1-deoxymannose crystal and method for crystallizing 1-deoxymannose

Abstract

To provide a method for crystallizing 1-deoxymannose that is safe for use in foods, and to provide a crystal thereof.SOLUTION: A crystalline 1-deoxymannose is obtained by concentrating a 1-deoxymannose solution and allowing it to stand.

Description

[Technical Field]

[0001] The present invention relates to novel 1-deoxymannose crystals precipitated from a hydrophilic solvent or an aqueous solvent, and to a method for crystallizing the same. [Background technology]

[0002] 1-Deoxymannose has the structure shown in Chemical Formula 1 and is also known as 1,5-anhydro-D-mannitol. Although there have been few reports of functional studies on 1-deoxymannose, it has recently been shown to bind to the fimbrial adhesin Fim H of pathogenic Escherichia coli, leading to expectations for its use as a preventative agent for urinary tract infections (Patent Document 1). 1-Deoxymannose is thus expected to be used as a supplement that claims health benefits.

[0003] [ka]

[0004] Several methods for preparing 1-deoxymannose have been reported. For example, it can be produced by contacting 1,5-anhydro-D-fructose with the yeast Saccharomyces cerevisiae or Wickermyces anomalus (Patent Documents 2 and 3). It can also be produced by catalytic reduction of 1,5-anhydro-D-fructose in the presence of a metal catalyst and hydrogen gas (Non-Patent Document 1). In methods using the yeast Saccharomyces cerevisiae or catalytic reduction, 1,5-anhydro-D-glucitol is also produced in addition to 1-deoxymannose. The purity ratio of 1-deoxymannose to 1,5-anhydro-D-glucitol is approximately 50:50 in the yeast Saccharomyces cerevisiae and 58:42 in the catalytic reduction method.

[0005] To precipitate sugar crystals dissolved in a hydrophilic solvent such as ethanol or methanol, one method is to gradually evaporate the solvent, ethanol, or methanol, to create a supersaturated solution. If the target sugar has low solubility in methanol or ethanol, it may be possible to precipitate the sugar by adding methanol or ethanol to the aqueous solvent in which it is dissolved.

[0006] When crystallizing sugar from an aqueous solvent, the target sugar purity (excluding water) is set to 90% or more, and after concentrating until supersaturation is reached, seed crystals are added and the temperature is lowered. Alternatively, the concentration is continued to maintain supersaturation while crystals are precipitated. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2024-024334 [Patent Document 2] Japanese Patent Application Publication No. 2023-182179 [Patent Document 3] Japanese Patent Application Laid-Open No. 2008-54531 [Non-patent literature]

[0008] [Non-Patent Document 1] Carbohydrate Research 337(2002),873-890 Summary of the Invention [Problem to be solved by the invention]

[0009] The 1-deoxymannose that exists to date has been obtained by chemical synthesis. 1-Deoxymannose prepared by chemical methods that have no proven track record in food manufacturing carries the risk of contamination with chemicals or organic solvents, making it unsuitable for food use. Since the expected use of 1-deoxymannose is food, the raw material for 1-deoxymannose must be food or a food additive, and the manufacturing process must be one that is proven in food manufacturing. Furthermore, safe, highly pure 1-deoxymannose crystals obtained from this crystallization stock solution are desired. [Means for solving the problem]

[0010] In the present invention, the raw materials are foods or food additives, and safe 1-deoxymannose crystals are provided, which are produced by a production process that has been proven in food production.

[0011] Suitable crystallization solutions for crystalline 1-deoxymannose are those obtained by contacting the food material 1,5-anhydro-D-fructose with the yeast Saccharomyces cerevisiae, or by contacting hydrogen gas with a metal catalyst that has been used to produce food-grade sugar alcohols. These solutions contain 1-anhydro-D-glucitol.

[0012] The solvent for crystallization is preferably a hydrophilic solvent or a water solvent, but these solvents may also contain other sugars, amino acids, salts, and the like.

[0013] The hydrophilic solvent or aqueous solvent in which 1-deoxymannose is dissolved serves as a crystallization stock solution, and the purity of 1-deoxymannose in the solid content of the crystallization stock solution must be 50% or higher, preferably 60% or higher, and more preferably 80% or higher, but is not limited to this range. The purity used here can be measured by high performance liquid chromatography (HPLC). The detailed conditions are shown below. The chromatogram obtained under these conditions is analyzed by the area percentage method, and the purity can be calculated using the following formula.

[0014] <Purity test> Purity [%]=PA / TA×100 PA: Peak area of ​​target substance TA: total peak area Analysis conditions Separation column: two connected MITSUBISHI MCIGEL CK08S columns (manufactured by Mitsubishi Chemical Corporation), Mobile phase: water, flow rate: 1.0 mL / min, Column temperature: 60°C, Detector: Differential refractive index detector, Sample volume: 20 μL

[0015] The sugar content at the time of crystallization is 65% or more, preferably 75% or more, and more preferably 80% or more, but is not limited to this range. The sugar content used here was defined as the Brix value measured using a Brix meter.

[0016] The temperature range for crystallization is preferably from 30° C. to 70° C., but is not limited to this range.

[0017] The resulting mixture of crystals and the crystal mother liquor can be collected by centrifugation or other methods. If necessary, the crystal surface can be washed with a solvent such as water. Finally, the crystals are dried in a dryer or similar device to obtain 1-deoxymannose crystals containing 1-anhydro-D-glucitol. [Effects of the Invention]

[0018] The 1-deoxymannose crystals of the present invention have high purity, safety and reliability, and can be used for food. [Brief explanation of the drawings]

[0019] [Figure 1] High-performance liquid chromatogram of crystalline 1-deoxymannose [Figure 2] X-ray diffraction profile of 1-deoxymannose crystals DETAILED DESCRIPTION OF THE INVENTION

[0020] The stock solution of the crystal of 1-deoxymannose can be prepared by the following method. An aqueous maltodextrin solution obtained by decomposing starch or starch with an enzyme such as alpha-amylase is reacted with alpha-1,4-glucan lyase to produce 1,5-anhydro-D-fructose. When isoamylase or pullulanase is used in combination during this enzymatic reaction, the purity of 1,5-anhydro-D-fructose increases. In addition, glucose is produced as an impurity, but by contacting yeast of the species Wickerhamomyces anomalus, the glucose can be removed and the purity of 1,5-anhydro-D-fructose can be increased. By contacting a highly pure 1,5-anhydro-D-fructose solution thus obtained with a metal catalyst such as sponge nickel and hydrogen gas, 1-deoxymannose and 1,5-anhydro-D-glucitol are produced. This mixture is concentrated, a small amount of seed crystal of 1-deoxymannose is added, and it is left at a predetermined temperature to obtain crystals of 1-deoxymannose.

[0021] <X-ray diffraction conditions> The crystal structure characteristics of the crystal can be analyzed by analyzing it with an X-ray diffractometer. The measurement conditions are shown below. Wide-angle X-ray diffractometer: RINT2500HL · Measurement conditions Measurement wavelength: Cukα (0.15418 nm), Scanning axis: 2θ / θ联动, X-ray output: 50 kV - 250 mA, Scanning method: Continuous scanning Optical system: Concentrated beam with monochromator Scanning range: 2 ≦ 2θ ≦ 80 deg Scanning speed: 0.5 deg / min, Sampling: 0.01 deg Slit: DS 0.5 deg + 5 mmH SS0.5 deg RS 0.15 mm · X-ray diffraction profile Vertical axis: Diffraction intensity (cps) Horizontal axis: Diffraction angle (deg) Diffraction condition: 2dsinθ = nλ (Bragg's equation) d: Lattice area spacing (nm) θ: Diffraction angle (deg) n: reflection order λ: X-ray wavelength during analysis Cukα1 (0.15406 nm) [Example]

[0022] <Seed crystal preparation> A 10% hydrolysis rate maltodextrin solution was dissolved in water to prepare 160 mL of a 14.7% concentration stock solution. To this was added 86 mg of an enzyme preparation (Cleistase PLF, manufactured by Yamato Kasei Co., Ltd.), and the mixture was allowed to react at 60°C for 15.5 hours. The temperature of the reaction mixture was then lowered to 45°C, and 2 g of 47 U / mL alpha-1,4-glucan lyase was added, followed by a 50.5-hour reaction at 45°C. After 50.5 hours of reaction, 46.5 g of an enzyme preparation (Fungamyl, manufactured by Novozymes) and 0.3 g of 47 U / mL alpha-1,4-glucan lyase were added, and the mixture was allowed to react for an additional 19 hours at 45°C, yielding a reaction mixture with 80% purity of 1,5-anhydro-D-fructose. The temperature of the reaction mixture was lowered to 35°C, and 3 ml of corn steep liquor and 9 ml of a culture medium of Wickerhamomyces anomalus were added to the mixture. The mixture was then cultured at 35°C for 25 hours while bubbling air into it. The purity of 1,5-anhydro-D-fructose reached 90% in this culture. To this culture solution, 4.7 g of sponge nickel activated with sodium hydroxide was added, and the mixture was reacted under hydrogen gas pressure (0.7 MPa) at 70°C for 3 hours to obtain a mixture of 1-deoxymannose and 1,5-anhydro-D-glucitol, with purities of 65.7% and 20.7%, respectively. This reaction mixture was subjected to HPLC multiple times under the conditions described below, and the eluate containing 1-deoxymannose was collected. The 1-deoxymannose purity of the collected eluate was 98.5%. This liquid was freeze-dried to obtain 1-deoxymannose powder. 100 mg of water was added to 400 mg of this powder, and the mixture was placed in a glass tube with a seal and completely dissolved in a boiling bath. After leaving the mixture at 30°C overnight, 1-deoxymannose crystals precipitated. The crystals were filtered through No. 1 qualitative filter paper, collected, and dried overnight at 50°C to obtain 1-deoxymannose crystals.

[0023] <HPLC Conditions> Separation column: Two connected MITSUBISHI MCIGEL C K08S (manufactured by Mitsubishi Chemical Corporation), Mobile phase: Water, Flow rate: 1.0 mL / min, Column temperature: 60 °C, Detector: Differential refractive index detector, Sample injection volume: 100 μL

[0024] <Preparation of Crystal Stock Solution> A maltodextrin solution with a decomposition rate of 10% was dissolved in water to prepare 1.6 L of a stock solution with a concentration of 14.7%. 0.8 g of an enzyme preparation (Cristase PLF, manufactured by Daiwa Kasei Co., Ltd.) was added thereto, and the mixture was reacted at 60 °C for 15.5 hours. The temperature of the reaction solution was lowered to 45 °C, 20 g of α-1,4-glucanase at 47 U / mL was added, and the mixture was reacted at 45 °C for 56.5 hours. After 56.5 hours of reaction, 0.46 g of an enzyme preparation (Fungamyl, manufactured by Novozymes) and 3 g of α-1,4-glucanase at 47 U / mL were added, and the mixture was further reacted at 45 °C for 19 hours to obtain a reaction solution with a 1,5-anhydro-fructose purity of 80%. The temperature of the reaction solution was lowered to 35 °C, 30 ml of corn steep liquor and 90 ml of a culture solution of Wickerhamomyces anomalus were added to the reaction solution, and the mixture was cultured at 35 °C for 25 hours while inserting air. After this main culture, the purity of 1,5-anhydro-D-fructose reached 90%. 47 g of sponge nickel activated with sodium hydroxide was added to this culture solution, and the mixture was reacted at 70 °C for 3 hours under hydrogen gas pressure (0.7 Mps) to obtain a mixture of 1-deoxymannose and 1,5-anhydro-D-glucitol. Their respective purities were 65.8% and 20.3%. It was concentrated with a vacuum evaporator while heating in a 70 °C water bath until the concentration reached 80%.

[0025] <Preparation of Crystals> It should be noted that in the original text, "反応50.5時間後に酵素製剤(ファンガミル ノボザイム社製)を0.46g及び47U / mLのアルファ-1,4-グルカンリアーゼを3g添加し、さらに45℃で19時間反応させ1,5-アンヒドロ-フルクトース純度80%の反応液を得た。" there seems to be an error in the time calculation. It should be 50.5 + 19 = 69.5 hours in total for this stage of reaction, but based on the task requirements, the text is translated as is. Also, in the translation of "反応液に対しコーンスティープリカー30ml、ウイッカーハモマイセス·アノマルス種の培養液を90ml添加し、空気を挿入しながら35℃で25時間培養した。本培養後に1,5-アンヒドロ-D-フルクトース純度が90%に達した。", the time for the first culture step is 29 hours in total considering the addition of two substances and then culturing, but again, translated as per the original text.0.01 g of 1-deoxymannose crystals was added to the concentrated solution, and the liquid temperature was gradually lowered to 40°C over 2 hours to precipitate the crystals. The resulting crystal mixture was subjected to solid-liquid separation in a basket centrifuge, and the resulting crystals were washed with cold water. The crystals were collected and dried in a dryer set at 50°C to obtain 1-deoxymannose crystals. <Crystal analysis> The obtained crystals were analyzed by high-performance liquid chromatography, and the purity of 1-deoxymannose was 98.8% and that of 1,5-anhydro-D-glucitol was 1.2% (Figure 1). The crystal was subjected to X-ray diffraction measurement under the above conditions. Figure 2 shows the X-ray crystal profile, and Table 1 shows the peak search results.

[0026] [Table 1]

[0027] <Comparative Example 1> Crystallization stock solutions with a solid content of 50 g and 1-deoxymannose purities ranging from 64% to 48% were concentrated under reduced pressure to a concentration of 80% while being heated in a 60°C water bath. After concentration, the pressure was returned to normal, 0.01 mg of 1-deoxymannose crystals were added, and the liquid temperature was then cooled to 30°C. The liquid was then continued to be heated at 30°C for 24 hours, and the presence or absence of an increase in 1-deoxymannose crystals was visually confirmed. The results are shown in Table 2. Crystals increased when the 1-deoxymannose purity was 50% or higher.

[0028] [Table 2] <Comparative Example 2>

[0029] A crystallization stock solution with a solid content of 5 g and a 1-deoxymannose purity of 64% was concentrated under reduced pressure while being heated in a 60°C water bath to concentrations of 50%, 60%, 65%, 70%, and 80%. After concentration, the pressure was returned to normal, and each concentrate was placed in a sealed test tube. 0.01 g of 1-deoxymannose crystals was added, followed by cooling to 30°C or 4°C and leaving the tube at the respective temperatures for 24 hours. The presence or absence of an increase in 1-deoxymannose crystals was then visually confirmed. The results are shown in Table 2. Crystals increased at concentrations of 65% or higher at both 30°C and 4°C.

[0030] [Table 3]

Claims

1. Crystals of 1-deoxymannose precipitated from a hydrophilic solvent or an aqueous solvent.

2. 2. The crystal of 1-deoxymannose according to claim 1, wherein a plurality of diffraction peaks obtained by powder X-ray diffraction are present between diffraction angles (2θ) of 12.89 and 48.

54.

3. The crystal of 1-deoxymannose according to claim 2, wherein the plurality of diffraction peaks include diffraction angles (2θ) of at least 14.26°, 17.08°, 20.16°, and 23.10°.

4. A crystallization method for precipitating 1-deoxymannose crystals from a crystallization stock solution having a 1-deoxymannose purity of 50% or more and a sugar content of 65% or more.

Images