A method for producing substantially pure D-thalitol or allitol

Abstract

[Problem] To provide a method for producing substantially pure D-talitol or allitol from a mixture of D-talitol and allitol. [Solution] The present invention pertains to (a) a method that comprises a step for performing a crystallization operation for precipitating, through condensation, crystals from an aqueous solution of a mixture (A) of D-talitol and allitol, to obtain a crystal (A-1) having a higher allitol proportion as compared to prior to crystallization and a mixture liquid (A-2) having a lower allitol content and an increased D-talitol percentage content as compared to prior to crystallization, and that is for obtaining substantially pure D-talitol (B) by causing the mixture liquid (A-2) to i) pass through a chromatography column to collect a D-talitol fraction not containing allitol or to ii) be treated with a microorganism that processes allitol but does not process D-talitol to break down allitol. Alternatively, the present invention pertains to (b) a method for obtaining substantially pure D-talitol (B) by producing D-allulose from allitol through processing of an aqueous solution of a mixture (A) of D-talitol and allitol using a microorganism having the ability to produce D-allulose from allitol, and by passing the solution through a chromatography column to collect a D-talitol fraction not containing D-allulose.

Description

[Technical Field]

[0001] The present invention relates to a method for producing substantially pure D-talitol or alitol from a mixture of D-talitol and alitol obtained by hydrogenating D-allulose. [Background technology]

[0002] D-allulose is the D-isomer of allulose, which is classified as a ketohexose. Also known as D-psicose, D-allulose is the epimer of D-fructose and is similar to D-fructose in terms of sweetness quality, but its sweetness is about 70% of that of sugar, and it has a refreshing, clean sweetness. D-allulose has been proven to be zero-calorie (Non-Patent Literature 3) in human studies (Non-Patent Literature 6) and animal studies (Non-Patent Literature 7), respectively. It has been shown to exhibit characteristics as a material for preventing lifestyle-related diseases, such as a postprandial blood glucose suppression effect (Non-Patent Literature 4), an effect of suppressing the rapid rise in blood glucose levels caused by D-glucose, which constitutes digestible sugars ingested through food and beverages (Patent Literature 1), and an anti-obesity effect (Non-Patent Literature 5).

[0003] Currently, D-allulose is available by any means, including extraction from nature and synthesis by chemical or biological methods. D-allulose is a rare sugar, and obtaining it in large quantities was extremely difficult. However, it is now possible to produce large quantities of high-purity D-allulose using reactions with epimerase. For example, D-allulose can be produced more efficiently than D-fructose using D-allulose 3-epimerase (DAE), and it is possible to produce large quantities of D-allulose with nearly 100% high-performance liquid chromatography (HPLC) purity (Non-Patent Literature 1). Ketose 3-epimerases are enzymes that catalyze the isomerization (epidynamic reaction) of the hydroxyl group at position 3 of ketohexoses. One such enzyme, D-allulose 3-epimerase (DAE, EC 5.1.3.30), is an enzyme that epiduralizes D-allulose to D-fructose and D-fructose to D-allulose. Several DAEs from microorganisms have been discovered with equilibrium reaction ratios of D-allulose:D-fructose ranging from 27:73 to 33:67. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2020 / 171144 [Patent Document 2] WO2020 / 195106 issue [Patent Document 3] Patent No. 4381684 [Non-patent literature]

[0005] [Non-Patent Document 1] Izumofleet Formula (Japanese Edition), by Ken Nanmori, first edition published November 2019, published by Rare Sugar Production Technology Research Institute Co., Ltd. [Non-Patent Document 2] Izumofleet Formula (English version), by Ken Nanmori, first edition published November 2019, published by Rare Sugar Production Technology Research Institute Co., Ltd. [Non-Patent Document 3] J. Nutr. Sci. Vitaminol. Vol.48, p.77-80 [Non-Patent Document 4] J. Nutr. Sci. Vitaminol. Vol.59, p.191-121 [Non-Patent Document 5] J. Clin. Biochem. Nutr. Vol.30, p.55-65 [Non-Patent Document 6] Metabolism(2010)Vol.59, p.206-214 [Non-Patent Document 7] Journal of the Japanese Society for Nutrition and Food Science, Vol. 63, pp. 17-19 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention aims to produce pure D-talitol from a mixture of D-talitol and allitol, which are sugar alcohols obtained by hydrogenating D-allulose. The aim is to efficiently mass-produce D-talitol in its pure form as a raw material for D-tagatose, while allitol, although a by-product of D-talitol production, can also be used as a raw material for D-allulose, and in recent years has been found to have anti-obesity effects equivalent to or greater than D-allulose, thus the aim is to efficiently mass-produce it in its pure form. [Means for solving the problem]

[0007] Hydrogenation of D-allulose yields a mixture of allitol and D-thalitol. While asymmetric reduction is possible through microbial reactions, efficient reduction from D-allulose to allitol is not feasible. Therefore, industrially inexpensive reduction (hydrogenation) is the optimal method. However, hydrogenation produces two polyols, allitol and D-thalitol, from the ketose, and their chromatographic elution sites are close together, making separation of allitol and D-thalitol difficult. Therefore, if (a) a method is adopted that utilizes the difference in solubility of the two polyols to crystallize and separate allitol, the sugar solution after the crystallization and separation of allitol will contain D-talitol and trace amounts of allitol. For D-talitol, this separation of allitol by crystallization is a process that reduces the allitol content. Thus, a mixture in which the D-thalitol content has increased and the allitol content has decreased compared to before separation can be passed through a chromatography column to collect the D-thalitol fraction that does not contain allitol, or the allitol can be broken down by treating it with microorganisms that act on allitol but not on D-thalitol to obtain substantially pure D-thalitol. Furthermore, (i) a mixture of D-talitol and allitol (A) can be treated with a microorganism capable of producing D-allulose from allitol to produce D-allulose from allitol, and the D-talitol fraction free of D-allulose can be collected by passing it through a chromatography column. D-talitol and D-allulose can be purified relatively easily because their chromatographic elution positions are farther apart than those of D-talitol and allitol.

[0008] The present invention is essentially a method for producing substantially pure D-talitol (B) or allitol (C) as described in (1) to (8) below. (1) A method for producing substantially pure D-talitol (B) or alitol (C) from a mixture of D-talitol and alitol (A), (a) A crystallization operation is performed by concentrating an aqueous solution of a mixture of D-talitol and allitol (A) to precipitate crystals, thereby obtaining crystals with a higher allitol ratio than before crystallization (A-1) and a mixture (A-2) in which the D-talitol content has increased and the allitol content has decreased compared to before crystallization. The aforementioned mixed liquid (A-2) i) Pass the sample through a chromatography column to collect the D-thalitol fraction that does not contain allitol, or ii) Decompose allitol by treating it with microorganisms that act on allitol but not on D-thalitol. By doing so, substantially pure D-thalitol (B) can be obtained, or (i) A microorganism capable of producing D-allulose from allitol is reacted with an aqueous solution of a mixture of D-talitol and allitol (A) to produce D-allulose from allitol, and the D-talitol fraction that does not contain D-allulose is collected by passing it through a chromatography column. A method characterized by obtaining substantially pure D-thalitol (B) by means of this. (2) The method according to (1) above, wherein the mixture of D-talitol and allitol (A) is obtained by a hydrogenation step in which D-allulose is used as a raw material and D-allulose is reduced with hydrogen under high temperature and pressure with a metal catalyst to produce a mixture of D-talitol and allitol. (3) The method according to (1) or (2) above, wherein substantially pure alitol is obtained by repeating the crystallization operation on crystals (A-1) with a higher alitol ratio than before crystallization, or by washing the crystallized crystals with a high alitol ratio with an alitol solution.

[0009] (4) The method according to any one of (1) to (3) above, wherein a microorganism (a) that acts on allitol but does not act on D-talitol is selected, which has the ability to produce D-allulose from allitol and simultaneously possesses a DAE, and the D-allulose converted from allitol by acting on allitol is converted to D-fructose by the action of the DAE, and is metabolized and degraded within the cell to degrade allitol, or a microorganism (b) that has the ability to produce D-allulose from allitol but does not possess a DAE is selected, immobilized DAE is present in the same location, and the D-allulose converted from allitol by acting on allitol is converted to D-fructose outside the cell by the action of the DAE, and is metabolized and degraded within the cell to degrade allitol. (5) The method according to any one of (1) to (4) above, wherein the microorganism having the ability to produce D-allose from the allitol does not have the ability to convert D-talitol, and is a microorganism selected from the group consisting of bacteria belonging to the genus Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, Lelliottia, and Pantoea. (6) The method according to (5) above, wherein the microorganism is selected from the group consisting of bacterial species of Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp.

[0010] (7) The method according to any one of (4) to (6) above, wherein the microorganism having the ability to produce D-allose from the allitol in (a) and simultaneously having DAE is selected from the group consisting of bacterial species of Burkholderia lata, Burkholderia diffusa, and Agrobacterium pusense. (8) The method according to any one of (4) to (6) above, wherein the microorganism having the ability to produce D-allose from the allitol of (b) and not having DAE is selected from the group consisting of bacterial species of Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Buttiauxella brennerae, Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp.

Advantages of the Invention

[0011] According to the present invention, D-talitol, which is a raw material for D-tagatose, one of the rare sugars, and allitol, a by-product in the D-talitol production stage, can be easily mass-produced in a pure form. In addition, the by-product allitol can be converted into D-allose, the starting material, and reused. Since hydrogenation is employed for mass production using D-allose as a raw material, allitol and D-talitol are obtained from D-allose, and a mixture of D-talitol and allitol becomes the starting material. By allowing a microorganism having the ability to produce D-allose from allitol and not having the ability to convert D-talitol to act on this allitol to convert allitol to D-allose and collecting the D-talitol fraction not containing D-allose by passing it through a chromatography column, mass production of D-talitol becomes possible. In addition, although allitol is a by-product in the D-talitol production stage, it can also be a raw material for D-allose. In recent years, it has been found that allitol has an anti-obesity effect equivalent to or greater than that of D-allose, and there is a technical contribution to efficiently mass-producing it in a pure form.

Brief Description of the Drawings

[0012] [Figure 1] This diagram illustrates the process of crystallizing and separating alitol from a mixture of D-talitol and alitol after the hydrogenation process, obtaining a mixture in which the proportion of D-talitol has increased and the proportion of alitol has decreased compared to before separation, and the process of crystallizing and separating alitol, using the Izumofleet structural formula. [Figure 2] This diagram illustrates the alitol degradation process using the Izumofleet formula, which includes a microbial alitol degradation step after the hydrogenation step, using microorganisms that decompose allitol but cannot decompose D-thalitol to obtain only pure D-thalitol, wherein the microorganism (a) has the ability to convert allitol to D-allulose and simultaneously possesses DAE. [Figure 3] This diagram illustrates the allitol degradation process using microorganisms that, after the hydrogenation step, decompose allitol but not D-thalitol, thereby obtaining only pure D-thalitol. Microorganism (b) has the ability to convert allitol to D-allulose and does not possess DAE. The diagram shows the allitol degradation process in the coexistence of immobilized DAE, illustrated by the Izumofleet structural formula.

[0013] [Figure 4]This document presents the fundamental principles of the Izumofleet formula as shown in Non-Patent Documents 1 and 2. Specifically, it compares the Fischer projection formula and the Izumofleet formula for aldoses, ketoses, and polyols, illustrating the principles of notation for the Izumofleet formula, which is a pattern figure. For example, D-glucose, represented by the Fischer projection formula, can be divided into six combinations: "CHO", "HC-OH", "HO-CH", "HC-OH", "HC-OH", and "CH2OH", depending on the combination of carbon atoms and other elements to which those carbon atoms are bonded. The lower part of Figure 4 shows the details of each corresponding pattern figure for each combination of carbon atoms and other elements to which those carbon atoms are bonded in the monosaccharides shown in the upper part of Figure 4. Figure 4 is a diagram showing the correspondence between each combination of carbon atoms and other elements to which those carbon atoms are bonded in sugars, and the corresponding patterns, where the corresponding patterns are predetermined for each combination of carbon atoms and other elements to which those carbon atoms are bonded. (a) The aldehyde group "CHO" combination corresponds to a pattern with a short vertical bar at the bottom of a circle. (b) The ketone group "C=O" combination corresponds to a pattern with short vertical bars at the top and bottom of a circle. (c) The "HO-CH" combination located in the middle of the chain in the Fischer projection corresponds to a pattern of the letter "T" with a bar sticking out rotated 90° clockwise. (d) Similarly, the "HC-OH" combination located in the middle of the chain corresponds to a pattern of the letter "T" with a bar sticking out rotated 270° clockwise. (e) Furthermore, the "CH2OH" combination placed at the bottom of the Fischer projection corresponds to a pattern of the letter "T" rotated 180°. (f) The "CH2OH" combination placed at the top of the Fischer projection corresponds to a pattern resembling the letter "T".

[0014] [Figure 5]As shown in Figure 3, the HPLC results (changes in sugar composition % of the reaction solution and sugar peak area over time) of Example 4, in which the microorganism Leliotia geogali (BDr27-3-2 cells), which converts allitol to D-allulose, is coexisted with immobilized DAE, and allitol is degraded using a 1% polyol mixture (alitol:D-talitol = 1:5). [Figure 6] As shown in Figure 3, the HPLC results (changes in sugar composition % of the reaction solution and sugar peak area over time) of Example 5, in which the microorganism Leliotia geogali (BDr27-3-2 cells), which converts allitol to D-allulose, is coexisted with immobilized DAE, and allitol is degraded using a 10% polyol mixture (alitol:D-talitol = 1:5) are shown. [Figure 7] As shown in Figure 3, the HPLC results (changes in sugar composition % of the reaction solution and sugar peak area over time) of Example 6, in which the microorganism Leliotia geogali (BDr27-3-2 cells), which converts allitol to D-allulose, is coexisted with immobilized DAE, and allitol is degraded using a 20% polyol mixture (alitol:D-talitol = 1:5) are shown. [Figure 8] As shown in Figure 3, the HPLC results (changes in sugar composition % of the reaction solution and sugar peak area over time) of Example 7, in which the microorganism Leliotia geogali (BDr27-3-2 cells), which converts allitol to D-allulose, is coexisted with immobilized DAE, and allitol is degraded using a 20% polyol mixture (alitol:D-talitol = 1:5) are shown.

[0015] [Figure 9] As shown in Figure 3, the HPLC results (changes over time in sugar composition % of the reaction solution and sugar peak area) of Example 8, in which the allitol degradation process was carried out using a polyol mixture after allitol crystallization, in which the proportion of D-talitol increased and the proportion of allitol decreased compared to before crystallization, by coexisting the microorganism Leliotia geogali (BDr27-3-2 cell), which converts allitol to D-allulose, with immobilized DAE, are shown.

[0016] [Figure 10] As shown in Figure 2, the HPLC results (changes in sugar composition % of the reaction solution and sugar peak area over time) for Example 9, in which allitol is converted to D-allulose and allitol is degraded using the DAE-containing microorganism Burkholderia diffuser (BCa7-2 cell), are shown, using a 10% polyol mixture (alitol:D-talitol = 1:5). [Modes for carrying out the invention]

[0017] [Essentially pure D-talitol or allitol] This process produces pure D-talitol, a raw material for D-tagatose, a rare sugar, and allitol, a by-product of D-talitol production. In a method for producing substantially pure D-talitol or allitol from a mixture of D-talitol and allitol, the mixture of D-talitol and allitol is obtained by a hydrogenation step in which D-allulose is used as a raw material and reduced with hydrogen under high temperature and pressure with a metal catalyst to produce a mixture of D-talitol and allitol. Furthermore, D-allulose is related to a mixture of D-talitol and allitol as a raw material, and also to allitol as a product when D-allulose is produced by using microorganisms capable of producing D-allulose from allitol when allitol is removed from the reaction system. When D-allulose is produced by treating allitol, a by-product of D-talitol production, with microorganisms capable of producing D-allulose from allitol, the resulting D-allulose is obtained as a by-product of D-tagatose, and is thus obtained as a mixture of D-tagatose and D-allulose. Chromatographic separation allows for the separation of D-allulose, yielding essentially pure D-tagatose, but the mixture can be used as is for food applications.

[0018] Allitol and D-talitol are polyols (sugar alcohols) and rare sugars. D-talitol is oxidized to D-tagatose by reacting with acetic acid bacteria. Therefore, in the production of D-tagatose, D-talitol is a raw material, while allitol is a by-product and is removed from the reaction system. Alitol is a six-carbon sugar alcohol produced by reducing D-allulose, and it is thought to exist in a dynamic alternation between D-allulose and alitol in the deciduous shrub Itea virginica. The plant itself, a natural product containing alitol, that is, the plant itself that produces and contains D-allulose and alitol, rare sugars produced in only very small amounts in nature, has already been verified to have a function of suppressing body fat accumulation and / or suppressing the rise in total cholesterol levels (Patent Document 2). It has also been found that alitol itself has an anti-obesity effect equivalent to or greater than that of D-allulose (Patent Document 3).

[0019] [Hydrogenation process] This invention employs a hydrogenation process for mass production, using D-allulose as a raw material to obtain a mixture of allitol and D-talitol. First, the raw material D-allulose is reduced with hydrogen under high temperature and pressure with a metal catalyst to produce a mixture of D-talitol and allitol (see Patent Document 3). A method of reducing (hydrogenating) the raw material D-allulose industrially and inexpensively is optimal for mass production and was therefore adopted.

[0020] This section describes a process for producing a mixture of D-thalitol and allitol by reducing D-allulose, a metal catalyst, under high temperature and high pressure using hydrogen. The metal catalyst used is a catalyst containing a metal selected from the elements of Group 8 to Group 10 of the periodic table. The elements of Group 8 to Group 10 of the periodic table are iron, cobalt, nickel, and platinum group elements. Here, the platinum group elements are the six elements ruthenium, rhodium, palladium, osmium, iridium, and platinum. Among the metals selected from the elements of Group 8 to Group 10 of the periodic table, nickel and metals selected from the platinum group elements are preferably used as catalysts in this invention. More preferably, the catalyst is selected from nickel, ruthenium, platinum, and palladium. Among these, ruthenium and platinum are preferred in terms of hydrogenation ability. It has been confirmed that ruthenium and platinum have higher hydrogenation ability than palladium, and that these reactions can be hydrogenated under low temperature and low pressure conditions, and that they have higher catalytic activity than copper-chromium and Raney-cobalt. Furthermore, so-called Raney nickel catalysts, obtained by treating an alloy of nickel and aluminum, etc., with a caustic alkali, are preferred because they increase the reaction rate by increasing the surface area, i.e., they have high catalytic activity. Furthermore, the properties of a catalyst used in a reduction reaction are not determined solely by the type of metal, but are also influenced by the support on which the metal is supported. Examples of supports on which metal catalysts are used include activated carbon, titanium oxide, metal oxides such as alumina, barium sulfate, and diatomaceous earth.

[0021] The manner in which this process is carried out is not particularly limited, but typically, the raw material D-allulose is dissolved in a solvent such as water, the above-mentioned metal catalyst is added to it, and the mixture is placed in a pressure vessel or the like, and hydrogen is injected into it under pressure to carry out the hydrogenation reaction of D-allulose. While water is typically used as the solvent, other solvents such as ethanol or other alcoholic solvents, methyl acetate, ethyl acetate, mixed solvents thereof, and mixed solvents of these with water can also be used. The concentration of D-allulose in the reaction solution is usually 1 to 60 w / v%, preferably 5 to 50 w / v%. The reaction temperature is usually 10 to 150°C, preferably 10 to 70°C, and more preferably 30 to 60°C, in order to minimize the formation of reaction by-products. The reaction pressure is usually 1 to 200 kg / cm².2 (Gauge pressure), preferably 5 to 100 kg / cm² 2 It will be carried out under these conditions.

[0022] The progress of this reaction can be confirmed by sampling the reaction solution at regular intervals and analyzing D-allulose and the resulting sugar alcohols D-thalitol and allitol. HPLC is preferably used for the analysis of D-allulose and the resulting sugar alcohols D-thalitol and allitol. In this process, the catalyst can be easily removed after the reaction by filtration, decantation, centrifugation, or other methods. By removing the catalyst in this way, a solution containing the desired sugar alcohol is obtained.

[0023] In this process, the production ratio of sugar alcohols, such as D-thalitol and allitol, can be changed by adding an optically active substance, i.e., a chiral source, to induce diastereoisomerism. Examples of such chiral sources include boric acid, cinconidine, cinconin, ephedrine, quinidine, brucine, alkaloids such as quinine and strychnine, sugars and their derivatives such as D-mannitol, menthol, camphor, terpenes, hydroxy acids such as L-tartaric acid, L-lactic acid, and L-malic acid, and amino acids such as L-leucine, L-cystine, and L-cysteine. Furthermore, the production ratio of sugar alcohols can also be changed by using a synthetic chiral source molecularly designed for diastereoisomeric reduction, together with a catalyst containing a metal selected from nickel, ruthenium, platinum, and palladium. Examples of such synthetic chiral sources include existing chiral phosphines such as BINAP.

[0024] Furthermore, in this process, the production ratio of D-thalitol and allitol can be changed depending on the type of metal catalyst used. In other words, the production ratio of two or more sugar alcohols changes depending on conditions such as the type of metal catalyst and the type of support material. In the production of D-thalitol and allitol, when Raney nickel is used, it is preferable to obtain a production ratio of approximately 50:50 to 40:60 for D-thalitol:allitol, and when platinum is used, it is preferable to obtain a production ratio of approximately 30:70 to 42:58 for D-thalitol:allitol. In this way, a mixture containing a large amount of D-talitol can be easily obtained, making it easy to obtain pure D-talitol if needed.

[0025] This section describes a process for adjusting the alitol content, which involves crystallizing and separating alitol from a mixture of D-talitol and alitol, thereby obtaining a mixture in which the D-talitol content has increased and the alitol content has decreased compared to before the separation. In the aforementioned hydrogenation process, two polyols are produced, and the problem is that it is difficult to separate allitol and D-thalitol. This is because their elution locations in chromatography are close together. Therefore, (a) employs a method that utilizes the difference in solubility of the two polyols to crystallize and separate allitol. Due to the difference in solubility between allitol and D-talitol, when the mixture is concentrated, allitol crystallizes, and the remaining mixture contains allitol at a lower concentration relative to D-talitol. For D-talitol, this separation of allitol through crystallization is a process that reduces its allitol content. By repeating this crystallization process, a mixture with a higher proportion of D-thalitol than before crystallization can be obtained, ultimately resulting in a mixture of 10% allitol and 90% D-thalitol.

[0026] The progress of this reaction can be monitored by taking a sample of the reaction solution at regular intervals and performing HPLC analysis to determine the ratio of allitol and D-thalitol in the crystal water and mother liquor. By repeating the crystallization operation from the mixed aqueous solution of allitol and D-thalitol, preferential crystallization of allitol is possible, and even purer allitol can be obtained. Furthermore, high-purity allitol can be obtained by washing the primary crystals obtained from the mixed aqueous solution of allitol and D-thalitol with an allitol solution. By repeating this crystallization process, crystals with a higher proportion of allitol than before crystallization can be obtained, and finally, 100% allitol crystals can be obtained. The obtained allitol can be used as a raw material for D-allulose, and in recent years, it has been reported to have anti-obesity effects equivalent to or greater than D-allulose, making it possible to use it as a functional substance.

[0027] On the other hand, for a mixture in which the D-thalitol content has increased and the allitol content has decreased compared to before crystallization, substantially pure D-thalitol can be obtained by i) passing it through a chromatography column to collect the D-thalitol fraction that does not contain allitol, or ii) treating it with microorganisms that act on allitol but not on D-thalitol to decompose allitol. In the above i), it becomes even easier to purify and separate D-thalitol and D-thalitol from the remaining mixture of allitol and D-thalitol by chromatography. This is because if the D-thalitol content in the mixture is high, the amount of pure D-thalitol fraction that leach out increases, and by collecting this fraction, pure D-thalitol can be produced efficiently. The chromatographic conditions can be the same as those used for normal separation.

[0028] Furthermore, in step ii) above, where allitol is broken down by treatment with microorganisms that act on allitol but not on D-thalitol, microorganisms that break down allitol but do not act on D-thalitol are used. For example, as microorganism (a), we select a microorganism that has the ability to produce D-allulose from allitol and also possesses DAE. As shown in Figure 2, when microorganism (a) is exposed to allitol, the D-allulose converted from allitol within the cell is further converted to D-fructose by DAE and metabolized and broken down within the cell, resulting in the breakdown of allitol. Furthermore, as microorganism (b), we select a microorganism (b) that has the ability to produce D-allulose from allitol and does not possess DAE. As shown in Figure 3, when microorganism (b) is exposed to allitol in the presence of immobilized DAE, the D-allulose converted from allitol inside the cell is converted to D-fructose outside the cell by the action of DAE, and then enters the cell and is metabolically broken down, resulting in the decomposition of allitol.

[0029] [A process for producing D-allulose from alitol by using microorganisms capable of producing D-allulose from alitol.] Furthermore, after the hydrogenation process, as (a), a microorganism capable of producing D-allulose from allitol can be reacted with an aqueous solution of a mixture of D-thalitol and allitol (A) to produce D-allulose from allitol. Microorganisms capable of producing D-allulose from allitol are selected from a group of bacteria belonging to the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, Lelliottia, and Pantoea, which do not have the ability to convert D-talitol.

[0030] The aforementioned microorganism is Burkholderia lata. The group is selected from the bacterial species consisting of (lata), Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp.

[0031] (b) If a microorganism capable of producing D-allulose from allitol does not possess a DAE, the process of producing D-allulose from allitol by reacting the microorganism with an aqueous solution of a mixture of D-talitol and allitol, and obtaining a mixture of D-talitol and D-allulose, will be described. The outline of this process is shown in Figure 3. When a microorganism capable of converting allitol (b) to D-allulose but lacking DAE acts on allitol in the presence of immobilized DAE, the allitol that enters the cell is converted to D-allulose. This D-allulose is then partially epilated by the immobilized DAE outside the cell and converted to D-fructose through an equilibrium reaction. This D-fructose enters the cell and is metabolized and broken down, so D-talitol and D-allulose are present outside the cell. This is then passed through a chromatography column to collect the D-talitol fraction that does not contain D-allulose. D-talitol and D-allulose can be purified relatively easily because their elution positions in chromatography are far apart.

[0032] Microorganisms capable of producing D-allulose from allitol and lacking DAE are selected from the group of bacteria belonging to the genera Burkholderia, Enterobacter, Buttiauxella, Lelliottia, and Pantoea, which, as described above, do not have the ability to convert D-talitol.

[0033] Furthermore, the bacterial species are selected from the group consisting of Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Buttiauxella brennerae, Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp., with Enterobacter soli, Lelliottia jeotgali, and Pantoea sp. being preferred.

[0034] (a) When a microorganism capable of producing D-allulose from allitol also possesses DAE, the process of reacting the microorganism with an aqueous solution of a mixture of D-talitol and allitol to produce D-allulose from allitol and obtain a mixture of D-talitol and D-allulose will be described. The outline of this process is shown in Figure 2. When a microorganism that has the ability to convert allitol (a) to D-allulose and also possesses DAE acts on allitol, the allitol that enters the cell is converted to D-allulose, which is then converted to D-fructose by DAE. D-fructose is then metabolized and broken down within the cell, so that only D-talitol remains outside the cell, and thus virtually pure D-talitol can be obtained.

[0035] Microorganisms that have the ability to produce D-allulose from allitol and simultaneously possess DAE are bacteria belonging to the genera Burkholderia or Agrobacterium that, as described above, do not have the ability to convert D-talitol. Examples include Burkholderia lata, Burkholderia diffusa, and Agrobacterium pusense.

[0036] The inventors used a soil library to search for microorganisms that can produce D-allulose from allitol when contacted with an aqueous solution containing allitol. As a result, any Burkholderia bacteria that have been confirmed to produce D-allulose from allitol can be used. Bacterial species of the genus Burkholderia that possess this ability include, for example, Burkholderia lata Y534-1=3 strains, Burkholderia lata U459-1-1=1 strain, Burkholderia multiborans Y488-4=4 strains, Burkholderia multiborans S332-4=1 strain, Burkholderia multiborans Y555-2d=2 strains, and Burkholderia diffusa Y452-1=3 strains, respectively, which were deposited on April 30, 2021, at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation, located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, with accession numbers NITE P-03413, NITE P-03410, NITE P-03412, NITE P-03408, and NITE The strain was deposited as P-03414 and NITE P-03411. Subsequently, on February 1, 2022, the strain was deposited again under accession numbers NITE BP-03413, NITE BP-03410, NITE BP-03412, NITE BP-03408, NITE BP-03414, and NITE BP-03411.

[0037] Furthermore, the Burkholderia diffusa BCa7-2 strain was deposited on September 9, 2021, at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, and received under receipt number NITE ABP-03531. Subsequently, the strain was deposited again on November 1, 2021, under accession number NITE BP-03531.

[0038] Furthermore, any Enterobacter species capable of producing D-allulose from allitol can be used. Examples of Enterobacter species with this capability include Enterobacter hormaechei and Enterobacter soli. Enterobacter formaekei BCr11-1, Enterobacter formaekei BCr11-2, and Enterobacter soli BDr27-1 strains, which were confirmed to produce D-allulose from allitol, were deposited on April 30, 2021, with accession numbers NITE P-03400, NITE P-03401, and NITE P-03405, respectively, at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation, located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture. Subsequently, the strains were deposited again on February 1, 2022, with accession numbers NITE BP-03400, NITE BP-03401, and NITE BP-03405.

[0039] Furthermore, bacteria belonging to the genera Agrobacterium, Buttiauxella, Lelliottia, and Pantoea that have the ability to produce D-allulose from allitol can be used, as long as they belong to the genera Agrobacterium, Buttiauxella, Lelliottia, and Pantoea. Examples of bacterial species in the genera Agrobacterium, Buttiauxella, and Lelliottia that possess this ability include Agrobacterium pusense, Buttiauxella brennerae, Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp.

[0040] The following strains, which have been confirmed to produce D-allulose from allitol, were deposited on April 30, 2021, at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, under accession numbers NITE P-03404, NITE P-03403, NITE P-03406, NITE P-03407, and NITE P-03409, respectively. Subsequently, the strains were deposited on February 1, 2022, under accession numbers NITE BP-03404, NITE BP-03403, NITE BP-03406, NITE BP-03407, and NITE BP-03409.

[0041] Furthermore, the Leliotia geogali BDr27-3-2 strain and the Pantoea sp. CCr79-1-1 strain were deposited on September 9, 2021, at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, and received as receipt numbers NITE ABP-03532 and NITE ABP-03533. Subsequently, the strains were deposited again on November 1, 2021, with accession numbers NITE BP-03532 and NITE BP-03533.

[0042] In this invention, these bacteria are first cultured in a conventional nutrient medium, preferably under aerobic conditions such as shaking and aeration stirring, to allow them to grow. During cultivation, or using the obtained viable cells, allitol in the mixture is converted to D-allulose. The bacteria used for D-allulose oxidation can be those present in the culture solution. Alternatively, viable cells isolated from the culture solution and dried cells can also be used. The culture method involves inoculating bacteria capable of producing D-allulose from allitol into a nutrient medium, preferably a liquid medium, that contains the nutrients these bacteria require, such as a carbon source, a nitrogen source, inorganic salts, and yeast extract, and culturing them under aerobic conditions at a temperature of 20-40°C for 1-10 days.

[0043] The viable cells obtained by this culture method are brought into contact with an aqueous solution containing allitol, preferably under predetermined conditions such as shaking, aeration and stirring, and oxygen injection, to convert allitol to D-allulose. When the reaction is carried out at 30°C under shaking, up to 100 w / w% of allitol is converted to D-allulose after 1 day. Furthermore, these bacteria can be used in an immobilized state, and highly active immobilized bacteria can be obtained through various immobilization methods, such as carrier binding, cross-linking, gel encapsulation, and microencapsulation. [Examples]

[0044] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In the following examples, HPLC analysis was performed using a GL-C611 column (Hitachi, Ltd.) and Prominence (Shimadzu Corporation), under the conditions of eluent 0.1 mM-NaOH, temperature 60°C, flow rate 1 mL / min, and detector (Shimadzu Corporation, product name "RID-20A"). In addition, OD, an indicator of bacterial cell concentration, was measured. 600 This was calculated by measuring the absorbance at a wavelength of 600 nm using a UV-Vis spectrophotometer UV-1800 (manufactured by Hitachi, Ltd.).

[0045] [Example 1] 100g of 20% NaOH aqueous solution was added to 10g of 50% Raney nickel (manufactured by Wako Pure Chemical Industries, Ltd.). After addition, the mixture was heated at 90°C for 1 hour. After confirming that the generation of bubbles had stopped, the catalyst was washed with distilled water by decantation. Washing was continued until the washing solution reached a pH of 9.2. In a 1 L glass autoclave equipped with a stirrer and thermometer, 300 g of an aqueous solution containing 100 g of D-allulose, to which 24 g of Raney nickel obtained by the above method was added. Water was then added to adjust the total volume of the reaction solution to 600 g. Calcium carbonate was added to adjust the pH of the reaction solution to 7. The temperature was set to 50°C, and the temperature was increased to 12 kg / cm². 2 The reaction was carried out under a hydrogen pressure of (gauge pressure) and a stirring speed of 700 rpm. At regular intervals, the reaction solution was sampled and analyzed for D-allulose, D-thalitol, and allitol to confirm the progress of the reaction. The analysis of D-allulose, D-thalitol, and allitol was performed using HPLC. The samples used for analysis were prepared by appropriately diluting samples of the reaction solution taken over time, and then desalting them by adding anion exchange resin and cation exchange resin. As a result, after 8 hours of reaction, the amount of D-allulose decreased to 1%, and D-thalitol and allitol were obtained in a production ratio of 55:45.

[0046] [Example 2] 2050 g of a mixed aqueous solution of allitol and D-talitol, obtained by 100% reduction of 270 g of D-allulose by hydrogenation using Raney nickel as a catalyst, was concentrated and crystallized. The crystals were separated from the mother liquor by vacuum suction filtration and recovered. These were designated as primary crystals. The primary crystals were recrystallized, and secondary and tertiary crystals were obtained by repeating the same method. 48 g of allitol was obtained from three crystallization operations. By the same analytical method as shown in Example 1, the obtained tertiary crystals were found to have a purity of 100% allitol. Meanwhile, in the above procedure, 217 g of a mixed sugar alcohol of allitol and D-thalitol was obtained from the solution mixed with the mother liquor obtained after separating the crystals. HPLC analysis was performed to determine the ratio of allitol and D-thalitol in the water of crystallization and the mother liquor. The results showed that allitol and D-thalitol were 91% and 9% in the primary crystals, 97% and 3% in the secondary crystals, 100% and 0% in the tertiary crystals, and 37% and 63% in the mixed mother liquor.

[0047] [Example 3] The fixed DAE was created as follows. D-allulose 3-epimerase (DAE) was obtained using recombinant crude enzyme derived from Arthrobacter histidinolovorans strain Y586-1, deposited under accession number NITE BP-02813. Recombinant Escherichia coli cells expressing DAE derived from strain Y586-1 were suspended in 10 ml of 50 mM Tris-HCl buffer (pH 7.5). The cell suspension was lysed using an ultrasonic homogenizer while cooling in ice water, and the lysates were centrifuged at 12,000 rpm for 30 minutes. The supernatant was used as the crude enzyme solution. The crude enzyme solution was heat-treated at 60°C for 10 minutes to inactivate contaminating proteins, which were then removed by centrifugation. The partially purified enzyme after heat treatment was added to a weakly basic anion exchange resin A111S (Purolite Co., Ltd.) ion exchange resin (immobilization carrier) that had been pre-equilibriumized with 50 mM Tris-HCl buffer (pH 7.5) to immobilize the enzyme. The resulting immobilized DAE was used in subsequent tests.

[0048] [Example 4] A reaction was observed when a 1% polyol mixture was treated with the microorganism Leliotia geogali, which has the ability to produce D-allulose from allitol. Leliotia geogali BDr27-3-2 strain cells cultured in TSB medium were collected by centrifugation and washed with sodium phosphate buffer (10 mM, pH 7.0). Polyol 1% (alitol:D-talitol = 1:5), sodium phosphate buffer 50 mM (pH 7.0), washed cells (OD 600 100 μL of immobilized DAE was added to a reaction mixture (10 mL) consisting of (20), and the mixture was shaken and stirred at 30°C. A portion of the reaction mixture was taken at various times, and after removing bacterial cells by centrifugation and desalting with ion exchange resin, the sugar composition was analyzed using HPLC. The results are shown in Figure 5. The allitol peak (retention time 19.2 min) disappeared after 18 hours of reaction, and a high-purity D-talitol solution (retention time 21.8 min) was obtained.

[0049] [Example 5] Reaction using a 10% polyol mixture BDR27-3-2 strain cells cultured in TSB medium were collected by centrifugation and washed with sodium phosphate buffer (10 mM, pH 7.0). Polyol 10% (alitol:D-talitol = 1:5), sodium phosphate buffer 50 mM (pH 7.0), washed cells (OD 600 100 μL of immobilized DAE was added to a reaction solution (10 mL) consisting of (20), and the mixture was shaken and stirred at 30°C. A portion of the reaction solution was taken at various points in time, and after removing bacterial cells by centrifugation and desalting with ion exchange resin, the sugar composition was analyzed using HPLC. The results are shown in Figure 6. As the reaction progressed, the allitol peak (retention time 19.2 min) decreased, and after 72 hours of reaction, the allitol peak disappeared, yielding a high-purity D-talitol solution (retention time 21.8 min).

[0050] [Example 6] Reaction using a 20% polyol mixture (1) BDR27-3-2 strain cells cultured in TSB medium were collected by centrifugation and washed with sodium phosphate buffer (10 mM, pH 7.0). Polyol 20% (alitol:D-talitol = 1:5), sodium phosphate buffer 50 mM (pH 7.0), washed cells (OD 600 400 μL of immobilized DAE was added to a reaction solution (10 mL) consisting of OD50, and bacterial cells equivalent to OD50 were added every 48 hours while shaking and stirring at 30°C. Parts of the reaction solution were taken at various points in time, and after removing bacterial cells by centrifugation and desalting with ion exchange resin, the sugar composition was analyzed using HPLC. The results are shown in Figure 7. As the reaction progressed, the allitol peak (retention time 19.2 min) decreased, and at 144 hours of reaction, the allitol peak disappeared, and a high-purity D-talitol solution (retention time 21.8 min) was obtained.

[0051] [Example 7] Reaction using a 20% polyol mixture (2) The cells of BDr27-3-2 strain cultured in TSB medium were collected by centrifugation and washed with sodium phosphate buffer (10 mM, pH 7.0). 400 μL of immobilized DAE was added to a reaction solution (10 mL) consisting of 20% polyol (aritol:D-talitol = 1:5), 50 mM sodium phosphate buffer (pH 7.0), and washed cells (OD 600 = 50), and the mixture was shaken and stirred at 30°C. The cells in the reaction solution were removed by centrifugation after 48 hours and 96 hours, and newly cultured cells were added to restart the reaction. A part of the reaction solution was sampled over time, and after removing the cells by centrifugation and desalting with an ion exchange resin, the sugar composition was analyzed using HPLC. The results are shown in Fig. 8. As the reaction progressed, the peak of aritol (retention time 19.2 min) decreased, and the aritol peak disappeared after 168 hours of reaction, and a high-purity D-talitol (retention time 21.8 min) solution was obtained.

[0052] [Example 8] Test using the D-allose hydrogenation reaction solution after aritol crystallization ((a) ii)) The cells of BDr27-3-2 strain cultured in TSB medium were collected by centrifugation and washed with sodium phosphate buffer (10 mM, pH 7.0). 5 mL of immobilized DAE was added to a reaction solution (500 mL) consisting of the D-allose hydrogenation reaction solution Brix10 after aritol crystallization, 50 mM sodium phosphate buffer (pH 7.0), and washed cells (OD 600 = 20), and the mixture was stirred at 30°C with aeration (0.8 L / min) using a jar fermenter. The reaction solution sampled over time was centrifuged to remove the cells, and after desalting, the sugar composition was analyzed using HPLC. The results are shown in Fig. The results are shown in Fig. 9. As the reaction progressed, the peak of aritol (retention time 19.2 min) decreased, and the aritol peak disappeared after 120 hours of reaction, and a high-purity D-talitol (retention time 21.8 min) solution was obtained.

[0053] [Example 9] Reaction using 10% polyol mixture BCa7-2 strain cells cultured in TSB medium were collected by centrifugation and washed with sodium phosphate buffer (10 mM, pH 7.0). Polyol 10% (alitol:D-talitol = 1:5), sodium phosphate buffer 50 mM (pH 7.0), washed cells (OD 600 A reaction solution (10 mL) consisting of (=50) was shaken and stirred at 30°C. A portion of the reaction solution was taken at various points in time, and after removing bacterial cells by centrifugation and desalting with ion exchange resin, the sugar composition was analyzed using HPLC. The results are shown in Figure 10. As the reaction progressed, the allitol peak (retention time 19.2 min) decreased, and a high-purity D-talitol solution (retention time 21.8 min) was obtained.

Claims

1. A method for producing substantially pure D-talitol (B) from a mixture of D-talitol and allitol (A), (a) A crystallization operation is performed by concentrating an aqueous solution of a mixture of D-thalitol and allitol (A) to precipitate crystals, thereby obtaining crystals with a higher allitol ratio than before crystallization (A-1) and a mixture (A-2) in which the D-thalitol content has increased and the allitol content has decreased compared to before crystallization. The aforementioned mixture (A-2) is treated with microorganisms that act on allitol but not on D-thalitol to decompose allitol, thereby obtaining substantially pure D-thalitol (B). It is characterized by the following: A method comprising selecting a microorganism (a) that acts on allitol but not on D-talitol, which has the ability to produce D-allulose from allitol and simultaneously possesses DAE, and acting on allitol to produce D-allulose, which is then converted to D-fructose by the action of DAE, which is then metabolized and degraded within the cell to degrade allitol, or selecting a microorganism (b) that has the ability to produce D-allulose from allitol but does not possess DAE, and coexisting with immobilized DAE, which acts on allitol to produce D-allulose, which is then converted to D-fructose outside the cell by the action of DAE, which is then metabolized and degraded within the cell to degrade allitol.

2. A method for producing substantially pure D-talitol (B) from a mixture of D-talitol and allitol (A), (i) The method is characterized by producing D-allulose from alitol by reacting an aqueous solution of a mixture of D-talitol and allitol (A) with a microorganism capable of producing D-allulose from allitol, passing the mixture through a chromatography column to collect a D-talitol fraction that does not contain D-allulose, thereby obtaining substantially pure D-talitol (B). The microorganism capable of producing D-allulose from allitol is a species of Burkholderia, Agrobacterium, Buttiauxella, Lelliottia, and Pantoea that does not have the ability to convert D-talitol. a) A method in which microorganisms are selected from the group consisting of bacteria belonging to (a).

3. The method according to claim 1 or 2, wherein the mixture of D-talitol and allitol (A) is obtained by a hydrogenation step in which D-allulose is used as a raw material and D-allulose is reduced with hydrogen under high temperature and high pressure with a metal catalyst to produce a mixture of D-talitol and allitol.

4. The method according to claim 1, wherein the microorganism that acts on allitol but does not act on D-thalitol is a microorganism selected from the group consisting of bacteria belonging to the genera Burkholderia, Enterobacter, Agrobacterium, Buttiauxella, Lelliottia, and Pantoea, which have the ability to produce D-allulose from allitol and do not have the ability to convert D-thalitol.

5. The aforementioned microorganisms include Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Agrobacterium pusense, Buttiauxella brennerae, and Buttiauxella sp. The method according to claim 2 or 4, selected from the group consisting of bacterial species Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp.

6. The method according to claim 4, wherein the microorganism having the ability to produce D-allulose from the alitol in (a) and simultaneously possessing a DAE is selected from the group consisting of bacterial species Burkholderia lata, Burkholderia diffusa, and Agrobacterium pusense.

7. Microorganisms that have the ability to produce D-allulose from allitol as described in (b) above and do not possess DAE include Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Butiauxella brennerae, and Butiauxella sp. The method according to claim 4, selected from the group consisting of bacterial species Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp.

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