Method for purifying 6-sialyllactose

Abstract

The invention relates to the technical field of biology, in particular to a method for purifying 6-sialic acid lactose. According to the 6-sialic acid lactase reaction liquid prepared based on multi-enzyme catalysis, the high-efficiency, high-purity and high-recovery-rate purification method of the 6-sialic acid lactase is developed by combining membrane separation and simulated moving bed chromatography technologies, meanwhile, less wastewater is generated, the cost benefit is high, and continuous large-scale production can be achieved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for purifying 6-sialyllactose. Background Art

[0002] Human milk oligosaccharides (HMOs) are a class of complex oligosaccharides, which are composed of monosaccharides and their derivatives, sialic acid and other structural units connected by glycosidic bonds. HMOs are the third largest solid component in human milk, second only to lactose and fat, and have many important physiological functions. Current studies have shown that 6-sialyllactose has good antibacterial activity and immunomodulatory function, and has a positive effect on improving intestinal flora microecology, maintaining intestinal barrier resistance to pathogen infection, etc., and provides important support for the brain development of infants and young children. The content of 6-sialyllactose has become a key nutritional indicator in infant formula.

[0003] Although 6-sialyllactose has important physiological effects, there is currently no rapid, large-scale, economical and safe production method in industry. Since the chemical synthesis of human milk oligosaccharides is full of challenges, multi-enzyme combined catalysis has been developed to produce human milk oligosaccharides. The process of multi-enzyme combined catalysis for the production of oligosaccharides is usually accompanied by the generation of a variety of side reaction products. The reaction solution usually includes unreacted substrates (lactose, glucose, fucose, galactose and acetylglucosamine, etc.), reaction intermediates (lactose-1-phosphate, galactose-1-phosphate and UDP-acetylglucosamine, etc.) and various side reaction products.

[0004] Especially in the multi-enzyme co-catalytic production process of 6-sialyllactose, due to the specificity of glycosyltransferase and the widespread presence of lactase in the crude enzyme solution, impurities such as 3-sialyllactose, 6-sialylgalactose, and 3-sialylgalactose are inevitably generated. In addition, lactose itself is easy to generate lactulose in the presence of isomerase and generate sialyllactulose under the action of subsequent glycosyltransferase. At the same time, sugars will also generate aldols or Maillard reactions at high temperatures. In most cases, the purification of human milk oligosaccharides is more difficult and the cost is relatively higher.

[0005] The existing process indicates that the aqueous medium containing the fermentation broth or the enzymatic reaction mixture can be pretreated by ultrafiltration, nanofiltration, activated carbon treatment or any combination thereof to obtain an aqueous solution containing the sialylated oligosaccharides, and the aqueous solution is treated with an H+-type strongly acidic cation exchange resin, and then treated with a free base type weakly basic anion exchange resin to obtain a sialylated oligosaccharide solution with higher purity. It is even simpler to purify / separate sialyllactose from contaminating carbohydrates by filtration using only two different membranes, and including two membrane filtration steps, one of which has a molecular weight cutoff of about 300 to about 500 Daltons, and the other of which has a molecular weight cutoff of about 600 to about 800 Daltons. In addition, simulated moving bed chromatography has certain advantages in the purification of human milk oligosaccharides. A patent indicates that the purification effect is achieved by combining membrane filtration and simulated moving bed chromatography on the fermentation broth.

[0006] At present, there are few studies on the downstream separation and purification of 6-sialyllactose in enzyme-catalyzed reaction liquids. The existing purification processes only use membrane separation and ion exchange to remove impurities such as sugars, so the purity is only about 90%, and there are problems such as a lot of wastewater, low cost-effectiveness, and difficulty in large-scale production. At the same time, a patent mentioned that human milk oligosaccharides can be finely purified by simulated moving bed chromatography, but the purity of the resulting product is also only 90%. Although simulated moving bed chromatography has a good separation effect, considering that the side reaction products and substrates in the enzyme-catalyzed liquid of 6-sialyllactose are highly similar to 6-sialyllactose in nature, the separation and purification effect of using a simulated moving bed alone is limited.

[0007] Therefore, the development of a high-purity and low-cost separation and purification method for 6-sialyllactose is of great significance for industrial production, product promotion, etc. Summary of the invention

[0008] In view of this, the present invention provides a method for purifying 6-sialyllactose.

[0009] The present invention provides a method for purifying 6-sialyllactose. Based on the 6-sialyllactase reaction solution prepared by multi-enzyme catalysis, the present invention develops a method for purifying 6-sialyllactose with high efficiency, high purity and high recovery rate by combining membrane separation and simulated moving bed chromatography technology, while generating less wastewater, being cost-effective and capable of continuous large-scale production.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides 1. A method for purifying 6-sialyllactose, characterized in that it comprises the following steps:

[0012] Step 1: heating the 6-sialyllactose enzyme reaction solution, centrifuging, and obtaining a 6-sialyllactose crude product reaction solution;

[0013] Step 2: taking the crude 6-sialyllactose reaction solution and subjecting it to ultrafiltration and nanofiltration to obtain a 6-sialyllactose filtrate;

[0014] Step 3: taking the 6-sialyllactose filtrate and subjecting it to simulated moving bed chromatography to obtain a 6-sialyllactose purified solution;

[0015] Step 4: taking the purified 6-sialyllactose solution and spray drying it to obtain a finished 6-sialyllactose product.

[0016] In some specific embodiments of the present invention, the centrifugation comprises: 8000 rpm, 30 min.

[0017] In some specific embodiments of the present invention, the 6-sialyllactose enzyme reaction solution in step 1 includes one or more of 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose or sialic acid.

[0018] In some specific embodiments of the present invention, the content of 6-sialyllactose is 81.2%; the content of 6-sialyllactulose is 7.5%; the content of 6-sialylgalactose is 6.9%; and the content of sialic acid is 4.4%.

[0019] In some specific embodiments of the present invention, the method for preparing the 6-sialyllactose enzyme reaction solution comprises the following steps:

[0020] Step (1): taking recombinant Escherichia coli BL21 cells containing exo-α-sialidase and mixing them with water, breaking the cells, and obtaining exo-α-sialidase enzyme solution;

[0021] Step (2): taking a mixture of the sialic acid solution and the lactose solution and mixing it with the exo-α-sialidase enzyme solution, stirring to obtain the 6-sialyllactose enzyme reaction solution.

[0022] In some specific embodiments of the present invention, the recombinant Escherichia coli BL21 cells containing exo-α-sialidase in step (1) are obtained according to the example in Chinese patent CN108220310A.

[0023] In some specific embodiments of the present invention, the cell disruption pressure in step (1) is 800 to 1000 bar.

[0024] In some specific embodiments of the present invention, the ratio of the recombinant Escherichia coli BL21 cells containing exo-α-sialidase to water in step (1) is 1:4 (m / m).

[0025] In some specific embodiments of the present invention, the concentration of the sialic acid solution in step (2) is 0.3 mol / L;

[0026] The concentration of the lactose solution is 0.4 mol / L.

[0027] In some specific embodiments of the present invention, the ratio of the sialic acid solution to the lactose solution in step (2) is 1:1 (v / v).

[0028] In some specific embodiments of the present invention, the method for preparing the 6-sialyllactose enzyme reaction solution specifically comprises:

[0029] (1) Preparation of exo-α-sialidase enzyme solution: according to the amount of enzyme required for the reaction, weigh an appropriate amount of recombinant Escherichia coli BL21 cells containing exo-α-sialidase, mix the cells with purified water at a ratio of 1:4 (m / m) and stir until there are no lumps, so that the bacterial solution is in a suspended liquid state; the bacterial solution is lysed twice at 5-10°C and a cell lysing pressure in the range of 800-1000 bar using a high-pressure homogenizer;

[0030] (2) Enzyme reaction: 0.3 mol / L sialic acid solution and 0.4 mol / L lactose solution were prepared and mixed in a ratio of 1:1 (v / v), and then exo-α-sialidase enzyme solution was added under stirring at 25°C, and the pH was adjusted to 6.5-7.5; the mixture was stirred at 25°C and 100 RPM for 6 h, and stirring was stopped after 6 h to terminate the reaction and obtain an enzyme reaction solution of 6-sialyllactose.

[0031] In some specific embodiments of the present invention, the heating temperature in step 1 is 40 to 90° C., and the heating time is 10 to 90 min.

[0032] In some specific embodiments of the present invention, the heating temperature is 50-70° C., and the heating time is 30-90 min.

[0033] In some specific embodiments of the present invention, the heating time is 30 to 60 minutes.

[0034] In some specific embodiments of the present invention, the molecular weight cutoff of the membrane used for ultrafiltration in step 2 ranges from 10 to 50 KDa.

[0035] In some specific embodiments of the present invention, the molecular weight cutoff of the membrane used for nanofiltration in step 2 ranges from 300D to 1000D.

[0036] In some specific embodiments of the present invention, the molecular weight cutoff of the membrane used in the nanofiltration is 500Da-1000Da.

[0037] In some specific embodiments of the present invention, the molecular weight cutoff of the membrane used in the nanofiltration is 600Da-1000Da.

[0038] In some specific embodiments of the present invention, the crude 6-sialyllactose reaction solution comprises 6-sialyllactose with a purity higher than 70-90%.

[0039] In some specific embodiments of the present invention, the crude 6-sialyllactose reaction solution comprises 6-sialyllactose with a purity higher than 80%.

[0040] In some specific embodiments of the present invention, the crude 6-sialyllactose reaction solution comprises 6-sialyllactose with a purity higher than 80-90%.

[0041] In some specific embodiments of the present invention, the simulated moving bed chromatography in step 3 comprises:

[0042] The 6-sialyllactose filtrate is loaded and eluted using at least 6 chromatographic columns;

[0043] The chromatographic column comprises at least two hydrogen-type cation chromatographic columns;

[0044] The loading speed includes 1 to 6 mL / min;

[0045] The eluent used for the elution includes a 5% to 10% potassium hydroxide aqueous solution and / or a 5% to 10% sodium hydroxide aqueous solution;

[0046] The elution rate includes 5 to 10 mL / min;

[0047] The simulated moving bed chromatography includes regions I, II, III and IV;

[0048] The flow rate of the region I includes 15 to 25 mL / min; the flow rate of the region II includes 13 to 20 mL / min; the flow rate of the region III includes 18 to 35 mL / min; the flow rate of the region IV includes 10 to 18 mL / min;

[0049] The switching time of the regions I, II, III and IV is comprised between 10 and 30 minutes.

[0050] In some specific embodiments of the present invention, the simulated moving bed chromatography in step 3 comprises:

[0051] The 6-sialyllactose filtrate is loaded and eluted using 6-8 chromatographic columns;

[0052] The chromatographic columns include 2 to 4 hydrogen-type cation chromatographic columns and / or 4 to 6 chloride-type anion chromatographic columns.

[0053] In some specific embodiments of the present invention, the hydrogen-type cation chromatography column comprises a sodium-type cation chromatography column and / or a calcium-type cation chromatography column.

[0054] In some specific embodiments of the present invention, the sodium type cationic chromatographic column comprises a combination of one or more of LX-160 cationic resin, LXP-08 cationic resin and LX-001 cationic resin.

[0055] The chloride-type anion chromatographic column comprises a combination of one or more of LXP-27 anion resin, LXP-207 anion resin and LXP-204 anion resin.

[0056] In some specific embodiments of the present invention, the elution liquid used is 10% potassium hydroxide aqueous solution and / or 5-10% sodium hydroxide aqueous solution.

[0057] In some specific embodiments of the present invention, the 6-sialyllactose filtrate comprises 6-sialyllactose with a purity higher than 85%.

[0058] In some specific embodiments of the present invention, the switching time of the four areas is 20 to 30 minutes.

[0059] In some specific embodiments of the present invention, the injection rate of the spray drying in step 4 includes 20 to 60 mL / min;

[0060] The inlet air temperature of the spray drying is 130-180°C, and the outlet air temperature is 80-120°C.

[0061] In some specific embodiments of the present invention, the injection speed of the spray drying in step 4 is 50 to 60 mL / min;

[0062] The inlet air temperature of the spray drying is 130-160°C, and the outlet air temperature is 80-100°C.

[0063] In some specific embodiments of the present invention, the pH value of the 6-sialyllactose filtrate is 3-7.

[0064] In some specific embodiments of the present invention, the pH of the 6-sialyllactose filtrate is 4-7.

[0065] In some specific embodiments of the present invention, the pH of the 6-sialyllactose filtrate is 6-7.

[0066] In some specific embodiments of the present invention, the purified 6-sialyllactose solution comprises 6-sialyllactose with a purity higher than 90% and / or higher than 95%.

[0067] In some specific embodiments of the present invention, the finished 6-sialyllactose product comprises 6-sialyllactose with a purity of ≥95% and / or ≥98%.

[0068] In some specific embodiments of the present invention, the finished 6-sialyllactose product comprises 6-sialyllactose with a purity of ≥98%.

[0069] The present invention provides a method for purifying 6-sialyllactose. The advantages of the present invention are as follows: (1) the heating pretreatment step after the enzyme reaction is completed can significantly reduce the occurrence of side reactions and the generation of impurities, such as significantly reducing the content of sialic acid, sialylgalactose and sialyllactulose, thereby reducing the pressure of subsequent purification;

[0070] (2) By adjusting the membrane separation process, the solution purity can be further improved, and the membrane separation itself has the ability to continuously process, meeting the requirements of low cost and high efficiency;

[0071] (3) The use of simulated moving bed chromatography can maintain high separation and purification effects while having the ability to process continuously, while generating less wastewater and reducing costs;

[0072] (4) Compared with the crystallization method, which is time-consuming, requires a large amount of organic solvent, and is accompanied by subsequent drying, the spray drying method for obtaining 6-sialyllactose has the advantages of low cost, high yield, and high processing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0074] Figure 1 6-sialyllactose purification steps are shown;

[0075] Figure 2 Shown is a physical picture of 6-sialyllactose. DETAILED DESCRIPTION

[0076] The present invention discloses a method for purifying 6-sialyllactose, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0077] The present invention is based on a 6-sialyllactose enzyme reaction solution prepared by multi-enzyme catalysis in the early stage. The preparation method of the enzyme reaction solution of 6-sialyllactose comprises: (1) preparation of exo-α-sialidase enzyme solution: according to the amount of enzyme required for the reaction, an appropriate amount of recombinant Escherichia coli BL21 bacteria containing exo-α-sialidase is weighed (the recombinant Escherichia coli BL21 bacteria containing exo-α-sialidase is obtained by the method in Chinese patent CN108220310A), and the bacteria are respectively mixed with purified water at a ratio of 1:4 (m / m) and stirred until there are no lumps, so that the bacterial solution is in a suspension state. The bacterial solution is lysed twice at 5-10° C. and a cell lysing pressure of 800-1000 bar using a high-pressure homogenizer. (2) Enzyme reaction: Prepare 0.3 mol / L sialic acid solution and 0.4 mol / L lactose solution, mix them in a ratio of 1:1 (v / v), then add exo-α-sialidase enzyme solution under stirring at 25°C, and adjust the pH to 6.5-7.5. Stir at 25°C and 100 RPM for 6 hours. After 6 hours, stop stirring to terminate the reaction and obtain 6-sialyllactose enzyme reaction solution.

[0078] By combining membrane separation and simulated moving bed chromatography technology, a method for purifying 6-sialyllactose with high efficiency, high purity and high recovery rate was developed, which also produces less wastewater, is cost-effective and can be produced on a continuous scale.

[0079] The invention provides a simple, high-yield and scalable method for obtaining high-purity 6-sialyllactose from an enzyme reaction solution, while removing other impurities in the reaction solution such as lactose, sialyllactulose and sialylgalactose.

[0080] The present invention provides a method for obtaining 6-sialyllactose from an enzyme reaction solution, wherein 6-sialyllactose with a purity of ≥95% and / or ≥98% is provided.

[0081] The present invention uses an enzyme reaction solution containing 6-sialyllactose and other impurities such as monosaccharides, disaccharides, sialylated monosaccharides and / or sialylated disaccharides as the initial material, and the enzyme reaction solution is obtained by the combined catalysis of multiple crude enzymes. Among them, the purity of 6-sialyllactose is 70%-90%. The enzyme reaction solution undergoes the following purification steps:

[0082] The solution needs to be pre-treated once, that is, heated, and filtered to remove solids to obtain a clear solution. This solution is an enzyme reaction solution, which contains a variety of crude enzymes and is not inactivated after the reaction. Side reactions will continue during the storage process. Therefore, heating the enzyme reaction solution after the reaction can reduce the purity of sialylated monosaccharide and sialylated disaccharide impurities to 5%, while the purity of 6-sialyllactose in the clear solution is higher than 80%;

[0083] The supernatant is subjected to at least two membrane filtration steps using an ultrafiltration membrane and a nanofiltration membrane. After standard operation, the purity of monosaccharides and disaccharides in the supernatant is reduced to 3%, while the purity of 6-sialyllactose in the supernatant is higher than 85%;

[0084] The filtrate after the membrane separation is subjected to a simulated moving bed chromatography step, wherein the purity of sialylated monosaccharide and sialylated disaccharide impurities in the obtained purified solution is reduced to less than 5%, and the purity of 6-sialyllactose is ≥95% and / or ≥98%;

[0085] The high-purity 6-sialyllactose purified solution is subjected to a spray drying step to obtain a white solid powder containing 6-sialyllactose;

[0086] The purity and content of 6-sialyllactose in the white solid powder are ≥95%, preferably ≥98%.

[0087] By the pretreatment step of heating and filtering as the first step of the inventive method, the enzyme is deactivated by heating, so as to reduce the generation of the side reaction product in the subsequent purification process, reduce the purification difficulty and improve the yield of 6-sialyllactose. Usually, due to the widespread existence of lactase and the lower substrate specificity and the lower substrate specificity of glycosyltransferase, the impurities such as sialic acid monosaccharide and sialic acid disaccharide will inevitably be introduced in the enzyme reaction liquid obtained by multi-crude enzyme combined catalysis, and along with the carrying out of the side reaction, the concentration and purity of 6-sialyllactose will also decline rapidly thereupon. Along with the gradual improvement of the reaction system, the material treatment time is extended, and the time when the side reaction occurs is also extended thereupon, and the content of by-product is gradually improved. By rapidly heating the enzyme reaction liquid after the reaction is terminated, the generation of side reactions can be effectively reduced, the decomposition rate of 6-sialyllactose is reduced and the purity of 6-sialyllactose is improved.

[0088] Subsequently, the filtered clear water is subjected to at least one ultrafiltration and nanofiltration by membrane separation to reduce high molecular weight polypeptides, polysaccharides, proteins and some solid insoluble particles from the desired medium molecular weight 6-sialyllactose, and can also reduce the content of lower molecular weight monosaccharides, disaccharides and amino acids. Different types of membrane materials can be used for membrane separation, and the membrane materials usually use organic polymer materials or inorganic materials such as ceramic membrane materials such as alumina.

[0089] In order to remove high molecular weight polypeptides, polysaccharides, proteins and solid insoluble particles, a membrane separation step is used, more specifically ultrafiltration separation. Ultrafiltration is a process in which higher molecular weight impurities are retained while medium or lower molecular weight sugars and amino acids are passed through by the pressure difference between the inside and outside of the membrane. The pore size of the ultrafiltration membrane in the present invention is between 10 kDa and 50 kDa.

[0090] In order to remove the content of monosaccharides, disaccharides and amino acid impurities with lower molecular weight, a membrane separation step is used, more specifically nanofiltration separation. Similarly, nanofiltration also allows sugars with higher molecular weight to be retained by the pressure difference between the inside and outside of the membrane, while monosaccharides, disaccharides and amino acid impurities with lower molecular weight are passed through. The pore size of the nanofiltration membrane in the present invention is between 300Da-1000Da, preferably between 500Da-1000Da, and more preferably between 600Da-1000Da.

[0091] Membrane separation is as second step of the present invention, wherein the purpose of the first step membrane separation is to use the ultrafiltration membrane of larger aperture to remove high molecular weight polypeptide, polysaccharide, protein and solid insoluble particles, retain medium or low molecular weight sugar. The purpose of the second step membrane separation is to use the nanofiltration membrane of smaller aperture to remove low molecular weight monosaccharide, disaccharide and amino acid substances, and retain the impurities such as 6-sialyllactose and sialic acid monosaccharide and sialic acid disaccharide of medium molecular weight, so that sialic acid monosaccharide and sialic acid disaccharide impurity can be removed in the subsequent simulated moving bed chromatographic separation step to obtain the higher 6-sialyllactose solution of purity. In the membrane separation process, along with the raising of membrane pore size, the removal effect of low molecular weight monosaccharide, disaccharide and amino acid substances strengthens gradually, but the yield of 6-sialyllactose also reduces thereupon.

[0092] After at least two steps of membrane separation, the resulting solution is subjected to simulated moving bed chromatography separation, which adjusts the content of different impurities such as sialic acid monosaccharide and sialic acid disaccharide impurities compared to 6-sialyllactose by adjusting relevant instrument and solution parameters. The solution used is a purified solution obtained by membrane separation, wherein the total content of sialic acid monosaccharide and sialic acid disaccharide impurities is less than 12%, preferably less than 8%, and the purity of 6-sialyllactose is higher than 80%.

[0093] Simulated moving bed chromatography separation as the third step of the present invention is to separate 6-sialyllactose from 6-sialyllactose and other sialic acid monosaccharides and sialic acid disaccharide impurities by continuous chromatography. Among them, with the adjustment of instrument and solution parameters, 6-sialyllactose may be present in the extract or the raffinate, and sialic acid monosaccharides and sialic acid disaccharide impurities are present in the raffinate or the extract. Through simulated moving bed chromatography separation, 6-sialyllactose with high purity can be provided in a continuous manner, which shows that the method is simple and easy to operate and has the potential for large-scale production. The purity of the obtained 6-sialyllactose is ≥95% and / or ≥98%.

[0094] The simulated moving bed chromatography separation step of this step includes:

[0095] (1) at least 6 chromatographic columns, preferably at least 8 chromatographic columns, of which at least two are hydrogen-type cation chromatographic columns, preferably sodium-type cation chromatographic columns;

[0096] (2) The flow rate is divided into four regions: I, II, III and IV, and the flow rates of the four regions I, II, III and IV are preferably: 15-25 mL / min for region I, 13-20 mL / min for region II, 18-35 mL / min for region III, and 10-18 mL / min for region IV;

[0097] (3) The eluent comprises part or all of the following substances: water, sodium chloride, potassium hydroxide, preferably 5%-10% potassium hydroxide, 90%-95% water, more preferably 5%-10% sodium hydroxide, 90%-95% water, the feed rate is 1-6 mL / min, and the eluent rate is 5-10 mL / min.

[0098] (4) The area switching time is 10-30 minutes, preferably 20-30 minutes.

[0099] (5) In the chromatographic column of simulated moving bed chromatography separation, cation and anion exchange resins are used as stationary phases. Preferably, the cation resin is selected from a combination of one or more of LX-160, LXP-08 and LX-001, and the anion resin is selected from a combination of one or more of LXP-27, LXP-207 and LXP-204.

[0100] Spray drying, as the last step of the present invention, mainly utilizes hot air to contact the material after physicalization to quickly vaporize the water and obtain a dry product. It is the first choice of solidification and drying process for the preparation of dairy products. Compared with crystallization, it does not require the use of organic solvents and can save the downstream drying and crushing steps. The solution used in this step is a purified solution obtained by simulated moving bed chromatography, wherein the purity of 6-sialyllactose is higher than 90% and / or higher than 95%. The inlet air temperature in the present invention is 130-180°C, preferably 130-160°C. The outlet air temperature is 80-120°C, preferably 80-100°C, and the flow rate is 20-60mL / min, preferably 50-60mL / min. Figure 2 As shown, the final product is a white solid powder, wherein the purity and content of 6-sialyllactose are ≥95%, preferably ≥98%.

[0101] 1. A method for obtaining high-purity 6-sialyllactose from an enzyme reaction solution, the method comprising:

[0102] - providing an enzyme reaction solution, wherein the enzyme reaction solution comprises 6-sialyllactose, non-6-sialyllactose sugars such as sialyllactulose, sialylgalactose, lactose, and a crude enzyme solution component;

[0103] -Heating the enzyme reaction solution and filtering to remove the solid precipitate to obtain a clear solution;

[0104] - using an ultrafiltration membrane and a nanofiltration membrane to perform at least two membrane filtration steps on the clear solution to provide a purified solution containing 6-sialyllactose for subsequent purification;

[0105] - subjecting the purified solution after membrane separation to at least one simulated moving bed chromatography step to obtain a high-purity purified solution of 6-sialyllactose

[0106] - subjecting the above-mentioned high-purity 6-sialyllactose purified solution to a spray drying step to obtain a white solid powder of 6-sialyllactose;

[0107] - The purity and content of 6-sialyllactose in the white solid powder are ≥95% and / or ≥98%.

[0108] 2. The purity of 6-sialyllactose in the enzyme reaction solution is 70%-90%, more preferably 80-90%;

[0109] 3. The temperature of the enzyme reaction solution is 40°C-90°C, preferably 50°C-70°C, and the heating time is 10min-90min, preferably 30-90min, and more preferably 30-60min.

[0110] 4. The membrane filtration step is a combination of ultrafiltration and nanofiltration, using a membrane with a molecular weight cutoff of 0.3-50 KDa, and the solution pH is adjusted to between 3-7, preferably between 4-6;

[0111] 5. The simulated moving bed chromatography step comprises:

[0112] At least 6 chromatographic columns, preferably at least 10 chromatographic columns, of which at least two are hydrogen-type cation chromatographic columns, preferably sodium-type cation chromatographic columns, and more preferably calcium-type cation chromatographic columns;

[0113] It is divided into four areas according to different flow rates: I, II, III and IV;

[0114] The eluent comprises part or all of the following substances: water, sodium chloride and potassium hydroxide, preferably 5%-10% potassium hydroxide and 90%-95% water, more preferably 5%-10% sodium hydroxide and 90%-95% water.

[0115] 6. The method steps of simulating moving bed chromatography include:

[0116] (1) The flow rates of the four regions I, II, III and IV are preferably: 15-25 mL / min in region I, 13-20 mL / min in region I, 18-35 mL / min in region I, and 10-18 mL / min in region I;

[0117] (2) the feed rate is 1-6 mL / min, and the eluent rate is 5-10 mL / min;

[0118] (3) The area switching time is 10-30 minutes, preferably 20-30 minutes.

[0119] 7. wherein the spray drying step comprises:

[0120] (1) The air inlet temperature is 130-180°C, preferably 130-160°C. The air outlet temperature is 80-120°C, preferably 80-100°C;

[0121] (2) The flow rate is 20-60 mL / min, preferably 20-35 mL / min.

[0122] 8. The purity and content of 6-sialyllactose in the white solid powder obtained by spray drying is ≥95%, preferably ≥98%.

[0123] (1) The inactivation of the enzyme solution after the reaction can be achieved by adjusting the pH value, but it is necessary to first centrifuge or filter to remove the precipitate and then adjust the pH value. The processing time will be correspondingly extended in the scale-up production;

[0124] (2) The solidification of the purified liquid obtained by simulated moving bed chromatography separation can be achieved by vacuum freeze drying, but it is time-consuming and costly. In addition, crystallization can be performed by referring to United States Patent 10745432, but it will increase the time cost and organic solvent cost.

[0125] The raw materials and reagents used in the method for purifying 6-sialyllactose provided by the present invention can be purchased from the market.

[0126] The present invention will be further described below in conjunction with embodiments:

[0127] Example 1

[0128] (1) Pretreatment: 3 L of 6-sialyllactose enzyme reaction solution (including 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose and sialic acid, with contents of 81.2%, 7.5%, 6.9% and 4.4%, respectively) was heated in a 40°C water bath for 90 min, and then centrifuged at 8000 rpm for 30 min to remove the supernatant to remove protein and other solid precipitates.

[0129] (2) Membrane separation: The pH of the reaction solution after pretreatment was adjusted to 3, and the permeate was collected by 10KD ultrafiltration. Then, the pH of the 10KD permeate was adjusted to 3, and the concentrate was collected by 300D nanofiltration to obtain 3.4 L of 6-sialyllactose filtrate.

[0130] (3) Simulated moving bed chromatography: The simulated moving bed chromatography column contains 2 sodium-type LX-160 cationic resins and 4 chloride-type LXP-207 anionic resins. 3.4 L of 6-sialyllactose filtrate was loaded at a rate of 6 mL / min, and the eluent was a 5% potassium hydroxide aqueous solution at an eluent rate of 10 mL / min. Region I was 15 mL / min, region II was 13 mL / min, region III was 18 mL / min, and region IV was 10 mL / min. The region switching time was 10 min, and 4.6 L of 6-sialyllactose purified solution was finally obtained.

[0131] (4) Spray drying: 4.6 L of purified 6-sialyllactose solution was injected at a rate of 20 mL / min, with the inlet and outlet temperatures being 180° C. and 120° C., respectively. After the spray drying, 2.4 kg of 6-sialyllactose white solid powder was obtained.

[0132] The yield and purity data during the purification process are shown in Table 1 below:

[0133] Table 1

[0134] step Volume L Yield % purity% Preprocessing 3.0 99.3 81.2 Membrane separation 3.4 98.5 86.5 Simulated Moving Bed Chromatography 4.6 92.3 95.5 Spray drying / 95.2 95.3

[0135] Example 2

[0136] (1) Pretreatment: 3 L of 6-sialyllactose enzyme reaction solution (including 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose and sialic acid, with contents of 82.5%, 7.1%, 6.2% and 4.2%, respectively) was heated in a 50°C water bath for 60 min, and then centrifuged at 8000 rpm for 30 min to remove the supernatant to remove protein and other solid precipitates.

[0137] (2) Membrane separation: The pH of the reaction solution after pretreatment was adjusted to 4, and the permeate was collected by 50KD ultrafiltration. Then, the pH of the 50KD permeate was adjusted to 4, and the concentrate was collected by 600D nanofiltration to obtain 3.5L of 6-sialyllactose filtrate.

[0138] (3) Simulated moving bed chromatography: The simulated moving bed chromatography column contains 4 sodium-type LX-160 cationic resins and 4 chloride-type LXP-207 anionic resins. 3.5 L of 6-sialyllactose filtrate was loaded at a rate of 4 mL / min, and the eluent was a 5% sodium hydroxide aqueous solution at an eluent rate of 5 mL / min. Region I was 25 mL / min, region II was 20 mL / min, region III was 35 mL / min, and region IV was 18 mL / min. The region switching time was 20 min, and 4.0 L of 6-sialyllactose purified solution was finally obtained.

[0139] (4) Spray drying: 4.0 L of the purified 6-sialyllactose solution was injected at a rate of 60 mL / min, with the inlet and outlet temperatures being 160° C. and 90° C., respectively. After the spray drying, 2.2 kg of 6-sialyllactose white solid powder was obtained.

[0140] The yield and purity data during the purification process are shown in Table 2 below:

[0141] Table 2

[0142] step Volume L Yield % purity% Preprocessing 3.0 99.3 82.5 Membrane separation 3.5 95.6 87.8 Simulated Moving Bed Chromatography 4.0 92.3 96.4 Spray drying / 95.0 96.5

[0143] Example 3

[0144] (1) Pretreatment: Take 3 L of 6-sialyllactose enzyme reaction solution (including 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose and sialic acid, with contents of 81.5%, 6.8%, 7.1% and 4.6%, respectively), heat it in a 70°C water bath for 30 min, and then centrifuge it at 8000 rpm for 30 min to remove the supernatant to remove protein and other solid precipitates.

[0145] (2) Membrane separation: The pH of the reaction solution after pretreatment was adjusted to 6, and the permeate was collected by 20KD ultrafiltration. Then, the pH of the 20KD permeate was adjusted to 6, and the concentrate was collected by 800D nanofiltration to obtain 3.4L 6-sialyllactose filtrate.

[0146] (3) Simulated moving bed chromatography: The simulated moving bed chromatography column contains 4 sodium-type LXP-08 cationic resins and 4 chloride-type LXP-204 anionic resins. 3.4L of 6-sialyllactose filtrate was loaded at a rate of 1mL / min, and the eluent was a 10% potassium hydroxide aqueous solution at an eluent rate of 7.5mL / min. Region I was 20mL / min, region II was 18mL / min, region III was 26mL / min, and region IV was 15mL / min. The region switching time was 30min, and finally 4.8L of 6-sialyllactose purified solution was obtained.

[0147] (4) Spray drying: 4.8 L of purified 6-sialyllactose solution was injected at a rate of 40 mL / min, with the inlet and outlet temperatures being 130° C. and 80° C., respectively. After the spray drying, 2.0 kg of 6-sialyllactose white solid powder was obtained.

[0148] The yield and purity data during the purification process are shown in Table 3 below:

[0149] Table 3

[0150] step Volume L Yield % purity% Preprocessing 3.0 99.3 81.5 Membrane separation 3.4 93.5 89 Simulated Moving Bed Chromatography 4.8 92.3 97.8 Spray drying / 94.3 97.9

[0151] Example 4

[0152] (1) Pretreatment: 3 L of 6-sialyllactose enzyme reaction solution (including 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose and sialic acid, with contents of 81.8%, 6.5%, 7.2% and 4.5%, respectively) was heated in a 90°C water bath for 10 min, and then centrifuged at 8000 rpm for 30 min to remove the supernatant to remove protein and other solid precipitates.

[0153] (2) Membrane separation: The pH of the reaction solution after pretreatment was adjusted to 7, and the permeate was collected by 10KD ultrafiltration. Then, the pH of the 10KD permeate was adjusted to 7, and the concentrate was collected by 1000D nanofiltration to obtain 3.7L 6-sialyllactose filtrate.

[0154] (3) Simulated moving bed chromatography: The simulated moving bed chromatography column contains 2 sodium-type LXP-08 cationic resins and 6 chloride-type LXP-204 anionic resins. 3.7L of 6-sialyllactose filtrate was loaded at a rate of 5mL / min, and the eluent was a 10% sodium hydroxide aqueous solution at an eluent rate of 10mL / min. Region I was 17.5mL / min, region II was 14mL / min, region III was 25mL / min, and region IV was 12mL / min. The region switching time was 20min, and finally 3.9L of 6-sialyllactose purified solution was obtained.

[0155] (4) Spray drying: 3.9 L of 6-sialyllactose purified solution was injected at a rate of 50 mL / min, with the inlet and outlet air temperatures being 160° C. and 100° C., respectively. After the spray drying, 2.38 kg of 6-sialyllactose white solid powder was obtained.

[0156] The yield and purity data during the purification process are shown in Table 4 below:

[0157] Table 4

[0158] step Volume L Yield % purity% Preprocessing 3.0 99.3 81.8 Membrane separation 3.7 91.8 92.1 Simulated Moving Bed Chromatography 3.9 92.3 98.9 Spray drying / 95.5 99.0

[0159] Comparative Example 1

[0160] (1) Pretreatment: Take 3 L of 6-sialyllactose enzyme reaction solution (including 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose and sialic acid, with contents of 81.2%, 7.5%, 6.9% and 4.4%, respectively), centrifuge at 8000 rpm for 30 min without heating, and take the supernatant to remove protein and other solid precipitates.

[0161] (1) Membrane separation: The pH of the reaction solution after pretreatment was adjusted to 3, and the permeate was collected by 10KD ultrafiltration. Then, the pH of the 10KD permeate was adjusted to 3, and the concentrate was collected by 300D nanofiltration to obtain 3.5L of 6-sialyllactose filtrate.

[0162] (3) Simulated moving bed chromatography: The simulated moving bed chromatography column contains 2 sodium-type LX-160 cationic resins and 4 chloride-type LXP-207 anionic resins. 3.5L of 6-sialyllactose filtrate was loaded at a rate of 6mL / min, and the eluent was a 5% potassium hydroxide aqueous solution at an eluent rate of 10mL / min. Region I was 15mL / min, region II was 13mL / min, region III was 18mL / min, and region IV was 10mL / min. The region switching time was 10min, and 4.4L of 6-sialyllactose purified solution was finally obtained.

[0163] (4) Spray drying: 4.4 L of purified 6-sialyllactose solution was injected at a rate of 20 mL / min, with the inlet and outlet temperatures being 180° C. and 120° C., respectively. After the spray drying, 2.4 kg of 6-sialyllactose white solid powder was obtained.

[0164] The yield and purity data during the purification process are shown in Table 5 below:

[0165] Table 5

[0166] step Volume L Yield % purity% Before pretreatment 3.0 100 81.2 After preprocessing 3.0 69.3 69.5 Membrane separation 3.5 98.2 74.2 Simulated Moving Bed Chromatography 4.4 92.5 84.5 Spray drying / 95.2 84.6

[0167] The existing patents do not mention the pretreatment of sialic acid oligosaccharide solution or other methods to prevent the formation of impurities. However, it can be seen from the results that removing the heating step during the pretreatment process will seriously affect the pretreatment yield and the purity of the subsequent product. Therefore, the heating pretreatment step is critical and indispensable for the purification of 6-sialyllactosidase reaction solution.

[0168] Comparative Example 2

[0169] (1) Pretreatment: 3 L of 6-sialyllactose enzyme reaction solution (including 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose and sialic acid, with contents of 82.5%, 7.1%, 6.2% and 4.2%, respectively) was heated in a 50°C water bath for 60 min, and then centrifuged at 8000 rpm for 30 min to remove the supernatant to remove protein and other solid precipitates.

[0170] (2) Membrane separation: The pH of the reaction solution after pretreatment was adjusted to 4, and the permeate was collected by 50KD ultrafiltration. Then, the pH of the 50KD permeate was adjusted to 4, and the concentrate was collected by 600D nanofiltration to obtain 3.5L of 6-sialyllactose filtrate.

[0171] (3) Ion exchange: 3.5 L of 6-sialyllactose filtrate was passed through a strong cation-type ion exchange column LXP-08 and a strong anion-type ion exchange column LXP-202 to obtain 5.0 L of 6-sialyllactose purified solution.

[0172] (4) Spray drying: 5.0 L of the purified 6-sialyllactose solution was injected at a rate of 60 mL / min, with the inlet and outlet temperatures being 160° C. and 90° C., respectively. After the spray drying, 2.2 kg of 6-sialyllactose white solid powder was obtained.

[0173] The yield and purity data during the purification process are shown in Table 6 below:

[0174] Table 6

[0175] step Volume L Yield % purity% Preprocessing 3.0 99.3 82.5 Membrane separation 3.5 95.6 87.8 Ion Exchange 5.0 98.3 89.2 Spray drying / 95.0 89.3

[0176] It can be seen that after replacing simulated moving bed chromatography with ion exchange in the reported purification process, although the yield was improved, the purity of 6-sialyllactose was limited and much lower than that of simulated moving bed chromatography, that is, it is difficult to obtain high-purity 6-sialyllactose from the 6-sialyllactase reaction solution only by simple multi-step membrane separation and ion exchange.

[0177] Comparative Example 3

[0178] (1) Pretreatment: Take 3 L of 6-sialyllactose enzyme reaction solution (including 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose and sialic acid, with contents of 81.5%, 6.8%, 7.1% and 4.6%, respectively), heat it in a 70°C water bath for 30 min, and then centrifuge it at 8000 rpm for 30 min to remove the supernatant to remove protein and other solid precipitates.

[0179] (2) Membrane separation: The pH of the reaction solution after pretreatment was adjusted to 6, and the permeate was collected by 20KD ultrafiltration, and then the pH of the 20KD permeate was adjusted to 6 to obtain 7.4L 6-sialyllactose filtrate.

[0180] (3) Simulated moving bed chromatography: The simulated moving bed chromatography column contains 4 sodium-type LXP-08 cationic resins and 4 chloride-type LXP-204 anionic resins. 7.4L of 6-sialyllactose filtrate was loaded at a rate of 1mL / min, and the eluent was a 10% potassium hydroxide aqueous solution at an eluent rate of 7.5mL / min. Region I was 20mL / min, region II was 18mL / min, region III was 26mL / min, and region IV was 15mL / min. The region switching time was 30min, and 5.8L of 6-sialyllactose purified solution was finally obtained.

[0181] (4) Spray drying: 5.8 L of purified 6-sialyllactose solution was injected at a rate of 40 mL / min, with the inlet and outlet temperatures being 130° C. and 80° C., respectively. After the spray drying, 2.0 kg of 6-sialyllactose white solid powder was obtained.

[0182] The yield and purity data during the purification process are shown in Table 7 below:

[0183] Table 7

[0184] step Volume L Yield % purity% Preprocessing 3.0 99.3 81.5 Membrane separation 7.4 98.5 81.8 Simulated Moving Bed Chromatography 5.8 92.3 87.8 Spray drying / 94.3 87.6

[0185] It can be seen that after eliminating one step of nanofiltration in membrane separation, the product purity cannot reach 90% even if simulated moving bed chromatography is used subsequently. Therefore, the combined use of two-step membrane separation and simulated moving bed chromatography is indispensable for high-purity 6-sialyllactose.

[0186] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for purifying 6-sialyllactose, characterized in that: The steps include: Step 1: heating the 6-sialyllactose enzyme reaction solution, centrifuging, and obtaining a 6-sialyllactose crude product reaction solution; Step 2: taking the crude 6-sialyllactose reaction solution and subjecting it to ultrafiltration and nanofiltration to obtain a 6-sialyllactose filtrate; Step 3: taking the 6-sialyllactose filtrate and subjecting it to simulated moving bed chromatography to obtain a 6-sialyllactose purified solution; Step 4: taking the purified 6-sialyllactose solution and spray drying it to obtain a finished 6-sialyllactose product.

2. The method according to claim 1, characterized in that The 6-sialyllactose enzyme reaction solution in step 1 includes one or more of 6-sialyllactose, 6-sialyllactulose, 6-sialylglucose or sialic acid.

3. The method according to claim 1 or 2, characterized in that The preparation method of the 6-sialyllactose enzyme reaction solution comprises the following steps: Step (1): taking recombinant Escherichia coli BL21 cells containing exo-α-sialidase and mixing them with water, breaking the cells, and obtaining exo-α-sialidase enzyme solution; Step (2): taking a mixture of the sialic acid solution and the lactose solution and mixing it with the exo-α-sialidase enzyme solution, stirring to obtain the 6-sialyllactose enzyme reaction solution.

4. The method according to any one of claims 1 to 3, characterized in that The heating temperature in step 1 is 40 to 90° C., and the heating time is 10 to 90 minutes.

5. The method according to any one of claims 1 to 4, characterized in that: The molecular weight cut-off of the membrane used for ultrafiltration in step 2 ranges from 10 to 50 KDa.

6. The method according to any one of claims 1 to 5, characterized in that: The molecular weight cut-off of the membrane used for nanofiltration in step 2 ranges from 300D to 1000D.

7. The method according to any one of claims 1 to 6, characterized in that: The simulated moving bed chromatography in step 3 comprises: The 6-sialyllactose filtrate is loaded and eluted using at least 6 chromatographic columns; The chromatographic column comprises at least two hydrogen-type cation chromatographic columns; The loading speed includes 1 to 6 mL / min; The eluent used for the elution includes a 5% to 10% potassium hydroxide aqueous solution and / or a 5% to 10% sodium hydroxide aqueous solution; The elution rate includes 5 to 10 mL / min; The simulated moving bed chromatography includes regions I, II, III and IV; The flow rate of the region I includes 15 to 25 mL / min; the flow rate of the region II includes 13 to 20 mL / min; the flow rate of the region III includes 18 to 35 mL / min; the flow rate of the region IV includes 10 to 18 mL / min; The switching time of the regions I, II, III and IV is comprised between 10 and 30 minutes.

8. The method according to claim 7, characterized in that The simulated moving bed chromatography in step 3 comprises: The 6-sialyllactose filtrate is loaded and eluted using 6-8 chromatographic columns; The chromatographic columns include 2 to 4 hydrogen-type cation chromatographic columns and / or 4 to 6 chloride-type anion chromatographic columns.

9. The method according to any one of claims 1 to 8, characterized in that The injection rate of the spray drying in step 4 includes 20 to 60 mL / min; The inlet air temperature of the spray drying is 130-180°C, and the outlet air temperature is 80-120°C.

10. The method according to any one of claims 1 to 9, characterized in that: The pH value of the 6-sialyllactose filtrate is 3-7.

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