Separation and preparation method of scyllo-inositol

Abstract

The invention discloses a separation and preparation method of scyllo-inositol, and relates to the technical field of scyllo-inositol production, myo-inositol solution is added into whole-cell wet thalli containing inositol dehydrogenase and scyllo-inositol dehydrogenase, conversion is carried out in PBS buffer solution to obtain conversion liquid, the conversion liquid is filtered and decolorized by a ceramic membrane and an ultrafiltration membrane, and the scyllo-inositol is obtained. Then concentrating and centrifuging to obtain a mixed crude product of scyllo-inositol and inositol, pulping and washing with purified water, and drying a wet product to obtain scyllo-inositol. In the separation and preparation process, boric acid, strong acid, strong alkali and organic solvent are not used, precipitation of composite salt is not needed, the process steps are reduced, the environment is not polluted, the operation is safe, the recovery cost is low, materials are recycled, a complex recovery system is not needed, the cost can be better reduced, and the method is environment-friendly.

Description

Technical Field

[0001] The invention relates to the technical field of scyllo-inositol production, and in particular to a separation and 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 myo-inositol. Studies have shown that scyllo-inositol has a certain effect on the treatment of brain diseases such as Alzheimer's disease, Down syndrome and Angelman syndrome. In addition, scyllo-inositol can reduce uric acid levels and is used to treat hyperuricemia.

[0003] In the traditional synthesis method, scyllo-inositol is mainly synthesized by chemical methods using myo-inositol as raw material through complex steps. However, this method requires the protection and deprotection of hydroxyl groups, and the steps are cumbersome and not conducive to large-scale production. In recent years, with the development of bioengineering technology, the biological preparation of scyllo-inositol has become a new development trend. The existing production process uses myo-inositol as raw material, first converts it into scyllo-inositol monoketone, and then most of the latter is converted into scyllo-inositol, but a significant part of unreacted scyllo-inositol monoketone is left to obtain a mixture. In addition, a significant amount of by-product scyllo-inositol is formed in this conversion. In order to separate the desired scyllo-inositol from the mixture of unreacted scyllo-inositol monoketone and by-product scyllo-inositol, scyllo-inositol is first chemically converted into scyllo-inositol-diboric acid-disodium complex, and then hydrolyzed with hydrochloric acid in a mixture of methane and water. This conversion requires the use of boric acid to form a scyllo-inositol-diboric acid-disodium complex salt, and the use of concentrated hydrochloric acid, which not only corrodes the instrument, but also requires special operating steps to ensure safety and stay away from harmful gases. In addition, since this method requires the use of boric acid in the process of recovering scyllo-inositol to chemically selectively derivatize it into a scyllo-inositol-diboric acid-disodium complex salt, and boric acid is a toxic substance, it is critical to remove the boric acid in the material to an appropriate level in the subsequent steps. Therefore, in view of the above technical problems, it is necessary to develop a method for the separation and preparation of scyllo-inositol. Summary of the invention

[0004] The technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a separation and preparation method of scyllo-inositol is provided, which is simple to operate, environmentally friendly and suitable for industrialization.

[0005] In order to solve the above technical problems, the technical solution of the present invention is:

[0006] A method for separating and preparing scyllo-inositol comprises the following steps:

[0007] A: Prepare myo-inositol solution, then add whole-cell wet bacteria containing inositol dehydrogenase and scyllo-inositol dehydrogenase, and transform in PBS buffer to obtain transformation solution;

[0008] B: The conversion liquid is filtered through a ceramic membrane, the clear liquid is collected, and the concentrated liquid is treated for environmentally friendly sewage treatment;

[0009] C: The filtrate is filtered through an ultrafiltration membrane to collect the ultrafiltration clear liquid, and the concentrated liquid is treated for environmentally friendly sewage treatment;

[0010] D: Add activated carbon to the ultrafiltrate, filter after decolorization, and collect the decolorized liquid;

[0011] E: The decolorized liquid is concentrated in a concentrator under the conditions of vacuum <-0.09Mpa and temperature 50-70°C until crystals are precipitated, then the concentration is stopped, the temperature is lowered to 5-10°C, and centrifuged to obtain a crude mixture of scyllo-inositol and inositol. The filtrate I is collected;

[0012] F: Add more than one times the weight of purified water to the mixed crude product, control the temperature at 20-25℃, beat and wash for 20-40min, centrifuge and filter, collect the wet product and filtrate II, and dry the wet product to obtain the finished scyllo-inositol product.

[0013] Preferably, the concentration of the myo-inositol solution in step A is 10-150 g / L, and the concentration of the PBS buffer is 40-60 mM.

[0014] Preferably, in step A, the ratio of inositol dehydrogenase to scyllo-inositol dehydrogenase is 1:1, and the weight of whole-cell wet bacteria added is 1-5% of the volume of the myo-inositol solution.

[0015] Preferably, the conversion temperature in step A is 35-45° C., pH 7-9, and the conversion time is 6-12 h.

[0016] Preferably, the pore size of the ceramic membrane in step B is 20-100 nm.

[0017] Preferably, the pore size of the ultrafiltration membrane in step C is 1000-10000 Da.

[0018] Preferably, the decolorization conditions of the ultrafiltration clear liquid in step D are: heating temperature 40-50° C., decolorization 20-40 min, and the amount of activated carbon added is 0.1-0.5% v / v of the volume of the ultrafiltration clear liquid.

[0019] Preferably, the mixed crude product is repeatedly washed with purified water for 2 to 4 times, and the filtrate I is mixed with the filtrate II obtained by multiple washings and then converted in step A for repeated use.

[0020] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0021] 1. In the separation and preparation process, no boric acid, strong acid, strong base and organic solvent are used, and no composite salt precipitation is required, which reduces the process steps, does not pollute the environment, is safe to operate, and has low recovery costs.

[0022] 2. The solubility of scyllo-inositol in water is very low, while the solubility of inositol is large. After concentration and crystallization, a mixture of inositol and scyllo-inositol comes out. The purpose of separation can be achieved by washing inositol with water, which greatly saves the separation cost, is simple to operate, and is easier to industrialize.

[0023] 3. Material recycling does not require a complicated recycling system, can better reduce costs and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a liquid chromatogram of scyllo-inositol in Example 1 of the present invention;

[0025] Figure 2 This is a crystal image of scyllo-inositol in Example 1 of the present invention under a 10x optical microscope. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the embodiments.

[0027] Example 1

[0028] 1. Prepare 50L of 100g / L myo-inositol solution, add whole-cell wet bacteria containing inositol dehydrogenase and scyllo-inositol dehydrogenase, the two whole-cell wet bacteria are added in a ratio of 1:1, and the added weight is 3% of the volume of the feed solution, and transform in 50mM PBS buffer at a transformation temperature of 40°C, pH 8, and a transformation time of 10h to obtain 50L of transformation solution, in which scyllo-inositol accounts for 34% and myo-inositol accounts for 66%;

[0029] 2. The conversion liquid is filtered through a ceramic membrane, and 55L of clear liquid is collected after top washing with water. The pore size of the ceramic membrane is 80nm, and the concentrated liquid is treated for environmentally friendly sewage discharge;

[0030] 3. The filtrate was filtered through an ultrafiltration membrane, and 61L of ultrafiltration clear liquid was collected after top washing with water. The pore size of the ultrafiltration membrane was 5000Da, and the concentrated liquid was treated for environmental protection;

[0031] 4. Heat the ultrafiltrate to 45°C, add 0.3% v / v activated carbon, stir for 30 min and filter, collect 61L of decolorized liquid;

[0032] 5. The decolorized liquid was concentrated in a concentrator under the conditions of vacuum <-0.09Mpa and temperature 60°C until crystals precipitated, then the concentration was stopped, the temperature was lowered to 8°C, and centrifuged to obtain a filter cake of 3100g, which was a crude mixed product of scyllo-inositol and inositol. 14L of filtrate I was collected;

[0033] 6. Add one times the weight of pure water to the mixed crude product, control the temperature at 22°C, beat and wash for 30 minutes, centrifuge and filter, collect the wet product and filtrate Ⅱ3L;

[0034] 7. Repeat the washing operation in step 6 for 3 times, collect the wet product and dry it to obtain 1580 g of scyllo-inositol finished product, with a scyllo-inositol separation yield of 92.94% and a purity of 99.7%. The filtrate 1 is mixed with 23 L of filtrate II washed multiple times and then converted in step 1 for repeated use.

[0035] Example 2

[0036] 1. Prepare 50L of 10g / L myo-inositol solution, add whole-cell wet bacteria containing inositol dehydrogenase and scyllo-inositol dehydrogenase, the two whole-cell wet bacteria are added in a ratio of 1:1, the added weight is 1% of the volume of the feed liquid, and the transformation is carried out in 40mM PBS buffer at a transformation temperature of 35°C, pH 7, and a transformation time of 6h to obtain 50L of transformation liquid, of which scyllo-inositol accounts for 36% and myo-inositol accounts for 64%;

[0037] 2. The conversion liquid is filtered through a ceramic membrane, and 54L of clear liquid is collected after top washing with water. The pore size of the ceramic membrane is 20-100nm, and the concentrated liquid is treated for environmentally friendly sewage discharge;

[0038] 3. The filtrate is filtered through an ultrafiltration membrane, and after top washing with water, 60L of ultrafiltration clear liquid is collected. The pore size of the ultrafiltration membrane is 1000Da, and the concentrated liquid is treated for environmentally friendly sewage discharge;

[0039] 4. Heat the ultrafiltrate to 40°C, add 0.1% v / v activated carbon, stir for 20 minutes and filter, collect 60L of decolorized liquid;

[0040] 5. The decolorized liquid was concentrated in a concentrator under the conditions of vacuum <-0.09Mpa and temperature 50°C until crystals precipitated, then the concentration was stopped, the temperature was lowered to 5°C, and centrifuged to obtain a filter cake of 310g, which was a crude mixed product of scyllo-inositol and inositol. 1.5L of filtrate I was collected;

[0041] 6. Add one times the weight of pure water to the mixed crude product, control the temperature at 20°C, beat and wash for 20 minutes, centrifuge and filter, collect the wet product and 0.4L of filtrate II;

[0042] 7. Repeat the washing operation in step 6 twice, collect the wet product and dry it to obtain 166 g of scyllo-inositol product, with a scyllo-inositol separation yield of 92.22% and a purity of 99.6%. The filtrate 1 is mixed with 2.3 L of filtrate II washed multiple times and then converted in step 1 for repeated use.

[0043] Example 3

[0044] 1. Prepare 50L of 150g / L myo-inositol solution, add whole-cell wet bacteria containing inositol dehydrogenase and scyllo-inositol dehydrogenase, add the two whole-cell wet bacteria at a ratio of 1:1, and the added weight is 5% of the volume of the feed solution, and transform in 60mM PBS buffer at a transformation temperature of 45°C, pH 9, and a transformation time of 12h to obtain 50L of transformation solution, in which scyllo-inositol accounts for 35% and myo-inositol accounts for 65%;

[0045] 2. The conversion liquid was filtered through a ceramic membrane, and 58L of clear liquid was collected. The pore size of the ceramic membrane was 100nm, and the concentrated liquid was treated for environmental protection;

[0046] 3. The filtrate was filtered through an ultrafiltration membrane to collect 64L of ultrafiltration clear liquid, the pore size of the ultrafiltration membrane was 10000Da, and the concentrated liquid was treated for environmental protection;

[0047] 4. Heat the ultrafiltrate to 50°C, add 0.5% v / v activated carbon, stir for 40 minutes and filter, collect 64L of decolorized liquid;

[0048] 5. The decolorized liquid was concentrated in a concentrator under the conditions of vacuum <-0.09Mpa and temperature 70°C until crystals precipitated, then the concentration was stopped, the temperature was lowered to 10°C, and centrifuged to obtain a filter cake of 5200g, which was a crude mixed product of scyllo-inositol and inositol. 16L of filtrate I was collected;

[0049] 6. Add one times the weight of pure water to the mixed crude product, control the temperature at 25°C, beat and wash for 40 minutes, centrifuge and filter, collect the wet product and filtrate II 5.2L;

[0050] 7. Repeat the washing operation in step 6 for 4 times, collect the wet product and dry it to obtain 2440 g of scyllo-inositol finished product, with a scyllo-inositol separation yield of 92.95% and a purity of 99.5%. The filtrate 1 is mixed with 36.8 L of filtrate II washed multiple times and then converted in step 1 for repeated use.

[0051] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A method for separating and preparing scyllo-inositol, characterized in that The following steps are involved: A: Prepare myo-inositol solution, then add whole-cell wet bacteria containing inositol dehydrogenase and scyllo-inositol dehydrogenase, and transform in PBS buffer to obtain transformation solution; B: The conversion liquid is filtered through a ceramic membrane, the clear liquid is collected, and the concentrated liquid is treated for environmentally friendly sewage treatment; C: The filtrate is filtered through an ultrafiltration membrane to collect the ultrafiltration clear liquid, and the concentrated liquid is treated for environmentally friendly sewage treatment; D: Add activated carbon to the ultrafiltrate, filter after decolorization, and collect the decolorized liquid; E: The decolorized liquid is concentrated in a concentrator under the conditions of vacuum <-0.09Mpa and temperature 50-70°C until crystals are precipitated, then the concentration is stopped, the temperature is lowered to 5-10°C, and centrifuged to obtain a crude mixture of scyllo-inositol and inositol. The filtrate I is collected; F: Add more than one times the weight of purified water to the mixed crude product, control the temperature at 20-25℃, beat and wash for 20-40min, centrifuge and filter, collect the wet product and filtrate II, and dry the wet product to obtain the finished scyllo-inositol product.

2. A method for separating and preparing scyllo-inositol as claimed in claim 1, characterized in that: In step A, the concentration of the myo-inositol solution is 10-150 g / L, and the concentration of the PBS buffer is 40-60 mM.

3. A method for separating and preparing scyllo-inositol according to claim 1, characterized in that: In step A, the ratio of inositol dehydrogenase to scyllo-inositol dehydrogenase is 1:1, and the weight of whole-cell wet bacteria added is 1-5% of the volume of the myo-inositol solution.

4. A method for separating and preparing scyllo-inositol as claimed in claim 1, characterized in that: The conversion temperature in step A is 35-45° C., pH 7-9, and the conversion time is 6-12 h.

5. A method for separating and preparing scyllo-inositol as claimed in claim 1, characterized in that: The pore size of the ceramic membrane in step B is 20-100 nm.

6. The method for separating and preparing scyllo-inositol according to claim 1, characterized in that: The pore size of the ultrafiltration membrane in step C is 1000-10000Da.

7. The method for separating and preparing scyllo-inositol according to claim 1, characterized in that: The decolorization conditions of the ultrafiltration clear liquid in step D are: heating temperature 40-50° C., decolorization 20-40 min, and the amount of activated carbon added is 0.1-0.5% v / v of the volume of the ultrafiltration clear liquid.

8. The method for separating and preparing scyllo-inositol according to claim 1, characterized in that: The mixed crude product is repeatedly washed with purified water for 2 to 4 times, and the filtrate I is mixed with the filtrate II obtained by multiple washings and then converted through step A for repeated use.

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