg Ala Thir Lieu. Asp Ser Trp Lieu. Ser 2O 25 3O Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Lieu. Asn. Asn. Ile Gly Ala 35 4 O 45 Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser SO 55 6 O Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser 65 70 7s 8O Gly Lieu Val Lieu Lys Thr Lieu Val Asp Lieu. Phe Arg Asn Gly Asp Thr 85 90 95 Ser Lieu. Lieu. Ser Thir Ile Glu Asn Tyr Ile Ser Ala Glin Ala Ile Val 1OO 105 11 O Glin Gly Ile Ser Asn Pro Ser Gly Asp Lieu. Ser Ser Gly Ala Gly Lieu. 115 12 O 125 Gly Glu Pro Llys Phe Asn Val Asp Glu Thir Ala Tyr Thr Gly Ser Trp 13 O 135 14 O Gly Arg Pro Glin Arg Asp Gly Pro Ala Lieu. Arg Ala Thr Ala Met Ile 145 150 155 160 Gly Phe Gly Gln Trp Leu Lleu. Asp Asn Gly Tyr Thr Ser Thr Ala Thr 1.65 17O 17s Asp Ile Val Trp Pro Lieu Val Arg Asn Asp Lieu. Ser Tyr Val Ala Glin 18O 185 19 O Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asin Gly Ser 195 2OO 2O5 Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser 21 O 215 22O Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Glin 225 23 O 235 24 O Ala Pro Glu Ile Lieu. Cys Tyr Lieu. Glin Ser Phe Trp Thr Gly Ser Phe 245 250 255 Ile Lieu Ala Asn. Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr 26 O 265 27 O Lieu. Lieu. Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp 27s 28O 285 Ser Thr Phe Glin Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu 29 O 295 3 OO Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Lieu. Asn Asp Gly Lieu. Ser 3. OS 310 315 32O Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr 3.25 330 335 Asn Gly Asn. Pro Trp Phe Lieu. Cys Thir Lieu Ala Ala Ala Glu Gln Lieu 34 O 345 35. O Tyr Asp Ala Lieu. Tyr Glin Trp Asp Llys Glin Gly Ser Lieu. Glu Val Thr 355 360 365 Asp Wal Ser Lieu. Asp Phe Phe Lys Ala Lieu. Tyr Ser Asp Ala Ala Thr 37 O 375 38O Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala 385 390 395 4 OO

Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala US 2013/0022711 A1 Jan. 24, 2013 62

- Continued

4 OS 41O 415 Ala Ser Asn Gly Ser Met Ser Glu Glin Tyr Asp Llys Ser Asp Gly Glu 42O 425 43 O Glin Lieu. Ser Ala Arg Asp Lieu. Thir Trp Ser Tyr Ala Ala Lieu. Lieu. Thir 435 44 O 445 Ala Asn. Asn Arg Arg Asn. Ser Val Val Pro Ala Ser Trp Gly Glu Thr 450 45.5 460 Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly 465 470 47s 48O Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr 485 490 495 Gly Gly. Thir Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr SOO 505 51O Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser Thr 515 52O 525 Ser Ser Thr Ser Cys Thir Thr Pro Thr Ala Val Ala Val Thr Phe Asp 53 O 535 54 O Lieu. Thir Ala Thr Thr Thr Tyr Gly Glu Asin Ile Tyr Lieu Val Gly Ser 5.45 550 555 560 Ile Ser Glin Lieu. Gly Asp Trp Glu Thir Ser Asp Gly Ile Ala Lieu. Ser 565 st O sts Ala Asp Llys Tyr Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr 58O 585 59 O Lieu Pro Ala Gly Glu Ser Phe Glu Tyr Llys Phe Ile Arg Ile Glu Ser 595 6OO 605 Asp Asp Ser Val Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val 610 615 62O Pro Glin Ala Cys Gly. Thir Ser Thr Ala Thr Val Thr Asp Thir Trp Arg 625 630 635 64 O

<210s, SEQ ID NO 7 &211s LENGTH: 2334 &212s. TYPE: DNA <213> ORGANISM: Flavobacterium sp. 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (2334)

<4 OO > SEQUENCE: 7 atg gaC cca cac gcc ccg cag cig caa ca agc ggg cag cqc ttg cc 48 Met Asp Pro His Ala Pro Glin Arg Glin Arg Ser Gly Glin Arg Lieu. Arg 1. 5 1O 15 gcc ct c goc ctg gcc gcg Ctg gCC to gog ctg agc ccg gcc cac goc 96 Ala Lieu Ala Lieu Ala Ala Lieu Ala Cys Ala Lieu. Ser Pro Ala His Ala 2O 25 3O gcc at C gat gcg cag cag Ctic ggc gcg cgc tac gac gcc gcc cag gCC 144 Ala Ile Asp Ala Glin Gln Lieu. Gly Ala Arg Tyr Asp Ala Ala Glin Ala 35 4 O 45 aac Ct c gCg tt C. c99 gtc. t at to C tog cqc gcg acc cqc gt c gag gtg 192 Asn Lieu Ala Phe Arg Val Tyr Ser Ser Arg Ala Thr Arg Val Glu Val SO 55 6 O ttic ctg tac aag aac Ccg acc ggc ticg cag gala gtc gcg cqg ctg gcg 24 O Phe Lieu. Tyr Lys Asn Pro Thr Gly Ser Glin Glu Val Ala Arg Lieu Ala 65 70 7s 8O

Ctg agc aag gaC ccg gcg acc cag gtg tdg tog Ctg tcg Ctg ccg acc 288 Lieu. Ser Lys Asp Pro Ala Thr Glin Val Trp Ser Leu Ser Leu Pro Thr

US 2013/0022711 A1 Jan. 24, 2013 65

- Continued gcc agc gcg at C tac gtc gcc tac aac ggc tigg to C ggc gcg gtC gac 216 O Ala Ser Ala Ile Tyr Val Ala Tyr Asn Gly Trp Ser Gly Ala Val Asp 7 Os 71O 71s 72O ttic aag ctg. cc.g. tig cc ggc acc ggc aag cag tig tac C9g gt C acc 22O8 Phe Llys Lieu Pro Trp Pro Gly Thr Gly Lys Gln Trp Tyr Arg Val Thr 72 73 O 73 gat acc gcg acc tig aac gaa ggc cc C aac gog gtg gcg ctg. CCC ggc 2256 Asp Thir Ala Thir Trp Asn. Glu Gly Pro Asn Ala Val Ala Lieu Pro Gly 740 74. 7 O agc gag acc ctg atc ggc ggc gag aac acc gtc tac ggc atg cag gcg 23O4. Ser Glu Thir Lieu. Ile Gly Gly Glu Asn Thr Val Tyr Gly Met Glin Ala 7ss 760 765 cgc ticg ctg. citg ttg Ctg atc gcg aag ta 2334 Arg Ser Lieu. Lieu Lleu Lieu. Ile Ala Lys 770 775

<210s, SEQ ID NO 8 211 LENGTH: 777 212. TYPE: PRT <213> ORGANISM: Flavobacterium sp. <4 OOs, SEQUENCE: 8 Met Asp Pro His Ala Pro Glin Arg Glin Arg Ser Gly Glin Arg Lieu. Arg 1. 5 1O 15 Ala Lieu Ala Lieu Ala Ala Lieu Ala Cys Ala Lieu. Ser Pro Ala His Ala 2O 25 3O Ala Ile Asp Ala Glin Gln Lieu. Gly Ala Arg Tyr Asp Ala Ala Glin Ala 35 4 O 45 Asn Lieu Ala Phe Arg Val Tyr Ser Ser Arg Ala Thr Arg Val Glu Val SO 55 6 O Phe Lieu. Tyr Lys Asn Pro Thr Gly Ser Glin Glu Val Ala Arg Lieu Ala 65 70 7s 8O Lieu. Ser Lys Asp Pro Ala Thr Glin Val Trp Ser Leu Ser Leu Pro Thr 85 90 95 Ser Thir Ile Lys Asn Thr Tyr Gly Ile Thr Gly Ala Val Tyr Tyr Gly 1OO 105 11 O Tyr Arg Ala Trp Gly Pro Asn Trp Pro Tyr Asp Ala Ala Trp Thir Lys 115 12 O 125 Gly Ser Ala Thr Gly Phe Val Ser Asp Wall Asp Asn Ala Gly Asn Arg 13 O 135 14 O Phe Asn Pro Asn Llys Lieu. Lieu. Lieu. Asp Pro Tyr Ala Arg Glu Ile Ser 145 150 155 160 Gln Asp Pro Asn Thr Ala Thr Cys Ala Asp Gly Thr Ile Tyr Ala Thr 1.65 17O 17s Gly Ala Ala His Arg Asn Lys Asp Ser Gly Lieu. Cys Ala Ser Lys Gly 18O 185 19 O Ile Ala Lieu Ala Ala Asp Ala Thir Ser Val Gly Ser Llys Pro Thr Arg 195 2OO 2O5 Ala Lieu Lys Asp Glu Val Ile Tyr Glu Val His Val Arg Gly Lieu. Thr 21 O 215 22O Arg Asn Asp Asp Ser Val Pro Ala Ala Glu Arg Gly. Thir Tyr Lys Gly 225 23 O 235 24 O Ala Ala Arg Lys Ala Ala Ala Lieu Ala Ala Lieu. Gly Val Thir Ala Val 245 250 255

Glu Phe Lieu Pro Val Glin Glu Thr Glin Asn Asp Glin Asn Asp Wall Asp US 2013/0022711 A1 Jan. 24, 2013 66

- Continued

26 O 265 27 O Pro Asn Ser Thr Ala Gly Asp Asn Tyr Trp Gly Tyr Met Thr Lieu. Asn 27s 28O 285 Tyr Phe Ala Pro Asp Arg Arg Tyr Ala Tyr Asp Llys Ser Ala Gly Gly 29 O 295 3 OO Pro Thr Arg Glu Trp Lys Ala Met Val Lys Ala Phe His Asp Ala Gly 3. OS 310 315 32O Ile Llys Val Tyr Ile Asp Val Val Tyr Asn His Thr Gly Glu Gly Gly 3.25 330 335 Pro Trp Ser Gly Thr Asp Gly Lieu Ser Val Tyr Asn Lieu. Leu Ser Phe 34 O 345 35. O Arg Gly Lieu. Asp ASn Pro Ala Tyr Tyr Ser Lieu. Ser Ser Asp Tyr Lys 355 360 365 Tyr Pro Trp Asp Asn Thr Gly Val Gly Gly Asn Tyr Asn Thr Arg His 37 O 375 38O Pro Ile Ala Glin Asn Lieu. Ile Val Asp Ser Lieu Ala Tyr Trp Arg Asp 385 390 395 4 OO Ala Lieu. Gly Val Asp Gly Phe Arg Phe Asp Lieu Ala Ser Val Lieu. Gly 4 OS 41O 415 Asn Ser Cys Glin His Gly Cys Phe Asin Phe Asp Lys Asn Asp Ser Gly 42O 425 43 O Asn Ala Lieu. Asn Arg Ile Val Ala Glu Lieu Pro Pro Arg Pro Ala Ala 435 44 O 445 Gly Gly Ala Gly Ala Asp Lieu. Ile Ala Glu Pro Trp Ala Ile Gly Gly 450 45.5 460 Asn Ser Tyr Glin Val Gly Gly Phe Pro Ala Gly Trp Ala Glu Trp Asn 465 470 47s 48O Gly Lieu. Tyr Arg Asp Ala Lieu. Arg Llys Lys Glin Asn Llys Lieu. Gly Val 485 490 495 Glu Thr Val Thr Pro Gly Thr Lieu Ala Thr Arg Phe Ala Gly Ser Asn SOO 505 51O Asp Lieu. Tyr Gly Asp Asp Gly Arg Llys Pro Trp His Ser Ile Asin Phe 515 52O 525 Val Val Ala His Asp Gly Phe Thr Lieu. Asn Asp Lieu. Tyr Ala Tyr Asn 53 O 535 54 O Asp Llys Glin Asn. Asn Gln Pro Trp Pro Tyr Gly Pro Ser Asp Gly Gly 5.45 550 555 560 Glu Asp His Asn Lieu. Ser Trp Asn Glin Gly Gly Ile Val Ala Glu Glin 565 st O sts Arg Lys Ala Ala Arg Thr Gly Lieu Ala Lieu. Lieu Met Lieu. Ser Ala Gly 58O 585 59 O Val Pro Met Ile Thr Gly Gly Asp Glu Ala Leu Arg Thr Glin Phe Gly 595 6OO 605 Asn Asn. Asn. Thir Tyr Asn Lieu. Asp Ser Ala Ala Asn Trp Lieu. Tyr Trip 610 615 62O Ser Arg Ser Ala Lieu. Glu Ala Asp His Glu Thir Tyr Thr Lys Arg Lieu. 625 630 635 64 O Ile Ala Phe Arg Lys Ala His Pro Ala Lieu. Arg Pro Ala Asn. Phe Tyr 645 650 655 Ser Ala Ser Asp Thr Asn Gly Asn. Wal Met Glu Glin Lieu. Arg Trp Phe 660 665 67 O

US 2013/0022711 A1 Jan. 24, 2013 70

- Continued

212. TYPE: PRT <213> ORGANISM: Pseudomonas amyloderamosa

<4 OOs, SEQUENCE: 10 Met Lys Cys Pro Llys Ile Lieu. Gly Ala Lieu. Lieu. Gly Cys Ala Val Lieu. 1. 5 1O 15 Ala Gly Val Pro Ala Met Pro Ala His Ala Ala Ile Asn Ser Met Ser 2O 25 3O Lieu. Gly Ala Ser Tyr Asp Ala Glin Glin Ala Asn. Ile Thr Phe Arg Val 35 4 O 45 Tyr Ser Ser Glin Ala Thr Arg Ile Val Lieu. Tyr Lieu. Tyr Ser Ala Gly SO 55 6 O Tyr Gly Val Glin Glu Ser Ala Thr Tyr Thr Lieu Ser Pro Ala Gly Ser 65 70 7s 8O Gly Val Trp Ala Val Thr Val Pro Val Ser Ser Ile Lys Ala Ala Gly 85 90 95 Ile Thr Gly Ala Val Tyr Tyr Gly Tyr Arg Ala Trp Gly Pro Asn Trp 1OO 105 11 O Pro Tyr Ala Ser Asn Trp Gly Lys Gly Ser Glin Ala Gly Cys Val Ser 115 12 O 125 Asp Wall Asp Ala Asn Gly Asp Arg Phe Asin Pro Asn Llys Lieu. Lieu. Lieu 13 O 135 14 O Asp Pro Tyr Ala Glin Glu Val Ser Glin Asp Pro Lieu. Asn. Pro Ser Asn 145 150 155 160 Gln Asn Gly Asn Val Phe Ala Ser Ala His Tyr Arg Thr Thr Asp Ser 1.65 17O 17s Gly Ile Tyr Ala Pro Lys Gly Val Val Lieu Val Pro Ser Thr Glin Ser 18O 185 19 O Thr Gly Thr Llys Pro Thr Arg Ala Glin Lys Asp Asp Val Ile Tyr Glu 195 2OO 2O5 Val His Val Arg Gly Phe Thr Glu Glin Asp Thir Ser Ile Pro Ala Glin 21 O 215 22O Tyr Arg Gly. Thir Tyr Tyr Gly Ala Gly Lieu Lys Ala Ser Tyr Lieu Ala 225 23 O 235 24 O Ser Leu Gly Val Thr Ala Val Glu Phe Leu Pro Val Glin Glu. Thr Glin 245 250 255 Asn Asp Ala Asn Asp Val Val Pro Asn. Ser Asp Ala Asn. Glin Asn Tyr 26 O 265 27 O Trp Gly Tyr Met Thr Glu Asn Tyr Phe Ser Pro Asp Arg Arg Tyr Ala 27s 28O 285 Tyr Asn Lys Ala Ala Gly Gly Pro Thr Ala Glu Phe Glin Ala Met Val 29 O 295 3 OO Glin Ala Phe His Asn Ala Gly Ile Llys Val Tyr Met Asp Val Val Tyr 3. OS 310 315 32O Asn His Thr Ala Glu Gly Gly. Thir Trp Thr Ser Ser Asp Pro Thr Thr 3.25 330 335 Ala Thir Ile Tyr Ser Trp Arg Gly Lieu. Asp Asn Ala Thr Tyr Tyr Glu 34 O 345 35. O Lieu. Thir Ser Gly Asn Glin Tyr Phe Tyr Asp Asn Thr Gly Ile Gly Ala 355 360 365 Asn Phe Asn. Thir Tyr Asn Thr Val Ala Glin Asn Lieu. Ile Val Asp Ser 37 O 375 38O US 2013/0022711 A1 Jan. 24, 2013 71

- Continued Val Ala Tyr Trp Ala Asn. Thir Met Gly Val Asp Gly Phe Arg Phe Asp 385 390 395 4 OO Lieu Ala Ser Val Lieu. Gly Asn. Ser Cys Lieu. Asn Ala Val His Ala Ser 4 OS 41O 415 Ala Pro Asn. Cys Pro Asn Gly Gly Tyr Asn. Phe Asp Ala Ala Asp Ser 42O 425 43 O Asn Val Ala Ile Asn Arg Ile Lieu. Arg Glu Phe Thr Val Arg Pro Ala 435 44 O 445 Ala Gly Gly Thr Val Trp Ile Cys Lieu. Arg Asn Lieu. Gly Pro Ser Ala 450 45.5 460 Ala Thr Arg Thir Ser Trp Val Asp Ser Arg Arg Val Val Arg Val Glu 465 470 47s 48O Trp Ser Val Pro Arg Glin Lieu. Arg Glin Ala Glin Asn. Glu Lieu. Gly Ser 485 490 495 Met Thr Ile Tyr Val Thr Glin Asp Ala Asn Asp Phe Ser Gly Ser Ser SOO 505 51O Asn Lieu. Phe Glin Ser Ser Gly Arg Ser Pro Trp Asn Ser Ile Asin Phe 515 52O 525 Ile Asp Val His Asp Gly Met Thir Lieu Lys Asp Val Tyr Ser Cys Asn 53 O 535 54 O Gly Ala Asn. Asn. Ser Glin Ala Ser Tyr Gly Pro Ser Asp Gly Gly. Thir 5.45 550 555 560 Ser Thr Asn Tyr Ser Trp Asp Gln Gly Met Ser Ala Gly Thr Gly Ala 565 st O sts Ala Val Asp Glin Arg Arg Ala Ala Arg Thr Gly Met Ala Phe Glu Met 58O 585 59 O Lieu. Ser Ala Gly Thr Pro Lieu Met Glin Gly Gly Asp Glu Tyr Lieu. Arg 595 6OO 605 Thir Lieu. Glin Cys Asn. Asn. Asn Ala Tyr Asn Lieu. Asp Ser Ser Ala Asn 610 615 62O Trp Lieu. Thr Tyr Ser Trp Thr Thr Asp Glin Ser Asn Phe Tyr Thr Phe 625 630 635 64 O Ala Glin Arg Lieu. Ile Arg Ser Ala Arg His Ile Pro Lieu. Arg Pro Ser 645 650 655 Ser Trp Tyr Ser Gly Ser Gln Lieu. Thir Trp Tyr Gln Pro Ser Gly Ala 660 665 67 O Val Ala Asp Ser Asn Tyr Trp Asn. Asn. Thir Ser Asn Tyr Ala Ile Ala 675 68O 685 Tyr Ala Ile Asin Gly Pro Ser Lieu. Gly Asp Ser Asn. Ser Ile Tyr Val 69 O. 695 7 OO Ala Tyr Asn Gly Trp Ser Ser Ser Val Thr Phe Thr Lieu Pro Ala Pro 7 Os 71O 71s 72O Pro Ser Gly Thr Gln Trp Tyr Arg Val Thr Asp Thr Cys Asp Trp Asn 72 73 O 73 Asp Gly Ala Ser Thr Phe Val Ala Pro Gly Ser Glu Thir Lieu. Ile Gly 740 74. 7 O Gly Ala Gly. Thir Thr Tyr Gly Glin Cys Gly Glin Ser Lieu. Lieu. Lieu. Lieu. 7ss 760 765 Ile Ser Lys 770

<210s, SEQ ID NO 11

US 2013/0022711 A1 Jan. 24, 2013 75

- Continued

&211s LENGTH: 865 212. TYPE: PRT <213> ORGANISM: Aspergillus niger

<4 OOs, SEQUENCE: 12 Met Lieu. Gly Ser Lieu Lleu Lleu Lieu. Lieu Pro Lieu Val Gly Ala Ala Val 1. 5 1O 15 Ile Gly Pro Arg Ala Asn. Ser Glin Ser Cys Pro Gly Tyr Lys Ala Ser 2O 25 3O Asn Val Glin Lys Glin Ala Arg Ser Lieu. Thir Ala Asp Lieu. Thir Lieu Ala 35 4 O 45 Gly Thr Pro Cys Asn. Ser Tyr Gly Lys Asp Lieu. Glu Asp Lieu Lys Lieu. SO 55 6 O Lieu Val Glu Tyr Glin Thr Asp Glu Arg Lieu. His Val Met Ile Tyr Asp 65 70 7s 8O Ala Asp Glu Glu Val Tyr Glin Val Pro Glu Ser Val Lieu Pro Arg Val 85 90 95 Gly Ser Asp Glu Asp Ser Glu Asp Ser Val Lieu. Glu Phe Asp Tyr Val 1OO 105 11 O Glu Glu Pro Phe Ser Phe Thir Ile Ser Lys Gly Asp Glu Val Leu Phe 115 12 O 125 Asp Ser Ser Ala Ser Pro Leu Val Phe Glin Ser Glin Tyr Val Asn Lieu. 13 O 135 14 O Arg Thr Trp Lieu. Pro Asp Asp Pro Tyr Val Tyr Gly Lieu. Gly Glu. His 145 150 155 160 Ser Asp Pro Met Arg Lieu Pro Thr Tyr Asn Tyr Thr Arg Thr Lieu. Trp 1.65 17O 17s Asn Arg Asp Ala Tyr Gly Thr Pro Asn. Asn. Thir Asn Lieu. Tyr Gly Ser 18O 185 19 O His Pro Val Tyr Tyr Asp His Arg Gly Lys Ser Gly Thr Tyr Gly Val 195 2OO 2O5 Phe Lieu. Lieu. Asn. Ser Asn Gly Met Asp Ile Lys Ile Asn Glin Thir Thr 21 O 215 22O Asp Gly Lys Glin Tyr Lieu. Glu Tyr Asn Lieu. Lieu. Gly Gly Val Lieu. Asp 225 23 O 235 24 O Phe Tyr Phe Phe Tyr Gly Glu Asp Pro Lys Glin Ala Ser Met Glu Tyr 245 250 255 Ser Lys Ile Val Gly Lieu Pro Ala Met Glin Ser Tyr Trp Thr Phe Gly 26 O 265 27 O Val Cys Pro Pro Pro Pro Asn Pro Ile Thr Val Arg Val Val Val Tyr 27s 28O 285 Asn Tyr Ser Glin Ala Lys Ile Pro Leu Glu Thir Met Trp Thr Asp Ile 29 O 295 3 OO Asp Tyr Met Asp Lys Arg Arg Val Phe Thr Lieu. Asp Pro Glin Arg Phe 3. OS 310 315 32O Pro Lieu. Glu Lys Met Arg Glu Lieu Val Thir Tyr Lieu. His Asn His Asp 3.25 330 335 Gln His Tyr Ile Val Met Val Asp Pro Ala Val Ser Val Ser Asn Asn 34 O 345 35. O Thir Ala Tyr Ile Thr Gly Val Arg Asp Asp Val Phe Lieu. His Asn Glin 355 360 365

Asn Gly Ser Leu Tyr Glu Gly Ala Val Trp Pro Gly Val Thr Val Phe 37 O 375 38O US 2013/0022711 A1 Jan. 24, 2013 76

- Continued

Pro Asp Trp Phe Asn Glu Gly Thr Glin Asp Tyr Trp Thr Ala Glin Phe 385 390 395 4 OO Glin Glin Phe Phe Asp Pro Llys Ser Gly Val Asp Ile Asp Ala Lieu. Trp 4 OS 41O 415 Ile Asp Met Asn. Glu Ala Ser Asn. Phe Cys Pro Tyr Pro Cys Lieu. Asp 42O 425 43 O Pro Ala Ala Tyr Ala Ile Ser Ala Asp Lieu Pro Pro Ala Ala Pro Pro 435 44 O 445 Val Arg Pro Ser Ser Pro Ile Pro Leu Pro Gly Phe Pro Ala Asp Phe 450 45.5 460 Glin Pro Ser Ser Lys Arg Ser Val Lys Arg Ala Glin Gly Asp Llys Gly 465 470 47s 48O Llys Llys Val Gly Lieu Pro Asn Arg Asn Lieu. Thir Asp Pro Pro Tyr Thr 485 490 495 Ile Arg Asn Ala Ala Gly Val Lieu. Ser Met Ser Thir Ile Glu Thir Asp SOO 505 51O Lieu. Ile His Ala Gly Glu Gly Tyr Ala Glu Tyr Asp Thr His Asn Lieu. 515 52O 525 Tyr Gly Thr Arg Lieu Val Met Ser Ser Ala Ser Arg Thr Ala Met Glin 53 O 535 54 O Ala Arg Arg Pro Asp Val Arg Pro Lieu Val Ile Thr Arg Ser Thr Phe 5.45 550 555 560 Ala Gly Ala Gly Ala His Val Gly. His Trp Lieu. Gly Asp Asn. Phe Ser 565 st O sts Asp Trp Val His Tyr Arg Ile Ser Ile Ala Glin Ile Lieu. Ser Phe Ala 58O 585 59 O Ser Met Phe Glin Ile Pro Met Val Gly Ala Asp Val Cys Gly Phe Gly 595 6OO 605 Ser Asn. Thir Thr Glu Glu Lieu. Cys Ala Arg Trp Ala Ser Lieu. Gly Ala 610 615 62O Phe Tyr Thr Phe Tyr Arg Asn His Asn Glu Lieu. Gly Asp Ile Ser Glin 625 630 635 64 O Glu Phe Tyr Arg Trp Pro Thr Val Ala Glu Ser Ala Arg Lys Ala Ile 645 650 655 Asp Ile Arg Tyr Llys Lieu. Lieu. Asp Tyr Ile Tyr Thr Ala Lieu. His Arg 660 665 67 O Gln Ser Glin Thr Gly Glu Pro Phe Leu Glin Pro Glin Phe Tyr Lieu. Tyr 675 68O 685 Pro Glu Asp Ser Asn Thr Phe Ala Asn Asp Arg Glin Phe Phe Tyr Gly 69 O. 695 7 OO Asp Ala Lieu. Lieu Val Ser Pro Val Lieu. Asn. Glu Gly Ser Thir Ser Val 7 Os 71O 71s 72O Asp Ala Tyr Phe Pro Asp Asp Ile Phe Tyr Asp Trp Tyr Thr Gly Ala 72 73 O 73 Val Val Arg Gly His Gly Glu Asn. Ile Thr Lieu. Ser Asn. Ile Asn. Ile 740 74. 7 O Thr His Ile Pro Lieu. His Ile Arg Gly Gly Asn Ile Ile Pro Val Arg 7ss 760 765 Thir Ser Ser Gly Met Thir Thr Thr Glu Val Arg Lys Glin Gly Phe Glu 770 775 78O

Lieu. Ile Ile Ala Pro Asp Lieu. Asp Asp Thir Ala Ser Gly Ser Lieu. Tyr

US 2013/0022711 A1 Jan. 24, 2013 80

- Continued

212. TYPE: PRT <213> ORGANISM: Paenibacillus macerans

<4 OOs, SEQUENCE: 14 Met Lys Ser Arg Tyr Lys Arg Lieu. Thir Ser Lieu Ala Lieu. Ser Lieu. Ser - 25 -2O - 15 Met Ala Leu Gly Ile Ser Leu Pro Ala Trp Ala Ser Pro Asp Thr Ser -10 - 5 - 1 1 5 Val Asp Asn Llys Val Asn. Phe Ser Thr Asp Val Ile Tyr Glin Ile Val 10 15 2O Thir Asp Arg Phe Ala Asp Gly Asp Arg Thr Asn. Asn Pro Ala Gly Asp 25 3O 35 Ala Phe Ser Gly Asp Arg Ser Asn Lieu Lys Lieu. Tyr Phe Gly Gly Asp 4 O 45 SO Trp Glin Gly Ile Ile Asp Llys Ile Asn Asp Gly Tyr Lieu. Thr Gly Met 55 6 O 65 Gly Val Thr Ala Leu Trp Ile Ser Glin Pro Val Glu Asn Ile Thr Ser 70 7s 8O 85 Val Ile Llys Tyr Ser Gly Val Asn Asn Thr Ser Tyr His Gly Tyr Trp 9 O 95 1OO Ala Arg Asp Phe Lys Glin Thr Asn Asp Ala Phe Gly Asp Phe Ala Asp 105 11 O 115 Phe Glin Asn Lieu. Ile Asp Thr Lieu. Thir Lieu. Ile Thir Ser Arg Ser Asp 12 O 125 13 O Arg Lieu. Arg Pro Gln Pro His Val Ser Gly Arg Ala Gly Thr Asn Pro 135 14 O 145 Gly Phe Ala Glu Asn Gly Ala Lieu. Tyr Asp Asn Gly Ser Lieu. Lieu. Gly 150 155 16 O 1.65 Ala Tyr Ser Asn Asp Thr Ala Gly Lieu Phe His His Asn Gly Gly Thr 17O 17s 18O Asp Phe Ser Thir Ile Glu Asp Gly Ile Tyr Lys Asn Lieu. Tyr Asp Lieu 185 19 O 195 Ala Asp Ile Asn His Asn. Asn. Asn Ala Met Asp Ala Tyr Phe Llys Ser 2OO 2O5 210 Ala Ile Asp Lieu. Trp Lieu. Gly Met Gly Val Asp Gly Ile Arg Phe Asp 215 22O 225 Ala Val Lys Glin Tyr Pro Phe Gly Trp Gln Lys Ser Phe Val Ser Ser 23 O 235 24 O 245 Ile Tyr Gly Gly Asp His Pro Val Phe Thr Phe Gly Glu Trp Tyr Lieu. 250 255 26 O Gly Ala Asp Glin Thr Asp Gly Asp Asn. Ile Llys Phe Ala Asn. Glu Ser 265 27 O 27s Gly Met Asn Lieu. Lieu. Asp Phe Glu Tyr Ala Glin Glu Val Arg Glu Val 28O 285 290 Phe Arg Asp Llys Thr Glu Thir Met Lys Asp Lieu. Tyr Glu Val Lieu Ala 295 3 OO 3. OS Ser Thr Glu Ser Glin Tyr Asp Tyr Ile Asin Asn Met Val Thr Phe Ile 310 315 32O 3.25 Asp Asn His Asp Met Asp Arg Phe Glin Val Ala Gly Ser Gly. Thir Arg 330 335 34 O Ala Thr Glu Glin Ala Lieu Ala Lieu. Thir Lieu. Thir Ser Arg Gly Val Pro 345 350 355 US 2013/0022711 A1 Jan. 24, 2013 81

- Continued

Ala Ile Tyr Tyr Gly Thir Glu Glin Tyr Met Thir Gly Asp Gly Asp Pro 360 365

Asn Asn Arg Ala Met Met Thir Ser Phe Asn Thir Gly Thir Thir Ala 375 385

Lys Wall Ile Glin Ala Lell Ala Pro Luell Arg Lys Ser Asn Pro Ala Ile 390 395 4 OO 4 OS

Ala Gly Thr Thir Thir Glu Arg Trp Wall ASn Asn Asp Wall Luell Ile 410 415

Ile Glu Arg Llys Phe Gly Ser Ser Ala Ala Luell Wall Ala Ile Asn Arg 425 43 O 435

Asn Ser Ser Ala Ala Pro Ile Ser Gly Luell Lell Ser Ser Luell Pro 44 O 445 450

Ala Gly Thr Tyr Ser Asp Wall Luell Asn Gly Luell Lell Asn Gly Asn Ser 45.5 460 465

Ile Thir Val Gly Ser Gly Gly Ala Wall Thir ASn Phe Thir Luell Ala Ala 470 485

Gly Gly Thir Ala Wall Trp Glin Thir Ala Pro Glu Thir Ser Pro Ala 490 495 SOO

Ile Gly Asn. Wall Gly Pro Thir Met Gly Glin Pro Gly Asn Ile Wall Thir 505 510 515

Ile Asp Gly Arg Gly Phe Gly Gly Thir Ala Gly Thir Wall Phe Gly 52O 525 53 O

Thir Thir Ala Wall Thir Gly Ser Gly Ile Wall Ser Trp Glu Asp Thir Glin 535 54 O 5.45

Ile Ala Wall Ile Pro Wall Ala Ala Gly Thir Gly Wall Ser 550 555 560 565

Wall Thir Ser Ser Gly Thir Ala Ser Asn Thir Phe Ser Phe Asn st O sts 58 O

Wall Luell Thr Gly Asp Glin Wall Thir Wall Arg Phe Lell Wall Asn Glin Ala 585 590 595

Asn Thir Asn Tyr Gly Thir Asn Wall Tyr Luell Wall Gly Asn Ala Ala Glu 6OO 605 610

Lell Gly Thir Trp Asp Pro Asn Ala Ile Gly Pro Met Asn Glin 615 625

Wall Ile Ala Lys Tyr Pro Ser Trp Tyr Asp Wall Ser Wall Pro Ala 630 635 64 O 645

Gly Thir Llys Lieu. Asp Phe Phe Ile Lys Gly Gly Gly Thir Wall 650 655 660

Thir Trp Glu Gly Gly Gly Asn His Thir Thir Thir Pro Ala Ser Gly 665 670 675

Wall Gly Thir Wall Thir Wall Asp Trp Glin Asn 68O 685

1.-18. (canceled) cooling the mixture containing the gelatinized enzyme 19. A method of producing a starch gel-containing food, treated Starch thereby gelling the starch to obtain a starch the method comprising the steps of gel-containing food, wherein treating starch granules with an enzyme at a temperature of the enzyme is selected from the group consisting of C.-amy about 10° C. or higher and about 70° C. or lower to lase having a characteristic capable of improving a gel obtain an enzyme-treated Starch; forming ability of a starch, amyloglucosidase, C-glu mixing a food material, the enzyme-treated Starch and cosidase, isoamylase, and cyclodextrin glucanotrans water to obtain a mixture; ferase. heating the mixture thereby gelatinizing the enzyme 20. The method according to claim 19, wherein the enzyme treated Starch in the mixture; and is selected from the group consisting of C.-amylase derived US 2013/0022711 A1 Jan. 24, 2013

from the genus Aspergillus, amyloglucosidase, C-glucosi (2) the enzyme is encoded by a nucleic acid molecule dase, isoamylase, and cyclodextrin glucanotransferase. which is capable of hybridizing under Stringent condi 21. The method according to claim 19, wherein the enzyme tions with a nucleic acid molecule consisting of a base is selected from the group consisting of C.-amylase derived sequence complementary to a base sequence of SEQID from Aspergillus Oryzae, C.-amylase derived from Aspergillus NO: 13, and has a transglycosylation activity; wherein niger, amyloglucosidase, C-glucosidase, isoamylase, and the stringent conditions are hybridization in a solution cyclodextrin glucanotransferase. containing 50% formamide, 5xSSC (750 mM. NaCl, 75 22. The method according to claim 19, wherein the enzyme mM trisodium citrate), 50 mM sodium phosphate (pH is selected from the group consisting of 7.6), 5xDenhardt’s solution (0.2% BSA, 0.2% Ficoll C-amylase derived from Aspergillus Oryzae or Aspergillus 400 and 0.2% polyvinylpyrrolidone), 10% dextran sul niger, fate and 20 ug/ml denatured sheared salmon sperm DNA amyloglucosidase derived from Aspergillus niger, Rhizo at 65°C., and Subsequent washing under the condition of pus niveus or Rhizopus Oryzae, 65° C. using an SSC solution having a 0.1 to 2-fold concentration (a composition of an SSC solution having C-glucosidase derived from Aspergillus niger, a 1-fold concentration is 150 mM sodium chloride and isoamylase derived from Flavobacterium sp. or 15 mM sodium citrate). Pseudomonas amyloderamosa; and 25. The method according to claim 19, wherein: cyclodextrin glucanotransferase derived from Bacillus (1) the enzyme has an amino acid sequence having at least licheniformis or Paenibacillus macerans (Bacillus mac 95% or more of homology with an amino acid sequence erans). of SEQ ID NO: 2, 4, 6, 12, 8, or 10, and has a starch 23. The method according to claim 19, wherein the enzyme hydrolysis activity; or is selected from the group consisting of C-amylase derived from Aspergillus Oryzae commercially (2) the enzyme has an amino acid sequence having at least available from Amano Enzyme as Biozyme A. C.-amy 95% or more of homology with an amino acid sequence lase derived from Aspergillus Oryzae commercially of SEQID NO: 14, and has a transglycosylation activity. available from SHIN NIHON CHEMICALS Corpora 26. The method according to claim 19, wherein the starch tion as Sumizyme L. C.-amylase derived from Aspergil granules are starch granules of an untreated Starch, a physi lus niger commercially available from Danisco as cally treated starch or a chemically modified starch. AMYLEX A3, O-amylase derived from Aspergillus 27. The method according to claim 19, wherein the starch niger commercially available from SHIN NIHON granules are starch granules of an untreated starch, and the CHEMICALS Corporation as Sumizyme AS, starch granules have been neither chemically modified nor amyloglucosidase derived from Aspergillus niger com physically treated in any stage until the starch gel-containing mercially available from Novozyme as AMG, amylo food is obtained by the method. glucosidase derived from Aspergillus niger commer 28. The method according to claim 19, wherein the starch cially available from Genencor as OPTIDEX L-400, granules are starch granules of an untreated Starch or a physi amyloglucosidase derived from Aspergillus niger com cally treated starch, the method further comprises the step of mercially available from DANISCO as DIAZYME chemically modifying the enzyme-treated Starch, and the X4NP amyloglucosidase derived from Aspergillus chemically modified enzyme-treated starch is mixed with the niger commercially available from Amano Enzyme as food material and water. glucoamylase "Amano' SD, amyloglucosidase derived 29. The method according to claim 19, wherein the starch from Rhizopus niveus commercially available from granules are starch granules of an untreated Starch or a chemi Amano Enzyme as Gluczyme AF6. cally modified starch, the method further comprises the step amyloglucosidase derived from Rhizopus Oryzae commer of physically treating the enzyme-treated Starch, and the cially available from SHIN NIHONCHEMICALS Cor physically treated enzyme-treated starch is mixed with the poration as Sumizyme, C-glucosidase derived from food material and water. Aspergillus niger commercially available from Amano 30. The method according to claim 19, further comprising Enzyme as transglucosidase L'Amano'. C-glucosidase the step of removing a carbohydrate eluted by enzymatic derived from Aspergillus niger commercially available hydrolysis, before the step of mixing the food material, the from Genencor as Transglucosidase L-500, enzyme-treated Starch and water to obtain the mixture. isoamylase derived from Pseudomonas amyloderamosa 31. A starch gel-containing food produced by the method commercially available from Sigma as isoamylase, according to claim 19. cyclodextrin glucanotransferase derived from Bacillus 32. The food according to claim 31, wherein the food is a licheniformis commercially available from Novozyme high moisture content type food and the amount of moisture as ToruZyme, and cyclodextrin glucanotransferase of the food is more than 40 g and less than 95g per 100 g of derived from Paenibacillus macerans (Bacillus macer the edible portion. ans) commercially available from Amano Enzyme as 33. The food according to claim 31, wherein the food is Cyclodextrin glucanotransferase “Amano'. selected from the group consisting of traditional Japanese 24. The method according to claim 19, wherein: style confectioneries, fat- or oil-containing foods, gelatinous (1) the enzyme is encoded by a nucleic acid molecule foods, fish meat and animal meat processed foods, Salsa and which is capable of hybridizing under Stringent condi sauces, and noodles. tions with a nucleic acid molecule consisting of a base 34. The food according to claim 31, wherein the food is a sequence complementary to a base sequence of SEQID low moisture content type food and the amount of moisture of NO: 1, 3, 5, 11, 7, or 9, and has a starch hydrolysis the food is 1 g or more and 40g or less per 100 g of the edible activity; or portion. US 2013/0022711 A1 Jan. 24, 2013 83

35. The food according to claim 31, wherein the food is Aspergillus Oryzae, C.-amylase derived from Aspergillus selected from the group consisting of bakeries, Western-style niger, amyloglucosidase, C-glucosidase, isoamylase, and confectioneries, and fried foods. cyclodextrin glucanotransferase. 36. The food according to claim 31, wherein the enzyme is selected from the group consisting of C.-amylase derived from 38. The food according to claim 31, wherein the starch is the genus Aspergillus, amyloglucosidase, C-glucosidase, derived from cassava, corn or wheat. isoamylase, and cyclodextrin glucanotransferase 37. The food according to claim 31, wherein the enzyme is selected from the group consisting of C.-amylase derived from k . . . .