Method for maintaining solution stability in separation and purification process of oligosaccharide fermentation liquor
By adjusting the pH value and temperature of the oligosaccharide fermentation broth in the sequential simulated mobile bed chromatography system, the problem of unstable oligosaccharide solution during the separation and purification process is solved, and a higher separation effect and product purity is achieved.
Patent Information
- Application Number
- CN202411799065.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-14
AI Technical Summary
During the separation and purification process of oligosaccharide fermentation broth, especially in simulated mobile bed chromatography systems, it is prone to instability due to changes in temperature and pH, which in turn affects the separation effect and product purity.
By adjusting the pH value of the oligosaccharide fermentation broth in the sequential simulated mobile bed chromatography system to 6 to 8, and controlling the temperature to 40°C to 100°C, to reduce the changes in oligosaccharides and other components and maintain the stability of the solution.
The stability of the oligosaccharide solution is achieved during the separation and purification process, reducing component changes, and improving separation effect and product purity.
Smart Images
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Abstract
Description
Technical Field
[0001] The present application relates to the field of biotechnology, and in particular to a method for maintaining solution stability during the separation and purification of oligosaccharide fermentation broth. Background Art
[0002] Human milk oligosaccharides (hereinafter referred to as oligosaccharides) are the third largest solid component in human milk after lactose and fat, with a content of about 5 to 15 g / L. Oligosaccharides contain five monomers, namely: D-glucose, D-galactose, N-acylglucosamine, L-fucose and N-acetylneuraminic acid (sialic acid). The above five monomers can be combined in different proportions to form thousands of oligosaccharides. Currently, more than 200 types have been successfully identified. Common oligosaccharides include 2'-fucosyllactose (2'-fucosyllactose, 2'-FL), 3-fucosyllactose (3-fucosyllactose, 3-FL) and 6'-sialyllactose (6'-sialyllactose, 6'-SL). The reducing end of oligosaccharides is a lactose structure, on which the sugar chain is extended to form oligosaccharides with diverse structures. Based on the connected monomers, they can be divided into three main categories: (1) fucosylated neutral oligosaccharides; (2) sialylated acidic oligosaccharides; and (3) non-fucosylated neutral oligosaccharides.
[0003] At present, oligosaccharides are mainly produced by microbial fermentation. The separation and purification process of oligosaccharide fermentation broth mainly includes the following steps: 1) Rough separation: First, the oligosaccharide fermentation broth is subjected to solid-liquid separation to remove the bacteria, and then heated and added with activated carbon for adsorption and filtration to decolorize and remove protein, and then ion exchange and desalination are performed to obtain a preliminary purified oligosaccharide clear liquid; 2) Further purification: In addition to the oligosaccharide target substance, the clear liquid obtained above also contains other impurities such as lactose, galactose, glucose, isomers, etc. These sugar substances are more difficult to further separate. It is difficult to separate oligosaccharides, so continuous simulated moving bed chromatography (SSMB) can be used for separation to obtain relatively pure oligosaccharides; however, during the use of simulated moving bed SSMB, there will be certain separation conditions, because oligosaccharides are a type of oligosaccharides with high viscosity. In order to improve industrial production efficiency, the concentration of oligosaccharide liquid entering SSMB is about 20-70% (w / w), and the viscosity is relatively high. Generally, when passing through SSMB, it is necessary to increase the liquid temperature to maintain good fluidity and separation performance of the material. The liquid temperature is generally selected to be 40-60°C. Too low is easy to grow bacteria, and too high is not economical. In addition, the pH of the initial solution of oligosaccharides is generally around 5, and after entering SSMB, because the resin in SSMB will contain a part of hydrogen resin, the pH of the solution coming out is generally less than 5. Under this condition, it is very unfavorable for the stability of oligosaccharides, especially under 40-60°C, it is easy to degrade or decompose into other small molecules or change to generate other macromolecules. Summary of the invention
[0004] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide a method for maintaining the stability of the solution during the separation and purification of oligosaccharide fermentation broth, thereby achieving a better separation effect and obtaining a product of higher purity. The method provided in this application solves the stability of human milk oligosaccharide solution during the separation and purification process, including but not limited to the SSMB process, and under the condition of temperature of 40-60°C.
[0005] To achieve the above purpose, the technical solution adopted by this application is:
[0006] The present application provides a method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth, comprising the following steps:
[0007] The oligosaccharide fermentation liquid is sequentially subjected to bacterial removal, decolorization, electrodialysis desalination, ion exchange resin desalination, concentration, sequential simulated moving bed chromatography system purification, ultrafiltration, re-concentration and spray drying;
[0008] The pH of the oligosaccharide fermentation liquid after sterilization, decolorization, electrodialysis desalination, ion exchange resin desalination and concentration is adjusted to 6-8 and the temperature is adjusted to 40-100°C when passing through a sequential simulated moving bed chromatography system.
[0009] After extensive research and experiments, the inventors of the present application found that during the purification and separation process of the oligosaccharide solution, the pH of the oligosaccharide fermentation broth after bacteria removal, decolorization, electrodialysis desalination, ion exchange resin desalination, and concentration is adjusted to 6-8 and the temperature is 40°C-100°C when passing through a sequential simulated moving bed chromatography system. This can reduce the changes in oligosaccharides and other components in the purification and separation process, and ultimately achieve the purpose of maintaining the stability of the oligosaccharide solution at a certain temperature.
[0010] In some specific embodiments, the proportions of lactose, fucose, glucose, etc. in the oligosaccharide solution (such as fucose-lactose solution) using the above conditions of the present application will not change significantly during the purification and separation process, indicating that the technical solution of the present application can achieve the technical effect of maintaining the stability of the oligosaccharide solution.
[0011] As a preferred embodiment of the method for maintaining solution stability during the separation and purification of oligosaccharide fermentation broth described in the present application, the pH of the oligosaccharide fermentation broth after removal of bacteria, decolorization, electrodialysis desalination, ion exchange resin desalination, and concentration is adjusted to 6 to 8 and the temperature is 40°C to 60°C when passing through a sequential simulated moving bed chromatography system.
[0012] Preferably, the pH of the oligosaccharide fermentation liquid after bacteria removal, decolorization, electrodialysis desalination, ion exchange resin desalination and concentration is adjusted to 6 and the temperature is 40°C when passing through the sequential simulated moving bed chromatography system.
[0013] In the technical solution of the present application, the above preferred conditions are adopted when adjusting the oligosaccharide fermentation broth to pass through the sequential simulated moving bed chromatography system, which can better reduce the changes of oligosaccharides and other components and better improve the stability of the oligosaccharide solution.
[0014] As a preferred embodiment of the method for maintaining solution stability during the separation and purification of oligosaccharide fermentation broth described in the present application, an alkaline solution is used to adjust the pH of the oligosaccharide fermentation broth after sterilization, decolorization, electrodialysis desalination, ion exchange resin desalination, and concentration when it passes through a sequential simulated moving bed chromatography system.
[0015] As a preferred embodiment of the method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth described in the present application, the alkaline solution includes but is not limited to at least one of sodium hydroxide, potassium hydroxide, potassium phosphate, and potassium dihydrogen phosphate.
[0016] As a preferred embodiment of the method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth described in the present application, the oligosaccharide fermentation broth after sterilization, decolorization, electrodialysis desalination, ion exchange resin desalination, and concentration is kept warm for 24 hours to 168 hours when passing through a sequential simulated moving bed chromatography system. The oligosaccharide fermentation broth is kept warm in a water bath, and the materials are fed while being kept warm, in order to achieve a better separation effect.
[0017] As a preferred embodiment of the method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth described in the present application, the conditions for purification in the sequential simulated moving bed chromatography system include:
[0018] The running time of the four steps A, B, C, and D were manually set on the operation interface of the sequential simulated moving bed chromatography system. The four steps included the following conditions: step A was 339 s, step B was 169 s, step C was 169 s, and step D was 106 s.
[0019] The sequential simulated moving bed chromatography system is equipped with 6 resin columns, which are divided into 4 zones, namely Z1 (1 column), Z2 (2 columns), Z3 (2 columns), and Z4 (1 column). The 4 zones circulate in sequence in the 6 resin columns, and the inlet and outlet positions change with the changes of the four zones; in step A, the 6 columns are operated in series; in step B and C, they are operated simultaneously, in step C, water is fed into Z1 and a slow component is discharged, and in step B, material is fed into Z3 and a fast component is discharged; in step D, water is fed into Z1 and a fast component is discharged from Z3.
[0020] The running time is set to achieve a better separation effect. If the running time is changed, lactose, fucose, glucose and lactulose isomers cannot be separated and the purification effect cannot be achieved.
[0021] As a preferred embodiment of the method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth described in the present application, the conditions for purification in the sequential simulated moving bed chromatography system also include:
[0022] Manually set the flow rates of the four steps A, B, C, and D on the high-pressure infusion pump. The flow rate of the circulation pump is 50 mL / min, the flow rate of the volumetric pump is 50 mL / min, and the flow rate of the sample pump is 25 mL / min.
[0023] The sequential simulated moving bed chromatography system is equipped with three high-pressure infusion pumps and six resin columns, wherein the sample pump delivers the liquid to be separated, the volumetric pump delivers pure water, and the circulation pump provides circulation power for the six resin columns connected in series during step A.
[0024] Changing the flow rate and changing the running time actually change the feed volume. Different volumes of feed have different separation effects after being separated by resin columns, and different pumps can set different flow rates. The ultimate goal is to achieve better separation effects.
[0025] The present application adopts the above-mentioned sequential simulated moving bed chromatography system purification conditions, which can better reduce the component changes in the oligosaccharide solution separation and purification process and maintain the stability of the oligosaccharide solution.
[0026] As a preferred embodiment of the method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth described in the present application, the sequential simulated moving bed chromatography system is equipped with a water bath for heat preservation of the oligosaccharide fermentation broth during separation and purification.
[0027] After adjusting the pH of the oligosaccharide solution to 6-8, place it in a water bath for heat preservation and feeding. Set the running time of the four steps A, B, C, and D during the operation of the equipment, A (339s), B (169s), C (169s), and D (106s). Manually set the flow rate of each step on the high-pressure infusion pump, the circulation pump (50mL / min), the volumetric pump (50mL / min), and the sample pump (25mL / min). When the sample pump is working, it will bring the oligosaccharide solution with a pH of 6-8 and a temperature of 40℃-60℃ into the resin column equipped with this equipment for separation to obtain the target product.
[0028] As a preferred embodiment of the method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth described in the present application, the ultrafiltration conditions are: the ultrafiltration mesh is 2000D, the membrane flux is 35L / h, and the membrane area is 2.0m 2, membrane temperature is <35℃, membrane inlet pressure is 4.2MPa, heat exchanger pressure is 0.4MPa, and permeate membrane pressure is -2.3MPa.
[0029] As a preferred embodiment of the method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth described in the present application, the spray drying conditions are: inlet air temperature of 150°C, outlet air temperature of 98-100°C, and feed speed of 30rpm.
[0030] The present application effectively maintains the stability of the oligosaccharide solution at a certain temperature during the separation and purification process. In some specific embodiments, the fucose lactose fermentation broth treated by the method of the present application does not show obvious changes in the fucose lactose during the separation and purification process, and can maintain stability for a long time, thereby achieving a better separation and purification effect.
[0031] Compared with the prior art, this application has the following beneficial effects:
[0032] The present application provides a method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth. During the purification and separation process of the oligosaccharide solution of the present application, the pH of the oligosaccharide fermentation broth (such as fucose lactose solution) is adjusted to 6 to 8 and the temperature is 40°C to 60°C when passing through a sequential simulated moving bed chromatography system. This can reduce changes in oligosaccharides and other components during the purification and separation process, thereby achieving the purpose of maintaining the stability of the oligosaccharide solution, and ultimately achieving better separation and purification effects, obtaining higher purity products, and having higher application value. DETAILED DESCRIPTION
[0033] 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.
[0034] In the following 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.
[0035] Example 1
[0036] This embodiment provides a method for maintaining the stability of a fucose lactose solution, comprising the following steps:
[0037] 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 during the fermentation process, the glycerol concentration was controlled at a low level to increase the accumulation of the product, the substrate lactose mass concentration was controlled to be about (10±0.5) g / L, and the dissolved oxygen in the fermentation tank was (30±5)%, to obtain a fermentation liquid;
[0038] 2) The fucose lactose fermentation broth (isomers accounted for 1.05% of the total sugar area) was sequentially subjected to debacterialization, decolorization, electrodialysis desalination, ion exchange resin desalination, concentration, sequential simulated moving bed chromatography purification, ultrafiltration, reconcentration and spray drying to detect changes in the fucose lactose components.
[0039] 1. Bacteria removal: The LNnT fermentation liquid released from the fermentation tank was added into the Shijie 30nm ceramic composite membrane for sterilization. The membrane flux was 20L / h and the membrane area was 1.7m 2 , membrane temperature <40℃.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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 concentrated to 200-300 g / L.
[0044] 6. Purification: The above concentrate is purified by moving bed chromatography (SSMB) to collect the required components.
[0045] 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.
[0046] 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 materials to produce a fast component, step C feeds in water to produce a slow component, and step D feeds in water to produce a fast component.
[0047] 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).
[0048] Manually set the flow rate of each step on the high-pressure infusion pump, including the circulation pump (50 mL / min), the volumetric pump (50 mL / min), and the 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 different pH, different temperatures, and different heating times (as shown in Table 1).
[0049] 7. Ultrafiltration: Add the collected components after passing through SSMB into Shijie multifunctional membrane equipment (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 permeate membrane pressure -2.3MPa.
[0050] 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.
[0051] 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.
[0052] 10. Testing: Take a small amount of sample for component analysis.
[0053] Detection of fucose lactose:
[0054] Instrument: Shimadzu high performance liquid chromatograph, UV detector;
[0055] Column temperature: 60°C;
[0056] 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;
[0057] Flow rate: 1ml / min;
[0058] Column model: TSKgel Amide-80 5μm 4.6mmI.D.*25cm. Detection of fucose and lactose:
[0059] Instrument: Shimadzu high performance liquid chromatograph, differential detector;
[0060] Column temperature: 60°C;
[0061] Mobile phase: 0.5 mol sulfuric acid;
[0062] Flow rate: 0.5ml / min;
[0063] The column model is Aminex HPX-87H Column 300*7.8mm. The test results are shown in Table 1.
[0064] Table 1
[0065]
[0066]
[0067]
[0068] The results are shown in Table 1. The proportions of the fucose lactose solutions before heating were 98.88%, 98.65%, 98.74%, 98.80%, 94.95% and 49.89% after the pH was adjusted to 2, 4, 6, 8, 10 and 12, respectively. After adjusting the pH to 6-8 and heating at 40°C-60°C for 168h, the proportion of fucose lactose did not change significantly and no other miscellaneous sugars were generated.
[0069] Fucose lactose of other pH values will produce miscellaneous sugars such as fucose and lactose when heated at this temperature. In addition, fucose lactose solution will produce miscellaneous sugars such as fucose and lactose when heated at 80℃~100℃ after adjusting pH to 2, 4, 6, 8, 10 and 12. Although the changes in its components are not obvious when the experiment is carried out at 25℃, the solution will deteriorate and grow bacteria under this condition. Therefore, the stability of the solution cannot be maintained under this condition. In summary, the stability of the solution can be maintained by adjusting the pH of the fucose lactose solution to 6~8 and keeping it warm at 40℃~60℃.
[0070] 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 maintaining solution stability during separation and purification of oligosaccharide fermentation broth, characterized in that: The following steps are involved: The oligosaccharide fermentation liquid is sequentially subjected to bacterial removal, decolorization, electrodialysis desalination, ion exchange resin desalination, concentration, sequential simulated moving bed chromatography system purification, ultrafiltration, re-concentration and spray drying; The pH of the oligosaccharide fermentation liquid after sterilization, decolorization, electrodialysis desalination, ion exchange resin desalination and concentration is adjusted to 6-8 and the temperature is adjusted to 40-100°C when passing through a sequential simulated moving bed chromatography system.
2. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 1, characterized in that: The pH of the oligosaccharide fermentation liquid after sterilization, decolorization, electrodialysis desalination, ion exchange resin desalination and concentration is adjusted to 6-8 and the temperature is 40-60°C when passing through a sequential simulated moving bed chromatography system.
3. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 1, characterized in that: The oligosaccharide fermentation liquid after sterilization, decolorization, electrodialysis desalination, ion exchange resin desalination and concentration was adjusted to pH 6 and temperature 40°C when passing through a sequential simulated moving bed chromatography system.
4. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to any one of claims 1 to 3, characterized in that: The alkaline solution is used to adjust the pH of the oligosaccharide fermentation liquid after the removal of bacteria, decolorization, electrodialysis desalination, ion exchange resin desalination and concentration when it passes through the sequential simulated moving bed chromatography system.
5. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 4, characterized in that: The alkaline solution includes at least one of sodium hydroxide, potassium hydroxide, potassium phosphate and potassium dihydrogen phosphate.
6. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 1, characterized in that: The oligosaccharide fermentation liquid after being removed from bacteria, decolorized, desalted by electrodialysis, desalted by ion exchange resin and concentrated is adjusted to have a heat preservation time of 24 hours to 168 hours when passing through a sequential simulated moving bed chromatography system.
7. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 1, characterized in that: The conditions for purification in the sequential simulated moving bed chromatography system include: The running time of the four steps A, B, C, and D were manually set on the operation interface of the sequential simulated moving bed chromatography system. The four steps included the following conditions: step A was 339 s, step B was 169 s, step C was 169 s, and step D was 106 s.
8. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 1, characterized in that: The sequential simulated moving bed chromatography system is equipped with a water bath for heat preservation of oligosaccharide fermentation liquid after bacteria removal, decolorization, electrodialysis desalination, ion exchange resin desalination and concentration in the separation and purification process.
9. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 1, characterized in that: The ultrafiltration conditions are as follows: the ultrafiltration mesh is 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.
10. The method for maintaining solution stability during separation and purification of oligosaccharide fermentation broth according to claim 1, characterized in that: The spray drying conditions are: inlet air temperature is 150°C, outlet air temperature is 98-100°C, and feed speed is 30rpm.