Gluconolactone and preparation method thereof

Abstract

The invention discloses glucurolactone and a preparation method thereof, belongs to the technical field of pharmacy, and aims to solve the technical problems that the existing industrial production of glucurolactone is low in yield and causes great pollution to the environment, and acetic acid causes great corrosion to equipment. The preparation method of the glucurolactone comprises the following steps: adding whole cells containing inositol oxidase into an inositol feed liquid for transformation, and adjusting the pH value to 7.0-8.0 by using a sodium hydroxide solution in the transformation process to obtain a transformation liquid; adjusting the pH value of the conversion liquid to 4.0-5.0 by using strong phosphoric acid, then carrying out first filtration, and collecting a clear liquid; desalting, decolorizing and concentrating the clear liquid until the solid content is 50-70% to obtain a concentrated solution; 5-10 g of citric acid is added into every 100 mL of the concentrated solution, a lactonization reaction is conducted at the temperature of 50-70 DEG C, and glucurolactone is obtained after a reaction product is subjected to cooling, second filtering and washing. The preparation method of glucurolactone used in the invention has the advantages of small corrosion to equipment, high yield, small pollution and high safety.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to glucuronolactone and a preparation method thereof. Background Art

[0002] The full name of glucuronolactone in Chinese is glucuronolactone, commonly known as liver taylor, with the molecular formula of C6H8O6, and its chemical composition is: D(+)-furanoglucuronic acid γ-lactone, English name: [D(+)-Glucofuranurono-6,3-lactone]. As a liver detoxifier and immune function regulator, glucuronolactone is a conventional liver protection medicine; glucuronolactone and its subsequent products are also the main additives of functional beverages and foods, weight loss drugs, cosmetics, etc., which have the effects of replenishing physical energy, improving hypoxia, nourishing skin, and delaying aging.

[0003] The main method for industrial production of glucuron lactone at present is the nitric acid oxidation method starting from starch, which includes: adding starch to nitric acid with a content of about 80% (V / V) to oxidize starch, heating and pressurizing the starch oxidation liquid under acidic conditions to hydrolyze the starch oxidation liquid, and then adding acetic acid to esterify the hydrolyzate when the hydrolyzate is concentrated to a Baume degree of 44-49, cooling the esterified mixture to 30-45°C, standing for 16-20 hours, and then concentrating it to a Baume degree of 40-45, gradually cooling it to -4 to -8°C, and the total cooling crystallization time is generally 70-80 hours, and the crystals are filtered by high-speed centrifugation to obtain a crude glucuron lactone with a purity of 80-90%. The yield of glucuron lactone obtained by this process is low, and its yield is only about 14%; acetic acid is added in the glucuron lactone production process, and acetic acid is easy to corrode the equipment, and the equipment control requirements are strict, and the energy consumption in the production process is large and the recovery of acetic acid is difficult; and the crude product extraction process has a large loss, the production cost is high, it is difficult to meet the needs of the market, and it is also highly polluting to the environment. Therefore, the current industrial production of glucuron-lactone has low purity and yield, causes great pollution to the environment, and the technical problems of acetic acid corrosion to equipment need to be solved urgently. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a glucuron lactone and a preparation method thereof, so as to solve the technical problems that the current industrial production of glucuron lactone has low purity and yield, causes great pollution to the environment, and causes great corrosion to equipment by acetic acid.

[0005] The purpose of the present invention is mainly achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing glucuronolactone, comprising the following steps:

[0007] Adding whole cells containing inositol oxidase to the inositol feed solution for conversion, adjusting the pH to 7.0-8.0 with sodium hydroxide solution during the conversion process, and obtaining a conversion solution;

[0008] The conversion solution is adjusted to pH 4.0-5.0 with concentrated phosphoric acid, and then filtered for the first time to collect the clear liquid;

[0009] The clear liquid is desalted, decolorized, and concentrated to a solid content of 50-70% to obtain a concentrated liquid;

[0010] 5-10 g of citric acid is added to every 100 mL of the concentrated solution, and a lactonization reaction is carried out at 50-70° C. The reaction product is cooled, filtered for a second time, and washed to obtain glucuron lactone.

[0011] Compared with the prior art, the conversion process of the present invention uses sodium hydroxide solution to adjust the pH to 7.0-8.0, and the pH adjustment ability of the sodium hydroxide solution is more stable and controllable, providing a precise reaction environment for the conversion reaction, which is conducive to improving the yield of the product, and the sodium hydroxide solution will not introduce additional ammonium ions, so that no high-ammonia nitrogen sewage will be generated during the subsequent desalination treatment, the sewage is easy to treat, and the pollution of ammonia volatilization to the environment is also reduced; after adjusting the pH to 4.0-5.0 with concentrated phosphoric acid, the first filtration is performed, and at this pH value, macromolecules such as proteins in the solution are easy to be denatured and aggregated, which is convenient for filtration and removal, which is conducive to improving the purity and stability of the product. In addition, it is found through research that adding phosphate ions to the feed liquid is more conducive to subsequent lactonization crystallization, so that the yield of glucuron lactone is increased; citric acid is used for lactonization reaction, the acidity of citric acid is weak, the acid resistance requirement of production equipment is low, and the citric acid is not easy to volatilize, which has less pollution to the environment; the reaction product is cooled, filtered and washed, and impurities in glucuron lactone can be further removed, which is conducive to increasing the purity and yield of glucuron lactone.

[0012] Furthermore, the inositol content in the inositol solution is 10-150 g / L; the whole cell addition amount is the system OD 600 =10-50; the conversion time is 2-6h.

[0013] Further, the sodium hydroxide solution has a concentration of 25 to 30 wt%, and / or

[0014] The concentration of the concentrated phosphoric acid is 80-85 wt %.

[0015] Furthermore, the first filtering includes:

[0016] The conversion liquid after adjusting the pH value is filtered through a ceramic membrane with a pore size of 20-100nm, a centrifuge or a plate and frame, and the filtrate is filtered through an ultrafiltration membrane with a pore size of 1000-10000Da to collect the clear liquid.

[0017] Further, the desalination comprises:

[0018] The clear liquid is desalted by a cation exchange resin at a column flow rate of 1-2 BV / h to obtain a desalted liquid.

[0019] Further, the decolorization comprises:

[0020] The desalted liquid is decolorized by a decolorizing resin under the condition of a column flow rate of 2-6 BV / h to obtain a decolorized liquid.

[0021] Further, the concentration includes:

[0022] The decolorized liquid is concentrated through a nanofiltration membrane with a pore size of 150 to 200 Da to obtain a nanofiltration concentrate with a solid content of 15 to 20%;

[0023] The nanofiltration concentrated liquid is further concentrated in a concentrator under the conditions of vacuum degree ≤-0.09Mpa and temperature 50-70°C to obtain a concentrated liquid with a solid content of 50-70%.

[0024] Furthermore, the lactonization reaction conditions also include: carrying out the lactonization reaction at a stirring speed of 50-200 rpm for 1-2 hours.

[0025] Furthermore, the reaction product is cooled, filtered for the second time, and washed to obtain glucuron lactone, which includes: the reaction product is cooled to 5-10°C at a rate of 1-5°C / h, filtered for the second time, and washed with 2-4°C cold water to obtain glucuron lactone crystals.

[0026] In a second aspect, the present invention further provides a glucuron-lactone prepared by the above-mentioned method for preparing glucuron-lactone.

[0027] Compared with the prior art, the glucuron lactone obtained by the glucuron lactone preparation method of the present invention has pure color, uniform particle size, higher purity and higher yield. DETAILED DESCRIPTION

[0028] 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 the 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.

[0029] The main method for industrial production of glucuron lactone is the nitric acid oxidation method starting from starch. The yield of glucuron lactone obtained by this process is low, and its yield is only about 14%. Acetic acid is added during the production of glucuron lactone, which is easy to corrode equipment and requires strict equipment control. The energy consumption in the production process is large and it is difficult to recover acetic acid. In addition, the crude product extraction process has a large loss, the production cost is high, it is difficult to meet the needs of the market, and it is also highly polluting to the environment.

[0030] The invention provides glucuronolactone and a preparation method thereof, which are used to solve the technical problems of low yield of glucuronolactone in current industrial production, great pollution to the environment and great corrosion of equipment by acetic acid.

[0031] In a first aspect, an embodiment of the present invention discloses a method for preparing glucuronolactone, comprising the following steps:

[0032] Adding whole cells containing inositol oxidase to the inositol feed solution for conversion, adjusting the pH to 7.0-8.0 with sodium hydroxide solution during the conversion process, and obtaining a conversion solution;

[0033] The conversion solution is adjusted to pH 4.0-5.0 with concentrated phosphoric acid, and then filtered for the first time to collect the clear solution;

[0034] The clear liquid is desalted, decolorized, and concentrated to a solid content of 50-70% to obtain a concentrated liquid;

[0035] 5-10 g of citric acid is added to every 100 mL of the concentrated solution, and a lactonization reaction is carried out at 50-70° C. The reaction product is cooled, filtered for a second time, and washed to obtain glucuron lactone.

[0036] By adopting the above-mentioned embodiment, the present invention adds whole cells containing inositol oxidase to the inositol feed liquid for conversion, and uses sodium hydroxide solution to adjust the pH to 7.0-8.0 in the conversion process, replacing the commonly used ammonia water to adjust the pH value. The pH adjustment ability of the sodium hydroxide solution is more stable and controllable, and it is easier to perform accurate pH control, providing an accurate reaction environment for the conversion reaction, which is conducive to improving the yield of the product, and the sodium hydroxide solution will not introduce ammonium ions, so that high ammonia nitrogen sewage will not be generated during desalination, and the sewage is easy to handle, reducing pollution to the environment; after adjusting the pH to 4.0-5.0 with concentrated phosphoric acid, the first filtration is performed. Under this pH condition, proteins in the solution can be denatured and aggregated, which is convenient for filtration and removal, which is conducive to improving the purity and stability of the product, and increasing the purity of glucuron-lactone. In addition, the addition of phosphate ions to the feed liquid is more conducive to the subsequent lactonization crystallization, thereby increasing the yield of glucuron lactone; the filtered clear liquid is subjected to desalting treatment, which is helpful to remove salts and ions in the clear liquid; the desalted liquid is subjected to decolorization treatment to remove pigments, impurities and organic substances in the desalted liquid, making the liquid transparent or clear; the decolorized liquid is subjected to concentration treatment to increase the solute concentration in the decolorized liquid, which is conducive to the subsequent lactonization reaction, thereby increasing the yield of glucuron lactone; citric acid is used for the lactonization reaction, and the acidity of citric acid is weak, and the acid resistance requirements of the production equipment are low. In addition, citric acid is not easy to volatilize, and compared with acetic acid, it has less environmental pollution; the reaction product is cooled, filtered for the second time, and washed to further remove impurities in glucuron lactone, which is conducive to increasing the purity and yield of glucuron lactone.

[0037] Further, in combination with the above embodiments, the inositol content of the inositol solution used in the embodiments of the present invention can be 10-150 g / L. Specifically, the inositol content of the inositol solution used in the present invention can be selected to be 10 g / L, 50 g / L, 80 g / L, 120 g / L or 150 g / L, etc.; the whole cell addition amount containing inositol oxidase can be the system OD 600 =10-50. Specifically, the amount of whole cells containing inositol oxidase can be selected as OD 600 =10, OD 600 =20, OD 600 =30, OD 600 =40 or OD 600 =50, etc.; the conversion time can be 2-6h, specifically, the conversion time can be selected as 2h, 3h, 4h, 5h or 6h, etc. The present invention can convert inositol to glucuronic acid by inositol oxidase without high pressure and high heat conditions, and the inositol solution with a concentration of 10-150g / L in the whole cell addition system is OD 600 =10-50, and the conversion time is 2-6h. The conversion efficiency of inositol to glucuronic acid is high, which is beneficial to improving the yield of glucuronolactone.

[0038] Further, in combination with the above embodiments, in the embodiments of the present invention, the concentration of sodium hydroxide solution can be 25-30wt%, and the concentration of concentrated phosphoric acid can be 80-85wt%. Specifically, the concentration of sodium hydroxide solution can be selected as 25wt%, 27wt%, 29wt% or 30wt%, etc., and the concentration of concentrated phosphoric acid can be selected as 80wt%, 81wt%, 83wt% or 85wt%, etc. By adjusting the sodium hydroxide solution at this concentration to an appropriate pH value, the activity of the enzyme can be ensured; by adjusting the phosphoric acid at this concentration to an appropriate pH value, denatured aggregation of proteins and the like can be made, which is convenient for filtration and removal, and is conducive to improving the purity and stability of the product. In addition, the inventors also found that too low pH has high requirements on equipment materials, while too high pH does not achieve the ideal effect. And the use of high-concentration sodium hydroxide solution and phosphoric acid solution can reduce the final volume of the feed liquid, and can increase the concentration of glucuronic acid in the solution, which is convenient for subsequent concentration.

[0039] Further, in combination with the above embodiment, in an embodiment of the present invention, the first filtering includes:

[0040] After adjusting the pH value, the conversion liquid is filtered through a ceramic membrane, a centrifuge or a plate and frame with a pore size of 20-100nm, and the filtrate is filtered through an ultrafiltration membrane with a pore size of 1000-10000Da to collect the clear liquid. It should be understood that the concentrated solution filtered here can be treated by environmental protection sewage discharge. For example, when selecting ceramic membrane filtration, the pore size of the ceramic membrane can be 20-100nm. Specifically, the pore size of the ceramic membrane can be selected as 20nm, 50nm, 80nm or 100nm, etc. For another example, when selecting a centrifuge for centrifugal separation, the speed of the centrifugal separation can be 3000rpm~5000rpm. For another example, when plate and frame filtration is selected, the filter pore size of the plate and frame filtration is 1000 mesh~2000 mesh. The present invention has no special requirements for the equipment of ceramic membrane, centrifuge or plate and frame filtration, and has no special requirements for other parameters of ceramic membrane or plate and frame filtration such as the number of filter layers, filtration pressure, water flow, etc., as long as the filtration under the above pore size can be achieved. Ceramic membrane, centrifuge or plate and frame filtration removes bacteria and most of the protein. When the filtrate is filtered again by ultrafiltration membrane, the pore size of the ultrafiltration membrane can be 1000-10000Da, and ultrafiltration removes other proteins and organic matter, such as some small molecule proteins, acid-resistant proteins, etc. Specifically, the pore size of the ultrafiltration membrane can be selected as 1000Da, 4000Da, 7000Da or 10000Da, etc. Ceramic membrane and ultrafiltration membrane can effectively remove impurities in the solution according to the size of the selected pore size. Through the first filtration, the impurities in the solution can be separated to obtain a purer glucuronic acid concentrate, which helps to improve the purity and yield of glucuron lactone in the subsequent lactone reaction.

[0041] Further, in combination with the above embodiment, in the embodiment of the present invention, the clear liquid is desalted by a cation exchange resin under the condition of a column flow rate of 1-2 BV / h to obtain a desalted liquid. Under the condition of a rate of 1-2 BV / h, it can be ensured that the resin particles have sufficient contact time to exchange ions in the liquid phase, thereby improving the desalting efficiency, and the cation exchange resin can more effectively remove the cationic salt in the liquid, which is beneficial to improve the purity of the subsequent glucuron-lactone.

[0042] Further, in combination with the above embodiment, in the embodiment of the present invention, the desalted liquid is decolorized by the decolorizing resin under the condition of a column feed rate of 2-6 BV / h to obtain a decolorized liquid. Under the rate condition of 2-6 BV / h, the contact time between the desalted liquid and the decolorizing resin is increased, which is conducive to removing the pigment and organic impurities in the desalted liquid, and can shorten the processing time of the entire decolorization process, improve production efficiency, and accelerate the production cycle of glucuron-lactone.

[0043] Further, in combination with the above embodiments, in the embodiments of the present invention, the decolorized liquid is concentrated through a nanofiltration membrane with a pore size of 150 to 200Da to obtain a nanofiltration concentrate with a solid content of 15 to 20%. The pore size of the nanofiltration membrane used in the nanofiltration membrane assembly can be 150 to 200Da. Specifically, the pore size of the nanofiltration membrane can be selected as 150Da, 170Da, 190Da or 200Da, etc. The solid content of the nanofiltration concentrate can be 15-20%. Specifically, the solid content of the nanofiltration concentrate can be selected as 15%, 17%, 19% or 20%, etc. By selecting a suitable pore size of the nanofiltration membrane, nanofiltration concentration can remove the solvent and small molecular impurities in the solvent. This helps to purify the target product, reduce the loss of the target product, and improve the purity and yield of glucuron lactone.

[0044] Further, in combination with the above embodiments, in the embodiments of the present invention, the nanofiltration concentrate is further concentrated in a concentrator under the conditions of vacuum degree ≤-0.09Mpa and temperature 50-70°C to obtain a concentrate with a solid content of 50-70%. Specifically, the concentration temperature of the nanofiltration concentrate can be selected to be 50°C, 60°C, 65°C or 70°C, etc. Specifically, the solid content of the concentrate can be selected to be 50%, 60%, 65% or 70%, etc. At this vacuum degree and concentration temperature, it is easy to obtain a glucuronic acid solution with the above-mentioned specific solid content, the concentration energy consumption is low, the lactonization reaction equilibrium time is further shortened, and the foundation for the lactonization reaction is laid.

[0045] Further, in combination with the above embodiment, in the embodiment of the present invention, the lactonization reaction is also carried out under the condition of a stirring speed of 50-200rpm, and the reaction is carried out for 1-2h. Specifically, the stirring speed can be selected as 50rpm, 100rpm, 150rpm or 200rpm, etc. Specifically, the reaction time can be selected as 1h, 1.5h or 2h, etc. Under the conditions of this stirring speed and reaction time, uniform mixing between the reactants can be maintained, which is conducive to improving the rate and efficiency of the reaction, and by uniform stirring, effective contact between the reactants can be ensured, the reaction is promoted, and the yield of glucuron lactone is thereby improved.

[0046] Further, in combination with the above embodiments, in the embodiments of the present invention, the reaction product is crystallized by dynamic gradient cooling, cooled to 5-10°C at a rate of 1-5°C / h, the crystallization mixture is filtered for the second time, and then washed with 2-4°C cold water to obtain glucuron lactone crystals. Specifically, the cooling rate can be selected as 1°C / h, 3°C / h, 4°C / h or 5°C / h, etc. Specifically, the crystallization termination temperature can be selected as 5°C, 7°C, 9°C or 10°C, etc. Specifically, the cold water temperature can be selected as 2°C / h, 3°C / h or 4°C / h, etc. The second filtration adopts vacuum filtration, and the pore size of the filter cloth is 400 mesh to 1000 mesh. Specifically, the pore size of the filter cloth can be selected as 400 mesh, 500 mesh, 600 mesh 700 mesh, 800 mesh or 1000 mesh, etc. The glucuron lactone crystals obtained by dynamic gradient cooling crystallization have uniform particle size, are not easily wrapped with impurities, and are easily filtered to obtain the glucuron lactone product. At the same time, vacuum filtration and cold water washing can help reduce the residue in the glucuron lactone crystal particles, thereby making the crystals cleaner and improving the purity and yield of glucuron lactone.

[0047] In a second aspect, an embodiment of the present invention further provides a glucuron-lactone, which is prepared by the above-mentioned glucuron-lactone preparation method.

[0048] Compared with the prior art, and relative to the glucuronolactone prepared by the nitric acid oxidation method starting from starch, the glucuronolactone provided by the present invention has pure color, uniform particle size, higher yield and higher purity, and can meet the production needs of glucuronolactone.

[0049] The following examples are used to further illustrate the glucuron lactone and its preparation method provided by the present invention. The following examples are merely explanations of the present invention, rather than limitations. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative labor are within the scope of protection of the present invention. The test methods used in the following embodiments are conventional methods unless otherwise specified; the raw materials used in the following embodiments are commercially available raw materials unless otherwise specified, and the selection of physical properties of each raw material can refer to the following:

[0050] The cation exchange resin is preferably SQD-67;

[0051] The decolorizing resin is preferably LS-109D.

[0052] Example 1

[0053] This embodiment provides a method for preparing glucuronolactone. Specifically, it comprises the following steps:

[0054] S1. Prepare 60L of inositol solution with an inositol content of 80g / L, add whole cells containing inositol oxidase for transformation, and make the bacterial concentration of the system reach OD 600 =20, during the conversion process, a 27wt% sodium hydroxide solution was used to adjust the pH to 7.0, and the reaction was performed for 4h to obtain a conversion solution;

[0055] After adding concentrated phosphoric acid with a concentration of 81wt% to the above conversion liquid to adjust the pH to 5.0, the conversion liquid was filtered through a ceramic membrane with a pore size of 50nm at an operating temperature of 30°C, a membrane inlet pressure of 0.4MPa, a membrane outlet pressure of 0.2MPa, and a membrane flux of 60L / h, and the filtrate was collected. The filtrate was filtered through an ultrafiltration membrane with a pore size of 7000Da, the ultrafiltration membrane operating temperature was 30°C, the membrane inlet pressure was 0.4MPa, the membrane outlet pressure was 0.2MPa, and the membrane flux was 50L / h, and the ultrafiltration membrane clear liquid was collected, and the concentrated liquid was treated for environmentally friendly sewage discharge;

[0056] S2. Under the condition of column flow rate of 2BV / h, the above clear liquid is desalted by 15L cation exchange resin to obtain desalted liquid, wherein the conductivity of the desalted liquid is 4735us / cm;

[0057] S3, under the condition of column flow rate of 3BV / h, the above desalted liquid is decolorized by decolorizing resin, and the decolorized liquid is collected after decolorization;

[0058] S4, concentrating the above decolorized liquid on a nanofiltration membrane with a pore size of 200Da, the nanofiltration membrane operating temperature is 30°C, the membrane inlet pressure is 3.0MPa, the membrane outlet pressure is 2.5MPa, the membrane flux is 30L / h, and concentrating to obtain a nanofiltration concentrate with a solid content of 19%;

[0059] S5, concentrating the nanofiltration concentrate in a concentrator at a vacuum degree of -0.09 MPa and a temperature of 60° C. to obtain 7.5 L of a concentrate with a solid content of 65%;

[0060] S6. Under the condition of a stirring speed of 150 rpm, citric acid was added to the above concentrated solution, with a mass of 8 g of citric acid added to every 100 mL of concentrated solution, and a lactonization reaction was carried out. The reaction temperature was set to 65°C. After the reaction was carried out for 2 hours, dynamic gradient cooling crystallization was carried out at a rate of 4°C / h. The crystallization termination temperature was 7°C to obtain a crystalline mixture.

[0061] S7. The crystallization mixture was vacuum filtered with a filter cloth having a pore size of 500 mesh, and washed with 2° C. cold water to obtain 3.4 kg of crude glucuronolactone (white crystalline particles). The purity was 98.8% as determined by liquid chromatography, and the crystallization yield was 83.4%.

[0062] Example 2

[0063] This embodiment provides a method for preparing glucuronolactone. Specifically, it comprises the following steps:

[0064] S1. Prepare 60L of inositol solution with an inositol content of 120g / L, add whole cells containing inositol oxidase for transformation, and make the bacterial concentration of the system reach OD 600 =30, during the conversion process, a 29wt% sodium hydroxide solution was used to adjust the pH to 7.5, and the reaction was performed for 3h to obtain a conversion solution;

[0065] After adding concentrated phosphoric acid with a concentration of 83wt% to the above conversion liquid to adjust the pH to 4.5, the conversion liquid was filtered through a ceramic membrane with a pore size of 80nm at an operating temperature of 32°C, a membrane inlet pressure of 0.4MPa, a membrane outlet pressure of 0.2MPa, and a membrane flux of 65L / h, and the filtrate was collected. The filtrate was filtered through an ultrafiltration membrane with a pore size of 1000Da, the ultrafiltration membrane operating temperature was 32°C, the membrane inlet pressure was 0.6MPa, the membrane outlet pressure was 0.4MPa, and the membrane flux was 45L / h, and the ultrafiltration membrane clear liquid was collected, and the concentrated liquid was treated for environmentally friendly sewage discharge;

[0066] S2. Under the condition of column flow rate of 1 BV / h, the above clear liquid is desalted by 15L cation exchange resin to obtain desalted liquid, wherein the conductivity of the desalted liquid is 4500us / cm;

[0067] S3, under the condition of column flow rate of 4BV / h, the desalted liquid is decolorized by decolorizing resin, and the decolorized liquid is collected after decolorization;

[0068] S4, concentrating the above decolorized liquid on a nanofiltration membrane with a pore size of 150Da, the nanofiltration membrane operating temperature is 31°C, the membrane inlet pressure is 3.2MPa, the membrane outlet pressure is 2.8MPa, the membrane flux is 28L / h, and concentrating to obtain a nanofiltration concentrate with a solid content of 17%;

[0069] S5, concentrating the nanofiltration concentrate in a concentrator at a vacuum degree of -0.09 MPa and a temperature of 50° C. to obtain 12 L of a concentrate with a solid content of 60%;

[0070] S6. Under the condition of a stirring speed of 60 rpm, citric acid was added to the above-mentioned concentrated solution, with a mass of 7 g of citric acid added to every 100 mL of concentrated solution, and a lactonization reaction was carried out. The reaction temperature was set to 50°C. After the reaction lasted for 1 hour, dynamic gradient cooling crystallization was carried out at a rate of 1°C / h. The crystallization termination temperature was 9°C to obtain a crystalline mixture.

[0071] S7. The crystallization mixture was vacuum filtered with a filter cloth having a pore size of 600 mesh, and washed with 3° C. cold water to obtain 5.28 kg of crude glucuronolactone (white crystalline particles). The purity was 98.9% as determined by liquid chromatography, and the crystallization yield was 84.1%.

[0072] Example 3

[0073] This embodiment provides a method for preparing glucuronolactone. Specifically, it comprises the following steps:

[0074] S1. Prepare 60L of inositol solution with an inositol content of 50g / L, add whole cells containing inositol oxidase for transformation, and make the bacterial concentration of the system reach OD 600 =40, during the conversion process, a 25wt% sodium hydroxide solution was used to adjust the pH to 8.0, and the reaction was performed for 5h to obtain a conversion solution;

[0075] After adding concentrated phosphoric acid with a concentration of 80wt% to the above conversion liquid to adjust the pH to 4.0, the conversion liquid was filtered through a ceramic membrane with a pore size of 100nm, at an operating temperature of 28°C, a membrane inlet pressure of 0.35MPa, a membrane outlet pressure of 0.2MPa, and a membrane flux of 58L / h, and the filtrate was collected. The filtrate was filtered through an ultrafiltration membrane with a pore size of 10000Da, the ultrafiltration membrane operating temperature was 28°C, the membrane inlet pressure was 0.4MPa, the membrane outlet pressure was 0.25MPa, and the membrane flux was 46L / h, and the ultrafiltration membrane clear liquid was collected, and the concentrated liquid was treated for environmental protection sewage discharge;

[0076] S2. Under the condition of column flow rate of 1.5 BV / h, the above clear liquid is desalted by 15 L cation exchange resin to obtain desalted liquid, wherein the conductivity of the desalted liquid is 5100 us / cm;

[0077] S3, under the condition of column flow rate of 6BV / h, the above desalted liquid is decolorized by decolorizing resin, and the decolorized liquid is collected after decolorization;

[0078] S4, concentrating the above decolorized liquid on a nanofiltration membrane with a pore size of 190Da, the nanofiltration membrane operating temperature is 28°C, the membrane inlet pressure is 3.5MPa, the membrane outlet pressure is 3.0MPa, the membrane flux is 26L / h, and concentrating to obtain a nanofiltration concentrate with a solid content of 20%;

[0079] S5, concentrating the nanofiltration concentrate in a concentrator at a vacuum degree of -0.08 MPa and a temperature of 70° C. to obtain 4.3 L of a concentrate with a solid content of 70%;

[0080] S6. Under the condition of a stirring speed of 190 rpm, citric acid was added to the above-mentioned concentrated solution, with a mass of 9 g of citric acid added to every 100 mL of concentrated solution, and a lactonization reaction was carried out. The reaction temperature was set to 60°C. After the reaction was carried out for 1.5 hours, dynamic gradient cooling crystallization was carried out at a rate of 1°C / h, and the crystallization termination temperature was 5°C to obtain a crystalline mixture.

[0081] S7. The crystallization mixture was vacuum filtered with a filter cloth having a pore size of 1000 mesh, and washed with 4° C. cold water to obtain 2.2 kg of crude glucuronolactone (white crystalline particles). The purity was 99.1% as determined by liquid chromatography, and the crystallization yield was 85.2%.

[0082] Example 4

[0083] This embodiment provides a method for preparing glucuronolactone. Specifically, it comprises the following steps:

[0084] S1. Prepare 60L of inositol solution with an inositol content of 150g / L, add whole cells containing inositol oxidase for transformation, and make the bacterial concentration of the system reach OD 600 =10, during the conversion process, a 30wt% sodium hydroxide solution was used to adjust the pH to 8.0, and the reaction was performed for 6h to obtain a conversion solution;

[0085] After adding concentrated phosphoric acid with a concentration of 85wt% to the above conversion liquid to adjust the pH to 4.0, the mixture was filtered through a ceramic membrane with a pore size of 20nm at an operating temperature of 35°C, a membrane inlet pressure of 0.6MPa, a membrane outlet pressure of 0.4MPa, and a membrane flux of 50L / h, and the filtrate was collected. The filtrate was filtered through an ultrafiltration membrane with a pore size of 4000Da, the ultrafiltration membrane operating temperature was 35°C, the membrane inlet pressure was 0.6MPa, the membrane outlet pressure was 0.4MPa, and the membrane flux was 48L / h, and the ultrafiltration membrane clear liquid was collected, and the concentrated liquid was treated for environmentally friendly sewage discharge;

[0086] S2. Under the condition of column flow rate of 2BV / h, the above clear liquid was desalted by 15L cation exchange resin SQD-67 to obtain desalted liquid, wherein the conductivity of the desalted liquid was 5140us / cm;

[0087] S3, under the condition of column flow rate of 4BV / h, the above desalted liquid is decolorized by decolorizing resin LS-109D, and the decolorized liquid is collected after decolorization;

[0088] S4, concentrating the above decolorized liquid on a nanofiltration membrane with a pore size of 150Da, the nanofiltration membrane operating temperature is 35°C, the membrane inlet pressure is 3.8MPa, the membrane outlet pressure is 3.3MPa, the membrane flux is 33L / h, and concentrating to obtain a nanofiltration concentrate with a solid content of 15%;

[0089] S5, concentrating the nanofiltration concentrate in a concentrator at a vacuum degree of -0.07 MPa and a temperature of 65° C. to obtain 18 L of a concentrate with a solid content of 50%;

[0090] S6. Under the condition of a stirring speed of 150 rpm, citric acid was added to the above concentrated solution, with a mass of 5 g of citric acid added to every 100 mL of concentrated solution, and a lactonization reaction was carried out. The reaction temperature was set to 70°C. After the reaction was carried out for 2 hours, dynamic gradient cooling crystallization was carried out at a rate of 2°C / h, and the crystallization termination temperature was 10°C to obtain a crystalline mixture.

[0091] S7. The crystallization mixture was vacuum filtered with a filter cloth having a pore size of 400 mesh, and washed with 2° C. cold water to obtain 6770 kg of crude glucuronolactone (white crystalline particles). The purity was 99.0% as detected by liquid chromatography, and the crystallization yield was 84.2%.

[0092] Example 5

[0093] This embodiment provides a method for preparing glucuronolactone. Specifically, it comprises the following steps:

[0094] S1. Prepare 60L of inositol solution with an inositol content of 10g / L, add whole cells containing inositol oxidase for transformation, and make the bacterial concentration of the system reach OD 600 =50, during the conversion process, a 25wt% sodium hydroxide solution was used to adjust the pH to 8.0, and the reaction was performed for 2h to obtain a conversion solution;

[0095] After adding concentrated phosphoric acid with a concentration of 80wt% to the above conversion liquid to adjust the pH to 4.5, centrifuge separation is performed at a speed of 4000rpm, and the filtrate is collected. The filtrate is filtered through an ultrafiltration membrane with a pore size of 10000Da, the ultrafiltration membrane operating temperature is 30°C, the membrane inlet pressure is 0.4MPa, the membrane outlet pressure is 0.3MPa, and the membrane flux is 40L / h. The ultrafiltration membrane clear liquid is collected, and the concentrated liquid is treated for environmental protection sewage discharge;

[0096] S2. Under the condition of column flow rate of 2BV / h, the above clear liquid is desalted by 15L cation exchange resin to obtain desalted liquid, wherein the conductivity of the desalted liquid is 5170us / cm;

[0097] S3, under the condition of column flow rate of 5BV / h, the desalted liquid is decolorized by decolorizing resin, and the decolorized liquid is collected after decolorization;

[0098] S4, concentrating the decolorized liquid on a nanofiltration membrane with a pore size of 170Da, the nanofiltration membrane operating temperature is 31°C, the membrane inlet pressure is 3.2MPa, the membrane outlet pressure is 2.8MPa, the membrane flux is 32L / h, and concentrating to obtain a nanofiltration concentrate with a solid content of 20%;

[0099] S5, concentrating the nanofiltration concentrate in a concentrator at a vacuum degree of -0.08 MPa and a temperature of 70° C. to obtain 1 L of a concentrate with a solid content of 70%;

[0100] S6. Under the condition of a stirring speed of 190 rpm, citric acid was added to the above-mentioned concentrated solution, with a mass of 10 g of citric acid added to every 100 mL of concentrated solution, and a lactonization reaction was carried out. The reaction temperature was set to 65°C. After the reaction was carried out for 1.5 hours, dynamic gradient cooling crystallization was carried out at a rate of 1°C / h, and the crystallization termination temperature was 5°C to obtain a crystalline mixture.

[0101] S7. The crystallization mixture was vacuum filtered with a filter cloth having a pore size of 700 mesh, and washed with 4° C. cold water to obtain 0.46 kg of crude glucuronolactone (white crystalline particles). The purity was 99.2% as determined by liquid chromatography, and the crystallization yield was 85.4%.

[0102] Example 6

[0103] This embodiment provides a method for preparing glucuronolactone. Specifically, it comprises the following steps:

[0104] S1. Prepare 60L of inositol solution with an inositol content of 80g / L, add whole cells containing inositol oxidase for transformation, and make the bacterial concentration of the system reach OD 600 =30, during the conversion process, a 27wt% sodium hydroxide solution was used to adjust the pH to 7.0, and the reaction was performed for 2h to obtain a conversion solution;

[0105] After adding 80wt% concentrated phosphoric acid to the above conversion liquid to adjust the pH to 4.0, the solution was filtered through a plate and frame with a pore size of 1500 mesh, and the filtrate was collected. The filtrate was filtered through an ultrafiltration membrane with a pore size of 10000Da. The ultrafiltration membrane operating temperature was 30°C, the membrane inlet pressure was 0.4MPa, the membrane outlet pressure was 0.2MPa, and the membrane flux was 48L / h. The ultrafiltration membrane clear liquid was collected, and the concentrated liquid was treated for environmental protection sewage discharge;

[0106] S2. Under the condition of column flow rate of 1 BV / h, the above clear liquid is desalted by 15L cation exchange resin to obtain desalted liquid, wherein the conductivity of the desalted liquid is 4780us / cm;

[0107] S3, under the condition of column flow rate of 4BV / h, the desalted liquid is decolorized by decolorizing resin, and the decolorized liquid is collected after decolorization;

[0108] S4, concentrating the above decolorized liquid on a nanofiltration membrane with a pore size of 150Da, the nanofiltration membrane operating temperature is 32°C, the membrane inlet pressure is 3.4MPa, the membrane outlet pressure is 3.0MPa, the membrane flux is 29L / h, and concentrating to obtain a nanofiltration concentrate with a solid content of 15%;

[0109] S5, concentrating the nanofiltration concentrate in a concentrator at a vacuum degree of -0.09 MPa and a temperature of 70° C. to obtain 9.5 L of a concentrate with a solid content of 50%;

[0110] S6. Under the condition of a stirring speed of 190 rpm, citric acid was added to the above-mentioned concentrated solution, with a mass of 6 g of citric acid added to every 100 mL of concentrated solution, and a lactonization reaction was carried out. The reaction temperature was set to 60°C. After the reaction lasted for 1 hour, dynamic gradient cooling crystallization was carried out at a rate of 1°C / h, and the crystallization termination temperature was 10°C to obtain a crystalline mixture.

[0111] S7. The crystallization mixture was vacuum filtered with a filter cloth having a pore size of 800 mesh, and washed with 2° C. cold water to obtain 3425 kg of crude glucuronolactone (white crystalline particles). The purity was 98.9% as detected by liquid chromatography, and the crystallization yield was 84.1%.

[0112] The present invention is further described below by using comparative examples.

[0113] Comparative Example 1

[0114] The difference between Comparative Example 1 and Example 1 of the present invention is that in step S6 of preparing glucuronolactone, citric acid is replaced by acetic acid in equal amounts, and other conditions remain unchanged. The purity detected by liquid chromatography is 86.7%, and the crystallization yield is 58.4%.

[0115] It is speculated that the reason may be that during the lactonization reaction and vacuum concentration process, due to the volatile nature of acetic acid, a large amount of acetic acid vapor is produced, causing serious corrosion to the equipment. At the same time, after the acetic acid evaporates, the amount of effective reactants is reduced. If acetic acid is not supplemented, the yield of the product is greatly reduced.

[0116] Comparative Example 2

[0117] The difference between Comparative Example 2 and Example 1 of the present invention is that in the step S1 of preparing glucuron-lactone, the 27wt% sodium hydroxide solution is replaced with 30wt% ammonia water to adjust the pH to 7.0, and other conditions remain unchanged. The purity detected by liquid chromatography is 89.7%, and the crystallization yield is 64.3%.

[0118] The speculated reason may be that after the ammonia water adjusts the pH, residual ammonia or ammonium ions are left in the conversion liquid and cannot be effectively removed. In addition, high-concentration ammonia water may cause corrosion to the equipment, thereby affecting the purity of the product.

[0119] Comparative Example 3

[0120] The difference between Comparative Example 3 and Example 1 of the present invention is that in the production step S1 of glucuron-lactone, the concentrated phosphoric acid with a concentration of 81wt% is replaced by hydrochloric acid with a concentration of 35wt% to adjust the pH to 5.0, and other conditions remain unchanged. The purity detected by liquid chromatography is 78.6%, and the crystallization yield is 59.2%.

[0121] It is speculated that the reason may be that after replacing with hydrochloric acid, the chloride ions present in the solution, on the one hand, have no promoting effect on subsequent crystallization, resulting in a decrease in the crystallization yield; on the other hand, the chloride ions, as impurities, cannot be effectively removed in the subsequent process and still exist in the crude extract of glucuronolactone until after crystallization, reducing the purity of the product and causing obvious color differences.

[0122] Comparative Example 4

[0123] The difference between Comparative Example 4 and Example 1 of the present invention is that in the preparation step S1 of glucuron-lactone, the pH is adjusted to 3.0 with concentrated phosphoric acid having a concentration of 81 wt %, and other conditions remain unchanged. The purity detected by liquid chromatography is 76.6%, and the crystallization yield is 61.4%.

[0124] The reason may be that the pH value is too low. The increase in the amount of acid used will increase the phosphate ions introduced into the glucuronic acid solution, and then increase the impurities in the generated glucuronolactone. In addition, too low a pH value may cause corrosion to the equipment, which will affect the product purity and reduce the yield of the product.

[0125] Comparative Example 5

[0126] The difference between Comparative Example 5 and Example 1 of the present invention is that in the preparation step S1 of glucuron-lactone, the pH is adjusted to 6.0 with concentrated phosphoric acid having a concentration of 81 wt %, and other conditions remain unchanged. The purity detected by liquid chromatography is 79.8%, and the crystallization yield is 69.3%.

[0127] The reason may be that the pH value is too high, which affects protein aggregation and leads to incomplete protein removal, making it difficult to separate or lost during the filtration process, reducing the purity and yield of the product.

[0128] Further, the liquid chromatography test conditions in Examples 1 to 6 of the present invention and Comparative Examples 1 to 5 can be referred to as follows:

[0129] Mobile phase: 10mmol / L formic acid aqueous solution;

[0130] Chromatographic column: calcium column (300*7.7 or similar column);

[0131] Flow rate: 0.5Ml / min;

[0132] Detector: differential detector;

[0133] Column temperature: 55°C;

[0134] Detector temperature: 45°C;

[0135] Solvent: 10mmol / L formic acid aqueous solution;

[0136] Standard concentration: 1.0 mg / mL (D-glucuronolactone [CAS No.] 32449-92-6, 99.9% in terms of C6H8O6, China Food and Drug Inspection Institutes);

[0137] Test sample concentration: 1.0 mg / mL;

[0138] Chromatographic conditions: run with 100% 10 mmol / L formic acid aqueous solution for 30 min.

[0139] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been 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 glucuronolactone, characterized in that: The steps include: Adding whole cells containing inositol oxidase to the inositol feed solution for conversion, adjusting the pH to 7.0-8.0 with sodium hydroxide solution during the conversion process, and obtaining a conversion solution; The conversion solution is adjusted to pH 4.0-5.0 with concentrated phosphoric acid, and then filtered for the first time to collect the clear liquid; The clear liquid is desalted, decolorized, and concentrated to a solid content of 50-70% to obtain a concentrated liquid; 5-10 g of citric acid is added to every 100 mL of the concentrated solution, and a lactonization reaction is carried out at 50-70° C. The reaction product is cooled, filtered for a second time, and washed to obtain glucuron lactone.

2. The method for preparing glucuronolactone according to claim 1, characterized in that: The inositol content in the inositol solution is 10-150 g / L; the whole cell addition amount is system OD600=10-50; and the conversion time is 2-6 h.

3. The method for preparing glucuronolactone according to claim 1 or 2, characterized in that: The concentration of the sodium hydroxide solution is 25-30 wt %, And / or, the concentration of the concentrated phosphoric acid is 80-85wt%.

4. The method for preparing glucuronolactone according to any one of claims 1 to 3, characterized in that: The first filtering includes: The conversion liquid after adjusting the pH value is filtered through a ceramic membrane with a pore size of 20-100nm, a centrifuge or a plate and frame, and the filtrate is filtered through an ultrafiltration membrane with a pore size of 1000-10000Da to collect the clear liquid.

5. The method for preparing glucuronolactone according to any one of claims 1 to 4, characterized in that: The desalination comprises: The clear liquid is desalted by a cation exchange resin at a column flow rate of 1-2 BV / h to obtain a desalted liquid.

6. The method for preparing glucuronolactone according to claim 5, characterized in that: The decolorization comprises: The desalted liquid is decolorized by a decolorizing resin under the condition of a column flow rate of 2-6 BV / h to obtain a decolorized liquid.

7. The method for preparing glucuronolactone according to any one of claims 1 to 6, characterized in that: The concentration includes: The decolorized liquid is concentrated through a nanofiltration membrane with a pore size of 150 to 200 Da to obtain a nanofiltration concentrate with a solid content of 15 to 20%; The nanofiltration concentrated liquid is further concentrated in a concentrator under the conditions of vacuum degree ≤-0.09Mpa and temperature 50-70°C to obtain a concentrated liquid with a solid content of 50-70%.

8. The method for preparing glucuronolactone according to claim 1, characterized in that: The lactonization reaction conditions also include: reacting for 1-2 hours at a stirring speed of 50-200 rpm to carry out the lactonization reaction.

9. The method for preparing glucuronolactone according to claim 1, characterized in that: The reaction product is cooled, filtered for the second time, and washed to obtain glucuron lactone, which includes: the reaction product is cooled to 5-10°C at a rate of 1-5°C / h, filtered for the second time, and washed with 2-4°C cold water to obtain glucuron lactone crystals.

10. A glucuronolactone, characterized in that The glucuronolactone is prepared by the method for preparing glucuronolactone according to any one of claims 1 to 9.