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CN-122011056-A - Separation preparation method of D-allose

CN122011056ACN 122011056 ACN122011056 ACN 122011056ACN-122011056-A

Abstract

The invention discloses a separation preparation method of D-allose, which relates to the technical field of D-allose production, wherein whole cell wet thalli containing recombinant ribose-5-phosphate isomerase are added into a D-allose solution for conversion, the collected conversion solution is sequentially treated by a ceramic membrane, an ultrafiltration membrane, a cation resin column and an anion resin column, desalted solution is collected, the desalted solution is concentrated to a solid content of 70-90% w/w, concentrated solution is collected, ethanol is added into the concentrated solution, the temperature is kept for 1-2h at 50-60 ℃, then the temperature is reduced to 22-28 ℃, and filter cakes are obtained after filtration, thus obtaining a D-allose crude product. The equipment investment is greatly reduced, and the industrialization threshold is greatly reduced. Meanwhile, the high-efficiency separation of the D-psicose and the D-psicose is realized by precisely controlling the proportion of ethanol and water and the crystallization condition.

Inventors

  • ZHU LIPING
  • XU LIANGPING
  • Huai Jianlu
  • SONG WENZHU
  • Qiu Chongshun

Assignees

  • 诸城市浩天药业有限公司

Dates

Publication Date
20260512
Application Date
20260127

Claims (10)

  1. 1. The separation and preparation method of the D-allose is characterized by comprising the following steps of: D-psicose solution is added with whole cell wet thalli containing recombinant ribose-5-phosphate isomerase for conversion, and the conversion liquid is collected; the conversion solution is treated by a ceramic membrane, an ultrafiltration membrane, a cation resin column and an anion resin column in sequence, and a desalting solution is collected; concentrating the desalted liquid to solid content of 70-90% w/w, and collecting the concentrated liquid; And D, adding ethanol into the concentrated solution, preserving heat for 1-2 hours at 50-60 ℃, then cooling to 22-28 ℃, and filtering to obtain a filter cake which is the crude product of D-allose.
  2. 2. The method for separating and preparing D-allose according to claim 1, wherein the conversion system in the step A further comprises a manganese chloride solution and a phosphate buffer solution, the conversion temperature is 60-80 ℃, and the conversion time is 4-6h; The concentration of the D-psicose solution is 600-700g/L, the addition amount of the whole cell wet thalli containing the recombinant ribose-5-phosphate isomerase is 50-100g/L, the addition amount of the manganese chloride solution is 0.5-1mM, the addition amount of the phosphate buffer solution is 20-50mM, and the pH value of the phosphate buffer solution is 7-8.
  3. 3. The method for separating and preparing D-allose as claimed in claim 1, wherein the ceramic membrane in the step B has a pore diameter of 20-100nm and an ultrafiltration membrane interception molecular weight of 2000-10000Da.
  4. 4. The method for preparing D-allose according to claim 1, wherein the flow rates of the cation resin column and the anion resin column in the step B are 1-3BV, and the electric conductance of the desalted liquid is less than 50 mu s/cm; Before the cationic resin column is used, the cationic resin column is soaked in 4-5 wt% hydrochloric acid solution for 1-2h, then is washed with deionized water until the cationic resin column is neutral for standby, and before the anionic resin column is used, the cationic resin column is soaked in 4-5 wt% sodium hydroxide solution for 1-2h, and then is washed with deionized water until the cationic resin column is neutral for standby.
  5. 5. The method for separating and preparing D-allose as claimed in claim 1, wherein the concentration temperature in the step C is 50-70 ℃ and the vacuum degree is < -0.09MPa.
  6. 6. The process for the isolation of D-allose according to claim 1, wherein the ethanol is added in an amount of 1 to 1.5 times the volume of the concentrate in step D.
  7. 7. The process for the isolated preparation of D-allose according to claim 1, wherein the crude D-allose product of step D has a D-allose content of >95%.
  8. 8. The method for separating and preparing D-allose as claimed in claim 1, wherein the crude D-allose in the step D is decolorized after being dissolved in purified water, ethanol is added into the decolorized solution, the temperature is reduced to 10-15 ℃, and the D-allose is obtained by suction filtration, ethanol washing and drying.
  9. 9. The method for separating and preparing D-allose as claimed in claim 8, wherein the purified water is added in an amount of 0.5-1 time by weight of the crude D-allose, the dissolution temperature is 60-70 ℃, the addition amount of the activated carbon during decolorization is 1-3% by weight of the crude D-allose, and the decolorization time is 30-60min.
  10. 10. The method for preparing D-allose according to claim 8, wherein the ethanol is added in an amount of 1-1.5 times the volume of the decolorized solution, and the content of D-allose in the obtained D-allose product is more than 99%.

Description

Separation preparation method of D-allose Technical Field The invention relates to the technical field of D-allose production, in particular to a separation preparation method of D-allose. Background D-allose is a rare sugar, has potential biological activity and application value, and is particularly widely focused in the fields of medicines, foods and functional health products. At present, the preparation of D-allose mainly depends on a biocatalysis method, wherein the allose is used as a substrate, and the D-allose is generated through ribose-5-phosphate isomerase catalytic conversion. However, the existing production process faces the problem of high separation and purification cost, the traditional method generally depends on chromatographic separation technology (such as simulated moving bed chromatography), and has the advantages of high equipment investment, complex operation, high solvent consumption, difficulty in realizing continuous and large-scale production, and high production cost of D-allose. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides the separation preparation method of the D-allose, which has good separation effect and low production cost. In order to solve the technical problems, the technical scheme of the invention is as follows: the separation and preparation method of the D-allose comprises the following steps: D-psicose solution is added with whole cell wet thalli containing recombinant ribose-5-phosphate isomerase for conversion, and the conversion liquid is collected; the conversion solution is treated by a ceramic membrane, an ultrafiltration membrane, a cation resin column and an anion resin column in sequence, and a desalting solution is collected; concentrating the desalted liquid to solid content of 70-90% w/w, and collecting the concentrated liquid; And D, adding ethanol into the concentrated solution, preserving heat for 1-2 hours at 50-60 ℃, then cooling to 22-28 ℃, and filtering to obtain a filter cake which is the crude product of D-allose. Preferably, the conversion system in the step A also comprises a manganese chloride solution and a phosphate buffer solution, the conversion temperature is 60-80 ℃, and the conversion time is 4-6h; The concentration of the D-psicose solution is 600-700g/L, the addition amount of the whole cell wet thalli containing the recombinant ribose-5-phosphate isomerase is 50-100g/L, the addition amount of the manganese chloride solution is 0.5-1mM, the addition amount of the phosphate buffer solution is 20-50mM, and the pH value of the phosphate buffer solution is 7-8. Preferably, the pore diameter of the ceramic membrane in the step B is 20-100nm, and the molecular weight cut-off of the ultrafiltration membrane is 2000-10000Da. Preferably, the flow rate of the cation resin column and the anion resin column in the step B is 1-3BV, and the electric conductivity of the desalted liquid is less than 50 mu s/cm; Before the cationic resin column is used, the cationic resin column is soaked in 4-5 wt% hydrochloric acid solution for 1-2h, then is washed with deionized water until the cationic resin column is neutral for standby, and before the anionic resin column is used, the cationic resin column is soaked in 4-5 wt% sodium hydroxide solution for 1-2h, and then is washed with deionized water until the cationic resin column is neutral for standby. Preferably, the concentration temperature in the step C is 50-70 ℃ and the vacuum degree is < -0.09MPa. Preferably, the ethanol is added in step D in an amount of 1 to 1.5 times the volume of the concentrate. Preferably, the D-allose content of the crude D-allose of step D is >95%. Preferably, the D-allose crude product in the step D is decolorized after being added with purified water for dissolution, ethanol is added into the decolorized solution, the temperature is reduced to 10-15 ℃, suction filtration and ethanol washing are carried out, and the D-allose finished product is obtained after drying. Preferably, the addition amount of the purified water is 0.5-1 time of the weight of the crude D-allose, the dissolution temperature is 60-70 ℃, the addition amount of the activated carbon during decolorization is 1-3% of the weight of the crude D-allose, and the decolorization time is 30-60min. Preferably, the ethanol is added in an amount which is 1 to 1.5 times the volume of the decolorized solution, and the content of D-allose in the obtained D-allose finished product is more than 99 percent. Due to the adoption of the technical scheme, the invention has the beneficial effects that: The solubility of D-allose in an ethanol-water system is smaller than that of D-allose, and the difference is largest at 25 ℃, so that the invention adopts a method of 'ethanol-water system selective crystallization separation' instead of chromatographic separation commonly relied on in the industry. The chromatographic equipment has high price, high operation energy consumpt