Preparation method of D-chiro-inositol

Abstract

The invention discloses a preparation method of D-chiro-inositol, and relates to the technical field of D-chiro-inositol production.The preparation method comprises the steps that whole cells expressing four enzymes including inositol-3-phosphate synthetase, inositol monophosphate, inositol dehydrogenase and keto isomerase are added into a substrate solution containing glucose for transformation, thalli are removed from the obtained transformation solution through a ceramic membrane, and the D-chiro-inositol is obtained. The method comprises the following steps: firstly, preparing a feed liquid, then removing macromolecular impurities such as protein in the feed liquid through an ultrafiltration membrane, finally, desalting through a cation exchange resin column and an anion exchange resin column, and finally, separating through a simulated moving bed to obtain a D-chiral inositol phase and a glucose phase, the content of D-chiral inositol in the D-chiral inositol phase is greater than or equal to 99%, and the content of glucose in the glucose phase is greater than or equal to 99%. According to the method, the D-chiro-inositol phase is concentrated, decolorized, cooled and crystallized, and finally filtered and dried to obtain the high-purity D-chiro-inositol, and the method is low in production cost and suitable for industrial production. In addition, the separated glucose phase can be reused for conversion, the utilization rate of raw materials is improved, and waste is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of D-chiro-inositol production, and in particular to a method for preparing D-chiro-inositol. Background Art

[0002] D-Chiro Inositol (DCI) is a white powder that is easily soluble in water and slightly soluble in organic solvents such as ethanol and methanol. It is one of the nine isomers of inositol that is optically active and is also the biologically active isomer of vitamin B8. It exists at relatively high levels in plants such as buckwheat seeds and soybeans, as well as some insects, mostly in the form of methylated or glycosylated derivatives. It is a biodegradable product of buckwheat sugar alcohol. It has the function of sensitizing insulin and promoting liver fat metabolism, can significantly reduce blood sugar content, and has a significant therapeutic effect on diabetes. In addition, DCI also has the effects of improving Polycystic Ovarian Syndrome (PCOS), anti-oxidation, anti-aging, and scavenging free radicals, and has good utilization value.

[0003] D-chiro-inositol can be extracted from plants such as buckwheat, but the content of D-chiro-inositol in these plants is not high, resulting in poor resource utilization and high separation and extraction costs. D-chiro-inositol can also be prepared by organic synthesis, but due to the cumbersome steps, by-products are difficult to separate, and there will be residual toxic substances, affecting the quality of the product; in addition, a large amount of organic solvents are needed, and the environmental friendliness is poor. In addition, D-chiro-inositol can be produced by hydrolyzing D-pine alcohol or kasugamycin, but the raw materials D-pine alcohol or kasugamycin are expensive, so the economy is poor.

[0004] In recent years, with the development of production engineering technology, biotransformation synthesis of D-chiro-inositol has become a new development trend. At present, it mainly transforms myo-inositol into D-chiro-inositol by modifying microorganisms. However, the raw material cost is high, the conversion rate is low, and the separation of D-chiro-inositol and myo-inositol is difficult, which limits the large-scale production of D-chiro-inositol. Therefore, it is urgent to develop a new method for preparing D-chiro-inositol with simple production process, low cost and high yield. Summary of the invention

[0005] The technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a method for preparing D-chiro-inositol is provided, which has low cost and high yield.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A method for preparing D-chiro-inositol comprises the following steps:

[0008] A: Take the glucose substrate solution, add whole cells expressing four enzymes: inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, and transform at a temperature of 35-40°C and a pH of 6-8 to obtain a transformation solution;

[0009] B: The conversion liquid is filtered through a ceramic membrane, and the ceramic membrane clear liquid is collected;

[0010] C: filtering the ceramic membrane clear liquid through an ultrafiltration membrane, and collecting the ultrafiltration membrane clear liquid;

[0011] D: The ultrafiltration membrane clear liquid first enters the cation exchange resin column, then enters the anion exchange resin column, and the desalted liquid is collected;

[0012] E: The desalted liquid is concentrated in a concentrator at -0.09~-0.1Mpa and 50-70℃ to obtain a concentrated liquid;

[0013] F: The concentrated liquid is separated in a simulated moving bed to obtain a D-chiro-inositol phase and a glucose phase, wherein the chiro-inositol content in the D-chiro-inositol component is ≥99%, and the glucose content in the glucose component is ≥98%;

[0014] G: Concentrate the collected D-chiro-inositol phase in a concentrator under vacuum conditions of -0.09 to -0.1 MPa and a temperature of 50 to 70° C. to obtain a re-concentrated solution;

[0015] H: Add 2-5% w / v activated carbon to the reconcentrated solution at 50-70°C for 20-40 min, and collect the decolorized solution by filtration;

[0016] I: The decolorizing solution is cooled to 5-10°C while stirring at 50-200 rpm, filtered, and the filter cake is dried at 40-50°C under vacuum at -0.09 to -0.1 MPa to obtain D-chiro-inositol.

[0017] Preferably, the content of glucose in the substrate solution in step A is 10-100 g / L, and the amount of wet bacteria expressing four enzymes, inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, added is 30-50 g / L.

[0018] Preferably, the pore size of the ceramic membrane in step B is 20-100 nm.

[0019] Preferably, the pore size of the ultrafiltration membrane in step C is 1000-10000 Da.

[0020] Preferably, in step D, the feed rate of the cation exchange resin column is 1-4 BV / h, and the feed rate of the anion exchange resin column is 2-5 BV / h.

[0021] Preferably, the solid content of the concentrate in step E is 50-55% w / w.

[0022] Preferably, the glucose phase obtained in step F is returned to step A for further conversion.

[0023] Preferably, the filler of the simulated moving bed in step F is DTF-01 calcium type separation resin, and the separation parameters are feed mass concentration of 50-55%, separation operating temperature of 55-65° C., and feed flow rate of 1.0-2.0 L / h.

[0024] Preferably, the solid content of the reconcentrated solution in step G is 40-50% w / w.

[0025] Preferably, the cooling rate of the decolorizing solution in step I is 3-5°C / h;

[0026] Preferably, the filtrate obtained after filtering the decolorized liquid is applied to step G and concentrated again.

[0027] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0028] 1. The whole cell expressing four enzymes, inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, is added to a substrate solution containing glucose for conversion, the obtained conversion solution is subjected to a ceramic membrane to remove the bacterial cells, and then the macromolecular impurities such as protein in the feed solution are removed by an ultrafiltration membrane, and finally the cation exchange resin column and the anion exchange resin column are used for desalination, and finally the simulated moving bed is used for separation to obtain a D-chiro-inositol phase and a glucose phase, wherein the content of D-chiro-inositol in the D-chiro-inositol phase is ≥99%, and the content of glucose in the glucose phase is ≥98%, and the D-chiro-inositol phase is concentrated, decolorized, cooled and crystallized, and finally filtered and dried to obtain high-purity D-chiro-inositol, and the production cost is low, and the product is suitable for industrial production. In addition, the separated glucose phase can be reused for conversion, thereby improving the utilization rate of raw materials and greatly reducing waste.

[0029] 2. Compared with the combination of myo-inositol and D-chiro-inositol, the separation between glucose and D-chiro-inositol is simpler, the obtained D-chiro-inositol has higher purity, and the quality of the product is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the liquid chromatogram of D-chiro-inositol in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the embodiments.

[0032] Example 1

[0033] A method for preparing D-chiro-inositol comprises the following steps:

[0034] A: Take 50L of substrate solution with a glucose content of 10g / L, add 1500g of whole cells expressing four enzymes: inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, and transform at 35°C and pH 6 to obtain 50L of transformation solution;

[0035] B: The conversion liquid was filtered through a ceramic membrane, and 50 L of the ceramic membrane clear liquid was collected. The pore size of the ceramic membrane was 20 nm.

[0036] C: Filter the ceramic membrane clear liquid through an ultrafiltration membrane, and collect 50L of ultrafiltration membrane clear liquid. The pore size of the ultrafiltration membrane is 1000Da;

[0037] D: The ultrafiltration membrane clear liquid first enters the cation exchange resin column at a rate of 1-4BV / h, and then enters the anion exchange resin column at a rate of 2BV / h, and 55L of desalted liquid is collected;

[0038] E: The desalted liquid was concentrated in a concentrator at -0.09 MPa and 50°C to obtain 1.2 L of concentrated liquid with a solid content of 50% w / w;

[0039] F: The concentrated liquid is separated in a simulated moving bed to obtain 2L of D-chiro-inositol phase and 8L of glucose phase, wherein the content of chiro-inositol in the D-chiro-inositol component is 99.3%, and the content of glucose in the glucose component is 98.9%; wherein the filler of the simulated moving bed is DTF-01 calcium-type separation resin, the separation parameters are feed mass concentration of 50-55%, separation operating temperature of 55-65°C, and feed flow rate of 1.0-2.0L / h. The glucose phase is returned to step A for re-conversion.

[0040] G: The collected D-chiro-inositol phase was concentrated in a concentrator under vacuum conditions of -0.09 MPa and a temperature of 50° C. to obtain 0.2 L of a re-concentrated solution having a solid content of 40% w / w;

[0041] H: Add 2% w / v activated carbon to the reconcentrated solution at 50°C for 20 min, and collect 0.2 L of the decolorized solution by filtration;

[0042] I: The decolorizing liquid was cooled to 10°C at a rate of 5°C / h while stirring at 50 rpm, and filtered to obtain 0.17 L of filtrate and 48 g of filter cake. The filter cake was dried at 40°C under vacuum at -0.09 MPa to obtain 45 g of D-chiro-inositol with a purity of 99.6% and a crystallization yield of 81.4%. The filtrate was applied to step G and concentrated again.

[0043] Example 2

[0044] A method for preparing D-chiro-inositol comprises the following steps:

[0045] A: Take 50L of substrate solution with a glucose content of 50g / L, add 2000g of whole cells expressing four enzymes: inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, and transform at 38°C and pH 7 to obtain 50L of transformation solution;

[0046] B: The conversion liquid was filtered through a ceramic membrane, and 50 L of the ceramic membrane clear liquid was collected. The pore size of the ceramic membrane was 70 nm.

[0047] C: Filter the ceramic membrane clear liquid through an ultrafiltration membrane, and collect 50L of ultrafiltration membrane clear liquid. The pore size of the ultrafiltration membrane is 5000Da;

[0048] D: The ultrafiltration membrane clear liquid first enters the cation exchange resin column at a rate of 2BV / h, and then enters the anion exchange resin column at a rate of 4BV / h, and 55L of desalted liquid is collected;

[0049] E: The desalted liquid was concentrated in a concentrator at -0.1 MPa and 60°C to obtain 6 L of concentrated liquid with a solid content of 52% w / w;

[0050] F: The concentrated liquid is separated in a simulated moving bed to obtain 13L of D-chiro-inositol phase and 32L of glucose phase, wherein the content of chiro-inositol in the D-chiro-inositol component is 99.2%, and the content of glucose in the glucose component is 98.7%; wherein the filler of the simulated moving bed is DTF-01 calcium-type separation resin, the separation parameters are feed mass concentration of 50-55%, separation operating temperature of 55-65°C, and feed flow rate of 1.0-2.0L / h. The glucose phase is returned to step A for further conversion.

[0051] G: The collected D-chiro-inositol phase was concentrated in a concentrator under vacuum conditions of -0.1 MPa and a temperature of 60° C. to obtain 0.6 L of a reconcentrated solution having a solid content of 45% w / w;

[0052] H: Add 3% w / v activated carbon to the reconcentrated solution at 60°C for 30 min, and collect 0.6 L of the decolorized solution by filtration;

[0053] I: The decolorizing liquid was cooled to 8°C at a rate of 4°C / h while stirring at 100 rpm, and filtered to obtain 0.45 L of filtrate and 220 g of filter cake. The filter cake was dried at 45°C under vacuum at -0.1 MPa to obtain 201 g of D-chiro-inositol with a purity of 99.7% and a crystallization yield of 80.2%. The filtrate was applied to step G and concentrated again.

[0054] Example 3

[0055] A method for preparing D-chiro-inositol comprises the following steps:

[0056] A: Take 50L of substrate solution with a glucose content of 100g / L, add 2500g of whole cells expressing four enzymes: inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, and transform at a temperature of 40°C and pH 8 to obtain 50L of transformation solution;

[0057] B: The conversion liquid was filtered through a ceramic membrane, and 50 L of the ceramic membrane clear liquid was collected. The pore size of the ceramic membrane was 100 nm.

[0058] C: Filter the ceramic membrane clear liquid through an ultrafiltration membrane, and collect 50L of ultrafiltration membrane clear liquid. The pore size of the ultrafiltration membrane is 10000Da;

[0059] D: The ultrafiltration membrane clear liquid first enters the cation exchange resin column at a rate of 4BV / h, and then enters the anion exchange resin column at a rate of 5BV / h, and 55L of desalted liquid is collected;

[0060] E: The desalted liquid was concentrated in a concentrator at -0.1 MPa and 70°C to obtain 11 L of concentrated liquid with a solid content of 55% w / w;

[0061] F: The concentrated liquid is separated in a simulated moving bed to obtain 31L of D-chiro-inositol phase and 46L of glucose phase, wherein the content of chiro-inositol in the D-chiro-inositol component is 99.6%, and the content of glucose in the glucose component is 99.0%; wherein the filler of the simulated moving bed is DTF-01 calcium-type separation resin, the separation parameters are feed mass concentration of 50-55%, separation operating temperature of 55-65°C, and feed flow rate of 1.0-2.0L / h. The glucose phase is returned to step A for further conversion.

[0062] G: The collected D-chiro-inositol phase was concentrated in a concentrator under vacuum conditions of -0.09 MPa and a temperature of 70° C. to obtain 1.1 L of a re-concentrated solution having a solid content of 50% w / w;

[0063] H: Add 5% w / v activated carbon to the reconcentrated solution at 70°C for 40 min, and collect 1.1 L of the decolorized solution by filtration;

[0064] I: The decolorizing liquid was cooled to 10°C at a rate of 5°C / h while stirring at 200 rpm, and filtered to obtain 0.8 L of filtrate and 480 g of filter cake. The filter cake was dried at 50°C under vacuum at -0.09 MPa to obtain 440 g of D-chiro-inositol with a purity of 99.8% and a yield of 81.1%. The filtrate was applied to step G and concentrated again.

[0065] 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 preparing D-chiro-inositol, characterized in that The following steps are involved: A: Take the glucose substrate solution, add whole cells expressing four enzymes: inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, and transform at a temperature of 35-40°C and a pH of 6-8 to obtain a transformation solution; B: The conversion liquid is filtered through a ceramic membrane, and the ceramic membrane clear liquid is collected; C: filtering the ceramic membrane clear liquid through an ultrafiltration membrane and collecting the ultrafiltration membrane clear liquid; D: The ultrafiltration membrane clear liquid first enters the cation exchange resin column, then enters the anion exchange resin column, and the desalted liquid is collected; E: The desalted liquid is concentrated in a concentrator at -0.09~-0.1Mpa and 50-70℃ to obtain a concentrated liquid; F: The concentrated liquid is separated in a simulated moving bed to obtain a D-chiro-inositol phase and a glucose phase, wherein the chiro-inositol content in the D-chiro-inositol component is ≥99%, and the glucose content in the glucose component is ≥98%; G: Concentrate the collected D-chiro-inositol phase in a concentrator under vacuum conditions of -0.09 to -0.1 MPa and a temperature of 50 to 70° C. to obtain a re-concentrated solution; H: Add 2-5% w / v activated carbon to the reconcentrated solution at 50-70°C for 20-40 min, and collect the decolorized solution by filtration; I: The decolorizing solution is cooled to 5-10°C while stirring at 50-200 rpm, filtered, and the filter cake is dried at 40-50°C under vacuum at -0.09 to -0.1 MPa to obtain D-chiro-inositol.

2. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: In step A, the content of glucose in the substrate solution is 10-100 g / L, and the amount of wet bacteria expressing four enzymes, inositol-3-phosphate synthase, inositol monophosphatase, inositol dehydrogenase and ketoisomerase, added is 30-50 g / L.

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

4. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: The pore size of the ultrafiltration membrane in step C is 1000-10000Da.

5. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: In step D, the feeding rate of the cation exchange resin column is 1-4 BV / h, and the feeding rate of the anion exchange resin column is 2-5 BV / h.

6. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: The solid content of the concentrate in step E is 50-55% w / w.

7. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: The glucose phase obtained in step F is returned to step A for further conversion.

8. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: The filler of the simulated moving bed in step F is DTF-01 calcium type separation resin, and the separation parameters are feed mass concentration of 50-55%, separation operating temperature of 55-65° C., and feed flow rate of 1.0-2.0 L / h.

9. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: The solid content of the reconcentrated solution in step G is 40-50% w / w.

10. A method for preparing D-chiro-inositol as claimed in claim 1, characterized in that: The cooling rate of the decolorizing solution in step I is 3-5°C / h; The filtrate obtained after filtering the decolorized liquid is used in step G and concentrated again.

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