A four-step method for the isolation and purification of glyceroglycosides

Abstract

The application discloses a method for separating and purifying glycerol glucoside. The method comprises the following steps: (1) sequentially performing microfiltration, ultrafiltration and nanofiltration on initial products of glycerol glucoside prepared by an enzymatic method, removing impurities in the initial products, and obtaining a filtration product with fructose and glycerol glucoside as main substances; and (2) separating the filtration product obtained in the step (1) by using a chromatographic separation method, separating the glycerol glucoside from the fructose, and collecting the separated and purified glycerol glucoside. The method is simple in operation and high in purity of the glycerol glucoside.

Description

Technical Field

[0001] This invention belongs to the field of separation and purification, specifically relating to a method for separating and purifying glycerol glucoside synthesized by enzymatic method. Background Technology

[0002] Glyceryl glucoside (GG) is a class of glycoside compounds formed by the linkage of glycerol and glucose molecules through glycosidic bonds. Due to its strong moisturizing, water-locking, and hydrating physiological effects, as well as its excellent effects in preventing tooth decay, inhibiting the accumulation of neutral lipids in adipocytes, and anti-allergic properties, glyceryl glucoside has significant application value and broad market prospects in the cosmetics, pharmaceutical, and food industries.

[0003] The enzymatic production process for synthesizing glycerol glucoside using sucrose phosphorylase as a substrate is green, efficient, and highly specific. The final reaction solution contains clearly defined components, mainly the enzyme catalyst, glycerol, glucoside (GG), and fructose, making it one of the main processes for GG production. This process requires the separation and purification of GG from the reaction solution. Currently, the most mature separation and purification technology for enzymatic GG production was developed by Bitop GmbH in Germany at the beginning of this century. This technology relies on immobilized enzymes to remove the enzyme catalyst by centrifugation after the reaction, followed by purification of GG using a fractional ethanol gradient elution with activated carbon and diatomaceous earth, achieving a high purity with a GG yield of 56%. Immobilized enzymes have relatively high production costs. Current mainstream research and development trends favor the synthesis of GG using whole-cell enzyme catalysts, but the corresponding downstream GG separation and purification processes are still in the exploratory stage, and no separation and purification process suitable for industrial-scale production has been developed.

[0004] Currently, there are two problems in separation and purification: (1) how to efficiently remove enzyme catalysts; (2) how to efficiently separate fructose and GG, which have very similar physicochemical properties, while ensuring a high recovery rate of GG. Summary of the Invention

[0005] The purpose of this invention is to provide a separation and purification process for separating and purifying glycerol glucoside from the enzymatic GG synthesis reaction solution.

[0006] The method for isolating and purifying glycerol glucoside of the present invention includes:

[0007] (1) The initial product of the enzymatic preparation of glycerol glucoside was filtered to remove impurities such as enzyme catalyst, glycerol and protein, and the filtered product was obtained with fructose and glycerol glucoside as the main substances.

[0008] (2) Separate the filtered product obtained in step (1) by chromatographic separation to separate glycerol glucoside from fructose, and collect the separated and purified glycerol glucoside.

[0009] In the method of the present invention, the filtration process includes microfiltration (or denaturing the proteins in the initial product and then centrifuging or filtering with filter paper to collect the filtrate), ultrafiltration, and nanofiltration.

[0010] In the method of this invention, microfiltration involves passing the initial product through a membrane and collecting the permeate. Ultrafiltration involves passing the microfiltration permeate through a membrane and collecting the permeate. Nanofiltration involves passing the ultrafiltration permeate through a membrane and collecting the retained product.

[0011] In the method of the present invention, the microfiltration membrane is a hollow fiber membrane or a ceramic membrane.

[0012] In the method of the present invention, the pore size of the microfiltration membrane is 0.1 μm or the molecular weight cutoff is 100 KD.

[0013] In the method of this invention, the membrane used for ultrafiltration has a molecular weight cutoff of 600-2500 Da.

[0014] In the method of this invention, the nanofiltration membrane has a molecular weight cutoff of 150-300 Da.

[0015] The viscosity of the feed solution used for microfiltration is 4 mPa.s-550 mPa.s, specifically 550 mPa.s, 440 mPa.s, 250 mPa.s, 150 mPa.s, 50 mPa.s, 12 mPa.s or 4 mPa.s.

[0016] Enzymatic preparation of glycerol glucosides refers to the use of sucrose phosphorylase to catalyze the reaction of glycerol and sucrose to produce glycerol glucosides.

[0017] The sucrose phosphorylase used in the enzymatic preparation of glycerol glucoside can be immobilized or non-immobilized. It can be pure sucrose phosphorylase or unpurified sucrose phosphorylase. It can also be recombinant cells expressing sucrose phosphorylase, lysate of recombinant cells expressing sucrose phosphorylase, or fermentation broth of recombinant cells expressing sucrose phosphorylase.

[0018] The reaction system for the enzymatic preparation of glycerol glucoside can be buffered or water-free.

[0019] In the method of the present invention, the packing material used in the chromatographic separation method is a strong acid cation exchange resin.

[0020] In the method of the present invention, the cation of the strong acid cation exchange resin is calcium or sodium.

[0021] In the method of this invention, the basic material of the strong acid cation exchange resin is, for example, styrene and divinylbenzene polymerized together.

[0022] In the method of the present invention, the strongly acidic reactive group in the strongly acidic cation exchange resin is a sulfonic acid group.

[0023] In the method of the present invention, the particle size of the filler is 0.20mm-0.35mm, preferably 0.25mm-0.30mm.

[0024] In the method of this invention, the mobile phase is water.

[0025] In the method of this invention, the chromatographic separation method is simulated moving bed chromatography (SMB).

[0026] In the method of this invention, the simulated moving bed can take any form disclosed in the prior art, such as sequential, four-zone, etc. The simulated moving bed can have any number of columns, such as 6, 8, or 12.

[0027] In the method of the present invention, the mass percentage concentration of solute in the feed solution during chromatographic separation is 10%-50%, specifically 10%, 20%, 30%, 40% or 50%.

[0028] In the method of this invention, when a sequential simulated moving bed is used, the flow rate of the injection liquid is 0.007 BV / min, and the flow rate of the mobile phase is 0.01 BV / min;

[0029] When using a four-zone simulated moving bed, the flow rate of the injection solution is 0.001 BV / min, the flow rate of the mobile phase is 0.01 BV / min, the flow rate of the extract is 0.001 BV / min, the flow rate of the raffinate is 0.002 BV / min, and the valve switching time is 8 min.

[0030] The permeate in the nanofiltration step is mainly glycerol, which can be recovered. The simulated moving bed step separates glycerol from fructose, allowing for the separate recovery of glycerol and fructose.

[0031] The separation and purification process of this invention requires only four steps and can efficiently remove impurities such as enzyme catalysts and proteins, separately purifying and harvesting glycerol, fructose, and GG. This method uses water throughout, eliminating the need for ion removal steps. The method is simple to operate and effectively separates and purifies GG produced by enzymatic synthesis scale-up, achieving high GG purity and high recovery rate. This method is safer and more environmentally friendly than the Bitop process. Attached Figure Description

[0032] Figure 1 This is the HPLC chromatogram of the ultrafiltration permeate.

[0033] Figure 2 This is the HPLC chromatogram of the nanofiltration concentrate.

[0034] Figure 3 HPLC chromatogram for purifying GG using a simulated moving bed. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Experimental methods

[0038] Protein detection: Protein concentration was determined using the Bradford Protein Assay Kit (Beyotime, P0006) according to the standard method listed in the kit instructions. This assay serves as an indicator of the removal rate of protein molecules, such as enzyme catalysts, from the reaction solution / sample. The standard curve for protein concentration was: Y (mg / ml) = 1.7566 × (A595nm) 样品 -A595nm 空白对照 -0.0364, R 2 =0.9953.

[0039] Bacterial count: The total bacterial count (CFU / ml) of the sample was determined according to the standard test method (total bacterial count test method) listed in the "2015 Cosmetic Safety Technical Specifications" as an indicator of the degree of separation and removal of bacterial cell catalyst in the reaction solution / sample.

[0040] Viscosity measurement: SNB-1 digital viscometer. 18℃, sample volume: 100ml, rotor number sp1, rotor speed 60±3.5%.

[0041] Detection of glucose, glycerol, fructose and sucrose:

[0042] HPLC detection conditions: Column: Shodex Asahipak NH2P (4.6×250mm); Column temperature: 30℃; Mobile phase: 80% acetonitrile; Flow rate: 1ml / min; Injection volume: 10μl; Run time: 16 minutes; Detector: RID differential detector.

[0043] GG standard (Qingdao Zhongke Lanzhi Biotechnology Development Co., Ltd., Spirulina GG, CAS: 22160-26-5) Peak elution time: 8.65 min, GG standard curve equation: Y(Area)=89393.96X(g / L)-464.39597, R 2=0.99546; Glycerin standard (Sinopharm Group, product number 10010618) peak elution time: 4.94 min, glycerin standard curve equation: Y(Area) = 80755.0103X(g / L) + 1868.00912, R 2 =0.99585; Fructose standard (Macklin, catalog number D809612, CAS: 57-48-7) peak time: 7.67 min, fructose standard curve equation: Y(Area) = 90397.00182X(g / L) + 1650.69614, R 2 =0.99536; Peak elution time of sucrose standard (Sinopharm Group, product number 10021463): 14.03 min; Equation of sucrose standard curve: Y(Area) = 95374.45721X(g / L) + 1288.14650, R 2 =0.99608; the elution times of GG, glycerol, fructose and sucrose in the reaction solution were consistent with those of the standard.

[0044] The simulated moving bed equipment consists of components such as an injection pump, elution pump, chromatographic column, solenoid valve, signal acquisition unit, and system controller computer. Operating pressure: 2-10 MPa; operating temperature: room temperature to 65℃.

[0045] Example 1: Selection of Separation Packing

[0046] The initial feed solution (hereinafter referred to as the blending solution) for packing material screening was prepared using GG and fructose standards. In this example, the blending solution used a solute concentration of 92 g / L GG and 40 g / L fructose.

[0047] The separation effects of different types of packing materials on fructose and glucose were explored using single-column experiments, including activated carbon, macroporous adsorption resins, and ion exchange resins.

[0048] For activated carbon packing materials, the main test was whether separation could be achieved by adsorbing either glucose (GG) or fructose. The test samples were from Shanghai Pustar Environmental Protection Technology Co., Ltd. (activated carbon model PSMD-1P) and Beijing Rixin Yuanwang Technology Development Co., Ltd. (activated carbon models RX-1 to RX-5, RX-8, RX-9). Static adsorption was used, with an activated carbon addition of 10% and a treatment time of 30 minutes. Samples were taken to measure the concentrations of GG and fructose, and the results were compared with the original solution to calculate the adsorption rate. The test results are shown in the table below:

[0049] Table: Adsorption effects of different activated carbons on GG and fructose

[0050]

[0051] The results show that the activated carbon samples above have the same adsorption effect on GG and fructose, therefore they have no separation effect on GG and fructose.

[0052] For resin-based fillers, the main test focused on whether glucose (GG) and fructose components would separate after passing through the filler. The resin type was selected based on the degree of separation. Resin samples included Lan-1 to Lan-7 (Xi'an Lanxiao Technology - macroporous adsorption resin), CSR-1Ca type (particle size: 0.2-0.25mm); CSR-2Ca type (particle size: 0.25-0.30mm); CSR-3Ca type (0.30-0.35mm) (Zibo Dongda Chemical - styrene-based gel-type strong acid (sulfonic acid group) cation exchange resin (calcium type)); SDM-04 and SDM-06 (Xiamen Shida Membrane Technology - styrene-based gel-type strong acid (sulfonic acid group) cation exchange resin (sodium type)); and Finex#001 (Finland Fennec Resin - styrene·diethylene copolymer strong acid cation exchange resin (sodium type)). The separation degree was calculated using the following formula:

[0053] Separation degree = 2*(T2-T1) / (W1+W2)

[0054] Where T1 and T2 are the retention times of peak 1 and peak 2, respectively; W1 and W2 are the baseline peak widths of peak 1 and peak 2, respectively. A higher resolution value indicates a better separation effect.

[0055] Resin testing employed dynamic separation mode with a column diameter-to-height ratio of 1:10, a flow rate of approximately 1 BV / h, and an injection volume of 0.5 BV. Samples were taken every 5 minutes for HPLC concentration analysis. BV represents column volume. The calculated resolutions for each resin are shown in the table below.

[0056] Table: Data on the separation efficiency of GG and fructose by different resins

[0057]

[0058] The results showed that the imported Finex#001 resin had the highest separation efficiency for GG and fructose, but its cost was relatively high. Both domestic Ca-type and sodium-type strong acid (sulfonic acid group) ion exchange resins could separate GG and fructose to a certain extent. In particular, the CSR-Ca resin had a higher separation efficiency, better separation effect for GG and fructose, and was also cheaper and easier to maintain.

[0059] The separation effect of CSR-2Ca type resin on a three-component mixture of GG, fructose, and glycerol standards was investigated using a single column. In this example, the solute concentrations in the three-component mixture were 140 g / L GG, 95 g / L fructose, and 80 g / L glycerol, respectively. The resin was tested in dynamic separation mode with a column diameter-to-height ratio of 1:30, a flow rate of approximately 0.18 BV / h, and an injection volume of 0.07 BV. Samples were taken every 10 minutes for HPLC concentration analysis. (BV: column volume).

[0060] The results showed that the separation degree between GG and fructose was 0.495, the separation degree between GG and glycerol was 0.72, and the separation degree between fructose and glycerol was 0.211, indicating that CSR-2Ca type resin still has a good separation effect on GG, fructose, and glycerol.

[0061] Example 2: Microfiltration

[0062] The inventors applied the above process to the scale-up production of GG, and purified and prepared GG from 100L of enzymatic GG production reaction solution.

[0063] I. Glyceryl glucoside enzymatic synthesis

[0064] 1. Induced expression of recombinant Escherichia coli sucrose phosphorylase

[0065] LmSP sucrose phosphorylase is derived from (Leuconostoc mesenteroides, Q59495), as disclosed in patent US10,683,525B2.

[0066] The gene encoding sucrose phosphorylase was introduced into *E. coli* and induced to express the enzyme, resulting in recombinant *E. coli* expressing sucrose phosphorylase. The vector used was pET28b, LB medium, and IPTG induction. The collected *E. coli* cells were then centrifuged and used as a whole-cell catalyst for subsequent enzymatic synthesis reactions.

[0067] 2. Utilizing LmSP whole-cell catalyst to catalyze GG synthesis

[0068] A 100L reaction system contained 480g / L glycerol, 650g / L sucrose, and 25g / L recombinant *E. coli* cells expressing sucrose phosphorylase, collected after centrifugation. The reaction was carried out at 37℃ and 40rpm for 10 days to obtain the enzyme reaction solution. The concentrations of the main components in the enzyme reaction solution were determined by HPLC, and the viscosity of the final enzyme reaction solution was measured using an SNB-1 digital viscometer.

[0069] Results: The final enzyme reaction solution contained 300 g / L glycerol, 340 g / L fructose, 430 g / L GG, and 25 g / L bacterial cells. The substrate sucrose was completely consumed, and the viscosity was 550 mPa·s, which is equivalent to the viscosity of a 95% glycerol aqueous solution under the same conditions.

[0070] II. Highly Efficient Removal of Whole-Cell Catalysts (Hollow Fiber Membrane Microfiltration Separation)

[0071] Take 10L of the above enzyme reaction solution and perform the following treatments. By comparing the removal effect on whole-cell bacteria and the removal effect on protein impurities, a highly efficient enzyme catalyst removal method can be screened:

[0072] (1) Hollow fiber membrane

[0073] Three hollow fiber membranes (PP material) with a pore size of 0.1 μm and molecular weight cutoffs of 100 KD and 50 KD, respectively, were selected for microfiltration. The operating pressure range was 0.05-0.4 MPa, and the operating temperature was 10-45℃. Permeate was obtained.

[0074] (2) Ceramic membrane

[0075] Two ceramic membranes (made of alumina) with pore sizes of 0.05 μm and 0.1 μm were selected for microfiltration treatment, with an operating temperature range of 10-45℃ and an operating pressure range of 0.05-0.5 MPa, to obtain the permeate.

[0076] (3) Centrifugation

[0077] The enzyme reaction solution was first denatured by heating at 95°C for 1 hour. After cooling to room temperature, it was centrifuged at 13000g for 40 minutes to obtain the supernatant.

[0078] (4) Filter paper filtration

[0079] The enzyme reaction solution was first heated at 95°C for 1 hour for heat denaturation, and then cooled to room temperature. It was then vacuum filtered using Whatman medium-speed double-loop qualitative filter paper (Hangzhou Wohua Filter Paper Co., Ltd., 9cm in diameter, 8μm in pore size, item number 99-102-090). The permeate was collected in the filtration flask.

[0080] Table 1. Statistical analysis of the treatment efficiency of enzyme reaction solution by different treatment methods.

[0081] Centrifugation 6L / h (excluding 1h of pretreatment heating and denaturation) Filter paper filtration method <![CDATA[2L / h / m 2 (Excluding 1 hour of pretreatment heating and denaturation) Ceramic membrane method (0.05 μm membrane pore size) 0 Ceramic membrane method (0.1 μm membrane pore size) <![CDATA[3L / h / m 2 ]]> Hollow fiber membrane method (0.1 μm membrane pore size) <![CDATA[4.5L / h / m 2 ]]> Hollow fiber membrane method (100KD retention range) <![CDATA[0.2L / h / m 2 ]]> Hollow fiber membrane method (50KD retention range) 0

[0082] Table 2. Protein content and colony count of samples obtained by different treatment methods.

[0083]

[0084] The results showed that although the recoveries of glycerol, fructose, and GG in the permeate samples were all above 98% after treatment by the four methods, the treatment efficiency and enzyme catalyst removal effects of the different methods differed significantly, as detailed in Table 1. Firstly, the treatment with 0.05 μm pore size ceramic membranes and hollow fiber membranes with a 50 KD cutoff could not separate and collect the permeate, and therefore could not be used for the removal of bacterial cells and proteins from the enzyme reaction solution. The other treatment methods were able to collect the separated solution, with the 0.1 μm pore size hollow fiber membrane treatment being the simplest to operate and the most efficient.

[0085] In addition, all four treatment methods mentioned above can effectively remove bacterial cell catalysts and protein impurities from the enzyme reaction solution (Table 2), but the treatment with a hollow fiber membrane with a pore size of 0.1 μm completely removes whole-cell bacteria and protein impurities from the feed solution, resulting in the best treatment effect.

[0086] III. Microfiltration of reaction solutions with different viscosities

[0087] The whole-cell enzyme catalytic reaction solution with a viscosity of 550 mPa·s from Example 4 was diluted with water to obtain 20 L of enzyme catalytic reaction solutions with the following viscosities: 440 mPa·s, 250 mPa·s, 150 mPa·s, 50 mPa·s, 12 mPa·s, and 4 mPa·s. These solutions were then treated with hollow fiber membranes with a pore size of 0.1 μm to evaluate the treatment effect of the hollow fiber membrane method on enzyme catalytic reaction solutions of different viscosities. The operating pressure range was 0.05–0.4 MPa, and the operating temperature range was 4–45 °C.

[0088] Results: Within a temperature range of 4-45℃, after treatment with hollow fiber membranes, the total bacterial count of the enzyme reaction solutions with different viscosities was 0 CFU / ml, and the protein content was also 0 mg / ml. Furthermore, the processing flux of the hollow fiber membrane significantly increased with decreasing enzyme reaction solution viscosity and increasing operating temperature. For an enzyme reaction solution with a viscosity of 12 mPa·s, the processing flux reached 30 L / h / m at an operating temperature of 40℃. 2 The enzyme reaction dilution with a viscosity of 4 mPa·s achieved a throughput of up to 70 L / h / m at an operating temperature of 40°C. 2 .

[0089] Example 3: Four-stage separation and purification of the reaction solution for enzymatic synthesis of glycerol glucoside: microfiltration-ultrafiltration-nanofiltration-SMB

[0090] The inventors used a four-stage separation and purification process of microfiltration-ultrafiltration-nanofiltration-SMB to purify and prepare GG from the enzymatic synthesis reaction solution produced in a 30L scale-up production line.

[0091] (1) Microfiltration

[0092] Take 30 L of the enzyme reaction solution from Example 2 above, dilute it with an equal volume of pure water, and then perform microfiltration using a hollow fiber membrane with a pore size of 0.1 μm. The operating pressure is 0.05-0.4 MPa, and the temperature is room temperature. Collect the permeate. The test results show that the protein concentration in the permeate is all within the range of 0.02 mg / ml, and the colony count is 0. Complete permeation of glycerol, fructose, and GG can be achieved, with recovery rates of up to 97%, 97%, and 98%, respectively.

[0093] The formula for calculating the microfiltration recovery rate Y is: Y = (concentration of permeate × volume of permeate) / (concentration of raw feed solution × volume of permeate).

[0094] (2) Ultrafiltration

[0095] Ultrafiltration membranes with a molecular weight cutoff of 600 Da were used for ultrafiltration separation of microfiltration products. The operating pressure was 1-2.4 MPa, and the operating temperature was 15-40℃. The permeate was collected. The results showed that the protein concentration in the permeate was 0 mg / mL, indicating that the ultrafiltration membrane completely removed residual proteins, peptides, and other impurities from the microfiltration solution. Complete permeation of glycerol and fructose was achieved, and 97% of GG was also permeated, with recoveries of 99.9%, 99.9%, and 97%, respectively. Figure 1 ).

[0096] The formula for calculating the ultrafiltration recovery rate Y is: Y = (concentration of permeate × volume of permeate) / (concentration of raw feed × volume of permeate); the formula for calculating the ultrafiltration rejection rate R is: Y = 1 - concentration of permeate / concentration of feed.

[0097] (3) Nanofiltration

[0098] Nanofiltration membranes with a molecular weight cutoff of 150-300 Da were used for nanofiltration separation of the ultrafiltration products. The operating pressure was 1.5-4 MPa, and the operating temperature was 4-40℃. The results showed that glycerol was recovered in the permeate, with a glycerol recovery rate of 99.2% and a purity of 80%. The concentrate did not contain glycerol, and therefore glycerol could be completely removed by nanofiltration. Analysis of the concentrate showed that the recovery rates of fructose and glycerol were as high as 76.4% and 98%, respectively (e.g., ...). Figure 2 (As shown). The solute mass concentration percentage in the collected 110L nanofiltration concentrate was 28%.

[0099] (4) Simulated moving bed separation

[0100] The nanofiltration concentrate was further concentrated by rotary evaporation to a solute mass concentration of 50%, and then purified by continuous separation using a simulated moving bed (SMB).

[0101] A 6-column sequential SMB system provided by Jiangsu Hanbang Co., Ltd. was used. Each column had a diameter of 3.5 cm and a length of 70 cm. The resin packing material was CSR-2Ca, and the total packing volume for all 6 columns was 4 L. The SMB separation parameters were as follows: pressure 2 MPa, injection flow rate 0.007 BV / min, injection volume 0.023 BV, eluent water flow rate 0.01 BV / min, circulation separation for 10 min, and operating temperature 55℃. Following the experimental method, HPLC was used to detect the GG collected after SMB separation and purification.

[0102] The results are as follows Figure 3 The purity of glucose (GG) reaches 99%, and the GG yield reaches 90%. This step also recovers the fructose component with a purity of 80% and a yield of 91%.

[0103] Example 4: Four-stage separation and purification of the reaction solution for enzymatic synthesis of glycerol glucoside: microfiltration-ultrafiltration-nanofiltration-SMB

[0104] The inventors used a four-stage separation and purification process of microfiltration-ultrafiltration-nanofiltration-SMB to purify and prepare GG from the enzymatic synthesis reaction solution produced in a 40L scale-up production line.

[0105] (1) Microfiltration

[0106] Take 40L of the enzyme reaction solution from Example 2 above, dilute it with an equal volume of pure water, and then perform microfiltration using a hollow fiber membrane with a pore size of 0.1μm. The operating pressure is 0.05-0.4MPa, and the operating temperature is between 20-65℃. Take the permeate.

[0107] The results showed that the protein concentration in the microfiltration permeate was 0.01 mg / ml, indicating that the microfiltration procedure effectively removed whole-cell enzyme catalysts and other protein impurities from the enzyme reaction solution. The recoveries of glycerol, fructose, and GG were all above 95%.

[0108] (2) Ultrafiltration

[0109] An ultrafiltration membrane with a molecular weight cutoff of 2500 Da was selected for ultrafiltration separation of the microfiltration permeate. The operating pressure was 0.05-2.4 MPa, and the operating temperature was between 8-30℃. The permeate was collected.

[0110] The results showed that the protein concentration in the permeate after ultrafiltration was 0 mg / ml, indicating that ultrafiltration membranes with a cutoff range of 2500 Da can effectively remove residual proteins and peptides. The recoveries of glycerol, fructose, and GG were 99%, 93%, and 86%, respectively.

[0111] (3) Nanofiltration

[0112] Nanofiltration membranes with a molecular weight cutoff of 150-300 Da were used to separate the permeate from the ultrafiltration membrane. The operating pressure was 0.05-4 MPa, and the operating temperature was between 15-35℃. The concentrated solution was collected.

[0113] The results showed that glycerol was present in the permeate, with a glycerol recovery rate of 99.2% and a purity of 81% in the nanofiltration permeate. The concentrate contained no glycerol, and the recoveries of fructose and glycerol (GG) were 77.1% and 95.4%, respectively. The solute mass concentration percentage in the collected 200 L nanofiltration concentrate was 20%.

[0114] (4) Simulated moving bed separation

[0115] The nanofiltration concentrate was diluted with water to a solute concentration of 10%, and GG was continuously purified using a simulated moving bed (SMB) system. An 8-column, four-zone SMB system was used, with each column measuring 2.0 cm in diameter and 36 cm in length. Finex #001 resin was used, with a total packing volume of 0.9 L for all 8 columns. The SMB separation parameters were as follows: pressure 2 MPa, injection flow rate 0.001 BV / min, eluent water flow rate 0.01 BV / min, extract flow rate 0.001 BV / min, retentate flow rate 0.002 BV / min, valve switching time 8 min, and operating temperature 60 °C. Following the experimental method, the GG collected after SMB purification was analyzed using HPLC.

[0116] Results: The purity of glucose (GG) reached 99%, and the yield was 85%. This method also yielded fructose, with a purity of 74% and a yield of 93%.

[0117] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for separating and purifying glycerol glucoside, comprising: (1) The initial product of the enzymatic preparation of glycerol glucoside was subjected to microfiltration, ultrafiltration and nanofiltration in sequence to remove impurities and obtain a filtered product whose main components were fructose and glycerol glucoside. (2) Separate the filtered product obtained in step (1) by chromatographic separation to separate glycerol glucoside from fructose and collect the separated and purified glycerol glucoside. The initial product contains glycerol glucoside, glycerol, and fructose; The viscosity of the feed solution used for microfiltration is 12 mPa·s-550 mPa·s; the microfiltration membrane is a hollow fiber membrane with a pore size of 0.1 μm; The packing material used in the chromatographic separation method is CSR-2Ca type resin; The microfiltration process involves passing the initial product through a membrane to remove proteins and colonies, and collecting the permeate; the ultrafiltration process involves passing the microfiltration permeate through a membrane to remove residual proteins and peptide impurities, and collecting the permeate; the nanofiltration process involves passing the ultrafiltration permeate through a membrane to remove glycerol, and collecting the retentate. The membranes used in ultrafiltration have a molecular weight cutoff of 600-2500 Da; Nanofiltration membranes have a molecular weight cutoff of 150-300 Da.

2. The method according to claim 1, characterized in that: The viscosity of the feed solution used for microfiltration is 550 mPa·s, 440 mPa·s, 250 mPa·s, 150 mPa·s, 50 mPa·s, or 12 mPa·s.

3. The method according to claim 1 or 2, characterized in that: The mobile phase in the chromatographic separation method is water; or, the mass percentage concentration of the solute in the feed solution in the chromatographic separation method is 10%-50%.

4. The method according to claim 1 or 2, characterized in that: The chromatographic separation method is a simulated moving bed chromatography method; When using a sequential simulated moving bed, the injection fluid flow rate is 0.007 BV / min, and the mobile phase flow rate is 0.01 BV / min. When using a four-zone simulated moving bed, the flow rate of the injection solution is 0.001 BV / min, the flow rate of the mobile phase is 0.01 BV / min, the flow rate of the extract is 0.001 BV / min, the flow rate of the raffinate is 0.002 BV / min, and the valve switching time is 8 min.

Images