A method for mass-producing a sweetener with sugar-like taste and functionality that is similar to D-allulose

Abstract

[Problem] To mass-produce, at a worldwide scale and at a competitive low cost, a sweetener that is very close to the sweetness and flavor quality of sugar and that contains D-allulose. [Solution] Provided is a method for mass-producing a sweetener having a flavor quality similar to that of sugar and having D-allulose functionality, the method being characterized in that, in a composition composed of rare sugar D-allulose, D-glucose, and D-fructose, the composition being obtained by using an inexpensive composition that contains D-glucose and D-fructose as a raw ingredient, causing isomerization of the D-fructose in the raw ingredient to advance through an isomerization reaction between D-fructose and rare sugar D-allulose by the action of D-allulose 3-epimerase, and ending the reaction at a stage in which the desired rare sugar D-allulose content of a target product has been reached, there is obtained a target product in which the D-fructose content is within a range such that the D-fructose supplements sweetness, which is the weak point of D-allulose, and imparts a flavor quality similar to that of sugar together with the D-allulose and D-glucose.

Description

[Technical Field]

[0001] The present invention relates to a method for mass-producing a sweetener containing three sugars consisting of D-glucose, D-fructose, and D-allulose (D-psicose) and having a taste similar to that of sugar with the functionality of D-allulose.In particular, the present invention relates to a method for improving inexpensive isomerized sugar or invert sugar, which is a sugar mixture containing D-glucose and D-fructose, by converting the isomerized sugar or invert sugar into a sweetener with the functionality of D-allulose while maintaining or improving the taste and imparting the function of exerting excellent physiological effects that do not cause diseases when continuously taken. [Background technology]

[0002] Carbohydrates, the most abundant organic compounds on Earth, are primarily composed of monosaccharides, which are crucial for living organisms as energy sources, particularly in the pursuit of "zero hunger." These monosaccharides typically have a ring structure consisting of three to eight carbon atoms. Structurally, they are broadly classified into two types: aldoses and ketoses. Aldoses and ketoses are further classified into trioses, tetroses, pentoses, and hexoses, depending on the number of carbon atoms. An industrially implemented reaction for these monosaccharides involves the isomerization of D-glucose to D-fructose, producing isomerized sugar. A typical method is to produce isomerized sugar from starches such as corn starch. With the increase in corn production, the large amount of cornstarch available on the market allowed for the inexpensive mass production of isomerized sugar, making it significantly cheaper than sugar and thus a substitute for sugar.

[0003] On the other hand, sugar, a disaccharide, was expensive before isomerized sugar became popular, and has tended to be replaced by isomerized sugar. However, in 2019, approximately 180 million tons were produced worldwide, and prices have been relatively stable. Using sugar as a raw material allows for the utilization of sugar's high-quality sweetness. Furthermore, because sugar is a disaccharide formed by the combination of glucose and fructose, it can also be used as a source of glucose and fructose, so depending on the price of sugar, it is thought to be cheaper than isomerized sugar in terms of cost. Furthermore, it has been reported that sweeteners containing rare sugars made from sugar have the advantage of having fewer unpleasant flavors than sweeteners made from isomerized sugar (Patent Document 1).

[0004] Based on these assimilable sugars, sweeteners excellent in sweetness and physical properties are industrially mass-produced. The typical method for producing isomerized sugar is to add water and a liquefying enzyme (alpha-amylase) to starch, such as corn starch, and hydrolyze it at around 95°C to produce dextrin. This process is called liquefaction. After liquefaction is complete, the starch is cooled to around 55°C, and a saccharifying enzyme (glucoamylase) is added to further break it down into D-glucose, producing a saccharified solution. In addition to D-glucose, this saccharified solution contains small amounts of oligosaccharides. The saccharified solution saccharified with glucoamylase is then continuously passed through an immobilized enzyme, in which an isomerizing enzyme (glucose isomerase) produced by a fungus belonging to the genus Streptomyces, for example, is immobilized by various methods, to isomerize the D-glucose to D-fructose. The above isomerization is an equilibrium reaction with an equilibrium point near 1. When equilibrium is reached, approximately 50% of D-glucose can be isomerized to D-fructose at a reaction temperature of approximately 60°C. However, it takes a considerable amount of time to achieve this level of isomerization, and this prolonged heating causes the reaction solution to discolor, increasing the costs of the purification and concentration processes required for commercialization. Therefore, the reaction is terminated when the D-fructose content reaches approximately 42%. Subsequent purification and concentration yields high-fructose corn syrup with a fructose content of 42%. The enzymes used in these production processes are all produced by safe microorganisms. The isomerization of D-glucose to D-fructose by glucose isomerase is an equilibrium reaction, and the ratio of D-glucose to D-fructose in isomerized sugar is usually about 58:42. The sweetness of this glucose-fructose syrup, which contains 42% D-fructose, is 70-90, with the sweetness of sugar being 100.

[0005] Furthermore, by using a separation device called chromatography, it is possible to produce a product with a 55% fructose content. Specifically, to convert the 42% isomerized sugar into 55% isomerized sugar, a D-fructose solution with a D-fructose content of approximately 95% is first obtained by continuous sugar separation using a large-scale facility such as a reaction column filled with cation exchange resin, and then this D-fructose solution is mixed with the 42% isomerized sugar. Alternatively, purified D-fructose may be added to compensate for the lack of sweetness, but in these cases the final D-glucose to D-fructose ratio is usually about 45:55. The sweetness of this high fructose syrup with a D-fructose content of 55% is 100-120, with the sweetness (strength of sweetness) of sugar being 100. If the sweetness (strength of sweetness) of sugar is 100, then the sweetness of D-glucose is 65-80, and D-fructose is 120-170. The order of sweetness is D-fructose (fruit sugar) > sucrose (sugar) > D-glucose (grape sugar), but D-fructose is only 60% as sweet as sucrose at high temperatures, is almost the same as sucrose at 40°C, and is not sweeter than sucrose unless the temperature is lower than that, so the sweetness of isomerized sugar is greatly affected by temperature. In the late 1970s, the advent of chromatographic fructose concentration technology made it possible to mass-produce isomerized sugar, which quickly spread. As consumption of isomerized sugar increased and consumption of sugar decreased, it can be said that it has become a substitute for sugar. High-fructose corn syrup (HFCS)55, which is made of 55% D-fructose and 42% D-glucose and has been adjusted to have the same sweetness as sucrose, is the most popular and can be considered a new type of sugar, as it is used in soft drinks.

[0006] High-fructose sugar or invert sugar, a mixed sugar composed primarily of D-glucose and D-fructose, is widely used due to its economic benefits, but is suspected to be a cause of hyperglycemia and overweight (obesity) ("metabolic syndrome") in developed countries such as Japan and the United States (Non-Patent Document 1). Compared with D-glucose, D-fructose metabolism in the liver is more likely to promote lipid production and induce hyperlipidemia and obesity, and therefore attention has been focused on the relationship between the intake of D-fructose, which is contained in high-fructose corn syrup, and the increase in obesity (lifestyle-related diseases). As mentioned above, the sweetness and quality of isomerized sugar differ from those of commonly used sugars, and although efforts have been made to bring the sweetness and quality closer to those of sugar by changing the ratio of D-glucose to fructose, it cannot be said that a satisfactory method has been established. Based on these assimilable sugars, which are extremely important for living organisms as energy sources, several sweeteners that are excellent in sweetness and physical properties and do not cause these diseases are currently being developed for commercialization.

[0007] D-allulose, which has zero energy and various functionalities, is attracting attention as a way to improve the functionality of assimilable sugars. D-Allulose is a rare sugar, and can be produced from D-fructose in a yield of 20 to 25% by treating D-fructose with D-ketohexose 3-epimerase (Patent Document 2). It has also been reported that D-allulose 3-epimerase (Non-Patent Document 2) can produce D-allulose in a yield of 40%. Pure rare sugar D-allulose (=D-psicose) is produced using, for example, ketose 3-epimerase (e.g., D-allulose 3-epimerase) and D-fructose as a raw material. The rare sugar D-allulose can be produced as a powdered crystal by treating D-fructose with epimerase to produce a mixture of D-fructose and D-allulose, which is then separated and purified. D-Allulose has been shown to exhibit properties useful as a preventative material for lifestyle-related diseases, such as zero kilocalories (Non-Patent Document 3), postprandial blood glucose suppression (Non-Patent Document 4), suppression of rapid blood glucose increases caused by D-glucose, a digestible sugar ingested through food and beverages (Patent Document 3), and anti-obesity effects (Non-Patent Document 5). D-allulose itself has applications as a sweetener and in medicine, and is being mass-produced and sold around the world. Because D-allulose has zero energy and various functionalities, it is being sold primarily as a sweetener. However, when producing purified D-fructose and then D-allulose, these processes are carried out in separate steps, which means that there are constraints on raw materials, transportation, reaction costs, and plant operating costs, and there are currently many economic issues that need to be resolved before D-allulose can be mass-produced industrially.

[0008] The rare sugar D-allulose, also known as D-psicose, is an epimer of D-fructose and has a sweetness similar to that of D-fructose. However, its sweetness is only about 70% of that of sugar, so it is necessary to bring its sweetness closer to that of sugar. A technology has been developed to mix the rare sugar D-allulose with isomerized sugar, which has the effect of suppressing the rapid rise in blood sugar levels caused by D-glucose, a digestible sugar contained in foods and beverages, when ingested with these foods and beverages. One method uses isomerized sugar as a raw material and uses a chemical reaction (processing in a system containing one or more selected from the group consisting of basic ion exchange resins, alkalis, and calcium salts) to produce a syrup containing rare sugars, called rare sugar sweet (RSS) (Patent Document 4).The syrup contains D-glucose and D-fructose as the main components, with approximately 15% rare sugars (D-allulose 5.4%, D-tagatose 2.0%, D-allose 1.4%, etc.). The second is aimed at solving the problem that the sweetness and quality of high fructose corn syrup, a conventional sweetener, differs from that of sugar, and aims to create a sweetener that is very close to the sweetness and taste of sugar. This sweetener is composed of D-fructose, D-glucose, and D-allulose and has a sweetness and taste similar to that of sugar, and is a sweetener that suppresses the obesity-inducing effects of isomerized sugar. Sweeteners have been developed that contain 9 or more parts by weight of D-allulose per 100 parts by weight of D-glucose and D-allulose, 34 to 55 parts by weight of D-fructose per 100 parts by weight of D-fructose, D-glucose, and D-allulose, and 66 to 45 parts by weight of D-glucose and D-allulose per 100 parts by weight of D-fructose, D-glucose, and D-allulose (Patent Document 5). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 6250946 [Patent Document 2] Japanese Patent Application Publication No. 6-125776 [Patent Document 3] Patent No. 5171249 [Patent Document 4] Patent No. 5715046 [Patent Document 5] Patent No. 5639759 [Patent Document 6] Patent No. 5997693 [Patent Document 7] Patent No. 5922880 [Patent Document 8] Japanese Patent Application Laid-Open No. 2017-36276 [Patent Document 9] Patent No. 4009720 [Patent Document 10] Japanese Patent Application Laid-Open No. 2007-91696 [Non-patent literature]

[0010] [Non-Patent Document 1] Am. J. Clin. Nutr. 2004, 79, 774-9. [Non-patent document 2] The 3rd Symposium of International Society of Rare Sugars [Non-patent document 3] J. Nutr.Sci. Vitaminol. 48,77-80. [Non-patent document 4] J. Nutr. Sci. Vitaminol.59, 191-121. [Non-patent document 5] J. Clin. Biochem. Nutr., 30,55-65. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0011] The target product produced by the above two methods is high-fructose sugar, which has excellent physiological effects, but it cannot be said to be price-competitive for mass production on a global scale. The challenge is how to reduce costs. In recent years, we have been faced with the new challenge of reducing CO2 emissions, and CO2 emissions have become a cost, and are expected to become an increasingly important part of the cost structure. There is a need to develop new manufacturing processes that make it possible to reduce CO2 emissions. We believe that the creation of a new manufacturing process that can compete on a global level and efficiently realizes the "manufacturing" that is theoretically possible using the above two methods will have a major impact on cost reduction. Corn and sugarcane have ideal characteristics as biomass. With the increase in corn production, the large amount of cornstarch on the market has been used to produce large quantities of low-fructose corn syrup at low cost, making it much cheaper than sugar and allowing it to replace sugar. However, we believe that a stable supply of corn-based high-fructose corn syrup can only be ensured by improving high-fructose corn syrup. This is because alcohol is produced from cornstarch and syrup, and ethylene is produced from this bioethanol through a dehydration reaction, resulting in the mass production of so-called biomass-based polyethylene. Because global polyethylene prices are determined based on expensive naphtha, biopolyethylene is considered price-competitive with fossil-fuel-based polyethylene. Biopolyethylene's greenhouse gas reduction effect is one-fifth that of petroleum-based polyethylene. Biopolyethylene is being used to appeal to consumers as a global warming-friendly material, and is therefore being used to partially or completely replace fossil-fuel-based polyethylene. This sudden increase in corn demand for ethanol could lead to price hikes due to shortages, and there is no guarantee that it will be used as a raw material for the greater amount of isomerized sugar required to achieve "Zero Hunger." Furthermore, all harvested sugarcane is collected at sugar factories and processed into sugar, and the collection system, which is the biggest problem in biomass utilization, has already been established. Due to its productivity and versatility, sugarcane has great advantages as biomass that are unmatched by other crops. As biomass is gaining attention as a measure against global warming, ethanol production using sugarcane is underway, and sugarcane, with its high biomass productivity, is becoming a trump card in global warming countermeasures. As with cornstarch, there are concerns that prices will rise due to shortages.

[0012] The problem with high-fructose corn syrup and invert sugar is that their continued consumption can lead to lifestyle-related diseases such as obesity. Because the lower the temperature of its component D-fructose, high-fructose corn syrup is, the sweeter it becomes. Approximately 70% of its consumption is added to beverages such as soft drinks, while the rest is used in general food products. Perhaps precisely because we live in an age where the sweetness of excessive assimilable sugars is readily available, the recent problem of carbohydrates (assimilable sugars) such as high-fructose corn syrup and sugar can lead to diabetes and obesity. Unless a sweetener based on these assimilable sugars has excellent sweetness and does not cause these diseases, it cannot be used as an energy source for the "Zero Hunger" initiative. Specifically, there is a need for a method for mass-producing, at low cost and on a global scale, a sweetener containing D-allulose, which has the effect of suppressing the rapid rise in blood glucose levels caused by D-glucose, a digestible sugar ingested through food and beverages, and which has a sweetness and taste very similar to that of sugar.Another object of the present invention is to provide foods and beverages, pharmaceuticals or quasi-drugs, and cosmetics that use a sweetener containing D-allulose produced at low cost, which has excellent sweetness and does not cause illness even with long-term intake. [Means for solving the problem]

[0013] The present inventors have produced D-allulose and expanded their research into rare sugars, which had previously focused solely on its effectiveness, by converting the energy of sunlight into the monosaccharide C6H6O7 in photosynthesis. 12I came up with the idea of ​​recycling O6 glucose and its polymerized starch, a disaccharide of glucose and fructose, on the energy flow that flows to humanity. Sugars play an important role as an energy source, are essential for humans and other living organisms, and have many challenges to overcome. However, this familiar glucose can be converted into isomerized sugar by isomerases such as D-xylose isomerase (glucose isomerase), and sugar can be converted into glucose and fructose by invertase. These sugars are beginning to be used not only as sweeteners but also as inexpensive raw materials in many organic chemistry fields. As isomerized sugar and sugar (invert sugar) become inexpensive and commonly used around the world, I recognized the global trends and the major challenges facing humanity, and discovered that the effectiveness of rare sugars could be put to good use. I was convinced that D-allulose would become a rare sugar that could be produced worldwide to solve the problems of isomerized sugar and sugar. The inventors were able to combine the effectiveness of D-allulose with inexpensive isomerized sugar and sugar (invert sugar) thanks to the enzyme ketose 3-epimerase, which is readily available (e.g., Patent Document 6, PCT / JP2019 / 045672). The new sugar industry, which has been producing sugar using fructose, which is already used on a global level, as a raw material, has made it possible to inexpensively produce valuable compositions using inexpensive isomerized sugar and sugar (invert sugar) by making full use of ketose 3-epimerase, which is readily available at the research level. In other words, by producing glucose, fructose, and D-allulose in the appropriate ratios, it became possible to mass-produce the desired product inexpensively. Due to issues such as obesity caused by isomerized sugar and sugar, the importance of these as energy sources for humanity has declined. As a result, society is increasingly using these sugars cheaply as bioethanol and other products. To solve this important issue related to carbohydrates, we utilize the power of bio (ketose 3-epimerase) to resolve the issues with these sugars and convert them into carbohydrates suitable for use as an energy source for humanity. This invention is intended to contribute to this goal of "zero hunger." Ketose 3-epimerase converts D-fructose into functional D-allulose. In this invention, any carbohydrate containing D-fructose can be used as a raw material. Typical examples are isomerized sugar and invert sugar. The composition is D-glucose, D-fructose, and D-allulose, and sugar with the desired composition can be produced using only ketose 3-epimerase. We have discovered an extremely simple method of utilizing the large-scale isomerization and sugar hydrolysis already performed in factories, such as grapes, by adding a new bioreactor containing only ketose 3-epimerase. The invention was completed by incorporating the reactions currently used in the sugar industry (i.e., creating a new manufacturing process). Furthermore, to improve the cost-effectiveness of the new production process, the inventors focused on the high durability (lifespan) of the enzyme ketose 3-epimerase. Focusing on bacterial species that are approved for use in food products not only in Japan but also in Europe and the United States and are considered to be nearly non-toxic, the inventors collected soil samples from various locations, isolated microorganisms from the soil, and continued searching for microorganisms with ketose 3-epimerase activity. As a result, among the many isolated strains, they discovered a microorganism that produces a novel ketose 3-epimerase. The microorganism was the Arthrobacter histidinolovorans Y586-1 strain, a microorganism of the genus Arthrobacter. This strain produces a novel, highly active and thermostable ketose 3-epimerase, and a separate international patent application was filed [PCT / JP2019 / 045672 (international filing date: November 21, 2019)]. Its high heat resistance and high productivity led us to believe that the overall productivity was guaranteed due to the enzyme's high durability (lifespan) rather than activity. As described above, in summary, the present inventor has conducted extensive research to solve the above problems, and has found the following: (A) We recognized that the characteristics of sweeteners containing D-glucose, D-fructose, and D-allulose are: (i) D-glucose is important as an energy source; (ii) D-fructose is important for providing sweetness and, together with D-glucose, provides a taste similar to that of sugar; however, as sugars, they have issues such as being the cause of obesity when taken continuously over a long period of time; and (iii) D-allulose has physiological functions such as anti-obesity. Based on this recognition, we discovered that (B) by optimizing the composition of the three sugars, it is possible to provide inexpensive sweeteners with compositions suited to specific purposes. The raw material for production of D-allulose needs to be an inexpensive raw material containing D-fructose. Regarding the inexpensive raw material containing D-fructose, (i) D-fructose can be easily produced from D-glucose using D-xylose isomerase, so D-glucose produced from starch can be used. (ii) Isomerized sugar (D-glucose + D-fructose) is commercially available at low cost in various compositions and can be used as a raw material. (iii) Sugar is an inexpensive sweetener formed by the combination of D-glucose and D-fructose, and is easily decomposed by invertase. Therefore, it can be used as an inexpensive raw material for bioethanol production. Based on the above findings (i) to (iii) regarding production raw materials, it was decided that the production raw material should be D-glucose or D-fructose, and that various types of isomerized sugars for which mass production technology has been established (production is possible using only an epimerase column, and by using various types appropriately, it is possible to create products with the desired composition) and inexpensive sugar (a D-glucose + D-fructose (1:1) composition that is mass-produced and used for bioethanol is available at low cost). Using such production raw materials makes it possible to produce a pure form free from by-products by appropriately connecting a conventional production line, and there is no need to add a special purification process. (C) Next, the present invention was arrived at by using an enzymatic reaction involving the highly thermostable and highly productive D-allulose 3-epimerase to achieve total productivity due to the high durability (lifespan) of the enzyme, and by producing the product without using simulated moving bed chromatography, which makes it possible to mass-produce the product on a global scale with sufficient price competitiveness.

[0014] The gist of the present invention is a method for mass-producing a sweetener with a taste similar to that of sugar and having the functionality of D-allulose described below in (1) to (10). (1) A method for mass-producing a sweetener containing three sugars consisting of D-glucose, D-fructose, and D-allulose and having the functionality of D-allulose and a taste similar to that of sugar, An inexpensive composition containing D-glucose and D-fructose is used as a raw material. D-allulose 3-epimerase is allowed to act to cause the isomerization of D-fructose in the raw material through an isomerization reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the target product reaches the desired rare sugar D-allulose content. A method for obtaining a target product, characterized by comprising a composition consisting of the rare sugars D-allulose, D-glucose and D-fructose, in which the content of D-fructose is in a range that compensates for the weak point of D-allulose, namely its sweetness, and, together with D-allulose and D-glucose, imparts a taste similar to that of sugar. (2) In the target product, D-glucose is important as an energy source, but its sweetness is 65-80, where the sweetness of sugar is 100. D-fructose has a sweetness level of 120-170 and is important for providing sweetness. Along with D-allulose and D-glucose, it provides a taste similar to sugar. However, continued intake of D-fructose can be problematic as it is the primary sugar causing obesity. The method described in (1) above, in which the content of each component is determined taking into consideration the characteristics of each component, namely, that D-allulose has a sweetness intensity of 70 and can provide physiological functions of anti-obesity and suppressing a sudden rise in blood sugar levels caused by digestible sugars, to obtain an appropriate composition according to the purpose.

[0015] (3) The method according to (1) or (2) above, wherein the raw material is a composition containing D-glucose and D-fructose, produced by using D-glucose produced from starch and treating it with D-xylose isomerase to promote isomerization through an equilibrium reaction between D-glucose and D-fructose, or by treating sugar with invertase to decompose it. (4) A method according to any one of (1) to (3) above, wherein the raw material is inexpensive isomerized sugar or invert sugar, and the target product is an improved isomerized sugar or invert sugar that has a taste similar to sugar with the functionality of D-allulose and exhibits excellent physiological effects that do not cause disease when taken continuously. (5) The method according to (4) above, wherein high-fructose corn syrup and / or high-fructose corn syrup is used as the inexpensive isomerized sugar.

[0016] (6) A method according to any one of (1) to (5) above, characterized in that the enzyme activity of the D-allulose 3-epimerase used is adjusted to achieve the desired content of rare sugar D-allulose in the target product. (7) A method according to any one of (1) to (6) above, characterized in that the isomerization reaction is carried out by passing a substrate solution through an immobilized enzyme column, and the composition of the target product is adjusted to the desired one by adjusting one or more of the following: the flow rate of the substrate, the reaction time with the substrate, the reaction temperature, the pH of the substrate solution, and the amount of enzyme. (8) A method according to any one of (1) to (7) above, characterized in that the raw material is used as a substrate in an immobilized enzyme reaction, and theoretically the desired product is obtained 100% without producing any by-products. (9) The method according to any one of (1) to (8) above, wherein the target product is not lost during the production process by simulated migration chromatography. (10) The D-allulose 3-epimerase is allowed to act to isomerize the D-fructose in the raw material through an isomerization reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the target rare sugar D-allulose content of the target product is reached. The method according to any one of (1) to (9) above, characterized in that a reaction step is added in which D-xylose isomerase, which does not act on the rare sugar D-psicose, is used to promote isomerization through an equilibrium reaction between D-fructose and D-glucose. (11) The method according to (10) above, wherein high fructose liquid sugar is used as the raw material, which is an inexpensive isomerized sugar. (12) The method according to any one of (1) to (11) above, which is an industrially practical, low-cost production method based on the total productivity derived from the high durability (lifespan) of the D-allulose 3-epimerase used due to its high thermostability. [Effects of the Invention]

[0017] The present invention provides a method for improving isomerized sugar or invert sugar, which are mixed sugars primarily composed of D-glucose and D-fructose, by maintaining or improving their taste and imparting them with functions that exert excellent physiological effects that do not cause disease when taken continuously. D-fructose (fruit sugar) has the advantage of being sweet and delicious, but its long-term intake can have adverse effects, such as the accumulation of neutral fats, which is why isomerized sugar containing D-fructose is said to be the cause of obesity and other problems. By adjusting the amount of D-fructose to minimize the drawbacks of long-term intake and by adjusting the composition to bring out the sweetness of D-fructose, it is possible to create a new sweetener that compensates for the weakness of D-allulose, its sweetness, and has the functionality of D-allulose. The rare sugar-containing composition of the present invention, which has the same taste as sugar, is a composition composed of D-glucose, D-fructose, and D-allulose, and can therefore be used in foods, beverages, and particularly functional foods (such as those for preventing obesity), as well as pharmaceuticals, cosmetics, feed, agricultural chemicals (such as plant growth regulators and plant disease resistance enhancers), and industrial applications.

[0018] The present invention provides a method for mass-producing a sweetener with the functionality of D-allulose and a taste similar to that of sugar in a simple manner without adding anything else, by performing one enzymatic reaction with raw materials and, if necessary, adding a second enzymatic reaction, and / or by adding an additional enzymatic reaction to the raw material production. This production method does not require new capital investment costs, is completed by reacting epimerase and, if necessary, isomerase under appropriate conditions, allows production using continuous or individual reactions as desired, and is characterized by high overall productivity due to the high durability (lifespan) of the enzymes.

[0019] When producing conventional sugar sweeteners such as HFCS and D-allulose, a separation step using simulated migration chromatography is essential. In the present invention, the raw material liquid sugar mixture can be completely converted into a product in theoretically 100% yield without this separation step.

[0020] Furthermore, the sweetness can be improved to be the same as that of sugar by increasing the D-fructose content, and production costs can be reduced by connecting it to a conventional production line in an appropriate manner.By constructing an unprecedented system that fully maintains the functionality of D-allulose, we can provide a complete production method.

[0021] By eliminating the separation step, there is almost no decrease in concentration due to separation, the concentration step can be omitted, and the product can be obtained by simply deionizing and concentrating the product of the enzymatic reaction, which is expected to result in significant cost reductions. Therefore, all the advantages of the already established production methods for isomerized sugar or invert sugar can be enjoyed, and by adding only the cheapest process, it is possible to provide a cost-competitive, low-cost production method for mass-producing on a global scale a sweetener that contains D-allulose, which has the effect of suppressing the rapid rise in blood sugar levels caused by D-glucose, a constituent of digestible sugars ingested through food and beverages, and has a sweetness and taste very similar to that of sugar. [Brief explanation of the drawings]

[0022] [Figure 1]In the case of using D-glucose as a raw material (1), D-glucose produced from starch is used, and D-xylose isomerase (DXI) is allowed to act to promote isomerization through an equilibrium reaction between D-glucose and D-fructose (=produced by a typical method for producing isomerized sugar). A composition containing D-glucose and D-fructose (isomerized sugar) is used as a raw material, and D-allulose 3-epimerase (DAE) is allowed to act to isomerize the D-fructose in the raw material to the rare sugar D-allulose, promoting isomerization through an equilibrium reaction between D-fructose and the rare sugar D-allulose. The reaction is terminated when the target product reaches the desired rare sugar D-allulose content. This is a diagram illustrating a method for obtaining a target product composed of the rare sugars D-allulose, D-glucose, and D-fructose. [Figure 2] In the case of using D-glucose as a raw material (2), a composition containing D-glucose and D-fructose (isomerized sugar) produced by a typical method for producing isomerized sugar is first separated into D-glucose and D-fructose using a separation device called chromatography to obtain a D-fructose liquid. This D-fructose liquid is then mixed with the isomerized sugar to produce various types of isomerized sugar depending on the amount of D-fructose added. This is used as a raw material and is treated with DAE to isomerize the D-fructose in the raw material to the rare sugar D-allulose, thereby progressing isomerization through an equilibrium reaction between D-fructose and the rare sugar D-allulose, thereby obtaining the desired product. [Figure 3]This figure explains the manufacturing process for various types of isomerized sugars (glucose-fructose corn syrup, fructose-glucose corn syrup, high-fructose corn syrup) that meet the Japanese Agricultural Standards, and a method for obtaining the target product by treating the various types of isomerized sugars obtained as a raw material with DAE to isomerize the D-fructose in the raw material to the rare sugar D-allulose through an equilibrium reaction between D-fructose and the rare sugar D-allulose. The leftmost section shows the same production method as in Figure 1 above, the middle section shows the same production method as in Figure 2, and the rightmost section shows a production method using high fructose liquid sugar as a raw material, similar to the production method (Figure 4) filed in a separate application (PCT / JP2019 / 043722), in which the D-allulose 3-epimerase is allowed to act to cause isomerization of D-fructose in the raw material through an isomerization reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the target product reaches the desired rare sugar D-allulose content.After that, a reaction step is added in which D-xylose isomerase, which does not act on the rare sugar D-psicose, is allowed to act to cause isomerization through an equilibrium reaction between D-fructose and D-glucose, and the D-fructose content is adjusted to the desired D-fructose content of the target product. [Figure 4] When D-fructose is used as a raw material, a method for which a separate application (PCT / JP2019 / 043722) has been filed is described. This method is characterized by the following: in the first step, D-fructose is mixed with D-allulose using ketose 3-epimerase (DAE); in the second step, D-fructose is converted to D-glucose using D-xylose isomerase (DXI), which does not act on D-allulose, thereby obtaining a target product with a new composition of D-glucose, D-fructose, and D-allulose. [Figure 5]When sugar is used as a raw material, a composition mainly composed of D-glucose and D-fructose is used as the raw material, which is produced by decomposing sugar with the action of invertase (produced by a typical method for producing invert sugar). D-allulose 3-epimerase (DAE) is then allowed to act to isomerize the D-fructose in the raw material to the rare sugar D-allulose, causing isomerization to proceed through an equilibrium reaction between D-fructose and the rare sugar D-allulose. The reaction is terminated when the desired rare sugar D-allulose content in the target product is reached, and this diagram explains a method for obtaining a target product composed of the rare sugars D-allulose, D-glucose, and D-fructose. [Figure 6] 1 is a line graph showing the change in sugar composition over time in a batch method when a sugar solution with a D-glucose:D-fructose ratio of 55:45 is used as the substrate in Example 1. [Figure 7] 1 is a line graph showing the change in sugar composition over time in a batch method when a sugar solution with a D-glucose:D-fructose ratio of 50:50 is used as the substrate in Example 1. [Figure 8] 1 is a line graph showing the change in sugar composition over time in a batch method when a sugar solution with a D-glucose:D-fructose ratio of 45:55 is used as the substrate in Example 1. BEST MODE FOR CARRYING OUT THE INVENTION

[0023] [Production raw materials] It is necessary to use inexpensive raw materials. To enable mass production on a global scale with sufficient price competitiveness, the raw materials used must first be inexpensive and suitable for mass production. To obtain the three sugars consisting of D-glucose, D-fructose, and D-allulose inexpensively, any inexpensive material containing D-glucose or D-fructose can be used as a raw material (see Figures 1 to 5). When producing D-allulose, it is necessary to produce it from an inexpensive raw material containing D-fructose, since D-allulose can be produced from D-fructose. <1> D-fructose can be easily produced from D-glucose using D-xylose isomerase, so D-glucose produced from starch can be used (see Figures 1 and 2). For example, in the case of using D-glucose as a raw material, D-glucose produced from starch is used, and D-xylose isomerase (DXI) is used to induce isomerization through an equilibrium reaction between D-glucose and D-fructose (=produced by a typical method for producing isomerized sugar). Alternatively, a composition containing D-glucose and D-fructose (isomerized sugar) is used as a raw material, and D-allulose 3-epimerase (DAE) is used to isomerize the D-fructose in the raw material to the rare sugar D-allulose, thereby accelerating isomerization through an equilibrium reaction between D-fructose and the rare sugar D-allulose. The reaction is terminated when the desired rare sugar D-allulose content in the target product is reached, resulting in the production of a target product composed of the rare sugars D-allulose, D-glucose, and D-fructose (Figure 1). Another example of using D-glucose as a raw material is to first obtain a D-fructose solution by separating a composition containing D-glucose and D-fructose (isomerized sugar) produced by a typical method for producing isomerized sugar using a separation device called chromatography to separate the D-glucose and D-fructose.This D-fructose solution is then mixed with the isomerized sugar to produce various types of isomerized sugar depending on the amount of D-fructose added.This is then used as a raw material and is subjected to the action of DAE, which isolating the D-fructose in the raw material into the rare sugar D-allulose.This allows the isomerization to proceed through an equilibrium reaction between D-fructose and the rare sugar D-allulose, and the desired product can be obtained (Figure 2). (2) Isomerized sugar, for which a method for inexpensive production has been established, is commercially available at low cost as a mixture of D-glucose and D-fructose in various compositions and can be used as a raw material. Figure 3 illustrates the manufacturing process for various types of isomerized sugars (glucose-fructose corn syrup, high-fructose corn syrup) that meet the Japanese Agricultural Standards (JAAS) and the method for using the resulting isomerized sugars as a raw material and treating them with DAE to isomerize the D-fructose in the raw material to the rare sugar D-allulose, thereby promoting isomerization through an equilibrium reaction between D-fructose and the rare sugar D-allulose to obtain the desired product. The left side of the figure shows a manufacturing method using the same glucose-fructose corn syrup with approximately 42% fructose as in Figure 1 above, the middle side shows a manufacturing method using the same fructose-glucose corn syrup with approximately 55% fructose as in Figure 2, and the right side shows a manufacturing method using high-fructose corn syrup with approximately 92% fructose as a raw material. When the D-fructose content of the desired product needs to be adjusted to an optimal range that provides the same taste as sugar and minimizes the drawbacks of long-term continuous consumption, D-fructose must be converted to D-glucose. A production method may be employed, as needed, in which D-allulose 3-epimerase is used to isomerize D-fructose in the raw material through an isomerization reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the target product reaches the desired rare sugar D-allulose content. After this, a reaction step is added in which D-xylose isomerase, which does not act on the rare sugar D-psicose, is used to promote isomerization through an equilibrium reaction between D-fructose and D-glucose, adjusting the target product to the desired D-fructose content. Such a two-step production method is similar to the production method (Fig. 4) in a separate application (PCT / JP2019 / 043722) using D-fructose as the raw material, as shown in Fig. 4. Figure 4 shows the method of the separately filed application, which uses D-fructose as a raw material, in which in the first step, D-fructose is mixed with D-allulose using ketose 3-epimerase (DAE), and in the second step, D-fructose is converted to D-glucose using D-xylose isomerase (DXI), which does not act on D-allulose, to obtain a target product with a new composition of D-glucose, D-fructose, and D-allulose. <3> Sugar is an inexpensive sweetener formed by the combination of D-glucose and D-fructose, and is easily decomposed by invertase, making it an inexpensive raw material that can also be used for bioethanol production (see Figure 5). Instead of using sugar directly as a raw material, a composition consisting primarily of D-glucose and D-fructose is used as the raw material, which is produced by decomposing sugar with the action of invertase (a typical method for producing invert sugar). D-allulose 3-epimerase (DAE) is then applied to the composition to isomerize the D-fructose in the raw material to the rare sugar D-allulose, and isomerization proceeds through an equilibrium reaction between D-fructose and the rare sugar D-allulose. The reaction is terminated when the target product reaches the desired rare sugar D-allulose content, yielding a target product composed of the rare sugars D-allulose, D-glucose, and D-fructose. In summary, it is preferable to use, as a raw material, isomerized sugar or invert sugar, which is a sugar mixture mainly composed of D-glucose and D-fructose.

[0024] [Details of high fructose sugar used as raw material] High-fructose corn syrup (HFCS) is a sugar made primarily from D-glucose corn syrup that has been isomerized using enzymes or alkalization to produce D-fructose and D-glucose. It is often labeled "fructose-glucose corn syrup" in Japanese food products, but isomerized corn syrup products are labeled as follows: glucose-fructose corn syrup (less than 50% fructose content, i.e., the proportion of fructose in the sugars) or high-fructose corn syrup (50% to 90% fructose content), or high-fructose corn syrup (90% or more fructose content). Also labeled isomerized corn syrup (mixed sugar corn syrup) is the syrup with 10% or more added sugar. If the syrup is glucose-fructose corn syrup, it is labeled "mixed sugar glucose-fructose corn syrup." High-fructose corn syrup is produced by an established method, as shown in Figure 3. Producing high-fructose corn syrup from starch requires three enzymatic reactions, purification, and concentration, as described below. (1) Liquefaction: Starch is added with water and the hydrolytic enzyme alpha amylase, and heated to about 95°C. This breaks down the high molecular weight starch into smaller pieces. (2) Saccharification: After liquefaction, the mixture is cooled to about 55°C and glucoamylase is added. In this reaction, the sugars are further broken down into glucose. (3) Isomerization: At 60°C, the isomerizing enzyme glucose isomerase is added to convert about half of the glucose into fructose. The name "isomerized sugar" comes from this reaction (the reaction in which glucose is isomerized into fructose). (4) Purification and concentration: After isomerization, the liquid sugar is purified using a filter or ion exchange device, and then evaporated and concentrated to produce glucose-fructose syrup with a fructose content of 42%. Furthermore, by increasing the purity of the fructose using chromatography, high-fructose syrup with a fructose content of 90-95% can be produced. Blending this with glucose-fructose syrup with a fructose content of 42% can produce high-fructose syrup with a fructose content of 55%. When high-fructose liquid sugar with a fructose content of 90-95% is used as the raw material, the D-allulose 3-epimerase is used to isomerize the D-fructose in the raw material through an isomerization reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the target rare sugar D-allulose content in the target product is reached. After this, it may be necessary to add a reaction step in which D-xylose isomerase, which does not act on the rare sugar D-psicose, is used to promote isomerization through an equilibrium reaction between D-fructose and D-glucose. This reaction step may be necessary to ensure that the target D-fructose content in the target product overcomes the issue of it being a sugar that causes obesity through continued intake, and to impart a taste similar to that of sugar, along with D-allulose and D-glucose.

[0025] [Details of invert sugar used as raw material] It is a sweetener made by hydrolyzing sugar (sucrose) into fructose and glucose using acid or an enzyme (invertase). Because it is sweeter than the same amount of sucrose, the amount of sugar can be reduced by inverting the sugar. It is also hygroscopic, which keeps sweets moist and prevents sugar crystallization. Sucrose and glucose are weakly right-handed, while fructose is strongly left-handed, so a solution of equal molecular weights of glucose and fructose will have weakly left-handed optical rotation while sucrose is weakly right-handed. It is named invert sugar because the plane of rotation for polarized light is reversed. While sucrose does not exhibit reducing properties, invert sugar exhibits reducing properties derived from glucose. Invertase is a yeast-derived enzyme that breaks down (inverts) sugar (sucrose) into its constituent glucose and fructose, creating invert sugar. Invert sugar is characterized by its elegant sweetness and mellow flavor, similar to that of honey, and it also imparts moisture and softness to foods, improving their color, luster, and texture. Invert liquid sugar can be produced, for example, by keeping 70% sucrose solution (300g warm water, 700g sucrose) at 60°C, adding 4.2g of invertase (0.6% of sucrose), and allowing it to react for about half a day while stirring.

[0026] [Enzymes used in the present invention] The enzyme D-allulose 3-epimerase (DAE) used in the present invention will be described, along with D-xylose isomerase (DXI), which is used as needed. In the case of monosaccharides, epimerase is an enzyme that moves the position of the -OH bonded to C to the opposite side. 12 Epimerases catalyze reactions that do not change the chemical formula of O6. Therefore, epimerases are enzymes that belong to the isomerase family. Isomerases catalyze reactions that do not change the chemical formula of O6. Isomerases that act on hexoses have the chemical formula C6H 12O6 is a catalyzer for reactions that do not change the molecule. For example, aldose isomerase catalyzes the intramolecular transfer of hydrogen and promotes the oxidation-reduction reaction between C1 and C2. Isomerases are superior to epimerases. The enzyme "isomerase" is well known in the industry, and examples include ketose 3-epimerase (D-allulose 3-epimerase), D-xylose isomerase, and glucose isomerase. However, glucose isomerase is not the official scientific name. Glucose isomerase is not included in the EC number registered by the International Union of Biochemistry and Molecular Biology. This is not the "scientific name of the enzyme." D-xylose isomerase is the scientific name, and glucose isomerase is commonly used as a commercial name. This is because D-xylose isomerase acts on the structurally similar D-glucose, and is only used by humans; nature does not convert D-glucose to D-fructose for use. Epimerases are a type of isomerase, and can therefore be described as (a) an enzyme that isomerizes D-glucose to D-fructose, or (b) an enzyme that isomerizes D-fructose to D-allulose. The above (a) "enzyme that isomerizes D-glucose to D-fructose" includes, for example, L-rhamnose isomerase from a certain microorganism that acts on D-glucose to convert it to D-fructose. It also includes all proteins contained in organisms such as microorganisms, plants, and animals that convert D-glucose to D-fructose. This also includes D-xylose isomerase used in a reaction step in which D-xylose isomerase, which does not act on the rare sugar D-psicose, acts on D-fructose to promote isomerization through an equilibrium reaction between D-fructose and D-glucose, and is employed as necessary to adjust the D-fructose content of the target product when high-fructose liquid sugar with a fructose content of 90-95% is used as a raw material. Furthermore, (b) "an enzyme that isomerizes D-fructose to D-allulose" includes proteins produced by any organism that convert D-fructose to D-allulose. Therefore, it includes all enzymes that belong to the EC number "EC.5.- (isomerase)." Even newly discovered enzymes or enzymes classified as other isomerases that have even a slight ability to catalyze these reactions are included. In this invention, the term "isomerase" is used to include a wide range of enzymes.

[0027] (D-allulose 3-epimerase) The isomerization of D-fructose in the raw material to the rare sugar D-allulose is promoted by an equilibrium reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the target product reaches the desired rare sugar D-allulose content. This ketohexose 3-epimerase (D-allulose 3-epimerase) can be of any origin or classification, as long as it is an enzyme that is capable of isomerizing D-fructose to D-allulose. A specific example is a D-ketohexose 3-epimerase derived from the Arthrobacter globiformis M30 strain (international deposit number NITE BP-1111), a bacterium belonging to the genus Pseudomonas (Patent Document 5) (Patent Document 6 and Non-Patent Document 2). The M30 strain was originally deposited as NITE P-1111 on June 22, 2011, with the Patent Organism Depositary of the National Institute of Technology and Evaluation, Japan (2-5-8 Higashikazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and was transferred to a deposit under the Budapest Treaty on May 2, 2012, and was internationally deposited under accession number NITE BP-1111. Furthermore, a preferred example is ketose 3-epimerase derived from Arthrobacter histidinolovorans Y586-1 strain (International Deposit Number NITE BP-02813) (International Application No. PCT / JP2019 / 045672). The Y586-1 strain was internationally deposited with the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on November 7, 2018, under accession number NITE BP-02813. Strain Y586-1 was initially identified as Arthrobacter protophormiae. Subsequent identification by the Japan Food Research Laboratories (JFRC) determined it to be Arthrobacter histidinolovorans. A complete genome sequence search of strain Y586-1 confirmed it to be Arthrobacter histidinolovorans. Therefore, a name change for the strain with accession number NITE BP-02813 was submitted to the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary, which was approved on October 4, 2019. The ketose 3-epimerase derived from the Y586-1 strain has approximately twice the activity of the enzyme produced per culture medium compared to the ketose 3-epimerase derived from Arthrobacter globiformis described in Patent Document 6, enabling efficient mass production of D-allulose. Furthermore, the enzyme has a high optimum temperature of 70°C, and even at 80°C, its relative activity is 59.4 (approximately 60%), with the activity at the optimum temperature of 70°C being taken as 100. Furthermore, the residual activity after incubation at 60°C for 1 hour is 49.4%, demonstrating its heat resistance, allowing for the production of a larger amount of product in reactions at the optimum temperature of 70°C. The excellent heat resistance of this enzyme can be further enhanced by creating amino acid substitution mutants of the enzyme. Specifically, by introducing mutations into the ketose 3-epimerase gene derived from the Y586-1 strain and substituting the corresponding amino acid residues with other amino acid residues by site-directed mutagenesis to prepare mutants with various amino acid substitutions, it is possible to obtain enzymes with higher thermostability than the wild-type enzyme without amino acid substitutions, and these mutants can be used. These mutants have thermostability, evaluated using two indices: the ratio of ketose 3-epimerase activity at reaction temperatures of 70°C and 50°C (T70 / T50), and the residual activity after incubation at 60°C for 1 hour. This allows for an industrially practical, low-cost production method based on total productivity derived from the high durability (lifespan) of the D-allulose 3-epimerase used, resulting from the high thermostability of the enzyme. In the examples of the present invention, various ketose 3-epimerase mutants were prepared by base substitution using site-directed mutagenesis. From among these, those with amino acid substitutions that would yield mutants with higher thermostability than the wild-type were selected, taking into consideration the general technical knowledge about enzymes that thermostability is an important property for extending the enzyme's lifespan. Of these, 20 mutant enzymes out of 37 were found to have a DAE higher than that of the parent wild-type recombinant enzyme, Y586-1 strain (1.78). Experiments were conducted using the amino acid substitution mutant listed at position 8 in Table 10 in the specification of the international application. The mutant is a protein consisting of (1) the name of the mutant sequence and (2) the amino acid sequence represented by SEQ ID NO:1. (1)Y586DAE_V129IA200VS218N (2) The amino acid sequence represented by SEQ ID NO: 1 is MKIGCHGLVWTGRFDAEGIRYSVQKTKEAGFDLIEFPLMDPFSFDVATAKSALAEHGLTASASLGLSEATDVSSEDPAIVKAGEELLNRALDVLAELGATDFCGVIYSAMKKYMEPATEQGLSNSKAAIGRVVDRAAGLGINVSLEVVNRYETNVLNTGRQALSYLSDVKRPNLGVHLDTYHMNIEESDMFSPVLDTAEVLKYVHIGESHRGYLGTGNVDFDNFFKALGRIGYDGPVVFESFSSAVVAPDLSRMLGIWRNLWADNEELGAHANAFIRDKLTAIKTIELH By using such a mutant with high thermostability, high thermostability and high productivity result in total productivity that is derived from the high durability (lifespan) of the enzyme rather than activity. (D-xylose isomerase) There are no restrictions on the origin or name of the D-xylose isomerase, as long as it is an enzyme capable of isomerizing D-glucose to D-fructose (see, for example, Patent Documents 5 and 7). An inexpensive composition primarily composed of D-glucose and D-fructose used as raw materials can be produced by using D-glucose produced from starch and allowing D-xylose isomerase to act on it to promote isomerization through an equilibrium reaction between D-glucose and D-fructose. Furthermore, when high-fructose liquid sugar with a fructose content of 90-95% is used as the raw material, D-xylose isomerase, which does not act on the rare sugar D-psicose and is used as needed to adjust the D-fructose content of the target product, can be used in a reaction step in which D-fructose is acted on to promote isomerization through an equilibrium reaction between D-fructose and D-glucose (see Figure 3). Commercially available D-xylose isomerase can be used. These enzymes may be purified enzymes or microorganisms that produce the enzymes. In the present invention, the purified enzymes or the microorganisms that produce the enzymes are used as immobilized enzymes or immobilized microorganisms.

[0028] [Enzyme immobilization] By using an immobilized enzyme, it becomes possible to carry out a continuous large-scale epimerization reaction, and if a heat-resistant enzyme is used as the enzyme, its life span is extended. For practical use, enzymes are immobilized on a suitable substrate such as an ion exchange resin. Ion exchange resins, such as basic anion exchange resins, can be either strongly basic anion exchange resins or weakly basic anion exchange resins. Examples of strongly basic anion exchange resins include HPA25L (manufactured by Mitsubishi Chemical Corporation) and IRA904CL (manufactured by Organo Corporation), while examples of weakly basic anion exchange resins include WA30 (manufactured by Mitsubishi Chemical Corporation), FPA54, and FPA95 (manufactured by Organo Corporation). In the examples, Purolite's weakly basic anion exchange resin A111S is used. When the immobilized enzyme is produced using a weakly basic anion exchange resin as a carrier, the immobilized ketose 3-epimerase can be easily eluted after its isomerization activity is lost, which makes it very easy to regenerate the carrier and reduces production costs. If a thermostable enzyme is used, its lifespan is extended, further reducing production costs. The entire production process can be carried out in either a batch or continuous manner. In the case of a continuous process, a raw material production isomerization step in which D-glucose produced from starch is used to produce a mixture of D-fructose and D-glucose by the action of D-xylose isomerase (glucose isomerase), and an isomerization reaction step in which D-fructose in the raw material is isomerized using D-allulose 3-epimerase are carried out in a single process. Furthermore, glucose isomerase and D-allulose 3-epimerase are immobilized separately. This type of production method, in which the epimerization reaction step using a mixture of D-fructose and D-glucose as a raw material and the raw material production isomerization step are carried out in a single process, and the enzymes used are immobilized and used separately for production, is a novel production method that has never been performed before. Enzymatic isomerization can be performed by immobilizing the enzyme, and various immobilization methods can produce stable, easily usable immobilized enzymes. The use of immobilized enzymes allows for continuous, large-scale isomerization and isomerization reactions. For example, immobilized enzymes with an activity of 1000 U / g wet weight resin are used. A crude enzyme solution is collected from the solution of disrupted cells obtained by disrupting bacterial cells. The crude enzyme solution is passed through a column-packed ion exchange resin at a low temperature (4°C) to bind the crude enzyme protein to the ion exchange resin, which is then washed with purified water to obtain the immobilized enzyme. An immobilized system capable of withstanding continuous production in terms of stability (activity retention) fully satisfactory for commercial production is obtained. The resulting immobilized enzyme can be used for continuous, large-scale isomerization (emerization) reactions. The support for immobilizing the enzyme can be any known support and is not limited to the resin supports exemplified above.

[0029] An overview of the production method of the present invention is shown in Figures 1 to 3. An inexpensive composition containing D-glucose and D-fructose is used as a raw material, and the raw material is passed through a single bioreactor (an immobilized epimerase (DAE) column) to convert the D-fructose contained in the raw material into D-allulose, thereby obtaining a target product with a new composition of D-glucose, D-fructose, and D-allulose. To explain the reaction in the immobilized epimerase (DAE) column, D-allulose 3-epimerase (DAE) is used to isomerize the D-fructose in the raw material into the rare sugar D-allulose, and the isomerization proceeds through an equilibrium reaction between D-fructose and the rare sugar D-allulose.The reaction is terminated when the desired product, improved isomerized sugar or invert sugar, with a rare sugar D-allulose content, is reached.In a composition consisting of the rare sugars D-allulose, D-glucose, and D-fructose, the D-fructose content compensates for the sweetness that is a weakness of D-allulose and gives it a taste close to that of sugar.A converted isomerized sugar or invert sugar with a taste close to that of sugar and the functionality of D-allulose comes out of the column.

[0030] [Target product] The resulting rare sugar-containing composition has an optimal range of D-glucose and D-fructose contents as energy sources, and the sweet and delicious D-fructose is adjusted in an amount that does not cause its drawbacks (such as the adverse effects of long-term continuous intake on the accumulation of neutral fats), but is in an amount sufficient to compensate for the weak point of D-allulose and D-glucose, which is its sweetness, and further contains an amount of D-allulose that allows the functionality of D-allulose to be exerted, resulting in a rare sugar-containing composition (sweetener) with the same taste as sugar. The content of D-allulose may need to be minimized to maximize its sweetening functionality. Sports drinks, for example, should contain a high content of D-glucose and D-fructose as energy sources. In such cases, the function of D-allulose is to suppress rapid sugar absorption and ensure sustained absorption, and it has already been demonstrated that this function is achieved at a content of 5% by mass or more. For people with obesity or high blood sugar levels, the higher the D-allulose content, the better for therapeutic or preventative intake, and continuous use is required. Therefore, the amount of D-allulose should vary between 5 and 30% by mass, preferably between 10 and 20% by mass. D-Allulose 3-epimerase is used to convert D-fructose in the raw material into the rare sugar D-allulose, yielding a mixture of D-fructose and the rare sugar D-allulose. The mixture of D-fructose and the rare sugar D-allulose is an equilibrium mixture of D-fructose and the rare sugar D-allulose, or a mixture of D-fructose and the rare sugar D-allulose adjusted to a desired concentration. By not allowing the reaction to reach equilibrium, the content of D-allulose in the target product can be changed. The raw material, D-glucose, is converted to D-fructose by the action of D-xylose isomerase, resulting in a mixture of D-glucose and D-fructose. This mixture of D-glucose and D-fructose is either an equilibrium mixture of D-fructose and D-glucose, or a mixture of D-glucose and D-fructose adjusted to the desired concentration. By not allowing the reaction to reach equilibrium, the D-glucose content in the target product can be altered. One of the characteristics of sweeteners containing the target products, D-glucose, D-fructose, and D-allulose, is their sugar-like taste. D-glucose is an important energy source, and while a high content is desirable for "zero hunger," its sweetness is only 65-80, based on sugar's sweetness index of 100. Since the sweetness of D-fructose is necessary to impart a sugar-like taste, the D-glucose content is determined by the D-fructose and D-allulose content. D-fructose has a sweetness index of 120-170 and is an important component for imparting sweetness, and together with D-glucose, it is a component that imparts a sugar-like taste. However, as a sugar, it has issues such as being a cause of obesity when taken over a long period of time. Therefore, the D-fructose content is determined by the D-glucose and D-allulose contents so that the target product has a sugar-like taste. The amount of D-fructose is adjusted to a level that does not have a negative effect on the accumulation of neutral fats, and to take advantage of the sweetness of D-fructose, which compensates for the weak sweetness of D-allulose. The sweetness of the target product can be expressed as a calculated value based on past performance, allowing it to be evaluated as a sweetener with the same taste quality as sugar. Therefore, in the present invention, expressions such as sugar-like taste, sugar-like taste, and sugar-same taste refer to a calculated value close to or equivalent to 100, with the sweetness (sweetness intensity) of sugar being 100, and are preferably used when the sweetness is supported by sensory testing.

[0031] [Features and advantages of the manufacturing method] [Feature 1] Inexpensive raw materials containing D-glucose and D-fructose can be used. A composition containing D-glucose and D-fructose as the main components can be used, produced by treating D-glucose produced from starch with D-xylose isomerase (DXI), or by decomposing sugar with invertase. [Feature 2] Isomerase (DXI) and epimerase (DAE) can be reacted in separate immobilized reaction columns, making it easy to carry out the desired reaction under appropriate conditions. [Feature 3] The two enzymes can be operated under appropriate reaction conditions, and if a decrease in activity occurs, it can be easily addressed by replacing them individually. [Feature 4] By performing the two enzyme reactions separately, it is possible to react only with the target sugar. By skillfully controlling the two specificities, the content of D-glucose as an energy source is optimal, and the amount of sweet and delicious D-fructose is adjusted to a level that does not exhibit its drawbacks (negative effects such as the accumulation of neutral fats when taken over a long period of time), but is sufficient to compensate for the weak point of D-allulose, its sweetness. Furthermore, a major feature of this product is that it is a rare sugar-containing composition (sweetener) that contains an amount of D-allulose that provides the functionality of D-allulose and has the same taste as sugar. [Advantage 1] By using various types of isomerized sugar for which mass production technology has been established (production is possible using only an epimerase column, and by using different types it is possible to create products with the desired composition) and inexpensive sugar (a D-glucose + D-fructose (1:1) composition that is mass-produced and used for bioethanol is available at low cost), it is possible to produce a pure form that does not contain by-products by connecting conventional production lines in an appropriate manner. [Advantage 2] The epimerase (DAE) column required to produce this rare sugar-containing composition can be simply connected to an isomerase (DXI) column that is generally used to produce isomerized sugar or invert sugar, making production very inexpensive. [Advantage 3] The composition of this rare sugar-containing composition can be easily kept constant or changed by adjusting the enzymatic reactions of isomerase (DXI) in the raw material production stage and epimerase (DAE) in the D-allulose production stage. [Advantage 4] The absence of a separation process in the manufacturing process simplifies the manufacturing process, which not only significantly reduces costs but also makes the manufacturing process easier to manage. [Advantage 5] During the manufacturing process, raw materials and products are not lost during separation operations, etc., and in principle, 100% of the raw materials can be obtained as products. [Advantage 6] The composition ratio of the products can be changed by adjusting the activity of the two enzymes or the rate at which they pass through the reaction column. In addition to the substrate flow rate, the composition of the target product can be adjusted by adjusting one or more of the following: reaction time with the substrate, reaction temperature, pH of the substrate solution, and amount of enzyme. [Advantage 7] An industrially practical, low-cost production method can be provided based on the total productivity derived from the high durability (lifespan) of the D-allulose 3-epimerase used, which is due to its high thermostability.

[0032] The novel sweetener thus obtained can be used in combination with other sweeteners such as sucrose, sugar alcohols, aspartame, stevia, etc. Furthermore, to add body to the sweetener, water-soluble dietary fiber with low sweetness (such as polydextrose, inulin, or indigestible dextrin) may be added as needed.

[0033] The sweetener of the present invention includes rare sugar-containing compositions with a D-glucose:D-fructose:D-allulose ratio of 58:29.4:12.6 or 45:38.5:16.5, but the content is not particularly limited when used to maximize sweetness intensity and sweetness quality. It can be adjusted appropriately depending on the desired degree of function, usage mode, amount used, etc.

[0034] The novel sweetener of the present invention, which has the same taste as sugar, contains D-allulose and has the effect of preventing obesity. It can also be used in combination with other active ingredients to prevent lifestyle-related diseases.

[0035] [Application] The sweetener composition of the present invention, which has the same taste as sugar and is made up of rare sugars D-allulose, D-glucose, and D-fructose, can be used in the same manner as known uses of rare sugar-containing compositions of D-glucose, D-fructose, and D-allulose. The sweetener composition of the present invention is characterized by being a product of raw materials such as isomerized sugar and invert sugar, without undergoing separation by simulated migration chromatography. It is also characterized by being a liquid sugar product of raw materials such as isomerized sugar and invert sugar that has passed through an immobilized enzyme reaction column. Because it is a composition of D-glucose, D-fructose, and D-allulose, it can also be used for the purposes described in Patent Document 7. Glucose, fructose, and D-allulose are all food-grade (in terms of approval and certification), and because they are a food mixture, they are extremely safe and easy to handle. D-Allulose (=D-psicose) is present in trace amounts in food ingredients, is highly safe, and, with the development of mass-production technologies, is highly cost-effective and valuable. It has already been shown to have an acute oral toxicity of 5,000 mg / kg or more in acute oral toxicity tests.

[0036] Examples of uses for sugar compositions containing D-allulose (=D-psicose) include foods, beverages, particularly functional foods (such as those for preventing obesity), pharmaceuticals, cosmetics, feed, agricultural chemicals (such as plant growth regulators and plant disease resistance enhancers), and industrial uses. Sweeteners with the same taste as sugar can be used in all products requiring sweetness, such as foods, health foods, foods for patients, food ingredients, health food ingredients, food ingredients for patients, food additives, health food additives, food additives for patients, beverages, health drinks, beverages for patients, drinking water, health drinking water, drinking water for patients, medicines, pharmaceutical ingredients, feed, patient animals and / or feed for patient animals.

[0037] When the sweetener of the present invention having the same taste as sugar is used in foods, it may be prepared in the form of its original form, diluted in water, suspended in oil, or in the form of an emulsion, or in the form of a carrier commonly used in the food industry. The beverage may be a non-alcoholic or alcoholic beverage. Examples of non-alcoholic beverages include non-carbonated beverages such as carbonated beverages, fruit juice drinks, and nectar drinks, as well as soft drinks, sports drinks, tea, coffee, and cocoa. Examples of alcoholic beverages include beer, happoshu (low-malt beer), third-grade beer, sake, plum wine, wine, champagne, liqueur, chuhai (Japanese shochu highball), and medicinal liquor. The rare sugar-containing composition of the present invention can be used as a food ingredient or food additive for the purpose of improving the above-mentioned carbohydrate metabolism disorders and / or lipid metabolism disorders in the form of tablets, capsules, solid preparations such as powders or granules to be dissolved in drinks, semi-solids such as jelly, liquids such as drinking water, and highly concentrated solutions to be used after dilution. Furthermore, the sweetener of the present invention, which has the same taste as sugar, can be appropriately added to foods to produce health foods or foods for patients with the purpose of improving carbohydrate metabolism disorders and / or lipid metabolism disorders. Optional ingredients such as vitamins, carbohydrates, colorings, and flavorings that are typically added to foods can be appropriately blended. The food can be consumed in any liquid or solid form. It can also be consumed as a soft capsule encapsulated in gelatin or the like. Capsules can be made, for example, by dissolving raw gelatin in water and adding a plasticizer (glycerin, D-sorbitol, etc.) to create a gelatin shell.

[0038] The sweetener of the present invention, which has the same taste quality as sugar, can be used in the same ways as sugar, including as a sweetener, in cooking, tea, coffee, seasonings (such as mirin), etc.

[0039] Specific examples of foods and beverages include the following: Western confectioneries (pudding, jelly, gummy candy, candy, drops, caramel, chewing gum, chocolate, pastries, butter cream, custard cream, cream puffs, pancakes, bread, potato chips, french fries, popcorn, crackers, pies, sponge cakes, castella cakes, waffles, cakes, donuts, biscuits, cookies, rice crackers, okaki (rice crackers), okoshi (rice crackers), okoshi (rice crackers), manju (buns), candy, etc.), dried noodle products (macaroni, pasta), egg products (mayonnaise, fresh cream), beverages (functional beverages, lactic acid beverages, lactic acid bacteria beverages, concentrated dairy beverages, fruit juice beverages, non-fruit juice beverages, pulp beverages, clear carbonated beverages, carbonated beverages with fruit juice, fruit-colored carbonated beverages), luxury items (green tea, black tea, instant coffee, cocoa, canned coffee drinks), dairy products (ice cream, yogurt, coffee milk, butter, butter sauce, cheese, fermented milk, processed milk), and pastes. (marmalade, jam, flower paste, peanut paste, fruit paste, fruit in syrup), meat products (ham, sausage, bacon, dry sausage, beef jerky, lard), seafood products (fish ham, fish sausage, kamaboko, chikuwa, hampen, dried fish, bonito flakes, mackerel flakes, dried sardines, sea urchin, salted squid, dried squid, mirin-boshi fish, dried shellfish, smoked salmon, etc.), tsukudani (food boiled in soy sauce) (small fish, shellfish, wild vegetables, mushrooms, kelp), Examples include curry pastes (instant curry, retort curry, canned curry), seasonings (miso, powdered miso, soy sauce, powdered soy sauce, moromi, fish sauce, sauce, ketchup, oyster sauce, solid bouillon, yakiniku sauce, curry roux, stew base, soup base, dashi base, paste, instant soup, furikake, dressing, salad oil), fried products (fried tofu, fried tofu snacks, instant ramen), soy milk, margarine, shortening, etc.

[0040] The above-mentioned foods and beverages can be processed and produced by blending the composition with general food ingredients according to a conventional method. The amount of the composition to be blended into the above-mentioned foods and beverages varies depending on the form of the food and is not particularly limited, but is usually preferably 0.1 to 50% by weight.

[0041] The above-mentioned foods and beverages can also be used as functional foods, nutritional supplements, or health foods. The form of the foods and beverages is not particularly limited. For example, in the production of foods, proteins with a well-balanced amino acid content and high nutritional value, such as milk protein, soy protein, and egg albumin, their hydrolyzates, egg white oligopeptides, soybean hydrolysates, and mixtures of simple amino acids can be used according to conventional methods. They can also be used in the form of soft capsules, tablets, etc.

[0042] Examples of nutritional supplements or functional foods include processed forms such as liquid diets, semi-digested nutritional foods, elemental nutritional foods, drinks, capsules, and enteral nutrients, which contain sugars, fats, trace elements, vitamins, emulsifiers, flavorings, etc. Among the above-mentioned various foods, for example, sports drinks, energy drinks, and other foods and beverages can further contain nutritional additives such as amino acids, vitamins, and minerals, as well as sweeteners, spices, flavorings, and colorings, in order to improve nutritional balance and flavor.

[0043] Functional foods are suitable for use in the fields of health foods and preventive medicines that prevent specific diseases (such as obesity). Health foods that prevent specific diseases can contain, in addition to a sugar composition containing the essential ingredient, the rare sugar D-allulose (=D-psicose), optional ingredients such as vitamins, carbohydrates, colorings, and flavorings that are typically added to foods. The food can be consumed in any liquid or solid form. It can also be consumed as a soft capsule encapsulated in gelatin or the like. Capsules are made, for example, by adding water to raw gelatin to dissolve it, and then adding a plasticizer (such as glycerin or D-sorbitol) to create a gelatin coating.

[0044] When the sweetener of the present invention, which has the same taste quality as sugar, is used as a food additive, its form can be selected appropriately. For example, the sweetener containing the rare sugar D-allulose (=D-psicose) of the present invention can be prepared as a food directly, added to other foods, or in any form commonly used in foods or health foods, such as capsules or tablets. Furthermore, when incorporated into food and administered, it can be mixed with appropriate excipients, bulking agents, binders, thickeners, emulsifiers, colorants, flavorings, food additives, seasonings, etc., and molded into powder, granules, tablets, etc. depending on the intended use. Furthermore, it can be incorporated into food ingredients to prepare food, which can then be commercialized as a functional food and consumed.

[0045] The sweetener composition of the present invention can be used in feed for livestock, poultry, and pets. For example, it can be incorporated into dry dog ​​food, dry cat food, wet dog food, wet cat food, semi-moist dog food, poultry feed, and feed for livestock such as cattle and pigs. The feed itself can be prepared according to a conventional method. These therapeutic and preventive agents can also be used in animals other than humans, for example, livestock mammals such as cows, horses, pigs, and sheep, poultry such as chickens, quails, and ostriches, pets such as reptiles, birds, and small mammals, and farmed fish. Pharmaceuticals that are intended to improve carbohydrate metabolism disorders and / or lipid metabolism disorders and improve obesity while taking advantage of the sweetness of the sweeteners of the present invention and are intended to exert physiological effects can be used alone or can be formulated with appropriate additives such as general excipients, stabilizers, preservatives, binders, and disintegrants, and can be formulated into an appropriate dosage form such as liquids, granules, fine granules, powders, tablets, capsules, pills, dusting agents, and sprays, and administered orally or nasally.

[0046] Pharmaceutical organic or inorganic solid, semi-solid, or liquid carriers, solubilizers, or diluents suitable for oral or nasal administration can be used to formulate the compositions of the present invention as pharmaceutical preparations. Water, gelatin, lactose, starch, magnesium stearate, talc, animal or vegetable oils, benzyl alcohol, gum, polyalkylene glycol, petroleum resin, coconut oil, lanolin, or other pharmaceutical carriers can all be used as pharmaceutical carriers, including the compositions of the present invention. Stabilizers, wetting agents, emulsifiers, and salts for adjusting the osmotic pressure or maintaining the appropriate pH of the formulation can also be used as auxiliary agents.

[0047] When used as a pharmaceutical composition, known methods can be used for formulation. The dosage form can be selected appropriately. For example, oral dosage forms include tablets, granules, powders, capsules, coatings, liquids, and suspensions. Parenteral dosage forms include injections, infusions, and suppositories. The dosage can be appropriately determined depending on the dosage form, administration method, intended use, and the age, weight, and symptoms of the recipient. In pharmaceutical preparations, sugar compositions containing the active ingredient, rare sugar D-allulose (=D-psicose), are used either as themselves or as pharmaceutically acceptable salts. The sweetener composition containing D-allulose (=D-psicose) can be used alone as a pharmaceutical preparation, or it can be processed into a pharmaceutical composition by adding a pharmaceutically acceptable carrier or diluent. Such preparations or pharmaceutical compositions can be administered orally or parenterally, as described above. For example, solid or fluid (gel and liquid) formulations or pharmaceutical compositions for oral administration may take the form of tablets, capsules, pills, powders, granules, or gel preparations. The exact dosage of a formulation or pharmaceutical composition varies depending on the intended use and duration of treatment, and is determined by the attending physician or veterinarian. The dosage and administration can be adjusted appropriately depending on the formulation. The daily dose may be administered once or several times in the form of oral solid preparations such as tablets or oral liquids. Furthermore, formulations such as syrups, lozenges, and chewable tablets, which are administered to infants as a single dose to exert both local and systemic effects, may be administered in a single dose of 1 / 2 to 1 / 10 of the daily dose, even if the total dose does not equal the daily dose. Conversely, if the dosage volume is not unreasonable given the dosage form, an amount equivalent to the daily dose may be formulated as a single dose. In preparing the formulation, commonly used diluents and excipients such as fillers, extenders, binders, disintegrants, surfactants, lubricants, coating agents, and sustained-release agents may be used. In addition, solubilizers, buffers, preservatives, solubilizers, isotonicity agents, emulsifiers, suspending agents, dispersants, thickeners, gelling agents, hardeners, absorbents, adhesives, elastic agents, plasticizers, adsorbents, fragrances, colorants, flavoring agents, antioxidants, moisturizers, light-blocking agents, glossing agents, antistatic agents, and the like may be used as needed.

[0048] The present invention provides an external skin preparation that utilizes the skin moisturizing, antioxidant, and anti-aging effects of the rare sugar D-allulose (D-psicose), i.e., an external skin preparation that has the effect of improving and preventing rough and chapped skin, for which therapeutic drugs, external skin preparations, cosmetics, etc. are known (Patent Document 8). The external skin preparation of the present invention contains a sugar composition containing the rare sugar D-allulose (=D-psicose) as an essential ingredient, and can also contain other ingredients commonly used in external skin preparations such as cosmetics and pharmaceuticals, such as aqueous components, oily components, powder components, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients, as needed. Other ingredients that may be added as appropriate include sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, verabamil, licorice extract, glabridin, hot water extract of Chinese quince, various herbal medicines, drugs such as tocopherol acetate, glycyrrhizinic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, and sugars such as D-glucose, D-fructose, and trehalose. Specifically, a topical skin preparation can be produced by appropriately blending a sugar composition containing the rare sugar D-allulose (=D-psicose) with ingredients commonly used in topical skin preparations, such as vegetable oils and other fats, waxes such as lanolin and beeswax, hydrocarbons, fatty acids, higher alcohols, esters, various surfactants, colorants, fragrances, vitamins, plant and animal extracts, UV absorbers, antioxidants, preservatives, and disinfectants. It can also be used with anti-inflammatory topical skin preparation ingredients such as licorice extract ingredients such as glycyrrhetinic acid, diphenhydramine hydrochloride, azulene, dl-α-tocopherol and its derivatives, vitamin B2, and vitamin B6. The topical skin preparation may be in any form commonly used in topical skin preparations, including ointments, creams, emulsions, lotions, packs, and bath additives, and the formulation is not particularly limited.That is, when used as an external preparation for skin, the dosage form can be selected appropriately, and examples thereof include a solution system, a solubilized system, an emulsion system, a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system, a gel, a mist, a spray, a mousse, a roll-on, a stick, and the like, as well as a preparation impregnated into or applied to a sheet such as a nonwoven fabric.

[0049] With the aim of providing a substance that has the effect of amplifying disease resistance in plants, which has the potential to dramatically reduce the amount of pesticides used, another group of the present inventors has already registered an invention for a plant disease resistance amplifier using rare sugars (Patent Document 9), and research and development has progressed dramatically since then, with multiple patents now available.

[0050] It can also be added to toothpaste and used as a sweetener. Furthermore, soluble films have come to be used in the formulation of cosmetics and the like. For example, edible soluble films are used as flavor films that retain fragrances and the like for the purposes of changing mood, preventing bad breath, and the like. Soluble films that exhibit excellent solubility and film properties and can be suitably used as packaging materials for foods, medicines, and the like, or as carriers for retaining active ingredients in foods, medicines, and the like, have been proposed (Patent Document 10). In this way, the sweetener of the present invention, which has the sweetness and taste of sugar, can be applied to medicines, quasi-drugs, and cosmetics.

[0051] The amount of D-allulose (=D-psicose) to be incorporated into foods, beverages, cosmetics, or feed is not particularly limited, but approximately 0.01 to 10% by weight is preferred. In the case of pharmaceuticals, it can be administered orally in the form of capsules, powder, tablets, etc., and because it dissolves in water, administration methods such as intravenous injection and intramuscular injection can be used in addition to oral administration. The dosage varies depending on, for example, the severity of diabetes symptoms, body weight, age, and gender, and it is desirable to determine the appropriate amount at the time of use based on the symptoms. The amount of D-allulose (=D-psicose) to be incorporated into pharmaceuticals is not particularly limited, but approximately 0.01 to 2,000 mg per kg of body weight is preferred for oral administration, 0.01 to 1,000 mg per kg for intravenous administration, and 0.01 to 1,000 mg per kg for intramuscular administration.

[0052] The present invention will be explained in more detail by way of examples, but the present invention is not limited to these examples in any way. [Example]

[0053] The D-allulose 3-epimerase used was a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The immobilization carrier used was a weakly basic anion exchange resin A111S manufactured by Purolite.

[0054] [Production of new sweeteners using the batch method] Three ml of the immobilized enzyme containing immobilized D-allulose 3-epimerase was weighed out, and a 60% sugar solution was added, followed by shaking at 60°C to carry out the enzymatic reaction. The sugar solutions used as substrates were D-glucose:D-fructose = 55:45, D-glucose:D-fructose = 50:50, and D-glucose:D-fructose = 45:55. When D-glucose:D-fructose = 55:45 was used, the equilibrium ratio was D-glucose:D-fructose:D-allulose = 55.6:32.1:12.3 (Figure 6). When D-glucose:D-fructose = 50:50 was used, the equilibrium ratio of D-glucose:D-fructose:D-allulose was 51.0:34.7:14.3 (Figure 7). Furthermore, when D-glucose:D-fructose = 45:55 was used, the equilibrium ratio was D-glucose:D-fructose:D-allulose = 45.6:38.8:15.6 (Figure 8).

[0055] [Production of new sweeteners using the column method] A bioreactor was constructed by packing approximately 250 mL of immobilized D-allulose 3-epimerase into a jacketed column. A 60% sugar solution was used as the substrate, and the enzymatic reaction was carried out by circulating 60°C hot water through the jacket and varying the flow rate from SV=1 to SV=5. The sugar solutions used as substrates were D-glucose:D-fructose = 58:42, D-glucose:D-fructose = 50:50, and D-glucose:D-fructose = 45:55. Table 1 shows the changes in sugar composition measured by the column method when the SV was changed using a sugar solution with a D-glucose:D-fructose ratio of 58:42 as the substrate. Table 2 shows the change in sugar composition measured by the column method when a sugar solution with a D-glucose:D-fructose ratio of 50:50 was used as the substrate and the SV was changed. Table 3 shows the change in sugar composition measured by the column method when a sugar solution with a D-glucose:D-fructose ratio of 42:58 was used as the substrate and the SV was changed.

[0056] [Table 1]

[0057] [Table 2]

[0058] [Table 3] [Industrial Applicability]

[0059] It is hoped that a sweetener with a sweetness and taste similar to that of sugar, which will not cause lifestyle-related diseases such as obesity even when widely used in the food industry, etc., can be mass-produced on a global scale at a sufficiently competitive cost. Successful mass production on a global scale at low cost will not only expand the uses of sugar compositions containing D-allulose (=D-psicose) to foods, beverages, particularly functional foods (for obesity prevention, etc.), pharmaceuticals, cosmetics, feed, pesticides (plant growth regulators, plant disease resistance enhancers, etc.), and industrial uses, but will also be supplied to the world as an energy source for the "Zero Hunger" campaign.

Claims

1. A method for mass-producing a sweetener containing three types of sugar consisting of D-glucose, D-fructose and D-allulose and having the functionality of D-allulose and a taste similar to that of sugar, comprising: An inexpensive composition containing D-glucose and D-fructose is used as a raw material, D-allulose 3-epimerase is allowed to act to cause the D-fructose in the raw material to be isomerized by an isomerization reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the desired rare sugar D-allulose content of the target product is reached; To obtain a target product, comprising a composition consisting of rare sugars D-allulose, D-glucose and D-fructose, in which the content of D-fructose is in a range that compensates for the weak point of D-allulose, namely, sweetness, and, together with D-allulose and D-glucose, gives a taste close to that of sugar. The method is characterized in that the D-allulose 3-epimerase is a highly thermostable and highly productive D-allulose 3-epimerase derived from Arthrobacter histidinolovorans, and that mass production on a global scale has been achieved, with total productivity depending on the high durability (lifespan) of the enzyme due to its high thermostability, and that production is performed without using simulated moving bed chromatography, making it possible to achieve mass production on a global scale with sufficient price competitiveness.

2. In the target product, D-glucose is important as an energy source, but its sweetness is 65-80, where the sweetness of sugar is 100. D-fructose has a sweetness level of 120-170 and is important for providing sweetness. Along with D-allulose and D-glucose, it provides a taste similar to sugar. However, continued intake of D-fructose can lead to obesity. The method according to claim 1, wherein the content of each component is determined taking into consideration the characteristics of each component, namely, that D-allulose has a sweetness intensity of 70 and can provide the physiological functions of anti-obesity and suppressing a sudden rise in blood glucose level caused by digestible sugar, to obtain an appropriate composition according to the purpose.

3. 3. The method according to claim 1 or 2, wherein the raw material is a composition containing D-glucose and D-fructose produced by treating D-glucose produced from starch with D-xylose isomerase to promote isomerization through an equilibrium reaction between D-glucose and D-fructose, or by decomposing sugar with invertase.

4. The method according to any one of claims 1 to 3, wherein the raw material is inexpensive isomerized sugar or invert sugar, and the target product is improved isomerized sugar or invert sugar which has a taste similar to sugar having the functionality of D-allulose and exhibits excellent physiological effects that do not cause disease when taken continuously.

5. 5. The method according to claim 4, wherein glucose-fructose syrup and / or fructose-glucose syrup is used as the raw material isomerized sugar, which is inexpensive.

6. The method according to any one of claims 1 to 5, characterized in that the enzyme activity of the D-allulose 3-epimerase used is adjusted to obtain the desired rare sugar D-allulose content of the product.

7. 7. The method according to claim 1, wherein the isomerization reaction is carried out by passing a substrate solution through an immobilized enzyme column, and the composition of the target product is adjusted to the desired one by adjusting one or more factors selected from the group consisting of substrate flow rate, reaction time with the substrate, reaction temperature, pH of the substrate solution, and amount of enzyme.

8. 8. The method according to claim 1, wherein an immobilized enzyme reaction is carried out using a raw material as a substrate, and theoretically, the desired product is obtained 100% without producing any by-products.

9. The D-allulose 3-epimerase is allowed to act on the D-fructose in the raw material to proceed with isomerization through an isomerization reaction between D-fructose and the rare sugar D-allulose, and the reaction is terminated when the target rare sugar D-allulose content of the target product is reached. The method according to any one of claims 1 to 8, further comprising a reaction step of allowing D-xylose isomerase, which does not act on the rare sugar D-allulose, to act to promote isomerization through an equilibrium reaction between D-fructose and D-glucose.

10. The method according to claim 9, wherein high fructose liquid sugar is used as the raw material of isomerized sugar, which is inexpensive.

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