Method for removing lactulose-based isomer in oligosaccharide separation and purification process

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for removing lactulose-based isomers in an oligosaccharide separation and purification process. The method comprises the following steps: taking oligosaccharide fermentation liquor for producing oligosaccharide through fermentation by using lactose as a substrate, sequentially carrying out thallus removal, decoloration, electrodialysis desalination, ion exchange resin desalination, concentration, sequential simulated moving bed chromatographic purification, ultrafiltration, re-concentration and spray drying, and detecting the oligosaccharide and the lactulose-based isomer in the oligosaccharide. According to the method disclosed by the invention, the content of lactulose-based isomers in the separation and purification process is greatly reduced, so that the product purity is effectively improved; the method can be applied to the synthesis process of other milk oligosaccharides such as fucose lactose (FL), lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT), lactose-N-fucopentasaccharide (LNPFI) and the like or other carbohydrates containing a lactosyl group, has a very high application value, and accords with laws and regulations.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for removing lactulose isomers during the separation and purification of oligosaccharides. Background Art

[0002] Common breast milk oligosaccharides include 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) and 6'-sialyllactose (6'-SL). Oligosaccharides are the third largest solid component in human milk after lactose and fat, with a content of about 5-15g / L. Oligosaccharides contain 5 monomers, namely: D-glucose, D-galactose, N-acylglucosamine, L-fucose and N-acetylneuraminic acid (sialic acid). The above 5 monomers can be combined in different proportions to form thousands of oligosaccharides, and currently more than 200 types have been successfully identified.

[0003] Breast milk oligosaccharides (hereinafter referred to as oligosaccharides) have four main functions: synergizing the development of microbiota, maintaining the integrity of the intestinal barrier, and promoting the development of the immune system and brain. Oligosaccharides in breast milk are an important factor affecting the composition and stability of infant intestinal flora. Infants who were breastfed in the early stage mainly showed a high abundance of Bifidobacteriaceae, while Actinobacteria and Bacteroidetes could be vertically transmitted from mother to infant. A Dutch study included 121 one-month-old infants who were exclusively breastfed. The study analyzed the relationship between oligosaccharides and infant intestinal bacteria and found that factors affecting the structure of infant fecal flora included gender, mode of birth, and the type and level of oligosaccharides, especially LNFPI and 2'-FL. Cesarean section affects the development of intestinal flora: especially the delay of bifidobacterium colonization in the first 3 months after birth, and fucosylated oligosaccharides in breast milk can alleviate the impact of cesarean section on infant intestinal microbiota.

[0004] Oligosaccharides have the function of maintaining the integrity of the intestinal barrier and intestinal health. A multicenter clinical study involving 789 infants compared the effects of different levels of HMO on intestinal development. The study was divided into four groups: breast milk group, formula milk group, formula milk + 1.5g / L oligosaccharide group and formula milk + 2.5g / L oligosaccharide group. The results showed that formula milk containing 5 types of oligosaccharides helps regulate immune function and intestinal barrier, and its intestinal bacteria composition is closer to that of the breastfed group.

[0005] Oligosaccharides can play a protective role by promoting the maturation of the intestinal mucosal immune system and maintaining the integrity of the gastrointestinal epithelial barrier of newborns. Studies have found that oligosaccharides can enhance the function of dendritic cells, thereby playing an important role in the development of the newborn immune system by recruiting regulatory T cells.

[0006] Nutrition is an indispensable condition for brain development, and breastfeeding provides adequate nutritional support for infants and young children. In addition, a large number of in vitro and in vivo studies have shown that human milk oligosaccharides are related to brain development in premature infants. Due to the immature brain development of premature infants, nutritional deficiencies are more likely to lead to poor brain and nerve development, which has become a new global public health issue. Since most oligosaccharides are not broken down by gastrointestinal digestive enzymes and maintain a high concentration in the colon, on the one hand, they promote the growth of beneficial bacteria, and on the other hand, they produce SCFAs to inhibit the growth of harmful bacteria, maintaining the homeostasis of the intestinal environment and affecting brain development and function through the brain-gut axis.

[0007] Breast milk is known as the gold standard for infant nutrition. Many infant formula companies have been conducting local breast milk research and continuously optimizing milk formula to make its nutritional level close to breast milk. Oligosaccharides, as an important nutrient in breast milk, have physiological functions such as antibacterial, antiviral, immune regulation and regulation of intestinal flora. Countries such as Europe and the United States have allowed the addition of oligosaccharides to milk powder. Although my country started late in the study of oligosaccharides, the efficacy research and industrial development of oligosaccharides have taken shape so far. The academic community has reached a scientific consensus on oligosaccharides. If oligosaccharides are successfully approved, they will surely lead to a major update of the formula of infant formula products in my country. In the future, it is still necessary to strengthen the research on the composition and content of oligosaccharides in regional breast milk to provide scientific guidance for fine formula design.

[0008] The current market requires oligosaccharide samples with high purity and lactulosyl isomer w / % ≤ 1%, but since oligosaccharides fermented with lactose as a substrate are generally accompanied by the production of lactulosyl isomers during the fermentation process, the purity of oligosaccharide samples and the content of lactulosyl isomers produced during the general separation and purification process are difficult to meet regulatory requirements. In order to produce oligosaccharide products that meet regulatory requirements, it is necessary to increase the purity of oligosaccharide products while reducing the content of lactulosyl isomers during the separation and purification process, so it is very necessary to optimize the separation and purification process. Summary of the invention

[0009] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a method for removing lactulose isomers during the separation and purification of oligosaccharides. The method of the present application greatly reduces the content of lactulose isomers in the oligosaccharide separation and purification process, thereby effectively improving the purity of the product.

[0010] To achieve the above purpose, the technical solution adopted by this application is:

[0011] The present application provides a method for removing lactulose isomers during oligosaccharide separation and purification, comprising the following steps:

[0012] Oligosaccharide fermentation liquid produced by fermentation with lactose as substrate is taken and sterilized, decolorized, desalted by electrodialysis, desalted by ion exchange resin, concentrated, purified by sequential simulated moving bed chromatography, ultrafiltered, re-concentrated and spray dried to detect oligosaccharides and lactulose isomers in oligosaccharides.

[0013] In the technical solution of the present application, a bacterial removal step is adopted to separate cells, bacterial bodies or other suspended particles; a decolorization step is adopted to remove pigments and residual proteins in the bacterial solution; an electrodialysis desalination step is adopted to utilize the selective permeability of ions in the solution to the ions of the ion exchange membrane under the action of an external DC electric field, so that anions and cations in the solution undergo ion migration and pass through the anion exchange membrane and cation exchange membrane respectively to achieve the purpose of desalination; an ion exchange resin desalination step is adopted to utilize the concentration difference to exchange ions to achieve the purpose of desalination; a concentration step is adopted to increase the concentration of the target product in the solution; a sequential simulated moving bed chromatography (SSMB) purification step is adopted to remove miscellaneous sugars such as lactose, fucose and lactulose isomers in the solution to obtain high-purity oligosaccharides; ultrafiltration is further adopted to purify the oligosaccharide solution; then a concentration step is adopted to obtain a high-concentration, high-purity oligosaccharide solution; and a spray drying step is adopted to finally obtain a high-purity oligosaccharide product.

[0014] After the oligosaccharide fermentation broth (lactulosyl isomers accounted for 1.05%) was separated and purified through processes such as sterilization, decolorization, and desalting, the proportions of oligosaccharide lactulosyl isomers were 1.25%, 1.43% and 1.94%, respectively, indicating that this conventional separation method cannot remove lactulosyl isomers in the oligosaccharide solution.

[0015] After extensive research and experiments, the inventors of the present application found that after the oligosaccharide fermentation broth was sterilized, deproteinized, decolorized, and desalted, an additional step of SSMB chromatography purification was added, in which the lactulose isomer accounted for 0.61%, which was 69% lower than before and met regulatory standards. Therefore, the method of the present application has a significant effect on reducing the lactulose isomers of oligosaccharides, improves the purity of oligosaccharides, and has strong application value.

[0016] As a preferred embodiment of the method for removing lactulose isomers in the oligosaccharide separation and purification process described in the present application, the step of removing bacteria includes: stirring the oligosaccharide fermentation liquid, passing it through a 30nm to 50nm ceramic membrane, and collecting the clear liquid.

[0017] The membrane flux of the ceramic membrane is 20 L / h and the membrane area is 1.7 m 2 , membrane temperature <40℃.

[0018] Preferably, the particle size of the ceramic membrane is 30 nm.

[0019] The present application adopts the above-mentioned step of removing bacteria, so that the oligosaccharides in the membrane can fully pass through the membrane pores to obtain a clear liquid, remove the bacteria, and improve the purity of the oligosaccharides.

[0020] As a preferred embodiment of the method for removing lactulose isomers during the separation and purification of oligosaccharides described in the present application, the decolorization step includes: concentrating the fermentation liquid after the bacteria have been removed, heating it to 60-90°C and keeping it warm for 30-60 minutes, adding activated carbon with a mass concentration of 1-4%, keeping it warm and stirring, and then filtering to obtain a decolorized liquid.

[0021] As a preferred embodiment of the method for removing lactulose isomers during the oligosaccharide separation and purification process described in the present application, the conditions for electrodialysis desalination are: the voltage is set to 35-45V (preferably 39V), and the electrodialysis ends when the conductivity of the liquid drops below 2000μs / cm.

[0022] As a preferred embodiment of the method for removing lactulose isomers in the oligosaccharide separation and purification process described in the present application, the ion exchange resin desalination step includes: passing the fermentation broth desalted by electrodialysis through an anion and cation exchange resin chromatography column at a flow rate of 1 to 5 BV / h (preferably 1.5 BV / h), first passing through the cationic resin and then passing through the anionic resin, and the conductivity of the obtained desalted solution is less than 20 μs / cm.

[0023] As a preferred embodiment of the method for removing lactulose isomers during the oligosaccharide separation and purification process described in the present application, the concentration conditions are: concentration temperature 55-65°C, tank pressure 0.09MPa, each concentration time ≤3h, and the fermentation broth desalted by ion exchange resin is concentrated to 200-300g / L.

[0024] As a preferred embodiment of the method for removing lactulosyl isomers in the oligosaccharide separation and purification process described in the present application, the steps of sequential simulated moving bed chromatography purification include:

[0025] The operation of the sequential simulated moving bed is divided into four steps: A, B, C, and D. Steps A and D are operated separately, and steps B and C are operated simultaneously. Step B is fed with material to produce a fast component, step C is fed with water to produce a slow component, and step D is fed with water to produce a fast component.

[0026] Manually set the running time of the four steps A, B, C, and D on the operation interface. Step A is 339s, step B is 169s, step C is 169s, and step D is 106s. Manually set the flow rate of each step on the high-pressure infusion pump. The flow rate of the circulation pump is 50mL / min, the flow rate of the volumetric pump is 50mL / min, and the flow rate of the sample pump is 25mL / min.

[0027] After all parameters are set correctly, switch to automatic mode and click on the system to start running; the feed is concentrated oligosaccharide fermentation broth.

[0028] Preferably, the solutes of the sequential simulated moving bed chromatography are fucose, lactose, glucose and lactulose isomers in the oligosaccharide fermentation broth; the stationary phase is an ion exchange resin (model 1840K or 1880K); and the mobile phase is pure water adjusted to pH 6-8.

[0029] The above-mentioned sequential simulated moving bed can utilize the differences in the forces between different solutes and the stationary phase and the mobile phase, such as distribution, adsorption, ion exchange, etc. When the two phases move relative to each other, each solute undergoes multiple equilibriums between the two phases, so that the solutes are separated from each other to obtain the target substance. The present application adopts the above-mentioned purification steps to significantly reduce the content of lactulose isomers in oligosaccharides.

[0030] As a preferred embodiment of the method for removing lactulose isomers in the oligosaccharide separation and purification process described in the present application, the ultrafiltration conditions are: the ultrafiltration mesh size is 1000D to 2000D (preferably 2000D), the membrane flux is 35L / h, and the membrane area is 1.7m 2 , membrane temperature is <35℃, membrane inlet pressure is 4.2MPa, heat exchanger pressure is 0.4MPa, and permeate membrane pressure is -2.3MPa.

[0031] As a preferred embodiment of the method for removing lactulose isomers during the oligosaccharide separation and purification process described in the present application, the re-concentration step includes: a concentration temperature of 50-60°C, a tank pressure of 0.09Mpa, each concentration time ≤3h, and concentrating the ultrafiltered fermentation broth to 200-300g / L.

[0032] As a preferred embodiment of the method for removing lactulose isomers during the oligosaccharide separation and purification process described in the present application, the spray drying conditions are: inlet air temperature is 120-180°C (preferably 150°C), outlet air temperature is 98-100°C, and feed speed is 30-40rpm (preferably 30rpm).

[0033] Compared with the prior art, this application has the following beneficial effects:

[0034] The present application provides a method for removing lactulose isomers during the separation and purification of oligosaccharides. The method of the present application can greatly reduce the content of lactulose isomers in the separation and purification process of oligosaccharides, thereby effectively improving the purity of the product. After the oligosaccharide fermentation broth (lactulose isomers account for 1.05%) is separated and purified through processes such as sterilization, decolorization, and desalting, the lactulose isomers in the oligosaccharide fermentation broth account for 1.25%, 1.43% and 1.94%, respectively. The inventors of the present application have found through extensive research and experiments that after the oligosaccharide fermentation broth is sterilized, deproteinized, decolorized, and desalted, an additional step of SSMB (sequential simulated moving bed) chromatography purification is added, in which the lactulose isomer accounts for 0.61%, which is 69% lower than before. Therefore, the method of the present application has a significant effect on reducing the lactulose isomers in oligosaccharides, better improves the product purity of oligosaccharides, has a strong application value, and meets regulatory standards. DETAILED DESCRIPTION

[0035] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0036] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0037] Example 1

[0038] This embodiment provides a method for removing lactulose isomers during the separation and purification of human milk oligosaccharides fucose and lactose, comprising the following steps:

[0039] 1) The seed liquid of the strain was inoculated into a fermentation tank medium containing lactose and glycerol (initial lactose mass concentration was 10 g / L, initial glycerol mass concentration was 20 g / L) according to an inoculum amount of 5% for fermentation culture, with a ventilation volume of 2 vvm, a rotation speed of 200 r / min, and a culture temperature of 37° C. During the fermentation process, the pH of the fermentation tank liquid was adjusted to 6.80 using 14% NH4OH by volume, and the glycerol concentration was controlled at a low level during the fermentation process to increase the accumulation of the product, the substrate lactose mass concentration was controlled at about (10±0.5) g / L, and the dissolved oxygen in the fermentation tank was (30±5)%, to obtain an oligosaccharide fermentation liquid;

[0040] 2) Taking the oligosaccharide fermentation broth (the area ratio of lactulosyl isomers to the total sugars is 1.05%), it is sequentially subjected to sterilization, decolorization, electrodialysis desalination, ion exchange resin desalination, concentration, sequential simulated moving bed chromatography purification, ultrafiltration, re-concentration and spray drying, and the oligosaccharides and oligosaccharide lactulosyl isomers are detected.

[0041] 1. Bacteria removal: The fucose lactose fermentation liquid released from the fermentation tank is added into the Shijie 30nm ceramic composite membrane for sterilization. The membrane flux is 20L / h and the membrane area is 1.7m 2 , membrane temperature <40℃.

[0042] 2. Protein removal and decolorization: After the sterilized fermentation liquid is concentrated, add it into a heating tank and heat it to 90°C for 30 minutes, add 4% (Jiangsu Zhuxi plant activated carbon) 305 wet carbon, keep it warm and stir for 30 minutes. Pass the decolorized liquid through a multi-layer filter to remove the activated carbon to obtain a decolorized liquid.

[0043] 3. Electrodialysis desalination: Add the decolorizing solution into the electrodialyzer for desalination, set the voltage to 39V, and end the electrodialysis when the liquid conductivity drops below 2000μs / cm.

[0044] 4. Desalination by ion exchange resin: The solution after electrodialysis desalination is passed through anion and cation exchange resin chromatography columns at a flow rate of 1.5 BV / h, first through the cation resin and then through the anion resin. The conductivity of the collected sample solution should be below 20μs / cm.

[0045] 5. Concentration: Concentrate the desalted liquid after ion exchange at a concentration temperature of 65°C and a tank pressure of 0.09 MPa. Each concentration time shall not exceed 3 hours and the concentration shall be 200-300 g / L.

[0046] 6. Purification: The above concentrate is purified by moving bed chromatography (SSMB) to collect the required components.

[0047] SSMB chromatography stands for Sequential Simulated Moving Bed. It utilizes the differences in the forces between different solutes and the stationary phase and the mobile phase, such as distribution, adsorption, and ion exchange. When the two phases move relative to each other, each solute undergoes multiple equilibriums between the two phases, allowing the solutes to be separated from each other to obtain the target substance.

[0048] The operation of the equipment is divided into four steps: A, B, C, and D. Steps A and D are operated separately, and steps B and C are operated simultaneously. Step B feeds in material and discharges fast components (for example, small molecular impurities, such as glucose and galactose), step C takes in water and discharges slow components (components with a larger molecular weight than fucose and lactose), and step D takes in water and discharges fast components.

[0049] Manually set the running time of the four steps A, B, C, and D on the operation interface: A (339s), B (169s), C (169s), and D (106s).

[0050] Manually set the flow rate of each step on the high-pressure infusion pump, including the circulation pump (50 mL / min), volumetric pump (50 mL / min), and sample pump (25 mL / min). After all parameters are set correctly, switch to automatic mode, click the system to start running, and feed the concentrated oligosaccharide fermentation broth with a pH of 6-8.

[0051] 7. Ultrafiltration: Add the collected components after passing through SSMB into the multifunctional membrane device (2000D) for ultrafiltration, and collect the membrane clear liquid. Membrane flux 35L / h, membrane area 1.7m 2 , membrane temperature <35℃, membrane inlet pressure 4.2MPa, heat exchanger pressure 0.4MPa, and membrane permeate pressure -2.3MPa.

[0052] 8. Concentration: Concentrate the membrane clear liquid after ultrafiltration in batches to 200g / L~300g / L, the concentration temperature is 50~60℃, the pressure in the tank is 0.09MPa, and the concentration time each time does not exceed 3h. Release the concentrated liquid for sampling and testing and prepare for spray drying.

[0053] 9. Spray drying: The above concentrate was spray dried with an inlet air temperature of 150°C, an outlet air temperature of 98-100°C, a feed speed of 30 rpm, and samples were collected and weighed approximately every 40 minutes.

[0054] 10. Detection: Take a small amount of sample for lactulose isomer detection.

[0055] Comparative Example 1

[0056] Comparative Example 1 directly uses the oligosaccharide fermentation broth obtained in Example 1 without performing step 2).

[0057] Comparative Example 2

[0058] Compared with Example 1, the difference is that only the sterilization step is performed on the oligosaccharide fermentation broth (the lactulose isomer accounts for 1.05% of the total sugar area), and steps 2-6 are not included. The remaining steps are the same as those in Example 1.

[0059] Comparative Example 3

[0060] Compared with Example 1, the difference is that only the steps of sterilization and decolorization are performed on the oligosaccharide fermentation broth (the area ratio of lactulosyl isomers to total sugars is 1.05%), and steps 3-6 are omitted. The remaining steps are the same as those in Example 1.

[0061] Comparative Example 4

[0062] Compared with Example 1, the difference is that the oligosaccharide fermentation broth (lactulosyl isomers account for 1.05% of the total sugar area) is only subjected to the steps of sterilization, decolorization, electrodialysis desalination, and ion exchange resin desalination, and does not contain steps 5-6. The remaining steps are the same as Example 1.

[0063] The above Example 1 and Comparative Examples 1-4 were subjected to oligosaccharide detection and lactulosyl isomer detection, and the specific detection conditions were as follows:

[0064] Oligosaccharide detection:

[0065] Instrument: Shimadzu high performance liquid chromatograph, UV detector;

[0066] Column temperature: 60°C;

[0067] Mobile phase: acetonitrile: 10 mM ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) mixed in a volume ratio of 7:3;

[0068] Flow rate: 1ml / min;

[0069] Column model: TSKgel Amide-80 5μm 4.6mmI.D.*25cm;

[0070] Lactulosyl isomer detection:

[0071] Instrument: Thermo Fisher high performance liquid chromatograph, charged aerosol detector (CAD);

[0072] Column temperature: 25°C; Nebulizer temperature: 35°C; Data acquisition rate: 20 Hz; Power function: 1; Filters: 5;

[0073] Flow rate: 1.1 mL / min;

[0074] Chromatographic column model: amino polymer column, 250mm×4.6mm, 5μm;

[0075] Mobile phase: A: water, B: acetonitrile (gradient elution);

[0076] The gradient elution conditions are shown in Table 1:

[0077] Table 1

[0078]

[0079] The results are shown in Table 2.

[0080] Table 2

[0081]

[0082]

[0083] By comparing the lactulose isomers in Example 1 and Comparative Examples 1-4 to the total sugar area ratio, it can be seen that the technical solution of the present application greatly reduces the content of lactulose isomers in the separation and purification process, thereby effectively improving the purity of the product. The lactulose isomers separated and purified by the method of Comparative Example 4 accounted for 1.94%, and the lactulose isomers separated and purified by the method of the present application accounted for only 0.61%, wherein the lactulose isomers accounted for 69%. Therefore, the effect of the present application is very significant and has a high application value.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A method for removing lactulose isomers during oligosaccharide separation and purification, characterized in that: The following steps are involved: Oligosaccharide fermentation liquid produced by fermentation with lactose as substrate is taken and sterilized, decolorized, desalted by electrodialysis, desalted by ion exchange resin, concentrated, purified by sequential simulated moving bed chromatography, ultrafiltered, re-concentrated and spray dried to detect oligosaccharides and lactulose isomers in oligosaccharides.

2. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process according to claim 1, characterized in that: The step of removing bacteria comprises: stirring the oligosaccharide fermentation liquid, passing it through a ceramic membrane with a diameter of 30nm to 50nm, and collecting the clear liquid.

3. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process as claimed in claim 1, characterized in that: The decolorization step includes: concentrating the fermentation liquid after removing bacteria, heating it to 60-90°C and keeping it warm for 30-60 minutes, adding activated carbon with a mass concentration of 1-4%, keeping it warm and stirring, and then filtering to obtain a decolorized liquid.

4. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process as claimed in claim 1, characterized in that: The conditions for the electrodialysis desalination are: the voltage is set at 35V to 45V, and the electrodialysis ends when the conductivity of the liquid drops below 2000μs / cm.

5. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process as claimed in claim 1, characterized in that: The ion exchange resin desalination step comprises: passing the fermentation liquid desalted by electrodialysis through an anion and cation exchange resin chromatography column at a flow rate of 1-5 BV / h, first through the cation resin and then through the anion resin, and the conductivity of the obtained desalted solution is less than 20 μs / cm.

6. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process according to claim 1, characterized in that: The concentration conditions are as follows: the concentration temperature is 55-65° C., the pressure in the tank is 0.09 MPa, and each concentration time is ≤3 hours, and the fermentation liquid desalted by the ion exchange resin is concentrated to 200-300 g / L.

7. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process according to claim 1, characterized in that: The steps of sequential simulated moving bed chromatography purification include: The operation of the sequential simulated moving bed is divided into four steps: A, B, C, and D. Steps A and D are operated separately, and steps B and C are operated simultaneously. Step B is fed with material to produce a fast component, step C is fed with water to produce a slow component, and step D is fed with water to produce a fast component. Manually set the running time of the four steps A, B, C, and D on the operation interface. Step A is 339s, step B is 169s, step C is 169s, and step D is 106s. Manually set the flow rate of each step on the high-pressure infusion pump. The flow rate of the circulation pump is 50mL / min, the flow rate of the volumetric pump is 50mL / min, and the flow rate of the sample pump is 25mL / min. After all parameters are set correctly, switch to automatic mode and click on the system to start running; the feed is concentrated oligosaccharide fermentation broth.

8. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process according to claim 1, characterized in that: The ultrafiltration conditions are as follows: the ultrafiltration mesh size is 1000D-2000D, the membrane flux is 35L / h, and the membrane area is 1.7m 2 , membrane temperature is <35℃, membrane inlet pressure is 4.2MPa, heat exchanger pressure is 0.4MPa, and permeate membrane pressure is -2.3MPa.

9. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process according to claim 1, characterized in that: The re-concentration step includes: a concentration temperature of 50-60° C., a tank pressure of 0.09 MPa, and a concentration time of ≤3 hours each time, and concentrating the ultrafiltered fermentation liquid to 200-300 g / L.

10. The method for removing lactulosyl isomers in the oligosaccharide separation and purification process according to claim 1, characterized in that: The spray drying conditions are: air inlet temperature of 120-180°C, air outlet temperature of 98-100°C, and feed speed of 30-40rpm.

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