USOO9005681 B2

(12) United States Patent (10) Patent No.: US 9,005,681 B2 Ichihara et al. (45) Date of Patent: Apr. 14, 2015

(54) FOOD PRODUCT CONTAINING STARCH (58) Field of Classification Search GEL STARCH GRANULE, PRODUCTION CPC ...... A23L 170522: A23L 1/095 METHOD AND USE THEREOF USPC ...... 426/48,578,549 See application file for complete search history. (75) Inventors: Takashi Ichihara, Osaka (JP); Junya Fukuda, Osaka (JP); Masakazu (56) References Cited Kimura, Osaka (JP); Kenichi Kurita, Osaka (JP) U.S. PATENT DOCUMENTS 5,445,950 A 8/1995 Kobayashi et al. (73) Assignee: Glico Nutrition Co., Ltd., Osaka (JP) 6,461,656 B1 * 10/2002 Bindzus et al...... 426.242 2005/0204425 A1 9/2005 Myers et al. (*) Notice: Subject to any disclaimer, the term of this 2007, 0110847 A1 5/2007 Okamoto et al. patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 13/383,060 CN 101028101 A 9, 2007 JP 59 196O72 11 1984 ...... A23L 1.325 JP 1-159047 A 6, 1989 (22) PCT Filed: Aug. 11, 2010 JP 5-112469 A 5, 1993 JP 06-269291. A 9, 1994 (86). PCT No.: PCT/UP2O1 O/OOSO46 JP 11-2558O2 A 9, 1994 JP 7-063324 B 7, 1995 S371 (c)(1), JP 8-277230 A 10, 1996 (2), (4) Date: Sep. 24, 2012 JP 10-215795 A 8, 1998 JP 11-192052 7, 1999 JP 2001-103991 A 4/2001 (87) PCT Pub. No.: WO2011/021372 JP 33 12225 8, 2002 JP 2003-2198.13 A 8, 2003 PCT Pub. Date: Feb. 24, 2011 JP 3723860 B2 12/2005 JP 2007-302767 A 11, 2007 (65) Prior Publication Data JP 417.0062 10, 2008 WO 96,03513 A2 2, 1996 US 2013/OO22711 A1 Jan. 24, 2013 WO 2005/096839 A1 10/2005 WO 2006/065579 A2 6, 2006 (30) Foreign Application Priority Data WO 2008/O59992 A1 5, 2008 Aug. 18, 2009 (JP) ...... 2009-189567 OTHER PUBLICATIONS (51) Int. Cl. JP-59-196072-Official Translation. A2.3L. I./0522 (2006.01) Protein Search Result-1. ID AAB84206 (May 7, 2013).* A2ID 2/18 (2006.01) Protein Search Result-1–IDAAY77741 (5-7-20130.* A23G 3/42 (2006.01) Chinese Office Action for corresponding Chinese Application No. A23G 9/34 (2006.01) 20108.0036978.3 issued Jan. 15, 2013 and English translation. A2.3L. I./6 (2006.01) (Continued) A2.3L I/31 (2006.01) A2.3L I/34 (2006.01) A2.3L I/37 (2006.01) Primary Examiner — Hamid R Badr CI2N 9/44 (2006.01) (74) Attorney, Agent, or Firm — Renner, Otto, Boisselle & A2.3L. I./00 (2006.01) Sklar, LLP A2.3L. I./87 (2006.01) A2.3L. I./22 (2006.01) (57) ABSTRACT A2.3L I/24 (2006.01) Here is provided a method of producing a starch gel-contain A2ID 8/04 (2006.01) ing food, the method comprising the steps of treating starch A2.3L I/325 (2006.01) granules with an enzyme at a temperature of about 10°C. or (52) U.S. Cl. higher and about 70° C. or lower to obtain an enzyme-treated CPC A2ID 2/186 (2013.01); A23G 3/42 (2013.01); starch; mixing a food material, the enzyme-treated Starch and A23G 9/34 (2013.01); A23L I/0522 (2013.01); water to obtain a mixture; heating the mixture thereby gela A2.3L 1/16 (2013.01); A23L I/31 (2013.01); tinizing the enzyme-treated Starch in the mixture; and cooling A2.3L I/31409 (2013.01); A23L I/31418 the mixture containing the gelatinized enzyme-treated Starch (2013.01); A23L I/317 (2013.01); C12N thereby gelling the starch to obtain a starch gel-containing 9/2451 (2013.01); C12N 9/246 (2013.01); food, wherein the enzyme is selected from the group consist CI2Y302/01033 (2013.01); C12Y302/01068 ing of amyloglucosidase, isoamylase, C-glucosidase, C.-amy (2013.01); A23L I/3175 (2013.01); A23L I/005 lase having a characteristic capable of improving a gel form (2013.01); A23L 1/1875 (2013.01); A23L ing ability of a starch, and cyclodextrin glucanotransferase. I/2128 (2013.01); A23L I/24 (2013.01); A2ID 8/042 (2013.01); A23L I/325 (2013.01); A23L I/05223 (2013.01) 36 Claims, No Drawings US 9,005,681 B2 Page 2

(56) References Cited Karim et al., “Dual Modification of Starch via Partial Enzymatic Hydrolysis in the Granular State and Subsequent Hydroxypropyla OTHER PUBLICATIONS tion”. J. Agric Food Chem, 2008, Vo. 56, No. 22, p. 10901-10907. Machida et al., “Genome sequencing and analysis of Aspergillus "Study on physic-chemical characteristics of potato gelatin starch'. oryzae', Nature (Lond), 2005, vol.438, No. 7071, p. 1157-1161. Nunberg et al., Molecular cloning and characterization of the China Academic Journal Electronic Publishing House. vol. 23, No. 8 glucoamylase gene of Aspergillus awamori, Mol Cell Biol. 1984, vol. 77, Dec. 31, 2002 and English translation. 4. No. 11, p. 2306-15. International Search Report and Written Opinion for corresponding Krohn et al., “An isoamylase with neutral pH optimum from a International Application No. PCT/JP2010/005046 mailed Nov.30, Flavobacterium species: cloning, characterization and expression of 2010. the iam gene”, Mol Gen Genet, 1997, vol. 254, No. 5, p. 469-478. Form PCT/ISA/237 for corresponding International Application No. Pel et al., “Genome sequencing and analysis of the versatile cell PCT/JP2010/005046 dated Nov.30, 2010. factory Aspergillus niger CBS 513.88”. Nat Biotechnol. 2007, vol. Japanese Office Action for corresponding Japanese Application No. 25, No. 2, p. 221-231. 2011-508150 dated Apr. 20, 2011. Japanese Office Action for corresponding Japanese Application No. Fukai et al., “Koso Shori ni yoru Kakushu Denpunryu no Tokusei 2011-508150 dated Apr. 20, 2011 (English translation provided here Kaihen (the 1 report) Characteristic Change of RiceStarch Granules with). by Enzymatic Treatment”, Journal of starch and its related carbohy Chinese Office Action for corresponding Chinese Application No. drates and enzymes, 1993, vol. 40, No. 3, pp. 263-269. 20108.0036978.3 issued Oct. 11, 2013 and English translation. Fukai et al., “Changes in Three Kinds of Starch Granules after Enzy Chinese Office Action for corresponding Chinese Application No. matic Treatment, part II”, Journal of the Agricultural Chemical Soci 20108.0036978.3 issued Jul. 2, 2014 and English translation. ety of Japan, 1994, vol. 68, No. 4, p. 793-800. Chinese Office Action for corresponding Chinese Application No. Absar et al., Enzymatic hydrolysis of potato starches containing 20108.0036978.3 issued Jan. 4, 2015 and partial English translation. different amounts of phosphorus, Food Chem. Jan. 1, 2009, Vo. 112, No. 1, p. 57-62. * cited by examiner US 9,005,681 B2 1. 2 FOOD PRODUCT CONTAINING STARCH degree is added to a food, since a large amount of the starch GEL STARCH GRANULE, PRODUCTION needs to be used so as to obtain the desired hardness, the METHOD AND USE THEREOF obtained food has increased powdery texture, thus causing deterioration of quality of a final product. Therefore, there is TECHNICAL FIELD a limit on the use amount of the starch having a low crosslink ing degree. In addition, processing of the starch utilizing a The present invention relates to a starch gel-containing chemical reaction also has such problems that there is a strict food, a starch having a high viscosity and a gel forming legal restriction on a processing method and a processing ability, a food containing the starch, and a method of produc degree so as to secure safety, and that it is not necessarily ing thereof. More particularly, the present invention relates to 10 Suited to needs of consumers who require security and safety. a method of producing a starch gel-containing food using an For the purpose of deigning these processed foods, it is enzyme capable of improving a gel forming ability of a urgently necessary to develop a processing technique to starch. obtain a processed Starch which exhibits various physical properties and has high safety. BACKGROUND ART 15 As a result of intensive studies, we have found that a food with rich elasticity, crispy sensation and the like can be pre With diversification of foods, foods having various shapes, pared by adopting the steps of treating starch granules with a physical properties and textures have been required. Particu starch hydrolase or a glycosyltransferase in advance; then larly, intense interest has recently been shown towards melt in mixing the resultant with a food material and water; and mouth and texture as important physical properties for the heating the mixture. purpose of designing foods. Also in the fields related to A starch is a material utilized for various purposes and the deglutition and care toward which intense interest has most important function thereof is the thickening function recently been shown, texture has been studied as important and the gel forming function. Particularly in the food industry, physical properties. the thickening function and the gel forming function of the In the case of designing processed foods, utilization of a 25 starch are widely utilized for forming the shape, physical gelling agent is important so as to improve texture and physi properties and texture of a food. The structure of a starch cal properties, and it is possible to develop various products delicately varies depending on plant from which the starch according to how to use. derived (for example, corn, potato, wheat, and cassava). As a For the purpose of altering physical properties of foods, result, the thickening function and gel forming function also various gelling agents have hitherto been added to food mate 30 vary depending on the plant from which the starch derived. rials in the case of preparing foods. Therefore, those skilled in the art have been selected a native In food processing, natural macromolecules such as agar, starch to be used for a long time depending on the purpose. gelatin, gellan gum, Xanthan gum, locust bean gum, carrag For example, a wheat starch has often been used in a fish paste eenan, pectin, Sodium alginate, Tamarind seed gum, psyllium product for a long time. The reason is that the wheat Starch is seed gum, microcrystalline cellulose, curdlan, and starch; or 35 excellent in gel forming function. For example, a cassava synthetic macromolecules Such as carboxymethyl cellulose starch is commonly utilized in a food which has high trans (CMC) or methyl cellulose are commonly used as gelling parency and requires Sticky texture. However, with the agents. advancement of characteristics required in the current food In the case of using these gelling agents, gelling agents may industry, it becomes impossible to cope with the advancement be sometimes used alone, however, in order to form gels 40 only by changing a native starch to be used. Therefore, there having more various characteristics, for example, use of two arises the need to alter the thickening function or the gel or more kinds of gelling agents such as native gellangum and forming function of a starch. guar gum in combination is studied and utilized (Patent Docu Means which are used most commonly to alter the thick ment 1). ening function or the gel forming function of a starch is a However, there are few combinations which can synergis 45 chemical modification of a starch. Above all, techniques of tically change the gel strength of foods. Even if it is possible applying a chemical treatment, Such as a technique of intro to synergistically change the gel strength, the gel obtained ducing a new crosslinking point between starch molecules thereby does not have nice physical properties. Mixing of two using a Suitable chemical crosslinking agent and a technique or more kinds of gelling agents is a defect due to being of introducing a suitable functional group have widely been complicated and that many materials are very expensive. 50 utilized so as to remarkably alter the thickening function or Furthermore, there is such a restriction on use in food the gel forming function. However, a starch Subjected to Such processing that, for example, a gelatin is inferior in resistance a chemical treatment has been specified as a food additive to an acid and an alkali, and also an agar is inferior in resis from October, 2008 in Japan, and thus restricted by law. tance to an acid. Therefore, there has been required a technique in which the Starches have successfully produced various physical 55 thickening function or the gel forming function of a starch is properties by adding not only raw starches but also a pro altered without a chemical treatment. cessed Starch obtained by chemically modifying starches The technique of altering a starch without a chemical treat (also referred to as a chemically modified Starch) such as ment includes a technique of an enzymatic treatment of a starch acetate and monostarch phosphate as agelling agent to starch. Since an enzyme commonly acts on a Substrate dis food materials. For example, Patent Documents 2, 3 and 4 60 Solved in water, an enzymatic treatment is usually carried out indicate examples in which a crosslinked Starch is utilized in after completely dissolving a starch in water. A hydrolytic a white table bread, confectioneries or noodles. However, in enzyme or a glycosyltransferase is allowed to act on a starch the case where a crosslinked starch having a high crosslinking dissolved in water to cleave the starch, thereby producing degree is added to a food, the hardness and the Viscosity of a molecules having a lower molecular weight such as dextrin, gel can be enhanced, but there is such a drawback that a final 65 starch syrup, maltooligosaccharide, maltose, and glucose. product has powdery texture and also is inferior in flavor. However, in the enzymatic treatment with a hydrolytic Also, in the case where a starch having a low crosslinking enzyme or a glycosyltransferase, a starch molecule is cleaved US 9,005,681 B2 3 4 to form low-molecular weight molecules. Therefore, it has Also, in the prior art, no attention was paid at all whether or been commonly considered that the thickening function and not an enzyme has characteristics capable of improving a gel the gel forming function of the obtained molecule deteriorate forming ability of a starch. It was not also found at all whether as compared with the thickening function and the gel forming or not industrial advantages are exerted by characteristics of function of the starch, or are lost. an enzyme capable of improving a gel forming ability of a Also, Patent Document 5 discloses, as a method of altering starch. physical properties of a starch, a technique in which an enzyme is allowed to act on a starch in the form of starch PRIOR ART DOCUMENTS granules in water without dissolving them in water. Patent Document 5 discloses that although a starch has convention 10 Patent Documents ally been dissolved in water before an enzymatic treatment in the case of subjecting the starch to the enzymatic treatment, it Patent Document 1: Japanese Laid-open Patent Publication is not necessarily required to dissolve the starch in water No. 10-215795 before the enzymatic treatment, and it is possible to subject Patent Document 2: Japanese Patent Gazette No. 3,723,860 starch granules, which are not dissolved in water but Sus 15 Patent Document 3: Japanese Patent Gazette No. 3,312,225 pended in water, to the enzymatic treatment. Specifically, it is Patent Document 4: Japanese Patent Publication for Opposi disclosed that a hydrolytic enzyme such as C.-amylase or tion No. 7-63324 glucoamylase can act on starch granules, which are not dis Patent Document 5: Japanese Laid-open Patent Publication Solved in water but Suspended in water, and thus a reducing No. 6-269291 Sugar can be produced. Patent Document 5 also discloses as a Patent Document 6: Japanese Laid-open Patent Publication result of this that the viscosity of the starch subjected to the No. 2003-2198.13 enzymatic treatment is lower than that of the starch which is Patent Document 7: Japanese Patent Gazette No. 4,170,062 not subjected to the enzymatic treatment. However, Patent Patent Document 8: Japanese Laid-open Patent Publication Document 5 neither suggests nor discloses that a starch hav No. 1-159047 ing improved thickening function or gel forming function as 25 Patent Document 9: Japanese Laid-open Patent Publication compared with the starch, which is not subjected to the enzy No. 5-112469 matic treatment, is obtained by allowing a hydrolytic enzyme Patent Document 10: Japanese Laid-open Patent Publication or a glycosyltransferase to act on starch granules. No. 8-27723O Patent Documents 6 to 10 also disclose a technique of allowing a hydrolytic enzyme to act on insoluble starch gran 30 SUMMARY OF THE INVENTION ules. These inventions disclose a technique in which the action of a hydrolytic enzyme on starch granules opens pores Problems to be Solved by the Invention on the Surfaces of starch granules to make porous starch granules, and the porous starch granules are utilized as a The present invention is intended to solve the above prob powdered base material or a porous carrier. However, Patent 35 lems, and it is an object of the invention to provide a food Documents 6 to 10 neither suggests nor discloses that a starch containing a starch gel having the desired degree of hardness having improved thickening function and gel forming func and a method of producing the same. In a specific embodi tion is obtained by allowing a hydrolytic enzyme or a glyco ment of the present invention, objects are to provide a starch Syltransferase to act on starch granules. An object of the excellent in thickening function or gel forming function with present invention is not to open pores on the Surfaces of 40 out utilizing a chemical modification of a starch; a food con enzyme-treated Starch granules, and there is not any relation taining the starch; and a method of producing the starch and ship between an improvement in thickening function and gel food. forming function, and whether or not pores are opened on the Surfaces of enzyme-treated Starch granules. If a heated food is Means for Solving the Problems produced using the enzyme-treated Starch of the present 45 invention, the enzyme-treated Starch forms a hard gel in the The present inventors have intensively studied so as to heated food. The enzyme-treated starch of the present inven solve the above problems and have found that a starch excel tion is usable in the heated food. On the other hand, in the lent in thickening function and gel forming function is prior art, it is important that pores are present on the Surfaces obtained by allowing a specific hydrolytic enzyme or glyco of starch granules. If starch granules after Subjected to the 50 Syltransferase having characteristics capable of improving a enzymatic treatment and water are mixed and then heated, gel forming ability of a starch to act on starch granules under starch granules are collapsed and pore-opened states thereof the condition where a starch is not dissolved, and thus have are lost. Therefore, those skilled in the art did not consider to completed the present invention based on this finding. It is use a pore-opened starch of the prior art in the heated food. In commonly considered that when a hydrolytic enzyme or a the present invention, it is possible to adjust the hardness of a 55 glycosyltransferase is allowed to act on a starch, the starch is gel to be formed using an enzyme-treated Starch by adjusting cleaved to form smaller molecules, and therefore the viscos the degree of the enzymatic treatment. The hardness of the gel ity and the gel forming ability of the obtained molecules exerts an influence on texture, chewiness, and the like of the deteriorate as compared with the viscosity and gel forming food. Therefore, use of the method of the present invention ability of the starch before being subjected to the enzymatic can exert an influence on texture of the food. As described 60 treatment, or are lost. Actually, when the same hydrolytic above, the enzyme-treated Starch granules of the prior art and enzyme or glycosyltransferase as the hydrolytic enzyme or the enzyme-treated Starch granules used in the present appli glycosyltransferase capable of producing such an excellent cation quite differ in application and usage. starch when being allowed to act on starch granules under the As described above, it was conventionally impossible to condition where a starch is not dissolved in water is allowed provide a starch excellent in thickening function or gel form 65 to act on a starchafter dissolving the starch in water, the starch ing function without utilizing a chemical modification of a Viscosity decreases, and thus a starch excellent in thickening starch. function or gel forming function cannot be obtained. As US 9,005,681 B2 5 6 described above, the present invention cannot be conceived Aspergillus Oryzae commercially available from SHIN from the conventionally general knowledge and technical NIHON CHEMICALS Corporation as Sumizyme L, C-amy common sense possessed by those skilled in the art. lase derived from Aspergillus niger commercially available The conditions of an enzymatic treatment of Starch gran from Danisco as AMYLEX A3, C.-amylase derived from ules can vary depending on the specificity of the enzyme and Aspergillus niger commercially available from SHIN the origin of Starch granules. For example, first, starch gran NIHON CHEMICALS Corporation as Sumizyme AS, ules are Suspended in ion-exchange water or a buffer Solution isoamylase derived from Pseudomonas amyloderamosa to prepare a starch Suspension. In the case where the pH commercially available from Sigma as isoamylase, cyclodex adjustment of the starch Suspension is required, the pH is trin glucanotransferase derived from Bacillus licheniformis adjusted to the optimum pH of the enzyme. While warming 10 this starch Suspension at the temperature at which starch commercially available from Novozyme as Toruzyme, and granules are not degraded (preferably from about 10° C. to cyclodextrin glucanotransferase derived from Paenibacillus about 70° C.), the enzyme is added and the reaction can be macerans (Bacillus macerans) commercially available from carried out, for example, within about 24 hours (preferably Amano Enzyme as Cyclodextrin glucanotransferase Amano’. from about for 1 hour to about 20 hours). Then, the enzyme 15 and a carbohydrate eluted by enzymatic hydrolysis are (Item 5) The method according to Item 1, wherein: removed by the washing and dehydration steps which are a (1) the enzyme is encoded by a nucleic acid molecule which conventional method of preparing a starch, followed by the is capable of hybridizing under stringent conditions with a drying step, and thus the objective enzyme-treated Starch nucleic acid molecule consisting of a base sequence comple granules can be obtained. mentary to a base sequence of SEQID NO: 1, 3, 5, 7, 9 or 11, The present invention is, for example, as follows: and has a starch hydrolysis activity; or (Item 1) A method of producing a starch gel-containing (2) the enzyme is encoded by a nucleic acid molecule which food, the method comprising the steps of: is capable of hybridizing under stringent conditions with a treating starch granules with an enzyme at a temperature of nucleic acid molecule consisting of a base sequence comple about 10°C. or higher and about 70° C. or lower to obtain an 25 mentary to a base sequence of SEQ ID NO: 13, and has a enzyme-treated Starch; transglycosylation activity; wherein the stringent conditions mixing a food material, the enzyme-treated Starch and are hybridization in a solution containing 50% formamide, water to obtain a mixture; 5xSSC (750 mM. NaCl, 75 mM trisodium citrate), 50 mM heating the mixture thereby gelatinizing the enzyme sodium phosphate (pH 7.6), 5xDenhardt’s solution (0.2% treated Starch in the mixture; and 30 cooling the mixture containing the gelatinized enzyme BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyrrolidone), 10% treated starch thereby gelling the starch to obtain a starch dextran sulfate and 20 g/ml denatured sheared salmon sperm gel-containing food, wherein DNA at 65°C., and subsequent washing under the condition the enzyme is selected from the group consisting of amy of 65° C. using an SSC solution having a 0.1 to 2-fold con loglucosidase, isoamylase, C-glucosidase, C.-amylase having 35 centration (a composition of an SSC solution having a 1-fold a characteristic capable of improving a gel forming ability of concentration is 150 mM sodium chloride and 15 mM sodium a starch, and cyclodextrin glucanotransferase. citrate). (Item 2) The method according to Item 1, wherein the (Item 6) The method according to Item 1, wherein: enzyme is selected from the group consisting of amyloglu (1) the enzyme has an amino acid sequence having at least cosidase, isoamylase, C-glucosidase, C.-amylase derived 40 95% or more of homology with an amino acid sequence of from the genus Aspergillus, and cyclodextrin glucanotrans SEQID NO: 2, 4, 6, 8, 10 or 12, and has a starch hydrolysis ferase. activity; or (Item 3) The method according to Item 1, wherein the (2) the enzyme has an amino acid sequence having at least enzyme is selected from the group consisting of amyloglu 95% or more of homology with an amino acid sequence of cosidase, isoamylase, C-glucosidase, C.-amylase derived 45 SEQID NO: 14, and has a transglycosylation activity. from Aspergillus Oryzae, C.-amylase derived from Aspergillus (Item 7) The method according to Item 1, wherein the niger, and cyclodextrin glucanotransferase. starch granules are starch granules of an untreated Starch, a (Item 4) The method according to Item 1, wherein the physically treated Starch or a chemically modified Starch. enzyme is selected from the group consisting of amyloglu (Item 8) The method according to Item 1, wherein the cosidase derived from Aspergillus niger commercially avail 50 starch granules are starch granules of an untreated Starch, and able from Novozyme as AMG, amyloglucosidase derived the starch granules have been neither chemically modified nor from Aspergillus niger commercially available from Genen physically treated in any stage until the starch gel-containing cor as OPTIDEX L-400, amyloglucosidase derived from food is obtained by the method. Aspergillus niger commercially available from DANISCO as (Item 9) The method according to Item 1, wherein the DIAZYMEX4NP amyloglucosidase derived from Aspergil 55 starch granules are starch granules of an untreated Starch or a lus niger commercially available from Amano Enzyme as physically treated starch, the method further comprises the glucoamylase "Amano' SD, amyloglucosidase derived from step of chemically modifying the enzyme-treated Starch, and Rhizopus niveus commercially available from Amano the chemically modified enzyme-treated starch is mixed with Enzyme as Gluczyme AF6, amyloglucosidase derived from the food material and water. Rhizopus oryzae commercially available from SHIN NIHON 60 (Item 10) The method according to Item 1, wherein the CHEMICALS Corporation as Sumizyme, C-glucosidase starch granules are starch granules of an untreated Starch or a derived from Aspergillus niger commercially available from chemically modified starch, the method further comprises the Amano Enzyme as transglucosidase L'Amano'. C-glucosi step of physically treating the enzyme-treated Starch, and the dase derived from Aspergillus niger commercially available physically treated enzyme-treated starch is mixed with the from Genencor as Transglucosidase L-500, C-amylase 65 food material and water. derived from Aspergillus Oryzae commercially available from (Item 11) A starch gel-containing food produced by the Amano Enzyme as Biozyme A, C.-amylase derived from method according to Item 1. US 9,005,681 B2 7 8 (Item 12) The food according to Item 11, wherein the food (Item 4A) The food according to Item 1A, wherein the is a high moisture content type food and the amount of mois untreated Starch is a untreated corn starch, the enzyme-treated ture of the food is more than 40 g and less than 95g per 100 starch is a enzyme-treated corn starch, and g of the edible portion. the enzyme-treated corn starch is capable of forming a gel (Item 13) The food according to Item 11, wherein the food having a Young's modulus which accounts for 110% or more is selected from the group consisting of traditional Japanese and 500% or less (110% or more and 330% or less in an style confectioneries, fat- or oil-containing foods, gelatinous embodiment) of the Young's modulus of the untreated corn foods, fish meat and animal meat processed foods, Salsa and starch, or a rupture stress which accounts for 110% or more sauces, and noodles. and 300% or less of the rupture stress of the untreated corn (Item 14) The food according to Item 11, wherein the food 10 starch, when measured by a rheometer. is a low moisture content type food and the amount of mois (Item 5A) A heat-cooked starch-containing food contain ture of the food is 1 g or more and 40g or less per 100 g of the ing an enzyme-treated wheat starch having high viscosity and edible portion. gel-forming ability, (Item 15) The food according to Item 11, wherein the food 15 the starch-containing food is a food produced by a method is selected from the group consisting of bakeries, Western comprising the steps of mixing a food material and the style confectioneries, and fried foods. enzyme-treated wheat starch and then heating them, (Item 16) The food according to Item 11, wherein the the enzyme-treated wheat starch is a starch obtained by enzyme is selected from the group consisting of amyloglu treating starch granules of untreated wheat starch with a cosidase, isoamylase, C-glucosidase, C.-amylase derived starch hydrolase under the condition where the starch gran from the genus Aspergillus, and cyclodextrin glucanotrans ules are not dissolved, ferase. the enzyme-treated wheat starch is not modified on (Item 17) The food according to Item 11, wherein the hydroxyl groups at the positions 2, 3 and 6 of the glucose enzyme is selected from the group consisting of amyloglu residues, cosidase, isoamylase, C-glucosidase, C.-amylase derived 25 the enzyme-treated wheat starch can form a gel having a from Aspergillus Oryzae, C.-amylase derived from Aspergillus Young's modulus of 5.0x10° dyn/cm or more and 8.0x10 niger, and cyclodextrin glucanotransferase. dyn/cm or less, or a rupture stress of 150 g or more and 450 (Item 18) The food according to Item 11, wherein the starch g or less, when measured by a rheometer. is derived from cassava, corn or wheat. (Item 6A) A heat-cooked starch-containing food contain In a specific embodiment, the present invention is, for 30 ing an enzyme-treated cassava starch having high viscosity example, as follows: and gel-forming ability, (Item 1A) A heat-cooked starch-containing food contain the starch-containing food is a food produced by a method ing an enzyme-treated Starch having high viscosity and gel comprising the steps of mixing a food material and the forming ability, enzyme-treated cassava starch and then heating them, the starch-containing food is a food produced by a method 35 the enzyme-treated cassava starch is a starch obtained by comprising the steps of mixing a food material and the treating starch granules of untreated cassava starch with an enzyme-treated Starch and then heating them, starch hydrolase under the condition where the starch gran the enzyme-treated Starch is a starch obtained by treating ules are not dissolved, starch granules of untreated Starch with a starch hydrolase the enzyme-treated cassava starch is not modified on under the condition where the starch granules are not dis 40 hydroxyl groups at the positions 2, 3 and 6 of the glucose solved, residues, the enzyme-treated starch is not modified on hydroxyl the enzyme-treated cassava starch can form a gel having a groups at the positions 2, 3 and 6 of the glucose residues, Young's modulus of 5.2x10 dyn/cm or more and 2.7x10° the enzyme-treated Starch can form a gel having a Young's dyn/cm or less (5.2x10 dyn/cm or more and 1.6x10' dyn/ modulus higher than that of the untreated Starch or a rupture 45 cm or less in one embodiment), or a rupture stress of 55 g or stress higher than that of the untreated Starch, when measured more and 150 g or less, when measured by a rheometer. by a rheometer. (Item 7A) A heat-cooked starch-containing food contain (Item 2A) The food according to Item 1A, wherein the ing an enzyme-treated corn starch having high viscosity and untreated Starch is a untreated wheat starch, the enzyme gel-forming ability, treated Starch is a enzyme-treated wheat starch, and 50 the starch-containing food is a food produced by a method the enzyme-treated wheat starch is capable of forming a gel comprising the steps of mixing a food material and the having a Young's modulus which accounts for 110% or more enzyme-treated corn starch and then heating them, and 500% or less (110% or more and 330% or less in an the enzyme-treated corn starch is a starch obtained by embodiment) of the Young's modulus of the untreated wheat treating starch granules of untreated corn starch with an starch, or a rupture stress which accounts for 110% or more 55 starch hydrolase under the condition where the starch gran and 300% or less of the rupture stress of the untreated wheat ules are not dissolved, starch, when measured by a rheometer. the enzyme-treated corn starch is not modified on hydroxyl (Item 3A) The food according to Item 1A, wherein the groups at the positions 2, 3 and 6 of the glucose residues, untreated Starch is a untreated cassava starch, the enzyme the enzyme-treated corn Starch can form a gel having a treated Starch is a enzyme-treated cassava starch, and 60 Young's modulus of 6.0x10° dyn/cm or more and 9.0x10° the enzyme-treated cassava starch is capable of forming a dyn/cm or less, or a rupture stress of 210 g or more and 450 gel having a Young's modulus which accounts for 110% or gorless (220g or more and 450g or less in one embodiment), more and 500% or less (110% or more and 330% or less in an when measured by a rheometer. embodiment) of the Young's modulus of the untreated cas (Item 8A) The food according to any one of Items 1A to sava starch, or a rupture stress which accounts for 110% or 65 7A, wherein the starch is forming a gel in the food. more and 300% or less of the rupture stress of the untreated (Item 9A) The food according to any one of Items 1A to cassava starch, when measured by a rheometer. 8A, wherein the food is a high moisture content type food and US 9,005,681 B2 10 the amount of moisture of the food is more than 40 g and less (Item 19A) The starch according to Item 17A, wherein the than 95g per 100 g of the edible portion. untreated Starch is a untreated cassava starch, the enzyme (Item 10A) The food according to any one of Items 1A to treated Starch is a enzyme-treated cassava starch, and 9A, wherein the food is selected from the group consisting of the enzyme-treated cassava starch is capable of forming a traditional Japanese-style confectioneries, fat- or oil-contain 5 gel having a Young's modulus which accounts for 110% or ing foods, gelatinous foods, fish meat and animal meat pro more and 500% or less (110% or more and 330% or less in an cessed foods, Salsa and sauces, and noodles. embodiment) of the Young's modulus of the untreated cas (Item 11A) The food according to any one of Items 1A to sava starch, or a rupture stress which accounts for 110% or 8A, wherein the food is a low moisture content type food and more and 300% or less of the rupture stress of the untreated 10 cassava starch, when measured by a rheometer. the amount of moisture of the food is 1 g or more and 40g or (Item 20A) The starch according to Item 17A, wherein the less per 100 g of the edible portion. untreated Starch is a untreated corn starch, the enzyme-treated (Item 12A) The food according to any one of Items 1A to starch is a enzyme-treated corn starch, and 8A and 11A, wherein the food is selected from the group the enzyme-treated corn starch is capable of forming a gel consisting of bakeries, Western-style confectioneries, and 15 having a Young's modulus which accounts for 110% or more fried foods. and 500% or less (110% or more and 330% or less in an (Item 13A) The food according to any one of Items 1A to embodiment) of the Young's modulus of the untreated corn 12A, wherein the starch hydrolase is selected from the group starch, or a rupture stress which accounts for 110% or more consisting of amyloglucosidase, isoamylase, C-glucosidase, and 300% or less of the rupture stress of the untreated corn and O-amylase having characteristics capable of improving a starch, when measured by a rheometer. gel-forming ability of a starch. (Item 21A) An enzyme-treated wheat starch having high (Item 14A) The food according to Item 13A, wherein the Viscosity and gel-forming ability, starch hydrolase is selected from the group consisting of the enzyme-treated wheat starch is a starch obtained by amyloglucosidase, isoamylase, C-glucosidase, and C.-amy treating starch granules of untreated wheat starch with an lase derived from the genus Aspergillus. 25 starch hydrolase under the condition where the starch gran (Item 15A) The food according to Item 13A, wherein the ules are not dissolved, starch hydrolase is selected from the group consisting of the enzyme-treated wheat starch is not modified on amyloglucosidase, isoamylase, C-glucosidase, C.-amylase hydroxyl groups at the positions 2, 3 and 6 of the glucose derived from Aspergillus Oryzae, and C-amylase derived from residues, Aspergillus niger: 30 the enzyme-treated wheat starch can form a gel having a (Item 16A) A method of producing a starch-containing Young's modulus of 5.0x10° dyn/cm or more and 8.0x10° food, the method comprising the steps of dyn/cm or less, or a rupture stress of 150 g or more and 450 adding and mixing an enzyme-treated Starch to a food g or less, when measured by a rheometer. material; and (Item 22A) An enzyme-treated cassava starch having high heat-cooking the mixture; 35 Viscosity and gel-forming ability, the enzyme-treated Starch is a starch obtained by treating the enzyme-treated cassava starch is a starch obtained by starch granules of untreated Starch with a starch hydrolase treating starch granules of untreated cassava starch with an under the condition where the starch granules are not dis starch hydrolase under the condition where the starch gran solved; ules are not dissolved, the enzyme-treated starch is not modified on hydroxyl 40 the enzyme-treated cassava starch is not modified on groups at the positions 2, 3 and 6 of the glucose residues, hydroxyl groups at the positions 2, 3 and 6 of the glucose the enzyme-treated Starch can form a gel having a Young's residues, modulus higher than that of the untreated Starch or a rupture the enzyme-treated cassava starch can form a gel having a stress higher than that of the untreated Starch, when measured Young's modulus of 5.2x10 dyn/cm or more and 2.7x10° by a rheometer. 45 dyn/cm or less (5.2x10 dyn/cm or more and 1.6x10° dyn/ (Item 17A) An enzyme-treated Starch having high viscos cm or less in one embodiment), or a rupture stress of 55g or ity and gel-forming ability, more and 150 g or less, when measured by a rheometer. the enzyme-treated Starch is a starch obtained by treating (Item 23A) An enzyme-treated corn starch having high starch granules of untreated Starch with an starch hydrolase Viscosity and gel-forming ability, under the condition where the starch granules are not dis 50 the enzyme-treated corn starch is a starch obtained by solved, treating starch granules of untreated corn starch with an the enzyme-treated starch is not modified on hydroxyl starch hydrolase under the condition where the starch gran groups at the positions 2, 3 and 6 of the glucose residues, ules are not dissolved, the enzyme-treated Starch can form a gel having a Young's the enzyme-treated corn starch is not modified on hydroxyl modulus higher than that of the untreated Starch or a rupture 55 groups at the positions 2, 3 and 6 of the glucose residues, stress higher than that of the untreated Starch, when measured the enzyme-treated corn Starch can form a gel having a by a rheometer. Young's modulus of 6.0x10° dyn/cm or more and 9.0x10° (Item 18A) The starch according to Item 17A, wherein the dyn/cm or less, or a rupture stress of 210 g or more and 450 untreated Starch is a untreated wheat starch, the enzyme gorless (220g or more and 450g or less in one embodiment), treated Starch is a enzyme-treated wheat starch, and 60 when measured by a rheometer. the enzyme-treated wheat starch is capable of forming a gel (Item 24A) The starch according to any one of Items 18A having a Young's modulus which accounts for 110% or more to 23A, wherein the starch hydrolase is selected from the and 500% or less (110% or more and 330% or less in an group consisting of amyloglucosidase, isoamylase, C-glu embodiment) of the Young's modulus of the untreated wheat cosidase, and C-amylase having characteristics capable of starch, or a rupture stress which accounts for 110% or more 65 improving a gel-forming ability of a starch. and 300% or less of the rupture stress of the untreated wheat (Item 25A). The starch according to Item 24A, wherein the starch, when measured by a rheometer. starch hydrolase is selected from the group consisting of US 9,005,681 B2 11 12 amyloglucosidase, isoamylase, C-glucosidase, and C.-amy ing under the condition of 65° C. using an SSC solution lase derived from the genus Aspergillus. having a 0.1 to 2-fold concentration (a composition of an SSC (Item 26A) The starch according to Item 24A, wherein the solution having a 1-fold concentration is 150 mM sodium starch hydrolase is selected from the group consisting of chloride and 15 mM sodium citrate). amyloglucosidase, isoamylase, C-glucosidase, C.-amylase 5 (Item 32A) The method according to any one of Items 27A derived from Aspergillus Oryzae, and C-amylase derived from to 30A, wherein the starch hydrolase has an amino acid Aspergillus niger: sequence having at least 95% or more of homology with an (Item 27A) A method of producing an enzyme-treated amino acid sequence of SEQID NO: 2, 4, 6, 8, 10 or 12, and starch having high viscosity and gel-forming ability, the has a starch hydrolysis activity. method comprising the step of 10 (Item 33A) An enzyme-treated Starch having high viscos treating starch granules of untreated Starch with a starch ity and gel-forming ability, hydrolase at a temperature of 10° C. or higher and 70° C. or the enzyme-treated Starch is a starch obtained by treating lower; starch granules of untreated Starch with a starch hydrolase the starch hydrolase is selected from the group consisting under the condition where the starch granules are not dis of amyloglucosidase, isoamylase, C-glucosidase, and 15 solved; C.-amylase having a characteristic capable of improving a gel the enzyme-treated starch is not modified on hydroxyl forming ability of a starch. groups at the positions 2, 3 and 6 of the glucose residues, (Item 28A) The method according to Item 27A, wherein the starch hydrolase is selected from the group consisting the starch hydrolase is selected from the group consisting of of amyloglucosidase derived from Aspergillus niger com amyloglucosidase, isoamylase, C-glucosidase, and C.-amy mercially available from Novozyme as AMG. amyloglucosi lase derived from the genus Aspergillus. dase derived from Aspergillus niger commercially available (Item 29A) The method according to Item 27A or 28A, from Genencor as OPTIDEX L-400, amyloglucosidase wherein the starch hydrolase is selected from the group con derived from Aspergillus niger commercially available from sisting of amyloglucosidase, isoamylase, C-glucosidase, DANISCO as DIAZYME X4NP. amyloglucosidase derived C.-amylase derived from Aspergillus Oryzae, and C-amylase 25 from Aspergillus niger commercially available from Amano derived from Aspergillus niger: Enzyme as glucoamylase "Amano' SD, amyloglucosidase (Item 30A). The method according to any one of Items 27A derived from Rhizopus niveus commercially available from to 29A, wherein the starch hydrolase is selected from the Amano Enzyme as Gluczyme AF6, amyloglucosidase group consisting of amyloglucosidase derived from Aspergil derived from Rhizopus Oryzae commercially available from lus niger commercially available from Novozyme as AMG, 30 SHIN NIHON CHEMICALS Corporation as Sumizyme, amyloglucosidase derived from Aspergillus niger commer C-glucosidase derived from Aspergillus niger commercially cially available from Genencor as OPTIDEXL-400, amylo available from Amano Enzyme as transglucosidase L glucosidase derived from Aspergillus niger commercially "Amano', C.-glucosidase derived from Aspergillus niger available from DANISCO as DIAZYME X4NP amyloglu commercially available from Genencor as Transglucosidase cosidase derived from Aspergillus niger commercially avail 35 L-50, C-amylase derived from Aspergillus Oryzae commer able from Amano Enzyme as glucoamylase "Amano' SD, cially available from Amano Enzyme as Biozyme A, C.-amy amyloglucosidase derived from Rhizopus niveus commer lase derived from Aspergillus Oryzae commercially available cially available from Amano Enzyme as Gluczyme AF6, from SHIN NIHON CHEMICALS Corporation as Sum amyloglucosidase derived from Rhizopus Oryzae commer izyme L. C.-amylase derived from Aspergillus niger commer cially available from SHIN NIHON CHEMICALS Corpora 40 cially available from Danisco as AMYLEX A3, C.-amylase tion as Sumizyme, C-glucosidase derived from Aspergillus derived from Aspergillus niger commercially available from niger commercially available from Amano Enzyme as trans SHIN NIHONCHEMICALS Corporation as Sumizyme AS, glucosidase L Amano'. C-glucosidase derived from and isoamylase derived from Pseudomonas amyloderamosa Aspergillus niger commercially available from Genencor as commercially available from Sigma as isoamylase. Transglucosidase L-50, C.-amylase derived from Aspergillus 45 (Item 34A) An enzyme-treated Starch having high viscos Oryzae commercially available from Amano Enzyme as ity and gel-forming ability, Biozyme A, C.-amylase derived from Aspergillus Oryzae com the enzyme-treated Starch is a starch obtained by treating mercially available from SHIN NIHON CHEMICALS Cor starch granules of untreated Starch with a starch hydrolase poration as Sumizyme L, C.-amylase derived from Aspergil under the condition where the starch granules are not dis lus niger commercially available from Danisco as AMYLEX 50 solved; A3, C.-amylase derived from Aspergillus niger commercially the enzyme-treated starch is not modified on hydroxyl available from SHIN NIHON CHEMICALS Corporation as groups at the positions 2, 3 and 6 of the glucose residues, Sumizyme AS, and isoamylase derived from Pseudomonas the starch hydrolase is encoded by a nucleic acid molecule amyloderamosa commercially available from Sigma as which is capable of hybridizing under Stringent conditions isoamylase. 55 with a nucleic acid molecule having a complementary (Item 31A) The method according to any one of Items 27A sequence of the base sequence of SEQID NO: 1, 3, 5, 7, 9 or to 30A, wherein the starch hydrolase is encoded by a nucleic 11, and has a starch hydrolysis activity; wherein the Stringent acid molecule which is capable of hybridizing understringent conditions are hybridization in a solution containing 50% conditions with a nucleic acid molecule having a complemen formamide, 5xSSC (750 mM. NaCl, 75 mM trisodium cit tary sequence of the base sequence of SEQID NO: 1, 3, 5, 7, 60 rate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt’s solu 9 or 11, and has a starch hydrolysis activity; wherein the tion (0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyrroli stringent conditions are hybridization in a solution containing done), 10% dextran sulfate and 20 lug/ml denatured sheared 50% formamide, 5xSSC (750 mM. NaCl, 75 mM trisodium salmon sperm DNA at 65°C., and subsequent washing under citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt's the condition of 65° C. using an SSC solution having a 0.1 to solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyr 65 2-fold concentration (a composition of an SSC solution hav rolidone), 10% dextran sulfate and 20 g/ml denatured ing a 1-fold concentration is 150 mM sodium chloride and 15 sheared salmon sperm DNA at 65°C., and subsequent wash mM sodium citrate). US 9,005,681 B2 13 14 (Item 35A) An enzyme-treated Starch having high viscos starch and is free from a forcible bond, the starch can suffi ity and gel-forming ability, ciently undergo gelatinization even at usual heating tempera the enzyme-treated Starch is a starch obtained by treating ture and can exhibit viscosity. Furthermore, the obtained starch granules of untreated Starch with a starch hydrolase starch paste has less spinnability regardless of being suffi under the condition where the starch granules are not dis ciently gelatinized. The gel obtained by using a high concen solved; tration of the starch of the present invention is very rich in the enzyme-treated starch is not modified on hydroxyl elasticity. That is, in the case where the starch of the present groups at the positions 2, 3 and 6 of the glucose residues, invention is added to a high moisture content type food, a the starch hydrolase has an amino acid sequence having at body can be imparted and also natural elasticity can be least 95% or more of homology with an amino acid sequence 10 imparted by a strong gel forming ability. On the other hand, in of SEQID NO: 2, 4, 6, 8, 10 or 12, and has a starch hydrolysis activity. the case where the starch of the present invention is added to a low moisture content type food, texture with nice melt in Effects of the Invention mouth can be imparted to a food. Furthermore, there is less 15 restriction even in the operation step due from the viewpoint According to the present invention, a starch “having a of gelatinization characteristics of them. strong gel forming ability and a high viscosity’ which have Even in the case where a processed starch or a physically never been achieved by a conventional starch has been Suc treated Starch is used as a raw material or a food is produced cessfully developed by using an enzyme having characteris under the condition where a chemical modification or a physi tics capable of improving a gel forming ability of a starch. cal treatment is applied in any stage of the production process Since a conventional starch having a strong gel forming of a food, a food of the present invention has a harder gel and ability cannot sufficiently undergo Swelling and gelatiniza has different texture as compared with the case where a food tion inausual heating temperature Zone, powderiness is likely is produced using a corresponding starch produced without to be left when added to a food. In order to sufficiently swell being Subjected to an enzymatic treatment. Therefore, and gelatinize the conventional starch having a strong gel 25 according to the present invention, it is possible to provide a forming ability, heating at higher temperature thanusual heat food having texture which is different from that of the prior ing temperature Zone of a food is required. As for a starch art. Subjected to an acid treatment and a starch having enriched in amylose fraction, they are excellent in gel forming ability, MODE FOR CARRYING OUT THE INVENTION however they do not exhibit viscosity or hardly exhibit vis 30 cosity, and thus the application of them has been limited. Hereinafter, the present invention will be described in Even in the case of such a starch subjected to an acid treat detail. ment, the gel forming ability can be improved as compared to (1. Materials) the prior art by the enzymatic treatment according to the (1.1 Starch Granules) method of the present invention while maintaining a certain 35 In the present description, the term “starch granules' refers degree of Viscosity. to starch molecules in a crystalline state. The starch granules Furthermore, although a chemically treated starch is often may be untreated Starch granules, or may be starch granules used for a bracken-starch dumpling (Warabimochi), it is nec obtained by a chemical modification or a physical treatment essary to use an acetylation treatment and a phosphate of untreated Starch granules. In the case where an enzyme crosslinking treatment in combination. 40 treated starch classified as a food is preferably used, starch The starch developed in this time is a starch in which these granules to be used are untreated Starch granules obtained defects have been improved. In the case where an untreated from plants. Plants store starch molecules as granules (i.e., as starch, a physically treated Starch, or a bleached starch is used a large crystal) in amyloplasts. The granules are called starch as a raw material, and the starch developed in this time is granules. In the starch granules, starch molecules are mutu produced under the condition where a chemical treatment is 45 ally bonded through a hydrogen bond or the like. Therefore, not applied in any stage of the production process, the addi starch granules are not easily dissolved in water as they are, tion to a usual food, or the application in a food containing a and are not also easily digested. When the starch granules are starch as a main raw material is not limited, and the starch can heated together with water, they are swollen and molecules be used in all foods “dealt as a food'. are disentangled to form a colloid. This change is called In the case where an untreated Starch, a physically treated 50 'gelatinization'. The size and shape of the starch granules starch or a bleached starch is used as a raw material and the vary depending on plants from which the starch granules are enzyme-treated Starch of the present invention is produced obtained. For example, an average granule size of corn starch under the condition where a chemical treatment is not applied granules (corn starch) is from about 12 um to about 15um and in any stage of the production process, the enzyme-treated is slightly smaller, and the size is relatively uniform, than that starch of the present invention prepared by using a starch 55 of other starch granules. Starch granules of wheat and barley hydrolase or a glycosyltransferase does not correspond to a are classified into two kinds in size: large-sized starch gran processed starch obtained by a chemical modification in a ules having a granule size of about 20 um to about 40 um, and food additive. Therefore, it is possible to prepare a food Small-sized starch granules having a granule size of several without the addition of a food additive if the enzyme-treated um. Rice has a compound starch granule structure in which starch of the present invention prepared by using a starch 60 many Small angular starch granules having a diameter of hydrolase or a glycosyltransferase is used. several um are accumulated in amyloplast. The average gran In the case where an untreated Starch is used as a raw ule size of potato starch granules is about 40 um and is the material and an enzyme-treated Starch is produced under the largest among those which are commonly used as a starch raw condition where neither a chemical treatment nor a physical material. In the present invention, commercially available treatment is applied in any stage of the production process, 65 various starch granules can be used. Starch granules may be since the enzyme-treated Starch used in the present invention prepared by the method of for example, purifying starch has a higher gel forming ability than that of the untreated granules from plants and used in the present invention. US 9,005,681 B2 15 16 Ina State of starch granules, the enzyme hardly acts on “distarch phosphate” refers to those obtained by esterifying a starch granules since starch molecules are strongly bonded to starch with sodium trimetaphosphate orphosphorus oxychlo each other. In a specific embodiment for obtaining an ride. The “monostarch phosphate' refers to those obtained by enzyme-treated starch to be treated as a food, the starch esterifying a starch with orthophosphoric acid, a potassium granules used in the present invention are isolated or purified 5 salt or a sodium salt thereof, or sodium tripolyphosphate. The from plants, but are not subjected to an acid treatment, a “phosphated distarch phosphate” refers to those obtained by chemical modification treatment and a heat treatment. In the esterifying a starch with orthophosphoric acid, a potassium present description, the term “untreated starch granules refer salt or a sodium salt thereof, or Sodium tripolyphosphate, and to starch granules which are naturally produced and are not esterifying it with sodium trimetaphosphate or phosphorus Subjected to a treatment other than treatments required to 10 oxychloride. separate starch granules from other components (for Examples of the types of the physically treated Starch gran example, protein and lipid) coexisting in a natural state. ules include a heat-moisture-treated Starch and a thermally Accordingly, the respective steps in the method of preparing inhibited starch. starch granules, such as the step of removing impurities from The starch granules used in the present invention may be plants or the like to purify a starch is not encompassed in a 15 either a aboveground starch or a underground starch. treatment of Starch granules in the present description. It is Examples of the underground starch include a cassava starch, possible to use, as starch granules, any starch granules as long a potato starch, a Sweet potato starch, and a kudzu Starch. as they are usually commercially available starch granules. Examples of the aboveground starch include a wheat starch, a In another specific embodiment, the starch granules used in corn Starch (for example, a high amylose corn starch, a usual the present invention may be starch granules treated by Sub corn starch, and a waxy corn starch), a rice starch (for jecting untreated Starch granules to a chemical modification example, a glutinous rice starch and a nonglutinous rice or a physical treatment. Examples of the chemically modified starch), a bean starch (for example, a green gram starch, a pea starch granules include an acetylated distarch adipate, an starch, an adzuki bean starch, and a fava bean starch), and an acetylated oxidized starch, an acetylated distarch phosphate, Amaranthus starch. The starch granules used in the present a starch sodium octenyl Succinate, a starch acetate, an oxi 25 invention are preferably starches derived from cassava, corn, dized starch, a bleached Starch, a hydroxypropyl distarch or wheat. In the case where the untreated starch is used as the phosphate, a hydroxypropyl Starch, a distarch phosphate, a starch granules, an untreated cassava starch, an untreated corn monostarch phosphate, and a phosphated distarch phosphate. starch or an untreated wheat starch is preferably used. In the The “acetylated distarch adipate” refers to those obtained by case where the chemically modified starch is used as the esterifying a starch with acetic anhydride and adipic anhy 30 starch granules, it is preferred to use an acetylated distarch dride. The “acetylated oxidized starch” refers to those adipate, an acetylated oxidized Starch, an acetylated distarch obtained by treating a starch with sodium hypochlorite and phosphate, a starch sodium octenyl succinate, a starch then esterifying it with acetic anhydride. The “acetylated acetate, an oxidized Starch, a bleached starch, a hydroxypro distarch phosphate” refers to those obtained by esterifying a pyl distarch phosphate, a hydroxypropyl Starch, a distarch starch with sodium trimetaphosphate orphosphorus oxychlo 35 phosphate, a monostarch phosphate or a phosphated distarch ride and acetic anhydride or vinyl acetate. The “starch sodium phosphate of a cassava starch, a corn starch or a wheat starch. octenyl succinate' refers to those obtained by esterifying a In the case where the physically treated starch is used, it is starch with octenyl succinic anhydride. The “starch acetate' preferred to use a heat-moisture-treated starch or a thermally refers to those obtained by esterifying a starch with acetic inhibited Starch of a cassava starch, a corn Starch or a wheat anhydride or vinyl acetate. The “oxidized starch” refers to 40 starch. those obtained by treating a starch with sodium hypochlorite, Since the structure of the starch delicately varies depending wherein the content of carboxyl groups is 1.1% or less when on the origin, features of physical properties vary depending carboxyl groups (also referred to as carboxyl groups) in a on the origin. For example, although the untreated wheat sample starch are analyzed in accordance with the method for starch has a high gel forming ability, the starch paste thereof the purity test described in Ministry of Health and Welfare 45 has a low viscosity and the starch paste is opaque. Although Notification No. 485. Provided that, even when the amount of the untreated cassava starch has a low gel forming ability, the a carboxyl group is within the above range, the "bleached starch paste thereof has a high viscosity and the starch paste starch' is not included in the definition of the “oxidized has high transparency and the degree of retrogradation is a starch”. The “bleached starch” refers to those obtained by middle degree. Particularly, although the untreated cassava treating a starch with sodium hypochlorite, wherein the con 50 starch is inexpensive, the starch paste thereof is transparent, tent of carboxyl groups is 0.1% or less when carboxyl groups and it therefore has a merit of being easily added, the appli in a sample starch are analyzed in accordance with the method cation thereof is limited because of their low gel forming for the purity test described in Ministry of Health and Welfare ability. Furthermore, the untreated native wheat starch could Notification No. 485, and wherein the test results of “Confir not be used in the application where viscosity is required mation test (3) of the oxidized starch described in Ministry 55 because of a low viscosity of the starch paste. Although the of Health and Welfare Notification No. 485 are negative and untreated corn starch has a high gel forming ability, the starch wherein it can be reasonably explained that a change in prop paste thereofhas slightly low viscosity, and the starch paste is erties, such as Viscosity, of the starch is not caused by oxida opaque and has high retrogradation property. tion. Those in which, even if the amount of carboxyl groups is The chemical modification alters physical properties of the 0.1% or less, properties such as Viscosity of the starch change 60 untreated Starch granules. Commonly, crosslinking Such as from those of the native starch are classified as the oxidized phosphate crosslinking or adipate crosslinking often makes starch, and are not dealt as a food in Japan but dealt as food the gel formed by using the obtained starch granules harder additives. The “hydroxypropyl distarch phosphate' refers to and higher turbidity than the gel formed by using the those obtained by esterifying a starch with sodium trimeta untreated Starch granules. Generally, hydroxypropylation, phosphate or phosphorus oxychloride and etherifying it with 65 acetylation and oxidation treatments often improve transpar propylene oxide. The “hydroxypropyl starch” refers to those ency of and make softer the gel formed by using the obtained obtained by etherifying a starch with propylene oxide. The starch granules as compared with the gel formed by using the US 9,005,681 B2 17 18 untreated Starch granules. Commonly, the treatment with before the treatment with the enzyme, when measured by the octenyl Succinic acid can make it possible for the gel formed judgment method described below. The starch hydrolase used using the obtained starch granules to contain oil. in the present invention is preferably an enzyme classified as The physical treatment also alters physical properties of C.-amylase, amyloglucosidase, isoamylase, or C-glucosidase. the untreated Starch granules. For example, commonly, the 5 The enzyme classified as B-amylase or pullulanase is not heat-moisture treatment often makes the gel formed by using preferable. It is considered that the enzyme classified as amy the obtained starch granule harder and the viscosity of the loglucosidase, isoamylase or C-glucosidase can produce an starch paste lower than those of the gel formed by using the enzyme-treated Starch having a high viscosity and a gel form untreated Starch granules. For example, commonly, the ther ing ability if these enzymes are allowed to act on starch mal inhibition treatment often makes the gel formed by using 10 granules. However, in the case of the enzyme classified as the obtained starch granules harder than the gel formed by C.-amylase, not all enzymes can be suitably utilized and using the untreated Starch granules. Also, when the time of the C.-amylase having characteristics capable of improving a gel dry heat treatment is long, the obtained starch often exhibits forming ability of a starch needs to be selected, and the starch low viscosity of the starch paste like a highly crosslinked of the present invention cannot be produced evenifan C.-amy starch. 15 lase not having this activity is used. It is preferred that the starch granules used in the present It is possible to judge whether or not the enzyme classified invention contain impurities as low as possible. The content as C.-amylase is C.-amylase having characteristics capable of of impurities in the starch granules is preferably about 10% by improving a gel forming ability of a starch, by the following weight or less, more preferably about 5% by weight or less, judgment method. and still more preferably about 1% by weight or less. Examples of the glycosyltransferase usable in the produc (1.2 Enzyme) tion of the starch of the present invention include cyclodextrin The enzyme usable in the present invention is a starch glucanotransferase. hydrolase or a glycosyltransferase. The starch hydrolase is (1.2.1 Method of Judging C.-Amylase Having Characteris roughly classified into C.-amylase, 3-amylase, amyloglucosi tics Capable of Improving Gel Forming Ability of Starch) dase, isoamylase, pullulanase, and C-glucosidase. However, 25 The C-amylase having characteristics capable of improv even in the enzymes classified as the same enzyme (for ing a gel forming ability of a starch can be judged by the example, C.-amylase), if the microorganisms producing the following method. To 400 g of a wheat starch, 900 g of enzyme are different, it is considered that features Such as ion-exchange water is added thereby Suspending the wheat reaction specificity and Substrate specificity of the enzymes starch, and each enzyme is added thereto. The amount of a are different. Since these starch hydrolases and glycosyltrans 30 reducing Sugar released in the Suspension by the reaction is ferase are very widely distributed in animals, microorganisms measured to determine a degradation ratio. When the degra and plants, it can be said that there are infinite kinds of starch dation ratio reaches 15%, starch granules are recovered by hydrolases and glycosyltransferases. filtration, washed with water and then dried. Using the The starch hydrolase usable in the production of the starch enzyme-treated Starch thus obtained, a Young's modulus and of the present invention is a starch hydrolase selected from the 35 a rupture stress are determined by rheometer analysis. In the group consisting of amyloglucosidase, isoamylase, C-glu case where theYoung's modulus or rupture stress of the starch cosidase, and C-amylase having characteristics capable of after treatment with the enzyme increases by 10% or more as improving a gel forming ability of a starch. In the present compared with the Young's modulus or rupture stress of the description, the “C.-amylase having characteristics capable of starch before treatment with the enzyme, the enzyme is improving a gel forming ability of a starch' is C.-amylase 40 judged as C.-amylase having characteristics capable of wherein the Young's modulus or rupture stress of the starch improving a gel forming ability of the starch. As an example, after the treatment with the enzyme is 10% or more higher the judgment results of various starch hydrolases are shown in than the Young's modulus or rupture stress of the starch Table 1A below. TABLE 1A Rupture stress Young's modulus Name of Measured Measured enzyme Product name value Relative value Relative group Origin (Selling agency) (g) %* (dyn/cm) 96** 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 US 9,005,681 B2 19 20 As described above, it is possible to easily decide whether lase, cyclodextrin glucanotransferase derived from Bacillus or not various C.-amylases have characteristics capable of licheniformis commercially available from Novozyme as improving a gel forming ability of a starch. It is noted that a ToruZyme, and cyclodextrin glucanotransferase derived from specific method of rheometer analysis is as described in 1.2.2 Paenibacillus macerans (Bacillus macerans) commercially below. available from Amano Enzyme as Cyclodextrin glucan (1.2.2 Specific Method of Rheometer Analysis) otransferase “Amano'. A starch paste is prepared so that the concentration of the In a specific preferred embodiment, the enzyme is a starch starch is 20% by weight on the dry matter basis, and then filled hydrolase, and the starch hydrolase is encoded by a nucleic in a Krehalon casing having a folding width of 45 mm. This acid molecule which is capable of hybridizing understringent starch paste filled in the casing is heated to 90° C. at 1° C./min 10 conditions with a nucleic acid molecule having a complemen and maintained at 90° C. for 30 minutes. Then the starch paste tary sequence to the base sequence of SEQID NO: 1, 3, 5, 7, is left to cool in a constant-temperature water bath at 20° C. 9 or 11 and has a starch hydrolysis activity; wherein the for 30 minutes and then cooled to 5°C. in a refrigerator. After stringent conditions are hybridization in a solution containing cooling, it is refrigeration stored at 5° C. for 16 hours, then it 50% formamide, 5xSSC (750 mM. NaCl, 75 mM trisodium is left at room temperature (about 25°C.) for 4 hours to return 15 citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt's the temperature of it to room temperature, and then measure solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyr ments by a rheometer (RT-2010J-CW) manufactured by rolidone), 10% dextran sulfate and 20 g/ml denatured Rheotech Inc. is performed. The measurement is carried out sheared salmon sperm DNA at 65°C., and subsequent wash under the measurement conditions of the rheometer: a test ing under the condition of 65° C. using an SSC solution item: a rupture test; a height of a sample: 25 mm; and a having a 0.1 to 2-fold concentration (a composition of an SSC movement rate (rupture rate) of a sample: 6 cm/min, using an solution having a 1-fold concentration is 150 mM sodium adapter of a spherical jig for measurement viscosity (25 (di chloride and 15 mM sodium citrate). ameter: 5 mm, area: 19.635 mm). At the measurement, the In a preferred embodiment, the starch hydrolase has an hardness of the starch gel is evaluated by a rupture stress (g) amino acid sequence having at least 95% or more of homol and a Young's modulus (dyn/cm). 25 ogy with an amino acid sequence of SEQID NO: 2, 4, 6, 8, 10 (1.2.3: Preferred Example Used in Present Application) or 12, and has a starch hydrolysis activity In order to produce the starch of the invention, an enzyme (1.2.4. C.-Amylase) selected from the group consisting of amyloglucosidase, C.-Amylases exist in many microorganisms, animals and isoamylase, C-glucosidase, C.-amylase having characteristics plants. Examples of microorganisms which produce an capable of improving a gel forming ability of a starch, and 30 C.-amylase include those of genus Aspergillus (for example, cyclodextrin glucanotransferase is used. Aspergillus Oryzae, Aspergillus niger, Aspergillus awamori, In a specific embodiment, the enzyme is selected from the Aspergillus flavus, Aspergillus kawachii, Aspergillus sclero group consisting of amyloglucosidase, isoamylase, C-glu tiorum and the like); those of genus Bacillus (for example, cosidase, C.-amylase derived from the genus Aspergillus, and Bacillus subtilis, Bacillus acidocaldarius, Bacillus amy cyclodextrin glucanotransferase. 35 loliquefaciens, Bacillus Stearothermophilus, Bacillus cereus, In a specific embodiment, the enzyme is selected from the Bacillus licheniformis and the like); those of genus Geoba group consisting of amyloglucosidase, isoamylase, C-glu cillus (for example, Geobacillus Stearothermophilus, Geoba cosidase, C.-amylase derived from Aspergillus Oryzae, cillus thermodenitrificans, Geobacillus thermodernitrificans C.-amylase derived from Aspergillus niger, and cyclodextrin and the like); those of genus Lactobacillus (for example, glucanotransferase. 40 Lactobacillus amylovorus, Lactobacillus cellobioses, Lacto In a preferred embodiment, the enzyme is selected from the bacillus manihotivorans and the like); further more, group consisting of amyloglucosidase derived from Aspergil Pseudomonas sp., Pyrococcus furiosus, Rhizopus lus niger commercially available from Novozyme as AMG, microsporus, Thermotoga maritima, Vibrio sp. and the like. amyloglucosidase derived from Aspergillus niger commer Furthermore, it is confirmed that the C.-amylase derived from cially available from Genencor as OPTIDEXL-400, amylo 45 animals exist in human pancreas, human saliva, human urine, glucosidase derived from Aspergillus niger commercially porcine pancreas, bovine pancreas, carp intestinal tract and available from DANISCO as DIAZYME X4NP amyloglu the like, and that the C-amylase derived from plants exist in cosidase derived from Aspergillus niger commercially avail barley, rice, wheat, oat, rye, soybean, and fava bean. The able from Amano Enzyme as glucoamylase "Amano' SD, organisms that produce an O-amylase are not limited to them. amyloglucosidase derived from Rhizopus niveus commer 50 C.-Amylase may be commercially available one or may be cially available from Amano Enzyme as Gluczyme AF6, prepared from these organisms by a method known in the art, amyloglucosidase derived from Rhizopus Oryzae commer or may be prepared by a genetic recombination method based cially available from SHIN NIHON CHEMICALS Corpora on an amino acid sequence or a base sequence of C.-amylase tion as Sumizyme, C-glucosidase derived from Aspergillus of these organisms, or may be chemically synthesized. Any niger commercially available from Amano Enzyme as trans 55 C.-amylase known in the art can be used as long as it has glucosidase L Amano'. C-glucosidase derived from properties of cleaving an O-1,4-glucoside bond in the end Aspergillus niger commercially available from Genencor as type. Transglucosidase L-50, C.-amylase derived from Aspergillus An O-amylase used in the present invention is preferably an Oryzae commercially available from Amano Enzyme as C.-amylase from genus Aspergillus, and most preferably an Biozyme A, C.-amylase derived from Aspergillus Oryzae com 60 C.-amylase derived from Aspergillus Oryzae or Aspergillus mercially available from SHIN NIHON CHEMICALS Cor niger: poration as Sumizyme L, C.-amylase derived from Aspergil A nucleotide sequence encoding typical C.-amylase lus niger commercially available from Danisco as AMYLEX derived from Aspergillus Oryzae is shown in SEQID NO: 1. A3, C.-amylase derived from Aspergillus niger commercially and its amino acid sequence is shown in SEQ ID NO: 2. A available from SHIN NIHON CHEMICALS Corporation as 65 nucleotide sequence encoding typical C.-amylase derived Sumizyme AS, isoamylase derived from Pseudomonas amy from Aspergillus niger is shown in SEQ ID NO: 3, and its loderamosa commercially available from Sigma as isoamy amino acid sequence is shown in SEQID NO: 4. It is consid US 9,005,681 B2 21 22 ered that C.-amylases of closely related species have a very Aspergillus phoenicis, Aspergillus Saitoi and the like); those high homology and exhibit the similar enzyme activities. of genus Candida (for example, Candida antarctica, Candida Therefore, it is considered that C.-amylases derived from tsukubaensis and the like); those of genus Rhizopus (for Aspergillus Oryzae have amino acid sequences having a very example, Rhizopus deleimar; Rhizopus delmar; Rhizopus jav high homology to SEQ ID NO: 2 and exhibit the similar anicus, Rhizopusniveus, Rhizopusniveus, Rhizopus Oli enzyme activities. Since it is shown that a commercially gosporus, Rhizopus Oryzae and the like); those of genus Sac available C-amylase derived from Aspergillus Oryzae has charomyces (for example, Saccharomyces cerevisiae, characteristics capable of improving a gel forming ability of Saccharomyces diastaticus, Saccharomyces diastaticus, Sac a starch, it is considered that C.-amylase having an amino acid charomyces fibuligera); further more, Clostridium ther sequence of SEQID NO: 2 and C.-amylase having an amino 10 moamylolyticum, Cladosporium resinae, Lentinus edodes, acid sequence which has a high homology thereto also have Mucor rouxianus, Magnaporthe grisea, Monascus kaoliang, characteristics capable of improving a gel forming ability of Paecilomyces varioti, Penicillium oxalicum, Thermomyces a starch. Similarly, since it is shown that a commercially lanuginosus, Trichoderma reesei and the like. Furthermore, it available C.-amylase derived from Aspergillus niger has char is confirmed that an amyloglucosidase derived from animals acteristics capable of improving a gel forming ability of a 15 exists in mucosa membrane of Small intestine of human, rat starch, it is considered that C.-amylase having an amino acid and mice, and that an amyloglucosidase derived from plants sequence of SEQID NO: 2 and C.-amylase having an amino exists in beet and the like. The organisms that produce an acid sequence which have a high homology thereto also have amyloglucosidase are not limited to them. characteristics capable of improving a gel forming ability of Amyloglucosidase may be commercially available one or a starch. may be prepared from these organisms by a method known in The C-amylase used in the present invention is not an the art, or may be prepared by a genetic recombination amylase derived from Bacillus amyloliquefaciens. The rea method based on an amino acid sequence or a base sequence son is that the amylase derived from Bacillus amyloliquefa of amyloglucosidase of these organisms, or may be chemi ciens cannot produce a starch having a high viscosity and a cally synthesized. Any amyloglucosidase known in the art gel forming ability. 25 can be used as long as it has properties of cleaving an O-1,4- A lot of C-amylase is commercially available. Examples of glucoside bond and an C-1,6-glucoside bond in an exotype the commercially available O.-amylase are described below: from a non-reducing terminal side in a glucose unit to produce Biozyme F1OSD (origin: Aspergillus Oryzae; Amano B-glucose. Enzyme Inc.), Biozyme A (origin: Aspergillus Oryzae; An amyloglucosidase used in the present invention is pref Amano Enzyme Inc.), Kokulase (origin: Aspergillus Oryzae; 30 erably an amyloglucosidase from genus Aspergillus or an Mitsubishi-Kagaku Foods Corporation), Sumizyme L (ori amyloglucosidase from genus Rizopus, and most preferably gin: Aspergillus oryzae; SHIN NIHON CHEMICALS Cor an amyloglucosidase derived from Aspergillus niger or an poration), AMYLEX A3 (origin: Aspergillus niger; Danisco amyloglucosidase derived from Rizopus niveus. Japan Ltd.), GRINDAMYLA (origin: Aspergillus oryzae: A nucleotide sequence encoding typical amyloglucosidase Danisco Japan Ltd.), VERON AX (origin: Aspergillus 35 derived from Aspergillus niger is shown in SEQ ID NO: 5, oryzae: HIGUCHI INC.), VERON GX (origin: Aspergillus and its amino acid sequence is shown in SEQID NO: 6. It is oryzae: HIGUCHI INC.), VERON M4 (origin: Aspergillus considered that amyloglucosidase of closely related species oryzae: HIGUCHI INC.), VERON ELS (origin: Aspergillus have a very high homology and exhibit the similar enzyme oryzae; HIGUCHI INC.), Sumizyme AS (origin: Aspergillus activities. Therefore, it is considered that amyloglucosidase niger; SHIN NIHON CHEMICALS Corporation), 40 derived from Aspergillus niger have amino acid sequences Bakezyme P500 (origin: Aspergillus oryzae: Nihon Siber having a very high homology to SEQID NO: 6 and exhibit the Hegner K.K.), and C.-Amylase (origin: Aspergillus Oryzae; similar enzyme activities. Since it is shown that a commer Sigma-Aldrich Corporation). cially available amyloglucosidase derived from Aspergillus Such commercially available C-amylase is subjected to niger has starch hydrolysis activity, it is considered that amy amino acid analysis to determine the amino acid sequence 45 loglucosidase having an amino acid sequence of SEQID NO: thereof, and a DNA sequence is designed based on the amino 6 and amyloglucosidase having an amino acid sequence acid sequence, and then the DNA sequence is introduced into which has a high homology thereto also have starch hydroly E. coli or the like, and thus C.-amylase having the same amino sis activity. acid sequence as that of the commercially available C.-amy The amyloglucosidase used in the present invention is not lase can be produced. 50 an amyloglucosidase derived from Candida tsukubaensis. (1.2.5 Amyloglucosidase) The reason is that the amyloglucosidase derived from Can Amyloglucosidase refers to an enzyme capable of produc dida tsukubaensis cannot produce a starch having a high ing B-D-glucose by hydrolyzing a 1,4-O bond at a non-reduc Viscosity and a gel forming ability. ing terminal of a carbohydrate chain of a starch or the like. A lot of amyloglucosidase is commercially available. The amyloglucosidase hydrolyzes an O-1,4-glucoside chain 55 Examples of the commercially available amyloglucosidase from a non-reducing terminal, and also an C-1,6-glucoside are described below: GlucS G (origin: Rhizopus niveus; chain, although the degradation rate is low. A systematic Amano Enzyme Inc.), Gluczyme AF6 (origin: Rhizopus name of the amyloglucosidase is glucan 1,4-O-glucosidase. niveus; Amano Enzyme Inc.), Gluczyme NL4.2 (origin: Another name of the amyloglucosidase is exo-1,4-O-D-glu Aspergillus niger, Amano Enzyme Inc.), Brewing glucoamy cosidase, 1,4-O-D-glucan glucohydrolase, glucoamylase, 60 lase "Amano' SD (origin: Aspergillus niger; Amano Enzyme Y-amylase, lysosomal C-glucosidase, or acidic maltase. The Inc.), GODO-ANGH (origin: Aspergillus niger; GODO amyloglucosidase is classified as EC 3.2.1.3. SHUSEICO., LTD.), OPTIDEXL-400 (origin: Aspergillus Amyloglucosidases exist in many microorganisms, ani niger; Genencor Kyowa), OPTIDEXL (origin: Aspergillus mals and plants. Examples of microorganisms which produce niger; Genencor Kyowa), Sumizyme (origin: Rhizopus an amyloglucosidase include those of genus Aspergillus (for 65 oryzae; SHINNIHON CHEMICALS Corporation), Sum example, Aspergillus niger, Aspergillus Oryzae, Aspergillus izyme SG (origin: Rhizopus sp.: SHIN NIHON CHEMI candidus, Aspergillus terreus, Aspergillus awamori, CALS Corporation), Sumizyme HG (origin: Rhizopus US 9,005,681 B2 23 24 oryzae; SHIN NIHON CHEMICALS Corporation), GLU enzyme activities. Since it is shown that a commercially COZYME #20000 (origin: Rhizopus sp.: Nagase Chemtex available isoamylase derived from Flavobacterium sp. has Corporation). AMG (origin: Aspergillus niger; Novozymes starch hydrolysis activity, it is considered that isoamylase Japan Ltd.), GLUTASEAN (origin: Aspergillus niger; HBI having an amino acid sequence of SEQID NO: 8 and isoamy Enzymes Ltd.), UNIASE K, 2K (origin: Rhizopus sp.: lase having an amino acid sequence which has a high homol YAKULT PHARMACEUTICAL INDUSTRY CO., LTD.), ogy thereto also have starch hydrolysis activity. Similarly, UNIASE 30 (origin: Rhizopus sp.: YAKULT PHARMA since it is shown that a commercially available isoamylase CEUTICAL INDUSTRY CO.,LTD.), UNIASE 60F (origin: derived from Pseudomonas amyloderamosa has starch Rhizopus sp.:YAKULT PHARMACEUTICAL INDUSTRY hydrolysis activity, it is considered that isoamylase having an CO., LTD.), MAGNUX JW-201 (origin: Rhizopus sp.: 10 amino acid sequence of SEQ ID NO: 10 and isoamylase Rakuto Kasei Industrial Co., Ltd.), GRINDAMYL AG (ori gin Aspergillus sp.; Danisco Japan Ltd.), DIAZYME X4NP having an amino acid sequence which has a high homology (origin: Aspergillus niger, Danisco Japan Ltd.), BakeZyme thereto also have starch hydrolysis activity. AG800 (origin: Aspergillus niger; Nihon Siber Hegner K.K.), A lot of isoamylase is commercially available. Examples Amyloglucosidase (origin: Aspergillus niger, Sigma-Aldrich 15 of the commercially available isoamylase are described Corporation), Amyloglucosidase (origin: Rhizopus sp.: below: GODO-FIA (origin: Flavobacterium odoratum; Sigma-Aldrich Corporation), and Glucoamylase (origin: GODO SHUSEI CO., LTD.), and Isoamylase (origin: Rhizopus sp., Toyobo Co., Ltd.). Pseudomonas sp.: Sigma-Aldrich Corporation). Such commercially available amyloglucosidase is Sub Such commercially available isoamylase is Subjected to jected to amino acid analysis to determine the amino acid amino acid analysis to determine an amino acid sequence sequence thereof, and a DNA sequence is designed based on thereof, and a DNA sequence is designed based on the amino the amino acid sequence, and then the DNA sequence is acid sequence, and then the DNA sequence is introduced into introduced into E. coli or the like, and thus amyloglucosidase E. coli or the like, and thus isoamylase having the same amino having the same amino acid sequence as that of the commer acid sequence as that of the commercially available isoamy cially available amyloglucosidase can be produced. 25 lase can be produced. (1.2.6 Isoamylase) (1.2.7 O-Glucosidase) Isoamylase refers to an enzyme which cleaves an O-1,6- C-Glucosidase refers to an enzyme which hydrolyzes an glucoside bond of at a branched point of amylopectin, glyco C-1,4-glucoside bond at a non-reducing terminal to produce gen, or the like to produce amylose-like linear polysaccha C-glucose. Systematic name of the C-glucosidase is C-D- rides. Another name of the isoamylase is glycogen 30 glucoside glucohydrolase. Another name of the C-glucosi 6-glucanohydrolase. The isoamylase is classified as dase is maltase, glucoinvertase, or glucoside Sucrase. The EC3.2.1.68. The isoamylase can be derived from any organ C-D-glucosidase is classified as EC 3.2.1.20. ism capable of producing isoamylase. C-Glucosidases exist in many microorganisms, animals Isoamylases exist in many microorganisms, animals and and plants. Examples of microorganisms which produce an plants. Examples of microorganisms which produce an 35 C-glucosidase include those of genus Aspergillus (for isoamylase include Flavobacterium sp.: Bacillus sp.: further example, Aspergillus Oryzae, Aspergillus niger, Aspergillus more, Pseudomonas amyloderamosa, Sulfolobus solfatari awamori, Aspergillus fumigatus, Aspergillus nidulans and cus and the like. Furthermore, it is confirmed that an isoamy the like); those of genus Bacillus (for example, Bacillus amy lase derived from animals exists in human pancreas and the loliquefaciens, Bacillus amylolyticus, Bacillus caldovelox, like, and that an isoamylase derived from plants exists in 40 Bacillus cereus, Bacillus licheniformis, Bacillus thermoglu Oryza sativa, potato (Solanum tuberosum) tuber, Arabidopsis cosidius, Bacillus sp., Bacillus subtilis, Bacillus brevis, thaliana and the like. The organisms that produce an isoamy Bacillus Stearothermophilus; those of genus Lactobacillus lase are not limited to them. (Lactobacillus acidophilus, Lactobacillus brevis and the Isoamylase may be commercially available or may be pre like); those of genus Penicillium (Penicillium brevicompac pared from these organisms by a method known in the art, or 45 tum, Penicilliumcitrinum, PenicilliumOxalicum, Penicillium may be prepared by a genetic recombination method based on purpurogenium); those of genus Pyrococcus (Pyrococcus an amino acid sequence or a base sequence of isoamylase of firiosus, Pyrococcus woesei and the like), those of genus these organisms, or may be chemically synthesized. Any Saccharomyces (Saccharomyces Carlsbergensis, Saccharo isoamylase known in the art can be used as long as it has myces cerevisiae, Saccharomyces fibuligera, Saccharomyces properties of cleaving an O-1,6-glucoside bond of amylopec 50 Oviformis, Saccharomyces Carlsbergensis, Saccharomyces tin in the end type. logos and the like); furthermore, Candida tropicalis, An isoamylase used in the present invention is preferably Schizosaccharomyces pombe, Sulfolobus solfataricus, Ther an isoamylase from genus Flavobacterium or genus motoga maritima, Escherichia coli and the like. It is con Pseudomonas, and more preferably an isoamylase derived firmed that the C-glucosidase derived from animals widely from Flavobacterium sp. or an isoamylase derived from 55 exist within a range from invertebrate animals such as mol Pseudomonas amyloderamosa. lusks, crustaceans, and insects to vertebrate animals such as A nucleotide sequence encoding typical isoamylase fishes, amphibians, reptiles, birds, and mammalians, and the derived from Flavobacterium sp. is shown in SEQID NO: 7. C-glucosidase derived from plants exist in beans, rice, buck and its amino acid sequence is shown in SEQID NO: 8. A wheat, corn, beet seeds and the like. It is noted that organisms nucleotide sequence encoding typical isoamylase derived 60 capable of producing O-glucosidase are not limited to them. from Pseudomonas amyloderamosa is shown in SEQID NO: C-Glucosidase may be commercially available one or may 9, and its amino acid sequence is shown in SEQID NO: 10. It be prepared from these organisms by a method known in the is considered that isoamylase of closely related species have art, or may be prepared by a genetic recombination method a very high homology and exhibit the similar enzyme activi based on an amino acid sequence or a base sequence of ties. Therefore, it is considered that isoamylase derived from 65 C-glucosidase of these organisms, or may be chemically Syn Flavobacterium sp. have amino acid sequences having a very thesized. Any O-glucosidase known in the art can be used as high homology to SEQ ID NO: 8 and exhibit the similar long as it has properties of cleaving an O-1,4-glucoside bond US 9,005,681 B2 25 26 and an C-1,6-glucoside bond in an exo type from a non may be allowed to act on in combination. Particularly, since reducing terminal side in a glucose unit to produce C-glucose. C-glucosidase alone does not easily react with starch gran An O-glucosidase used in the present invention is prefer ules, it is preferred to use in combination with C.-amylase. ably an O-glucosidase from genus Aspergillus, and more pref (1.2.10 Common Explanation about Enzymes) erably an O-glucosidase derived from Aspergillus niger. In the present description, the fact that the enzyme is A nucleotide sequence encoding typical C-glucosidase "derived from certain organisms means not only the fact that derived from Aspergillus niger is shown in SEQID NO: 11, the enzyme is directly isolated from the organisms, but also and its amino acid sequence is shown in SEQID NO: 12. It is the fact that an enzyme having the same amino acid sequence considered that C-glucosidase of closely related species have is produced from another organisms based on an amino acid a very high homology and exhibit the similar enzyme activi 10 sequence of the enzyme possessed by the organisms, or a base ties. Therefore, it is considered that C-glucosidase derived sequence encoding the amino acid sequence. For example, from Aspergillus niger have amino acid sequences having a also in the case of introducing a gene encoding the enzyme very high homology to SEQID NO: 12 and exhibit the similar obtained from the organisms into E. coli and isolating the enzyme activities. Since it is shown that a commercially enzyme from the E. coli, it is said that the enzyme is "derived available C-glucosidase derived from Aspergillus niger has 15 from the organisms. starch hydrolysis activity, it is considered that C-glucosidase In the present description, a large excess amount of the having an amino acid sequence of SEQID NO: 12 and C-glu enzyme is added to starch granules. Therefore, the amount of cosidase having an amino acid sequence which has a high the enzyme is represented by % by weight. It is not necessary homology thereto also have starch hydrolysis activity. to represent it by the unit (U). A lot of C-glucosidase is commercially available. Many C.-amylases, amyloglucosidases, isoamylases, Examples of the commercially available C-glucosidase are C-glucosidases and cyclodextrin glucanotransferases are described below: Transglucosidase L 500 (origin: Aspergil known, and, therefore, many natural base sequences and lus; Genencor Kyowa), Transglucosidase L'Amano' (origin: amino acid sequences of these enzymes are known. It is Aspergillus niger, Amano Enzyme Inc.), O-Glucosidase (ori known that variants (so-called allele variants) having a gin: Bacillus Stearothermophilus; Sigma-Aldrich Corpora 25 slightly different sequence from the natural sequences can tion), O-Glucosidase (origin: rice; Sigma-Aldrich Corpora occur naturally. Such naturally occurring variants and vari tion), O-Glucosidase (origin: Saccharomyces cerevisiae: ants created by artificially mutating the natural enzymes, in Sigma-Aldrich Corporation), O-Glucosidase (origin: addition to the enzymes exemplified above, can be used in the Aspergillus niger, Sigma-Aldrich Corporation), and O-Glu method of the present invention insofar as they have a desired cosidase (origin: Microorganism: Toyobo Co., Ltd.). 30 activity. Variant enzymes preferably have activity equal to, or Such commercially available C-glucosidase is subjected to higher than, that of the enzyme before modification. For amino acid analysis to determine the amino acid sequence example, the amino acid sequence of a starch hydrolase used thereof, and a DNA sequence is designed based on the amino in the present invention, in a certain embodiment, may be acid sequence, and then the DNA sequence is introduced into identical with (that is, 100% identical with) an amino acid E. coli or the like, and thus C-glucosidase having the same 35 sequence (that is, a reference amino acid sequence) of the amino acid sequence as that of the commercially available starch hydrolase used in the Examples of the present appli C-glucosidase can be produced. cation, or the amino acid sequence of SEQID NO: 2, SEQID (1.2.8 Cyclodextrin Glucanotransferase) NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, or Cyclodextrin glucanotransferase is also called CGTase and SEQID NO: 12; or this amino acid sequence may, in another is classified as EC2.4.1.19. CGTase is an enzyme capable of 40 embodiment, be altered in up to a certain number of amino catalyzing a transglycosylation reaction (i.e., disproportion acids compared with a reference amino acid sequence. Such ation reaction) of maltooligosaccharide. CGTase is an alterations can be selected from the group consisting of a enzyme which performs a transfer reaction so as to recognize deletion, a Substitution (including conservative and non-con 6 to 8 glucose-chain at a non-reducing terminal of donor servative substitution), or an insertion of at least 1 (preferably molecules thereby cyclizing this portion to produce cyclodex 45 1 or several; there is no specific upper limit, for example, trin having a degree of polymerization of 6 to 8 and noncyclic about 50 or less, about 40 or less, about 30 or less, about 20 or limit dextrin. As an example of CGTase usable in the present less, about 10 or less, or the like) amino acids. This alteration invention, CGTase derived from well-known microorganisms may occur at a position of an amino terminus or a carboxyl or a commercially available CGTase can be used. CGTase is terminus of a reference amino acid sequence, or may occurat preferably selected from the group consisting of cyclodextrin 50 any position other than these termini. Alteration of an amino glucanotransferase derived from Bacillus licheniformis com acid residue may be interspersed with one residue, or a few mercially available from Novozyme as Toruzyme, and cyclo residues may be contiguous. Those skilled in the art can easily dextrin glucanotransferase (optimum pH 6.0) derived from selecta objective enzyme having a desired property. Alterna Paenibacillus macerans (also classified as Bacillus macer tively, a gene encoding the objective enzyme may be directly ans) commercially available from Amano Enzyme as Cyclo 55 chemically synthesized. Methods for such chemical synthesis dextrin glucanotransferase "Amano'. are well-known in the art. CGTase may be commercially available or may be pre Modification to enzyme can be carried out using a method pared from CGTase producing organisms by a method known well-known in the art, for example, by carrying out site in the art, or may be prepared by a genetic recombination directed mutagenesis, mutagenesis with a mutagen (treat method based on an amino acid sequence or a base sequence 60 ment of a subject gene with a mutagenic agent such as nitrite, of CGTase of CGTase producing organisms, or may be or treatment with UV rays), or error-prone PCR. It is prefer chemically synthesized. Any CGTase known in the art can be able to use site-directed mutagenesis from the viewpoint that used as long as it has a transglycosylation activity, and an the objective mutation is easily obtained, because the objec activity capable of improving agel forming ability of a starch. tive modification can be introduced at an objective site when (1.2.9 Use of Enzymes in Combination) 65 site-directed mutagenesis is used. Alternatively, a nucleic In the case of producing the starch of the present invention, acid molecule having an objective sequence may be directly multiple kinds of Starch hydrolases or glycosyltransferases synthesized. Such chemical synthesis methods are well US 9,005,681 B2 27 28 known in the art. Techniques of site-directed mutagenesis are be obtained by combining fractionation with ion-exchange described in, for example, Nucl. Acid Research, Vol. 10, pp. chromatography on Q-Sepharose or the like, fractionation 6487-6500 (1982). with gel filtration chromatography on Sephacryl S-200HR Upon design of the aforementioned modification, the (manufactured by Pharmacia) or the like and fractionation hydrophobicity index of an amino acid can be considered. with hydrophobic chromatography on Phenyl-TOYOPEARL Significance of a hydrophobic amino acid index upon impar 650M (manufactured by Tosoh Corporation) or the like, if tation interacting biological function to a protein is generally necessary. recognized in the art (Kyte.J and Doolittle, R. F. J. Mol. Biol. Alternatively, the enzyme used in the method of the present 157 (1): 105-132, 1982). The hydrophobic nature of an amino invention can be obtained by introducing a nucleic acid mol acid contributes to the secondary structure of a produced 10 ecule containing a base sequence encoding enzyme of interest protein and, then, defines interaction between the protein and into a suitable host cell, to express the enzyme, and purifying other molecule (e.g. starch hydrolase or glycosyltransferase, the expressed enzyme from the host cell or its culture liquid. Substrate, receptor, DNA, antibody, antigen and the like). An Purified enzyme obtained resultingly is treated with amino acid is assigned a hydrophobicity index based on trypsin, the resulting trypsin treated fragment is separated by hydrophobicity and a nature of a charge thereof. They are: 15 HPLC, and the amino acid sequence of the N-terminus of any isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylala of the separated peptide fragments is determined using a nine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); ala peptide sequencer. Then, using synthetic oligonucleotide nine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); probes prepared based on the identified amino acid sequence, tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine a suitable genome library or a cDNA library is screened, (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid thereby, a nucleic acid molecule (also referred to as a gene) (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). comprising a base sequence encoding natural enzyme can be It is well-known in the art to substitute a certain amino acid obtained. Fundamental strategies for preparing the oligo with another amino acid having a similar hydrophobicity nucleotide probes and DNA libraries, and screening them by index, thereby, a protein still having Substantially similar hybridization of nucleic acids, are well-known to those biological functions (e.g. protein Substantially equivalent in 25 skilled in the art. For example, see Sambrooketal. Molecular enzyme activity) can be produced. In Such an amino acid Cloning: A Laboratory Manual (1989); DNA Cloning, Vol substitution, a hydrophobicity index is preferably within +2, umes I and II (edited by D. N. Glover, 1985); Oligonucleotide more preferably within +1, further preferably within +0.5. It Synthesis (edited by M. J. Gait, 1984); and Nucleic Acid is understood in the art that Such the Substitution of an amino Hybridization (edited by B. D. Hames & S.J. Higgins, 1984). acid based on hydrophobicity is efficient. As described in U.S. 30 Alternatively, based on homology to a base sequence of a Pat. No. 4,554,101, the following hydrophilicity index is certain enzyme gene for which a base sequence encoding assigned to amino acid residues: arginine (+3.0); lysine enzyme is known, screening can be conducted by hybridiza (+3.0); aspartic acid (+3.0+1); glutamic acid (+3.0+1); serine tion using nucleic acid probes containing at least apart of this (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); base sequence, thereby, a nucleic acid molecule containing threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine 35 another kind of the enzyme gene may be acquired. Such (-0.5); cysteine (-1.0); methionine (-1.3): valine (-1.5); leu methods are known in the art. cine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine Alternatively, degenerate primers corresponding to a (-2.5); and tryptophan (-3.4). It is understood that an amino region which is conserved in the amino acid sequence of acid can be substituted with another amino acid which has a various enzymes are prepared, and PCR is performed, and the similar hydrophilicity index, and can still provide a biological 40 base sequence of the enzyme may be acquired. Such methods equivalent. In Such the amino acid Substitution, the hydrophi are known in the art. licity index is preferably within +2, more preferably within When a genome library is screened, the resulting nucleic +1, and further preferably within +0.5. acid molecule can be subcloned using methods well-knownto In the present invention, "conservative substitution” refers those skilled in the art. For example, by mixing w phage to substitution in which a hydrophilicity index or/and a hydro 45 containing an objective gene, Suitable Escherichia coli and phobicity index are similar, as described above, between the Suitable helper phage, a plasmid containing an objective gene original amino acid and an amino acid to be substituted, in can be easily obtained. Thereafter, by transforming suitable amino acid Substitution. Examples of conservative Substitu Escherichia coli using a solution containing the plasmid, an tion are well-knownto those skilled in theart, and include, but objective gene can be subcloned. By culturing the resulting are not limited to Substitution among the following each 50 transformant, a plasmid DNA may be obtained, for example, group, for example: arginine and lysine; glutamic acid and by an alkaline SDS method, and the base sequence of the aspartic acid; serine and threonine; glutamine and aspar objective gene can be determined. A method of determining a agines; and valine, leucine, and isoleucine. base sequence is well-known to those skilled in the art. Fur The enzyme used in the method of the present invention ther, using primers synthesized based on a base sequence of a may be isolated from naturally occurring microorganisms 55 DNA fragment, and using a polymerase chain reaction (PCR) producing the above-mentioned enzyme of interest. For employing, for example, the genomic DNA of Aquifex aeoli example, firstly, a microorganism producing the enzyme of cus, Rhodothermus Obamensis, Bacillus Stearothermophilus, interest is inoculated into a Suitable medium (for example, L Bacillus caldovelox, Bacillus thermocatenulatus, Bacillus broth (1% Bacto-Tryptone (Difco Laboratories, Detroit, caldolyticus or the like as a template, an enzyme gene may be Mich., USA), 0.5% Bacto-Yeast Extract (Difco), 0.5% NaCl, 60 directly amplified. pH 7.3) and cultured at appropriate temperature (for example, Alternatively, the enzyme gene can be chemically synthe about 30° C. to about 40°C.) overnight with shaking. Then, sized based on a known base sequence. this culture is centrifuged to precipitate the microbial cells A base sequence encoding an amino acid sequence of the and then obtained a culture supernatant. The obtained culture enzyme used in the method of the present invention may be Supernatant is concentrated with UF membrane to obtain an 65 altered in up to certain number of nucleotides as compared enzyme liquid of interest. When further purification is neces with the nucleotide sequence (that is, the reference nucleotide sary, a solution containing a purified enzyme of interest can sequence) encoding the reference amino acid sequence US 9,005,681 B2 29 30 described above. Such alterations can be selected from the 99% identity with a comparison Subject sequence, when com group consisting of a deletion of at least one nucleotide, pared in maximum matching in for example GENETYX Substitution with at least one nucleotide, including transition WINVer, 4.0 under the conditions described above. and transversion, or an insertion of at least one nucleotide. A starch hydrolase, which is encoded by a nucleic acid This alteration may occurataposition of the 5' terminus or the molecule which is capable of hybridizing under stringent 3' terminus of a reference nucleotide sequence, or may occur conditions with a nucleic acid molecule having a complemen at any position other than these termini. Alteration of a base tary sequence of a base sequence (for example, SEQID NO: may be interspersed with one base, or a few bases may be 1, 3, 5, 7, 9 or 11) encoding a natural known starch hydrolase, contiguous. can be used in the method of the present invention as long as A nucleotide alteration can generate a nonsense, missense 10 orframe shift mutation in a code sequence, and thus alteration it has a starch hydrolysis activity (characteristics of improv of the enzyme encoded by Sucha altered base sequence can be ing a gel forming ability of a starch in a specific case). A starch effected. hydrolase, which is encoded by a nucleic acid molecule con In the case where the enzyme used in the present invention taining an altered base sequence obtained by altering a is a starch hydrolase, it is preferred that this enzyme has at 15 nucleic acid molecule which is capable of hybridizing under least about 20%, preferably at least about 30%, more prefer stringent conditions with a nucleic acid molecule having a ably at least about 40%, still more preferably at least about complementary sequence of a base sequence encoding a natu 50%, and particularly preferably at least about 60%, about ral known starch hydrolase can also be used in the method of 70%, about 80%, about 90%, about 95%, about 96%, about the present invention as long as it has an ability capable of 97%, about 98% or about 99% of identity against an amino producing a high viscosity starch having agel forming ability. acid sequence of a starch hydrolase used in Examples, or an Those skilled in the art can easily select a desired starch amino acid sequence of SEQID NO: 2, 4, 6, 8, 10 or 12, and hydrolase gene. has a starch hydrolysis activity (characteristics capable of A transglycosylase, which is encoded by a nucleic acid improving a gel forming ability of a starch in a specific case). molecule which is capable of hybridizing under stringent In the case where the enzyme used in the present invention 25 conditions with a nucleic acid molecule having a complemen is a glycosyltransferase, it is preferred that this enzyme has at tary sequence of a base sequence (for example, SEQID NO: least about 20%, preferably at least about 30%, more prefer 13) encoding a natural known transglycosylase, can be used ably at least about 40%, still more preferably at least about in the method of the present invention as long as it has a 50%, and particularly preferably at least about 60%, about transglycosylase activity (characteristics of improving a gel 70%, about 80%, about 90%, about 95%, about 96%, about 30 forming ability of a starch in a specific case). A transglyco 97%, about 98% or about 99% of identity against an amino Sylase, which is encoded by a nucleic acid molecule contain acid sequence of a glycosyltransferase used in Examples, or ing an altered base sequence obtained by altering a nucleic an amino acid sequence of SEQID NO: 14, and has a trans acid molecule which is capable of hybridizing understringent glycosylation activity (characteristics capable of improving a conditions with a nucleic acid molecule having a complemen gel forming ability of a starch in a specific case). 35 tary sequence of a base sequence encoding a natural known In the present specification, the identity of sequences is transglycosylase can also be used in the method of the present calculated using maximum matching of GENETYX-WIN invention as long as it has an ability to produce a high viscos Ver. 4.0 (Genetics Co., Ltd.). This program aligns sequence ity starch having a gel forming ability. Those skilled in the art data to be analyzed, and sequence data to be compared so that can easily select a desired transglycosylase gene. amino acid pairs matched between sequences become great 40 As used in the present description, the term "stringent est while substitution and deletion are considered, and there conditions' refers to conditions under which a sequence upon, gives a score to each of Matches, Mismatches, and hybridizes with a specific sequence, but not with a non-spe Gaps, calculates a Sum, outputs alignment at the Smallest cific sequence. Selection of appropriate Stringent conditions Sum, and calculates identity thereupon (Reference: Takashi, is well-known to those skilled in the art, and is described, for K., and Gotoh, O. 1984. Sequence Relationships among Vari 45 example, in Molecular Cloning (Sambrook, et al., Supra). For ous 4.5 S RNA Species J. Biochem. 92:1173-1177). In the example, “stringent conditions' are hybridization in a solu present specification, the percentage identity of sequences is tion containing 50% formamide, 5xSSC (750 mM NaCl, 75 calculated using maximum matching of GENETYX-WIN mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Ver, 4.0 under the condition of Matches=-1; Mismatches=1; 5xDenhardt’s solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% Gaps=1; *N+=2. 50 polyvinylpyrrolidone), 10% dextran sulfate and 20 ug/ml As a natural enzyme or nucleic acid molecule, an enzyme denatured sheared salmon sperm DNA at 65° C., and subse or nucleic acid molecule having a sequence that is not iden quent washing under the condition of 65° C. using an SSC tical with, but is homologous to, the amino acid sequence of (saline-sodium citrate) solution having a 0.1 to 2-fold con the enzyme or the base sequence encoding the amino acid centration (a composition of an SSC solution having a 1-fold sequence of the enzyme can also be used. Such an enzyme or 55 concentration is 150 mM sodium chloride and 15 mM sodium nucleic acid molecule having homology with the natural citrate). Therefore, for example, a polynucleotide being enzyme or nucleic acid molecule includes, but are not limited capable to hybridize under Stringent conditions means, spe to, in the case of a nucleic acid, nucleic acid molecules con cifically, a polynucleotide which can be identified using the taining a base sequence having at least about 30%, about conditions under which hybridization is performed at 65° C. 35%, about 40%, about 45%, about 50%, about 55%, about 60 in a solution containing 50% formamide, 5xSSC (750 mM 60%, about 65%, about 70%, about 75%, about 80%, about NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate 85%, about 90%, about 95%, or about 99% identity with a (pH 7.6), 5xDenhardt’s solution (0.2% BSA, 0.2% Ficoll 400 comparison Subject sequence, and, in the case of an enzyme, and 0.2% polyvinyl pyrrolidone), 10% dextran sulfate, and 20 includes, but are not limited to, enzymes having an amino ug/ml denatured sheared salmon sperm DNA using a filter on acid sequence having at least about 40%, about 45%, about 65 which a DNA derived from a colony or a plaque has been 50%, about 55%, about 60%, about 65%, about 70%, about immobilized, and a filter is washed under the condition of 65° 75%, about 80%, about 85%, about 90%, about 95%, or about C. using an SSC (Saline-sodium citrate) solution having a 0.1 US 9,005,681 B2 31 32 to 2-fold concentration (a composition of an SSC solution published codon usage table (for example, Sharp, et al., having a 1-fold concentration is 150 mM sodium chloride, 15 Nucleic Acids Research 16, No. 17, p. 8207 (1988)). mM sodium citrate). An expression vector can be made using a nucleic acid Specifically, the conditions mean, for example, that molecule comprising the base sequence modified as A nucleic acid molecule used for producing an enzyme described above. A method for preparing an expression vector used in the present method may be a nucleic acid molecule using a particular nucleic acid sequence is well-known to which was conservatively modified relative to a nucleic acid those skilled in the art. molecule comprising a base sequence encoding a natural When a nucleic acid molecule is referred to in the present enzyme. The “nucleic acid molecule which was conserva specification, a “vector” refers to a nucleic acid molecule 10 which can transfer an objective base sequence into an objec tively modified relative to a nucleic acid molecule comprising tive cell. Examples of such vectors include a vector which can a base sequence encoding natural enzyme” refers to a nucleic autonomously replicate in an objective cell, or can be incor acid molecule comprising a base sequence encoding an porated into a chromosome of an objective cell, and has a amino acid sequence which is the same or essentially the promoter at a position Suitable for transcribing a modified same as an amino acid sequence of the natural enzyme. The 15 base sequence. In the present specification, the vector may be “amino acid sequence which is essentially the same as an a plasmid. amino acid sequence of the natural enzyme” refers to an As used in the present description, an “expression vector” amino acid sequence having essentially the same enzyme refers to a vector which can express a modified base sequence activity as that of the natural enzyme. Due to the degeneracy (i.e. base sequence encoding modified enzyme) in an objec of the genetic code, many functionally equivalent base tive cell. An expression vector contains, in addition to a modi sequences encode any prescribed amino acid sequence. For fied base sequence, various regulation elements such as a example, codons GCA, GCC, GCG and GCU all encode the promoter regulating expression thereofand, if necessary, fac amino acid alanine. Therefore, at all positions where alanine tors necessary for replication in an objective cell and selection is specified by a GCA codon, the codon can be changed to of a recombinant (e.g. origin of replication (ori), and a select GCC, GCG or GCU without changing the encoded alanine. 25 able marker Such as a drug resistant gene). In an expression Similarly, regarding an amino acid which can be encoded by vector, a modified base sequence is operably linked so that it a plurality of codons, at all positions where the amino acid is is transcribed and translated. Regulation elements include a specified by a codon, the codon can be changed to any another promoter, a terminator and an enhancer. In addition, when codon encoding the amino acid without changing the particu secretion of an expressed enzyme outside a cell is intended, a lar amino acid coded. Such a variation in a base sequence is a 30 base sequence encoding a secretion signal peptide is linked “silent mutation” which is one kind of conservatively modi upstream of a modified base sequence in the correct reading fied mutation. All base sequences in the present specification frame. It is well-known to those skilled in the art that both the which encode a polypeptide also include all possible silent type of an expression vector used for introduction into a mutations of the nucleic acid. Silent mutation includes “silent particular organism (e.g. bacterium), and the kind of a regu Substitution' in which a coded amino acid is not changed, and 35 lation element and other factors used in the expression vector, the case where a nucleic acid does not originally encode an can vary depending on an objective cell. amino acid (for example, a mutation at an intron portion, a As used in the present description, a “terminator is a mutation at other untranslated region and the like). When a sequence which is situated downstream of a protein coding certain nucleic acid encodes an amino acid, silent mutation region, and is involved in termination of transcription upon has the same meaning as that of silent Substitution. In the 40 transcription of a base sequence into an mRNA, and in the present specification, “silent substitution” refers to substitu addition of a poly A sequence. It is known that a terminator tion of a base sequence encoding a certain amino acid with influences the expression level of a gene by involving the another base sequence encoding the same amino acid, in a stability of an mRNA. base sequence. Based on the phenomenon of degeneracy in As used in the present description, a “promoter refers to a the genetic code, in the case where there area plurality of base 45 region on a DNA which determines a transcription initiation sequences encoding a certain amino acid (for example, gly site of a gene, and directly regulates the transcription fre cine and the like), such silent substitution is possible. There quency, and is a base sequence to which a RNA polymerase fore, a polypeptide having an amino acid sequence encoded binds, thereby, initiating transcription. Since the region of a by a base sequence produced by silent Substitution has the promoter is usually a region about 2 kbp or less upstream of same amino acid sequence as that of the original polypeptide. 50 a first exon of a putative protein coding region in many cases, In the art, it is understood that each codon in a nucleic acid when a protein coding region in a genome base sequence is (except for AUG which is the only codon usually encoding predicted using a DNA analyzing software, a promoter region methionine, and TGG which is the only codon usually encod can be putative. A putative promoter region varies with every ing tryptophan) can be modified in order to produce function structural gene, and is usually upstream of a structural gene ally the same molecule. Therefore, each silent mutation of a 55 without limitation, and may be downstream of a structural nucleic acid encoding a polypeptide is implicitly included in gene. Preferably, a putative promoter region is present about each described sequence. Preferably, such a modification can 2 kbp or less upstream of a first exon translation initiation be performed so that substitution of cysteine, which is an point. amino acid that greatly influences the conformation of a As used in the present description, an "enhancer can be polypeptide, is avoided. 60 used for enhancing the expression efficiency of an objective A base sequence encoding enzyme used in the present gene. Such an enhanceris well-known in the art. A plurality of invention can be changed in conformity with a codon usage in enhancers can be used, or only one may be used, or may not an organism into which the sequence is introduced for expres be used at all. Sion. Codon usage reflects the usage in a gene which is highly As used in the present description, “operably linked’ refers expressed in the organism. For example, when expression is 65 to when a desired base sequence is placed under the control of intended in Escherichia coli, the sequence can be made to be a transcription and translation regulating sequence (e.g. pro optimal for expression in Escherichia coli according to the moter, enhancer and the like) or a translation regulating US 9,005,681 B2 33 34 sequence which effect expression (i.e. operation). In order calcium chloride, or magnesium chloride is added, increased that a promoter is operably linked to a gene, usually, a pro by 1.5 to 2 times as compared with the system in which no moter is disposed immediately upstream of the gene, but it is metalion is added. not necessary that the promoter is disposed adjacent to the (2. Method for Producing Enzyme-Treated Starch Gran gene. ule) In order to operably link a modified nucleic acid sequence Enzyme-treated Starch granules are produced by treating to the aforementioned regulation element, an enzyme gene starch granules with a starch hydrolase or a glycosyltrans should be processed in Some cases. Examples include the case ferase. Details of each step will be described below. where the distance between a promoter and a coding region is (2.1 Preparation of Suspension) too long, and reduction in a transcription efficiency is pre 10 In the production method of the present invention, for dicted, the case where the distance between a ribosome bind example, starch granules, a starch hydrolase or a glycosyl ing site and a translation initiation codon is not suitable, and transferase, a buffer agent, and a solvent dissolving them are the like. Examples of processing mean include digestion with used as main materials. Although all of these materials are a restriction enzyme, digestion with an exonuclease such as usually added at the time of initiation of a reaction, any Bal31 and ExoIII, or introduction of site-directed mutation 15 material among these materials may be further added during using a single-stranded DNA such as M13 or PCR. the reaction. The solvent used in the production method of the Then, the expression vector prepared as described above is present invention can be any solvent as long as it is a solvent introduced into a cell, thereby, the objective enzyme is which does not impair an enzyme activity of the enzyme to be expressed. used. The typical solventis water (for example, ion-exchange As used in the present description, "expression of an water, purified water, and tap water). The solvent may be enzyme refers to in vivo or in vitro transcription and transla moisture of a crushed cell liquid obtained in association with tion of a base sequence encoding the enzyme, and production the enzyme upon preparing the enzyme. of the encoded enzyme. In the production method of the present invention, first, a A cell into which an expression vector is introduced (also reaction Solution is prepared. The reaction Solution can be referred to as a host) includes prokaryotes and eukaryotes. A 25 obtained, for example, by adding starch granules and a starch cell into which an expression vector is introduced can be hydrolase or a glycosyltransferase to a suitable solvent. For easily selected, taking various conditions such as ease of example, the enzyme may be added after preparing a starch expression of objective enzyme, ease of culturing, growth Suspension by Suspending starch granules in the solvent (for rate, and safety into consideration. Examples of Such cells example, water or buffer solution). Alternatively, the reaction include microorganisms such as bacteria and fungi. Examples 30 Solution may be prepared by mixing a Suspension containing of more preferable cells include mesophilic microorganisms starch granules with a solution containing an enzyme. To this (e.g. yeast, mold, Escherichia coli, Bacillus subtilis). A cell reaction solution, any buffer agent may be optionally added may be a microorganism cell, or may be a plant or animal cell. for the purpose of adjusting the pH as long as it does not Depending on the cell to be used, a starch hydrolase can be an inhibit the enzyme reaction. It is noted that although starch enzyme which has undergone post-translational processing. 35 granules are not dissolved but Suspended in the reaction solu In the method of the present invention, the technique of tion, it is called as reaction solution, since other components introducing an expression vector into a cell may be any tech Such as an enzyme are dissolved therein. nique known in the art. Examples of such techniques include, The pH of the reaction solution can be arbitrarily set as long for example, transformation, transduction, and transfection. as it is the pH at which the enzyme to be used can exert an Such techniques of introducing a nucleic acid molecule are 40 activity. The pH of the reaction solution is preferably around well-known in the art, and are conventional, and are the optimum pH of the enzyme to be used. The pH of the described, for example, in Ausubel F. A., et al. ed. (1988), reaction solution is typically about 2 or more, preferably Current Protocols in Molecular Biology, Wiley, New York, about 3 or more, still more preferably about 4 or more, par N.Y.; Sambrook J, et al. (1987) Molecular Cloning: A Labo ticularly preferably about 5 or more, particularly preferably ratory Manual, 2nd Ed., Cold Spring Harbor Laboratory 45 about 6 or more, and most preferably about 7 or more. The pH Press, Cold Spring Harbor, N.Y., and Bessatsu Jikken-igaku of the reaction solution is typically about 13 or less, prefer “Idenshidounyu & Hatsugen kaiseki jikkenhou, Yodosha, ably about 12 or less, still more preferably about 11 or less, 1997. particularly preferably about 10 or less, particularly prefer (1.3 Other Materials) ably about 9 or less, and most preferably about 8 or less. In an In the production of enzyme-treated Starch granules, any 50 embodiment, the pH of the reaction solution is typically material used usually in an enzymatic treatment can be used within the optimum pH +3, preferably within the optimum pH as long as it does not obstruct an action of the enzyme. +2, still more preferably within the optimum pH +1, and most Examples of such other material include salts and buffer preferably within the optimum pH +0.5, of the enzyme to be agents. Since it is commonly known that a rate of an enzyme used. reaction can be drastically improved by adding a specific salt 55 The amount of the starch granules in the reaction Solution Suitable to each enzyme, it is preferred to add Such a specific can be arbitrarily set as long as it is the amount which enables salt. It is possible to shorten the treatment time by adding Such the enzyme reaction. The amount of the starch granules in the a suitable salt to each enzyme. Examples of the combination reaction solution is preferably about 5% by weight or more, of the enzyme and the salt include a combination of amylo more preferably about 10% by weight or more, still more glucosidase and a metalion (for example, Sodium ion, potas 60 preferably about 20% by weight or more, and most preferably sium ion, calcium ion, or magnesium ion). As a result of a test about 30% by weight or more. The amount of the starch by the present inventors, for example, in the case of treating granules in the reaction solution is preferably about 60% by an untreated native cassava starch with amyloglucosidase (for weight or less, more preferably about 50% by weight or less, example, “OPTIDEXL-400' derived from Aspergillus niger, still more preferably about 40% by weight or less, and most manufactured by Genencor), a degradation rate of the starch 65 preferably about 35% by weight or less. in the system, in which 100 ppm (in terms of a metal ion) of The amount of the enzyme in the reaction Solution can be Sodium chloride, or Sodium Sulfate, or potassium chloride, or arbitrarily set as long as it is the amount which enables the US 9,005,681 B2 35 36 enzyme reaction. The amount of the enzyme is preferably the more, it is preferred to obtain the objective enzyme-treated amount enough to carryout the reaction within a reasonable starch granules by drying the starch after dehydration. Drying time. As the amount of the enzyme increases, the time of the starch after dehydration can be carried out by any required to the reaction becomes shorter. As the amount of the method known in the art. enzyme decreases, the time required to the reaction becomes (2.4 Chemical Modification) longer. When the amount of the enzyme is too large, the cost The starch granules Subjected to the enzymatic treatment increases too much and the enzyme may be sometimes aggre can be subjected to a chemical modification, if desired. Not gated to form a precipitate. Therefore, it is preferred to appro only in the case where the starch granules used in the enzy priately set the amount of the enzyme. matic treatment are untreated Starch granules or starch gran The amount of the enzyme in the reaction solution is pref 10 ules Subjected to a physical treatment, but also in the case erably about 0.01% by weight or more, more preferably about where starch granules of some chemically modified Starch are 0.05% by weight or more, and still more preferably about used, it is possible to be subjected to a chemical modification 0.1% by weight or more, based on the solid content of the which is different from various chemical modifications starch granules. The amount of the enzyme in the reaction applied to the chemically modified starch. Examples of the solution is preferably about 10% by weight or less, more 15 chemical modification include acetylation, adipate crosslink preferably about 5% by weight or less, and still more prefer ing, oxidation, bleaching, phosphate crosslinking, treatment ably about 1% by weight or less, based on the solid content of with octenyl Succinic acid, hydroxypropylation, phosphory the starch granules. The amount of the enzyme in the reaction lation, and phosphoric acid monoesterification. These chemi Solution may be the amount enough to enable proceeding of cal modification methods are well known in the art. These the enzyme reaction. Therefore, it is not necessary to examine chemical modifications can be carried out to any degree as in detail about an activity (number of units) of the enzyme. long as they are within the scope permitted by the Food (2.2 Enzyme Reaction) Sanitation Law of Japan. In Japan, in order that the chemi Next, the reaction Solution is reacted optionally by heating cally modified Starch is approved as a food additive, it is using a method known in the art. The solution temperature in essential that various chemical Substances in a sample starch the reaction step can be any temperature as long as it is the 25 are analyzed in accordance with a method for a purity test temperature at which the starch granules are not substantially described in Ministry of Health and Welfare Notification No. collapsed. The reaction temperature is preferably the tem 485 and the obtained analytical results meet the following perature at which an enzyme to be used can Sufficiently exert standards: an activity and Sufficiently retain an activity (that is, less (a) Acetylated distarch adipate: the content of adipic acid likely to be inactivated). The temperature of the solution in 30 groups shall be 0.135% or less and the content of acetyl this reaction step is preferably the temperature at which about groups shall be 2.5% or less; 50% or more, and more preferably about 80% or more of the (b) Acetylated oxidized starch: the content of acetyl groups activity of the enzyme contained in this solution before the shall be 2.5% or less and the content of carboxyl groups shall reaction remains after a predetermined reaction time. For be 1.3% or less; example, this temperature can be an optimum temperature 35 (c) Acetylated distarch phosphate: the content of acetyl +10° C., more preferably an optimum temperature +5, still groups shall be 2.5% or less and the content of phosphorus more preferably an optimum temperature t1° C., and most shall be 0.14% or less in terms of P: preferably an optimum pH +0.5°C., of the enzyme to be used. (d) Starch sodium octenyl Succinate: the content of octenyl The reaction temperature is preferably about 10°C. or higher, succinic acid groups shall be 3.0% or less; more preferably about 10°C. or higher, still more preferably 40 (e) Starch acetate: the content of acetyl groups shall be 2.5% about 15° C. or higher, further more preferably about 20°C. or less; or higher, particularly preferably about 30°C. or higher, and (f) Oxidized starch: the content of carboxyl groups shall be most preferably 40°C. or higher. The reaction temperature is 1.1% or less; preferably about 70° C. or lower, more preferably about 65° (g) Hydroxypropyl distarch phosphate: the content of C. or lower, particularly preferably about 60°C. or lower, and 45 hydroxypropyl groups shall be 7.0% or less and the content of most preferably 55° C. or lower. phosphorus shall be 0.14% or less in terms of P: The reaction time can be arbitrarily set taking the reaction (h) Hydroxypropyl starch: the content of hydroxypropyl temperature, the amount of the enzyme to starch granules into groups shall be 7.0% or less; consideration. The reaction time can be preferably for about 1 (i) Distarch phosphate: the content of phosphorus shall be hour or more, for example, about 2 hours or more, about 3 50 0.5% or less in terms of P: hours or more, about 6 hours or more, and about 12 hours or (j) Monostarch phosphate: the content of phosphorus shall be more. Although there is no particular upper limit of the reac 0.5% or less in terms of P: tion time, the reaction time is preferably about 72 hours or (k) Phosphated distarch phosphate: the content of phosphorus less, more preferably about 48 hours or less, still more pref shall be 0.5% or less in terms of P: erably about 36 hours or less, particularly preferably about 24 55 (1) Bleached starch; the content of carboxyl groups shall be hours or less, and most preferably about 20 hours or less. 0.1% or less, the test results of “Confirmatory test (3) of the (2.3 Post-Treatment) oxidized starch described in Ministry of Health and Welfare The starch granules Subjected to the enzymatic treatment Notification No. 485 shall be negative, and it shall be reason can be used as they are depending on the application. How ably explained that a change in properties, such as Viscosity, ever, it is preferred that the enzyme used and glucide eluted by 60 of the starch is not caused by oxidation. Regarding the coun enzymatic hydrolysis are removed by washing the starch tries other than Japan, any degree of a chemical treatment can granules Subjected to the enzymatic treatment, and followed be carried out as long as it is within the scope permitted in that by dehydration. Washing and dehydration of the starch gran country. Some kinds of chemical modifications can be used in ules Subjected to the enzymatic treatment can be carried out combination. by any method known in the art. Washing and dehydration of 65 (2.5 Physical Treatment) the starch granules are conventional methods used for prepa The starch granules Subjected to the enzymatic treatment ration of a starch, and are commonly carried out. Further can be subjected to a physical treatment, if desired. Not only US 9,005,681 B2 37 38 in the case where the starch granules used in the enzymatic becomes higher, a more highly inhibited Starch is obtained. treatment are untreated Starch granules or a chemically modi As the time of the heat treatment becomes longer, a more fied Starch, but also in the case where the starch granules highly inhibited starch is obtained. The thermal treatment Subjected to Some physical treatment are used, it is possible to time for a thermal inhibition treatment can be, for example, be subjected to a physical treatment which is different from about 3 hours or more, and preferably about 20 hours or less. the physical treatment. Examples of the physical treatment The thermal inhibition treatment is described in various docu include a heat-moisture treatment and a thermal inhibition ments and can be carried out in accordance with any thermal treatment. inhibition treatment method known in the art. The thermal The “heat-moisture treatment” refers to heating to a tem inhibition treatment is described, for example, in U.S. Pat. perature of about 95 to about 125°C. in a low moisture state 10 No. 6,221,420, Pamphlet of International Publication No. where a starch is not gelatinized in a closed container under WO95/04082, and Japanese Laid-open Patent Publication the condition of a relative humidity of about 100%. The “low No. 2008-223,032. The temperature, time, and the like of the moisture state where a starch is not gelatinized' indicates, for thermal inhibition treatment can be appropriately set depend example, the moisture content of about 50% or less. The low ing on the objective starch and physical properties thereof. moisture state where a starch is not gelatinized may be, for 15 The physical treatment can be carried out in accordance with example, the moisture content of about 35% or less, about the method well known in the art. 30% or less, about 25% or less, or about 20% or less. The Examples of the heat-moisture-treated starch include, for heating time of the heat-moisture treatment can vary depend example, “Delicastar series”, “Naturastar series', and “AMY ing on the method of the heat-moisture treatment. For LOGEL manufactured by SANWA CORNSTARCH CO., example, a heat-moisture treatment is carried out in accor LTD.; and “ROADSTER’ manufactured by Nihon Shokuhin dance with the method described in Japanese Laid-open Kako Co., Ltd. Examples of the thermally inhibited starch Patent Publication No. 6-145203, a heat treatment is carried include “NOVATION series' manufactured by National out by first decompressing to a pressure of about 0 to 500 torr Starch Corp. (about 0 to 66.661 kPa) and then introducing pressurized (3. Characteristics of Enzyme-Treated Starch Granules of steam, followed by retention at about 100° C. to about 150° C. 25 the Present Invention) for about 2 minutes to about 120 minutes. The heat-moisture In a specific embodiment, the enzyme-treated starch of the treatment is described in various documents and can be car present invention is an enzyme-treated Starch having a high ried out in accordance with any heat-moisture treatment Viscosity and a gel forming ability, and the enzyme-treated method known in the art. The heat-moisture treatment is starch is an enzyme-treated Starch obtained by treating starch described, for example, in Japanese Laid-open Patent Publi 30 granules with an enzyme at a temperature of about 10°C. or cation No. 6-145203, Japanese Laid-open Patent Publication higher and about 70° C. or lower. No. 4-1301.02, A Technical Journal on Food Chemistry & In another specific embodiment, the enzyme-treated starch Chemicals 2010-2 (P. 37-42) and the like. The temperature, of the present invention is an enzyme-treated Starch having a time and the like of the heat-moisture treatment can be appro high viscosity and a gel forming ability; the enzyme-treated priately set depending on the objective starch and physical 35 starch is a starch obtained by treating starch granules of an properties thereof. untreated starch with a starch hydrolase under the condition The “thermal inhibition treatment” refers to the fact that a where the starch granules are not dissolved; the enzyme crystal structure of starch granules is reinforced by Subjecting treated Starch is not modified on hydroxyl groups at the posi starch granules dried to extremely low water content to a dry tions 2, 3 and 6 of the glucose residues; and the enzyme heat treatment. The “starch granules dried to extremely low 40 treated Starch can forma gel having aYoung's modulus higher water content” refers to starch granules whose moisture con than that of the untreated Starch or a rupture stress higher than tent is less than about 1%. The moisture content of the starch that of the untreated starch, when measured by a rheometer. granules Subjected to a thermal inhibition treatment is pref (3.1 Viscosity) erably about 0%. The method of drying starch granules to It is well known that when a starch is heated together with extremely low water content is described, for example, in 45 a predetermined amount or more of water, starch granules JP-A-2008-223,032 and can be, for example, a method in generally cause a gelatinization phenomenon Such as Swell which the pH of starch granules is adjusted to the pH of 7.0 or ing, an increase in transparency, and an increase in Viscosity. more and then dehydration is carried out until the moisture The starch granules are collapsed by further heating. In order content reaches less than about 1%. In the case of drying to to measure a change in Viscosity associated with a series of low water content, the pH is preferably 7 or more, more 50 these events, an amylograph manufactured by Brabender Inc. preferably more than 8, still more preferably from 7.5 to 10.5, is practical and is widely used, although there are some meth and further more preferably from 8 to 9.5. The dehydration ods. The amylograph is that in which the object is heated at a may be either thermal dehydration or nonthermal dehydra predetermined rate and a relationship between the tempera tion. In the case of a dry heat treatment, a heat treatment is ture and the viscosity of the object is recorded. That is, starch carried out at a Sufficient temperature for the time enough to 55 granules undergo swelling with heating, while manifestation inhibit a starch. Preferably, a heat treatment is carried out at a of Viscosity and an increase in Viscosity arise in the amylo Sufficient temperature for the time enough to make a starch graph. Then, when the Swelling of the starch granules non-aggregative. The heating temperature for a thermal inhi becomes to maximum, the Viscosity also reaches a peak. This bition treatment is preferably higher than about 100°C. The Viscosity is called maximum viscosity. Furtherheating causes heat treatment temperature is preferably about 200° C. or 60 collapse of the starch granules and simultaneously causes a lower. The heating temperature for a thermal inhibition treat decrease in Viscosity. This degree of the decrease in Viscosity ment is more preferably from about 120° C. to about 180°C., is called breakdown. A viscosity curve obtained by this amy particularly preferably from about 140°C. to about 160° C. lograph varies depending on the origin and production and most preferably from about 160° C. The level of inhibi method of the starch, and is a measuring method showing tion depends on the pH, heating temperature and heating 65 features of the starch. time. As the pH becomes higher, a more highly inhibited For example, the measurement by the amylograph is car starch is obtained. As the temperature of the heat treatment ried out as follows. A starch suspension is prepared in 450 ml US 9,005,681 B2 39 40 of water so as to obtain a predetermined amount of enzyme For example, regarding the corn starch, a maximum vis treated Starch granules (for example, the concentration of a cosity of the native corn starch when measured by an amylo wheat starch is 8.5% by weight, the concentration of a corn graph under the above conditions is from about 400 BU to starch is 7.0% by weight, and the concentration of a cassava about 500 BU. On the other hand, in the case where the starch is 6.0% by weight, on the dry matter basis), put in a enzyme-treated Starch granules of the present invention are sample container, and then warmed to 50° C. while rotating prepared from an untreated corn starch and have not been them. Then the suspension is heated to 95°C. at 1.5°C./min Subjected to either a chemical modification or a physical and maintained at 95°C. for 15 minutes, followed by cooling treatment, a maximum viscosity of the enzyme-treated corn at 1.5° C./min. The measurement is carried out using an starch granules of the present invention when measured by an 10 amylograph under the above conditions may be preferably amylograph VISCOGRAPH-E manufactured by Brabender about 250 BU or more, more preferably about 270 BU or Inc. under the conditions of a rotation number of a sample more, particularly preferably about 300 BU or more, most container of 75 rpm and a measuring cartridge of 700 cmg. preferably about 350 BU or more, for example, about 400 BU Wherein, the Viscosity reached to a peak is regarded as a or more, about 420 BU or more, about 440 BU or more, or maximum viscosity, and a difference between this maximum 15 about 450 BU or more. The maximum viscosity of the Viscosity and a viscosity at the point after maintaining at 95° enzyme-treated corn starch granules of the present invention C. for 15 minutes is regarded as breakdown. This difference is when measured by an amylograph under the above conditions also called as a breakdown viscosity. When the difference can be, for example, about 600 BU or less, about 550 BU or between the maximum viscosity and the Viscosity at the point less, about 520 BU or less, or about 500 BU or less. after maintaining at 95°C. for 15 minutes is less than 100 BU, For example, regarding the cassava starch, a maximum it is said that the starch has no “breakdown'. Viscosity of the native cassava starch when measured by an In the case where the enzyme-treated Starch granules of the amylograph under the above conditions is from about 700 BU present invention are prepared from an untreated Starch and to about 800 BU. On the other hand, in the case where the have not been subjected to either a chemical modification or enzyme-treated Starch granules of the present invention are a physical treatment, it is preferred that the enzyme-treated 25 prepared from an untreated cassava starch and have not been starch granules of the present invention have a maximum Subjected to either a chemical modification or a physical viscosity which accounts for about 50% or more (more pref treatment, a maximum viscosity of the enzyme-treated cas erably about 60% or more, particularly preferably about 70% sava starch granules of the present invention when measured or more, and most preferably about 80% or more, about 90% by an amylograph under the above conditions may be prefer or more, or about 100% or more) of the maximum viscosity of 30 ably about 500 BU or more, more preferably about 520 BU or the untreated Starch, when measured by an amylograph under more, particularly preferably about 530 BU or more, most the above conditions. There is no particular upper limit of the preferably about 550 BU or more, for example, about 600 BU maximum viscosity of the enzyme-treated Starch of the or more, about 620 BU or more, about 630 BU or more, or present invention. For example, the maximum viscosity of the about 650 BU or more. The maximum viscosity of the enzyme-treated Starch of the present invention can be about 35 enzyme-treated cassava starch granules of the present inven 300% or less, about 250% or less, about 200% or less, about tion when measured by an amylograph under the above con 150% or less, about 110% or less, and about 100% or less of ditions can be, for example, about 900 BU or less, about 850 the maximum viscosity of the untreated Starch, when mea BU or less, about 800 BU or less, or about 770 BU or less. Sured by an amylograph under the above conditions. For In the case where the enzyme-treated Starch granules of the example, it is preferred that the enzyme-treated wheat starch 40 present invention are prepared from an untreated Starch and can form a gel having a viscosity which accounts for 70% or have not been subjected to either a chemical modification or more and 200% or less (more preferably 80% or more and a physical treatment, the enzyme-treated Starch granules of 200% or less) of the viscosity of the untreated wheat starch. the present invention have breakdown when measured by an For example, regarding the wheat starch, a maximum vis amylograph. Some conventional starches have no break cosity of the native wheat starch when measured by an amy 45 down, whereas, the enzyme-treated Starch granules of the lograph under the above conditions is from about 550 BU to present invention have breakdown. about 650 BU. On the other hand, in the case where the For example, in the case where the untreated Starch is a enzyme-treated Starch granules of the present invention are wheat starch, a corn starch or a cassava starch and neither a prepared from an untreated wheat starch and have not been chemical modification nor a physical treatment is carried out, Subjected to either a chemical modification or a physical 50 the obtained enzyme-treated Starch has a breakdown viscos treatment, a maximum viscosity of the enzyme-treated wheat ity of about 100 BU or more. starch granules of the present invention when measured by an In the case where the untreated starch is a wheat starch and amylograph under the above conditions may be preferably neither a chemical modification nor a physical treatment is about 400 BU or more, more preferably about 420 BU or carried out, the breakdown viscosity of the obtained enzyme more, particularly preferably about 450 BU or more, most 55 treated starch is preferably about 100 BU or more, more preferably about 500 BU or more, for example, about 550 BU preferably about 120 BU or more, still more preferably about or more, about 570 BU or more, about 600 BU or more, or 130 BU or more, and most preferably about 150 BU or more. about 650 BU or more. In a specific embodiment, it is possible In the case where the untreated starch is a wheat starch and to make the maximum viscosity of the enzyme-treated wheat neither a chemical modification nor a physical treatment is starch granules of the present invention when measured by an 60 carried out, although there is no particular upper limit of the amylograph under the above conditions to about 660 BU or breakdown viscosity of the obtained enzyme-treated starch, more, about 670 BU or more, or about 700 BU or more. The the breakdown viscosity of the obtained enzyme-treated maximum viscosity of the enzyme-treated wheat starch gran starch can be, for example, about 500 BU or less, about 450 ules of the present invention when measured by an amylo BU or less, about 400 BU or less, about 350 BU or less, or graph under the above conditions can be, for example, about 65 about 300 BU or less. 900 BU or less, about 850 BU or less, about 800 BU or less, In the case where the untreated Starch is a corn starch and or about 750 BU or less. neither a chemical modification nor a physical treatment is US 9,005,681 B2 41 42 carried out, the breakdown viscosity of the obtained enzyme enzyme-treated cassava starch has a rupture stress which treated starch is preferably about 100 BU or more, more accounts for 110% or more and 300% or less of the rupture preferably about 110 BU or more, still more preferably about stress of the untreated cassava starch, or has a Young's modu 120 BU or more, and most preferably about 150 BU or more. lus which accounts for 110% or more and 500% or less (33.0% In the case where the untreated Starch is a corn starch and 5 or less in an embodiment) of the untreated cassava starch. neither a chemical modification nor a physical treatment is In the case where the enzyme-treated Starch granules of the carried out, although there is no particular upper limit of the present invention are prepared from an untreated wheat starch breakdown viscosity of the obtained enzyme-treated starch, and have not been subjected to either a chemical modification the breakdown viscosity of the obtained enzyme-treated or a physical treatment, and the untreated Starch is a wheat starch can be, for example, about 300 BU or less, about 290 10 starch, the rupture stress of the obtained enzyme-treated BU or less, about 280 BU or less, 200 BU or less, about 190 starch is preferably about 150 g or more, more preferably BU or less, or about 180 BU or less. about 160 g or more, still more preferably about 170 g or In the case where the untreated Starch is a cassava starch more, particularly preferably about 180g or more, and most and neither a chemical modification nor a physical treatment preferably about 200 g or more. In the case where the is carried out, the breakdown viscosity of the obtained 15 untreated Starch is a wheat starch, although there is no par enzyme-treated starch is preferably about 300 BU or more, ticular upper limit of the rupture stress of the obtained more preferably about 320 BU or more, still more preferably enzyme-treated starch, the rupture stress of the obtained about 330 BU or more, and most preferably about 350 BU or enzyme-treated Starch can be, for example, about 450 g or more. In the case where the untreated Starch is a cassava less, about 440 g or less, about 430 g or less, about 420 g or starch and neither a chemical modification nor a physical less, about 410 g or less, or about 400 g or less. treatment is carried out, although there is no particular upper In the case where the untreated Starch is a corn starch and limit of the breakdown viscosity of the obtained enzyme neither a chemical modification nor a physical treatment is treated starch, the breakdown viscosity of the obtained carried out, the rupture stress of the obtained enzyme-treated enzyme-treated starch can be, for example, about 550 BU or starch is preferably about 210 g or more, more preferably less, about 540 BU or less, about 530 BU or less, about 500 25 about 220 g or more, still more preferably about 230 g or BU or less, about 480 BU or less, or about 470 BU or less. more, and most preferably about 240 g or more, and, in one (3.2 Gel Forming Ability) embodiment, is 250 g or more. In the case where the untreated It is well known that when the concentration of a starch of starch is a corn starch and neither a chemical modification nor a starch paste reaches a predetermined concentration or more, a physical treatment is carried out, although there is no par a starch gel is formed by cooling it. Similarly to the Viscosity, 30 ticular upper limit of the rupture stress of the obtained physical properties of this starch gel vary depending on the enzyme-treated starch, the rupture stress of the obtained origin and production method of the starch, and the starch is enzyme-treated starch can be, for example, about 450 g or used in various foods taking features of this gelling physical less, about 440 g or less, about 430 g or less, about 420 g or properties into consideration. Some methods of measuring less, about 410 g or less, or about 400 g or less. physical properties of the gel are practically used, and one of 35 In the case where the untreated Starch is a cassava starch them is a method of measuring using a rheometer. The gel and neither a chemical modification nor a physical treatment forming ability can be measured by the following method is carried out, the rupture stress of the obtained enzyme using a rheometer. For example, a starch paste is filled in a treated starch is preferably about 55 g or more, more prefer casing, heated, and then refrigerated for 16 hours or 21 days ably about 60 g or more, still more preferably about 65g or (for example, at about 5° C.) and, after returning to room 40 more, and most preferably about 70 g or more. In the case temperature (for example, at about 25°C.), physical proper where the untreated Starch is a cassava starch and neither a ties of the gel are measured by a rheometer. chemical modification nor a physical treatment is carried out, The specific measuring method using a rheometer is as although there is no particular upper limit of the rupture stress described in the aforementioned 1.2.2. In the case where the of the obtained enzyme-treated starch, the rupture stress of enzyme-treated Starch granules of the present invention are 45 the obtained enzyme-treated Starch can be, for example, prepared from an untreated wheat starch and have not been about 150 g or less, about 140 g or less, about 130 g or less, Subjected to either a chemical modification or a physical about 120 g or less, about 110 g or less, or about 100 g or less. treatment, it is preferred that the enzyme-treated wheat starch In the case where the untreated starch is a wheat starch and has a rupture stress which accounts for 110% or more and neither a chemical modification nor a physical treatment is 300% or less of the rupture stress of the untreated wheat 50 carried out, the Young's modulus of the obtained enzyme starch, or has a Young's modulus which accounts for 110% or treated starch is preferably about 5.0x10' dyn/cm or more, more and 500% or less (110% or more and 330% or less in an more preferably about 5.2x10° dyn/cm or more, still more embodiment) of the Young's modulus of the untreated wheat preferably about 5.4x10° dyn/cm or more, and most prefer starch. ably about 5.6x10° dyn/cm or more. In the case where the In the case where the enzyme-treated Starch granules of the 55 untreated Starch is a wheat starch and neither a chemical present invention are prepared from an untreated corn starch modification nor a physical treatment is carried out, although and have not been subjected to either a chemical modification there is no particular upper limit of the Young's modulus of or a physical treatment, it is preferred that the enzyme-treated the obtained enzyme-treated starch, the Young's modulus of corn starch has a rupture stress which accounts for 110% or the obtained enzyme-treated Starch can be, for example, more and 300% or less of the rupture stress of the untreated 60 about 8.0x10° dyn/cm or less, about 7.5x10° dyn/cm or less, corn starch, or has a Young's modulus which accounts for about 7.0x10° dyn/cm or less, about 6.9x10° dyn/cm or less, 110% or more and 500% or less (330% or less in an embodi about 6.8x10° dyn/cm or less, or about 6.7x10° dyn/cm or ment) of the Young's modulus of the untreated corn starch. less. In the case where the enzyme-treated Starch granules of the In the case where the untreated Starch is a corn starch and present invention are prepared from an untreated cassava 65 neither a chemical modification nor a physical treatment is starch and have not been subjected to either a chemical modi carried out, the Young's modulus of the obtained enzyme fication or a physical treatment, it is preferred that the treated starch is preferably about 6.0x10' dyn/cm or more, US 9,005,681 B2 43 44 more preferably about 6.2x10' dyn/cm or more, still more are modified, refers to a modified starch (also referred to as a preferably about 6.3x10° dyn/cm or more, and most prefer chemically modified starch) subjected to so-called chemical ably about 6.5x10° dyn/cm or more. In the case where the modification by an industrial process. According to the min untreated Starch is a corn starch and neither a chemical modi isterial ordinance to revise a part of the Ordinance For fication nor a physical treatment is carried out, although there Enforcement of the Food Sanitation Act notified in Ministry is no particular upper limit of the Young's modulus of the of Health and Welfare Notification No. 485 dated Oct. 1, obtained enzyme-treated starch, the Young's modulus of the 2008, the following 11 items of modified starches will be obtained enzyme-treated Starch can be, for example, about dealt as an additive: 9.0x10° dyn/cm or less, about 8.9x10° dyn/cm or less, about acetylated distarch adipate; 8.8x10° dyn/cm or less, about 8.7x10° dyn/cm or less, about 10 acetylated oxidized starch; 8.6x10° dyn/cm or less, or about 8.5x10° dyn/cm or less. acetylated distarch phosphate: In the case where the untreated Starch is a cassava starch starch sodium octenyl Succinate; and neither a chemical modification nor a physical treatment starch acetate; is carried out, the Young's modulus of the obtained enzyme oxidized starch; treated starch is preferably about 5.2x10 dyn/cm or more, 15 hydroxypropyl distarch phosphate; more preferably about 5.4x10 dyn/cm or more, still more hydroxypropyl Starch; preferably about 5.6x10f dyn/cm or more, and most prefer distarch phosphate: ably about 5.8x10 dyn/cm or more. In the case where the monostarch phosphate; and untreated Starch is a cassava starch and neither a chemical phosphated distarch phosphate. In Ministry of Health and modification nor a physical treatment is carried out, although Welfare Notification No. 485, a method for a purity test of there is no particular upper limit of the Young's modulus of these starches is described. Therefore, it is possible to judge the obtained enzyme-treated starch, the Young's modulus of that a sample starch is not a starch Subjected to a chemical the obtained enzyme-treated Starch can be, for example, modification, for example, by analyzing various chemical about 2.7x10° dyn/cm or less, about 2.5x10'dyn/cm or less, Substances in the sample starch, Such as adipic acid groups, about 2.4x10° dyn/cm or less, about 2.3x10'dyn/cm or less, 25 acetyl groups, and carboxyl groups in accordance with a about 2.2x10° dyn/cm or less, about 2.0x10° dyn/cm or less, method for a purity test of the above various modified starches about 1.8x10" dyn/cm or less, about 1.6x10'dyn/cm or less, described in Ministry of Health and Welfare Notification No. about 1.5x10° dyn/cm or less, about 1.4x10° dyn/cm or less, 485 dated Oct. 1, 2008, comparing with the results of analysis about 1.3x10° dyn/cm or less, about 1.2x10'dyn/cm or less, of a raw material native starch carried out for comparison or about 1.1x10° dyn/cm or less. 30 reference, and confirming there is no increase in the content In a specific embodiment, in the case where the untreated of corresponding various chemical Substances. Particularly, it starch is a wheat starch and neither a chemical modification is possible to judge that a sample starch is not a starch sub nor a physical treatment is carried out, the obtained enzyme jected to a chemical modification, by measuring the content treated starch has breakdown (about 100 BU or more), and a of adipic acid groups, the content of acetyl groups, the content rupture stress of about 150 to about 450 (g) or a Young's 35 of carboxyl groups, the content of vinyl acetate, the content of modulus of about 5,000,000 to about 8,000,000 (dyn/cm). octenyl Succinic acid groups, the content of hydroxypropyl In a specific embodiment, in the case where the untreated groups, and the content of propylene chlorohydrins, and con starch is a corn starch and neither a chemical modification nor firming that the contents of them do not increase as compared a physical treatment is carried out, the obtained enzyme with those of the raw material native starch. It is preferred to treated starch has breakdown (about 100 BU or more), and a 40 use the content of adipic acid groups, the content of acetyl rupture stress of about 210 to about 450 (g) (about 220 to groups, the content of carboxyl groups, the content of octenyl about 450 (g) in one embodiment) or a Young's modulus of Succinic acid groups, the content of hydroxypropyl groups, about 6,000,000 to about 9,000,000 (dyn/cm). and the content of propylene chlorohydrins as evaluation In a specific embodiment, in the case where the untreated criteria. It is recognized that a bleached starch Subjected to a starch is a cassava starch and neither a chemical modification 45 bleaching treatment using sodium hypochlorite is distributed nor a physical treatment is carried out, the obtained enzyme as a food. It is also possible to judge this bleached starch by treated starch has breakdown (about 100 BU or more), and a measuring the content of carboxyl groups using a method for rupture stress of about 55 to about 150 (g) or a Young's a purity test similar to that in the above oxidized starch. The modulus of about 520,000 to about 2,700,000 (dyn/cm) chemical modified starch other than the above modified (about 520,000 to about 1,600,000 (dyn/cm) in one embodi 50 starches of 11 items cannot be used in a food since it is not ment). recognized by the Food Sanitation Law of JAPAN. Therefore, Also, in the case where a chemically modified Starch or a the chemically modified starch other than the above 11 items physically treated Starch is used as starch granules, or in the are not basically used in JAPAN and are not distributed. case where a chemical modification or a physical treatment is Accordingly, practically, in the case of confirming whether or carried out after an enzymatic treatment, an improvement in 55 not hydroxyl groups at the 2-, 3- and 6-positions of a glucose gel forming ability can be obtained similarly to the above. residue of the starch of the invention of the present application (3.3 Enzyme-Treated Starch in which Hydroxyl Groups at are modified, it is not necessary to confirm whether or not a Positions 2, 3 and 6 of Glucose Residues are not Modified) chemical modification other than the above chemical modi In the case where the enzyme-treated Starch granules of the fication has been subjected. present invention are prepared from an untreated Starch, a 60 In the present description, in the case where “hydroxyl physically treated starch or a bleached starch and have not groups at the positions 2, 3 and 6 of glucose residues are not been subjected to chemical modification, since the enzyme modified, it is preferred that all hydroxyl groups at the posi treated Starch of the present invention is not subjected to an tions 2, 3 and 6 of glucose residues are not modified. How artificial chemical treatment, hydroxyl groups at the positions ever, in the case where hydroxyl groups are Subjected to some 2, 3 and 6 of glucose residues are not modified as compared 65 modification in a natural state, Some modifications may be with a native starch (i.e., untreated Starch). A starch, in which contained. In this case, based on the total number of hydroxyl hydroxyl groups at the positions 2, 3 and 6 of glucose residues groups at the positions 2, 3 and 6 of glucose residues, prefer US 9,005,681 B2 45 46 ably about 70% or more, more preferably about 80% or more, In the case where the enzyme-treated starch of the present still more preferably about 90% or more, particularly prefer invention is utilized in a low moisture content type food, it is ably about 95% or more, about 96% or more, about 97% or possible to impart smooth texture with nice melt in mouth. more, about 98% or more, about 99% or more, or about The low moisture content type food refers to a food in which 99.5%, and most preferably about 100% of hydroxyl groups 5 the amount of moisture per 100 g of the edible portion is 40 g are not modified. or less in a state at the time of eating. Examples of the low (4. Food of the Present Invention) moisture content type food include, for example, bakeries, In a specific embodiment, the food of the present invention Western-style confectioneries, fried foods, and jelly candies. is a food produced by a method including the steps of treating As described above, the high moisture content type food starch granules with an enzyme at a temperature of about 10 10 C. or higher and about 70° C. or lower to obtain an enzyme and the low moisture content type food are classified by the treated Starch, mixing a food material, the enzyme-treated amount of moisture, per 100g of the edible portion, which is starch and water to obtain a mixture; heating the mixture more than 40g, or 40g or less. Provided that the food in which thereby gelatinizing the enzyme-treated Starch in the mixture; the amount of moisture per 100 g of the edible portion is and cooling the mixture containing the gelatinized enzyme 15 around 40 g (35 to 50 g) may sometimes exhibit contradicting treated Starch thereby gelling the starch to obtain a starch physical properties depending on the form, even in the case of gel-containing food. the same amount of moisture. Also, in the case of the fried In another specific embodiment, the food of the present food, it is judged by the amount of moisture for the coating invention is a heat cooked starch-containing food containing part in which core food materials have been removed. an enzyme-treated Starch having a high Viscosity and a gel The amounts of water per 100 g of the edible portion of forming ability. In another specific embodiment, the starch various foods are exemplified below (extract from Standard containing food of the present invention is a food produced by Tables of Food Composition in Japan (Fifth Revised and a method including mixing a food material with the enzyme Enlarged Edition); the number in parenthesis denotes the treated Starch, and then heating the mixture. amount of moisture): In the present description, the starch gel-containing food 25 (1) Bakeries: white table bread (38.0 g), hard biscuit (2.6 g), refers to a food containing a starch gel. If the food contains the pie pastry (32.0 g), Eisei-boro (4.5 g); starch gel, it is not necessary for the food to be entirely in a gel (2) Traditional Japanese-style confectioneries: Uiro (54.5 g), form. For example, in the case of gelatinous foods such as Kudzu-manju (45.0 g), Daifuku-mochi (41.5 g); custard pudding; and gel-like traditional Japanese-style con (3) Western-style confectioneries: sponge cake (32.0 g), fectioneries Such as kudzu Starch cake and Uiro, entire foods 30 Kasutera (25.6 g), hot cake (40.0 g); form a gel. In the case of fat or oil-containing foods such as (4) Fat- or oil-containing foods: whipping cream (milk fat whipping cream and ice cream; and sauces such as meat type, 42.1 g), whipping cream (vegetable fattype, 41.2g), ice sauce, foods are not entirely in a gel form but contain a micro creams (ice milk: 65.6 g., lactic ice: 60.4 g); starch gel, and are therefore included in the starch gel-con (5) Gelatinous foods: custard pudding (74.1 g); taining food of the present invention. Also, bakeries and West 35 (6) Fish meat and animal meat processed foods: Sumaki ern-style confectioneries are included in the starch gel-con kamaboko (75.8 g), Yakinuki-kamaboko (72.8g), Vienna sau taining food of the present invention since they contain a sage (53.0 g); starch gel with the decreased water content which was (7) Salsa and sauces: Worcester sauce (61.7 g), meat sauce obtained by once forming a gel during the production process, (78.8 g), Thousand Island dressing (44.1 g); and and baking the gel. 40 (8) Jelly candies: jelly candy (16 g), jellybeans (9.5 g). In a specific embodiment, the food of the present invention By using the enzyme-treated Starch of the present invention can be prepared by using enzyme-treated Starch granules. The in these foods, the following physical properties, for example, starch produced by the method of the present invention can be are improved as compared with the case of using a conven utilized in the same application as in a conventional starch. By tional starch: utilizing the enzyme-treated Starch of the present invention in 45 (1) In bakeries, textures with softness and nice melt in mouth a food, physical properties and texture of the food are altered. is imparted. Examples of bakeries include breads, cookies, The enzyme-treated starch of the present invention can be biscuits, pizza crusts, pie pastries, corn cups for ice creams, used in almost all of compositions for eating and drinking or pastries of Monaka, and puff of cream puff. compositions for food additives prepared by utilizing a con (2) In traditional Japanese-style confectioneries, appropriate ventional starch. 50 hardness, brittleness, and appropriate viscoelasticity and In the food of the present invention, any material used Sticky textures are imparted. Examples of traditional Japa usually in the objective composition and food can be used as nese-style confectioneries include kudzu Starch cake, Uiro, long as an excellent effect obtained by the enzyme-treated and Manju. starch granules is not impaired. In a preferred embodiment, (3) In Western-style confectioneries, improvement of Vol the starch of the present invention forms a gel in the food of 55 umes by nice puffing after baking as well as Soft and nice the present invention. textures are imparted. Examples of Western-style confection In the case where the enzyme-treated starch of the present eries include sponge cake, chiffon cake, Kasutera, Madeleine, invention is utilized in a high moisture content type food, it financier, pound cake, and Swiss roll. imparts a body, imparts natural elasticity by a strong gel (4) In fat- or oil-containing foods, while maintaining appro forming ability, and also imparts appropriate Smooth texture 60 priate body and shape retention, nice melt in mouth and in mouth. The high moisture content type food refers to a food Smooth texture is imparted. Examples of the fat- or oil-con in which the amount of moisture per 100 g of the edible taining food include custard cream, flour paste, filling, whip portion is more than 40 g in a state at the time of eating. ping cream, and ice creams (for example, ice milk, lactic ice). Examples of the high moisture content type food include, for (5) In gelatinous foods, while maintaining sticky and chewy, example, traditional Japanese-style confectioneries, fat or 65 nice melt in mouth and Smooth texture is imparted. Examples oil-containing foods, gelatinous foods, fish meat and animal of the gelatinous food include jelly, pudding, mousse, yogurt, meat processed foods, Salsa and sauces, and noodles. and goma-dofu. US 9,005,681 B2 47 48 (6) In fish meat and animal meat processed foods, while cally modified Starch, the step of physically treating the having elasticity with nice chewiness, the effect of small enzyme-treated Starch is further included, and the physically change with time is imparted. Examples of fish meat and meat treated enzyme-treated starch is mixed with the food material processed foods include kamaboko and sausage. and water. (7) In Salsa and sauces, while having nice body and shape Next, a mixture is obtained by mixing a food material, the retention, properties of being less likely to cause dropping enzyme-treated Starch and water. A mixing method and a because of nice adhesion onto a food as well as less stickiness mixing ratio of the food material, the enzyme-treated Starch and thread-forming sensation, and Smooth textures are and water can be a mixing method and a mixing ratio in imparted. Examples of Salsa and sauces include Salsa for split accordance with a usual method for producing the objective and broiled fish, glaze formitarashi dango, fruit sauce, white 10 sauce, and dressing. food. (8) In fried foods, crispy light texture is imparted. Examples Next, the mixture is heated thereby gelatinizing the of fried foods include tempura and fried prawn. enzyme-treated Starch in the mixture. The heating can be heat (9) In noodles, Sticky texture rich in chewiness is imparted. cooking. Heating can be carried out under the same condi Examples of noodles include udon, Somen, hiyamugi, Chi 15 tions as those of heat cooking in a usual method for producing nese noodles, buckwheat noodles, macaroni, and spaghetti. the objective food. (10) Injelly candies, while having appropriate elasticity, nice Next, the mixture containing the gelatinized enzyme melt in mouth and Smooth texture is imparted. Examples of treated Starch is cooled, thereby gelling the starch to obtain a jelly candies include jelly candy and jellybeans. starch gel-containing food. Cooling may be carried out by In the food of the present invention, the enzyme-treated leaving the mixture after heating at room temperature, or starch of the present invention can be used in the same amount carried out in a refrigerator or the like. as that of the starch which has been conventionally used in the In the embodiment in which the enzyme-treated starch of food. A conventional starch may be used as a part and the the present invention is used, the food of the present invention remainder may be replaced by the enzyme-treated starch of can be produced in the same method as in the case of a usual the present invention. The enzyme-treated starch of the 25 starch, except that the enzyme-treated Starch is used. The present invention preferably accounts for about 50% by method for producing the starch-containing food of the weight or more, more preferably about 60% by weight or present invention includes the steps of adding an enzyme more, still more preferably about 70% by weight or more, treated Starch to a food material and mixing them; and heat further preferably about 80% by weight or more, particularly cooking the mixture. preferably about 90% by weight or more, and most preferably 30 The enzyme-treated starch of the present invention has 100% by weight, of a usual use amount of the starch. In other excellent viscosity and gel forming ability as compared with word, most preferably, the entire amount of a conventional a conventional untreated starch. Therefore, by adding the starch is replaced by the enzyme-treated starch of the present enzyme-treated starch of the present invention to the food invention. material, mixing them and heat cooking the mixture, this (5. Method for Producing Starch Gel-Containing Food) 35 enzyme-treated Starch is gelatinized and then cooled to form In a specific embodiment, the method for producing a a gel. Accordingly, the obtained heat cooked material is pro starch gel-containing food of the present invention includes vided with excellent physical properties (for example, excel the steps of treating starch granules with an enzyme at a lent body, natural elasticity, nice melt in mouth, Smooth tex temperature of about 10° C. or higher and about 70° C. or ture, Sticky texture, and soft texture) as compared with the lower to obtain an enzyme-treated Starch; mixing a food 40 heat cooked material in which a conventional untreated Starch material, the enzyme-treated Starch and water to obtain a is used. In the present description, the food may also be a mixture; heating the mixture thereby gelatinizing the beverage. enzyme-treated Starch in the mixture; and cooling the mixture In the present description, "heat cooking refers to heating containing the gelatinized enzyme-treated Starch thereby gel of a mixture of a food material and a starch. Preferably, heat ling the starch to obtain a starch gel-containing food. In the 45 cooking can be heating at a collapse temperature or higher of production of a conventional food, starch granules are not starch granules. For example, the mixture of a food material Subjected to an enzymatic treatment during the food produc and a starch can be heated at about 70° C. or higher, about 80° tion process. C. or higher, about 90° C. or higher or about 95°C. or higher. The step of treating starch granules with an enzyme at a Preferably, heat cooking is carried out at a temperature at temperature of about 10° C. or higher and about 70° C. or 50 which excess denaturation of the food material and the starch lower to obtain an enzyme-treated Starch can be carried out as does not arise. For example, the mixture of a food material described in detail in the aforementioned “2.2 Enzyme Reac and a starch can be heated at about 200° C. or lower, about tion'. As described above, the starch granules can be starch 150° C. or lower, about 130° C. or lower or about 110° C. or granules of an untreated Starch, a physically treated Starch or lower. Heat cooking is carried out for a usual heat cooking a chemically modified starch. In the case where it is prefer 55 time of the objective food. able to obtain an enzyme-treated Starch which is dealt as a Heat cooking is preferably carried out in the presence of food, starch granules are starch granules of an untreated Some degree of moisture. Usually, when starch granules are starch, a physically treated Starch orableached starch, and the heated in the presence of a predetermined amount or more of starch granules are not subjected to a chemical modification water, Swelling arises, transparency increases and Viscosity in any stage until a starch gel-containing food is obtained 60 increases. When the food material contains too much mois using the starch granules. In a specific embodiment, the starch ture, it is not necessary to add water to the mixture of a food granule is a starch granule of an untreated Starch or a physi material and a starch. However, when the food material con cally treated Starch, the step of chemically modifying the tains Small amount of moisture, it is preferred to add water to enzyme-treated starch is further included, and the chemically the mixture of a food material and a starch. It is noted that in modified enzyme-treated starch is mixed with the food mate 65 the case of a food which does not contain food materials other rial and water. In another specific embodiment, the starch than water and a starch, like a Sugar-free kuZuyu, water is granules are starch granules of an untreated Starch or a chemi considered as the food material. US 9,005,681 B2 49 50 Heat cooking can be a part of the method for producing the basis, and then filled in a Krehalon casing having a folding objective food. For example, in the case of a gelatinous food width of 45 mm. This starch paste filled in the casing was Such as jelly, it can be heat cooled after cooking at a tempera heated to 90° C. at 1° C./min and maintained at 90° C. for 30 ture of, for example, about 5 to 10°C. minutes. Then, the starch paste was left to cool in a constant (6. Explanation of Sequence) temperature water bath at 20° C. for 30 minutes and then SEQID NO: 1 is a nucleotide sequence encoding C.-amy cooled to 5°C. in a refrigerator. After cooling, it was refrig lase derived from Aspergillus Oryzae, eration storaged at 5°C. for 16 hours, then it was left at room SEQ ID NO: 2 is an amino acid sequence of O-amylase temperature (about 25°C.) for 4 hours to return the tempera derived from Aspergillus Oryzae, ture of it to room temperature, and then physical properties of SEQID NO:3 is a nucleotide sequence encoding C.-amy 10 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 SEQ ID NO. 4 is an amino acid sequence of O-amylase under the measurement conditions of the rheometer: a test derived from Aspergillus niger, item: a rupture test; a height of a sample: 25 mm; and a SEQID NO: 5 is a nucleotide sequence encoding amylo movement rate (rupture rate) of a sample: 6 cm/min, using an glucosidase derived from Aspergillus niger, 15 adapter of a spherical jig for measurement viscosity (p5 (diam SEQID NO: 6 is an amino acid sequence of amyloglucosi eter: 5 mm, area: 19.635 mm). At the measurement, the dase derived from Aspergillus niger, hardness of the starch gel was evaluated by a rupture stress (g) SEQID NO: 7 is a nucleotide sequence encoding isoamy and a Young's modulus (dyn/cm). lase derived from Flavobacterium sp.: (3. Method for Measurement of Degradation Ratio of SEQ ID NO: 8 is an amino acid sequence of isoamylase Starch Granules) derived from Flavobacterium sp.: A degradation ratio of starch granules was measured by the SEQID NO: 9 is a nucleotide sequence encoding isoamy following method. The amount (g) of released reducing Sug lase derived from Pseudomonas amyloderamosa, ars contained in the Supernatant obtained by centrifugation (at SEQID NO: 10 is an amino acid sequence of isoamylase 3,000 rpm for 5 minutes) of a starch degraded suspension derived from Pseudomonas amyloderamosa, 25 after Subjecting to an enzyme reaction was measured by a SEQID NO: 11 is a nucleotide sequence encoding O-glu phenol-sulfuric acid method. The percentage of the amount of cosidase derived from Aspergillus niger, the released reducing Sugars to the total amount of the starch SEQID NO: 12 is an amino acid sequence of C-glucosi (g) before subjecting to an enzyme reaction was determined. dase derived from Aspergillus niger, SEQID NO: 13 is a nucleotide sequence encoding cyclo 30 Degradation ratio (%) of starch granules={(amount dextrin glucanotransferase derived from Paenibacillus mac (g) of released reducing Sugars)x100}/{(total erans (also classified as Bacillus macerans). amount (g) of starch before enzymatic reaction)}. Equation 1 SEQID NO: 14 is an amino acid sequence of cyclodextrin glucanotransferase derived from Paenibacillus macerans Test Example 1 (also classified as Bacillus macerans). 35 Comparison Between Liquid Reaction and Solid EXAMPLES Reaction Next, the present invention will be described in more detail 1. Liquid Reaction by way of Examples, but the present invention is not limited 40 To 15 g (dry weight) of an untreated native wheat starch, to these Examples. It is noted that in the Examples, a viscosity 250 g of ion-exchange water was added and, after adjusting was measured by an amylograph from Brabender Inc., and the pH of the mixture to 5.0, the mixture was warmed in a physical properties of a gel were measured by a rheometer boiled water bath to prepare a starch paste in which a starch from Rheotech Inc. was completely dissolved. To this starch paste, 0.1% by (1. Method for Measurement of Viscosity) 45 weight (based on starch Solid content) of C-amylase (origin: A viscosity was measured by the following method. A Aspergillus oryzae) was added to make the total weight to 300 starch suspension was adjusted in 450 ml of water so that the g, and stirred at 50° C. to carry out an enzyme reaction. After concentration of a wheat starch was 8.5% by weight, the 30 minutes, this was left in a boiled water bath for 10 minutes concentration of a corn starch was 7.0% by weight and the to deactivate the enzyme and thereby obtained a sample 1. concentration of a cassava starch was 6.0% by weight, on the 50 Using the obtained sample 1, physical properties of the gel dry matter basis and, put in a sample container, and then were measured and evaluated by a rupture stress and a warmed to 50° C. while rotating them. Then the suspension Young's modulus. was heated to 95°C. at 1.5°C/min, and maintained at 95°C. 2. Solid Reaction for 15 minute, followed by cooling at 1.5°C/min. The mea To 400 g of an untreated native wheat starch (dry weight), Surement was carried out using an amylograph VISCO 55 900 g of ion-exchange water was added to prepare a starch GRAPH-Emanufactured by Brabender Inc. under the condi Suspension. After adjusting the pH of the Suspension to 5.0, tions of a rotation number of the sample container of 75 rpm 1% by weight (based on starch solid content) of C.-amylase and a measuring cartridge of 700 cmg. Wherein, the Viscosity (origin: Aspergillus Oryzae) was added and stirred at 50° C. reached to a peak was regarded as a maximum viscosity, and for 18 hours to carry out an enzyme reaction. After comple a difference between this maximum viscosity and a viscosity 60 tion of the reaction, an enzyme-treated Starch was recovered soon after maintaining at 95°C. for 15 minutes was regarded by centrifugal filtration and blow drying. To 15 g (dry weight) as breakdown. of this enzyme-treated Starch, ion-exchange water was added (2. Method for Measurement of Physical Properties of a to make the total weight to 300g. This was warmed in a boiled Gel) water bath to prepare a starch paste in which the starch was Physical properties of a gel were measured by the follow 65 completely dissolved, as a sample 2. Using the obtained ing method. A starch paste was prepared so that the concen sample 2, physical properties of the gel were measured and tration of the starch was 20% by weight on the dry matter evaluated by a rupture stress and a Young's modulus. US 9,005,681 B2 52 TABLE 1. After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch solid content) of C-amylase (AMYLEX Sample 1 Sample 2 Physical (those reacted (those reacted Untreated A3' derived from Aspergillus niger, manufactured by properties in a form of in a form of wheat DANISCO: optimum pH of 5.0) was added and stirred at 50° of Gel liquid) solid) starch C. for 18 hours to carry out an enzyme reaction. After comple Rupture Not measurable 206 g 141 g tion of the reaction, an enzyme-treated Starch was recovered StreSS since gel is by centrifugal filtration and blow drying. Viscosity character Young's not formed 5,533,540 4,601,665 istics of the obtained enzyme-treated starch were analyzed by modulus because of dyn/cm’ dyn/cm. the amylograph and the rheometer. After completion of the being too soft 10 reaction, a degradation ratio was determined using a part of the reaction solution. The results are shown in Table 2-2. When an enzyme was allowed to act on the starch after gelling, a remarkable decrease in Viscosity was confirmed in Example 2B the obtained sample 1, and the sample did not retain viscosity physical properties of the starch anymore and thus a gel was 15 To 400 g of an untreated native wheat starch, 900 g of not formed. On the other hand, when the enzyme is reacted ion-exchange water was added to prepare a starch Suspension. keeping the starch granules as it is, it was confirmed that the After adjusting the pH of the suspension to 5.0, 1% by weight obtained sample 2 retained Viscosity physical properties of (based on starch Solid content) of C.-amylase ("Sumizyme the starch and a hard gel was formed. AS” derived from Aspergillus niger, manufactured by SHIN NIHONCHEMICALS Corporation; optimum pH of 4.5) was Comparative Example 1 added and stirred at 50° C. for 18 hours to carry out an enzyme Viscosity characteristics were analyzed by the amylograph reaction. After completion of the reaction, an enzyme-treated and the rheometer without Subjecting an untreated native starch was recovered by centrifugal filtration and blow dry wheat starch to an enzymatic treatment. The results are shown ing. Viscosity characteristics of the obtained enzyme-treated in Table 2-2. 25 starch were analyzed by the amylograph and the rheometer. After completion of the reaction, a degradation ratio was Examples 1-1 and 1-2 determined using a part of the reaction Solution. The results are shown in Table 2-2. To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspension. 30 Comparative Examples 2, 3, 4-3, and 5 to 6 After adjusting the pH of the suspension to 5.0, 0.1% by weight (based on starch solid content) of O-amylase To 400 g of an untreated native wheat starch, 900 g of (“Biozyme A' derived from Aspergillus Oryzae, manufac ion-exchange water was added to prepare a starch Suspension. tured by Amano Enzyme Inc.; optimum pH of 5.0) was added After adjusting the pH of the suspension to 5.0, 1% by weight and stirred at 50° C. for 1 hour to carry out an enzyme reaction 35 (based on starch Solid content) of C.-amylase ("C.-amylase and resulted in preparation of a sample having a degradation 3A' derived from Bacillus subtilis, manufactured by HBI. ratio of about 5% (Example 1-1). Using a similar amount of Inc., optimum pH of 5.9; Comparative Example 2), C.-amy the enzyme, stirring was carried out at 50° C. for 3 hours to lase (“Novamyl derived from Bacillus subtilis, manufac prepare a sample having a degradation ratio of about 10% tured by Novo: optimum pH of 5.0; Comparative Example 3), (Example 1-2). After completion of the reaction, an enzyme 40 C.-amylase (“O-amylase' derived from Bacillus amylolique treated starch was recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained enzyme faciens, manufactured by Sigma-Aldrich Corporation, opti treated Starch were analyzed by the amylograph and the rhe mum pH of 6.0; Comparative Example 4-3), C.-amylase ometer. After completion of the reaction, a degradation ratio (“TERMAMYL 120L derived from Bacillus licheniformis, was determined using a part of the reaction solution. The manufactured by Novo: optimum pH of 6.0; Comparative results are shown in Table 2-2. 45 Example 5), or O-amylase (“Maltogenase L' derived from Bacillus sp., manufactured by Novo: optimum pH of 5.0; Example 1-3 Comparative Example 6) was added and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of To 400 g of an untreated native wheat starch, 900 g of the reaction, an enzyme-treated Starch was recovered by cen ion-exchange water was added to prepare a starch Suspension. 50 trifugal filtration and blow drying. Viscosity characteristics of After adjusting the pH of the suspension to 5.0, 1% by weight the obtained enzyme-treated starch were analyzed by the (based on starch solid content) of C.-amylase (“Biozyme A” amylograph and the rheometer. After completion of the reac derived from Aspergillus Oryzae, manufactured by Amano tion, a degradation ratio was determined by a part of the Enzyme Inc.; optimum pH of 5.0) was added and stirred at reaction solution. The results are shown in Table 2-2. 50° C. for 18 hours to carryout an enzyme reaction. After 55 completion of the reaction, an enzyme-treated Starch was Comparative Examples 4-1 and 4-2 recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained enzyme-treated starch were To 400 g of an untreated native wheat starch, 900 g of analyzed by the amylograph and the rheometer. Also, after ion-exchange water was added to prepare a starch Suspension. completion of the reaction, a degradation ratio was deter 60 After adjusting the pH of the suspension to 5.0, 0.01% by mined using a part of the reaction solution. The results are weight (based on starch Solid content) of C-amylase ("C.- shown in Table 2-2. amylase' derived from Bacillus amyloliquefaciens, manufac tured by Sigma-Aldrich Corporation; optimum pH of 6.0) Example 2A was added and stirred at 50° C. for 30 minutes to prepare a 65 sample having a degradation ratio of about 5%. Also, using a To 400 g of an untreated native wheat starch, 900 g of similar amount of the enzyme, stirring was carried out at 50° ion-exchange water was added to prepare a starch Suspension. C. for 1.5 hours to prepare a sample having a degradation ratio US 9,005,681 B2 53 54 of about 10%. After completion of the reaction, an enzyme recovered by centrifugal filtration and blow drying. Viscosity treated starch was recovered by centrifugal filtration and blow characteristics of the obtained enzyme-treated starch were drying. Viscosity characteristics of the obtained enzyme analyzed by the amylograph and the rheometer. After comple treated Starch were analyzed by the amylograph and the rhe tion of the reaction, a degradation ratio was determined using ometer. The results are shown in Table 2-2. 5 a part of the reaction solution. The results are shown in Table 2-2. Examples 3A-1 and 3A-2 Example 3D To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspension. 10 To 400 g of an untreated native wheat starch, 900 g of After adjusting the pH of the suspension to 5.0, 0.1% by ion-exchange water was added to prepare a starch Suspension. weight (based on starch Solid content) of amyloglucosidase After adjusting the pH of the suspension to 5.0, 1% by weight (AMG' derived from Aspergillus niger, manufactured by (based on starch Solid content) of amyloglucosidase (glu Novozymes; optimum pH of 4.5) was added and stirred at 50° coamylase Amano SD derived from Aspergillus niger, C. for 2 hours to prepare a sample having a degradation ratio 15 manufactured by Amano Enzyme Inc.; optimum pH of 4.5) of about 5% (Example 3A-1). Also, 0.5% by weight (based on was added and stirred at 50° C. for 18 hours to carry out an starch Solid content) of the similar enzyme was added and enzyme reaction. After completion of the reaction, an stirred at 50° C. for 3 hours to prepare a sample having a enzyme-treated starch was recovered by centrifugal filtration degradation ratio of about 10% (Example 3A-2). After and blow drying. Viscosity characteristics of the obtained completion of the reaction, an enzyme-treated Starch was enzyme-treated Starch were analyzed by the amylograph and recovered by centrifugal filtration and blow drying. Viscosity the rheometer. After completion of the reaction, a degradation characteristics of the obtained enzyme-treated starch were ratio was determined using a part of the reaction solution. The analyzed by the amylograph and the rheometer. results are shown in Table 2-2. Example 3A-3 25 Example 3E To 400 g of an untreated native wheat starch, 900 g of To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspension. ion-exchange water was added to prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 1% by weight After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch solid content) of amyloglucosidase (AMG' 30 (based on starch Solid content) of amyloglucosidase (“Gluc derived from Aspergillus niger, manufactured by Zyme AF6” derived from Rhizopus niveus, manufactured by Novozymes; optimum pH of 4.5) was added and stirred at 50° Amano Enzyme Inc.; optimum pH of 4.5) was added and C. for 18 hours to carry out an enzyme reaction. After comple stirred at 50° C. for 18 hours to carry out an enzyme reaction. tion of the reaction, an enzyme-treated Starch was recovered After completion of the reaction, an enzyme-treated Starch by centrifugal filtration and blow drying. Viscosity character- 35 was recovered by centrifugal filtration and blow drying. Vis istics of the obtained enzyme-treated starch were analyzed by cosity characteristics of the obtained enzyme-treated Starch the amylograph and the rheometer. After completion of the were analyzed by the amylograph and the rheometer. After reaction, a degradation ratio was determined using a part of completion of the reaction, a degradation ratio was deter the reaction solution. The results are shown in Table 2-2. mined using a part of the reaction solution. The results are 40 shown in Table 2-2. Example 3B Example 3F To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspension. To 400 g of an untreated native wheat starch, 900 g of After adjusting the pH of the suspension to 5.0, 1% by weight 45 ion-exchange water was added to prepare a starch Suspension. (based on starch solid content) of amyloglucosidase (“OPTI After adjusting the pH of the suspension to 5.0, 1% by weight DEXL-400' derived from Aspergillus niger, manufactured (based on starch Solid content) of amyloglucosidase ("Sum by Genencor; optimum pH of 4.4) was added and stirred at izyme” derived from Rhizopus oryzae, manufactured by 50° C. for 18 hours to carry out an enzyme reaction. After SHIN NIHON CHEMICALS Corporation; optimum pH of completion of the reaction, an enzyme-treated Starch was 50 5.0) was added and stirred at 50° C. for 18 hours to carry out recovered by centrifugal filtration and blow drying. Viscosity an enzyme reaction. After completion of the reaction, an characteristics of the obtained enzyme-treated starch were enzyme-treated starch was recovered by centrifugal filtration analyzed by the amylograph and the rheometer. After comple and blow drying. Viscosity characteristics of the obtained tion of the reaction, a degradation ratio was determined using enzyme-treated Starch were analyzed by the amylograph and a part of the reaction solution. The results are shown in Table 55 the rheometer. After completion of the reaction, a degradation 2-2. ratio was determined using a part of the reaction solution. The results are shown in Table 2-2. Example 3C Comparative Example 8 To 400 g of an untreated native wheat starch, 900 g of 60 ion-exchange water was added to prepare a starch Suspension. To 400 g of an untreated native wheat starch, 900 g of After adjusting the pH of the suspension to 5.0, 1% by weight ion-exchange water was added to prepare a starch Suspension. (based on starch solid content) of amyloglucosidase (“DIAZ After adjusting the pH of the suspension to 5.0, 1% by weight YMEX4NP' derived from Aspergillus niger, manufactured (based on starch Solid content) of amyloglucosidase ("Re by DANISCO; optimum pH of 4.0) was added and stirred at 65 agent” derived from Candida tsukubaensis, manufactured by 50° C. for 18 hours to carry out an enzyme reaction. After Sigma-Aldrich Corporation; optimum pH of 2.5) was added completion of the reaction, an enzyme-treated Starch was and stirred at 50° C. for 18 hours to carry out an enzyme US 9,005,681 B2 55 56 reaction. After completion of the reaction, an enzyme-treated Klebsiella pneumoniae, manufactured by Amano Enzyme starch was recovered by centrifugal filtration and blow dry Inc.; optimum pH of 6.0) was added and stirred at 50° C. for ing. Viscosity characteristics of the obtained enzyme-treated 18 hours to carry out an enzyme reaction. After completion of starch were analyzed by the amylograph and the rheometer. the reaction, an enzyme-treated Starch was recovered by cen After completion of the reaction, a degradation ratio was trifugal filtration and blow drying. Viscosity characteristics of determined using a part of the reaction Solution. The results the obtained enzyme-treated starch were analyzed by the are shown in Table 2-2. amylograph and the rheometer. After completion of the reac tion, a degradation ratio was determined using a part of the Example 4 reaction solution. The results are shown in Table 2-2. 10 To 400 g of an untreated native wheat starch, 900 g of Comparative Example 12 ion-exchange water was added to prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 0.1% by Viscosity characteristics were analyzed by the amylograph weight (based on starch solid content) of isoamylase (“Re and the rheometer without Subjecting a corn starch to an agent” derived from Pseudomonas amyloderamosa, manu 15 enzymatic treatment. The results are shown in Table 3-2. factured by Sigma-Aldrich Corporation; optimum pH of 3.0) was added and stirred at 50° C. for 18 hours to carry out an Example 6 enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration To 400 g of an untreated native corn starch, 900 g of and blow drying. Viscosity characteristics of the obtained ion-exchange water was added to prepare a starch Suspension. enzyme-treated Starch were analyzed by the amylograph and After adjusting the pH of the suspension to 5.0, 1% by weight the rheometer. After completion of the reaction, a degradation (based on starch solid content) of C.-amylase (“Biozyme A” ratio was determined using a part of the reaction Solution. The derived from Aspergillus Oryzae, manufactured by Amano results are shown in Table 2-2. Enzyme Inc.; optimum pH of 5.0) was added and stirred at 25 50° C. for 18 hours to carryout an enzyme reaction. After Example 5A completion of the reaction, an enzyme-treated Starch was recovered by centrifugal filtration and blow drying. Viscosity To 400 g of an untreated native wheat starch, 900 g of characteristics of the obtained enzyme-treated starch were ion-exchange water was added to prepare a starch Suspension. analyzed by the amylograph and the rheometer. Also, after After adjusting the pH of the suspension to 5.0, 1% by weight 30 completion of the reaction, a degradation ratio was deter (based on starch Solid content) of C-glucosidase ("Transglu mined using a part of the reaction solution. The results are cosidase L Amano” derived from Aspergillus niger, manu shown in Table 3-2. factured by Amano Enzyme Inc.; optimum pH of 5.0) was added and stirred at 50° C. for 18 hours to carry out an enzyme Comparative Examples 13-1 and 13-2 reaction. After completion of the reaction, an enzyme-treated 35 starch was recovered by centrifugal filtration and blow dry To 400 g of an untreated native corn starch, 900 g of ing. Viscosity characteristics of the obtained enzyme-treated ion-exchange water was added to prepare a starch Suspension. starch were analyzed by the amylograph and the rheometer. After adjusting the pH of the suspension to 5.0, 0.01% by After completion of the reaction, a degradation ratio was weight (based on starch Solid content) (Comparative Example determined using a part of the reaction Solution. The results 40 13-1) of O-amylase (“Reagent” derived from Bacillus amy are shown in Table 2-2. loliquefaciens, manufactured by Sigma-Aldrich Corporation; optimum pH of 6.0) was added and stirred at 50° C. for 30 Example 5B minutes, or 1% by weight (based on starch solid content) (Comparative Example 13-2) of the C-amylase was added To 400 g of an untreated native wheat starch, 900 g of 45 and stirred at 50° C. for 18 hours, to carryout an enzyme ion-exchange water was added to prepare a starch Suspension. reaction. After completion of the reaction, an enzyme-treated After adjusting the pH of the suspension to 5.0, 1% by weight starch was recovered by centrifugal filtration and blow dry (based on starch Solid content) of C-glucosidase ("Transglu ing. Viscosity characteristics of the obtained enzyme-treated cosidase L-500' derived from Aspergillus niger, manufac starch were analyzed by the amylograph and the rheometer. tured by Genencor; optimum pH of 5.0) was added and stirred 50 Also, after completion of the reaction, a degradation ratio was at 50° C. for 18 hours to carry out an enzyme reaction. After determined by a part of the reaction solution. The results are completion of the reaction, an enzyme-treated Starch was shown in Table 3-2. recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained enzyme-treated starch were Examples 7-1 and 7-2 analyzed by the amylograph and the rheometer. After comple 55 tion of the reaction, a degradation ratio was determined using To 400 g of an untreated native corn starch, 900 g of a part of the reaction solution. The results are shown in Table ion-exchange water was added to prepare a starch Suspension. 2-2. After adjusting the pH of the suspension to 5.0, 0.5% by weight (based on starch solid content) (Example 7-1) of amy Comparative Examples 10 and 11 60 loglucosidase (AMG' derived from Aspergillus niger, manufactured by Novozymes; optimum pH of 4.5) was added To 400 g of an untreated native wheat starch, 900 g of and stirred at 50° C. for 3 hours, or 1% by weight (based on ion-exchange water was added to prepare a starch Suspension. starch Solid content) (Example 7-2) of the amyloglucosidase After adjusting the pH of the suspension to 5.0, 1% by weight was added and stirred at 50° C. for 18 hours, to carry out an (based on starch solid content) of B-amylase (“OPTIMALT 65 enzyme reaction. After completion of the reaction, an BBA derived from barley, manufactured by Genencor; opti enzyme-treated starch was recovered by centrifugal filtration mum pH of 5.0) or pullulanase (“Pullulanase” derived from and blow drying. Viscosity characteristics of the obtained US 9,005,681 B2 57 58 enzyme-treated Starch were analyzed by the amylograph and After completion of the reaction, a degradation ratio was the rheometer. Also, after completion of the reaction, a deg determined using a part of the reaction Solution. The results radation ratio was determined by a part of the reaction solu are shown in Table 3-2. tion. The results are shown in Table 3-2. Comparative Example 15 Comparative Example 14 Viscosity characteristics were analyzed by the amylograph To 400 g of an untreated native corn starch, 900 g of and the rheometer without Subjecting an untreated native ion-exchange water was added to prepare a starch Suspension. cassava starch to an enzymatic treatment. The results are After adjusting the pH of the suspension to 5.0, 1% by weight 10 shown in Table 4-2. (based on starch solid content) of B-amylase (“OPTIMALT BBA derived from barley, manufactured by Genencor; opti Example 9 mum pH of 5.0) was added and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of the To 400 g of an untreated native cassava starch, 900 g of reaction, an enzyme-treated Starch was recovered by centrifu 15 ion-exchange water was added to prepare a starch Suspension. gal filtration and blow drying. Viscosity characteristics of the After adjusting the pH of the suspension to 5.0, 1% by weight obtained enzyme-treated Starch were analyzed by the amylo (based on starch solid content) of C.-amylase (“Biozyme A” graph and the rheometer. After completion of the reaction, a derived from Aspergillus Oryzae, manufactured by Amano degradation ratio was determined using a part of the reaction Enzyme Inc.; optimum pH of 5.0) was added and stirred at solution. The results are shown in Table 3-2. 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an enzyme-treated Starch was Example 8A recovered by centrifugal filtration and blow drying. Viscosity To 400 g of an untreated native corn starch, 900 g of characteristics of the obtained enzyme-treated starch were ion-exchange water was added to prepare a starch Suspension. 25 analyzed by the amylograph and the rheometer. After comple After adjusting the pH of the suspension to 5.0, 0.1% by tion of the reaction, a degradation ratio was determined using weight (based on starch solid content) of isoamylase (“Re a part of the reaction solution. The results are shown in Table agent” derived from Pseudomonas amyloderamosa, manu 4-2. factured by Sigma-Aldrich Corporation; optimum pH of 3.0) was added and stirred at 50° C. for 18 hours to carry out an 30 Comparative Examples 16-1 and 16-2 enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration To 400 g of an untreated native cassava starch, 900 g of and blow drying. Viscosity characteristics of the obtained ion-exchange water was added to prepare a starch Suspension. enzyme-treated Starch were analyzed by the amylograph and After adjusting the pH of the suspension to 5.0, 0.01% by the rheometer. After completion of the reaction, a degradation 35 weight (based on starch Solid content) (Comparative Example ratio was determined using a part of the reaction Solution. The 16-1) of O-amylase (“Reagent” derived from Bacillus amy results are shown in Table 3-2. loliquefaciens, manufactured by Sigma-Aldrich Corporation; optimum pH of 6.0) was added and stirred at 50° C. for 30 Example 8B minutes, or 1.0% by weight (based on starch solid content) 40 (Comparative Example 16-2) of the C-amylase was added To 400 g of an untreated native corn starch, 900 g of and stirred at 50° C. for 18 hours, to carryout an enzyme ion-exchange water was added to prepare a starch Suspension. reaction. After completion of the reaction, an enzyme-treated After adjusting the pH of the suspension to 5.0, 1% by weight starch was recovered by centrifugal filtration and blow dry (based on starch solid content) of C-amylase (AMYLEX ing. Viscosity characteristics of the obtained enzyme-treated A3' derived from Aspergillus niger, manufactured by 45 starch were analyzed by the amylograph and the rheometer. DANISCO: optimum pH of 5.0) was added and stirred at 50° Also, after completion of the reaction, a degradation ratio was C. for 18 hours to carry out an enzyme reaction. After comple determined by a part of the reaction solution. The results are tion of the reaction, an enzyme-treated Starch was recovered shown in Table 4-2. by centrifugal filtration and blow drying. Viscosity character istics of the obtained enzyme-treated starch were analyzed by 50 Examples 10-1 and 10-2 the amylograph and the rheometer. After completion of the reaction, a degradation ratio was determined using a part of To 400 g of an untreated native cassava starch, 900 g of the reaction solution. The results are shown in Table 3-2. ion-exchange water was added to prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 0.5% by Example 8C 55 weight (based on starch solid content) (Example 10-1) of amyloglucosidase (AMG' derived from Aspergillus niger, To 400 g of an untreated native corn starch, 900 g of manufactured by Novozymes; optimum pH of 4.5) was added ion-exchange water was added to prepare a starch Suspension. and stirred at 50° C. for 3 hours, or 1% by weight (based on After adjusting the pH of the suspension to 5.0, 1% by weight starch Solid content) (Example 10-2) of the amyloglucosidase (based on starch Solid content) of C-glucosidase ("Transglu 60 was added and stirred at 50° C. for 18 hours, to carry out an cosidase L Amano’ derived from Aspergillus niger, manu enzyme reaction. After completion of the reaction, an factured by Amano Enzyme Inc.; optimum pH of 5.0) was enzyme-treated starch was recovered by centrifugal filtration added and stirred at 50° C. for 18 hours to carry out an enzyme and blow drying. Viscosity characteristics of the obtained reaction. After completion of the reaction, an enzyme-treated enzyme-treated Starch were analyzed by the amylograph and starch was recovered by centrifugal filtration and blow dry 65 the rheometer. Also, after completion of the reaction, a deg ing. Viscosity characteristics of the obtained enzyme-treated radation ratio was determined by a part of the reaction solu starch were analyzed by the amylograph and the rheometer. tion. The results are shown in Table 4-2. US 9,005,681 B2 59 60 Comparative Example 17 characteristics can be prepared by Subjecting to an enzymatic treatment. Further, using C-amylase derived from Bacillus To 400 g of an untreated native cassava starch, 900 g of amyloliquefaciens used in Comparative Examples 4-1 and ion-exchange water was added to prepare a starch Suspension. 4-2, 13-1, 13-2, 16-1, 16-2 at a degradation ratio of 40% or After adjusting the pH of the suspension to 5.0, 1% by weight less, it was impossible to prepare a starch having both a high (based on starch solid content) of B-amylase (“OPTIMALT Viscosity and strong gel characteristics, which is the object of BBA derived from barley, manufactured by Genencor; opti the present inventors. Therefore, it has been proved that the mum pH of 5.0) was added and stirred at 50° C. for 18 hours starch developed by the present inventors is a Substance to carry out an enzyme reaction. After completion of the which is different from the starch prepared by Japanese Patent reaction, an enzyme-treated Starch was recovered by centrifu 10 Gazette No. 2,615,398. gal filtration and blow drying. Viscosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylo Example 12A graph and the rheometer. After completion of the reaction, a degradation ratio was determined using a part of the reaction Method for Preparation of C.-Amylase derived from solution. The results are shown in Table 4-2. 15 Aspergillus Oryzae Example 1 1A A double stranded DNA was chemically synthesized by To 400 g of an untreated native cassava starch, 900 g of adding an EcoRI recognition site (GAATTC) to both termi ion-exchange water was added to prepare a starch Suspension. nals of a base sequence of SEQ ID NO: 1 in the sequence After adjusting the pH of the suspension to 5.0, 0.1% by listing. This synthetic DNA was completely cleaved by a weight (based on starch solid content) of isoamylase (“Re restriction enzyme EcoRI, mixed with pYCDE1 (Method in agent” derived from Pseudomonas amyloderamosa, manu Enzymology, 101, pp. 192-201 (1983)) which was previously factured by Sigma-Aldrich Corporation; optimum pH of 3.0) completely cleaved by EcoRI, and then ligation was carried was added and stirred at 50° C. for 18 hours to carry out an 25 out. E. coli TG1 was transformed with the ligation reaction enzyme reaction. After completion of the reaction, an Solution and a transformant into which a synthetic gene was enzyme-treated starch was recovered by centrifugal filtration properly introduced was selected. Plasmid pYAMY1 held by and blow drying. Viscosity characteristics of the obtained this transformant was prepared. enzyme-treated Starch were analyzed by the amylograph and In accordance with the method of Ito et al. (J. bacterial. the rheometer. After completion of the reaction, a degradation 30 Vol. 153, 163-168 (1983)), pYAMY1 was introduced into a ratio was determined using a part of the reaction Solution. The yeast host DBY746, and obtained a transformant capable of results are shown in Table 4-2. growing in a tryptophan-free culture medium by complemen tation of the tryptophan requirement. This transformant was Example 11B inoculated in 100 ml of a synthetic culture medium consisting 35 of 2% glucose, 0.67% yeast nitrogen base, 24 mg/l L-uracil, To 400 g of an untreated native cassava starch, 900 g of 24 mg/l L-histidine and 36 mg/l L-leucine at pH 5.7 and then ion-exchange water was added to prepare a starch Suspension. cultured with shaking at 30° C. for 120 hours. After adjusting the pH of the suspension to 5.0, 1% by weight The supernatant obtained by centrifugation (at 5,000 rpm (based on starch solid content) of C-amylase (AMYLEX for 10 minutes) of the culture was concentrated using a hol A3' derived from Aspergillus niger, manufactured by 40 low fiber type UF membrane module having a molecular DANISCO: optimum pH of 5.0) was added and stirred at 50° weight cut-off of 10,000 to prepare C-amylase derived from C. for 18 hours to carry out an enzyme reaction. After comple Aspergillus Oryzae. This C.-amylase has an amino acid tion of the reaction, an enzyme-treated Starch was recovered sequence of SEQID NO: 2. by centrifugal filtration and blow drying. Viscosity character istics of the obtained enzyme-treated starch were analyzed by 45 Example 12B the amylograph and the rheometer. After completion of the reaction, a degradation ratio was determined using a part of Method for Preparation of C.-Amylase derived from the reaction solution. The results are shown in Table 4-2. Aspergillus niger

Example 11C 50 A double stranded DNA was chemically synthesized by adding an EcoRI recognition site (GAATTC) to both termi To 400 g of an untreated native cassava starch, 900 g of nals of a base sequence of SEQ ID NO: 3 in the sequence ion-exchange water was added to prepare a starch Suspension. listing. This synthetic DNA was completely cleaved by a After adjusting the pH of the suspension to 5.0, 1% by weight restriction enzyme EcoRI, mixed with pYCDE1 (Method in (based on starch Solid content) of C-glucosidase ("Transglu 55 Enzymology, 101, pp. 192-201 (1983)) which was previously cosidase L Amano’ derived from Aspergillus niger, manu completely cleaved by EcoRI, and then ligation was carried factured by Amano Enzyme Inc.; optimum pH of 5.0) was out. E. coli TG1 was transformed with the ligation reaction added and stirred at 50° C. for 18 hours to carry out an enzyme Solution and a transformant into which a synthetic gene was reaction. After completion of the reaction, an enzyme-treated properly introduced was selected. Plasmid pYAMY2 held by starch was recovered by centrifugal filtration and blow dry 60 this transformant was prepared. ing. Viscosity characteristics of the obtained enzyme-treated In accordance with the method of Ito et al. (J. bacterial, Vol. starch were analyzed by the amylograph and the rheometer. 153, 163-168 (1983)), pYAMY2 was introduced into a yeast After completion of the reaction, a degradation ratio was host DBY746, and obtained a transformant capable of grow determined using a part of the reaction Solution. The results ing in a tryptophan-free culture medium by complementation are shown in Table 4-2. 65 of the tryptophan requirement. This transformant was inocu As a result, in Examples 1 to 11C, it was confirmed that a lated in 100 ml of a synthetic culture medium consisting of novel starch having both a high viscosity and strong gel 2% glucose, 0.67% yeast nitrogen base, 24 mg/l L-uracil, 24 US 9,005,681 B2 61 62 mg/l L-histidine and 36 mg/l L-leucine at pH 5.7 and then The supernatant obtained by centrifugation (at 5,000 rpm cultured with shaking at 30° C. for 120 hours. for 10 minutes) of the culture was concentrated using a hol The Supernatant obtained by centrifugation (at 5,000 rpm low fiber type UF membrane module having a molecular for 10 minutes) of the culture was concentrated using a hol weight cut-off of 10,000 to prepare isoamylase derived from low fiber type UF membrane module having a molecular 5 Flavobacterium sp. This isoamylase has an amino acid weight cut-off of 10,000 to prepare C-amylase derived from sequence of SEQID NO: 8. Aspergillus niger. This C.-amylase has an amino acid sequence of SEQID NO: 4. Example 12E Example 12C 10 Method for Preparation of Isoamylase Derived from Pseudomonas Amyloderamosa Method for Preparation of Amyloglucosidase Derived from Aspergillus niger A double stranded DNA was chemically synthesized by adding an EcoRI recognition site (GAATTC) to both termi A double stranded DNA was chemically synthesized by 15 nals of a base sequence of SEQ ID NO: 9 in the sequence listing. This synthetic DNA was completely cleaved by a adding an EcoRI recognition site (GAATTC) to both termi restriction enzyme EcoRI, mixed with pYCDE1 (Method in nals of a base sequence of SEQ ID NO. 5 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 pYISO2 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 In accordance with the method of Ito et al. (J. bacterial. properly introduced was selected. Plasmid pYGLU1 held by 25 Vol. 153, 163-168 (1983)), pYISO2 was introduced into a this transformant was prepared. yeast host DBY746, and obtained a transformant capable of In accordance with the method of Ito et al. (J. bacterial, Vol. growing in a tryptophan-free culture medium by complemen 153, 163-168 (1983)), pYGLU1 was introduced into a yeast tation of the tryptophan requirement. This transformant was host DBY746, and obtained a transformant capable of grow inoculated in 100 ml of a synthetic culture medium consisting ing in a tryptophan-free culture medium by complementation 30 of 2% glucose, 0.67% yeast nitrogen base, 24 mg/l L-uracil, of the tryptophan requirement. This transformant was inocu 24 mg/l L-histidine and 36 mg/l L-leucine at pH 5.7 and then lated in 100 ml of a synthetic culture medium consisting of cultured with shaking at 30° C. for 120 hours. 2% glucose, 0.67% yeast nitrogen base, 24 mg/l L-uracil, 24 The supernatant obtained by centrifugation (at 5,000 rpm mg/l L-histidine and 36 mg/l L-leucine at pH 5.7 and then for 10 minutes) of the culture was concentrated using a hol cultured with shaking at 30° C. for 120 hours. 35 low fiber type UF membrane module having a molecular The Supernatant obtained by centrifugation (at 5,000 rpm weight cut-off of 10,000 to prepare isoamylase derived from for 10 minutes) of the culture was concentrated using a hol Pseudomonas amyloderamosa. This isoamylase has an low fiber type UF membrane module having a molecular amino acid sequence of SEQID NO: 10. weight cut-off of 10,000 to prepare amyloglucosidase derived from Aspergillus niger. This amyloglucosidase has an amino 40 Example 12A-1 acid sequence of SEQID NO: 6. To 400 g of an untreated native wheat starch, 900 g of Example 12D ion-exchange water was added to prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 1% by weight Method for Preparation of Isoamylase Derived from 45 (based on starch solid content) of C.-amylase (derived from Flavobacterium sp. Aspergillus Oryzae) prepared in Example 12A was added and stirred at 50° C. for 18 hours to carryout an enzyme reaction. A double stranded DNA was chemically synthesized by After completion of the reaction, an enzyme-treated Starch adding an EcoRI recognition site (GAATTC) to both termi was recovered by centrifugal filtration and blow drying. Vis nals of a base sequence of SEQ ID NO: 7 in the sequence 50 cosity characteristics of the obtained enzyme-treated Starch listing. This synthetic DNA was completely cleaved by a were analyzed by the amylograph and the rheometer. After restriction enzyme EcoRI, mixed with pYCDE1 (Method in completion of the reaction, a degradation ratio was deter Enzymology, 101, pp. 192-201 (1983)) which was previously mined using a part of the reaction solution. The results are completely cleaved by EcoRI, and then ligation was carried shown in Table 5-2 below. out. E. coli TG1 was transformed with the ligation reaction 55 Solution and a transformant into which a synthetic gene was Example 12B-1 properly introduced was selected. Plasmid pYISO1 held by this transformant was prepared. To 400 g of an untreated native wheat starch, 900 g of In accordance with the method of Ito et al. (J. bacterial. ion-exchange water was added to prepare a starch Suspension. Vol. 153, 163-168 (1983)), pYISO1 was introduced into a 60 After adjusting the pH of the suspension to 5.0, 1% by weight yeast host DBY746, and obtained a transformant capable of (based on starch solid content) of C.-amylase (derived from growing in a tryptophan-free culture medium by complemen Aspergillus niger) prepared in Example 12B was added and tation of the tryptophan requirement. This transformant was stirred at 50° C. for 18 hours to carry out an enzyme reaction. inoculated in 100 ml of a synthetic culture medium consisting After completion of the reaction, an enzyme-treated Starch of 2% glucose, 0.67% yeast nitrogen base, 24 mg/l L-uracil, 65 was recovered by centrifugal filtration and blow drying. Vis 24 mg/l L-histidine and 36 mg/l L-leucine at pH 5.7 and then cosity characteristics of the obtained enzyme-treated Starch cultured with shaking at 30° C. for 120 hours. were analyzed by the amylograph and the rheometer. After US 9,005,681 B2 63 64 completion of the reaction, a degradation ratio was deter Flavobacterium sp.) prepared in Example 12D was added and mined using a part of the reaction solution. The results are stirred at 50° C. for 18 hours to carry out an enzyme reaction. shown in Table 5-2 below. After completion of the reaction, an enzyme-treated Starch was recovered by centrifugal filtration and blow drying. Vis Example 12C-1 5 cosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and the rheometer. After To 400 g of an untreated native wheat starch, 900 g of ion-exchange water was added to prepare a starch Suspension. completion of the reaction, a degradation ratio was deter After adjusting the pH of the suspension to 5.0, 1% by weight mined using a part of the reaction solution. The results are (based on starch Solid content) of amyloglucosidase (derived 10 shown in Table 5-2 below. from Aspergillus niger) prepared in Example 12C was added Example 12E-1 and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration and blow dry To 400 g of an untreated native wheat starch, 900 g of ing. Viscosity characteristics of the obtained enzyme-treated 15 ion-exchange water was added to prepare a starch Suspension. starch were analyzed by the amylograph and the rheometer. After adjusting the pH of the suspension to 5.0, 1% by weight After completion of the reaction, a degradation ratio was (based on starch solid content) of isoamylase (derived from determined using a part of the reaction Solution. The results Pseudomonas amyloderamosa) prepared in Example 12E are shown in Table 5-2 below. was added and stirred at 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an Example 12D-1 enzyme-treated starch was recovered by centrifugal filtration and blow drying. Viscosity characteristics of the obtained To 400 g of an untreated native wheat starch, 900 g of enzyme-treated Starch were analyzed by the amylograph and ion-exchange water was added to prepare a starch Suspension. the rheometer. After completion of the reaction, a degradation After adjusting the pH of the suspension to 5.0, 1% by weight ratio was determined using a part of the reaction solution. The (based on starch solid content) of isoamylase (derived from results are shown in Table 5-2 below. 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 amyloiquefaciens Reagent (Sigma-Aldrich Corporation) Comp. Ex. 4-2 C-amylase Bacilius amyloiquefaciens Reagent (Sigma-Aldrich Corporation) Comp. Ex. 4–3 C-amylase Bacilius amyloiquefaciens 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 OPTIDEXL-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 Candida tsukubaensis Reagent (Sigma-Aldrich Corporation) Example 4 isoamylase Pseudamonas amyloderamosa 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 US 9,005,681 B2 65 66 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 15 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 O6 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 O3 16S 218 155 5,520,234 2O USable Example 3A-2 15 719 16 229 263 187 5,890,552 28 USable Example 3A-3 29 727 17 267 311 221 6,356,475 38 USable Example 3B 2O 858 38 385 307 218 6,731,469 46 Usable Example 3C 26 873 41 394 313 222 6489,069 41 Usable Example 3D 2O 867 40 369 242 172 5,998.440 3O USable Example 3E 42 806 30 407 283 2O1 5,581,328 21. Usable Example 3F 43 808 30 403 286 203 5.941241 29 USable Comp. Ex. 8 5 736 19 263 119 84 4,096,046 89 Not usable Example 4 6 828 33 3OO 297 211 6,987,728 52 USable Example 5A 4 746 2O 291 166 118 5,142,993 12 Usable Example SB 5 554 89 274 18O 128 6.418,528 39 USable Comp. Ex. 10 7 757 22 256 145 103 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 12 Usable Example 13-2 12 475 76 460 159 113 5,291,915 15 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) 9% (%) (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 US 9,005,681 B2 67 68 TABLE 3-2-continued 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 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) 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 55 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 Example Name Origins (Manufacturer) 60 Product name Example Name Origins (Manufacturer) Comparative Untreated Example 1 wheat starch Example 12D-1 isoamylase Flavobacterium sp. SEQID NO: 8 Example 12A-1 C-amylase Aspergillus oryzae SEQID NO: 2 Example 12E-1 isoamylase Pseudomonas SEQ ID NO: 10 Example 12B-1 C-amylase Aspergilius niger SEQ ID NO:4 amyloderanosa Example 12C-1 amylogluco- Aspergilius niger SEQ ID NO: 6 65 sidase US 9,005,681 B2 69 70 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 26 806 130 407 378 268 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

Trial Production Examples mixed. Furthermore, white soft Sugar and common salt were Next, the present invention will be described in more detail added, well mixed, and then ammonium hydrogen carbonate by way of Trial Production Examples, but the present inven previously dissolved in water was added and well mixed. tion is not limited to the following Trial Production Example. Unless otherwise specified, “parts' means “parts by mass”. Finally, a powder sample obtained by previously mixing soft wheat flour, a starch and baking Soda (Sodium hydrogen car Trial Production Example 1 25 bonate) was added, followed by well mixing until a mass of a Preparation of Cookie dough was formed. The mass of the dough was spread thinly Among the formulations shown in Table 10 below, salt-free using a rollingpin, cut using a mold and thenbaked in an oven butter and shortening were put in a mixer and then well (at 200°C. for 15 minutes) to prepare cookies. 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 1SO 150 150 150 150 150 Chemically unmodified 1SO cassava starch'' (' 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. Note (3) 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, US 9,005,681 B2 71 72 The obtained cookies showed the following results. That is, The obtained sponge cakes showed the following results. both the cookies of Trial Production Examples 1-1 and 1-2, in That is, all the sponge cakes of Trial Production Example 2-1 to 2-4, in which any one of the starches prepared in Examples which any one of the starches prepared in Examples 9 and 1-3, 2A, 3 A-3 and 5A was added, showed nice swelling after 10-2 was added, were soft and had texture with nice melt in 5 baking and had a large Volume, and also had soft and puffy mouth as compared with the cookies of Comparative Trial nice texture as compared with the sponge cakes of Compara Production Examples 1 to 4, which were hard and crunchy, tive Trial Production Example 2-1 and Comparative Trial and also had texture with poor melt in mouth. In particular, the Production Example 2-2. cookies of Trial Production Examples 1-1 and 1-2 had very light texture and were readily edible. Regarding the dough at 10 Trial Production Example 3 the time of shaping, both the doughs of Trial Production Examples 1-1 and 1-2 were very dry and non-sticky as com Preparation of Custard Cream pared with the doughs of Comparative Trial Production 15 Among the formulations shown in Table 12 below, granu Examples 1-1 to 1-4, and did not stick to hands, rolling pin lated Sugar was added to egg yolk beaten well by a beater, and the like, and also showed very nice operability. followed by mixing by the beater. To the mixture, a powder sample obtained by previously mixing soft wheat flour and a starch was added through sieving, followed by mixing. Fur Trial Production Example 2 20 thermore, warmed milk was added and mixed with them, the mixture was filtered and put in a pan, and then heated. The mixture was stirred by a wooden spatula until the mixture Preparation of Sponge Cake become to a mixture that has a viscosity and a smooth state. Finally, butter, a food color and vanilla essence were added Among the formulations shown in Table 11 below, whole 25 and mixed with them to prepare a custard cream. egg and granulated Sugar were warmed to around a body temperature while mixing using a hand mixer. Furthermore, TABLE 12 the mixture was stirred by a hand mixer until the mixture Comparative Comparative Trial Trial 30 Trial Trial Production Production become to a mixture that has a viscosity, fine bubbles and Formulation Production Production Example Example wholly whitish state. To the mixture, a powder sample (Parts) Example 3-1 Example 3-2 3-1 3-2 obtained by previously mixing soft wheat flour, a starch and Milk 300 3OO 3OO 300 wheat gluten was added through sieving, followed by mixing Egg yolk 35 35 35 35 Granulated Sugar 60 60 60 60 using a spatula. Finally, a mixture of melted butter and milk 35 Soft wheat flour 10 10 10 10 Chemically 10 was added and mixed. The obtained mixture was poured into unmodified wheat a mold and then baked in an oven (at 200° C. for 15 minutes, starch Note (3) then at 190° C. for 18 minutes) to prepare a sponge cake. 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 starche () 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'e (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: “M DSOL 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

E99Fsdie CO.,LTD. Chemically unmodified wheat starch: untreated native wheat starch. US 9,005,681 B2 73 TABLE 12-continued TABLE 13 Comparative Comparative Trial Trial Comparative Comparative Trial Trial Trial Trial Production Production Trial Trial Production Production Formulation Production Production Example Example Formulation Production Production Example Example (Parts) Example 3-1 Example 3-2 3-1 3-2 5 (Parts) Example 4-1 Example 4-2 4-1 4-2 Chemically 10 Milk 170 170 170 170 modified wheat Granulated Sugar 10 10 10 10 starch Note (1) Chemically 10 Starch prepared in 10 unmodified wheat Example 1-3 10 starch Noe (2) Starch prepared in 10 Chemically 10 Example 3A-3 Modified wheat Salt-free butter 15 15 15 15 starch Yo'e () KUCEHINA COLOR appro- appro- appro- appro Starch prepared in 10 40OLS Note (2) priate priate priate priate Example 1-3 amount amount amount amount 15 Starch prepared in 10 Vanilla essence appro- appro- appro- appro Example 3A-3 priate priate priate priate amount amount amount amount Note (1) Chemically modified wheat starch: “MIDSOL 1020”, manufactured by GLICO FOODS Note (1) Note.)CO.,LTD. Chemically modified wheat starch: “MIDSOL 1020”, manufactured by GLICO FOODS Chemically unmodified wheat starch: Untreated native wheat starch. CO.,LTD.Note 25 KUCHINA COLOR 400LS: Gardenia yellow food color. “KUCHINA COLOR is a reg istered trademark of GLICO FOODS CO.,LTD. The obtained milk puddings showed the following results. Note (3) That is, both the milk puddings of Trial Production Examples Chemically unmodified wheat starch: Untreated native wheat starch. 4-1 and 4-2, in which any one of the starches prepared in The obtained custard creams showed the following results. 25 Examples 1-3 and 3A-3 was added, had sticky and chewy and That is, both the custard creams of Trial Production Example also had nice melt in mouth and smooth texture. On the other 3-1 and 3-2, in which any one of starches prepared in hand, the milk pudding of Comparative Trial Production Examples 1-3 and 3A-3 was added, had appropriate body and Example 4-1 was sticky but had texture with hard yogurt-like shape retention, and had nice melt in mouth and Smooth hardness and was therefore inferior in both melt in mouth and texture. On the other hand, the custard cream of Comparative 30 smoothness as compared with those of Trial Production Trial Production Example 3-1 had gel-like physical proper Examples. Also, the milk pudding of Comparative Trial Pro ties and heavy texture, and also had poor melt in mouth and duction Example 4-2 was not firmly gelled and had a texture poor Smoothness. Also, the custard cream of Comparative with Stickiness, and also had poor melt in mouth. Trial Production Example 3-2 had poor body and shape reten tion, and had texture with Stickiness and poor melt in mouth. 35 Trial Production Example 5 Trial Production Example 4 Preparation of Milk Pudding Preparation of Kudzu Starch Cake 40 Among the formulations shown in Table 13 below, granu Among the formulations shown in Table 14 below, a mix lated Sugar was added to milk and mixed well using a wooden ture of a starch sample and white Soft Sugar was added to spatula to dissolve the granulated Sugar. To the mixture, a water and white Soft Sugar was dissolved by well mixing starch sample was added and mixed well using the wooden using a wooden spatula. The mixture was heated while stir spatula. The mixture was heated while stirring by the wooden 45 ring using the wooden spatula until the mixture become a spatula until the mixture become to a mixture having a vis pasty mixture with a viscosity and a transparency state. The cosity and a Smooth state. The mixture was filled in a jelly cup mixture was poured into a mold and quenched in an ice bath and quenched in an ice bath to prepare a milk pudding. 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 starche () Starch prepared in Example 1-3 Starch prepared in Example 2A Starch prepared in 56 Example 3A-3 Starch prepared in 56 Example 5A US 9,005,681 B2

TABLE 14-continued 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 White soft Sugar 32 32 32 32 32 32 32 Water 28O 28O 28O 28O 28O 28O 28O

Note (1) Chemically modified wheat starch: “M DSOL 1020”, manufactured by GLICO FOODS CO.,LTD. Note (2) Chemically unmodified wheat starch: Untreated native wheat starch.

The obtained kudzu starch cakes showed the following TABLE 15-continued results. That is, all the kudzu starch cakes of Trial Production 15 Comparative Comparative Trial Trial Example 5-1 to 5-4, in which any one of the starches prepared Trial Trial Production Production in Examples 1-3, 2A, 3 A-3 and 5A was added, had a appro Formulation Production Production Example Example priate hardness and brittleness, and had appropriate vis (Parts) Example 6-1 Example 6-2 6-1 6-2 coelasticity and Sticky texture. As compared with a kudzu Starch prepared in 30 Example 3A-3 starch cake so called in the Kanto area in Comparative Trial Water 300 3OO 3OO 300 Production Example 5-1, in which a fermented wheat starch Note (1) obtained by fermenting for a long period, for example, one or Chemically modified wheat starch: “MIDSOL 1020”, manufactured by GLICO FOODS more years was used, the kudzu Starch cakes having the CO.,LTD. 25 Note.) identical texture could be prepared without requiringfermen Chemically unmodified wheat starch: Untreated native wheat starch. tation for a long period in Trial Production Examples. Fur thermore, the obtained kudzu starch cakes had nice flavor The obtained goma-dofus showed the following results. without having peculiar flavor derived from a fermented That is, both the goma-dofus of Trial Production Examples wheat starch and fermentation odor. On the other hand, the 30 6-1 and 6-2, in which anyone of Starches prepared in kudzu starch cake of Comparative Trial Production Example Examples 1-3 and 3A-3 was added, had appropriately sticky 5-2 was hard and brittle, and had texture with stickiness in the texture and appropriate crispy sensation in contrast to a tex mouth. Also, the kudzu starch cake of Comparative Trial ture with rich elasticity like texture obtained by the addition of a kudzu powder, and had readily edible texture with less Production Example 5-3 had soft and brittle texture and 35 showed texture far different from that of the kudzu starch cake Stickiness and Sticking in the mouth. Thus, it could be so called in the Kanto area, together with those of Compara expected for the obtained goma-dofus to be applied to foods tive Trial Production Example 5-2 and Comparative Trial for advanced aged persons, for example. On the other hand, Production Example 5-3. the goma-dofu of Comparative Trial Production Example 6-1 40 has soft and strong Sticky texture and the goma-dofu of Com Trial Production Example 6 parative Trial Production Example 6-2 had hard and brittle texture, but had neither elasticity nor sticky texture, and thus Preparation of Goma-dofu both goma-dofus of Comparative Trial Production Example 45 6-1 and Comparative Trial Production Example 6-2 were Among the formulation shown in Table 15 below, a starch inferior in deliciousness and ease of eating. sample was added to water and the mixture was heated while stirring using a wooden spatula until the mixture become a Trial Production Example 7 pasty mixture with a viscosity and a transparency state. A sesame paste was added to them and mixed well. The mixture 50 Preparation of Kamaboko was filled in a container and then cooled to obtain a goma dofu. Among the formulations shown in Table 16 below, a fish TABLE 1.5 paste, common salt, Sugar, monosodiumglutamate and potas 55 sium sorbate were put in a (silent) mixer and well mixed until Comparative Comparative Trial Trial the mixture had a viscosity. In order to inhibit a temperature Trial Trial Production Production Formulation Production Production Example Example rise of the fish paste, a half amount of moisture with ice was (Parts) Example 6-1 Example 6-2 6-1 6-2 added to them and mixed. Then, egg white, Mirin and a starch Sesame paste 50 50 50 50 previously suspended in the remaining water with ice were Chemically 30 60 added to them and mixed well until a homogeneous mixture unmodified wheat starch Noe (2) was obtained. Indication of the temperature of the fish paste Chemically 30 after mixing was within a range from 10 to 15°C. The mixed modified wheat fish paste was deaerated and filled in a cage. After filling, the starch Yo'e () Starch prepared in 30 65 cage filled with a mixed fish paste was subjected to a steril Example 1-3 ization step (at 90° C. for 40 minutes) and cooled to prepare a kamaboko. US 9,005,681 B2 77 78 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 100 100 100 1OO Chemically unmodified 15 wheat starche () Chemically modified 15 wheat starch 1 '' () Chemically modified 15 wheat starch 2^e (2) Starch prepared in 15 Example 1-3 Starch prepared in 15 Example 3A-3 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: “M DSOL 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.

On the next day of production and after one week, the TABLE 17-continued obtained kamabokos were subjected to a sensory test. The 30 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 Mirin 35 35 35 35 eration for one week, and thus the kamaboko showed dry and 35 Starch syrup 8 8 8 8 Chemically 10 tasteless texture with water separation. The kamaboko of unmodified cassava Comparative Trial Production Example 7-3 was less likely to starch Noe (2) cause change with time due to retrogradation because of the Chemically 10 structure of the starch, but showed greasy texture with poor modified cassava elasticity. As compared with these Comparative Trial Produc 40 starch Note (1) tion Examples, both the kamabokos of Trial Production Starch prepared in 10 Examples 7-1 and 7-2, in which any one of the starches Example 9 Starch prepared in 10 prepared in Examples 1-3 and 3A-3 was added, had elasticity Example 10-2 with nice chewiness and also caused less change with time. Water 70 70 70 70 45 Trial Production Example 8 Note (1) Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Preparation of Glaze for Mitarashi Dango Note (2) Chemically unmodified cassava starch: Untreated native cassava starch, Among the formulation shown in Table 17 below, a starch sample was previously Suspended in a part of water. The total 50 The obtained glaze formitarashidango showed the follow amount of white Soft Sugar, dark soy sauce, Mirin, starch ing results. That is, both the glazes for mitarashi dango of syrup and the remaining water were put in a pan and then Trial Production Example 8-1 and 8-2, in which any one of mixed well by a wooden spatula. Furthermore, the starch the starches prepared in Examples 9 and 10-2 was added, had sample previously suspended in water was added to them and nice body and shape retention, and were less likely to drop heated while stirring using the wooden spatula. The mixture 55 because of nice adhesion onto the dango, and also had less was heated until the mixture becomes a pasty mixture with a Stickiness and thread-forming sensation and had smooth tex Viscosity and a transparency state, to prepare a glaze for ture. On the other hand, the glaze for mitarashi dango of mitarashi dango. Comparative Trial Production Example 8-1 had gel-like physical properties and heavy texture, and also had poor melt TABLE 17 60 in mouth and no Smoothness. The glaze for mitarashi dango Comparative Comparative Trial Trial of Comparative Trial Production Example 8-2 had poor body Trial Trial Production Production and poor shape retention and caused dropping because of Formulation Production Production Example Example poor adhesion onto a dango, and also had texture with Sticki (Parts) Example 8-1 Example 8-2 8-1 8-2 ness and poor melt in mouth. For example, in freezing distri White soft Sugar 95 95 95 95 65 bution of a split and broiled eel, in order to prevent a salsa for Dark soy sauce 8O 8O 8O 8O the split and broiled eel from dropping at the time of thawing, a Salsa having a high viscosity and nice body and shape US 9,005,681 B2 79 80 retention may be sometimes used in the final step of baking. 90° C. for 10 minutes. Brewed vinegar, common salt, lemon However there is a problem that the salsa having a high juice, and seasonings including monosodium glutamate and Viscosity usually has strong Stickiness or in a gel-like form, the like were added and further heated with stirring for 5 and also has jellied fish-like physical properties and heavy minutes. After cooling it to room temperature, egg yolk was texture. Use of the enzyme-treated starch in the present inven- 5 added to them and mixed well. Using a homomixer manufac tion makes it possible to prepare a split and broiled fish which tured by Tokushu Kika Kogyo Co., Ltd. (Now in the name of: is less likely to drop because of nice adhesion onto the eel and PRIMIX Corporation), salad oil was slowly added dropwise the like, and has less Stickiness and thread-forming sensation, while m1X1ng with stirring at 8,000 rpm. After dropwise addi and has Smooth texture tion of the entire amount of salad oil, the mixture was mixed 10 with stirring at 8,000 rpm further for 5 minutes to prepare a Trial Production Example 9 dressing. Preparation of Fruit Sauce TABLE 19 15 Comp arative Comparative Among the formulations shown in Table 18 below, a starch Trial Trial Trial Trial sample was previously suspended in a part of water. Fruit Formulati ritute ritute ritute Pitt" puree, white soft Sugar, lemon juice and the total amount of (Parts)Ormulation xample1O-1 xample10-2 xample1O-1 xample10-2 the remaining water were put in a pan and heated with stirring using the wooden spatula. Furthermore, the starch sample Salad oil 38 38 38 38 previously Suspended in water was added to them. The mix- 2O Sysia 10 10 10 10 ture was heated until the mixture become a pasty mixture with Egg yolk 5 5 5 5 a viscosity and a transparency state to prepare a fruit sauce. White soft Sugar 5 5 5 5 Common Salt 3 3 3 3 Lemon juice 2 2 2 2 TABLE 18 25 Chemically 2.5 Comparative Comparative Trial Trial Alissassive Trial Trial Production Production Chemicall 2.5 Formulation Production Production Example Example modified essa (Parts) Example 9-1 Example 9-2 9-1 9-2 starch Yo'e () Fruit puree 100 1OO 1OO 100 30 Starch prepared in 2.5 White soft Sugar 10 10 10 10 Example 9 Chemically 3 Starch prepared in 2.5 unmodified cassava Example 10-2 starch Noe (2) Monosodium O.2 O.2 O.2 O.2 Chemically 3 glutamate modified cassava 35 Pepper Suitable Suitable Suitable Suitable starch Note (1) amount amount amount amount Starch prepared in 3 Mustard Suitable Suitable Suitable Suitable Example 9 amount amount amount amount Starch prepared in 3 Water 33.8 33.8 33.8 33.8 Example 10-2 Note (1) Lemon juice 2 2 2 2 Water 10 10 10 10 40 Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Note (2) Note (1) Chemically unmodified cassava starch: Untreated native cassava starch, Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Note (2) The obtained dressings showed the following results. That Chemically unmodified cassava starch: Untreated native cassava starch, 45 is, both the dressings of Trial Production Examples 10-1 and The obtained fruit sauces showed the following results. 10-2, in which each of the starches prepared in Examples 9 That is, both the fruit sauces of Trial Production Example 9-1 and 10-2 was added, had nice body and nice shape retention, and 9-2, in which any one of the starches prepared in and were less likely to drop because of nice adhesion onto Examples 9 and 10-2 was added, had nice body and shape Vegetables and the like, and caused less stickiness and thread retention, and had nice adhesion onto a food Such as dessert, 50 forming sensation, and had smooth texture. On the other and had less Stickiness and thread-forming sensation, and hand, the dressing of Comparative Trial Production Example smooth texture. On the other hand, the fruit sauce of Com 10-1 had gel-like physical properties and heavy texture, and parative Trial Production Example 9-1 had gel-like physical also had poor melt in mouth and no Smoothness. Also, the properties and heavy texture, and also had poor melt in mouth dressing of Comparative Trial Production Example 10-2 had and no smoothness. The fruit sauce of Comparative Trial 55 poor body and poor shape retention and caused dropping Production Example 9-2 had poor body and poor shape reten because of poor adhesion onto vegetables and the like, and tion, and caused dropping because of poor adhesion onto a also had texture with Stickiness, and poor melt in mouth. food Such as dessert, and also had texture with Stickiness, and poor melt in mouth. Trial Production Example 11 60 Trial Production Example 10 Preparation of Batter for Deep-Fried Food Preparation of Dressing Among the formulations shown in Table 20 below, soft Among the formulations shown in Table 19 below, white 65 wheat flour and a starch sample previously mixed in a powder Soft Sugar and a starch sample which are previously mixed in state were Suspended in cold water and mixed well to prepare powder state was added to water, and heated with stirring at a batter for deep-fried food. US 9,005,681 B2 82 TABLE 20 TABLE 21-continued 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 5 Formulation Example Example Example Example (Parts) 11-1 11-2 11-1 11-2 (Parts) 12-1 12-2 12-1 12-2 Soft wheat flour 150 95 95 95 Chemically 3 Chemically 30 modified cassava unmodified cassava starch Yo'e () starch Noe (2) 10 Starch prepared in 3 Chemically 30 Example 9 modified cassava Starch prepared in 3 starch Yo'e () Example 10-2 Starch prepared in 30 Water with ice 25 25 25 25 Example 9 Common salt 1.4 1.4 1.4 1.4 Starch prepared in 30 15 White soft Sugar 1 1 1 1 Example 10-2 Seasoning O.3 O.3 O.3 O.3 Cold water 230 230 230 230 pickle solution O.S O.S O.S O.S Pork powder 1 1 1 1 Note (1) Spice O.S O.S O.S O.S Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Potassium sorbate O.15 O.15 O.15 O.15 Note (2) pH adjusting agent O.15 O.15 O.15 O.15 Chemically unmodified cassava starch: Untreated native cassava starch, Creation Color appro- appro- appro- appro RC. Note (2) priate priate priate priate The obtained batters for deep-fried food showed the fol amount amount amount amount lowing results. That is, both the tempuras obtained by dipping Note (1) ingredients such as prawn in the batters for deep-fried food of Chemically modified cassava starch: “RK-08”, manufactured by GLICOFOODS CO.,LTD. Trial Production Examples 11-1 and 11-2, in which the starch 25 Note (2) prepared in Example 9 or 10-2 was added, thereby coating the Creation Color RC: Cochineal food color, “Creation” is a registered trademark of GLICO ingredients with the batters, and frying in oil, or the fried FORSdie CO.,LTD. foods obtained by dipping ingredients such as prawn in the Chemically unmodified cassava starch: Untreated native cassava starch, batters for deep-fried food of Trial Production Examples 11-1 On the next day of production and after one week, the and 11-2, in which the starch prepared in Example 9 or 10-2 30 obtained sausages were subjected to a sensory test. The sau was added, thereby coating the ingredients with the batters, sage of Comparative Trial Production Example 12-1 had tex further coating this with a breadcrumbs and frying in oil, had ture with slightly poor elasticity and had no good chewiness. a crispy and light texture. On the other hand, tempuras and The sausage of Comparative Trial Production Example 12-2 fried foods, in which the batters for deep-fried food of Com had hardness but had stiff texture, and retrogradation of the parative Trial Production Example 11-1 and Comparative 35 starch arose in a sensory test after refrigeration for one week, Trial Production Example 11-2 were used, had poor crispy and thus the sausage showed dry and tasteless texture with texture, and hardly made users feel lightness. water separation. As compared with those of these Compara tive Trial Production Examples, both the sausages of Trial Trial Production Example 12 Production Examples 12-1 and 12-2, in which the starch 40 prepared in Example 9 or 10-2 was added, had elasticity with Preparation of Sausage nice chewiness and also caused less change with time. Among the formulations shown in Table 21 below, pork Trial Production Example 13 arm meat was put in a silent cutter, while cutting the pork arm 45 Preparation of Raw Udon meat at a high speed, casein Sodium, common salt, white soft Sugar, a seasoning, a pickle solution, a pork powder, spice, To a powder mixture obtained by mixing a starch, medium potassium Sorbate, a pH adjusting agent and a food color were wheat flour and a powdered gluten in the following ratio in added and mixed well. When the mixture was formed into a accordance with the formulation shown in Table 22 below, paste, water with ice and lard were added and cutting was water for kneading obtained by dissolving 2 parts of common continued. Finally, a starch sample was added to them and 50 salt in 40 parts of water was added, followed by kneading in mixed well to give a homogeneous paste. The paste was filled a vacuum mixer for 12 minutes. Using a noodle making in a casing and then sterilized at 80° C. for 40 minutes to machine, the obtained kneaded mixture was subjected to prepare a sausage by cooling with running water. compound and rolling to obtain a noodle strip, which was cut 55 using a cutting-tooth No. 10 to obtain a raw udon. TABLE 21 TABLE 22 Comparative Comparative Trial Trial Trial Trial Comparative Comparative Comparative Production Production Production Production Trial Trial Trial Trial Formulation Example Example Example Example 60 Production Production Production Production (Parts) 12-1 12-2 12-1 12-2 Formulation Example Example Example Example (Parts) 13-1 13-2 13-3 13-1 Pork arm meat 60 60 60 60 Lard 10 10 10 10 Wheat flour 8O 8O 8O 8O Casein sodium 1 1 1 1 Chemically 2O Chemically 3 unmodified unmodified cassava 65 C3SS8W8 starch Note (3) starch Note (3) US 9,005,681 B2 83 84 TABLE 22-continued 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 5 Formulation Example Example Example Example (Parts) 13-1 13-2 13-3 13-1 (Parts) 14-1 14-2 14-1 14-2 Chemically 2O Starch prepared in 21 modified Example 10-2 C3SS8W8 Water 15 15 15 15 starch 1 Noe () 10 Chemically 2O Note (1) modified Chemically modified cassava starch: "CHEMISTAR 300S” manufactured by GLICO C3SS8W8 NoteFOODS (2) CO.,LTD. “CHEMISTAR is a registered trademark of GLICO FOODS CO.,LTD. starch 2 Noe (2) Starch prepared in 2O Chemically unmodified cassava starch: Untreated native cassava starch, Example 10-2 15 Powdered gluten 2 2 2 2 The obtained jelly candies showed the following results. Salt 2 2 2 2 That is, both the jelly candies of Trial Production Example Water 40 40 40 40 14-1 and 14-2 had appropriate viscoelasticity and nice melt texture in mouth. On the other hand, the jelly candy of Com Note (1) parative Trial Production Example 14-1 had strong elastic Chemically modified cassava starch 1: “CHEMISTAR 280, manufactured by GLICO F92PS CO.,LTD. “CHEMISTAR” is a registered trademark of GLICO FOODS CO.,LTD. sensation, and the jelly candy of Comparative Trial Produc die tion Example 14-2 had strong sticky textures, but both the Chemically modified cassava starch 2: “RK-08”, manufactured by GLICO FOODS CO., jelly candies had strong pasty sensation and poor melt in EP) mouth. Chemically unmodified cassava starch: Untreated native cassava starch, 25 Trial Production Example 15 The obtained raw udon was boiled in boiling water for 10 minutes and dipped in a hot Soup, and then texture was evalu Preparation of Frozen Dessert ated. The udon of Comparative Trial Production Example 13-1 and Comparative Trial Production Example 13-2 was In accordance with the formulation shown in Table 24 poor in elasticity and texture was hardly improved. Regarding 30 below, while raw materials and water were mixed with stir the udon of Comparative Trial Production Example 13-3, a ring in the following ratio, the mixture was dissolved by slight effect of imparting elasticity was recognized. However, heating up to Bx (Brix) 40. The obtained solution was put in rigid hardness was merely imparted and this effect may have an ice cream maker and cooled with stirring for 35 minutes. a bad effect on noodles. On the other hand, regarding the udon The obtained materials was transferred to a container and then of Trial Production Example 13-1, the effect of imparting 35 frozen to obtain a frozen dessert. Sticky texture with excellent chewiness was recognized. TABLE 24 Comparative Comparative Trial Production Example 14 Trial Trial Trial Trial 40 Production Production Production Production Formulation Example Example Example Example Preparation of Jelly Candy (Parts) 15-1 15-2 15-1 15-2 Starch syrup 18 18 18 18 In accordance with the formulations shown in Table 23 Granulated Sugar 12 12 12 12 below, white Sugar, starch syrup, a starch and water were 45 Fresh cream 12 12 12 12 Vegetable oil 6 6 6 6 mixed with stirring in the following ratio, the mixture was and fat dissolved by heating up to Bx (Brix) 75. The obtained solu Chemically 2.4 tion was filled in a mold, and left at normal temperature for 24 unmodified cassava hours. After confirming that the solution has been solidified, starch Note (2) 50 Chemically 2.4 it was removed from the mold to obtain a jelly candy. modified cassava starch Note (1) TABLE 23 Starch prepared in 2.4 Example 9 Comparative Comparative Starch prepared in 2.4 Trial Trial Trial Trial 55 Example 10-2 Production Production Production Production Guar gum 0.4 0.4 0.4 0.4 Formulation Example Example Example Example Emulsifier O.2 O.2 O.2 O.2 (Parts) 14-1 14-2 14-1 14-2 Water 49 49 49 49 Note (1) Sugar 34 34 34 34 Starch syrup 30 30 30 30 Chemically modified cassava starch: “CH EMISTAR 300S, manufactured by GLICO 60 FOODS CO.,LTD.CH EMISTAR” is a registered trademark of GLICO FOODS CO.,LTD. Chemically 21 Note (2) unmodified cassava Chemically unmodifie cassava starch: Untreated native cassava starch, starch Noe (2) Chemically 21 modified cassava The obtained frozen desserts showed the following results. starch Yo'e () That is, both the frozen desserts of Trial Production Examples Starch prepared in 21 65 15-1 and 15-2 had appropriate viscoelasticity and sticky tex Example 9 ture, and had nice melt texture in mouth. On the other hand, the frozen dessert of Comparative Trial Production Example US 9,005,681 B2 85 86 15-1 had sticky texture and the frozen dessert of Comparative and stirred at 50° C. for 18 hours to carry out an enzyme Trial Production Example 15-2 also had sticky texture and reaction. After completion of the reaction, an enzyme-treated spinnability. However, both the frozen desserts had strong starch was recovered by centrifugal filtration and blow dry pasty sensation and poor melt in mouth. ing. Viscosity characteristics of the obtained enzyme-treated starch were analyzed by the amylograph and the rheometer. Example 13-1 Also, after completion of the reaction, a degradation ratio was determined by a part of a reaction solution. The results are To 400 g of an untreated native wheat starch, 900 g of shown in Table 4-2. As a result, the setback viscosity of it was ion-exchange water was added to prepare a starch Suspension. 2 (BU). After adjusting the pH of the suspension to 5.0, 1% by weight 10 (based on starch Solid content) of cyclodextrin glucanotrans Comparative Example 18 ferase (“Toruzyme 3.0 L derived from Bacillus lichenifor mis, manufactured by Novo: optimum pH of 6.0) was added To 500 g of an untreated native cassava starch, 750 g of an and stirred at 50° C. for 18 hours to carry out an enzyme aqueous 6.7% (w/w) sodium sulfate solution was added to reaction. After completion of the reaction, an enzyme-treated 15 prepare a starch Suspension. After adjusting the pH of the starch was recovered by centrifugal filtration and blow dry Suspension to 8.5, 7.36 g of a vinyl acetate monomer was ing. Viscosity characteristics of the obtained enzyme-treated added and stirred at 30° C. for 40 minutes to allow a reaction starch were analyzed by the amylograph and the rheometer. proceed. After 40 minutes, the pH of the suspension was Also, after completion of the reaction, a degradation ratio was adjusted to 6.0 and the reaction was terminated. After comple determined by a part of a reaction solution. The results are tion of the reaction, starch acetate was recovered by centrifu shown in Table 2-2. As a result, the setback viscosity of it was gal filtration and blow drying. Viscosity characteristics of the 7.0 (BU). obtained starch acetate were analyzed by the amylograph and Example 13-2 the rheometer. 25 To 400 g of an untreated native wheat starch, 900 g of Comparative Example 19 ion-exchange water was added to prepare a starch Suspension. After adjusting the pH of the suspension to 6.0, 1% by weight To 500 g of an untreated native cassava starch, 785 g of an (based on starch Solid content) of cyclodextrin glucanotrans aqueous 11% (w/w) sodium sulfate solution was added to ferase (Cyclodextrin Cycrodextrin glucanotransferase 30 prepare a starch Suspension. After adjusting the pH of the Amano’ derived from Paenibacillus macerans (Bacillus Suspension to 11.0, 24 g of propylene oxide was added and macerans), manufactured by Amano Enzyme) was added and stirred at 42°C. for 16 hours to allow a reaction proceed. After stirred at 50° C. for 18 hours to carry out an enzyme reaction. 16 hours, the pH of the suspension was adjusted to 6.0 and the After completion of the reaction, an enzyme-treated Starch reaction was terminated. After completion of the reaction, a was recovered by centrifugal filtration and blow drying. Vis 35 hydroxypropyl starch was recovered by centrifugal filtration cosity characteristics of the obtained enzyme-treated Starch and blow drying. Viscosity characteristics of the obtained were analyzed by the amylograph and the rheometer. Also, hydroxypropyl Starch were analyzed by the amylograph and after completion of the reaction, a degradation ratio was the rheometer. determined by a part of a reaction solution. The results are shown in Table 2-2. 40 Comparative Example 20 Example 14 To 500 g of an untreated native cassava starch, 750 g of an aqueous 6.7% (w/w) sodium sulfate solution was added to To 400 g of an untreated native corn starch, 900 g of prepare a starch Suspension. After adjusting the pH of the ion-exchange water was added to prepare a starch Suspension. 45 Suspension to 11.0, 10 ul of phosphorus oxychloride was After adjusting the pH of the suspension to 5.0, 1% by weight added and stirred at 30° C. for 1 hour to allow a reaction (based on starch Solid content) of cyclodextrin glucanotrans proceed. After 1 hour, the pH of the suspension was adjusted ferase (“Toruzyme 3.0 L derived from Bacillus lichenifor to 6.0 and the reaction was terminated. After completion of mis, manufactured by Novo: optimum pH of 6.0) was added the reaction, a distarch phosphate was recovered by centrifu and stirred at 50° C. for 18 hours to carry out an enzyme 50 gal filtration and blow drying. Viscosity characteristics of the reaction. After completion of the reaction, an enzyme-treated obtained distarch phosphate were analyzed by the amylo starch was recovered by centrifugal filtration and blow dry graph and the rheometer. ing. Viscosity characteristics of the obtained enzyme-treated starch were analyzed by the amylograph and the rheometer. Comparative Example 21 Also, after completion of the reaction, a degradation ratio was 55 determined by a part of a reaction solution. The results are To 500 g of an untreated native cassava starch, 910 g of an shown in Table 3-2. As a result, the setback viscosity of it was aqueous 10% (w/w) sodium sulfate solution was added to 0 (BU). prepare a starch Suspension. After adjusting the pH of the Suspension to 11.0, 16 g of propylene oxide was added and Example 15 60 stirred at 42°C. for 16 hours to allow an etherification reac tion proceed. After 16 hours, the temperature of the starch To 400 g of an untreated native cassava starch, 900 g of Suspension was adjusted to 30°C., 5 Jul of phosphorus oxy ion-exchange water was added to prepare a starch Suspension. chloride was added and stirred at 30°C. for 1 hour to carry out After adjusting the pH of the suspension to 5.0, 1% by weight a crosslinking reaction proceed. After 1 hour, the pH of the (based on starch Solid content) of cyclodextrin glucanotrans 65 Suspension was adjusted to 6.0 and the entire reaction was ferase (“Toruzyme 3.0 L derived from Bacillus lichenifor terminated. After completion of the reaction, a hydroxypro mis, manufactured by Novo: optimum pH of 6.0) was added pyl distarch phosphate was recovered by centrifugal filtration US 9,005,681 B2 87 88 and blow drying. Viscosity characteristics of the obtained to prepare a starch Suspension. After adjusting the pH of the hydroxypropyl distarch phosphate were analyzed by the amy suspension to 5.0, 1% by weight (based on starch solid con lograph and the rheometer. tent) of C.-amylase (AMYLEXA3’ derived from Aspergillus niger, manufactured by DANISCO; optimum pH of 5.0) was Example 16 added and stirred at 50° C. for 18 hours to carry out an enzyme To 4 Kg of an untreated native cassava starch, 9 Kg of reaction. After completion of the reaction, an enzyme-treated ion-exchange water was added to prepare a starch Suspension. starch was recovered by centrifugal filtration and blow dry After adjusting the pH of the suspension to 5.0, 1% by weight ing. Viscosity characteristics of the obtained enzyme-treated (based on starch solid content) of amyloglucosidase (“OPTI starch were analyzed by the amylograph and the rheometer. DEXL-400' derived from Aspergillus niger, manufactured 10 Also, after completion of the reaction, a degradation ratio was by Genencor; optimum pH of 4.4) was added and stirred at determined using a part of the reaction solution. 50° C. for 18 hours to carry out an enzyme reaction. After completion of the reaction, an enzyme-treated Starch was Example 21 recovered by centrifugal filtration and blow drying. It is noted that the degradation ratio of the obtained sample was 21%. 15 To 400 g of the distarch phosphate prepared in Compara tive Example 20, 900 g of ion-exchange water was added to Example 17 prepare a starch Suspension. After adjusting the pH of the suspension to 5.0, 1% by weight (based on starch solid con To 400 g of the starch acetate prepared in Comparative Example 18,900 g of ion-exchange water was added to pre tent) of amyloglucosidase (“OPTIDEXL-400' derived from pare a starch Suspension. After adjusting the pH of the Sus Aspergillus niger, manufactured by Genencor, optimum pH pension to 5.0, 1% by weight (based on starch solid content) of 4.4) was added and stirred at 50° C. for 18 hours to carry out of amyloglucosidase (“OPTIDEX L-400' derived from an enzyme reaction. After completion of the reaction, an Aspergillus niger, manufactured by Genencor, optimum pH enzyme-treated starch was recovered by centrifugal filtration of 4.4) was added and stirred at 50° C. for 18 hours to carry out and blow drying. Viscosity characteristics of the obtained an enzyme reaction. After completion of the reaction, an 25 enzyme-treated Starch were analyzed by the amylograph and enzyme-treated starch was recovered by centrifugal filtration the rheometer. Also, after completion of the reaction, a deg and blow drying. Viscosity characteristics of the obtained radation ratio was determined using a part of the reaction enzyme-treated Starch were analyzed by the amylograph and Solution. the rheometer. After completion of the reaction, a degradation ratio was determined using a part of the reaction solution. 30 Example 22 Example 18 To 400 g of the distarch phosphate prepared in Compara tive Example 20, 900 g of ion-exchange water was added to To 400 g of the starch acetate prepared in Comparative prepare a starch Suspension. After adjusting the pH of the Example 18,900 g of ion-exchange water was added to pre 35 suspension to 5.0, 1% by weight (based on starch solid con pare a starch Suspension. After adjusting the pH of the Sus tent) of C.-amylase (AMYLEXA3’ derived from Aspergillus pension to 5.0, 1% by weight (based on starch solid content) niger, manufactured by DANISCO; optimum pH of 5.0) was of C.-amylase (AMYLEX A3' derived from Aspergillus added and stirred at 50° C. for 18 hours to carry out an enzyme niger, manufactured by DANISCO; optimum pH of 5.0) was reaction. After completion of the reaction, an enzyme-treated added and stirred at 50° C. for 18 hours to carryout an enzyme 40 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 determined using a part of the reaction solution. Also, after completion of the reaction, a degradation ratio was 45 determined using a part of the reaction solution. Example 23 Example 19 To 400g of the hydroxypropyl distarch phosphate prepared To 400 g of the hydroxypropyl starch prepared in Com in Comparative Example 21, 900 g of ion-exchange water parative Example 19,900 g of ion-exchange water was added 50 was added to prepare a starch Suspension. After adjusting the to prepare a starch Suspension. After adjusting the pH of the pH of the suspension to 5.0, 1% by weight (based on starch suspension to 5.0, 1% by weight (based on starch solid con solid content) of amyloglucosidase (“OPTIDEX L-400' tent) of amyloglucosidase (“OPTIDEXL-400' derived from derived from Aspergillus niger, manufactured by Genencor, Aspergillus niger, manufactured by Genencor, optimum pH optimum pH of 4.4) was added and stirred at 50° C. for 18 of 4.4) was added and stirred at 50° C. for 18 hours to carry out 55 hours to carry out an enzyme reaction. After completion of the an enzyme reaction. After completion of the reaction, an reaction, an enzyme-treated Starch was recovered by centrifu enzyme-treated starch was recovered by centrifugal filtration gal filtration and blow drying. Viscosity characteristics of the and blow drying. Viscosity characteristics of the obtained obtained enzyme-treated Starch were analyzed by the amylo enzyme-treated Starch were analyzed by the amylograph and graph and the rheometer. Also, after completion of the reac the rheometer. Also, after completion of the reaction, a deg 60 tion, a degradation ratio was determined using a part of the radation ratio was determined using a part of the reaction reaction Solution. Solution. Example 24 Example 20 65 To 400g of the hydroxypropyl distarch phosphate prepared To 400 g of the hydroxypropyl starch prepared in Com in Comparative Example 21, 900 g of ion-exchange water parative Example 19,900 g of ion-exchange water was added was added to prepare a starch Suspension. After adjusting the US 9,005,681 B2 89 90 pH of the suspension to 5.0, 1% by weight (based on starch characteristics of the obtained enzyme-treated distarch phos solid content) of C.-amylase (AMYLEX A3' derived from phate were analyzed by the amylograph and the rheometer. Aspergillus niger, manufactured by DANISCO: optimum pH Also, after completion of the reaction, a degradation ratio was of 5.0) was added and stirred at 50° C. for 18 hours to carry out determined by a part of the reaction solution. an enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration Example 28 and blow drying. Viscosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and To 500g of the enzyme-treated starch prepared in Example the rheometer. Also, after completion of the reaction, a deg 16, 910 g of an aqueous 10% (w/w) sodium sulfate solution radation ratio was determined using a part of the reaction 10 was added to prepare a starch Suspension. After adjusting the Solution. pH of the suspension to 11.0, 16 g of propylene oxide was Example 25 added and stirred at 42° C. for 16 hours to carry out an etherification reaction. After 16 hours, the temperature of the To 500g of the enzyme-treated starch prepared in Example 15 starch suspension was adjusted to 30° C. and 5 ul of phos 16, 750 g of an aqueous 6.7% (w/w) sodium sulfate solution phorus oxychloride was added and stirred at 30°C. for 1 hour was added to prepare a starch Suspension. After adjusting the to carry out a crosslinking reaction. After 1 hour, the pH of the pH of the suspension to 8.5, 7.36 g of a vinyl acetate monomer Suspension was adjusted to 6.0 and the entire reaction was was added and stirred at 30° C. for 40 minutes to carry out a terminated. After completion of the reaction, an enzyme reaction. After 40 minutes, the pH of the suspension was treated hydroxypropyl distarch phosphate was recovered by adjusted to 6.0 and the reaction was terminated. After comple centrifugal filtration and blow drying. Viscosity characteris tion of the reaction, an acetic acid enzyme-treated Starch was tics of the obtained enzyme-treated hydroxypropyl distarch recovered by centrifugal filtration and blow drying. Viscosity phosphate were analyzed by the amylograph and the rheom characteristics of the obtained acetic acid enzyme-treated eter. Also, after completion of the reaction, a degradation ratio starch were analyzed by the amylograph and the rheometer. 25 was determined by a part of the reaction Solution. Also, after completion of the reaction, a degradation ratio was The measurement results of Comparative Examples 18 to determined by apart of the reaction solution. 21 and Examples 17 to 28 are shown in Table 25-2. It is noted that in an analysis by the rheometer of the present starch Example 26 which used the chemical modification and the enzymatic 30 treatment in combination, after refrigeration storage at 5°C. To 500g of the enzyme-treated starch prepared in Example for 16 hours the gel did not have the hardness sufficient for the 16, 785 g of an aqueous 11% (w/w) sodium sulfate solution measurement. Therefore, it was difficult to compare physical was added to prepare a starch Suspension. After adjusting the properties of the gels. Therefore, confirmation was carried out pH of the Suspension to 11.0, 24 g of propylene oxide was after refrigeration storage at 5°C. for 21 days. The details are added and stirred at 42°C. for 16 hours to carry out a reaction. 35 as follows. After 16 hours, the pH of the suspension was adjusted to 6.0 A starch paste was prepared so that the concentration of the and the reaction was terminated. After completion of the starch was 20% by weight on the dry matter basis, and then reaction, a hydroxypropyl enzyme-treated Starch was recov filled in a Krehalon casing having a folding width of 45 mm. ered by centrifugal filtration and blow drying. Viscosity char This starch paste filled in the casing was heated to 90° C. at 1 acteristics of the obtained hydroxypropyl enzyme-treated 40 C./min and maintained at 90° C. for 30 minutes. Then, the starch were analyzed by the amylograph and the rheometer. starch paste was left to cool in a constant-temperature water Also, after completion of the reaction, a degradation ratio was bath at 20° C. for 30 minutes, and then it was cooled to 5°C. determined by a part of the reaction solution. in a refrigerator. After cooling, it was refrigerated at 5°C. for 21 days, then it was left at room temperature (about 25°C.) Example 27 45 for 4 hours to return the temperature of it to room temperature, and then the measurement was carried out with a rheometer To 500g of the enzyme-treated starch prepared in Example (RT-2010J-CW) manufactured by Rheotech Inc. The mea 16, 750 g of an aqueous 6.7% (w/w) sodium sulfate solution Surement was carried out under the measurement conditions was added to prepare a starch Suspension. After adjusting the of the rheometer: a test item: a rupture test; a height of a pH of the suspension to 11.0, 10ul of phosphorus oxychloride 50 sample: 25 mm; and a movement rate (rupture rate) of a was added and stirred at 30° C. for 1 hour to carry out a sample: 6 cm/min, using an adapter of a spherical jig for reaction. After 1 hour, the pH of the Suspension was adjusted measurement viscosity (p5 (diameter: 5 mm, area: 19.635 to 6.0 and the reaction was terminated. After completion of mm). At the measurement, the hardness of the starch gel was the reaction, an enzyme-treated distarch phosphate was evaluated by a rupture stress (g) and a Young's modulus recovered by centrifugal filtration and blow drying. Viscosity (dyn/cm). 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 US 9,005,681 B2 91 92 TABLE 25-1-continued 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 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 OO 2,499,026 OO Example17 7 727 81 433 145 2O 2,923,860 17 Example18 9 693 77 429 142 17 3,023,821 21 Example25 21 797 89 472 215 78 4,169,554 67 Comp. Ex. 19 1004 OO 670 33 OO 373,715 OO Example19 2O 845 84 550 47 42 483,149 29 Example20 21 661 66 470 43 30 669,986 79 Example26 21 824 82 525 71 215 573,625 53 Comp. Ex. 20 903 OO O 170 OO 3,943,691 OO Example21 21 908 O1 O 283 66 5,319,388 35 Example22 15 871 96 O 337 98 7,529,620 91 Example27 21 898 99 O 554 326 10,027,924 2S4 Comp. Ex. 21 769 OO 403 31 OO 250,328 OO Example23 19 740 96 413 45 45 391,117 56 Example24 21 524 68 356 64 2O6 1,218,285 487 Example28 21 784 O2 2O7 125 403 1497,293 598 Comp. Ex. 22 317 OO 227 91 OO 4,468,130 OO Example29 7 419 32 324 119 31 5,953,997 33 Example30 9 411 30 315 101 11 5,048,987 13 Comp. Ex. 23 715 OO 181 137 OO 4494,603 OO Example31 22 673 94 138 224 64 5,293,378 18 Example32 31 630 88 143 237 73 6,764,052 50 * Degradation ratios in Examples 25 to 28 each refer to a degradation ratio of an enzyme-treated starch used as a base material,

It was confirmed that when the chemical modification and Comparative Example 22 the enzymatic treatment are used in combination, particularly when the distarch phosphate is subjected to the enzymatic 55 Oxidized Starch treatment, gel forming ability can be enhanced while main taining a maximum viscosity. This is an extremely excellent To 500 g of an untreated native cassava starch, 750 g of advantage as compared with the fact that when phosphate ion-exchange water was added to prepare a starch Suspension. crosslinking is increased in a conventional chemical modifi After adjusting the pH of the suspension to 10.0, 2.5 g of cation, the gel becomes harder but the maximum viscosity 60 sodium hypochlorite whose effective chlorine amount is 10% was added and stirred at 30° C. for 2 hours to carry out a drastically decreases, thus leading to the cause of powderi reaction, while maintaining the pH of the Suspension at 10.0. ness. It was also confirmed for not only the distarch phosphate After 2 hours, the pH of the suspension was adjusted to 6.0 but also other chemically modified starches that, by carrying and then 2 g of sodium hydrogen sulfite was added. Immedi out an enzymatic treatment, it is possible to enhance gel 65 ately after stirring, the pH of the Suspension was adjusted to forming ability while relatively maintaining the viscosity as 6.0 and the reaction was terminated. After completion of the compared with a conventional chemically modified Starch. reaction, the oxidized starch was recovered by centrifugal US 9,005,681 B2 93 94 filtration and blow drying. Viscosity characteristics of the Aspergillus niger, manufactured by Genencor, optimum pH obtained oxidized starch were analyzed by the amylograph of 4.4) was added and stirred at 50° C. for 18 hours to carry out and the rheometer. an enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration Comparative Example 23 and blow drying. Viscosity characteristics of the obtained enzyme-treated Starch were analyzed by the amylograph and Bleached Starch the rheometer. Also, after completion of the reaction, a deg radation ratio was determined using a part of the reaction To 500 g of an untreated native cassava starch, 700 g of Solution. ion-exchange water was added to prepare a starch Suspension. 10 After adjusting the pH of the suspension to 11.0, 2.5 g of Example 32 sodium hypochlorite whose effective chlorine amount is 10% was added and stirred at 30°C. for 5 minutes while maintain In the Case where a Bleached Starch was Treated ing the pH of the suspension at 11. Then, 0.25 g of sodium with an O-Amylase metabisulfite was added and stirred for 10 minutes, the pH of 15 the Suspension was adjusted to 6.0 and the reaction was ter To 400 g of the bleached starch prepared in Comparative minated. After completion of the reaction, the bleached starch Example 23,900 g of ion-exchange water was added to pre was recovered by centrifugal filtration and blow drying. Vis pare a starch Suspension. After adjusting the pH of the Sus cosity characteristics of the obtained bleached starch were pension to 5.0, 1% by weight (based on starch solid content) analyzed by the amylograph and the rheometer. of C.-amylase (AMYLEX A3' derived from Aspergillus Example 29 niger, manufactured by DANISCO; optimum pH of 5.0) was added and stirred at 50° C. for 18 hours to carry out an enzyme In the Case where an Oxidized Starch was Treated reaction. After completion of the reaction, an enzyme-treated with an Amyloglucosidase starch was recovered by centrifugal filtration and blow dry 25 ing. Viscosity characteristics of the obtained enzyme-treated To 400 g of the oxidized starch prepared in Comparative starch were analyzed by the amylograph and the rheometer. Example 22,900 g of ion-exchange water was added to pre Also, after completion of the reaction, a degradation ratio was pare a starch Suspension. After adjusting the pH of the Sus determined using a part of the reaction Solution. The mea pension to 5.0, 1% by weight (based on starch solid content) surement results of Comparative Examples 22 to 23 and of amyloglucosidase (“OPTIDEX L-400' derived from 30 Examples 29 to 32 are shown in Table 25-2. Aspergillus niger, manufactured by Genencor, optimum pH of 4.4) was added and stirred at 50° C. for 18 hours to carry out Comparative Example 24 an enzyme reaction. After completion of the reaction, an enzyme-treated starch was recovered by centrifugal filtration To 2 kg of an untreated native corn starch, ion-exchange and blow drying. Viscosity characteristics of the obtained 35 enzyme-treated Starch were analyzed by the amylograph and water was added thereby adjusting the water content to 21%. the rheometer. Also, after completion of the reaction, a deg The resultant was filled in a 3 L glass beaker in a state where radation ratio was determined using a part of the reaction blank space was as Small as possible, and the upper portion Solution. was covered with an aluminum foil, and then heated at 120° C. for 15 minutes to carry out aheat-moisture treatment. After Example 30 40 completion of the heat-moisture treatment, the heat-mois ture-treated starch was recovered by blow drying. In the Case where an Oxidized Starch was Treated with an O-Amylase Example 33 To 400 g of the oxidized starch prepared in Comparative 45 To 4 Kg of an untreated native corn starch, 9 Kg of ion Example 22,900 g of ion-exchange water was added to pre exchange water was added to prepare a starch Suspension. pare a starch Suspension. After adjusting the pH of the Sus After adjusting the pH of the suspension to 5.0, 1% by weight pension to 5.0, 1% by weight (based on starch solid content) (based on starch solid content) of amyloglucosidase (“OPTI of C.-amylase (AMYLEX A3' derived from Aspergillus DEXL-400' derived from Aspergillus niger, manufactured niger, manufactured by DANISCO; optimum pH of 5.0) was 50 by Genencor; optimum pH of 4.4) was added and stirred at added and stirred at 50° C. for 18 hours to carry out an enzyme 50° C. for 18 hours to carry out an enzyme reaction. After reaction. After completion of the reaction, an enzyme-treated completion of the reaction, an enzyme-treated Starch was starch was recovered by centrifugal filtration and blow dry recovered by centrifugal filtration and blow drying. It is noted ing. Viscosity characteristics of the obtained enzyme-treated that the degradation ratio of the obtained sample was 34%. starch were analyzed by the amylograph and the rheometer. 55 Also, after completion of the reaction, a degradation ratio was Example 34 determined using a part of the reaction solution. To 4 Kg of an untreated native corn starch, 9 Kg of ion Example 31 exchange water was added to prepare a starch Suspension. In the Case where a Bleached Starch was Treated 60 After adjusting the pH of the suspension to 5.0, 1% by weight with an Amyloglucosidase (based on starch solid content) of C-amylase (AMYLEX A3' derived from Aspergillus niger, manufactured by To 400 g of the bleached starch prepared in Comparative DANISCO: optimum pH of 5.0) was added and stirred at 50° Example 23, 900 g of ion-exchange water was added to C. for 18 hours to carry out an enzyme reaction. After comple prepare a starch Suspension. After adjusting the pH of the 65 tion of the reaction, an enzyme-treated Starch was recovered suspension to 5.0, 1% by weight (based on starch solid con by centrifugal filtration and blow drying. It is noted that the tent) of amyloglucosidase (“OPTIDEXL-400' derived from degradation ratio of the obtained sample was 28%. US 9,005,681 B2 95 96 Example 35 Example 38 To 400 g of the heat-moisture-treated starch prepared in Comparative Example 24, 900 g of ion-exchange water was To 400g of the enzyme-treated starch prepared in Example added to prepare a starch Suspension. After adjusting the pH 34, ion-exchange water was added thereby adjusting the of the suspension to 5.0, 1% by weight (based on starch solid water content to 20%. The resultant was filled in a 1 L. glass content) of amyloglucosidase (“OPTIDEX L-400 derived beaker in a state where blank space was as Small as possible, from Aspergillus niger, manufactured by Genencor, optimum and the upper portion was covered with an aluminum foil, and pH of 4.4) was added and stirred at 50° C. for 18 hours to carry then heated at 120° C. for 15 minutes to carry out a heat out an enzyme reaction. After completion of the reaction, an 10 moisture treatment. After completion of the heat-moisture enzyme-heat-moisture-treated Starch was recovered by cen- treatment, the heat-moisture-enzyme-treated starch was trifugal filtration and blow drying. Viscosity characteristics of recovered by blow drying. Viscosity characteristics of the the obtained enzyme-heat-moisture-treated Starch were ana- obtained heat-moisture-enzyme-treated Starch were analyzed lyzed by the amylograph and the rheometer. Also, after by the amylograph and the rheometer. Also, after completion completion of the reaction, a degradation ratio was deter- 15 of the reaction, a degradation ratio was determined by a part mined using a part of the reaction solution. of the reaction solution. The measurement results of Com parative Example 24 and Examples 35 to 38 are shown in Example 36 Table 26-2. TO 400 g of the heat-moisture-treated starch prepared in 20 TABLE 26-1 Comparative Example 24, 900 g of ion-exchange water was added to prepare a starch Suspension. After adjusting the pH Summary of Names, Origins and Product Names of Enzymes of the suspension to 5.0, 1% by weight (based on starch solid Used for a Treatment of a Heat-moisture-treated Starch content) of C-amylase (AMYLEX A3” derived from Prod Aspergillus niger, manufactured by DANISCO: optimum pH 25 Name of time of 5.0) was added and Stirred at 50° C. for 18 hours to carry out Example enzyme Origin (manufacture) an enzyme reaction. After completion of the reaction, an enzyme-heat-moisture-treated Starch was recovered by cen- Comparative — Untreated corn trifugal filtration and blow drying. Viscosity characteristics of Example 24 starch the obtained enzyme-heat-moisture-treated Starch were ana- 30 Example 35 amyloglucosidase Aspergillus niger OPTIDEXL-400 lyzed by the amylograph and the rheometer. Also, after (Genencor) completion of the reaction, a degradation ratio was deter- Example 36 C-amylase Aspergillus niger AMYLEXA3(DANISCO) mined uS1ng a part of the reaction solution. Example 37 amyloglucosidase Aspergillus niger OPTIDEXL-400 (Genencor) Example 37 35 Example 38 C-amylase Aspergillus niger AMYLEXA3 (DANISCO) To 400g of the enzyme-treated starch prepared in Example 33, ion-exchange water was added thereby adjusting the 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) % (%) (BU) (g) % (%) (dyn/cm) % (%) Comparative 4O1 100 108 230 100 6,091,460 100 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.

55 water content to 21%. The resultant was filled in a 1 L. glass As described above, the present invention has been exem beaker in a state where blank space was as Small as possible, plified using a preferred embodiment of the present invention, and the upper portion was covered with an aluminum foil, and but the present invention should not be construed to be limited then heated at 120° C. for 15 minutes to carry out a heat 60 to this embodiment. It is understood that the present invention moisture treatment. After completion of the heat-moisture should be construed for its scope only by the claims. It is treatment, the heat-moisture-enzyme-treated Starch was understood that those skilled in the art can practice an equiva recovered by blow drying. Viscosity characteristics of the lent range based on the description of the invention and the obtained heat-moisture-enzyme-treated Starch were analyzed technical common knowledge, from the description of the by the amylograph and the rheometer. Also, after completion 65 specific preferable embodiment of the present invention. It is of the reaction, a degradation ratio was determined by a part understood that patents, patent applications and publications of the reaction solution. cited in the present specification should be herein incorpo US 9,005,681 B2 97 98 rated by reference for the content thereofas if the contents be further increased, thus making it possible to increase a themselves were specifically described in the present speci yield factor. Furthermore, for a food such as kudzu starch fication. cake so-called in the Kanto area, which requires longtime and much labor heretofore because the preparation of a wheat INDUSTRIAL APPLICABILITY 5 starch as a raw material requires a fermentation process for a long period Such as one or more years, use of an enzyme As described above, the present invention provides various treated wheat starch of the present invention makes it possible industrial advantages by using an enzyme having character- to easily prepare a kudzu Starch cake so-called in the Kanto istics capable of increasing a maximum viscosity of a starch. area which does not have fermentation odor derived from a According to the present invention, it becomes possible to 10 fermented wheat starch and has nice flavor, without requiring provide a food having new textures which could not be Such long time and much labor. obtained by a conventional chemically unmodified Starch and Furthermore, when the enzyme-treated cassava starch of a chemically modified starch. For example, use of the the present invention is used in noodles, for example, raw enzyme-treated cassava starch in the present invention makes udon, the texture improving effect of impairing Sticky texture it possible to prepare a cookie which is very light and soft and 15 with rich chewiness is recognized, and no adverse influence has texture with nice melt in mouth, and to provide a cookie was exerted on factors of the quality of noodles. Such as having readily edible texture which is also suited for persons 'slippery and Smooth” and 'sogginess'. Thus, it has been of advanced age and infants. In addition, since the dough at found that the addition of this enzyme-treated starch easily the time of shaping a cookie is very dry and not sticky, the improve the texture of the noodle to those favored by Japa water addition amount at the time of preparing the dough can S.

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 cc 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 gaa aat cag 144 Met Met Thr His Pro Ser Tyr Ile Trp Lieu. Tyr Lieu Ser Glu Asn Gln 35 4 O 45

citt titt citt gtt ggg tat titt cat cag titt cac ttic gta gcc tict cat 192 Leu Phe Leu Val Gly Tyr Phe His Glin Phe His Phe Val Ala Ser His SO 55 60

agc ggc titt citt tot att caa goc atc aat gtt acc cat tcc atc ctd 24 O Ser Gly Phe Leu Ser Ile Glin Ala Ile Asn Val Thr His Ser Ile Leu 65 70 7s 8O

gtt tot citt act tct ct a titc aca agc ggit cqg tot gtg gat act aaa 288 Val Ser Lieu. Thir Ser Leu Phe Thr Ser Gly Arg Ser Val Asp Thr Lys 85 90 95

tac gtg gtt aaa at a gaa gag goa cag cat citt act aaa citc cca toa 336 Tyr Val Val Lys Ile Glu Glu Ala Glin His Lieu. Thir Lys Lieu Pro Ser 1OO 105 110

tgg gat acc cct gac aat tog citc atg cta caa ggit titt gaa togg cat 384 Trp Asp Thr Pro Asp Asn Ser Leu Met Leu Gln Gly Phe Glu Trp His 115 12O 125

gtt C ca gat gat caa ggg cat tdg aaa cqt ctt Caa cqc to a cta gtg 432 Val Pro Asp Asp Glin Gly His Trp Lys Arg Lieu. Glin Arg Ser Lieu Val 13 O 135 14 O

agt cta aaa tog att ggt gtc gac agt att tog att coa ccg gga tigt 48O Ser Lieu Lys Ser Ile Gly Val Asp Ser Ile Trp Ile Pro Pro Gly Cys 145 15 O 155 16 O

US 9,005,681 B2 101 102 - Continued ggit at C aga cgc ggc gtc a.a.a. aat cc c atg act C Ca t cc tgt ggc gga 488 Gly Ile Arg Arg Gly Wall Asn Pro Met Thir Pro Ser Cys Gly Gly 485 490 495 aag citt CC a gtt citt gca cgg gct cgt aag citt tat gct tac ggc gala 536 Lys Luell Pro Wall Lell Ala Arg Ala Arg Lys Luell Ala Tyr Gly Glu SOO 505 51O

Cala tgc gac tat titt gat Cala gcc aat tgc atc. gga tto gtC cgt tat 584 Glin Cys Asp Phe Asp Glin Ala Asn Cys Ile Gly Phe Wall Arg 515 52O 525 ggc aac ttg Cat CaC cc.g t cc ggt Cta gca tgc atc. atg agc aac 999 632 Gly Asn Luell His His Pro Ser Gly Luell Ala Cys Ile Met Ser Asn Gly 53 O 535 54 O ggit gcg tot cag a.a.a. cgt atg tac gt C gga cgg agc Cat gcc aag gag Gly Ala Ser Glin Lys Arg Met Wall Gly Arg Ser His Ala Lys Glu 5.45 550 555 560 cga tgg aca gac att 999 tgg Cat CC a aag a Ca gtt at C at C gat 728 Arg Trp Thir Asp Ile Lell Gly Trp His Pro Lys Thir Wall Ile Ile Asp 565 st O sts aag a.a.a. ggt tat 999 ata titt cott gtt tot gca atg Cag gtt agt gt C 776 Lys Gly Tyr Gly Ile Phe Pro Wall Ser Ala Met Glin Wall Ser Wall 58O 585 59 O tgg gtg aac tog gcc gca gaa gcg aga gala agt citt Cala gag cott ttic 824 Trp Wall Asn Ser Ala Ala Glu Ala Arg Glu Ser Lell Glin Glu Pro Phe 595 6OO 605 gag gag aag att tac gag aat tga 848 Glu Glu Lys Ile Tyr Glu Asn 610 615

SEQ ID NO 2 LENGTH: 615 TYPE : PRT ORGANISM: Aspergillus oryzae

< 4 OOs SEQUENCE: 2

Met Pro Ser Lys Wall Thir Glin Luell Thir Gly Wall Pro His Thir Asp 1. 5 15

Cys Luell Gly Thir Glu Ala Wall Asp Thir Ser Ile Glu Wall Glu Arg Arg 25

Met Met Thir His Pro Ser Ile Trp Luell Lell Ser Glu Asn Glin 35 4 O 45

Lell Phe Luell Wall Gly Phe His Glin Phe His Phe Wall Ala Ser His SO 55 6 O

Ser Gly Phe Luell Ser Ile Glin Ala Ile Asn Wall Thir His Ser Ile Luell 65 70

Wall Ser Luell Thir Ser Lell Phe Thir Ser Gly Arg Ser Wall Asp Thir 85 90 95

Wall Wall Lys Ile Glu Glu Ala Glin His Luell Thir Luell Pro Ser 105 11 O

Trp Asp Thir Pro Asp Asn Ser Luell Met Luell Glin Gly Phe Glu Trp His 115 12 O 125

Wall Pro Asp Asp Glin Gly His Trp Arg Luell Glin Arg Ser Luell Wall 13 O 135 14 O

Ser Luell Ser Ile Gly Wall Asp Ser Ile Trp Ile Pro Pro Gly Cys 145 150 155 160

Ala Met Asn Pro Ser Gly Asn Gly Tyr Asp Ile Asp Luell Tyr 1.65 17O 17s

Asp Luell Gly Glu Phe Asp Glin Gly Ser Arg Ser Thir Lys Trp Gly 18O 185 19 O US 9,005,681 B2 103 104 - Continued

Ser Thir Glu Lell Glin Ser Luell Ala Ser Ala Arg Asn Luell Gly 195 2OO 2O5

Ile Gly Ile Trp Asp Ala Wall Luell Asn His Lys Ala Gly Ala Asp 21 O 215 22O

Tyr Thir Glu Arg Phe Ser Ala Wall Wall Asp Pro Lys Asp Arg Ser 225 23 O 235 24 O

Wall Glu Ile Phe Ala Ala Arg Glu Ile Glu Gly Trp Wall Gly Phe Ser 245 250 255

Phe Pro Gly Arg Gly Gly Ile Ser Ser Met Ser Trp His 26 O 265 27 O

His Phe Ser Gly Wall Asp Trp Asp Glu Ala Arg Lys Asn Ala Ile 27s 285

Arg Wall Ala Ser Arg Trp Ser Asp Asp Wall Ala His Glu 29 O 295 3 OO

Gly Asn Tyr Asp Tyr Lell Met Phe Ala Asp Luell Asp Ser Asn Luell 3. OS 310 315

Glu Wall Glin Asp Wall Lell Arg Trp Gly Glu Trp Ile Gly Ser Glin 3.25 330 335

Lell Pro Luell Trp Gly Met Arg Luell Asp Ala Ser His Tyr Ser Ala 34 O 345 35. O

Asp Phe Glin Lys Phe Wall Asn His Wall Arg Ala Thir Wall Gly Pro 355 360 365

Glin Ile Phe Phe Wall Ala Glu Trp Ser Gly Asp Wall Arg Wall Luell 37 O 375 38O

Met His Luell Glin Lys Met Asp Glin Luell Ser Lell Phe Asp Ala 385 390 395 4 OO

Pro Luell Wall Gly Arg Phe Ser Arg Ile Ser Arg Thir Gly Glu Asp Luell 4 OS 415

Arg Glu Ile Phe Asp Asp Thir Luell Wall Gly ASn Pro Ala His Ala 425 43 O

Ile Thir Luell Wall Met Asn His Asp Thir Wall Arg Glu Arg Glin Ser Luell 435 44 O 445

Glu Ala Pro Ile Ala Ser Phe Phe Pro Luell Ala Ala Luell Ile 450 45.5 460

Lell Luell Arg Asp Gly Glin Pro Ile Phe Gly Asp Luell Tyr 465 470

Gly Ile Arg Arg Gly Wall Asn Pro Met Thir Pro Ser Gly Gly 485 490 495

Luell Pro Wall Lell Ala Arg Ala Arg Luell Tyr Ala Tyr Gly Glu SOO 505 51O

Glin Asp Phe Asp Glin Ala Asn Ile Gly Phe Wall Arg 515 525

Gly Asn Luell His His Pro Ser Gly Luell Ala Cys Ile Met Ser Asn Gly 53 O 535 54 O

Gly Ala Ser Glin Arg Met Wall Gly Arg Ser His Ala Glu 5.45 550 555 560

Arg Trp Thir Asp Ile Lell Gly Trp His Pro Lys Thir Wall Ile Ile Asp 565 st O sts

Gly Gly Ile Phe Pro Wall Ser Ala Met Glin Wall Ser Wall 585 59 O

Trp Wall Asn Ser Ala Ala Glu Ala Arg Glu Ser Lell Glin Glu Pro Phe 595 6OO 605

US 9,005,681 B2 107 108 - Continued

Ala Thir Cys Pro Glin Asn Wall Met Asp Gly Val Lieu. Asn 26 O 265 27 O c cc att tac tat C Ca citc. citc. aac gcc ttic aag toa a CC to C ggc agc 864 Pro Ile Tyr Pro Lell Lell Asn Ala Phe Lys Ser Thir Ser Gly Ser 27s 28O 285 atg gac gac citc. tac aac atg at C aac acc gtc a.a.a. gac tdt CC a 912 Met Asp Asp Luell Tyr Asn Met Ile Asn Thir Wall Lys Ser Asp Cys Pro 29 O 295 3 OO gac to a aca citc. Ctg ggc a Ca ttic gt C gag aac CaC gac aac Coa. cgg 96.O Asp Ser Thir Luell Lell Gly Thir Phe Wall Glu ASn His Asp As Pro Arg 3. OS 310 315 32O tto gct tot tac a CC aac gac at a gcc citc. gcc aag aac gtC goa gca OO8 Phe Ala Ser Thir Asn Asp Ile Ala Luell Ala Lys Asn Wall Ala Ala 3.25 330 335 tto at C at C citc. aac gac gga at C cc c at C atc. tac gcc ggc cala gala Phe Ile Ile Luell Asn Asp Gly Ile Pro Ile Ile Ala Gly Glin Glu 34 O 345

Cag CaC tac gcc ggc gga aac gac cc c gcg aac cgc gaa gca acc tgg 104 Glin His Tyr Ala Gly Gly Asn Asp Pro Ala ASn Arg Glu Ala Thr Trp 355 360 365 citc. tog ggc tac cc.g a CC gac agc gag Ctg tac aag tta att gcc to c 152 Lell Ser Gly Pro Thir Asp Ser Glu Luell Tyr Lys Lell Ile Ala Ser 37 O 375 38O gcg aac gca at C cgg aac tat gcc att agc a.a.a. gat a Ca gga titc gtg 2OO Ala Asn Ala Ile Arg Asn Ala Ile Ser Lys Asp Thir Gly Phe Wall 385 390 395 4 OO a CC tac aag aac tgg c cc atc. tac a.a.a. gac gac a Ca acg atc gcc atg 248 Thr Lys Asn Trp Pro Ile Asp Asp Thr Thr Ile Ala Met 4 OS 41O 415 cgc aag ggc aca gat 999 tcg cag at C gtg act atc. ttg to C aac aag 296 Arg Lys Gly Thir Asp Gly Ser Glin Ile Wall Thir Ile Lell Ser Asn Lys 42O 425 43 O ggit gct tog ggt gat tcg tat acc citc. to c ttg agt ggit gcg ggt tac 344 Gly Ala Ser Gly Asp Ser Thir Luell Ser Luell Ser Gly Ala Gly 435 44 O 445 a Ca gcc ggc cag Cala ttg acg gag gt C att ggc tgc acg acc gtg acg 392 Thir Ala Gly Glin Glin Lell Thir Glu Wall Ile Gly Cys Thir Thir Wall Thir 450 45.5 460 gtt ggt tog gat gga aat gtg cott gtt cott atg gca ggit ggg ct a cott 44 O Wall Gly Ser Asp Gly Asn Wall Pro Wall Pro Met Ala Gly Gly Lieu. Pro 465 470 47s 48O agg gta ttg tat cc.g act gag aag ttg gca ggt agc aag atc tdt agt 488 Arg Wall Luell Pro Thir Glu Lys Luell Ala Gly Ser Lys Ile Cys Ser 485 490 495 agc tog tga 497 Ser Ser

SEQ ID NO 4 LENGTH: 498 TYPE : PRT ORGANISM: Aspergillus niger

< 4 OOs SEQUENCE: 4.

Met Val Ala Trp Trp Ser Lieu. Phe Luell Tyr Gly Lieu. Glin Wall Ala Ala 1. 5 15

Pro Ala Lieu Ala Ala Thr Pro Ala Asp Trp Arg Ser Glin Ser Ile Tyr 25 3O

Phe Lieu. Lieu. Thir Asp Arg Phe Ala Arg Thir Asp Gly Ser Th Thr Ala 35 4 O 45 US 9,005,681 B2 109 110 - Continued

Thir Cys Asn Thir Ala Asp Glin Cys Gly Gly Thir Trp Glin Gly SO 55 6 O

Ile Ile Asp Lell Asp Ile Glin Gly Met Gly Phe Thir Ala Ile 65 70

Trp Ile Thir Pro Wall Thir Ala Glin Luell Pro Glin Thir Thir Ala Tyr Gly 85 90 95

Asp Ala His Gly Trp Glin Glin Asp Ile Ser Luell Asn Glu 105 11 O

Asn Gly Thir Ala Asp Asp Luell Ala Luell Ser Ser Ala Luell His 115 12 O 125

Glu Arg Gly Met Tyr Lell Met Wall Asp Wall Wall Ala Asn His Met Gly 13 O 135 14 O

Tyr Asp Gly Ala Gly Ser Ser Wall Asp Ser Wall Phe 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 Thir Glin Wall Glu Asp Trp Luell Gly Asp Asn Thir Wall Ser 18O 185 19 O

Lell Pro Asp Luell Asp Thir Thir Lys Asp Wall Wall Asn Glu Trp 195

Asp Trp Wall Gly Ser Lell Wall Ser Asn Ser Ile Asp Gly Luell Arg 21 O 215

Ile Asp Thir Wall Lys His Wall Glin Asp Phe Trp Pro Gly Asn 225 23 O 235 24 O

Ala Ala Gly Wall Ile Gly Glu Wall Lieu Asp Gly Asp Pro 245 250 255

Ala Thir Cys Pro Glin Asn Wall Met Asp Gly Wall Luell Asn 26 O 265 27 O

Pro Ile Tyr Pro Lell Lell Asn Ala Phe Ser Thir Ser Gly Ser 27s 285

Met Asp Asp Luell Tyr Asn Met Ile Asn Thir Wall Lys Ser Asp Pro 29 O 295 3 OO

Asp Ser Thir Luell Lell Gly Thir Phe Wall Glu ASn His Asp Asn Pro Arg 3. OS 310 315

Phe Ala Ser Thir Asn Asp Ile Ala Luell Ala Asn Wall Ala Ala 3.25 330 335

Phe Ile Ile Luell Asn Asp Gly Ile Pro Ile Ile 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

Lell Ser Gly Pro Thir Asp Ser Glu Luell Lys Lell Ile Ala Ser 37 O 375

Ala Asn Ala Ile Arg Asn Ala Ile Ser Lys Asp Thir Gly Phe Wall 385 390 395 4 OO

Thir Asn Trp Pro Ile Asp Asp Thir Thir Ile Ala Met 4 OS 415

Arg Gly Thir Asp Gly Ser Glin Ile Wall Thir Ile Lell Ser Asn 425 43 O

Gly Ala Ser Gly Asp Ser Thir Luell Ser Luell Ser Gly Ala Gly 435 44 O 445

Thir Ala Gly Glin Glin Lell Thir Glu Wall Ile Gly Cys Thir Thir Wall Thir 450 45.5 460

Wall Gly Ser Asp Gly Asn Wall Pro Wall Pro Met Ala Gly Gly Luell Pro

US 9,005,681 B2 115 116 - Continued atc. tot cag Ctg ggit gac tgg gala acc agc gac ggc ata gct Ctg agt 728 Ile Ser Glin Luell Gly Asp Trp Glu Thir Ser Asp Gly Ile Ala Luell Ser 565 st O sts gct gac aag tac act t cc agc gac cc.g citc. tgg tat gtc act gtg act 776 Ala Asp Lys Tyr Thir Ser Ser Asp Pro Luell Trp Wall Thir Wall Thir 58O 585 59 O

Ctg cc.g gct ggt gag tcg titt gag tac aag titt atc. cgc att gag agc 824 Lell Pro Ala Gly Glu Ser Phe Glu Lys Phe Ile Arg Ile Glu Ser 595 6OO 605 gat gac to c gtg gag tgg gag agt gat cc c aac cga gaa tac acc gtt 872 Asp Asp Ser Wall Glu Trp Glu Ser Asp Pro ASn Arg Glu Thir Wall 610 615 62O cott cag gcg tgc gga acg tcg acc gcg acg gtg act gac acc tgg cgg 92 O Pro Glin Ala Cys Gly Thir Ser Thir Ala Thir Wall Thir Asp Thir Trp Arg 625 630 635 64 O tag 923

SEQ ID NO 6 LENGTH: 64 O TYPE : PRT ORGANISM: Aspergillus niger

< 4 OOs SEQUENCE: 6

Met Ser Phe Arg Ser Lell Lell Ala Luell Ser Gly Lell Wall Thir Gly 1. 5 1O 15

Lell Ala Asn Wall Ile Ser Arg Ala Thir Luell Asp Ser Trp Luell Ser 25

Asn Glu Ala Thr Wall Ala Arg Thr Ala Ile Lieu. Asn Asn Ile Gly Ala 35 4 O 45

Asp Gly Ala Trp Wall Ser Gly Ala Asp Ser Gly Ile Wall Wall Ala Ser SO 55 6 O

Pro Ser Thir Asp Asn Pro Asp Phe Thir Trp Thir Arg Asp Ser 65 70

Gly Luell Wall Luell Lys Thir Lell Wall Asp Luell Phe Arg Asn Gly Asp Thir 85 90 95

Ser Luell Luell Ser Thir Ile Glu Asn Tyr Ile Ser Ala Glin Ala Ile Wall 105 11 O

Glin Gly Ile Ser Asn Pro Ser Gly Asp Luell Ser Ser Gly Ala Gly Luell 115 12 O 125

Gly Glu Pro Phe Asn Wall Asp Glu Thir Ala Tyr Thir Gly Ser Trp 13 O 135 14 O

Gly Arg Pro Glin Arg Asp Gly Pro Ala Luell Arg Ala Thir Ala Met Ile 145 150 155 160

Gly Phe Gly Glin Trp Lell Lell Asp Asn Gly Thir Ser Thir Ala Thir 1.65 17O

Asp Ile Wall Trp Pro Lell Wall Arg Asn Asp Luell Ser Wall Ala Glin 18O 185 19 O

Trp Asn Glin Thir Gly Asp Luell Trp Glu Glu Wall Asn Gly Ser 195 2O5

Ser Phe Phe Thir Ile Ala Wall Glin His Arg Ala Lell Wall Glu Gly Ser 21 O 215

Ala Phe Ala Thir Ala Wall Gly Ser Ser Ser Trp Asp Ser Glin 225 23 O 235 24 O

Ala Pro Glu Ile Lell Luell Glin Ser Phe Trp Thir Gly Ser Phe 245 250 255

Ile Luell Ala Asn Phe Asp Ser Ser Arg Ser Gly Asp Ala Asn Thir US 9,005,681 B2 117 118 - Continued

26 O 265 27 O

Lell Luell Gly Ser Ile His Thir Phe Asp Pro Glu Ala Ala Asp Asp 28O 285

Ser Thir Phe Glin Pro Ser Pro Arg Ala Luell Ala Asn His Glu 29 O 295 3 OO

Wall Wall Asp Ser Phe Arg Ser Ile Thir Luell Asn Asp Gly Luell Ser 3. OS 310 315

Asp Ser Glu Ala Wall Ala Wall Gly Arg Tyr Pro Glu Asp Thir Tyr Tyr 3.25 330 335

Asn Gly Asn Pro Trp Phe Lell Thir Luell Ala Ala Ala Glu Glin Luell 34 O 345 35. O

Asp Ala Luell Tyr Glin Trp Asp Glin Gly Ser Lell Glu Wall Thir 355 360 365

Asp Wall Ser Luell Asp Phe Phe Ala Luell Ser Asp Ala Ala Thir 37 O 375

Gly Thir Ser Ser Ser Ser Ser Thir Ser Ser Ile Wall Asp Ala 385 390 395 4 OO

Wall Thir Phe Ala Asp Gly Phe Wall Ser Ile Wall Glu Thir His Ala 4 OS 415

Ala Ser Asn Gly Ser Met Ser Glu Glin Tyr Asp Ser Asp Gly Glu 425 43 O

Glin Luell Ser Ala Arg Asp Lell Thir Trp Ser Ala Ala Luell Luell Thir 435 44 O 445

Ala Asn Asn Arg Arg Asn Ser Wall Wall Pro Ala Ser Trp Gly Glu Thir 450 45.5 460

Ser Ala Ser Ser Wall Pro Gly Thir Ala Ala Thir Ser Ala Ile Gly 465 470 47s

Thir Ser Ser Wall Thir Wall Thir Ser Trp Pro Ser Ile Wall Ala Thir 485 490 495

Gly Gly Thir Thir Thir Thir Ala Thir Pro Thir Gly Ser Gly Ser Wall Thir SOO 505

Ser Thir Ser Thir Thir Ala Thir Ala Ser Thir Ser Thir Ser Thir 515 525

Ser Ser Thir Ser Cys Thir Thir Pro Thir Ala Wall Ala Wall Thir Phe Asp 53 O 535 54 O

Lell Thir Ala Thir Thir Thir Gly Glu Asn Ile Lell Wall Gly Ser 5.45 550 555 560

Ile Ser Glin Luell Gly Asp Trp Glu Thir Ser Asp Ile Ala Luell Ser 565 st O sts

Ala Asp Tyr Thir Ser Ser Asp Pro Luell Trp Wall Thir Wall Thir 585 59 O

Lell Pro Ala Gly Glu Ser Phe Glu Phe Ile Arg Ile Glu Ser 595 6OO 605

Asp Asp Ser Wall Glu Trp Glu Ser Asp Pro ASn Arg Glu Thir Wall 610 615 62O

Pro Glin Ala Cys Gly Thir Ser Thir Ala Thir Wall Thir Asp Thir Trp Arg 625 630 635 64 O

SEO ID NO 7 LENGTH: 2334 TYPE: DNA ORGANISM: Flavobacterium sp. FEATURE: NAME/KEY: CDS LOCATION: (1) ... (2334)

US 9,005,681 B2 123 124 - Continued

agc cgc agc gcg citc. gag gcc gac CaC gag acc tac a CC aag cgc Ctg 1920 Ser Arg Ser Ala Lell Glu Ala Asp His Glu Thir Thir Lys Arg Luell 625 630 635 64 O atc. gcg ttic cgc aag gcg CaC cc.g gcg Ctg cgc cc.g gcg aac ttic tat 1968 Ile Ala Phe Arg Lys Ala His Pro Ala Luell Arg Pro Ala Asn Phe Tyr 645 650 655 tcg gcc agc gac a CC aac ggc aac gtg atg gag Cag ttg cgc tgg ttic Ser Ala Ser Asp Thir Asn Gly Asn Wall Met Glu Glin Lell Arg Trp Phe 660 665 67 O aag cc c gac ggc gcg Cag gcc gac agc gcc tac tto aac ggc gcc gac Lys Pro Asp Gly Ala Glin Ala Asp Ser Ala Tyr Phe Asn Gly Ala Asp 675 68O 685 aac CaC gcc Ctg gcc tgg cgc at C gac ggc agc gag tto ggc gac agc 2112 Asn His Ala Luell Ala Trp Arg Ile Asp Gly Ser Glu Phe Gly Asp Ser 69 O. 695 7 OO gcc agc gcg at C tac gtc gcc tac aac ggc tgg t cc ggc gcg gt C gac 216 O Ala Ser Ala Ile Tyr Wall Asn Gly Trp Ser Gly Ala Wall Asp 7 Os 71O 72O tto aag Ctg cc.g tgg cc.g acc ggc aag cag tgg tac cgg gt C acc Phe Lys Luell Pro Trp Pro Thir Gly Lys Glin Trp Arg Wall Thir 72 73 O 73 gat acc gcg acc tgg aac ggc cc c aac gC9 gtg gcg Ctg cc c ggc 2256 Asp Thir Ala Thir Trp Asn Gly Pro Asn Ala Wall Ala Luell Pro Gly 740 74. 7 O agc gag acc atc. ggc gag aac acc gtc tac ggc atg cag gcg 23O4. Ser Glu Thir Luell Ile Gly Glu Asn Thir Wall Gly Met Glin Ala 7ss 760 765 cgc tog Ctg Ctg ttg Ctg gcg aag tga 2334 Arg Ser Luell Luell Lell Lell Ala Lys 770 775

<210s, SEQ ID NO 8 &211s LENGTH: 777 212. TYPE : PRT &213s ORGANISM: Flavobacterium sp.

<4 OOs, SEQUENCE: 8

Met Asp Pro His Ala Pro Glin Arg Glin Arg Ser Gly Glin Arg Luell Arg 1. 15

Ala Luell Ala Luell Ala Ala Lell Ala Cys Ala Luell Ser Pro Ala His Ala 2O 25

Ala Ile Asp Ala Glin Glin Lell Gly Ala Arg Asp Ala Ala Glin Ala 35 4 O 45

Asn Luell Ala Phe Wall Tyr Ser Ser Arg Ala Thir Arg Wall Glu Wall SO 55 6 O

Phe Luell Asn Pro Thir Gly Ser Glin Wall Ala Arg Luell Ala 65 70

Lell Ser Asp Pro Ala Thir Glin Wall Trp Ser Lell Ser Luell Pro Thir 85 90 95

Ser Thir Ile Lys Asn Thir Gly Ile Thir Gly Ala Wall Tyr Gly 105 11 O

Arg Ala Trp Gly Pro Asn Trp Pro Asp Ala Ala Trp Thir Lys 115 12 O 125

Gly Ser Ala Thir Gly Phe Wall Ser Asp Wall Asp Asn Ala Gly Asn Arg 13 O 135 14 O

Phe Asn Pro Asn Lys Lell Lell Luell Asp Pro Tyr Ala Arg Glu Ile Ser 145 150 155 160 US 9,005,681 B2 125 126 - Continued

Glin Asp Pro Asn Thir Ala Thir Ala Asp Gly Thir Ile Tyr Ala Thir 1.65 17O 17s

Gly Ala Ala His Arg Asn Asp Ser Gly Luell Cys Ala Ser Lys Gly 18O 185 19 O

Ile Ala Luell Ala Ala Asp Ala Thir Ser Wall Gly Ser Lys Pro Thir Arg 195

Ala Luell Asp Glu Wall Ile Glu Wall His Wall Arg Gly Luell Thir 21 O 215 22O

Arg Asn Asp Asp Ser Wall Pro Ala Ala Glu Arg Gly Thir Gly 225 23 O 235 24 O

Ala Ala Arg Ala Ala Ala Luell Ala Ala Luell Gly Wall Thir Ala Wall 245 250 255

Glu Phe Luell Pro Wall Glin Glu Thir Glin Asn Asp Glin Asn Asp Wall Asp 26 O 265 27 O

Pro Asn Ser Thir Ala Gly Asp Asn Trp Gly Met Thir Luell Asn 27s 285

Phe Ala Pro Asp Arg Arg Ala Asp Lys Ser Ala Gly Gly 29 O 295 3 OO

Pro Thir Arg Glu Trp Lys Ala Met Wall Ala Phe His Asp Ala Gly 3. OS 310 315

Ile Wall Ile Asp Wall Wall Asn His Thir Gly Glu Gly Gly 3.25 330 335

Pro Trp Ser Gly Thir Asp Gly Luell Ser Wall Asn Lell Luell Ser Phe 34 O 345 35. O

Arg Gly Lieu Asp Asn Pro Ala Tyr Ser Lieu. Ser Ser Asp 355 360 365

Pro Trp Asp Asn Thir Gly Wall Gly Gly ASn Tyr Asn Thir Arg His 37 O 375

Pro Ile Ala Glin Asn Lell Ile Wall Asp Ser Luell Ala Trp Arg Asp 385 390 395 4 OO

Ala Luell Gly Wall Asp Gly Phe Arg Phe Asp Luell Ala Ser Wall Luell Gly 4 OS 415

Asn Ser Glin His Gly Phe Asn Phe Asp Asn Asp Ser Gly 425 43 O

Asn Ala Luell Asn Arg Ile Wall Ala Glu Luell Pro Pro Arg Pro Ala Ala 435 44 O 445

Gly Gly Ala Gly Ala Asp Lell Ile Ala Glu Pro Trp Ala Ile Gly Gly 450 45.5 460

Asn Ser Glin Wall Gly Gly Phe Pro Ala Gly Trp Ala Glu Trp Asn 465 470

Gly Luell Tyr Arg Asp Ala Lell Arg Lys Glin Asn Luell Gly Wall 485 490 495

Glu Thir Wall Thir Pro Gly Thir Luell Ala Thir Arg Phe Ala Gly Ser Asn SOO 505

Asp Luell Tyr Gly Asp Asp Gly Arg Pro Trp His Ser Ile Asn Phe 515 525

Wall Wall Ala His Asp Gly Phe Thir Luell Asn Asp Lell Tyr Ala Asn 53 O 535 54 O

Asp Glin Asn Asn Glin Pro Trp Pro Tyr Gly Pro Ser Asp Gly Gly 5.45 550 555 560

Glu His Asn Lell Ser Trp Asn Glin Gly Gly Ile Wall Ala Glu Glin 565 st O sts

Arg Ala Ala Arg Thir Gly Luell Ala Luell Luell Met Lell Ser Ala Gly US 9,005,681 B2 127 128 - Continued

585 59 O

Wall Pro Met Ile Thir Gly Gly Asp Glu Ala Luell Arg Thir Glin Phe Gly 595 605

Asn Asn Asn Thir Tyr Asn Lell Asp Ser Ala Ala Trp Luell Tyr Trp 610 615

Ser Arg Ser Ala Lell Glu Ala Asp His Glu Thir Thir Arg Luell 625 630 635 64 O

Ile Ala Phe Arg Lys Ala His Pro Ala Luell Arg Pro Ala Asn Phe Tyr 645 650 655

Ser Ala Ser Asp Thir Asn Gly Asn Wall Met Glu Glin Lell Arg Trp Phe 660 665 67 O

Pro Asp Gly Ala Glin Ala Asp Ser Ala Tyr Phe Asn Gly Ala Asp 675 685

Asn His Ala Luell Ala Trp Arg Ile Asp Gly Ser Glu Phe Gly Asp Ser 69 O. 695 7 OO

Ala Ser Ala Ile Tyr Wall Ala Asn Gly Trp Ser Gly Ala Wall Asp 7 Os

Phe Luell Pro Trp Pro Gly Thir Gly Lys Glin Trp Arg Wall Thir 72 73 O 73

Asp Thir Ala Thir Trp Asn Glu Gly Pro Asn Ala Wall Ala Luell Pro Gly 740 74. 7 O

Ser Glu Thir Luell Ile Gly Gly Glu Asn Thir Wall Gly Met Glin Ala 760 765

Arg Ser Luell Luell Lell Lell Ile Ala 770 775

SEO ID NO 9 LENGTH: 2316 TYPE: DNA ORGANISM: Pseudomonas amyloderamosa FEATURE: NAME/KEY: CDS LOCATION: (1) . . (2316)

<4 OOs, SEQUENCE: atg aag tgc CC a att citc. ggc gcg Ctg citt ggc tgc gcg gtg citc. 48 Met Lys Cys Pro Ile Lell Gly Ala Luell Luell Gly Cys Ala Wall Luell 1. 1O 15 gct ggt gtg cc c gca atg cc.g gcg Cat gcg gcc atc. aac agc atg agc 96 Ala Gly Wall Pro Ala Met Pro Ala His Ala Ala Ile Asn Ser Met Ser 2O 25 3O

Ctg ggc gcg agc tac gac gcg Cala cag gcc aac atc. a CC titt cgc gtt 144 Lell Gly Ala Ser Tyr Asp Ala Glin Glin Ala ASn Ile Thir Phe Arg Wall 35 4 O 45 tac to c tog cag gcc acg cgc at C gtg Ctg tac citc. tat tog gca ggt 192 Ser Ser Glin Ala Thir Arg Ile Wall Luell Tyr Lell Tyr Ser Ala Gly SO 55 6 O tac ggt gtg cag gag tcg gcc acc tac acg Ctg agc C Ca gcg ggc agt 24 O Tyr Gly Wall Glin Glu Ser Ala Thir Thir Luell Ser Pro Ala Gly Ser 65 70 7s 8O ggit gta tgg gcg gtg acg gtg cc.g gtg tog tog atc. aag gcg gcc ggc 288 Gly Wall Trp Ala Wall Thir Wall Pro Wall Ser Ser Ile Ala Ala Gly 85 90 95 atc. acg 999 gcg gtg tac tac 999 tat gcc tgg 999 cc.g aat tgg 336 Ile Thir Gly Ala Wall Gly Tyr Arg Ala Trp Gly Pro Asn Trp 1OO 105 11 O cott tat gcc agc aac tgg ggc aag ggt tog cag gcg ggc gtt to c 384 Pro Ala Ser Asn Trp Gly Gly Ser Glin Ala Gly Wall Ser

US 9,005,681 B2 133 134 - Continued ggc gcg acc acc tat ggg caa to ggit caa ticg Ctg ctg. Ctg ttg 23O4. Gly Ala Gly. Thir Thr Tyr Gly Glin Cys Gly Glin Ser Lieu. Leu Lleu Lieu 760 765 atc. to c aag tag 2316 Ile Ser Lys 770

SEQ ID NO 10 LENGTH: 771 TYPE : PRT ORGANISM: Pseudomonas amyloderamosa

< 4 OOs SEQUENCE: 10

Met Lys Cys Pro Lys Ile Lell Gly Ala Luell Luell Gly Ala Wall Luell 1. 5 15

Ala Gly Wall Pro Ala Met Pro Ala His Ala Ala Ile Asn Ser Met Ser 25

Lell Gly Ala Ser Tyr Asp Ala Glin Glin Ala ASn Ile Thir Phe Arg Wall 35 4 O 45

Ser Ser Glin Ala Thir Arg Ile Wall Luell Tyr Lell Tyr Ser Ala Gly SO 55 6 O

Tyr Gly Wall Glin Glu Ser Ala Thir Thir Luell Ser Pro Ala Gly Ser 65 70

Gly Wall Trp Ala Wall Thir Wall Pro Wall Ser Ser Ile Ala Ala Gly 85 90 95

Ile Thir Gly Ala Wall Gly Tyr Arg Ala Trp Gly Pro Asn Trp 105 11 O

Pro Ala Ser Asn Trp Gly Lys Gly Ser Glin Ala Gly Cys Wall Ser 115 12 O 125

Asp Wall Asp Ala Asn Gly Asp Arg Phe Asn Pro Asn Luell Luell Luell 13 O 135 14 O

Asp Pro Ala Glin Glu Wall Ser Glin Asp Pro Lell Asn Pro Ser Asn 145 150 155 160

Glin Asn Asn Wall Phe Ala Ser Ala His Arg Thir Thir Asp Ser 1.65 17s

Gly Ile Ala Pro Gly Wall Wall Luell Wall Pro Ser Thir Glin Ser 18O 185 19 O

Thir Gly Thir Pro Thir Arg Ala Glin Asp Asp Wall Ile Glu 195

Wall His Wall Arg Gly Phe Thir Glu Glin Asp Thir Ser Ile Pro Ala Glin 21 O 215 22O

Tyr Arg Gly Thir Tyr Tyr Gly Ala Gly Luell Lys Ala Ser Luell Ala 225 23 O 235 24 O

Ser Luell Gly Wall Thir Ala Wall Glu Phe Luell Pro Wall Glin Glu Thir Glin 245 250 255

Asn Asp Ala Asn Asp Wall Wall Pro Asn Ser Asp Ala Asn Glin Asn Tyr 26 O 265 27 O

Trp Gly Tyr Met Thir Glu Asn Tyr Phe Ser Pro Asp Arg Arg Ala 27s 285

Asn Ala Ala Gly Gly Pro Thir Ala Glu Phe Glin Ala Met Wall 29 O 295 3 OO

Glin Ala Phe His Asn Ala Gly Ile Wall Tyr Met Asp Wall Wall Tyr 3. OS 310 315 32O

Asn His Thir Ala Glu Gly Gly Thir Trp Thir Ser Ser Asp Pro Thir Thir 3.25 330 335 US 9,005,681 B2 135 136 - Continued

Ala Thir Ile Tyr Ser Trp Arg Gly Luell Asp ASn Ala Thir Tyr Tyr Glu 34 O 345 35. O

Lell Thir Ser Gly Asn Glin Phe Asp ASn Thir Gly Ile Gly Ala 355 360 365

Asn Phe Asn Thir Tyr Asn Thir Wall Ala Glin ASn Lell Ile Wall Asp Ser 37 O 375

Wall Ala Trp Ala Asn Thir Met Gly Wall Asp Gly Phe Arg Phe Asp 385 390 395 4 OO

Lell Ala Ser Wall Lell Gly Asn Ser Luell ASn Ala Wall His Ala Ser 4 OS 415

Ala Pro Asn Cys Pro Asn Gly Gly Tyr Asn Phe Asp Ala Ala Asp Ser 425 43 O

Asn Wall Ala Ile Asn Arg Ile Luell Arg Glu Phe Thir Wall Arg Pro Ala 435 44 O 445

Ala Gly Gly Thir Wall Trp Ile Luell Arg ASn Lell Gly Pro Ser Ala 450 45.5 460

Ala Thir Arg Thir Ser Trp Wall Asp Ser Arg Arg Wall Wall Arg Wall Glu 465 470

Trp Ser Wall Pro Arg Glin Lell Arg Glin Ala Glin Asn Glu Luell Gly Ser 485 490 495

Met Thir Ile Tyr Wall Thir Glin Asp Ala Asn Asp Phe Ser Gly Ser Ser SOO 505

Asn Luell Phe Glin Ser Ser Gly Arg Ser Pro Trp Asn Ser Ile Asn Phe 515 525

Ile Asp Wall His Asp Gly Met Thr Lieu Asp Wall Ser Asn 53 O 535 54 O

Gly Ala Asn Asn Ser Glin Ala Ser Gly Pro Ser Asp Gly Gly Thir 5.45 550 555 560

Ser Thir Asn Ser Trp Asp Glin Gly Met Ser Ala Gly Thir Gly Ala 565 st O sts

Ala Wall Asp Glin Arg Arg Ala Ala Arg Thir Gly Met Ala Phe Glu Met 58O 585 59 O

Lell Ser Ala Gly Thir Pro Lell Met Glin Gly Gly Asp Glu Luell Arg 595 605

Thir Luell Glin Asn Asn Asn Ala Asn Luell Asp Ser Ser Ala Asn 610 615

Trp Luell Thir Ser Trp Thir Thir Asp Glin Ser Asn Phe Thir Phe 625 630 635 64 O

Ala Glin Arg Luell Ile Arg Ser Ala Arg His Ile Pro Lell Arg Pro Ser 645 650 655

Ser Trp Ser Gly Ser Glin Luell Thir Trp Glin Pro Ser Gly Ala 660 665 67 O

Wall Ala Asp Ser Asn Trp Asn Asn Thir Ser Asn Tyr Ala Ile Ala 675 685

Ala Ile Asn Gly Pro Ser Luell Gly Asp Ser Asn Ser Ile Wall 69 O. 695 7 OO

Ala Asn Gly Trp Ser Ser Ser Wall Thir Phe Thir Lell Pro Ala Pro 7 Os 71O

Pro Ser Gly Thir Glin Trp Arg Wall Thir Asp Thir Asp Trp Asn 72 73 O 73

Asp Gly Ala Ser Thir Phe Wall Ala Pro Gly Ser Glu Thir Luell Ile Gly 740 74. 7 O

Gly Ala Gly Thir Thir Gly Glin Gly Glin Ser Lell Luell Luell Luell US 9,005,681 B2 137 138 - Continued

760 765 Ile Ser Lys 770

SEQ ID NO 11 LENGTH: 2598 TYPE: DNA ORGANISM: Aspergillus niger FEATURE: NAME/KEY: CDS LOCATION: (1) ... (2598)

<4 OOs, SEQUENCE: 11 atg ttg 999 tot ttg citt tta citc. tta cc c citt gtg ggc gct gct gt C 48 Met Luell Gly Ser Lell Lell Lell Luell Luell Pro Luell Wall Gly Ala Ala Wall 1. 5 1O 15 att gga cc c agg gca aac agt cag agt tgc CCa 999 tat aag gcg to c 96 Ile Gly Pro Arg Ala Asn Ser Glin Ser Cys Pro Gly Lys Ala Ser 2O 25 3O aac gt C Cala aag Cag gct agg to a Ctg act gC9 gat Ctg act Cta gct 144 Asn Wall Glin Lys Glin Ala Arg Ser Luell Thir Ala Asp Lell Thir Luell Ala 35 4 O 45 ggit acg cott tgt aat agc tat ggc aag gat ttg gaa gac citc. aag Ctg 192 Gly Thir Pro Cys Asn Ser Tyr Gly Lys Asp Luell Glu Asp Luell Lys Luell SO 55 6 O citt gtg gala tat Cag act gat gala cgg tta Cat gtt atg at C tac gat 24 O Lell Wall Glu Glin Thir Asp Glu Arg Luell His Wall Met Ile Asp 65 70 7s 8O gCC gac gag gala gtc tat ca gtt cct gala tca gtc citt cct cgc gtg 288 Ala Asp Glu Glu Wall Glin Wall Pro Glu Ser Wall Lell Pro Arg Wall 85 90 95 ggit agt gac gag gac tot gag gac agt gtt ttg gaa titt gac tat gtg 336 Gly Ser Asp Glu Asp Ser Glu Asp Ser Wall Luell Glu Phe Asp Wall 1OO 105 11 O gaa gala cc.g titt toa tto a CC at C to c aag gga gat gag gtC Ctg titt 384 Glu Glu Pro Phe Ser Phe Thir Ile Ser Lys Gly Asp Glu Wall Luell Phe 115 12 O 125 gac tot tog gca toa C Ca Cta gtt titt cag tog Cala tat gtg aac citt 432 Asp Ser Ser Ala Ser Pro Lell Wall Phe Glin Ser Glin Wall Asn Luell 13 O 135 14 O cgc acc tgg ttg c cc gat gat cc c gtg tat ggit citc. gga gag Cat Arg Thir Trp Luell Pro Asp Asp Pro Wall Tyr Gly Lell Gly Glu His 145 150 155 160 tot gac cott atg cgc ttg C Ca aca aat tac acg cgg acc citt tgg 528 Ser Asp Pro Met Arg Lell Pro Thir Asn Thir Arg Thir Luell Trp 1.65 17O 17s aac cgc gac gcg tat ggc act CC a aac aac acc aac ttg tac ggt agt 576 Asn Arg Asp Ala Tyr Gly Thir Pro Asn Asn Thir Asn Lell Tyr Gly Ser 18O 185 19 O

Cat cott gt C tac tat gat CaC gga aag to c gga act tat gga gt C 624 His Pro Wall Tyr Asp His Arg Gly Ser Gly Thir Gly Wall 195 2OO 2O5 tto Ctg Ctg aac tot aat ggit atg gac at C aag atc. aac Cala acg aca 672 Phe Luell Luell Asn Ser Asn Gly Met Asp Ile Ile Asn Glin Thir Thir 21 O 215 22O gat gga aag cag tac ttg gaa tac aat citt citc. ggc ggit gtt Ctg gac 72 O Asp Gly Glin Tyr Lell Glu Asn Luell Luell Gly Gly Wall Luell Asp 225 23 O 235 24 O tto tac ttic ttic tac gga gaa gat cott aag Cala gcg agc atg gala tac 768 Phe Phe Phe Tyr Gly Glu Asp Pro Lys Glin Ala Ser Met Glu 245 250 255

US 9,005,681 B2 143 144 - Continued

<210s, SEQ ID NO 12 &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 Leu 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 9,005,681 B2 145 146 - Continued

Pro Asp Trp Phe Asn Glu Gly Thir Glin Asp Tyr Trp Thir Ala Glin Phe 385 390 395 4 OO

Glin Glin Phe Phe Asp Pro Ser Gly Wall Asp Ile Asp Ala Luell Trp 4 OS 415

Ile Asp Met Asn Glu Ala Ser Asn Phe Pro Pro Cys Luell Asp 425 43 O

Pro Ala Ala Ala Ile Ser Ala Asp Luell Pro Pro Ala Ala Pro Pro 435 44 O 445

Wall Arg Pro Ser Ser Pro Ile Pro Luell Pro Gly Phe Pro Ala Asp Phe 450 45.5 460

Glin Pro Ser Ser Lys Arg Ser Wall Arg Ala Glin Gly Asp Gly 465 470

Wall Gly Lell Pro Asn Arg Asn Luell Thir Asp Pro Pro Tyr Thir 485 490 495

Ile Arg Asn Ala Ala Gly Wall Luell Ser Met Ser Thir Ile Glu Thir Asp SOO 505

Lell Ile His Ala Gly Glu Gly Tyr Ala Glu Asp Thir His Asn Luell 515 525

Gly Thir Arg Lell Wall Met Ser Ser Ala Ser Arg Thir Ala Met Glin 53 O 535 54 O

Ala Arg Arg Pro Asp Wall Arg Pro Luell Wall Ile Thir Arg Ser Thir Phe 5.45 550 555 560

Ala Gly Ala Gly Ala His Wall Gly His Trp Luell Gly Asp Asn Phe Ser 565 st O sts

Asp Trp Wall His Tyr Arg Ile Ser Ile Ala Gln Ile Lieu Ser Phe Ala 585 59 O

Ser Met Phe Glin Ile Pro Met Wall Gly Ala Asp Wall Cys Gly Phe Gly 595 605

Ser Asn Thir Thir Glu Glu Lell Ala Arg Trp Ala Ser Luell Gly Ala 610 615

Phe Thir Phe Tyr Arg Asn His Asn Glu Luell Gly Asp Ile Ser Glin 625 630 635 64 O

Glu Phe Arg Trp Pro Thir Wall Ala Glu Ser Ala Arg Ala Ile 645 650 655

Asp Ile Arg Tyr Lys Lell Lell Asp Tyr Ile Thir Ala Luell His Arg 660 665 67 O

Glin Ser Glin Thir Gly Glu Pro Phe Luell Glin Pro Glin Phe Luell 675 685

Pro Glu Asp Ser Asn Thir Phe Ala Asn Asp Arg Glin Phe Phe Gly 69 O. 695 7 OO

Asp Ala Luell Luell Wall Ser Pro Wall Luell Asn Glu Gly Ser Thir Ser Wall 7 Os 72O

Asp Ala Phe Pro Asp Asp Ile Phe Tyr Asp Trp Thir Gly Ala 72 73 O 73

Wall Wall Arg Gly His Gly Glu Asn Ile Thir Luell Ser Asn Ile Asn Ile 740 74. 7 O

Thir His Ile Pro Lell His Ile Arg Gly Gly ASn Ile Ile Pro Wall Arg 760 765

Thir Ser Ser Gly Met Thir Thir Thir Glu Wall Arg Lys Glin Gly Phe Glu 770 775 78O

Lell Ile Ile Ala Pro Asp Lell Asp Asp Thir Ala Ser Gly Ser Luell Tyr 78s 79 O 79.

Lell Asp Asp Gly Asp Ser Lell Asn Pro Ser Ser Wall Thir Glu Luell Glu

US 9,005,681 B2 153 154 - Continued

-10

Wall Asp Asn Wall Asn Phe Ser Thir Asp Wall Ile Glin Ile Wall 10 15

Thir Asp Arg Phe Ala Asp Gly Asp Arg Thir ASn Asn Pro Ala Gly Asp 25 35

Ala Phe Ser Gly Asp Arg Ser Asn Luell Luell Phe Gly Gly Asp 4 O 45 SO

Trp Glin Gly Ile Ile Asp Lys Ile Asn Asp Gly Tyr Lell Thir Gly Met 55 6 O 65

Gly Wall Thir Ala Lell Trp Ile Ser Glin Pro Wall Glu Asn Ile Thir Ser 70 85

Wall Ile Ser Gly Wall Asn Asn Thir Ser His Gly Tyr Trp 9 O 95

Ala Arg Asp Phe Lys Glin Thir Asn Asp Ala Phe Gly Asp Phe Ala Asp 105 11 O 115

Phe Glin Asn Luell Ile Asp Thir Luell Thir Luell Ile Thir Ser Arg Ser Asp 12 O 125 13 O

Arg Luell Arg Pro Glin Pro His Wall Ser Gly Arg Ala Gly Thir Asn Pro 135 14 O 145

Gly Phe Ala Glu Asn Gly Ala Luell Asp ASn Gly Ser Luell Luell Gly 150 155 16 O 1.65

Ala Ser Asn Asp Thir Ala Gly Luell Phe His His Asn Gly Gly Thir 17O 17s 18O

Asp Phe Ser Thir Ile Glu Asp Gly Ile Asn Lell Tyr Asp Luell 185 19 O 195

Asp Ile Asn His Asn Asn Asn Ala Met Asp Ala Tyr Phe Ser 210

Ile Asp Luell Trp Lell Gly Met Gly Wall Asp Gly Ile Arg Phe Asp 215 22O 225

Wall Glin Tyr Pro Phe Gly Trp Glin Lys Ser Phe Wall Ser Ser 235 24 O 245

Gly Gly Asp His Pro Wall Phe Thir Phe Gly Glu Trp Tyr Luell 250 255 26 O

Ala Asp Glin Thir Asp Gly Asp Asn Ile Phe Ala Asn Glu Ser 265 27 O 27s

Met Asn Luell Lell Asp Phe Glu Ala Glin Glu Wall Arg Glu Wall 285 290

Phe Arg Asp Thir Glu Thir Met Asp Luell Tyr Glu Wall Luell Ala 295 3 OO 3. OS

Ser Thir Glu Ser Glin Tyr Asp Ile Asn ASn Met Wall Thir Phe Ile 310 315 3.25

Asp Asn His Asp Met Asp Arg Phe Glin Wall Ala Gly Ser Gly Thir Arg 330 335 34 O

Ala Thir Glu Glin Ala Lell Ala Luell Thir Luell Thir Ser Arg Gly Wall Pro 345 350 355

Ala Ile Tyr Gly Thir Glu Glin 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 Thir Thir Thir Glu Arg Trp Wall ASn Asn Asp Wall Luell Ile 410 415 42O US 9,005,681 B2 155 156 - Continued

Ile Glu Arg Llys Phe Gly Ser Ser Ala Ala Lieu Val Ala Ile Asn Arg 425 43 O 435

Asn Ser Ser Ala Ala Tyr Pro Ile Ser Gly Lieu Lleu Ser Ser Leul Pro 44 O 445 450

Ala Gly. Thir Tyr Ser Asp Val Lieu. Asn Gly Lieu. Lieu. Asn Gly Asn. Ser 45.5 460 465

Ile Thr Val Gly Ser Gly Gly Ala Val Thr Asn Phe Thr Lieu. Ala Ala 470 47s 48O 485

Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Pro Glu Thir Ser Pro Ala 490 495 SOO

Ile Gly Asn Val Gly Pro Thr Met Gly Glin Pro Gly Asn Ile Wall. Thir 505 510 515 Ile Asp Gly Arg Gly Phe Gly Gly Thr Ala Gly Thr Val Tyr Phe Gly 52O 525 53 O

Thir Thr Ala Val Thr Gly Ser Gly Ile Val Ser Trp Glu Asp Thr Gin 535 54 O 5.45

Ile Lys Ala Val Ile Pro Llys Val Ala Ala Gly Llys Thr Gly Wall Ser 550 555 560 565

Val Lys Thir Ser Ser Gly Thr Ala Ser Asn Thr Phe Lys Ser Phe Asn st O sts 58 O

Val Lieu. Thr Gly Asp Glin Val Thr Val Arg Phe Lieu Val Asn Glin Ala 585 590 595

Asn Thr Asn Tyr Gly Thr Asn Val Tyr Lieu Val Gly Asn Ala Ala Glu 6OO 605 610

Lieu. Gly. Thir Trp Asp Pro ASn Lys Ala Ile Gly Pro Met Tyr ASn Glin 615 62O 625

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

Gly. Thir Lys Lieu. Asp Phe Llys Phe Ile Llys Lys Gly Gly Gly Thir Wall 650 655 660 Thir Trp Glu Gly Gly Gly Asn His Thr Tyr Thr Thr Pro Ala Ser Gly 665 670 675 Val Gly Thr Val Thr Val Asp Trp Glin Asn 68O 685

The invention claimed is: 45 mosa; and cyclodextringlucanotransferase derived from 1. A method of producing a starch gel-containing food, the Bacillus licheniformis or Paenibacillus macerans (Ba method comprising the steps of cillus macerans), provided that said starch granule is not treating starch granules with an enzyme at a temperature of a starch granule of high amylose starch containing 30% about 10° C. or higher and about 70° C. or lower to or more amylose. obtain enzyme-treated Starch granules having improved 50 2. A method of producing a starch gel-containing food, the gel forming ability; method comprising the steps of removing a carbohydrate produced by enzymatic hydroly treating starch granules with an enzyme at a temperature of sis from the enzyme-treated Starch granules; about 10° C. or higher and about 70° C. or lower to mixing a food material, the enzyme-treated Starch granules 55 obtain enzyme-treated Starch granules having improved and water to obtain a mixture; gel forming ability; heating the mixture thereby gelatinizing the enzyme removing a carbohydrate produced by enzymatic hydroly treated Starch granules in the mixture; and sis from the enzyme-treated Starch granules; cooling the mixture containing the gelatinized enzyme mixing a food material, the enzyme-treated Starch granules treated Starch granules thereby gelling the starch gran- 60 and water to obtain a mixture; ules to obtain a starch gel-containing food, wherein heating the mixture thereby gelatinizing the enzyme the enzyme is selected from the group consisting of C.-amy treated Starch granules in the mixture; and lase derived from Aspergillus Oryzae or Aspergillus cooling the mixture containing the gelatinized enzyme niger; amyloglucosidase derived from Aspergillus niger; treated Starch granules thereby gelling the starch gran Rhizopus niveus or Rhizopus Oryzae, C-glucosidase 65 ules to obtain a starch gel-containing food, wherein: (1) derived from Aspergillus niger, isoamylase derived the enzyme is encoded by a nucleic acid molecule which from Flavobacterium sp. or Pseudomonas amylodera is capable of hybridizing under stringent conditions with US 9,005,681 B2 157 158 a nucleic acid molecule consisting of a base sequence style confectioneries, fat- or oil-containing foods, gelatinous complementary to a base sequence of SEQID NO: 1, 3, foods, fish meat and animal meat processed foods, Salsa and 5, 7, or 9, and has a starch hydrolysis activity; sauces, and noodles. wherein the stringent conditions are hybridization in a 11. The food according to claim8, wherein the food is a low solution containing 50% formamide, 5xSSC (750 mM moisture content type food and the amount of moisture of the NaCl, 75 mM trisodium citrate), 50 mM sodium phos food is 1 g or more and 40 g or less per 100 g of the edible phate (pH 7.6), 5xDenhardt’s solution (0.2% BSA, portion. 0.2% Ficoll 400 and 0.2% polyvinylpyrrolidone), 10% 12. The food according to claim 8, wherein the food is dextran Sulfate and 20 ug/ml denatured sheared salmon selected from the group consisting of baked foods, Western sperm DNA at 65°C., and subsequent washing under the 10 style confectioneries, and fried foods. condition of 65°C. using an SSC solution having a 0.1 to 13. The food according to claim 8, wherein the starch is 2-fold concentration (a composition of an SSC solution derived from cassaya, corn or wheat. having a 1-fold concentration is 150 mM sodium chlo 14. The method according to claim 2, wherein the starch ride and 15 mM sodium citrate), provided that said 15 granules are starch granules of an untreated Starch, a physi starch granule is not a starch granule of high amylose cally treated starch or a chemically modified starch. starch containing 30% or more amylose. 15. The method according to claim 2, wherein the starch 3. A method of producing a starch gel-containing food, the granules are starch granules of an untreated Starch, and the method comprising the steps of starch granules have been neither chemically modified nor treating starch granules with an enzyme at a temperature of physically treated in any stage until the starch gel-containing about 10° C. or higher and about 70° C. or lower to food is obtained by the method. obtain enzyme-treated Starch granules having improved 16. The method according to claim 2, wherein the starch gel forming ability; granules are starch granules of an untreated Starch or a physi removing a carbohydrate produced by enzymatic hydroly cally treated starch, the method further comprises the step of sis from the enzyme-treated Starch granules; 25 chemically modifying the enzyme-treated Starch, and the mixing a food material, the enzyme-treated Starch granules chemically modified enzyme-treated starch is mixed with the and water to obtain a mixture; food material and water. heating the mixture thereby gelatinizing the enzyme 17. The method according to claim 2, wherein the starch treated Starch granules in the mixture; and granules are starch granules of an untreated Starch or a chemi cooling the mixture containing the gelatinized enzyme 30 cally modified starch, the method further comprises the step of physically treating the enzyme-treated Starch, and the treated Starch granules thereby gelling the starch gran physically treated enzyme-treated starch is mixed with the ules to obtain a starch gel-containing food, wherein: food material and water. (1) the enzyme has an amino acid sequence having at least 18. A starch gel-containing food produced by the method 95% or more of sequence identity with an amino acid 35 according to claim 2. sequence of SEQID NO: 2, 4, 6, 8, or 10, and has a starch 19. The food according to claim 18, wherein the food is a hydrolysis activity, provided that said starch granule is high moisture content type food and the amount of moisture not a starch granule of high amylose starch containing of the food is more than 40 g and less than 95g per 100 g of 30% or more amylose. the edible portion. 4. The method according to claim 1, wherein the starch 40 20. The food according to claim 18, wherein the food is granules are starch granules of an untreated Starch, a physi selected from the group consisting of traditional Japanese cally treated starch or a chemically modified starch. style confectioneries, fat- or oil-containing foods, gelatinous 5. The method according to claim 1, wherein the starch foods, fish meat and animal meat processed foods, Salsa and granules are starch granules of an untreated Starch, and the sauces, and noodles. starch granules have been neither chemically modified nor 45 21. The food according to claim 18, wherein the food is a physically treated in any stage until the starch gel-containing low moisture content type food and the amount of moisture of food is obtained by the method. the food is 1 g or more and 40g or less per 100 g of the edible 6. The method according to claim 1, wherein the starch portion. granules are starch granules of an untreated Starch or a physi 22. The food according to claim 18, wherein the food is cally treated starch, the method further comprises the step of 50 selected from the group consisting of baked foods, Western chemically modifying the enzyme-treated Starch, and the style confectioneries, and fried foods. chemically modified enzyme-treated starch is mixed with the 23. The food according to claim 18, wherein the starch is food material and water. derived from cassaya, corn or wheat. 7. The method according to claim 1, wherein the starch 24. The method according to claim 3, wherein the starch granules are starch granules of an untreated Starch or a chemi 55 granules are starch granules of an untreated Starch, a physi cally modified starch, the method further comprises the step cally treated starch or a chemically modified starch. of physically treating the enzyme-treated Starch, and the 25. The method according to claim 3, wherein the starch physically treated enzyme-treated starch is mixed with the granules are starch granules of an untreated Starch, and the food material and water. starch granules have been neither chemically modified nor 8. A starch gel-containing food produced by the method 60 physically treated in any stage until the starch gel-containing according to claim 1. food is obtained by the method. 9. The food according to claim8, wherein the food is a high 26. The method according to claim 3, wherein the starch moisture content type food and the amount of moisture of the granules are starch granules of an untreated Starch or a physi food is more than 40 g and less than 95 g per 100 g of the cally treated starch, the method further comprises the step of edible portion. 65 chemically modifying the enzyme-treated Starch, and the 10. The food according to claim 8, wherein the food is chemically modified enzyme-treated starch is mixed with the selected from the group consisting of traditional Japanese food material and water. US 9,005,681 B2 159 160 27. The method according to claim 3, wherein the starch 32. The food according to claim 28, wherein the food is granules are starch granules of an untreated Starch or a chemi selected from the group consisting of baked foods, Western cally modified starch, the method further comprises the step of physically treating the enzyme-treated Starch, and the style confectioneries, and fried foods. physically treated enzyme-treated starch is mixed with the 33. The food according to claim 28, wherein the starch is food material and water. derived from cassaya, corn or wheat. 28. A starch gel-containing food produced by the method 34. The method according to claim 1, wherein the enzyme according to claim 3. treated Starch granules can form a gel having a rupture stress 29. The food according to claim 28, wherein the food is a higher than that of the starch granules before the treatment high moisture content type food and the amount of moisture with the enzyme when measured by a rheometer. of the food is more than 40 g and less than 95g per 100 g of 10 the edible portion. 35. The method according to claim 2, wherein the enzyme 30. The food according to claim 28, wherein the food is treated Starch granules can form a gel having a rupture stress selected from the group consisting of traditional Japanese higher than that of the starch granules before the treatment style confectioneries, fat- or oil-containing foods, gelatinous with the enzyme when measured by a rheometer. foods, fish meat and animal meat processed foods, Salsa and 15 36. The method according to claim3, wherein the enzyme sauces, and noodles. treated Starch granules can form a gel having a rupture stress 31. The food according to claim 28, wherein the food is a low moisture content type food and the amount of moisture of higher than that of the starch granules before the treatment the food is 1 g or more and 40g or less per 100 g of the edible with the enzyme when measured by a rheometer. portion. k k k k k