Modifier for defective rice cooking, method for producing cooked rice, method for modifying cooked rice, and cooked rice

Abstract

Provided is a technology capable of modifying cooked rice cooked using defective rice.　The present technology provides a modifier for defective rice cooking, the modifier comprising a starch degrading enzyme and 4-α-glucanotransferase. The present technology also provides: a method for producing cooked rice, the method involving a rice cooking step for cooking defective rice to which a starch degrading enzyme and 4-α-glucanotransferase have been added; and a method for modifying cooked rice. The present technology further provides cooked rice containing defective rice, wherein the modifier for defective rice cooking according to the present technology is used.

Description

Modifier for cooking rice with poor-quality rice, method for producing cooked rice, method for modifying cooked rice, and cooked rice

[0001] This technology relates to a modifier for cooking rice with poor-quality rice. More specifically, this technology relates to a modifier for cooking rice with poor-quality rice using a specific enzyme, a method for producing cooked rice, a method for modifying cooked rice, and cooked rice.

[0002] For the purpose of improving the quality of cooked rice, various technologies have been studied. For example, Patent Document 1 discloses a method for producing cooked rice using 4-α-glucanotransferase for the purpose of obtaining cooked rice whose quality does not deteriorate even after a long period of time. Patent Document 2 discloses a method for suppressing the aging of cooked rice by adding maltotriosyltransferase to the soaking water before cooking and then cooking the rice for the purpose of suppressing the aging of rice. Patent Document 3 discloses a method for producing cooked rice using two or more of branching enzyme, exo-type amylase, and 4-α-glucanotransferase for the purpose of obtaining a cooked rice food or a processed cooked rice product that can suppress the rise in blood glucose level.

[0003] International Publication No. 2022 / 004402 International Publication No. 2011 / 001722 International Publication No. 2021 / 210626

[0004] In recent years, due to the influence of global warming, high-temperature damaged rice has appeared on the market, and its low quality has become a problem. In addition to global warming, there are also problems with poor-quality rice whose quality has deteriorated due to wind damage such as typhoons, rain damage such as continuous rain, and insufficient sunlight. If these poor-quality rice can be utilized, it will also contribute to a sustainable society.

[0005] Therefore, the main purpose of this technology is to provide a technology that can modify cooked rice using poor-quality rice.

[0006] In this technology, first, a modifier for cooking rice with poor-quality rice containing a starch-degrading enzyme and 4-α-glucanotransferase is provided. In this technology, a method for producing cooked rice and a method for modifying cooked rice are also provided, including a cooking step of cooking poor-quality rice to which a starch-degrading enzyme and 4-α-glucanotransferase are added. In this technology, further, cooked rice containing poor-quality rice using the modifier for cooking rice with poor-quality rice according to this technology is provided.

[0007] The following describes preferred embodiments for implementing this technology. Note that the embodiments described below are merely examples of typical embodiments of this technology, and this should not be interpreted as narrowing the scope of this technology.

[0008] 1. Rice Cooking Modifier The rice cooking modifier related to this technology contains a starch-degrading enzyme and 4-α-glucanotransferase. It may also contain other enzymes or other components as needed.

[0009] In this technology, "defective rice" refers to rice that is of lower quality than typical ordinary rice. Examples include germinated grains, diseased grains, bud-rotted grains, insect-damaged grains, cracked grains, deformed grains (cut grains, twisted grains, and other deformed grains), brown rice, broken grains, spotted grains, germ-deficient grains, peeled grains, and other damaged grains; dead rice such as green dead rice and white dead rice; discolored grains such as fully discolored grains, partially discolored grains, and red rice; immature green grains, immature grains with immature bases, immature grains with white bellies, immature grains with white backs, other immature grains, immature grains with powdery texture, and water-soaked cracked grains.

[0010] The causes of poor quality are not limited to one specific factor, and include, for example, damage from high temperatures due to global warming, wind damage from typhoons, rain damage from prolonged rain, and insufficient sunlight. Among these, the rice cooker modifier related to this technology exhibits a particularly high modification effect on rice damaged by high temperatures. Rice damaged by high temperatures is characterized by a phenomenon where the starch supply function does not work properly due to exposure to high temperatures during the ripening period, resulting in cloudy rice grains that easily break down, and rapid starch aging.

[0011] The specific physical properties of "defective rice" are not particularly limited, but for example, the proportion of powdery grains (grains with a powdery or semi-powdered texture) in the milled rice can be 50% by weight or less, 40% by weight or less, 30% by weight or less, or 20% by weight or less. Preferably, the defective rice cooking modifier according to this technology can exert its modifying effect on rice in which the proportion of powdery grains (grains with a powdery or semi-powdered texture) in the milled rice is 15% by weight or less, 10% by weight or less, or 8% by weight or less. The lower limit of the proportion of powdery grains (grains with a powdery or semi-powdered texture) in the milled rice is not particularly limited, but for example, it can be 3.0% by weight or more, 4.0% by weight or more, or 5.0% by weight or more.

[0012] Specifically, poor quality rice may have air gaps within its cells (inside the rice grain, between starch granules), making the rice brittle and resulting in a soft texture and poor grain separation during cooking. Furthermore, poor quality rice may have longer amylopectin chains, making it more susceptible to starch aging compared to normal rice. In contrast, this technology successfully suppresses starch aging and improves grain separation by using starch-degrading enzymes and 4-α-glucanotransferase during cooking.

[0013] The origin of the defective rice is not particularly limited; for example, it could be Japonica rice, Indica rice, Javanica rice, etc., and these may be blended. It could also be either non-glutinous rice or glutinous rice, or a blend of non-glutinous and glutinous rice. Furthermore, the degree of milling is not particularly limited; it could be brown rice, germinated rice, partially milled rice, 70% milled rice, polished rice, pre-washed rice, etc., and these may be blended. In addition, it could be new rice, old rice, very old rice, or a blend of these. The following describes in detail each component that can be used in this technology.

[0014] (1) Starch-degrading enzymes The starch-degrading enzymes that can be used in this technology may be freely selected from one or more enzymes that have the effect of degrading starch, as long as they do not impair the action or effect of this technology. Examples include α-amylase, β-amylase, maltotriose-producing enzyme, transglucosidase, glucoamylase, cyclodextrin-producing enzyme, etc.

[0015] [α-Amylase] The α-amylase that can be used in this technology is an endo-type enzyme that acts on starch and randomly hydrolyzes α-1,4 glycosidic bonds. The α-amylase that can be used in this technology may also be an enzyme that has other functions, as long as it has α-amylase activity.

[0016] The origin of the α-amylase that can be used in this technology is not particularly limited, but examples include α-amylases derived from microorganisms of the Aspergillus genus (e.g., Aspergillus oryzae, Aspergillus niger, etc.) and the Bacillus genus (e.g., Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, etc.), preferably α-amylases derived from microorganisms of the Bacillus genus, and more preferably α-amylases derived from Bacillus amyloliquefaciens.

[0017] Here, "α-amylase derived from Bacillus amyloriquefaciens" refers to α-amylase produced by microorganisms classified as Bacillus amyloriquefaciens (whether wild-type or mutant), or α-amylase obtained by genetic engineering using the α-amylase gene. Therefore, recombinant organisms produced by host microorganisms into which the α-amylase gene obtained from Bacillus amyloriquefaciens (or a modified version of that gene) has been introduced also fall under the category of "α-amylase derived from Bacillus amyloriquefaciens."

[0018] The α-amylase used in this technology can be prepared from the culture medium of the microorganism from which the α-amylase originates. Specific preparation methods include recovering α-amylase from the culture medium or cells of the above-mentioned microorganism. For example, when using α-amylase-secreting microorganisms, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using α-amylase-non-secreting microorganisms, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the cells can be crushed by pressurization, sonication, etc. to extract the enzyme, and then the enzyme can be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying method. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0019] In this technology, commercially available α-amylase can also be used, and a preferred example of a commercially available product is α-amylase (derived from Bacillus amyloriquefaciens) manufactured by Amano Enzyme Co., Ltd.

[0020] The α-amylase content in the rice cooking modifier related to this technology can be freely set as long as it does not impair the effects of this technology. The α-amylase content can be set to, for example, 0.022 U or more per gram of raw rice used in the production of cooked rice, and from the viewpoint of further enhancing the cooked rice modification effect, it can be set to preferably 0.11 U or more, and more preferably 0.22 U or more.

[0021] The upper limit of the α-amylase content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 450U or less, 300U or less, 100U or less, 90U or less, 70U or less, 50U or less, 45U or less, 30U or less, 10U or less, 5U or less, 3U or less, 1U or less, 0.9U or less, 0.8U or less, 0.7U or less, 0.6U or less, 0.5U or less, or 0.45U or less per gram of raw rice used in the production of cooked rice.

[0022] In this technology, the α-amylase activity is the value measured using the measurement method described in the examples below.

[0023] [β-Amylase] The β-amylase that can be used in this technology is an enzyme that acts on starch and primarily hydrolyzes α-1,4 glycosidic bonds from the non-reducing end to the exo disaccharide unit. The β-amylase that can be used in this technology may also be an enzyme that has other functions, as long as it has β-amylase activity.

[0024] The origin of the β-amylase that can be used in this technology is not particularly limited, but examples include β-amylase derived from plants (wheat, soybeans) and β-amylase derived from the genus Bacillus. Preferably, β-amylase derived from the genus Bacillus is preferred, and more preferably, β-amylase derived from Bacillus Flexus is preferred.

[0025] Here, "Bacillus flexus-derived β-amylase" refers to β-amylase produced by microorganisms classified as Bacillus flexus (whether wild-type or mutant strains), or β-amylase obtained through genetic engineering using the β-amylase gene. Therefore, recombinant organisms produced by host microorganisms into which a β-amylase gene obtained from Bacillus flexus (or a modified version of that gene) has been introduced also fall under the category of "Bacillus flexus-derived β-amylase."

[0026] The β-amylase used in this technology can be prepared from the culture medium of the microorganism from which the above-mentioned β-amylase originates. Specific preparation methods include recovering β-amylase from the culture medium or cells of the above-mentioned microorganism. For example, when using β-amylase-secreting microorganisms, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using non-β-amylase-secreting microorganisms, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the cells can be crushed by pressurization, sonication, etc. to extract the enzyme, and then the enzyme can be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying method. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0027] In this technology, commercially available β-amylase can also be used. A preferred example of a commercially available product is β-amylase derived from the genus Bacillus Flexus, manufactured by Amano Enzyme Co., Ltd.

[0028] The β-amylase content in the rice cooking modifier related to this technology can be freely set as long as it does not impair the effects of this technology. The β-amylase content can be set to, for example, 0.003 U or more per gram of raw rice used in the production of cooked rice. From the viewpoint of further enhancing the cooked rice modification effect, it can be preferably set to 0.01 U or more, more preferably 0.03 U or more, 0.05 U or more, even more preferably 0.1 U or more, and even more preferably 0.2 U or more.

[0029] The upper limit of the β-amylase content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 350U or less, 300U or less, 100U or less, 60U or less, 50U or less, 30U or less, 10U or less, 5U or less, 1U or less, 0.5U or less, 0.4U or less, or 0.3U or less per gram of raw rice used in the production of cooked rice.

[0030] In this technology, the β-amylase activity is the value measured using the measurement method described in the examples below.

[0031] [Maltotriose-producing enzyme] The maltotriose-producing enzyme that can be used in this technology is an enzyme that acts on starch and primarily produces maltotriose. The maltotriose-producing enzyme that can be used in this technology may also be an enzyme that has other functions, as long as it has the activity to produce maltotriose.

[0032] The origin of the maltotriose-producing enzymes that can be used in this technology is not particularly limited, but examples include maltotriose-producing enzymes derived from microorganisms of the genera Streptomyces, Bacillus, Microbacterium, and Cellulosimicrobium. These maltotriose-producing enzymes may be used individually or in combination of multiple types. Among these maltotriose-producing enzymes, maltotriose-producing enzymes derived from microorganisms of the genus Microbacterium are preferred, and maltotriose-producing enzymes derived from Microbacterium sp. are more preferred. Specifically, enzymes that exhibit substrate specificity acting on amylose, amylopectin, glycogen, and starch can be used.

[0033] Here, "maltotriose-producing enzyme derived from Microbacterium sp." refers to maltotriose-producing enzyme produced by microorganisms classified as Microbacterium sp. (whether wild-type or mutant), or maltotriose-producing enzyme obtained through genetic engineering using the maltotriose-producing enzyme gene. Therefore, recombinant organisms produced by host microorganisms into which the maltotriose-producing enzyme gene obtained from Microbacterium sp. (or a modified version of that gene) has been introduced also fall under the category of "maltotriose-producing enzyme derived from Microbacterium sp.".

[0034] The maltotriose-producing enzyme used in this technology can be prepared from the culture medium of the microorganism from which the maltotriose-producing enzyme originates. Specific preparation methods include recovering the maltotriose-producing enzyme from the culture medium or cells of the above-mentioned microorganism. For example, when using a maltotriose-secreting microorganism, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using a non-secreting maltotriose-producing microorganism, the cells can be recovered from the culture medium beforehand, as needed, and then the cells can be crushed by pressurization, sonication, etc., to extract the enzyme, and then the enzyme can be separated and / or purified. As for the enzyme separation and / or purification method, any known protein separation and / or purification method can be used without particular limitation, such as centrifugation, UF concentration, salting-out, and various chromatography methods using ion exchange resins. The separated and / or purified enzymes can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying process. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0035] In this technology, commercially available products can be used as maltotriose-producing enzymes. A preferred example of a commercially available product is the maltotriose-producing enzyme derived from Microbacterium sp. manufactured by Amano Enzyme Co., Ltd.

[0036] The content of maltotriose-producing enzyme in the rice cooking modifier related to this technology can be freely set as long as it does not impair the effects of this technology. The content of maltotriose-producing enzyme can be set to, for example, 0.006 U or more per gram of raw rice used in the production of cooked rice, and from the viewpoint of further enhancing the cooked rice modification effect, it can be preferably set to 0.03 U or more, more preferably 0.06 U or more, even more preferably 0.3 U or more, and even more preferably 0.5 U or more.

[0037] The upper limit of the maltotriose-producing enzyme content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 600U or less, 400U or less, 300U or less, 200U or less, 150U or less, 100U or less, 60U or less, 30U or less, 10U or less, 5U or less, 3U or less, 2U or less, 1U or less, or 0.6U or less per gram of raw rice used in the production of cooked rice.

[0038] In this technology, the activity of the maltotriose-producing enzyme is the value measured using the measurement method described in the examples below.

[0039] [Transglucosidase] The transglucosidase that can be used in this technology is an enzyme that catalyzes the hydrolysis of α-1,4-glucosidic bonds. The transglucosidase that can be used in this technology may also be an enzyme that has other functions, as long as it has transglucosidase activity.

[0040] The origin of the transglucosidase that can be used in this technology is not particularly limited, but examples include transglucosidases from plants (rice, millet, leeks, mung beans, buckwheat, etc.), and from microorganisms, for example, transglucosidases from filamentous fungi of the genus Aspergillus, yeasts of the genus Saccharomyces, and bacteria of the genus Bacillus. Preferably, transglucosidases from the genus Aspergillus are used, and more preferably, transglucosidases from Aspergillus niger are used.

[0041] Here, "Aspergillus niger-derived transglucosidase" refers to transglucosidase produced by microorganisms classified as Aspergillus niger (whether wild-type or mutant), or transglucosidase obtained through genetic engineering using the transglucosidase gene. Therefore, recombinant organisms produced by host microorganisms into which the transglucosidase gene obtained from Aspergillus niger (or a modified version of that gene) has been introduced also fall under the category of "Aspergillus niger-derived transglucosidase."

[0042] The transglucosidase used in this technology can be prepared from the culture medium of the microorganism from which the transglucosidase originates. Specific preparation methods include recovering the transglucosidase from the culture medium or cells of the microorganism. For example, when using transglucosidase-secreting microorganisms, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using non-transglucosidase-secreting microorganisms, the cells can be recovered from the culture medium by pressurization, sonication, etc., to extract the enzyme, and then the enzyme can be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method without particular limitation, such as centrifugation, UF concentration, salting-out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying method. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0043] In this technology, commercially available transglucosidases can also be used. A preferred example of a commercially available product is transglucosidase derived from Aspergillus niger, manufactured by Amano Enzyme Co., Ltd.

[0044] The content of transglucosidase in the rice modifier according to the present technology can be freely set as long as the effects of the present technology are not impaired. The content of transglucosidase can be set, for example, at 0.00055 U or more per 1 g of raw rice used in the production of cooked rice. From the perspective of further enhancing the rice modification effect, it is preferably 0.002 U or more, 0.005 U or more, or 0.02 U or more, preferably 0.04 U or more, more preferably 1 U or more, still more preferably 10 U or more, even more preferably 50 U or more, still more preferably 70 U or more, even more preferably 100 U or more, still more preferably 110 U or more, particularly preferably 130 U or more.

[0045] The upper limit of the content of transglucosidase is not particularly limited as long as the effects of the present technology are not impaired. However, it can be set, for example, at 10000 U or less, 5000 U or less, 2000 U or less, 1000 U or less, 800 U or less, 600 U or less, 400 U or less, 300 U or less, 250 U or less, 200 U or less, 180 U or less, 170 U or less, 160 U or less per 1 g of raw rice used in the production of cooked rice.

[0046] In the present technology, the activity of transglucosidase is the value measured by the measurement method described in the examples below.

[0047] [Glucoamylase] The glucoamylase that can be used in the present technology is an enzyme having an activity of hydrolyzing the α-1,4-glucoside bond of carbohydrates such as starch into glucose units from the non-reducing end. As the glucoamylase that can be used in the present technology, an enzyme having other actions may be used as long as it has glucoamylase activity.

[0048] The origin of the glucoamylase that can be used in this technology is not particularly limited, but examples include glucoamylases derived from the genera Aspergillus and Rhizopus. These glucoamylases may be used individually or in combination of multiple species. Among these glucoamylases, glucoamylase derived from the genus Rhizopus is preferred, and more preferably from Rhizopus oryzae.

[0049] Here, "glucoamylase derived from Rhizopus oryzae" refers to glucoamylase produced by microorganisms classified as Rhizopus oryzae (whether wild-type or mutant), or glucoamylase obtained by genetic engineering using the glucoamylase gene. Therefore, recombinant organisms produced by host microorganisms into which the glucoamylase gene obtained from Rhizopus oryzae (or a modified version of that gene) has been introduced also fall under the category of "glucoamylase derived from Rhizopus oryzae."

[0050] The glucoamylase used in this technology can be prepared from the culture medium of the microorganism from which the glucoamylase originates. Specific preparation methods include recovering glucoamylase from the culture medium or cells of the microorganism. For example, when using glucoamylase-secreting microorganisms, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using non-glucoamylase-secreting microorganisms, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the cells can be crushed to extract the enzyme, and then the enzyme can be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying method. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0051] In this technology, commercially available glucoamylase can also be used. A preferred example of a commercially available product is glucoamylase derived from Rhizopus oryzae, manufactured by Amano Enzyme Co., Ltd.

[0052] The glucoamylase content in the rice cooking modifier related to this technology can be freely set as long as it does not impair the effects of this technology. The glucoamylase content can be set to, for example, 0.008 U or more per gram of raw rice used in the production of cooked rice, and from the viewpoint of further enhancing the cooked rice modification effect, it can be preferably set to 0.04 U or more, more preferably 0.08 U or more, even more preferably 0.4 U or more, and even more preferably 0.7 U or more.

[0053] The upper limit of the glucoamylase content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 800U or less, 500U or less, 300U or less, 150U or less, 100U or less, 80U or less, 50U or less, 30U or less, 10U or less, 5U or less, 3U or less, 1.5U or less, 1U or less, 0.8U or less, or 0.5U or less per gram of raw rice used in the production of cooked rice.

[0054] In this technology, the glucoamylase activity is the value measured using the measurement method described in the examples below.

[0055] [Cyclodextrin-producing enzyme] The cyclodextrin-producing enzyme (EC 2.4.1.19) that can be used in this technology is an enzyme that has the activity to produce cyclodextrins with a degree of polymerization of 6 to 8 using a polymer consisting of α-1,4 linked glucose such as starch as a substrate. The cyclodextrin-producing enzyme that can be used in this technology may also be an enzyme that has other functions, as long as it has the activity to produce cyclodextrins.

[0056] The origin of the cyclodextrin-producing enzymes that can be used in this technology is not particularly limited, but for example, the genus Bacillus includes Bacillus stearothermophilus, Bacillus megaterium, Bacillus circulans, Bacillus macerans, Bacillus ohbensis, and Bacillus clarkii; the genus Geobacillus includes Geobacillus stearothermophilus; and the genus Paenibacillus includes Paenibacillus macerans. Examples of cyclodextrin-producing enzymes include those derived from microorganisms of the genera *Macerans*, *Klebsiella* (specifically *Klebsiella pneumoniae*), *Anoxybacillus* (specifically *Anoxybacillus caldiproteolyticus*, *Anoxybacillus pushchinoensis*, and *Anoxybacillus flavithermus*), and *Thremoanaerobacter* and *Brevibacterium*. These cyclodextrin-producing enzymes may be used individually or in combination. In particular, from the perspective of the rice modification effect, the cyclodextrin-producing enzyme is preferably a cyclodextrin-producing enzyme derived from microorganisms of the genus Anoxybacillus, and in this technology, Anoxybacillus caldiproteolyticus (including what was formerly called Geobacillus stearothermophilus) is preferred.It is more preferable that the enzyme is a cyclodextrin-producing enzyme derived from ).

[0057] Here, "cyclodextrin-producing enzyme derived from Anoxybacillus cardiprotheolitics" refers to cyclodextrin-producing enzyme produced by microorganisms classified as Anoxybacillus cardiprotheolitics (whether wild-type or mutant), or cyclodextrin-producing enzyme obtained by genetic engineering using the cyclodextrin-producing enzyme gene. Therefore, recombinant organisms produced by host microorganisms into which the cyclodextrin-producing enzyme gene (or a modified version thereof) obtained from Anoxybacillus cardiprotheolitics has been introduced also fall under the category of "cyclodextrin-producing enzyme derived from Anoxybacillus cardiprotheolitics."

[0058] The cyclodextrin-producing enzyme used in this technology can be prepared from the culture medium of the microorganism from which the above-mentioned cyclodextrin-producing enzyme originates. Specific preparation methods include recovering the cyclodextrin-producing enzyme from the culture medium or cells of the above-mentioned microorganism. For example, when using a cyclodextrin-secreting microorganism, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using a non-secreting cyclodextrin-producing microorganism, the cells can be recovered from the culture medium beforehand, as needed, and then the cells can be crushed by pressurization, sonication, etc., to extract the enzyme, and then the enzyme can be separated and / or purified. As for the enzyme separation and / or purification method, any known protein separation and / or purification method can be used without particular limitation, such as centrifugation, UF concentration, salting-out, and various chromatography methods using ion exchange resins. The separated and / or purified enzymes can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying process. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0059] In this technology, commercially available products can be used as cyclodextrin-producing enzymes. Preferred examples of commercially available products include cyclodextrin glucanotransferase derived from Anoxybacillus caldiproteolyticus (Geobacillus stearothermophilus) manufactured by Amano Enzyme Co., Ltd.

[0060] The content of cyclodextrin-producing enzyme in the rice cooking modifier related to this technology can be freely set as long as it does not impair the effects of this technology. The content of cyclodextrin-producing enzyme can be set to, for example, 0.002 U or more per gram of raw rice used in the production of cooked rice, and from the viewpoint of further enhancing the rice modification effect, it can be set to preferably 0.01 U or more, more preferably 0.02 U or more, and even more preferably 0.2 U or more.

[0061] The upper limit of the cyclodextrin-producing enzyme content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 200U or less, 100U or less, 80U or less, 60U or less, 40U or less, 20U or less, 10U or less, 5U or less, 1U or less, 0.8U or less, 0.6U or less, 0.5U or less, 0.4U or less, or 0.2U or less per gram of raw rice used in the production of cooked rice.

[0062] In this technology, the activity of the cyclodextrin-producing enzyme is the value measured using the measurement method described in the examples below.

[0063] (2) 4-α-glucanotransferase The 4-α-glucanotransferase (EC 2.4.1.25) used in this technology is an enzyme that catalyzes a chemical reaction in which a portion of 1,4-α-glucan is transferred to another portion of a hydrocarbon such as glucose or 1,4-α-D-glucan. The 4-α-glucanotransferase used in this technology may also be an enzyme that has other functions, as long as it acts on polysaccharides and oligosaccharides having α-1,4-glucosidic bonds and has the activity to transfer maltotriose units to sugars.

[0064] The 4-α-glucanotransferase can be derived from either plants or microorganisms. Plant-derived sources include, for example, 4-α-glucanotransferase from potato (Solanum tuberosum L.) tubers, while microorganism-derived sources include, for example, actinomycetes (Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, and Streptomyces violaceoruber) or bacteria (Agrobacterium radiobacter). Radiobacter, Arthrobacter, Bacillus, Erwinia, Geobacillus pallidus, Geobacillus stearothermophilus, Gluconobacter oxydans, Leuconostoc mesenteroides, Paenibacillus alginolyticus, Pimelobacter, Protaminobacter, Pseudomonas, Serratia, Sporosarcina globispora Examples include 4-α-glucanotransferases derived from the genera *globispora*, *Thermus*, and *Aeribacillus pallidus*. These 4-α-glucanotransferases may be used individually or in combination.In particular, from the viewpoint of the rice modification effect, it is preferable to use 4-α-glucanotransferase derived from microorganisms, more preferably 4-α-glucanotransferase derived from Aeribacillus, and even more preferably 4-α-glucanotransferase derived from Aeribacillus pallidus (including what was formerly called Geobacillus pallidus).

[0065] Here, "4-α-glucanotransferase derived from Aeribacillus paridus" refers to 4-α-glucanotransferase produced by microorganisms classified as Aeribacillus paridus (whether wild-type or mutant), or 4-α-glucanotransferase obtained by genetic engineering using the 4-α-glucanotransferase gene. Therefore, recombinant organisms produced by host microorganisms into which the 4-α-glucanotransferase gene obtained from Aeribacillus paridus (or a modified version of that gene) has been introduced also fall under the category of "4-α-glucanotransferase derived from Aeribacillus paridus."

[0066] The 4-α-glucanotransferase used in this technology can be prepared from the culture medium of the microorganism from which the above-mentioned 4-α-glucanotransferase originates. Specific preparation methods include recovering the 4-α-glucanotransferase from the culture medium or cells of the above-mentioned microorganism. For example, when using a 4-α-glucanotransferase-secreting microorganism, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using a 4-α-glucanotransferase-non-secreting microorganism, the cells can be recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme can be extracted by crushing the cells by pressurization, sonication, etc., and then the enzyme can be separated and / or purified. As for the enzyme separation and / or purification method, any known protein separation and / or purification method can be used without particular limitation, such as centrifugation, UF concentration, salting-out, and various chromatography methods using ion exchange resins. The separated and / or purified enzymes can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying process. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0067] In this technology, commercially available 4-α-glucanotransferases can also be used. Preferred examples of commercially available products include 4-α-glucanotransferase derived from Aeribacillus pallidus (formarly Geobacillus pallidus) manufactured by Amano Enzyme Co., Ltd.

[0068] The content of 4-α-glucanotransferase in the rice cooking modifier related to this technology can be freely set as long as it does not impair the effects of this technology. The content of 4-α-glucanotransferase can be set to, for example, 0.015 U or more per gram of raw rice used in the production of cooked rice, and from the viewpoint of further enhancing the cooked rice modification effect, it can be preferably set to 0.05 U or more, 0.075 U or more, more preferably 0.15 U or more, even more preferably 1.5 U or more, and even more preferably 2.7 U or more.

[0069] The upper limit of the 4-α-glucanotransferase content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 3000U or less, 1000U or less, 800U or less, 600U or less, 300U or less, 100U or less, 50U or less, 10U or less, 5U or less, 4U or less, or 3U or less per gram of raw rice used in the production of cooked rice.

[0070] In this technology, the activity of 4-α-glucanotransferase is the value measured using the measurement method described in the examples below.

[0071] (3) Other Components The rice cooking modifier for defective rice related to this technology may be used in combination with other components as long as it does not impair the action or effect of this technology. Other components may include, for example, excipients, pH adjusters, colorants, flavoring agents, disintegrants, lubricants, stabilizers, enzymes, etc., which are commonly used in formulations. Furthermore, known or future discovered functional components may be used in combination as appropriate for the purpose.

[0072] 2. Method for producing cooked rice and method for modifying cooked rice The method for producing cooked rice and method for modifying cooked rice according to this technology include a cooking step in which defective rice to which starch-degrading enzymes and 4-α-glucanotransferases have been added is cooked. The addition of each enzyme and the cooking may be carried out at the same institution or at different institutions. For example, one institution may carry out the enzyme addition step and cook the enzyme-added rice obtained, or one institution may carry out the enzyme addition step and distribute the enzyme-added rice obtained, and another institution may carry out the cooking step of the enzyme-added rice.

[0073] In the rice modification method relating to this technology, in addition to the enzyme addition step and / or cooking step, steps such as hydration, soaking, storage, freezing, thawing, processing, recovery, and other steps commonly used in rice production can be appropriately selected and carried out. The raw materials used in this technology and each step will be described in detail below.

[0074] (1) Raw materials This technology is characterized by the use of substandard rice. Details of substandard rice are as described above, so we will omit the explanation here. In this technology, as long as substandard rice is used, it is also possible to blend it with ordinary rice. The type of ordinary rice is not particularly limited, and for example, one or more types of rice such as Japonica rice, Indica rice, and Javanica rice can be used. In addition, either non-glutinous rice or glutinous rice can be used, and non-glutinous rice and glutinous rice can also be blended. Furthermore, the degree of milling is not particularly limited, and one or more types such as brown rice, germinated rice, partially milled rice, 70% milled rice, polished rice, and rinse-free rice can be used in combination. In addition, new rice, old rice, very old rice, or a blend of these may also be used.

[0075] Additionally, if necessary, it is possible to create a mixed grain rice blend with other grains (for example, barley, sprouted brown rice, adzuki beans, black beans, glutinous millet, sorghum, glutinous foxtail millet, amaranth, quinoa, Job's tears, barnyard millet, corn, black sesame seeds, white sesame seeds, etc.), and various ingredients and seasonings can be used according to the purpose and use.

[0076] (2) Enzyme addition process The enzyme addition process involves adding starch-degrading enzyme and 4-α-glucanotransferase to the defective rice. These enzymes may be added to the defective rice simultaneously or separately. There is no particular order in which two or more enzymes are added separately. Details of each enzyme and the amount to be added are the same as those used in the defective rice cooking modifier mentioned above, so an explanation is omitted here.

[0077] The specific method of enzyme addition is not limited; it can be added to the defective rice in any way and at any time, as long as the enzyme can be added before the cooking process described later. For example, methods include adding the enzyme to raw defective rice, adding the enzyme to washed defective rice, adding water with added enzymes to the defective rice, adding the enzyme when soaking the defective rice in water, or adding the enzyme to the defective rice immediately before cooking. It is also possible to add the enzyme in two or more stages.

[0078] (3) Water Addition Process The water addition process is the process of adding water to the defective rice. The amount of water added in the water addition process can be freely set as long as it does not impair the function and effect of this technology. The lower limit of the amount of water added in this technology is, for example, 1.40 g or more, preferably 1.45 g or more, more preferably 1.50 g or more, even more preferably 1.55 g or more, and particularly preferably 1.60 g or more per gram of defective rice. Cooked rice made from defective rice tends to spoil easily and harden after storage, but hardening after storage can be prevented by setting the lower limit of the amount of water added within this range.

[0079] The upper limit of the amount of water added in this technology is not particularly limited, but is, for example, 2.0 g or less, preferably 1.80 g or less, and more preferably 1.70 g or less, per 1 g of defective rice.

[0080] (4) Soaking Process The soaking process is a process in which defective rice is soaked in water. The soaking process is not an essential process in this technology, but it is preferable to perform it from the viewpoint of smoothly carrying out the enzyme treatment on defective rice. The timing of applying the enzyme to the defective rice may be during the soaking process, during the cooking process described later, or during both the soaking and cooking processes.

[0081] The soaking process may be performed simultaneously with or in any order with the enzyme addition process. That is, defective rice to which enzymes have been added may be soaked in water, or defective rice that has been soaked in water may be to which enzymes have been added. Furthermore, when soaking defective rice to which enzymes have been added in water, enzymes may be added again. Moreover, the same or different enzymes may be added to the rice that has been soaked in defective rice in multiple stages.

[0082] The various conditions in the immersion process can be freely set as long as they do not impair the effectiveness of this technology. For example, when an enzyme is used in the immersion process, the pH, temperature, and action time can be set according to the physicochemical properties of the enzyme used, such as its optimal pH, stable pH range, optimal temperature, and temperature stability. The optimal conditions can be determined through preliminary experiments. The following are examples of immersion conditions when using various enzymes.

[0083] [α-Amylase] When immersing defective rice to which α-amylase has been added in water, the pH can be set to, for example, pH 3.0 to 10.0, preferably pH 4.5 to 8.0, and more preferably pH 5.0 to 7.0. The temperature can be set to, for example, 10°C to 80°C, preferably 20°C to 70°C, and more preferably 30°C to 50°C. The soaking time can be set to, for example, 10 minutes to 12 hours, preferably 15 minutes to 6 hours, and more preferably 30 minutes to 4 hours.

[0084] [β-Amylase] When immersing defective rice to which β-amylase has been added in water, the pH can be set to, for example, pH 5 to 9, preferably pH 6 to 8, and more preferably pH 6.5 to 7.5. The temperature can be set to, for example, 20 to 80°C, preferably 30 to 70°C, and more preferably 40 to 60°C. The reaction time can be set to, for example, 5 minutes to 24 hours, preferably 15 minutes to 12 hours, and more preferably 30 minutes to 2 hours. The optimal reaction conditions can be determined through preliminary experiments.

[0085] [Maltotriose-producing enzyme] When immersing defective rice to which maltotriose-producing enzyme has been added in water, the pH can be set to, for example, pH 4.0 to 10.0, preferably pH 4.5 to 9.0, and more preferably pH 5.0 to 7.0. The temperature can be set to, for example, 20°C to 70°C, preferably 25°C to 60°C, and more preferably 25°C to 50°C. The soaking time can be set to, for example, 10 minutes to 12 hours, preferably 15 minutes to 6 hours, and more preferably 30 minutes to 4 hours.

[0086] [Transglucosidase] When soaking defective rice to which transglucosidase has been added in water, the pH can be set to, for example, pH 3.0 to 8.0, preferably pH 4.0 to 7.5, and more preferably pH 5.0 to 7.0. The temperature can be set to, for example, 20°C to 70°C, preferably 25°C to 60°C, and more preferably 25°C to 50°C. The soaking time can be set to, for example, 10 minutes to 30 hours, preferably 15 minutes to 20 hours, and more preferably 30 minutes to 4 hours.

[0087] [Glucoamylase] When soaking defective rice to which glucoamylase has been added in water, the pH can be set to, for example, pH 3.0 to 9.0, preferably pH 3.5 to 8.0, and more preferably pH 5.0 to 7.0. The temperature can be set to, for example, 10°C to 70°C, preferably 20°C to 65°C, and more preferably 30°C to 65°C. The soaking time can be set to, for example, 10 minutes to 2 hours, preferably 15 minutes to 2 hours, and more preferably 30 minutes to 2 hours.

[0088] [Cyclodextrin-producing enzyme] When immersing defective rice to which cyclodextrin-producing enzyme has been added in water, the pH can be set to, for example, pH 3.0 to 11.0, preferably pH 4.0 to 9.0, and more preferably pH 5.0 to 7.0. The temperature can be set to, for example, 30°C to 90°C, preferably 30°C to 80°C, and more preferably 30°C to 50°C. The soaking time can be set to, for example, 10 minutes to 48 hours, preferably 15 minutes to 24 hours, and more preferably 30 minutes to 4 hours.

[0089] [4-α-glucanotransferase] When immersing defective rice to which 4-α-glucanotransferase has been added in water, the pH can be set to, for example, pH 3.0 to 11.0, preferably pH 4.0 to 9.0, and more preferably pH 5.0 to 7.0. The temperature can be set to, for example, 20°C to 80°C, preferably 25°C to 70°C, and more preferably 25°C to 60°C. The soaking time can be set to, for example, 10 minutes to 48 hours, preferably 15 minutes to 24 hours, and more preferably 30 minutes to 4 hours.

[0090] Other components may be added to the water used in the immersion process, as long as they do not impair the function or effect of this technology. Examples of other components include various seasonings, various ingredients, pH adjusters, colorants, flavoring agents, stabilizers, and enzymes. Furthermore, known or future-discovered functional components may be added as appropriate for the purpose.

[0091] (5) Rice Cooking Process The rice cooking process is the process of cooking defective rice to which starch-degrading enzymes and 4-α-glucanotransferase have been added.

[0092] In this technology, rice cooking refers to heating rice while adding water. The heating method is not particularly limited and can include boiling, steaming, baking, microwave heating, etc., and these can be used in combination. Rice can also be cooked using a rice cooker or rice pot.

[0093] The cooking conditions can be freely set as long as they do not impair the effectiveness of this technology. For example, they can be freely set according to the type and amount of rice used, the type and amount of ingredients used in combination, and the intended use of the cooked rice.

[0094] (6) Preservation process The preservation process is the process of preserving cooked rice. The preservation process is not an essential step in this technology, but as shown in the examples described later, cooked rice prepared using this technology can maintain good quality even after preservation.

[0095] The various conditions in the storage process can be freely set as long as they do not impair the effectiveness of this technology. The storage temperature can be set, for example, to -40°C to 40°C, preferably to -30°C to 30°C, and more preferably to -25°C to 25°C. The storage time can be set, for example, to 1 hour or more, preferably to 6 hours or more, more preferably to 12 hours or more, and even more preferably to 20 hours or more. The upper limit of the storage time can be appropriately set depending on the storage temperature and storage method. For example, when storing at freezing temperatures, it can be set to 1 year or less, 6 months or less, 3 months or less, etc. When storing at chilling temperatures, it can be set to 7 days or less, 5 days or less, 3 days or less, etc. When storing at refrigeration temperatures, it can be set to 3 days or less, 2 days or less, etc.

[0096] Furthermore, in the processing steps described later, when processing into vacuum-packed rice or retort-packed rice, the storage time can be freely set according to the processing method.

[0097] The preservation process may be carried out by the same institution that performs the enzyme addition and cooking processes, or by different institutions. For example, one institution can cook and preserve the rice, or one institution can cook the rice, distribute it, and another institution can preserve it.

[0098] (7) Freezing process The freezing process is the process of freezing cooked rice. The freezing process is not an essential process in this technology, but it can be performed, for example, when storing the rice at a freezing temperature range in the storage process.

[0099] The timing of the freezing process is not particularly limited, as long as it does not impair the function or effect of this technology, and is performed after the rice cooking process. The freezing process can be performed after the storage process, or the storage process can be performed at freezing temperatures after the freezing process. Furthermore, the freezing process can be performed after the storage process at chilled or refrigerated temperatures, and then the storage process can be performed again at freezing temperatures.

[0100] The conditions in the freezing process are not particularly limited as long as they do not impair the function or effect of this technology, and general freezing methods can be freely adopted. For example, a method of freezing at around -20°C can be used.

[0101] The freezing process may be carried out by the same institution that performs the enzyme addition process, the rice cooking process, and the storage process, or it may be carried out by different institutions. For example, one institution can freeze cooked rice and then store it, or one institution can freeze cooked rice and distribute it as frozen rice, while another institution stores the frozen rice.

[0102] (8) Thawing process The thawing process is the process of thawing the frozen cooked rice. The thawing process is not an essential process in this technology, but for example, if the freezing process is performed, thawing can be done before consumption.

[0103] The specific method used in the thawing process is not particularly limited as long as it does not impair the function or effect of this technology, and any general thawing method can be freely adopted. Examples include thawing at room temperature, thawing at refrigeration temperature, thawing under running water, microwave heating, water bath, steaming, boiling, baking, and other heating methods.

[0104] The thawing process may be carried out by the same institution as the enzyme addition process, the rice cooking process, the storage process, and the freezing process, or it may be carried out by different institutions. For example, one institution may freeze cooked rice and then thaw it, or one institution may distribute cooked rice, another institution may freeze the rice and distribute it in a frozen state, and yet another institution may thaw the frozen rice. It is also possible for consumers who purchase the distributed frozen rice to thaw it themselves.

[0105] (9) Processing Process The processing process is the process of processing the cooked rice obtained through the rice cooking process according to the purpose and use. The processing process is not an essential step in this technology, but it can be performed as needed.

[0106] The specific methods used in the processing steps can be one or more general rice processing methods selected and used, as long as they do not impair the function or effect of this technology. Examples include methods for processing rice into vacuum-packed rice, retort-packed rice, dried rice, etc., and methods for cooking rice into dishes such as seasoned rice, fried rice, pilaf, gratin, paella, rice porridge, rice balls, and sushi.

[0107] The timing of the processing steps is not particularly limited, as long as it does not impair the function or effect of this technology, and is performed after the enzyme addition step. For example, cooked rice obtained through the rice cooking step can be processed, or, for example, when used in dishes such as mixed rice, pilaf, or paella, the rice cooking step and processing step can be performed simultaneously. Furthermore, the processing step can be performed after thawing frozen cooked rice, or the processed cooked rice can be frozen for storage.

[0108] The processing steps—enzyme addition, rice cooking, storage, freezing, and thawing—may be carried out by the same institution or by different institutions. For example, one institution may process cooked rice, or one institution may distribute cooked rice while another institution processes it. It is also possible for consumers who purchase the distributed rice to process it themselves.

[0109] (10) Recovery Process The recovery process is a process for recovering cooked rice or processed cooked rice obtained through the enzyme addition process and / or the cooking process, and, if necessary, the soaking process, storage process, freezing process, thawing process, processing process, etc. Depending on the state and type of cooked rice obtained, one or more recovery methods used in the production of general cooked rice can be freely combined and used.

[0110] The method relating to the present technology described above may include the following steps A to K: A: Step of preparing defective rice and other raw materials as needed B: Step of adding starch-degrading enzyme and 4-α-glucanotransferase to the prepared defective rice, etc. C: Step of adding water D: Step of soaking the defective rice in water E: Step of cooking the defective rice to which starch-degrading enzyme and 4-α-glucanotransferase have been added F: Step of storing the cooked rice G: Step of deactivating the enzyme H: Step of freezing the cooked rice I: Step of thawing the frozen cooked rice J: Step of processing the cooked rice K: Step of recovering the cooked rice

[0111] Step C may be performed before, after, or simultaneously with one or more steps selected from steps A, B, and D, and step C may be performed multiple times. Steps B and E may be performed individually or both. Steps D, F, G, H, I, J, and K are not mandatory and can be performed as needed. Furthermore, step G may be performed multiple times as needed: after step D, during step D, before or after step E, simultaneously with step E, or before or after step F. In addition, when steps E and G are performed simultaneously, the enzymes can be deactivated by heating during the rice cooking process. That is, step E can be performed instead of step G. Moreover, when multiple enzymes are used, enzyme deactivation can be performed between the actions of the enzymes. For example, when multiple enzymes are applied in immersion step D, it is possible to perform enzyme deactivation step G as appropriate during immersion step D.

[0112] 3. The cooked rice according to this technology is characterized by containing defective rice that has been treated with a defective rice cooking modifier. Details of the defective rice cooking modifier are as described above, so we will omit the explanation here.

[0113] The cooked rice according to this technology may be cooked using only defective rice to which the defective rice cooking modifier has been added, or it may be cooked using a mixture of defective rice and cooked regular rice. The mixing of defective rice and regular rice can be done before or after cooking. When mixing defective rice and regular rice, there are no particular restrictions on the timing of adding the defective rice cooking modifier according to this technology. For example, one method is to add the defective rice cooking modifier according to this technology after mixing the defective rice and regular rice before cooking, or to mix defective rice to which the defective rice cooking modifier according to this technology has been added beforehand with regular rice, and then cook the rice.

[0114] The distribution method for the cooked rice related to this technology is not particularly limited. For example, it can be distributed frozen, refrigerated, chilled, or at room temperature. Alternatively, cooked rice that has been stored frozen, refrigerated, chilled, or at room temperature may be heated or cooked at a store or other location and then distributed as pre-cooked processed cooked rice.

[0115] The present invention will be described in more detail below based on the following examples. The examples described below are merely representative examples of the present invention and should not be interpreted as narrowing the scope of the invention.

[0116] 1. In the material examples, rice with a powdery grain content of 7.6% by weight was used as an example of defective rice, and rice with a powdery grain content of 1.0% by weight was used as an example of normal rice. The powdery grain content was measured using a grain sorter (manufactured by Satake Corporation). The enzymes used in the examples are shown in Table 1 below.

[0117]

[0118] 2. Method for Measuring Enzyme Activity [Method for Measuring α-Amylase Activity] 10 mL of 1% potato starch substrate solution (0.1 mol / L acetic acid (pH 5.0)) was heated at 37°C for 10 minutes, then 1 mL of the sample solution containing α-amylase was added and immediately mixed. After letting this solution stand at 37°C for 10 minutes, 1 mL of this solution was added to 10 mL of 0.1 mol / L hydrochloric acid solution and immediately mixed. Next, 0.5 mL of this solution was measured, 10 mL of 0.0002 mol / L iodine solution (Japanese Pharmacopoeia) was added and mixed, and the absorbance (AT) at a wavelength of 660 nm was measured using water as a control. Separately, the same procedure was performed by adding 1 mL of water instead of the sample solution, and the absorbance (AB) was measured. The 0.0002 mol / L iodine reagent (Japanese Pharmacopoeia) was prepared by adding 12.7 g of iodine and 25 g of potassium iodide to 10 mL of water, mixing thoroughly, adding water to make a total volume of 100 mL, and then diluting it 2500 times with water. The α-amylase activity was calculated using the following formula. One unit (1 U) of enzyme was defined as the amount that reduces the color development of potato starch by iodine by 10% in one minute. α-amylase activity (U / g, U / mL) = {(AB - AT) / AB} × 1 / W AT: absorbance of the reaction solution AB: absorbance of the blank solution W: amount of sample in 1 mL of sample solution (g or mL)

[0119] [β-Amylase] The β-amylase was measured in accordance with the method described in the Compendium of Food Additives (9th edition). The specific method is as follows: Potato starch was used as the substrate, and it was dried at 105°C for 2 hours beforehand. 1.0 g of the dried product was weighed out, 20 mL of water was added, and 5 mL of sodium hydroxide solution (2 mol / L) was gradually added while stirring until it became a paste. Next, it was heated in a water bath for 3 minutes while stirring, and then 25 mL of water was added. After cooling, hydrochloric acid solution (2 mol / L) and hydrochloric acid solution (0.1 mol / L) were added to neutralize it, 10 mL of 1 mol / L acetic acid / sodium acetate buffer (pH 5.0) was added, and then water was added to make a total volume of 100 mL, which was the substrate solution. 10 mL of substrate solution was weighed out and heated at 37°C for 10 minutes. 1 mL of sample solution was added and immediately mixed. After heating at the same temperature for 10 or 30 minutes, 4 mL of Fehling's reagent was added and gently mixed. The mixture was then heated in a water bath for 15 minutes, cooled to below 25°C, and 2 mL of 30% potassium iodide solution and 2 mL of sulfuric acid (1→6) were added to prepare the test solution. Fehling's reagent was prepared immediately before use by weighing out 34.66 g of fine crystals of copper(II) sulfate pentahydrate, dissolving them in water to make a copper solution of 500 mL, and weighing out 173 g of (+)-potassium sodium tartrate tetrahydrate and 50 g of sodium hydroxide, dissolving them in water to make an alkaline tartrate solution of 500 mL, and mixing them in a ratio of 1 volume of copper solution to 1 volume of alkaline tartrate solution. Separately, a comparison solution was prepared using 10 mL of water instead of substrate solution, following the same procedure as for the test solution. The test solution and the comparison solution were titrated with a 0.05 mol / L sodium thiosulfate solution to release iodine. The endpoint was determined when 1-2 drops of soluble starch solution were added near the endpoint of the titration, and the resulting blue color disappeared. One unit (1 U) of enzyme was defined as the amount of enzyme that produces an increase in reducing power equivalent to 1 mg of glucose per minute, and was calculated using the following formula.β-amylase activity (U / g, U / mL) = amount of glucose (mg) × 1 / 10 × 1 / M Amount of glucose (mg) = (b - a) × 1.6 × f a: titration value of enzyme reaction solution (mL) b: titration value of blank solution (mL) 1.6: 1 mL of 0.05 mol / L sodium thiosulfate solution corresponds to 1.6 mg of glucose 1 / 10: unit conversion factor for reaction time (minutes) M: amount of sample in 1 mL of sample solution (g or mL) f: factor of 0.05 mol / L sodium thiosulfate solution (for quantitative analysis).

[0120] [Maltotriose-producing enzyme] The activity of the maltotriose-producing enzyme was measured using a method based on the maltotriose hydrolase activity test method in the 9th edition of the Compendium of Food Additives. The specific method is as follows: The enzyme was reacted with soluble starch as the substrate, and the resulting reducing sugar was colorimetrically determined by the Somogyi-Nelson method. 0.5 mL of soluble starch solution and 0.1 mol / L acetic acid / sodium acetate buffer (pH 6.0) (0.05 mol / L CaCl) were placed in a 50 mL Nessler tube. ２ 0.4 mL of (containing) was measured, shaken well, and left at 40 ± 0.5°C for 10 to 15 minutes. Then, 0.1 mL of the sample solution was added and immediately shaken. After this solution was left at 40 ± 0.5°C for exactly 15 minutes, 1 mL of alkaline copper reagent was added, shaken, and the container was stoppered. The container was heated in a boiling water bath for exactly 20 minutes and immediately cooled. After cooling, 1 mL of Nelson's solution was added and shaken well until the red precipitate of cuprous oxide was completely dissolved. Then, the container was left at room temperature for 20 minutes, and 22 mL of water was added and shaken well. The absorbance of this solution at a wavelength of 520 nm was measured using water as a control. One unit was defined as the amount of reducing sugar equivalent to 1 μmol of glucose produced per minute.

[0121] [Transglucosidase] (1) 2.0 g of α-methyl-D-glucoside was weighed out, dissolved in water to make 100 mL, and an α-methyl-D-glucoside solution was prepared. 1 mL of the α-methyl-D-glucoside solution and 1 mL of 0.02 mol / L acetic acid / sodium acetate buffer (pH 5.0) were weighed into a test tube, left at 40°C for 10 to 15 minutes, then 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) was added and shaken well. This was left at 40°C for exactly 60 minutes. After exactly 60 minutes, it was placed in a boiling water bath and heated for exactly 5 minutes, then cooled under running water. (2) 3 mL of the color-developing solution of Glucose CII-Test Wako (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed into a test tube, 0.2 mL of the reaction solution obtained in (1) was added and shaken well, then left at 40°C for exactly 5 minutes. For this solution, the absorbance (E60) at a wavelength of 505 nm was measured with water as a control. Separately, as a blank, 1 mL of 0.02 mol / L acetic acid / sodium acetate buffer (pH 5.0) and 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) were measured into test tubes, heated in a boiling water bath for exactly 5 minutes, and then cooled under running water. After cooling, 1 mL of α-methyl-D-glucoside solution was added, and the absorbance (E0) was measured in the same manner as above. (3) Glucose CII Test Wako's glucose standard solution I or II was diluted with water to the specified concentration (20 mg / dL, 40 mg / dL). 3 mL of Glucose CII Test Wako color development solution was measured into a test tube, 0.2 mL of the above glucose solution was added to each, and after shaking well, it was left to stand at 40°C for exactly 5 minutes. For this solution, the absorbance (ES) at a wavelength of 505 nm was measured with water as a control. Separately, absorbance (EB) was measured using 0.2 mL of water instead of glucose solution as a blank, in the same procedure as above. A glucose calibration curve was created from the obtained absorbances, and the amount of glucose (μg) (G) at an absorbance difference of 1.000 was determined. (4) The amount of enzyme that produces 1 μg of glucose in 60 minutes was defined as 1 unit (1 U), and was calculated using the following formula.Transglucosidase activity (U / g, U / mL) = (E60 - E0) × G × 2.5 / 0.1 × n / 0.5 E60: Absorbance of reaction solution E0: Absorbance of blank solution G: Amount of glucose (μg) when absorbance difference is 1.000 2.5: Volume of reaction system (mL) 0.1: Volume of reaction solution taken (mL) 0.5: Volume of enzyme solution taken (mL) n: Dilution factor per 1 g or 1 mL of sample.

[0122] [Glucoamylase] The glucoamylase activity test was performed using the following method in accordance with Method 4 of the 9th edition of the Japanese Food Additives Compendium. The specific method is as follows: 0.50 g of enzyme sample was weighed and diluted with water to an appropriate concentration to prepare the sample solution. Potato starch was pre-dried at 105°C for 2 hours, 1.0 g of the dried product was weighed, 20 mL of water was added, and 5 mL of sodium hydroxide solution (2 mol / L) was gradually added while stirring to form a paste. The paste-like starch was heated in a water bath for 3 minutes while stirring, then 25 mL of water was added, and after cooling, hydrochloric acid solution (2 mol / L) and hydrochloric acid solution (0.1 mol / L) were added to neutralize it, 10 mL of 1 mol / L acetic acid / sodium acetate buffer (pH 4.5) was added, and water was added to make a total volume of 100 mL to prepare the substrate solution. 10 mL of substrate solution was weighed out and heated at 37°C for 10 minutes. 1 mL of sample solution was added and immediately mixed. After heating at 37°C for 10 minutes, 4 mL of Fehling's reagent was added and gently mixed. After heating in a water bath for 15 minutes, the mixture was cooled to below 25°C. 2 mL of potassium iodide reagent and 2 mL of sulfuric acid (6 parts by volume of sulfuric acid diluted with water) were added to prepare the test solution. Separately, 10 mL of water was used instead of the substrate solution and the same procedure was followed to prepare the comparison solution. The liberated iodine in both the test solution and the comparison solution was titrated with a 0.05 mol / L sodium thiosulfate solution. The endpoint was determined when 1-2 drops of soluble starch reagent were added near the endpoint of the titration, and the resulting blue color disappeared. Under these conditions, the amount of enzyme that produces an increase in reducing power equivalent to 1 mg of glucose per minute was defined as 1 unit (1 U), and the glucoamylase activity was calculated using the following formula. Glucoamylase activity (U / g) = Amount of glucose (mg) × 1 / 10 × 1 / M Amount of glucose (mg) = (b - a) × 1.6 × f a: Titration value of the test solution (mL) b: Titration value of the comparison solution (mL) 1.6: 1 mL of 0.05 mol / L sodium thiosulfate solution corresponds to 1.6 mg of glucose 1 / 10: Unit conversion factor for reaction time (minutes) M: Amount of enzyme sample in 1 mL of sample solution (g or mL) f: Factor of 0.05 mol / L sodium thiosulfate solution

[0123] [Cyclodextrin-producing enzyme] The activity of the cyclodextrin-producing enzyme was measured using a method based on the cyclodextrin-producing enzyme activity test method in the 9th edition of the Japanese Food Additives Compendium. The specific method is as follows: 1.0 g of potato starch was mixed with 20 mL of water, and 5 mL of sodium hydroxide solution (1 mol / L) was gradually added while stirring until it became a paste. After heating in a boiling water bath for 3 minutes while stirring, 25 mL of water was added, and after cooling with running water, the pH was adjusted to 5.5 with acetic acid solution (1 mol / L), and water was added to make a total volume of 100 mL, which was the substrate solution. 10 mL of the substrate solution was measured out and heated at 40°C for 10 minutes, and 1 mL of enzyme solution was added and mixed. After incubation at 40°C for 10 minutes, the reaction was stopped by adding 10 mL of hydrochloric acid solution (0.1 mol / L). To 1 mL of this reaction solution, 10 mL of iodine-potassium iodide reagent (0.4 mmol / L) was added and mixed to prepare the test solution. The iodine-potassium iodide reagent was prepared by dissolving 10.0 g of potassium iodide and 1.0 g of iodine in water, making a total volume of 100 mL, and then diluting it 200-fold with water. A comparison solution was prepared using water instead of the reaction solution, and the absorbance at 660 nm of the test solution and the comparison solution was measured. One unit (U) of enzyme was defined as the amount that reduced the blue iodine color of starch by 1% per minute.

[0124] [Method for measuring the activity of 4-α-glucanotransferase] 180 mg of maltotetraose (manufactured by Nagase Vita Co., Ltd.) was weighed out and placed in a 20 mL volumetric flask. Approximately 15 mL of 10 mmol / L MES buffer (pH 6.5) was added and dissolved. After dissolution, 10 mmol / L MES buffer (pH 6.5) was added to bring the volume to 20 mL, which was used as the substrate solution. 2 mL of the substrate solution was weighed into a test tube and left at 40 ± 0.5°C for 10 to 15 minutes. Then, 0.5 mL of the sample solution was added, and the mixture was shaken well and left at 40 ± 0.5°C for exactly 60 minutes. After standing, the test tube was placed in a boiling water bath and heated for exactly 5 minutes, then cooled under running water. The generated glucose was quantified using Lab Assay Glucose (Fujifilm Wako Pure Chemical Industries, Ltd.). The Lab Assay Glucose kit consists of a chromogenic agent containing mutarotase, glucose oxidase, peroxidase, 4-aminoantipyrine, and ascorbate oxidase, and a buffer containing phosphate buffer pH 7.1 and phenol. The glucose concentration can be determined by measuring the red pigment produced by the oxidative condensation of phenol and 4-aminoantipyrine. Under these conditions, one unit of enzyme was defined as the amount of glucose produced per minute at a rate of 1 μmol.

[0125] 3. Preliminary Study (1) Method 100g of defective rice or regular rice was weighed and placed in a colander. The rice was then gently stirred 10 times clockwise by hand in a bowl filled with purified water that had been brought back to room temperature. The purified water was changed and the same process was repeated 4 times. After washing the rice, it was drained and transferred to a rice cooker. 140g of water was added to the rice cooker, and the rice cooker was placed in the rice cooker and soaked at room temperature for 1 hour. After soaking, the rice was cooked in the rice cooker (Panasonic: SR-03GP) using the normal method. After steaming for 10 minutes, the cooked rice was placed in a container, spread out, and allowed to cool to room temperature. An example using regular rice was used as a control example.

[0126] (2) Sensory evaluation 20g of cooled cooked rice was weighed out, and rice balls were made using commercially available rice ball molds (mini size), and a sensory evaluation was conducted. For the rice balls made as described above, the ease of breaking apart and the texture of the grains were evaluated according to the evaluation criteria shown in Table 2 below, both at the time of completion and after storing the cooked rice at 10°C for one day. The control example was given a score of 0, and the evaluation was conducted by five panelists using a scoring system from -3 to 3 points.

[0127]

[0128] (3) Results The results are shown in Table 3 below.

[0129]

[0130] (4) Discussion: When the inferior rice was cooked under the same cooking conditions as regular rice, it was found that it was inferior to regular rice in terms of both looseness and grain separation.

[0131] 4. Experimental Example (1) Method 100g of defective rice was weighed and placed in a colander. The polished rice was then gently stirred 10 times clockwise by hand in a bowl filled with purified water that had been brought back to room temperature. The purified water was changed and the same process was repeated 4 times. After washing the rice, it was drained and transferred to a rice cooker. The enzymes shown in Table 4 were dissolved in the amount of water shown in Table 4 and added to the rice cooker. The rice cooker was then placed in the rice cooker and soaked at room temperature for 1 hour. After soaking, the rice was cooked in the rice cooker (Panasonic: SR-03GP) using the normal method. After steaming for 10 minutes, the cooked rice was placed in a container, spread out, and allowed to cool to room temperature.

[0132] (2) Sensory evaluation The evaluation was carried out using the same method as in the preliminary study described above.

[0133] (3) Results The results are shown in Table 4 below.

[0134]

[0135] (4) Discussion: Compared to the comparative example in which poor quality rice was cooked using only 4-α-glucanotransferase, Examples 1 to 8, in which 4-α-glucanotransferase and starch-degrading enzyme were used in combination for cooking, showed superiority in almost all evaluations regarding both looseness and grain separation.

[0136] Furthermore, since substandard rice is inherently prone to stickiness, increasing the amount of water generally results in even more stickiness immediately after cooking, and after storage, it rapidly deteriorates and hardens. However, in Examples 1 to 8, where 4-α-glucanotransferase and starch-degrading enzymes were used in combination during cooking, the rice did not become excessively soft immediately after cooking, stickiness was suppressed, and the rice maintained its softness even after storage.

Claims

1. A rice cooker for defective rice, containing starch-degrading enzymes and 4-α-glucanotransferase.

2. A method for producing cooked rice, comprising a cooking step of cooking defective rice to which starch-degrading enzymes and 4-α-glucanotransferases have been added.

3. A method for improving cooked rice, comprising a cooking step of cooking defective rice to which starch-degrading enzymes and 4-α-glucanotransferases have been added.

4. Cooked rice containing defective rice that has been prepared using the defective rice cooking modifier described in claim 1.

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