Preparation method of scyllo-inositol

Abstract

The invention provides a preparation method of scyllo-inositol. The preparation method comprises the following steps: (1) preparing raw materials: preparing a crude enzyme solution of inositol dehydrogenase and scyllo-inositol dehydrogenase, preparing a muscle inositol solution, adding the muscle inositol solution into the crude enzyme solution, and controlling the temperature of a raw material system to be 40-50 DEG C; (2) conversion reaction: adjusting the pH value to 7-10, controlling the temperature at 40-50 DEG C, carrying out conversion reaction to obtain conversion liquid A, carrying out first cooling crystallization on the conversion liquid A, filtering, and collecting filtrate and a filter cake; and (3) adding water and a myo-inositol solid into the filtrate, repeating the step (2) and the step (3) until no crystal is separated out, and respectively carrying out primary ethanol washing, secondary cooling crystallization, filtration, secondary ethanol washing and drying on a filter cake collected each time to obtain scyllo-inositol. According to the method, the use amount of crude enzyme liquid is reduced, the conversion performance is good, the production period of scyllo-inositol is shortened, the production cost is remarkably reduced, and the generation of waste liquid and the pollution to the environment are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of scyllo-inositol, and in particular to a preparation method of scyllo-inositol. Background Art

[0002] Scyllo-inositol (SI) is a diastereomer of myo-inositol (MI) and exists in plants, animals and some bacteria, but its content in nature is much lower than that of myo-inositol.

[0003] In the traditional synthesis method, scyllo-inositol is mainly synthesized by chemical methods using myo-inositol as raw material through complex steps. The main disadvantages are as follows: (1) the inositol feed concentration is low and the production cycle is long; (2) the bacterial culture time is long, the utilization efficiency of inositol dehydrogenase and scyllo-inositol dehydrogenase is not high, and they cannot be recycled; (3) a large amount of phosphoric acid, boric acid or borate is required in the production process, which is not environmentally friendly; (4) the amount of buffer salt used in the conversion process is large. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing scyllo-inositol, which is used to overcome the problems in the prior art of low inositol feed concentration, long production cycle, low utilization efficiency of inositol dehydrogenase and scyllo-inositol dehydrogenase, use of a large amount of phosphoric acid, boric acid or borate, and environmental unfriendliness.

[0005] In a first aspect, the present invention provides a method for preparing scyllo-inositol, comprising the following steps:

[0006] (1) preparing raw materials: preparing crude enzyme solutions of inositol dehydrogenase and scyllo-inositol dehydrogenase, preparing a myo-inositol solution, adding the myo-inositol solution to the crude enzyme solution, and controlling the temperature of the raw material system between 40° C. and 50° C.;

[0007] (2) conversion reaction: adjusting the pH value to 7-10, controlling the temperature to 40-50° C., performing conversion reaction to obtain conversion liquid A, performing first cooling crystallization and filtering on the conversion liquid A, and collecting the filtrate and filter cake respectively;

[0008] (3) adding water and myo-inositol solid to the filtrate, repeating steps (2) and (3) until no crystals are precipitated, washing the filter cake collected each time with ethanol for the first time, cooling and crystallizing for the second time, filtering, washing with ethanol for the second time, and drying to obtain the scyllo-inositol finished product.

[0009] Compared with the prior art, firstly, after the crude enzyme solution and the myo-inositol solution are reacted to obtain the conversion solution, the conversion solution is directly cooled and crystallized, replacing the boric acid extraction of the scyllo-inositol in the conversion solution commonly used in the art, avoiding the use of acids such as boric acid, not only simplifying the reaction steps, but also improving production safety, and being able to obtain a scyllo-inositol product with a purity of up to 99.8%; secondly, water and myo-inositol solid are continuously added to the filtrate of the conversion solution after cooling, crystallizing and filtering, preferably ensuring that the concentration of myo-inositol in the cyclic conversion process is consistent with the concentration of myo-inositol solution in the raw material system, and cyclic conversion is performed, so that the inositol dehydrogenase and scyllo-inositol dehydrogenase in the filtrate can be recycled, reducing the amount of crude enzyme solution, and having good conversion performance. The preparation method of the present invention greatly shortens the scyllo-inositol production cycle, significantly reduces production costs, and reduces the generation of waste liquid and pollution to the environment.

[0010] Furthermore, in step (1), the preparation method of the crude enzyme solution is: resuspending the bacterial cells containing inositol dehydrogenase and the bacterial cells containing scyllo-inositol dehydrogenase respectively, mixing the two obtained resuspensions, homogenizing the mixed resuspension, breaking the cell wall, centrifuging, and taking the supernatant as the crude enzyme solution; the OD of the mixed resuspension is 600 The OD of the resuspension of the bacterial cells containing inositol dehydrogenase and the resuspension of the bacterial cells containing scyllo-inositol dehydrogenase is 150-200. 600 The value ratio is 1:(1-3).

[0011] Furthermore, the concentration of the myo-inositol solution is 160-200 g / L.

[0012] Compared with the prior art, the technical scheme limits the concentration of the myo-inositol solution to 160-200 g / L. The solubility of myo-inositol in water is relatively low, with a solubility of about 140 g / L at room temperature (25° C.) and a solubility of 170-310 g / L at a temperature of 40-50° C. The myo-inositol solution used in the present invention is a solution with a concentration of 160-200 g / L prepared at 40-50° C. The use of a higher concentration of myo-inositol solution combined with a cyclic conversion process can reduce the amount of enzyme solution used, and greatly improve the conversion rate of subsequent conversion reactions, so that the yield of the prepared scyllo-inositol is higher.

[0013] Furthermore, in step (1), the crude enzyme solution accounts for 10-20% of the volume of the myo-inositol solution.

[0014] Furthermore, in step (2), the conversion reaction time is 8-18 hours.

[0015] Furthermore, in step (2), the cooling rate of the cooling crystallization is 3-5°C / h, and the crystallization is stopped when the temperature drops to 5-15°C.

[0016] Compared with the prior art, the above technical scheme limits the cooling rate of the conversion liquid and the temperature of cooling and crystallization. At the above cooling rate and temperature of cooling and crystallization, the high concentration of scyllo-inositol in the conversion liquid can be precipitated more efficiently. If the cooling rate is too fast and the cooling temperature is too low, the crystals cannot be fully precipitated and the crystal shape of the product will be affected. If the cooling rate is too slow and the cooling temperature is too high, the purpose of precipitating crystals cannot be achieved and energy is wasted.

[0017] Furthermore, in step (2), the filtration method is to complete the filtration by suction filtration through a suction filtration bottle.

[0018] Furthermore, in step (3), the purity of the added myo-inositol solid is above 99wt%; after the addition of water and myo-inositol solid, the total volume of the circulating solution is the same as the total volume of the raw material system in step (1), and the concentration of myo-inositol in the circulating solution is the same as the concentration of the myo-inositol solution in step (1).

[0019] Furthermore, in step (3), during washing, the volume fraction of the ethanol is 95wt%, and the mass ratio of the added ethanol to the filter cake is (0.5-1):1.

[0020] Furthermore, in step (3), the cooling rate of the cooling crystallization is 3-5°C / h, and the crystallization is stopped when the temperature drops to 5-15°C.

[0021] Compared with the prior art, the above technical solution limits the temperature and cooling rate of the cooling crystallization when purifying the obtained scyllo-inositol, and its beneficial effects are the same as those of the cooling crystallization in step (2), which will not be repeated here. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] In a first aspect, an embodiment of the present invention provides a method for preparing scyllo-inositol, comprising the following steps:

[0024] Step S1, preparing raw materials: preparing crude enzyme solutions of inositol dehydrogenase and scyllo-inositol dehydrogenase, and adding myo-inositol solution to the crude enzyme solutions.

[0025] In some embodiments, the preparation method of the crude enzyme solution is: resuspending the bacterial cells containing inositol dehydrogenase and the bacterial cells containing scyllo-inositol dehydrogenase respectively, mixing the two obtained resuspensions, homogenizing the mixed resuspension, breaking the cell wall, centrifuging, and taking the supernatant as the crude enzyme solution; the OD of the mixed resuspension is 600The OD of the mixed resuspension is 150-200, for example 600 The preferred range is 165-180, and specific examples include 150, 165, 170, 180, and 200.

[0026] In some embodiments, the OD of the bacterial cells containing inositol dehydrogenase in the resuspension and the OD of the bacterial cells containing scyllo-inositol dehydrogenase in the resuspension are 600 The ratio is 1:(1-3). For example, the OD of the bacteria containing inositol dehydrogenase in the resuspension and the OD of the bacteria containing scyllo-inositol dehydrogenase in the resuspension are 600 The specific value ratio can be selected as 1:1, 1:2, 1: 2.5 , 1:3 The OD of the bacteria containing inositol dehydrogenase and the bacteria containing scyllo-inositol dehydrogenase in the resuspension 600 When the ratio is within the above range, the crude enzyme solution can exert good activity and promote the conversion of myo-inositol.

[0027] For example, the method for preparing the crude enzyme solution is as follows: the bacteria containing inositol dehydrogenase and the bacteria containing scyllo-inositol dehydrogenase are resuspended in PBS (PH7.0, 50 mM) respectively, the two resuspensions are mixed, the mixed resuspension is homogenized and broken for 3 times at a pressure of 40-120 MPa and a temperature of 4-16°C, and the supernatant is taken as the crude enzyme solution.

[0028] In some embodiments, the concentration of the myo-inositol solution is 160-200 g / L. For example, the concentration of the myo-inositol solution can be specifically selected as 160 g / L, 165 g / L, 170 g / L, 180 g / L, 190 g / L, or 200 g / L.

[0029] It should be noted that when preparing the myo-inositol solution, it needs to be prepared at 40-50°C. After the crude enzyme solution is mixed with the myo-inositol solution, the overall temperature of the raw material system is maintained between 40-50°C.

[0030] In some embodiments, the crude enzyme solution accounts for 10-20% of the volume of the myo-inositol solution. For example, the crude enzyme solution can be selected to account for 10%, 12%, 15%, 17%, 18%, or 20% of the volume of the myo-inositol solution.

[0031] Step S2, conversion reaction: adjusting the pH value to 7-10 and the temperature to 35-50° C. to carry out a conversion reaction to obtain a conversion liquid A, subjecting the conversion liquid A to a first cooling crystallization and filtering, and collecting the filtrate and the filter cake respectively.

[0032] In some embodiments, the cooling rate of the first cooling crystallization can be 3-5°C / h, and the crystallization is stopped when the temperature drops to 5-15°C. For example, the cooling rate can be 3°C / h, 3.5°C / h, 4°C / h, 5°C / h; the temperature for stopping crystallization can be 5°C, 6°C, 8.5°C, 10°C, 12°C, 15°C. It should be understood that myo-inositol has a low solubility in water. Therefore, when preparing scyllo-inositol in the prior art, the concentration of the myo-inositol solution generally used is low, and the prepared scyllo-inositol cannot be precipitated from the solution by cooling crystallization, and boric acid is required to extract scyllo-inositol. In the present invention, the myo-inositol solution used is a solution with a concentration of 160-200g / L prepared at 40-50°C, and the concentration is higher than that of the myo-inositol solution used in the prior art. Under the joint action of the myo-inositol solution of the present invention and the crude enzyme solution, after the myo-inositol solution completes the conversion, the content of scyllo-inositol in the solution is high, and the scyllo-inositol crystal can be precipitated by cooling, and the process is simple, which greatly shortens the scyllo-inositol production cycle and is suitable for industrial production. After the conversion reaction is completed, the conversion liquid contains not only scyllo-inositol, but also other substances such as crude enzyme solution. By setting the cooling rate and the temperature of stopping crystallization to the above range, the conversion liquid can quickly and fully complete the crystallization process to obtain scyllo-inositol crystals. If the cooling rate is too fast, scyllo-inositol crystals cannot be completely precipitated; if the cooling rate is too slow, the overall time for scyllo-inositol crystals to precipitate is too long, which increases the crystal precipitation time and increases the preparation time.

[0033] In some embodiments, the product after the first cooling crystallization can be filtered using conventional filtering methods in the prior art, as long as the purpose of separating the filtrate and the filter cake can be achieved. For example, filter paper is placed in a Buchner funnel and moistened with a small amount of water; the Buchner funnel is connected to a suction filtration bottle, and the suction filtration bottle is connected to a vacuum pump; the vacuum pump is turned on and air is pumped; the scyllo-inositol mixed liquid to be filtered is poured in, and the liquid enters the suction filtration bottle through the filter paper, and when the separation is complete, the filtrate and the filter cake are collected separately.

[0034] Step S3, adding water and myo-inositol solid to the filtrate, repeating steps (2) and (3) until no crystals are precipitated, washing the filter cakes collected each time with ethanol for the first time, cooling and crystallizing for the second time, filtering, washing with ethanol for the second time, and drying to obtain the scyllo-inositol finished product.

[0035] In some embodiments, water and myo-inositol solid are added to the filtrate, and the purity of the myo-inositol solid is 99wt% or more; after the water and myo-inositol solid are added, the total volume of the circulating solution is the same as the total volume of the raw material system in step (1). It should be understood that the total volume of the circulating solution here is the total volume of the filtrate, water and myo-inositol solid, and the total volume of the raw material system is the total volume of the crude enzyme solution and the myo-inositol solution. After the myo-inositol solid and water are added to the filtrate, the amount of water added is to make the total volume of the circulating solution the same as the total volume of the raw material system in step (1). The amount of myo-inositol solid added needs to ensure that the concentration of myo-inositol in the circulating solution is the same as the concentration of myo-inositol solution in step (1). At this time, the concentration of myo-inositol in the circulating solution is slightly higher than the concentration of myo-inositol in the raw material system in step (1). Combined with the circulation conversion process, the amount of raw materials used in the enzyme solution is reduced, costs are saved, the preparation cycle is shortened, and the conversion efficiency is improved. For example, after adding solid myo-inositol, the concentration of myo-inositol in the circulating solution can be 160 g / L, 165 g / L, 170 g / L, 180 g / L, 190 g / L, 200 g / L. The concentration of myo-inositol in the circulating solution is within the above range, so that the crude enzyme solution in the filtrate can fully react with the myo-inositol solution for conversion.

[0036] In some embodiments, the ethanol used in the first ethanol washing and the second ethanol washing in the above steps is 95% ethanol by volume. Scyllo-inositol has extremely low solubility in ethanol, but has a certain solubility in water. Compared with the use of purified water washing in the prior art, the use of ethanol washing can remove impurities to a greater extent while reducing the loss of scyllo-inositol products.

[0037] In some embodiments, during the first ethanol washing and the second ethanol washing, the mass ratio of the amount of ethanol added to the filter cake is (0.5-1):1, for example, 0.5:1, 0.6:1, 0.8:1, 0.85:1, 1:1 can be selected. When the amount of ethanol added to the filter cake is within the above mass ratio range, impurities in the filter cake can be removed to the greatest extent, and the filter cake can be purified.

[0038] In some embodiments, the cooling rate of the second cooling crystallization is 3-5°C / h, for example, 3°C / h, 3.5°C / h, 4°C / h, 4.5°C / h, 5°C / h; the temperature drops to 5-15°C to stop crystallization, and the temperature at which crystallization stops is, for example, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C. Its beneficial effects are the same as those of the cooling crystallization in step (2), and will not be repeated here.

[0039] The above filtration can be performed in the same manner as in step S2, or in other manners, as long as the crystals can be separated from the solution.

[0040] In some embodiments, in step S3, the crystals obtained after the second ethanol washing can be dried in a conventional drying method. For example, an oven can be used to dry the crystals at a temperature of 60 to 80° C. to finally obtain a scyllo-inositol product.

[0041] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0042] Embodiment 1:

[0043] A method for preparing scyllo-inositol comprises the following steps:

[0044] (1) Prepare raw materials:

[0045] Prepare 2L of myo-inositol solution with a concentration of 160g / L, set the preparation temperature to 40°C, stir thoroughly, add myo-inositol solution to the crude enzyme solution of inositol dehydrogenase and scyllo-inositol dehydrogenase, and control the overall temperature at 40°C. The preparation method of the crude enzyme solution is as follows: resuspend the bacteria containing inositol dehydrogenase and the bacteria containing scyllo-inositol dehydrogenase with PBS (PH7.0, 50mM), respectively, mix the two resuspended solutions, and homogenize the mixed resuspended solution at a pressure of 40Mpa and a temperature of 16°C for 3 times, then centrifuge, and take the supernatant as the crude enzyme solution. The OD of the mixed resuspended solution is 600 The OD of the resuspension of the bacterial cells containing inositol dehydrogenase and the resuspension of the bacterial cells containing scyllo-inositol dehydrogenase is 150. 600 The value ratio is 1:2, the amount of crude enzyme solution added accounts for 20% of the volume of the myo-inositol solution, and the total volume of the raw material system is 2.4L.

[0046] (2) Conversion reaction:

[0047] Sodium hydroxide was used to adjust the pH of the system to 7.5, the conversion temperature was 40°C, the conversion time was 8h, and the conversion reaction was carried out to obtain 2.2L of conversion liquid;

[0048] After the first conversion, the conversion solution was cooled to 5°C at a rate of 5°C / h, stirred for 1h and filtered, and the filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 95.8g;

[0049] At room temperature, 95 wt% ethanol was added to the filter cake for the first washing, the mass ratio of the added amount of 95 wt% ethanol to the filter cake was 0.5:1, the temperature was lowered to 5°C at a rate of 5°C / h, and the solid crude product was filtered to obtain the solid crude product. The solid crude product was then washed with 95 wt% ethanol for the second time, and the filter cake was filtered to obtain the filter cake, which was dried to obtain 93.4 g of white granular scyllo-inositol finished product, with a scyllo-inositol content of 99.5%.

[0050] (3) Add myo-inositol solid and water to the collected filtrate, stir until the solid is completely dissolved, the solution volume is 2.4 L, the concentration of myo-inositol in the solution is 160 g / L, repeat steps (2) and (3), cycle four times, and obtain white granular scyllo-inositol finished products with solid dry weight masses of 94.1 g, 92.6 g, 90.5 g, and 90.6 g, respectively, and scyllo-inositol contents of 99.8%, 99.2%, 99.1%, and 99.7%, respectively.

[0051] Example 2

[0052] A method for preparing scyllo-inositol comprises the following steps:

[0053] (1) Prepare raw materials:

[0054] Prepare 3L of myo-inositol solution with a concentration of 180g / L, set the preparation temperature to 42°C, stir thoroughly, add myo-inositol solution to the crude enzyme solution of inositol dehydrogenase and scyllo-inositol dehydrogenase, and control the overall temperature at 42°C. The preparation method of the crude enzyme solution is as follows: resuspend the bacteria containing inositol dehydrogenase and the bacteria containing scyllo-inositol dehydrogenase with PBS (PH7.0, 50mM), respectively, mix the two resuspended solutions, homogenize and break the mixed resuspended solution at a pressure of 120Mpa and a temperature of 4°C for 3 times, and then centrifuge to take the supernatant as the crude enzyme solution. The OD of the mixed resuspended solution is 600 The OD of the resuspension of the bacterial cells containing inositol dehydrogenase and the resuspension of the bacterial cells containing scyllo-inositol dehydrogenase is 200. 600 The value ratio is 1:1, the amount of crude enzyme solution added accounts for 10% of the volume of the myo-inositol solution, and the total volume of the raw material system is 3.3L.

[0055] (2) Conversion reaction:

[0056] Sodium hydroxide was used to adjust the pH of the system to 7.5, the conversion temperature was 42°C, the conversion time was 10 hours, and the conversion reaction was carried out to obtain 3.1 L of conversion liquid;

[0057] After the first conversion, the conversion solution was cooled to 10°C at a rate of 3°C / h, stirred for 1.5h and filtered, and the filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 106.8g;

[0058] At room temperature, 95 wt% ethanol was added to the filter cake for the first washing, the mass ratio of the added amount of 95 wt% ethanol to the filter cake was 0.5:1, the temperature was lowered to 10°C at a rate of 3°C / h, and the solid crude product was filtered to obtain the solid crude product. The solid crude product was then washed with 95 wt% ethanol for the second time, and the filter cake was filtered to obtain the filter cake, which was dried to obtain 104.3 g of white granular scyllo-inositol finished product, with a scyllo-inositol content of 99.2%.

[0059] (3) Add myo-inositol solid and water to the collected filtrate and stir until the solid is completely dissolved. The volume of the solution is 3.3 L and the concentration of myo-inositol in the solution is 180 g / L. Repeat steps (2) and (3) for five cycles to obtain white granular scyllo-inositol finished products with solid dry weight masses of 108.2 g, 101.4 g, 109.5 g, 104.7 g and 99.6 g, respectively, and scyllo-inositol contents of 99.1%, 99.2%, 99.1%, 99.3% and 99.0%, respectively.

[0060] Example 3

[0061] A method for preparing scyllo-inositol comprises the following steps:

[0062] (1) Prepare raw materials:

[0063] Prepare 2.5L of myo-inositol solution with a concentration of 200g / L, set the preparation temperature to 47°C, stir thoroughly, add myo-inositol solution to the crude enzyme solution of inositol dehydrogenase and scyllo-inositol dehydrogenase, and control the overall temperature at 47°C. The preparation method of the crude enzyme solution is as follows: resuspend the bacteria containing inositol dehydrogenase and the bacteria containing scyllo-inositol dehydrogenase with PBS (PH7.0, 50mM), mix the two resuspended solutions, and homogenize the mixed resuspended solution at a pressure of 80Mpa and a temperature of 10°C for 3 times, then centrifuge and take the supernatant as the crude enzyme solution. The OD of the mixed resuspended solution is 600 The OD of the resuspension of the bacterial cells containing inositol dehydrogenase and the resuspension of the bacterial cells containing scyllo-inositol dehydrogenase is 180. 600 The value ratio is 1:3, the amount of crude enzyme solution added accounts for 10% of the volume of the myo-inositol solution, and the total volume of the raw material system is 2.75L.

[0064] (2) Conversion reaction:

[0065] Sodium hydroxide was used to adjust the pH of the system to 8.0, the conversion temperature was 47°C, the conversion time was 12h, and the conversion reaction was carried out to obtain 2.6L of conversion liquid;

[0066] After the first conversion, the conversion solution was cooled to 15°C at a rate of 5°C / h, stirred for 1.5h and filtered, and the filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 125.4g;

[0067] At room temperature, 95 wt% ethanol was added to the filter cake for the first washing, the mass ratio of the added amount of 95 wt% ethanol to the filter cake was 1:1, the temperature was lowered to 15°C at a rate of 5°C / h, and the solid crude product was obtained by filtration. The solid crude product was then washed with 95 wt% ethanol for the second time, and the filter cake was filtered to obtain a filter cake, which was dried to obtain 120.9 g of a white granular scyllo-inositol product with a scyllo-inositol content of 99.3%.

[0068] (3) Add myo-inositol solid and water to the collected filtrate and stir until the solid is completely dissolved. The volume of the solution is 2.75 L and the concentration of myo-inositol in the solution is 200 g / L. Repeat steps (2) and (3) for six cycles to obtain white granular scyllo-inositol finished products with solid dry weight masses of 126.3 g, 122.4 g, 118.5 g, 119.9 g, 117.4 g and 115.6 g, respectively, and scyllo-inositol contents of 99.0%, 99.2%, 99.1%, 99.2%, 99.1% and 99.5%, respectively.

[0069] Example 4

[0070] A method for preparing scyllo-inositol comprises the following steps:

[0071] (1) Prepare raw materials:

[0072] Prepare 2L of myo-inositol solution with a concentration of 160g / L, set the temperature to 50°C, stir thoroughly, add myo-inositol solution to the crude enzyme solution of inositol dehydrogenase and scyllo-inositol dehydrogenase, and control the overall temperature at 50°C. The preparation method of the crude enzyme solution is as follows: resuspend the bacteria containing inositol dehydrogenase and the bacteria containing scyllo-inositol dehydrogenase with PBS (PH7.0, 50mM), mix the two resuspended solutions, homogenize and break the mixed resuspended solution at a pressure of 60Mpa and a temperature of 8°C for 3 times, and then centrifuge to take the supernatant as the crude enzyme solution. The OD of the mixed resuspended solution is 0.04477 / ml. 600 The OD of the resuspension of the bacterial cells containing inositol dehydrogenase and the resuspension of the bacterial cells containing scyllo-inositol dehydrogenase is 180. 600 The value ratio is 1:2, the amount of crude enzyme solution added accounts for 15% of the volume of the myo-inositol solution, and the total volume of the raw material system is 2.3L.

[0073] (2) Conversion reaction:

[0074] Sodium hydroxide was used to adjust the pH of the system to 10, the conversion temperature was 50°C, the conversion time was 18h, and the conversion reaction was carried out to obtain 2.1L of conversion liquid;

[0075] After the first conversion, the conversion solution was cooled to 12°C at a rate of 4°C / h, stirred for 1.3h and filtered, and the filtrate and filter cake were collected separately. The solid wet product in the filter cake weighed 95.2g;

[0076] At room temperature, 95 wt% ethanol was added to the filter cake for the first washing, the mass ratio of the added amount of 95 wt% ethanol to the filter cake was 0.8:1, the temperature was lowered to 12°C at a rate of 4°C / h, and filtered to obtain a solid crude product. The solid crude product was then washed with 95 wt% ethanol for the second time, and the filter cake was filtered to obtain a filter cake, which was dried to obtain 90.1 g of a white granular scyllo-inositol product with a scyllo-inositol content of 99.2%.

[0077] (3) Add myo-inositol solid and water to the collected filtrate and stir until the solid is completely dissolved. The volume of the solution is 2.3 L and the concentration of myo-inositol in the solution is 160 g / L. Repeat steps (2) and (3) for six cycles to obtain white granular scyllo-inositol finished products with solid dry weights of 89.5 g, 91.3 g, 90.5 g, 91.1 g, 92.4 g and 91.9 g, respectively, and scyllo-inositol contents of 99.5%, 99.1%, 99.1%, 99.3%, 99.7% and 99.7%, respectively.

[0078] Comparative Example 1

[0079] A method for preparing scyllo-inositol comprises the following steps:

[0080] (1) Prepare raw materials:

[0081] Prepare 2L of myo-inositol solution with a concentration of 50g / L, set the preparation temperature to 40°C, stir thoroughly, add myo-inositol solution to the crude enzyme solution of inositol dehydrogenase and scyllo-inositol dehydrogenase, and control the overall temperature at 40°C. The preparation method of the crude enzyme solution is as follows: resuspend the bacteria containing inositol dehydrogenase and the bacteria containing scyllo-inositol dehydrogenase with PBS (PH7.0, 50mM), mix the two resuspended solutions, homogenize the mixed resuspended solution at a pressure of 40Mpa and a temperature of 16°C for 3 times, and then centrifuge to take the supernatant as the crude enzyme solution. The OD of the mixed resuspended solution is 600 The OD of the resuspension of the bacterial cells containing inositol dehydrogenase and the resuspension of the bacterial cells containing scyllo-inositol dehydrogenase is 150. 600 The value ratio is 1:2, the amount of crude enzyme solution added accounts for 20% of the volume of the myo-inositol solution, and the total volume of the raw material system is 2.4L.

[0082] (2) Conversion reaction:

[0083] Sodium oxide was used to adjust the pH of the system to 7.5, the conversion temperature was 40°C, the conversion time was 8 hours, and the conversion reaction was carried out to obtain 2.2L of conversion liquid;

[0084] After the conversion was completed, the conversion solution was cooled to 5°C at a rate of 5°C / h, and no crystals were precipitated.

[0085] It can be seen from the above-mentioned Examples 1 to 4 and Comparative Example 1 that the content of scyllo-inositol in the scyllo-inositol product prepared by the preparation method of the present invention is above 99.1%, and can reach up to 99.8%. Using the preparation method of the present invention, the prepared product has high purity, short production cycle, is convenient for large-scale production, and has high utilization efficiency of inositol dehydrogenase and scyllo-inositol dehydrogenase, which replaces the large amount of phosphoric acid, boric acid or borate used in the prior art, and is environmentally friendly.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing scyllo-inositol, characterized in that: The following steps are involved: (1) preparing raw materials: preparing crude enzyme solutions of inositol dehydrogenase and scyllo-inositol dehydrogenase, preparing a myo-inositol solution, adding the myo-inositol solution to the crude enzyme solution, and controlling the temperature of the raw material system between 40° C. and 50° C.; (2) conversion reaction: adjusting the pH value to 7-10, controlling the temperature to 40-50° C., performing conversion reaction to obtain conversion liquid A, performing first cooling crystallization and filtering on the conversion liquid A, and collecting the filtrate and filter cake respectively; (3) adding water and myo-inositol solid to the filtrate, repeating steps (2) and (3) until no crystals are precipitated, washing the filter cake collected each time with ethanol for the first time, cooling and crystallizing for the second time, filtering, washing with ethanol for the second time, and drying to obtain the scyllo-inositol finished product.

2. The preparation method according to claim 1, characterized in that: In step (1), the preparation method of the crude enzyme solution is: resuspending the bacterial cells containing inositol dehydrogenase and the bacterial cells containing scyllo-inositol dehydrogenase respectively, mixing the two obtained resuspensions, homogenizing the mixed resuspensions, breaking the cell walls, centrifuging, and taking the supernatant as the crude enzyme solution; The OD of the mixed resuspension 600 The OD of the resuspension of the bacterial cells containing inositol dehydrogenase and the resuspension of the bacterial cells containing scyllo-inositol dehydrogenase is 150-200. 600 The value ratio is 1:(1-3).

3. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the myo-inositol solution is 160-200 g / L.

4. The preparation method according to claim 1, characterized in that: In step (1), the crude enzyme solution accounts for 10-20% of the volume of the myo-inositol solution.

5. The preparation method according to claim 1, characterized in that: In step (2), the conversion reaction time is 8-18 hours.

6. The preparation method according to claim 1, characterized in that: In step (2), the cooling rate of the cooling crystallization is 3-5°C / h, and the crystallization stops when the temperature drops to 5-15°C.

7. The preparation method according to claim 1, characterized in that: In step (2), the filtering method is to filter by suction through a suction bottle to complete the filtering.

8. The preparation method according to claim 1, characterized in that: In step (3), the purity of the added myo-inositol solid is above 99wt%; after the addition of water and myo-inositol solid, the total volume of the circulating solution is the same as the total volume of the raw material system in step (1), and the concentration of myo-inositol in the circulating solution is the same as the concentration of the myo-inositol solution in step (1).

9. The preparation method according to claim 1, characterized in that: In step (3), during washing, the volume fraction of the ethanol is 95%, and the mass ratio of the added ethanol to the filter cake is (0.5-1):

1.

10. The preparation method according to claim 1, characterized in that: In step (3), the cooling rate of the cooling crystallization is 3-5°C / h, and the crystallization stops when the temperature drops to 5-15°C.