Method for separating and purifying galactooligosaccharide through sequential simulated moving bed two-step coupling

Abstract

The invention relates to a method for separating and purifying galactooligosaccharide through two-step coupling of a sequential simulated moving bed, which comprises the following steps: (1) determining basic parameters of a chromatographic column of a fixed bed, (2) screening stationary phase resin through a pulse experiment method, (3) determining adsorption isotherms of galactose, glucose and galactooligosaccharide through a pulse experiment, and (4) determining the adsorption isotherms of galactose, glucose and galactooligosaccharide through a two-step coupling method. (4) determining adsorption isotherms of galactose, glucose and galactooligosaccharide by a leading edge analysis method; (5) determining kinetic parameters of galactose, glucose and galactooligosaccharide; (6) process simulation of the two-step coupling experiment of the sequential simulation moving bed is carried out, and experiment verification is carried out; and (7) performing multi-objective optimization based on a genetic algorithm to obtain two-step coupled operation parameters of the sequential simulated moving bed. Through the SSMB two-step coupling design, simultaneous separation and purification of galactooligosaccharide and galactose can be realized, the purity of galactooligosaccharide can reach 98%, and the purity of galactose can reach 95%.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification, and in particular relates to a method for separating and purifying oligosaccharides by two-step coupling of a sequential simulated moving bed. Background Art

[0002] Galacto-oligosaccharides (GOS) are naturally occurring sugars, which are composed of galactose molecules connected by β-1,6 glycosidic bonds. They are an important class of functional oligosaccharides, widely present in human milk and some plants, and have attracted attention for their unique physiological functions. Galacto-oligosaccharides have a variety of biological activities, including promoting the growth of beneficial bacteria in the intestine, enhancing immunity, regulating intestinal function, etc. They can be used by certain probiotics in the intestine, such as bifidobacteria, to help maintain the balance of intestinal microecology. In the food industry, galacto-oligosaccharides are used as food additives to increase the nutritional value of food and improve its health benefits. They are widely used in infant formula to simulate the oligosaccharide components in breast milk and promote the healthy growth of infants. With the increasing attention paid to health and nutrition, the research and application of galacto-oligosaccharides are deepening, and their potential in the fields of functional foods, health products and medicine is gradually being explored and utilized.

[0003] At present, the separation methods of galacto-oligosaccharides (GOS) mainly include: column chromatography, ion exchange resin, membrane separation, and nanofiltration. However, traditional methods may not always meet the requirements of high purity, especially when separating complex sugar mixtures, there may be cross-contamination or other impurities. Some traditional purification technologies may use a large amount of solvents or produce a large amount of wastewater, which may have an adverse impact on the environment.

[0004] The concentration of crude industrial galacto-oligosaccharide products is within the range of (57%-65%), wherein the main impurities are galactose and glucose. Since galacto-oligosaccharide (GOS) is a mixture of oligosaccharides of different polymerization degrees formed by galactose connected by β-glycosidic bonds, its main chain is composed of galactose as a constituent unit, and the chain end often ends with a single glucose; galactose and glucose are both hexoses, but have different stereostructures and are isomers of each other. Since GOS, galactose and glucose have a high degree of similarity in molecular structure, they are difficult to be effectively distinguished and separated in traditional separation technology. The use of ordinary one-step sequential simulated moving bed (SSMB) cannot meet the high purity requirements and still cannot obtain high-purity galacto-oligosaccharides. Therefore, how to obtain high-purity galacto-oligosaccharide products is a technical problem to be solved in this field. Summary of the invention

[0005] In the first aspect, the present invention relates to a sequential simulated moving bed (SSMB) two-step coupled separation and purification of oligogalactose, which is particularly suitable for the separation and purification of industrial oligogalactose (GOS). Specifically, the following steps are included: (1) determining the basic parameters of the fixed bed chromatographic column, wherein the basic parameters include dead volume and void ratio;

[0006] ①Determination of dead volume: Use blue glucan that is not adsorbed by the stationary phase column resin as a tracer, prepare a blue glucan solution, disconnect the detector from the column, and connect the chromatographic pump and the detector directly through a pipeline. Perform five groups of pulse experiments at different flow rates (such as 2mL / min, 4mL / min, 6mL / min, 8L / min, 10L / min), and observe the elution curve to obtain the retention time of blue glucan at different flow rates. After the flow rate and retention time are known, the average dead volume of the chromatographic system can be calculated using the following formula.

[0007] V D =Qt 0 (1)

[0008] Where V D is the dead volume of the system, in mL, t 0 is the system dead time, in min; Q is the chromatographic pump flow rate corresponding to the dead time, in mL / min.

[0009] ②Porosity determination

[0010] Blue dextran which is not adsorbed by the stationary phase column resin is used as a tracer. The retention volume is calculated from the retention time of blue dextran at different flow rates, and the pore volume in the column is calculated. The porosity of the column is calculated when the column volume is known.

[0011]

[0012] Where, t R,i is the retention time of component i, in min; L is the length of the chromatographic column, in cm; u is the flow rate, in mL / min; ε is the porosity of the chromatographic column (the ratio of pore volume to total volume); Vc is the volume of the chromatographic column, in cm 3 ; Q is the flow rate of the system, in mL / min.

[0013] ③Establish standard curve

[0014] When using HPLC for quantitative analysis, it is necessary to establish standard concentration samples of galactose, glucose and galacto-oligosaccharides of different concentrations and to establish a standard curve.

[0015] In one embodiment of the present invention, the concentration of galactose is 0.2, 0.25, 0.3, 0.35 and 0.4 g / L, the concentration of glucose is 0.2, 0.3, 0.4, 0.5, 0.6 g / L, and the concentration of galacto-oligosaccharide is 1.2, 1.3, 1.4, 1.5, 1.6 g / L.

[0016] (2) The stationary phase resin is screened by pulse experiment method, and the advantages and disadvantages of different resins in separation system in terms of separation degree, selectivity, porosity and retention time are compared to determine the stationary phase resin.

[0017] The separation degree is defined as the ratio of the difference between the retention values ​​of two adjacent chromatographic peaks to half the sum of the base widths of the two chromatographic peaks.

[0018]

[0019] Where t R1 and t R2 are the retention times of the two components, W t1 and W t2 is the base width of the chromatographic peak of the corresponding component.

[0020] The selectivity can be used to measure the difference in adsorption capacity of two components. The selectivity of different resins can be calculated by the following formula.

[0021]

[0022] Where k 1 , k 2 is the retention factor of different substances, where k 2 Greater than k 1 . Where t R,i is the retention time of component i, t 0 It is the time for the mobile phase without retention time to pass through the chromatographic column, also known as the dead time. This formula is used to evaluate the separation effect of two different compounds in the chromatographic process. The larger the selectivity α value, the better the separation between the two compounds.

[0023] When the selectivity, retention time and half-peak width are known, the column efficiency under different resins can be calculated by the following formula.

[0024]

[0025] Where N i is the column efficiency, α is the selectivity, t R,i is the retention time, W h / 2,i is the half peak width.

[0026] In one embodiment of the present invention, the resin is a strongly acidic cation exchange resin ZGSPC106K + .

[0027] (3) Pulse experiment to determine the adsorption isotherms of galactose, glucose and galacto-oligosaccharides

[0028] Generally speaking, the concentration of industrial galacto-oligosaccharide crude products is within the range of (57%-65%), in which the main impurities are galactose and glucose. Combined with the operability of the experiment, 40g / L galacto-oligosaccharide, 10g / L galactose and 10g / L glucose solutions were prepared for the experiment.

[0029] The temperature condition is 35-50℃; the mobile phase is ultrapure water; the flow rate is 10mL / min to flush the chromatographic column until the baseline of the differential detector is flushed, and the baseline is zeroed. For the accuracy of the experiment, the quantitative loop is flushed with a pre-configured solution of the same concentration so that the six-way valve will not reduce the injection concentration due to the ultrapure water, causing errors in the experiment. The manual valve of the six-way valve is rotated and the timer button of the differential detector is pressed at the same time. The elution curves of oligogalactose, galactose and glucose are recorded at five flow rates of 2mL / min, 4mL / min, 6mL / min, 8mL / min and 10mL / min. According to the retention time of the elution curve at different flow rates, the adsorption isotherm parameters are calculated using the following formula (7).

[0030]

[0031] In the formula, H i is the Henry constant of component i, u is the flow rate, ε is the void ratio, t R is the retention time, in min, V c is the volume of the chromatographic column, in cm 3 , L is the length of the chromatographic column, in cm, and r is the inner radius of the chromatographic column, in cm.

[0032] (4) Determination of adsorption isotherms of galactose, glucose and galacto-oligosaccharides by frontal analysis

[0033] The present invention selects pulse experiment and pre-analysis method to combine the two methods to achieve comprehensive and accurate determination of adsorption isotherm, so as to better verify the accuracy of kinetic parameters and adsorption isotherm.

[0034] First, the mobile phase balances the chromatographic system, and different concentrations of galacto-oligosaccharide solutions are configured with ultrapure water. In one embodiment of the present invention, 55, 60, 65, 70, 75, and 80 g / L galacto-oligosaccharide solutions are configured. Ultrapure water rinses the chromatographic column at a flow rate of 8-10 mL / min, and the differential detector purge key is turned on to rinse the reference cell until the baseline is stable. The minimum concentration (55 g / L) galacto-oligosaccharide solution is switched as the mobile phase to start the experiment. The chromatographic pump flow rate is 8-10 mL / min, and the platform rises to a stable 15-20 min or more to obtain a breakthrough curve of the current concentration. It is believed that the chromatographic column has reached equilibrium, and the next concentration sample is switched to detect 60, 65, 70, 75, and 80 g / L galacto-oligosaccharide solutions respectively. Stop recording, save data, open the purge valve, and rinse the pipeline, RI detection cell, and sample cell with deionized water.

[0035] Different concentrations of glucose and galactose were prepared and the same experimental steps were used to perform frontal analysis. The adsorption isotherm was calculated using formula (8):

[0036]

[0037] In the formula, q j is the mass of adsorbed substance per unit volume of resin in step j, in g / L, V c is the volume of the chromatographic column in cm 3 , t 0 is the system dead time, in min, t B is the breakthrough time of the jth step, and ε is the void fraction of the chromatographic column, which is dimensionless.

[0038] In one embodiment of the present invention, the concentration of glucose is 5, 10, 15, 20, 25, 30 g / L, and the concentration of galactose is 0.5, 1, 1.5, 2, 2.5, 3 g / L.

[0039] (5) Determination of kinetic parameters of galactose, glucose and galacto-oligosaccharides

[0040] After the dead volume, void ratio and related column parameters were determined, the transfer behavior of oligogalactose, galactose and glucose was fully considered in the modeling process, and the kinetic parameters of the separation system were determined and calculated. m (mass transfer coefficient) and N L (axial diffusion coefficient). According to the single-component pulse experiments of oligogalactose, galactose and glucose, the retention time, half-peak width of the chromatographic peak and the porosity at the corresponding concentration and flow rate can be obtained. The kinetic parameters are fitted according to formula (9), using uλ, N at different flow rates. L The slope and intercept obtained by fitting are used to obtain k m,i and N L .

[0041]

[0042] Where, t R is the retention time, W 2 / h H is the half peak width. i is the Henry constant for component i, D L,i is the diffusion coefficient of component i in the mobile phase, N i The column efficiency of component i represents the efficiency of the chromatographic column, u is the linear velocity, L is the length of the chromatographic column, is the void ratio (the ratio of pore volume to solid particle volume), λ i is the resistance coefficient, K m is the mass transfer coefficient, N L is the axial diffusion coefficient. When the flow rate is within a reasonable range, 1 / N L,i It is linearly related to u.

[0043] (6) Process simulation of the two-step coupling experiment of the sequential moving bed and experimental verification

[0044] By measuring the chromatographic model parameters of oligosaccharides, galactose and glucose, the complete separation area is determined according to the triangle theory, the optimal operating point is selected in the complete separation area, the operating parameters of the sequential simulated moving bed SSMB are preliminarily designed, and the corresponding two-step coupling experiment is completed. The experimental results (purity and yield of the target product, etc.) are determined by HPLC. At the same time, a mathematical model is established for the entire separation process, the process simulation is completed, and the simulation results are compared with the experimental results to verify the accuracy and reliability of the calculation model.

[0045] The triangle theory is to use Henry's constant and m Ⅱ and m Ⅲ , an intersecting triangular area can be drawn, which represents the complete separation area, meaning that the value (m value) selected in this area can produce relatively pure extract and raffinate. On the contrary, the area outside the triangle indicates that the purity of the extract or raffinate is insufficient. Fig. 9 As shown, the triangular area formed can help narrow the range of parameter selection in advance when designing SSMB experimental parameters.

[0046] Specifically, the range of m values ​​is determined by the Henry constants of the two substances to be separated, and the m values ​​in different regions are screened. The screening rules are: H A <m Ⅰ <5 (according to the upper limit of the pump flow), H B <m Ⅱ <H A , H B <m Ⅲ <HA , m Ⅳ <H B , m Ⅱmin <m Ⅱ <m Ⅲ <m Ⅲmax ; After determining the m value of different regions by screening, calculate the switching time. Among them, H A , H B are the Henry constants of substances A and B to be separated respectively.

[0047] The triangle theory helps determine the complete separation area, and the switching time (t 1 ,t 2 and t 3 ) and feed flow rate (Q F ). This theoretical tool narrowed the range of parameter selection in advance when designing SSMB experimental parameters, ensuring the accuracy and efficiency of the experimental design stage. The accuracy of the measured chromatographic model parameters and adsorption isotherm parameters was verified by comparison between simulation and experiment. At the same time, the accuracy of the numerical simulation was verified, proving that the multi-objective optimization results in the next step have practical guiding significance.

[0048] This experiment adopts a two-step coupling process. In the first step, SSMB separates the three components of galacto-oligosaccharides, galactose and glucose to obtain the target product galacto-oligosaccharides. In the second step, SSMB further recovers the galactose in the extracted impurity components to obtain high-purity galacto-oligosaccharides.

[0049] The first step of simulation experiment is mainly to obtain the target product galacto-oligosaccharide. Through SSMB technology, galacto-oligosaccharide is separated from other components (galactose and glucose).

[0050] In the sequential simulated moving bed (SSMB) system, where Q i represents the flow rate of the i-th section in mL / min; V is the column volume in cm 3 ; ε represents the void ratio; t 1 ,t 2 ,t 3 are the switching time of the feeding phase, circulation phase and elution phase, in min; t s is the complete switching time, in min; m i It represents the net flow ratio of the four different areas of SSMB, a dimensionless parameter, Q F is the feed flow rate, in mL / min; Q E is the circulation flow rate in mL / min.

[0051]

[0052] ts=t 1 +t 2 +t 3 (16)

[0053]

[0054] According to the Henry constants of oligogalactose, galactose and glucose obtained in pulse experiment and frontier analysis experiment, m can be determined by triangle theory. i According to formulas (12)-(16), we can get t 1 ,t 2 ,t 3 ,t s According to formula (17), the feed flow rate Q is obtained F .

[0055] Complete separation meets the switching time t s It is greater than the retention time of galacto-oligosaccharides and less than the retention time of galactose and glucose, that is, formula (18). This conditional limitation ensures that high-purity galacto-oligosaccharides can be obtained at the residue outlet, and galactose and glucose are completely flushed out at the extraction outlet.

[0056] ts>t GOS ,ts <t glucose ,ts <t galactose (18)

[0057] Substituting the above parameters into the Fortran program simulation, the purity (Pur), unit throughput (UT), water consumption (WC), yield (Rec), raffinate outlet flow rate (Q raf ) and other parameters. Experiment with the simulated parameters and determine the accuracy of the model by comparing the simulation results with the experimental results.

[0058]

[0059]

[0060] Where c is the concentration of the substance in g / L.

[0061] The second step is simulation experiment: based on the first step, the target product, high-purity oligosaccharide, is obtained. In this step, the Henry constants of galactose and glucose obtained in the pulse experiment and frontier analysis experiment are used to determine m through triangle theory for different components. i 'Value range, filtering rules are the same as above.

[0062] According to formula (12), we can calculate m Ⅰ '、mⅡ '、m Ⅲ '、m Ⅳ 'The flow rate of each area is m i ', according to formula (12)-(16), we can calculate t1', t2', t3', t s ', according to formula (17) the feed flow rate Q is calculated F '.

[0063] Complete separation requires that the switching time ts' is greater than the retention time of galactose (t galactose ) and is less than the retention time of glucose (t glucose ), that is, formula (23). This design ensures that high-purity galactose can be obtained at the raffinate outlet, and glucose is completely flushed out at the extraction outlet.

[0064] Glucose retention time (t glucose )>Switching time ts'>Retention time of galactose (t galactose )(twenty three)

[0065] By bringing the above-obtained parameters into the simulation program, the results of the second step simulation experiment process (Pur'), ​​unit treatment volume (UT'), water consumption (WC'), and yield (Rec') can be obtained according to formulas (19)-(22), and these results can be verified with the experimental results.

[0066] Furthermore, the optimal solution obtained by simulation in step (6) is experimentally verified to ensure the accuracy and practicality of the model and provide a basis for step (7).

[0067] The specific simulated moving bed experiment used a four-column system, which was carried out at a column temperature of 50°C and a circulation flow rate of 10 mL / min (based on the maximum pressure of the system). The automatic control of the experiment was achieved through Wincc Runtime Start software.

[0068] The experimental operation of SSMB is divided into three stages: feeding stage, circulation stage and elution stage (such as Figure 1). In the feeding stage, by turning the feeding valve, the sample is pumped into the simulated moving bed using a feeding pump, while the feeding is completed in column 1 and the eluent is introduced into column 3. In the circulation stage, the feed solution circulates in the column and is fully distributed between the stationary phase and the mobile phase, which is a key step in saving water consumption. In the elution stage, the 4 columns are disconnected to connect columns 1, 2, and 3, so that a high-purity oligosaccharide sample can be taken at the raffinate port. The experiment is planned to carry out 16 cycles, and the number of cycles is adjusted by dynamically monitoring the purity of the extract port and the raffinate port. After the first step is stable, the solution at the extract port in the first step is introduced into the feed port for a second separation, and the above operation is repeated. Before the experiment starts, the system needs to be flushed, and the switching time and method are input into the control system. The experiment starts feeding from the second cycle, and samples are collected from the tenth cycle, and then every two cycles. The collected samples will be tested by HPLC until the purity of the samples is no longer improved for two consecutive times. At this time, the entire SSMB system will be flushed again, and the solution obtained from the extraction port in the first step will be fed into the second cycle of the experiment. The above operation will be repeated to test the collected samples by HPLC until the purity is no longer improved. These steps are all to ensure the optimization of experimental parameters to achieve an efficient separation process.

[0069] In one embodiment of the present invention, the process simulation is close to the experimental results, and the two are highly consistent, which shows the correctness of the program simulation method of the present invention.

[0070] (7) Based on the multi-objective optimization of genetic algorithm, the optimal operating range of the two-step coupled separation and purification of oligosaccharides by sequential simulated moving bed was determined.

[0071] The present invention is based on the multi-objective optimization of genetic algorithm, and the specific process is as follows:

[0072] The specific parameters of the genetic algorithm in the program are: PARAMETER (MXPARM = 50, NSECT = 200, N = 3200, NSWIT = 80); MXPARM is a basic data structure used to store numerical data; NSECT is the number of cycles; N is the number of theoretical plates; NSWIT is the number of switches.

[0073] 1) Initialize the population: Randomly generate a set of initial solutions, each of which represents a possible operating condition configuration of SSMB.

[0074] 2) Define the objective function: Determine the objective function to be optimized, such as purity (Pur), unit throughput (UT), water consumption (WC), and recovery rate (Rec).

[0075] Preferably, the purity (Pur) and recovery (Rec) of galacto-oligosaccharides are maximized and the water consumption (WC) is minimized simultaneously.

[0076] 3) Constraint setting: Constraints are set according to experimental requirements and process limitations, such as purity must be greater than 90%, recovery rate must be greater than 80%, etc.

[0077] 4) Fitness evaluation: The NSGA-II algorithm is used to evaluate the fitness of each solution, i.e., the objective function value, while considering the constraints.

[0078] 5) Selection operation: Select excellent solutions to enter the next generation based on fitness.

[0079] 6) Crossover and mutation: Perform crossover operations on the selected solutions to generate new solutions. Perform mutation operations on the new solutions to increase the diversity of the population.

[0080] 7) Non-dominated sorting: Perform non-dominated sorting on the newly generated solutions to determine their advantages over other solutions in the current population.

[0081] 8) Crowding calculation: Calculate the crowding degree of the solution to maintain population diversity.

[0082] 9) Select a new population: Select a new generation of population based on the non-dominated sorting and crowding calculation results.

[0083] 10) Iterative process: Repeat the selection, crossover, mutation and non-dominated sorting process until the termination condition is met (such as reaching the maximum number of iterations or the quality of the solution reaches a predetermined standard).

[0084] 11) Output Pareto optimal solutions: After the algorithm terminates, the final set of Pareto optimal solutions is output, which provide the best trade-off between multiple objectives.

[0085] 12) Result analysis: Analyze the solutions in the Pareto optimal solution set to determine the optimal operating range of SSMB.

[0086] In a second aspect, the present invention relates to a method for purifying galacto-oligosaccharides by two-step coupled separation and purification in a sequential simulated moving bed, and its application in the separation and purification of industrial galacto-oligosaccharides.

[0087] Beneficial effects of the present invention:

[0088] The present invention determines the chromatographic model parameters of oligogalactose, galactose and glucose, preliminarily designs the SSMB operating parameters according to the triangle theory and completes the corresponding two-step coupling experiment, and verifies the experimental results through process simulation in Fortran. It can be obtained that the simulation results are in good agreement with the experimental results, which proves the accuracy of the determination of the chromatographic model parameters and the adsorption isotherm parameters, and further verifies the reliability of the process simulation.

[0089] The SSMB two-step coupled separation technology of the present invention can more accurately control the operating parameters to meet the separation requirements of different components, thereby improving the purity and yield of the target product to achieve more efficient separation; for complex sugar mixtures, a single SSMB step may not be able to meet the high purity requirements, and the two-step coupling process can be gradually purified to ultimately obtain high-purity galacto-oligosaccharides, thereby achieving an efficient separation process of substances with similar structures such as lacto-oligosaccharides, galactose and glucose.

[0090] The present invention simulates the moving bed coupling process by regulating specific variables, uses a more advanced genetic algorithm multi-objective optimization method to explore conditions, takes the final performance of the product (purity, yield, processing volume, recovery rate, etc.) as a direct target, uses Fortran programming to simulate the process, screens operating conditions and simulates the moving bed experiment design, so that the entire separation process is faster and more efficient, and the process exploration process is simplified. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 This is a flow chart of the SSMB two-step coupling separation of the present invention;

[0092] Figure 2 Standard curves for galacto-oligosaccharides, glucose, and galactose;

[0093] Figure 3 is the adsorption isotherm of galacto-oligosaccharide;

[0094] Figure 4 is the glucose adsorption isotherm;

[0095] Figure 5 is the galactose adsorption isotherm;

[0096] Figure 6 is the fitting diagram of galacto-oligosaccharide 1 / Ni and uλi;

[0097] Figure 7 is the fitting diagram of glucose 1 / Ni and uλi;

[0098] Figure 8 is the fitting diagram of galactose 1 / Ni and uλi;

[0099] Fig. 9 It is the separation area of ​​triangle theory under linear and nonlinear isotherms;

[0100] Fig.10 Pareto diagram (UT) for improving the purity of galacto-oligosaccharide with the constraints of recovery greater than 80% and purity greater than 90%;

[0101] Fig.11 is the Pareto chart (m value) with the constraints that the recovery is greater than 80% and the purity is greater than 90%;

[0102] Fig.12 The Pareto diagram (t and Q) with the constraints that the recovery rate is greater than 80% and the purity is greater than 90% F ). DETAILED DESCRIPTION

[0103] Example 1

[0104] A sequential simulated moving bed two-step coupled separation and purification of oligogalactose comprises the following steps:

[0105] (1) determining basic parameters of a fixed bed chromatographic column, wherein the basic parameters include dead volume and void ratio;

[0106] ①Determination of dead volume

[0107] The determination method is to use blue glucan that is not adsorbed by the stationary phase chromatographic column resin as a tracer, prepare a 1.0g / L blue glucan solution, disconnect the detector from the chromatographic column, connect the chromatographic pump and the detector directly through a pipeline, and conduct five sets of pulse experiments at flow rates of 2mL / min, 4mL / min, 6mL / min, 8L / min, and 10L / min, respectively. The retention time of blue glucan at the above five flow rates can be obtained by observing the elution curve. After the flow rate and retention time are known, the average dead volume of the chromatographic system can be calculated by the following formula.

[0108] V D =Qt 0 (1)

[0109] Where V D is the dead volume of the system, in mL, t 0 is the dead time of the system, in min; Q is the chromatographic pump flow rate corresponding to the dead time, in mL / min. As shown in Table 1, the dead time and dead volume at different flow rates give an average dead volume of 4.73 mL.

[0110] Table 1 Summary of system dead volume data

[0111]

[0112] ②Porosity determination

[0113] The determination method is to use blue dextran that is not adsorbed by the stationary phase chromatographic column resin as a tracer, calculate the retention volume based on the blue dextran retention time at different flow rates, calculate the pore volume in the chromatographic column, and calculate the porosity of the chromatographic column if the column volume of the chromatographic column is known.

[0114]

[0115] Where, t R,iis the retention time corresponding to the dead time after removing the dead volume, in min, L is the length of the chromatographic column, in cm, u is the flow rate, in mL / min, ε is the porosity of the chromatographic column, V c is the volume of the column in cm 3 , Q is the flow rate of the system, in mL / min. The calculation results are shown in Table 2.

[0116] Table 2 Summary of column porosity data

[0117]

[0118]

[0119] ③Establish standard curve

[0120] When using HPLC for quantitative analysis, it is necessary to establish a standard curve for five standard samples of galactose solution with concentrations of 0.2, 0.25, 0.3, 0.35 and 0.4 g / L, and a standard curve for five standard samples of glucose with concentrations of 0.2, 0.3, 0.4, 0.5 and 0.6 g / L. A standard curve for five standard samples of galacto-oligosaccharide with concentrations of 1.2, 1.3, 1.4, 1.5 and 1.6 g / L is required. Figure 2 shown.

[0121] Under the selected standard concentration, the relationship between concentration and peak area can be established, and a fitting curve is formed with the peak area of ​​the chromatogram as the horizontal axis and the sample injection concentration as the vertical axis.

[0122] (2) Screening the stationary phase resins, and comparing the advantages and disadvantages of different resins in the separation system in terms of separation degree, selectivity, porosity and retention time through the pulse experiment method to determine the stationary phase resin.

[0123] The separation degree is defined as the ratio of the difference between the retention values ​​of two adjacent chromatographic peaks to half the sum of the base widths of the two chromatographic peaks.

[0124]

[0125] Where t R1 and t R2 are the retention times of the two components, W t1 and W t2 is the base width of the chromatographic peak of the corresponding component.

[0126] The selectivity can be used to measure the difference in adsorption capacity of two components. The selectivity of different resins can be calculated by the following formula.

[0127]

[0128] Where k 1 , k2 is the retention factor of different substances, where k 2 Greater than k 1 . Where t R,i is the retention time of compound i, t 0 is the dead time. The formula is used to evaluate the separation effect of two different compounds in the chromatographic process. The larger the selectivity α value, the better the separation between the two compounds.

[0129] When the selectivity, retention time and half-peak width are known, the column efficiency under different resins can be calculated by the following formula.

[0130]

[0131] Where N i is the column efficiency, α is the selectivity, t R,i is the retention time of the substance, W h / 2,i is the half peak width.

[0132] The separation, selectivity, column efficiency, stability and other parameters of oligogalactose, galactose and glucose on different resins were investigated, and the strong acid cation exchange resin ZGSPC106K was selected. + Resins are suitable for use in the present invention.

[0133] (3) Pulse experiment to determine the adsorption isotherm

[0134] Generally speaking, the concentration of industrial galacto-oligosaccharide crude products is within the range of (57%-65%), in which the main impurities are galactose and glucose. Combined with the operability of the experiment, 40g / L galacto-oligosaccharide, 10g / L galactose and 10g / L glucose solutions were prepared for the experiment.

[0135] The temperature condition is 50℃; the mobile phase is ultrapure water; the flow rate is 10mL / min to flush the chromatographic column until the baseline of the differential detector is flushed and the baseline is zeroed. For the accuracy of the experiment, the quantitative loop is flushed with a pre-configured solution of the same concentration so that the six-way valve will not reduce the injection concentration due to the ultrapure water, causing errors in the experiment. The manual valve of the six-way valve is rotated and the timer button of the differential detector is pressed at the same time. The elution curves of oligogalactose, galactose and glucose are recorded at five flow rates of 2mL / min, 4mL / min, 6mL / min, 8mL / min and 10mL / min. According to the retention time of the elution curve at different flow rates, the adsorption isotherm parameters are calculated using the following formula (7).

[0136]

[0137] In the formula, H i is the Henry constant of component i, u is the linear velocity (or flow rate) in mL / min, ε is the porosity, tR is the retention time, in min, V c is the volume of the chromatographic column, in cm 3 , L is the length of the chromatographic column, in cm, and r is the inner radius of the chromatographic column, in cm. The results of the pulse experiments of galacto-oligosaccharides, galactose and glucose are shown in Table 3-5.

[0138] Table 3 Results of galacto-oligosaccharide pulse experiment

[0139] 2 4 6 8 10 <![CDATA[u i ]]> 0.773 1.547 2.320 3.094 3.867 <![CDATA[t R,i (Retention time)]]> 155.097 78.097 51.863 38.824 31.203 <![CDATA[W h / 2,i (half peak width)]]> 38.113 21.701 15.786 12.908 11.433 <![CDATA[N i (Column Efficiency)]]> 23.267 18.572 15.975 13.959 11.940 <![CDATA[H i (Henry's constant)]]> 0.288 0.294 0.291 0.289 0.293 <![CDATA[λ i ]]> 0.00555 0.00559 0.00557 0.00548 0.00540

[0140] Table 4 Glucose pulse test results

[0141] 2 4 6 8 10 <![CDATA[u i ]]> 0.773 1.547 2.320 3.094 3.867 <![CDATA[t R,i (Retention time)]]> 178.288 90.740 60.617 45.283 35.123 <![CDATA[W h / 2,i (half peak width)]]> 17.222 10.712 8.512 7.266 6.403 <![CDATA[N i (Column Efficiency)]]> 150.575 102.898 75.056 59.930 48.234 <![CDATA[H i (Henry's constant)]]> 0.418 0.436 0.438 0.434 0.423 <![CDATA[λ i ]]> 0.00609 0.00613 0.00613 0.00612 0.00610

[0142] Table 5 Galactose pulse experimental results

[0143] 2 4 6 8 10 <![CDATA[u i ]]> 0.773 1.547 2.320 3.094 3.867 <![CDATA[t R,i (Retention time)]]> 193.642 96.725 64.628 47.832 38.380 <![CDATA[W h / 2,i (half peak width)]]> 17.283 11.819 9.601 8.155 7.718 <![CDATA[N i (Column Efficiency)]]> 210.387 109.706 74.220 59.826 45.600 <![CDATA[H i (Henry's constant)]]> 0.503 0.502 0.505 0.491 0.494 <![CDATA[λ i ]]> 0.00622 0.00622 0.00622 0.00621 0.00621

[0144] Table 3-5 is based on ZGSPC106K + The basic parameters of the three components obtained from the pulse experiment at different flow rates under the resin include H i and λ i The accurate determination of these parameters is crucial for simulating and optimizing the SSMB purification process. In addition, the Henry constants of each component are relatively stable at different flow rates, and it can be preliminarily inferred that the oligogalactose system belongs to linear adsorption.

[0145] (4) Determination of adsorption isotherms by frontal analysis

[0146] The present invention selects pulse experiment and pre-analysis method to combine the two methods to achieve comprehensive and accurate determination of adsorption isotherm, so as to better verify the accuracy of kinetic parameters and adsorption isotherm.

[0147] First, balance the chromatographic system with the mobile phase, and use ultrapure water to prepare 55, 60, 65, 70, 75, and 80 g / L oligosaccharide solutions. Use ultrapure water to flush the chromatographic column at a flow rate of 8 mL / min, open the differential detector purge button to flush the reference cell until the baseline is stable, switch to the lowest concentration (55 g / L) oligosaccharide solution as the mobile phase to start the experiment, the chromatographic pump flow rate is 8 mL / min, the platform rises to stabilize for more than 15 minutes, and the breakthrough curve of the current concentration is obtained. It is considered that the chromatographic column has reached equilibrium, and the next concentration sample is switched to detect 60, 65, 70, 75, and 80 g / L oligosaccharide solutions respectively. Stop recording, save data, open the purge valve, and flush the pipeline, RI detection cell, and sample cell with deionized water.

[0148] The concentrations of glucose were 5, 10, 15, 20, 25, and 30 g / L, and the concentrations of galactose were 0.5, 1, 1.5, 2, 2.5, and 3 g / L. The same experimental steps were used to perform frontal analysis.

[0149] The adsorption isotherm was calculated using formula (8):

[0150]

[0151] In the formula, q j is the mass of adsorbed substance per unit volume of resin in the jth step, in g, V c is the column volume in cm 3 , t 0 is the system dead time, in min, t R is the breakthrough time of the jth step, and ε is the void fraction of the chromatographic column, dimensionless. The calculation results are shown in Table 6.

[0152] Table 6 Summary of c and q* of galacto-oligosaccharides / glucose / galactose

[0153]

[0154]

[0155] In the aforementioned pulse experiment, the adsorption isotherm parameters were obtained by measurement, and a preliminary fitting was performed based on them. The results show that galacto-oligosaccharides, galactose and glucose exhibit linear adsorption characteristics on the screened stationary phase. Therefore, a linear adsorption model can be applied to fit the adsorption isotherms of these substances in the frontier analysis method, as shown in formula (24). In the frontier analysis method, by configuring galacto-oligosaccharides, galactose and glucose at different concentrations, a specific mathematical model can be used to describe the adsorption process. Substituting these parameters and different concentration values ​​into the adsorption isotherm equation of formula (8), the adsorption amount at different concentrations can be calculated.

[0156] q i =H i c i (twenty four)

[0157] In the formula, H i is the Henry constant for component i, q i is the mass of adsorbed substance per unit volume of resin in step j, in g / L, c i It is the concentration of substance in g / L.

[0158] exist Figure 3-5 are respectively the adsorption amounts of galacto-oligosaccharide, glucose and galactose q i With concentration c iThe Henry constant H can be obtained from the slope of the fitting curve (i.e. adsorption isotherm). i The fitting results obtained Henry constants for galacto-oligosaccharides (H=0.2915), galactose (H=0.496), and glucose (H=0.43046), and the accuracy of the adsorption isotherms was further verified by comparing with the pulse experiment.

[0159] (5) Determination of kinetic parameters

[0160] After the dead volume, void ratio and related column parameters were determined, the transfer behavior of oligogalactose, galactose and glucose was fully considered in the modeling process, and the kinetic parameters of the separation system were determined and calculated. m (mass transfer coefficient) and N L (axial diffusion coefficient). According to the single-component pulse experiments of oligogalactose, galactose and glucose, the retention time, half-peak width of the chromatographic peak and the porosity at the corresponding concentration and flow rate can be obtained. The kinetic parameters are fitted according to formula (9), using uλ, N at different flow rates. L The slope and intercept obtained by fitting are used to obtain k m,i and N L .

[0161]

[0162] Where, t R is the retention time, W 2 / h H is the half peak width. i is the Henry constant for component i, D L,i is the diffusion coefficient of component i in the mobile phase, N i The column efficiency of component i (indicating the efficiency of the chromatographic column), u is the linear velocity, L is the length of the chromatographic column, is the void ratio, λ i is the resistance coefficient, K m is the mass transfer coefficient, N L is the axial diffusion coefficient. When the flow rate is within a reasonable range, 1 / N L,i It is linearly related to u.

[0163] According to the previous single-component pulse experiments of oligogalactose, galactose and glucose, the retention time, half-peak width of the chromatographic peak and the void fraction at the corresponding concentration and flow rate can be obtained. i is the horizontal axis, 1 / N i Plot the graph on the ordinate and perform fitting of kinetic parameters. Figure 6-8 The 1 / Ni and uλi fitting diagrams of oligogalactose, glucose and galactose are shown respectively. The mass transfer coefficient k can be solved by using the intercept and slope of the fitting formula respectively. m and the axial diffusion coefficient N L:Among which galacto-oligosaccharide: k m =0.418, N L =30.769; galactose k m =1.144, N L =1666.67; glucose k m =1.344, N L =344.828.

[0164] (6) Process simulation of the two-step coupling experiment of the sequential moving bed and experimental verification

[0165] Through the determination of chromatographic model parameters for galacto-oligosaccharides, galactose and glucose, the SSMB operating parameters were preliminarily designed according to the triangle theory and the corresponding two-step coupling experiment was completed. The experimental results were verified by process simulation in Fortran.

[0166] This experiment adopts a two-step coupling process. The first step is to separate the three components of galacto-oligosaccharides, galactose and glucose, mainly to obtain the target product galacto-oligosaccharides; the second step is to further recover the galactose in the extracted impurity components.

[0167] The first step of simulation experiment is mainly to obtain the target product galacto-oligosaccharide. Through SSMB technology, galacto-oligosaccharide is separated from other components (galactose and glucose).

[0168] Based on the Henry constants of oligogalactose (H = 0.2915), galactose (H = 0.496), and glucose obtained in pulse experiments and frontier analysis experiments, m can be determined by triangle theory. i Value range: filter m in different regions i Value, screening rule: 0.496<m Ⅰ <5 (according to the upper limit of the pump flow), 0.2915<m Ⅱ <0.496,0.2915<m Ⅲ <0.496,m Ⅳ <0.2915,m Ⅱmin <m Ⅱ <m Ⅲ <m Ⅲmax .

[0169] The switching time t of each region at different stages is calculated by (12)-(17) 1 ,t 2 ,t 3 , and the complete switching time t s and feed flow rate Q F In a sequential simulated moving bed (SSMB) system, Q i represents the flow rate in the i-th segment, Vc is the column volume, ε represents the void fraction, t 1 ,t 2 ,t 3 is the switching time. m i It is a dimensionless parameter that represents the net flow ratio of the four different areas of SSMB.

[0170]

[0171] ts=t 1 +t 2 +t 3 (16)

[0172]

[0173] In order to completely separate oligolactose from galactose + glucose, the total switching time t s It is greater than the retention time of galacto-oligosaccharides and less than the retention time of galactose and glucose, i.e., formula (18), which ensures that high-purity galacto-oligosaccharides can be obtained at the residue outlet and galactose and glucose can be completely flushed out at the extraction outlet.

[0174] ts>t GOS ,ts <t glucose ,ts <t galactose (18)

[0175] Among them, t GOS ,t glucose ,t galactose They are the retention time of galacto-oligosaccharide, glucose and galactose respectively, in min.

[0176] Substituting the above parameters into the Fortran program simulation, according to the running formulas (19)-(22), the purity (Pur), unit treatment capacity (UT), water consumption (WC), yield (Rec), and raffinate outlet flow rate (Q) in the first step of the SSMB simulation experiment can be obtained. raf ) and other parameters, where c is the substance concentration in g / L.

[0177]

[0178] The second step is simulation experiment: based on the first step, the target product, high-purity oligosaccharide, is obtained. For different components, the Henry constants of galactose and glucose obtained in the pulse experiment and frontier analysis experiment can be used to determine m i The range of values. Through the Henry constants of galactose and glucose, the m values ​​in different regions are screened: 0.496<m Ⅰ <5,0.430<m Ⅱ <0.496,0.430<mⅢ <0.496,m Ⅳ <0.430,m Ⅱmin <m Ⅱ <m Ⅲ <m Ⅲmax .

[0179] According to equations (12)-(17), we can calculate m Ⅰ 、m Ⅱ 、m Ⅲ 、m Ⅳ The flow ratio of each area m i ', complete switching time t s '、t 1 '、t 2 '、t 3 'Switching time and feed flow Q F '.

[0180] In order to completely separate galactose and glucose, the complete switching time t s 'Need to be greater than the retention time of galactose (t galactose ) and is less than the retention time of glucose (t glucose ). Through this design (Formula 23), it is ensured that high-purity galactose can be obtained at the raffinate outlet, and glucose can be completely flushed out at the extraction outlet.

[0181] Glucose retention time (t glucose )>ts'>Retention time of galactose (t galactose ) (twenty three)

[0182] The above parameters are introduced into the simulation program. According to equations (19)-(22), the second step simulation experiment process (Pur'), ​​unit treatment capacity (UT'), water consumption (WC'), yield (Rec'), and raffinate outlet flow rate (Q raf ') and other results, and verified with the experimental results.

[0183] The specific simulated moving bed experiment used a four-column system, which was carried out at a column temperature of 50°C and a circulation flow rate of 10 mL / min. The automatic control of the experiment was achieved through Wincc Runtime Start software.

[0184] Five groups of experiments were designed based on the triangle theory. The specific experimental conditions are shown in Tables 7-8. The experimental operation of SSMB is divided into three stages: feeding stage, circulation stage and elution stage (e.g. Figure 1). In the feeding stage, by turning the feeding valve, the sample is pumped into the simulated moving bed using a feeding pump, while the feeding is completed in column 1 and the eluent is introduced into column 3. In the circulation stage, the feed solution circulates in the column and is fully distributed between the stationary phase and the mobile phase, which is a key step in saving water consumption. In the elution stage, the 4 columns are disconnected to connect columns 1, 2, and 3, so that a high-purity oligosaccharide sample can be taken at the raffinate port. The experiment is planned to carry out 16 cycles, and the number of cycles is adjusted by dynamically monitoring the purity of the extract port and the raffinate port. After the first step is stable, the solution at the extract port in the first step is introduced into the feed port for a second separation, and the above operation is repeated. Before the experiment starts, the system needs to be flushed, and the switching time and method are input into the control system. The experiment starts feeding from the second cycle, and samples are collected from the tenth cycle, and then every two cycles. The collected samples will be tested by HPLC until the purity of the samples does not increase for two consecutive times. At this time, the entire SSMB system will be flushed again, and the solution obtained from the extraction port in the first step will be fed into the second cycle of the experiment. The above operation will be repeated and the collected samples will be tested by HPLC until the purity does not increase.

[0185] Table 7 Comparison of SSMB first step separation experiment and process simulation results

[0186]

[0187] Table 8 Comparison of SSMB second step separation experiment and process simulation results

[0188]

[0189] The SSMB separation experimental results in Tables 7-8 show that by optimizing the operating parameters, the purity of oligogalactose can reach 98%, and the purity of galactose can reach 95%, and the parameter optimization results are close to the experimental results, so the experimental operating conditions can be accurately determined. The simultaneous separation and purification of oligogalactose and galactose can be achieved through this two-step coupled design. It is proved that the present invention uses the preset parameters of the trigonometric theory, and uses the Fortran program simulation method to verify the guiding value of the experimental results in practical applications.

[0190] (7) Determine the optimal operating range of SSMB based on multi-objective optimization of genetic algorithm

[0191] After verifying the accuracy of the model, a genetic algorithm was used to perform multi-objective optimization in Fortran based on process simulation. Through efficient numerical screening, the optimal operating conditions of the separation process were obtained, providing better guidance for the experiment.

[0192] Specifically, a set of multi-objective optimization results are obtained by simulation using Fortran program. Fig.10It can be seen that by taking the simultaneous improvement of galacto-oligosaccharide purity and raw material processing volume as the optimization goal, and taking the recovery rate greater than 80% and the purity greater than 90% as the constraints, the purity of galacto-oligosaccharide exceeding 90% can be successfully achieved by appropriately selecting decision variables in SSMB through genetic algorithm.

[0193] Figure 11-12 The constraints are the value of m with a recovery greater than 80% and a purity greater than 90%, and the corresponding operating condition t 1 ,t 2 ,t 3 and Q F .from Fig.11 It can be seen that all operating points are within the parameter range preset by the simulation program in step (6). I It increases slowly with the increase of purity, indicating that the total switching time t s It increases gradually. IV The increase in the cycle step time t 2 Since the separation is mainly carried out in the circulation step, m IV The increase in helps to separate the target product more thoroughly, thereby improving its purity. Fig.12 In the process, the feed flow rate Q F As purity increases, it decreases, which may be Fig.12 Medium III The decrease of t 2 As purity increases, this is related to m IV The trend is consistent, which also indirectly reflects the accuracy of the simulation program of the present invention.

[0194] In summary, the two-step coupled sequential simulated moving bed two-step coupled separation and purification method of oligogalactose of the present invention, through process simulation and parameter optimization of the two-step coupling experiment, uses experiments to determine the accuracy of the simulation, and performs multi-objective optimization design of operating conditions to determine the optimal operating range of the separation process, which not only provides an efficient and clean separation and purification method for the industrial production of oligogalactose and galactose, but also provides a reference for the separation and purification of other biochemical products and has broad application prospects.

Claims

1. A method for separating and purifying oligogalactose by two-step coupling of a sequential simulated moving bed, characterized in that: The steps include: (1) determining basic parameters of a fixed bed chromatographic column, wherein the basic parameters include dead volume and void ratio, and establishing standard curves of galactose, glucose and galacto-oligosaccharide; (2) Screening stationary phase resins by pulse experiment method; (3) Pulse experiments to determine the adsorption isotherms of galactose, glucose and galacto-oligosaccharides; (4) Determination of adsorption isotherms of galactose, glucose and galacto-oligosaccharides by frontier analysis; (5) Determination of kinetic parameters of galactose, glucose and galacto-oligosaccharides; (6) Process simulation of the sequential moving bed two-step coupling experiment and experimental verification; Among them, in the first step of the simulation experiment: the switching time is greater than the retention time of galacto-oligosaccharide, and less than the retention time of galactose and glucose; The second step of simulation experiment: the switching time is greater than the retention time of galactose and less than the retention time of glucose; (7) Based on the multi-objective optimization of genetic algorithm, the optimal operating range of the two-step coupled separation and purification of oligosaccharides by sequential simulated moving bed was determined.

2. The method according to claim 1, characterized in that The dead volume in step (1) is calculated according to formula (1): V D =Q0t0 (1) Where V D is the dead volume of the system, in mL; t0 is the dead time of the system, in min; Q0 is the flow rate of the chromatographic pump corresponding to the dead time, in mL / min; The porosity of the chromatographic column is calculated according to formula (2): Where, t R,i is the retention time of component i, in min; L is the length of the chromatographic column, in cm; u is the flow rate, in mL / min; ε is the porosity; V c is the volume of the chromatographic column in cm 3 ; Q is the flow rate, the unit is mL / min.

3. The method according to claim 1, characterized in that The stationary phase resin is a strongly acidic cation exchange resin; Step (3) uses formula (7) to calculate the adsorption isotherm parameters, In the formula, H i is the Henry constant of component i, u is the flow rate in mL / min, ε is the porosity, t R,i is the retention time of component i, in min, V c is the volume of the chromatographic column, in cm 3 , L is the length of the chromatographic column, in cm, and r is the inner radius of the chromatographic column, in cm.

4. The method according to claim 1 or 2, characterized in that: Step (4) uses formula (8) to calculate the adsorption isotherm: In the formula, q j is the mass of adsorbed substance per unit volume of resin in step j, in g / L, V c is the volume of the column in cm 3 , t0 is the system dead time, unit is min, t B is the breakthrough time of the jth step, in min.

5. The method according to claim 1, characterized in that Step (5) uses formula (9) to fit the kinetic parameters, using uλ, N at different flow rates L Fit the slope and intercept to get k m,i and N L : Where W 2 / h H is the half peak width; i is the Henry constant of component i; D L,i is the diffusion coefficient of component i in the mobile phase; N i is the column efficiency of component i; u is the flow rate, in mL / min; L is the length of the chromatographic column, in cm; is the void ratio; i is the resistance coefficient; k m is the mass transfer coefficient; N L is the axial diffusion coefficient.

6. The method according to claim 1, characterized in that Step (6) The first step of simulation experiment: Based on the Henry constants of oligogalactose, galactose and glucose, the complete separation area is determined according to the triangle theory to preliminarily design the operating parameters of the sequential simulated moving bed, and screen different areas m Ⅰ 、m Ⅱ 、m Ⅲ 、m Ⅳ The net flow ratio m i , and calculate the switching time t1, t2, t3, and complete switching time t of the feeding stage, circulation stage, and elution stage in the first step of the sequential simulation moving bed according to formulas (12)-(17). s And the feed flow rate Q F , and the system complete switching time t s Satisfies formula (18): ts=t1+t2+t3 (16) ts>t GOS ,ts<t glucose ,ts<t galactose (18) In the formula, Q i is the flow rate of the i-th segment, in mL / min; V c is the volume of the chromatographic column in cm 3 ; ε is the void ratio; t1, t2, t3 are the switching time of the feeding stage, circulation stage and elution stage, respectively, in min; t s is the switching time, in min; t GOS ,t glucose ,t galactose are the retention times of oligogalactose, glucose and galactose, respectively, in min; m i is m Ⅰ 、m Ⅱ 、m Ⅲ 、m Ⅳ Net flow ratio of four different areas, Q F is the feed flow rate, in mL / min; Q E is the circulation flow rate in mL / min.

7. The method according to claim 6, characterized in that The relevant parameters were introduced into the Fortran program for simulation, and the purity (Pur), unit throughput (UT), water consumption (WC), yield (Rec) and raffinate outlet flow rate (Q) were obtained according to formulas (19)-(22). raf ): Where c is the concentration of the substance in g / L.

8. The method according to claim 7, characterized in that The second step of simulation experiment: Based on the Henry constants of galactose and glucose, the net flow ratio m of different regions was screened according to the triangle theory. i ', according to formulas (12)-(17), the switching time t1', t2', t3' of the feeding stage, circulation stage and elution stage and the feed flow rate Q are calculated respectively F ', the switching time ts' satisfies formula (23), t glucose >ts’>t galactose (23) The relevant parameters were brought into the Fortran program for simulation, and the purity (Pur'), ​​unit throughput (UT'), water consumption (WC') and yield (Rec') were obtained according to equations (19)-(22).

9. The method according to claim 1, characterized in that: In step (6), a four-column system was used to simulate the moving bed experiment, and 40 g / L galacto-oligosaccharide, 10 g / L galactose and 10 g / L glucose solution were configured. The experiment was carried out at a column temperature of 50° C. and a circulation flow rate of 10 mL / min.

10. The method according to claim 6, characterized in that Step (6) Determine the complete separation area according to the triangle theory to preliminarily design the operating parameters of the sequential simulated moving bed and screen the flow ratio m of different areas. i : H A <m Ⅰ <5,H B <m Ⅱ <H A ,H B <m Ⅲ <H A ,m Ⅳ <H B ,m Ⅱmin <m Ⅱ <m Ⅲ <m Ⅲmax ; Among them, H A , H B are the Henry constants of substances A and B to be separated respectively; Preferably, in the first simulation experiment, H A , H B are the Henry constants of oligogalactose and galactose, respectively; in the second step simulation experiment, H A , H B are the Henry constants for galactose and glucose, respectively.

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