Purification method of sialylated oligosaccharide

Abstract

The invention relates to the technical field of substance purification, in particular to a purification method of sialylated oligosaccharide. The purification method of the sialylated oligosaccharide, provided by the invention, not only is simple and feasible in process, but also reduces dissociation and browning of the sialylated oligosaccharide in the purification process, effectively reduces the loss of the sialylated oligosaccharide and improves the yield of the sialylated oligosaccharide; the sialylated oligosaccharide prepared by the purification method can realize higher purity and yield, and has better application prospect in industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of substance purification, and in particular to a method for purifying sialylated oligosaccharides. Background Art

[0002] Sialyl human milk oligosaccharides (SHMOs) are important oligosaccharides in breast milk. They contain sialic acid (SA) in their molecules, usually in the form of N-acetylneuraminic acid (Neu5Ac). They play an important role in infant brain development, neurotransmission and synapse formation, and also have immunomodulatory functions. Sialyl lactose (SL) is a sialylated human milk oligosaccharide with important physiological functions, among which 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are two particularly important sialylated human milk oligosaccharides in breast milk.

[0003] At present, the synthesis methods of sialylated human milk oligosaccharides mainly include chemical synthesis and biosynthesis. The chemical synthesis method usually synthesizes sialylated human milk oligosaccharides through enzymatic reactions, and the biosynthesis method uses microorganisms such as Escherichia coli to synthesize sialylated human milk oligosaccharides. In the above method, purifying the target product from the fermentation broth or enzymatic reaction liquid is also a key step in the preparation of sialylated human milk oligosaccharides. The main purification process currently used is: using glycosidase to hydrolyze lactose into galactose, using anion and cation exchange columns to remove excess salt compounds, and then removing galactose and other miscellaneous sugar substances through two nanofiltration or chromatographic separations; for example, the method disclosed in Chinese patent CN111447845B first uses glycosidase to hydrolyze lactose to form galactose, and then uses 200L volume of H +-type strong cation exchanger to remove cationic contaminants; then use NaOH to adjust the pH of the resulting solution to 7.0; then use a strong anion exchanger in the form of chloride to remove anions and colorant impurities from the solution, and after nanofiltration and decolorization, use chromatography to remove other contaminating sugars. Among them, the purpose of purification with a strong anion exchanger in the form of chloride is to remove excess heteroanions. This method requires the simultaneous addition of substances that inhibit the adsorption of sialylated oligosaccharides by anion exchangers (such as co-anions). The removal of heterosaccharides is not concerned in the ion exchange purification step, and the heterosaccharides are mainly removed in the chromatography step. Similarly, the purification methods disclosed in CN112203519B and CN116964066A all adopt this process route, but the removal of heterosaccharides adopts a membrane filtration process. Although the method disclosed in patent application WO2017152918A1 does not use glycosidase to hydrolyze lactose, according to the description in its specification, "a method for allowing negatively charged materials to be absorbed onto an anion exchange resin, including sialylated oligosaccharides / sialylated lactose. The liquid obtained after contact with the anion exchange resin as the eluate mainly contains water, cations and neutral carbohydrates," it can be inferred that the process does not separate lactose and sialyllactose in the anion exchange resin purification step, and it has no special requirements for the anion exchange resin. At the same time, according to another process route provided by it (purification only using a cation exchange column), it can be seen that the purpose of the ion exchange purification step in this process is mainly to prepare the product into a salt.

[0004] In summary, the current purification process of sialylated human milk oligosaccharides is still relatively complicated, and it needs to rely on chromatographic separation to remove impurities in the later stage, which increases the complexity of the process and the purification cost. Summary of the invention

[0005] The present invention provides a method for purifying sialylated oligosaccharides.

[0006] In order to simplify the purification process of sialylated oligosaccharides and improve the purification efficiency, the present invention attempts to use a simplified ion exchange process to remove excess acid, lactose and small molecule salts. In this process, the inventor unexpectedly found that while using ion exchange technology to remove excess acid, lactose and small molecule salts, sialyllactose will lose about 10-15%. The main reason is that the elution time experienced by sialyllactose in anion exchange resin is long, resulting in the dissociation and browning of sialyllactose, which reduces the content of sialyllactose in the purified product or requires the introduction of a decolorization step to solve the browning problem, thereby reducing the yield. After continuous attempts, the inventor found that the use of a chlorine-type weakly basic anion exchange resin can better solve the above problems, reduce the loss of sialyllactose in the process of removing excess acid, lactose and small molecule salts using ion exchange technology, and improve its yield. Although ion exchange technology is a known technical means for purifying sialylated oligosaccharides, in which weakly basic anion exchange resins have also been used, the prior art mostly selects weakly basic anion exchange resins because of their advantage that the counter ions are basically not released into the eluent compared with strongly basic anion exchange resins, and their main purpose is to remove anionic impurities, which is obviously different from the purpose and function of the chloride-type weakly basic anion exchange resin used in the present invention.

[0007] Specifically, the present invention provides the technical solution described below.

[0008] The present invention provides a method for purifying sialylated oligosaccharides, the method comprising: treating a solution containing sialylated oligosaccharides with an ion exchange resin; the ion exchange resin treatment comprising: treating with a chlorine-type weakly basic anion exchange resin so that the sialylated oligosaccharides are adsorbed on the chlorine-type weakly basic anion exchange resin, and collecting the eluate containing the sialylated oligosaccharides after elution.

[0009] In the present invention, the chloride-type weakly basic anion exchange resin is a weakly basic anion exchange resin modified by chloride ions. It is significantly different from the free-base type weakly basic anion exchange resin and the strong anion exchange resin in the form of chloride used in the prior art in terms of structure and properties. The weakly basic anion exchange resin usually contains a base with a lone pair of electrons that attracts protons, such as certain nitrogen-containing groups, which include, for example, primary amines, secondary amines, tertiary amines (free amine groups), guanidine groups or nitrogen-containing heteroaromatic groups (such as pyridine, pyrimidine, etc.), preferably including tertiary amine groups. In the free-base form, the base is not in a protonated form, in other words, there is no counter anion in the base. Therefore, the group can adsorb acids as a whole, i.e., acid cations (H + ) and acid anions (X - ) can be removed from the feed solution without ion exchange (without releasing counterions).

[0010] In addition, chloride-type weakly basic anion exchange resins have significantly different binding forces for sialylated oligosaccharides and impurities such as lactose, sialic acid, and pigments. By utilizing this characteristic, impurities such as lactose, sialic acid, and pigments can be efficiently removed during the treatment with chloride-type weakly basic anion exchange resins. At the same time, the dissociation and browning of sialylated oligosaccharides during the elution process can be effectively reduced, thereby reducing the loss of sialylated oligosaccharides and reducing the amount of pigment impurities eluted (generally speaking, the decolorization step before ion exchange resin treatment cannot completely remove pigment impurities, and some pigments will be adsorbed by the anion exchange resin. Therefore, it is necessary to reduce the possibility of pigments being eluted together with sialylated oligosaccharides). During the column and elution process, impurities such as sialic acid and lactose are removed. First, lactose directly passes through the chloride-type weakly basic anion exchange resin, and then is eluted with the eluent, and sialylated oligosaccharides are eluted. At this time, sialic acid and pigment impurities are still bound to the chloride-type weakly basic anion exchange resin, which can be removed in the resin purification and regeneration steps (for example, when sodium hydroxide is used to purify the anion exchange resin, all the pigments and sialic acid therein are eluted, and then converted with sodium chloride to regain the chloride-type weakly basic anion exchange resin).

[0011] In some embodiments of the present invention, the chloride-type weakly basic anion exchange resin is obtained by modifying the weakly basic anion exchange resin with sodium chloride.

[0012] The present invention does not impose any special restrictions on the structural skeleton of the chloride-type weakly basic anion exchange resin, including but not limited to styrene series, acrylic acid series, acetate series, epoxy series, vinylpyridine series, urea-formaldehyde series, ethylene oxide series, etc.

[0013] The above-mentioned ion exchange resin treatment also includes treatment with an acidic cation exchange resin. Acidic cation exchange resin treatment is beneficial for removing inorganic cationic impurities and impurities such as organic amines, amino acids and short peptides. The resin eluate obtained by treatment with an acidic cation exchange resin contains acidic sialylated oligosaccharides, and the pH of the resin eluate is usually 1-2, but the material residence time in this step is very short, and the sialylated oligosaccharides are not easily hydrolyzed.

[0014] Preferably, the acidic cation exchange resin is H + Type strong acid cation exchange resin.

[0015] For H + The structural skeleton of the type strong acid cation exchange resin is not particularly limited in the present invention, and includes but is not limited to styrene type, acrylic type, etc.

[0016] In some specific embodiments of the present invention, the above-mentioned ion exchange resin treatment includes: first treating the solution containing sialylated oligosaccharides with an acidic cation exchange resin, and then treating it with a chloride-type weakly basic anion exchange resin.

[0017] In other specific embodiments of the present invention, the above-mentioned ion exchange resin treatment includes: first treating the solution containing sialylated oligosaccharides with a strong basic anion exchange resin, then treating it with an acidic cation exchange resin, and then treating it with a chloride-type weakly basic anion exchange resin. By introducing the strong basic anion exchange resin treatment before the acidic cation exchange resin treatment, acidic impurities such as sialic acid can be adsorbed on the strong basic anion exchange resin, and then acidic impurities such as sialic acid can be removed in advance. The present invention has no special restrictions on the structural framework of the strong basic anion exchange resin, and various known strong basic anion exchange resins for purification can be used.

[0018] Preferably, after the acidic cation exchange resin treatment, the eluate containing sialylated oligosaccharides is collected and directly treated with a chloride-type weakly basic anion exchange resin without pH adjustment of the eluate obtained by the acidic cation exchange resin treatment.

[0019] In the above method, the eluent used in the treatment process of the chloride-type weakly basic anion exchange resin is a neutral salt and / or a weakly acidic salt.

[0020] Preferably, the concentration of the eluent is 0.01-0.05M.

[0021] The present invention finds that when the eluent concentration is higher than 0.05M, sialic acid will be eluted together with sialic acid oligosaccharides, while when the eluent concentration is 0.01-0.05M, sialic acid oligosaccharides will be eluted first and sialic acid will not be eluted, and the eluate containing sialic acid oligosaccharides can be collected.

[0022] In some specific embodiments of the present invention, the concentration of the eluent is 0.03-0.05M.

[0023] Preferably, the eluent is a sodium salt.

[0024] In some embodiments of the present invention, the eluent is sodium chloride and / or sodium acetate.

[0025] In the above method, after the treatment with the chloride-type weakly basic anion exchange resin, no chromatography step is included, especially no ion exchange resin treatment step. The present invention realizes the effective removal of impurities such as sialic acid, lactose, pigments, etc. through the above ion exchange resin treatment process, and can ensure a high purity of sialic acid oligosaccharides, without the need for other chromatography steps to remove impurities such as miscellaneous sugars as in the prior art such as CN111447845B.

[0026] In order to remove the eluent used in the treatment process of the chlorine-type weakly basic anion exchange resin, the method further comprises a step of removing the eluent after the treatment of the chlorine-type weakly basic anion exchange resin.

[0027] Preferably, nanofiltration is used to remove the eluent.

[0028] After removing the eluent, the obtained sialylated oligosaccharide solution can be spray-dried, freeze-dried, vacuum-dried or crystallized to obtain sialylated oligosaccharides in solid form; alternatively, the obtained sialylated oligosaccharide solution can be prepared as a concentrated aqueous solution or syrup by removing water by distillation (e.g., vacuum distillation) or nanofiltration.

[0029] In the above method, the solution containing sialylated oligosaccharides is an aqueous solution containing sialylated oligosaccharides.

[0030] Preferably, the pH of the aqueous solution containing sialylated oligosaccharides is 6-7.

[0031] Preferably, the concentration of the aqueous solution containing sialylated oligosaccharides is 30-50 g / L.

[0032] The solution containing sialylated oligosaccharides is obtained by subjecting a fermentation solution or an enzymatic reaction solution containing sialylated oligosaccharides to one or more pretreatment steps.

[0033] For sialylated oligosaccharides produced by fermentation or in vitro enzymatic processes, in addition to the produced sialylated oligosaccharides, the fermentation broth usually contains biomass of the microbial cells used for fermentation, as well as proteins, protein fragments, nucleic acids, inorganic salts, endotoxins, biogenic amines, unreacted carbohydrates (e.g., lactose), sugar byproducts, sialic acid, pigments, etc.; in vitro enzymatic reaction liquid usually contains, in addition to the produced sialylated oligosaccharides, proteins, protein fragments, inorganic salts, unreacted carbohydrate acceptors (e.g., lactose), sugar byproducts, sialic acid and its precursors, etc. Therefore, it is preferred to pretreat the fermentation broth or enzymatic reaction liquid containing sialylated oligosaccharides before ion exchange resin treatment.

[0034] Preferably, the pretreatment comprises one or more selected from centrifugation, ultrafiltration, concentration, desalting and decolorization.

[0035] In the above pretreatment, ultrafiltration or centrifugation is preferably set as the first step. Among them, ultrafiltration is to separate biomass and high molecular weight suspended solids from soluble components in aqueous media such as fermentation broth and enzymatic reaction liquid, and the soluble components are separated into the permeate through an ultrafiltration membrane. Any conventional ultrafiltration membrane can be used for ultrafiltration, and the molecular weight cutoff (MWCO) range is about 1~500kDa, such as 10~250kDa, 50~100kDa, 200~500kDa, 100~250kDa, 1~100kDa, 1~50kDa, 10~25kDa, 1~5kDa, or any other suitable sub-range. The membrane material can be ceramic or made of synthetic or natural polymers, such as polysulfone, polypropylene, cellulose acetate or polylactic acid. Ultrafiltration can be performed more than once using ultrafiltration membranes with different MWCO, wherein the MWCO of the membrane used for the first ultrafiltration is higher than that of the second. The permeate obtained by the above ultrafiltration contains sialylated oligosaccharides.

[0036] In some specific embodiments of the present invention, a 20-50 nm ceramic membrane is used to ultrafilter the fermentation broth or enzymatic reaction liquid containing sialylated oligosaccharides. Preferably, two ultrafiltrations are performed, and a high temperature inactivation and decolorization filtration step is provided between the two ultrafiltrations; the second ultrafiltration uses an ultrafiltration membrane with a MWCO of 2-4 kDa.

[0037] The above-mentioned pretreatment may also include a concentration step, or a concentration and desalination step, wherein the concentration or the concentration and desalination may be achieved by nanofiltration.

[0038] The nanofiltration step can be performed after the ultrafiltration step or the optional decolorization step. Nanofiltration can play a role in concentration and / or desalination (mainly monovalent ions). The MWCO of the nanofiltration membrane can ensure the retention of the target sialylated oligosaccharide, and its MWCO is lower than the MWCO of the ultrafiltration membrane used above, and is about 25-50% of the molecular weight of the target sialylated oligosaccharide. For example, a nanofiltration membrane with a MWCO of about 150-300Da is suitable for retaining sialyllactose. Nanofiltration can be used in combination with water diafiltration to more effectively remove permeable molecules until the conductivity of the permeate shows no salt or very low salt content.

[0039] The above-mentioned pretreatment may also include a decolorization step, and the decolorization is preferably carried out by activated carbon treatment. The decolorization may be arranged after the ultrafiltration step or the nanofiltration step. The activated carbon treatment helps to remove or reduce pigments and water-soluble pollutants (e.g., salts).

[0040] Carbohydrates such as sialylated oligosaccharides can also be bound to the surface of activated carbon particles from their aqueous solution, while pigments can also be adsorbed to the activated carbon. When carbohydrates and pigments are adsorbed, water-soluble materials that are not bound to the activated carbon or are weakly bound to the activated carbon can be eluted with water. By changing the eluent from water to ethanol-water solution, the adsorbed sialylated oligosaccharides can be easily eluted while the pigments remain adsorbed on the activated carbon, thereby achieving decolorization and desalination at the same time. Of course, under certain conditions, sialylated oligosaccharides can be not or at least substantially not adsorbed on the activated carbon particles, while the pigments are still adsorbed.

[0041] In the present invention, sialylated oligosaccharides can be synthesized by an in vitro enzymatic reaction or produced by culturing a microorganism capable of synthesizing sialylated oligosaccharides.

[0042] Preferably, the sialylated oligosaccharide is produced by microbial fermentation. The microorganisms used for fermentation include but are not limited to Escherichia coli and the like.

[0043] In some embodiments of the present invention, the method for preparing the solution containing sialylated oligosaccharides comprises: removing bacteria and proteins from the fermentation broth containing sialylated oligosaccharides, decolorizing the broth, and concentrating and desalting the clear solution obtained by decolorization.

[0044] The concentration and desalination may adopt nanofiltration technology; the removal of bacteria and proteins may adopt ultrafiltration technology.

[0045] In the present invention, the sialylated oligosaccharide contains at least one sialic acid group.

[0046] Preferably, the sialylated oligosaccharide is sialylated human milk oligosaccharide; more preferably, it is sialyllactose.

[0047] The above-mentioned sialylated human milk oligosaccharide is selected from 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), LSTa, fucosyl-LSTa (FLSTa), LSTb, fucosyl-LSTb (FLSTb), LSTc, fucosyl-LSTc (FLSTc), sialyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) or disialyl-lacto-N-tetraose (DS-LNT).

[0048] In some embodiments of the present invention, the sialylated oligosaccharide is 3'-sialyllactose or 6'-sialyllactose. The purification method of the present invention is particularly suitable for the purification of 3'-sialyllactose or 6'-sialyllactose produced by fermentation.

[0049] In some embodiments of the present invention, a method for purifying sialyllactose from a fermentation broth is provided, wherein the fermentation broth is obtained by culturing a microorganism capable of producing sialyllactose, and the method comprises the following steps: (1) ultrafiltration of the fermentation broth to obtain an ultrafiltration permeate; (2) subjecting the ultrafiltration permeate to a temperature-raising inactivation treatment to obtain an inactivated product; (3) decolorizing the inactivated material with activated carbon to obtain a decolorized liquid; (4) ultrafiltration of the decolorized liquid to obtain an ultrafiltration permeate; (5) subjecting the ultrafiltration permeate to nanofiltration to obtain a nanofiltration retentate; (6) The nanofiltration retentate is first treated with an acidic cation exchange resin, and the eluate containing sialylated oligosaccharides is collected. The eluate containing sialylated oligosaccharides is further treated with a chloride-type weakly basic anion exchange resin so that the sialylated oligosaccharides are adsorbed on the chloride-type weakly basic anion exchange resin, and then eluted with an eluent to collect the eluate containing sialylated oligosaccharides.

[0050] The beneficial effects of the present invention include at least: the purification method of sialylated oligosaccharides provided by the present invention is not only simple and easy to operate, but also reduces the dissociation and browning of sialylated oligosaccharides during the purification process, effectively reduces the loss of sialylated oligosaccharides, and improves the yield of sialylated oligosaccharides; the purification method does not need to rely on subsequent chromatographic separation to remove miscellaneous sugars, and uses an ion exchange process to effectively remove impurities such as miscellaneous sugars. The sialylated oligosaccharides prepared by the purification method can achieve higher purity and yield, and have good application value in industrial production. DETAILED DESCRIPTION

[0051] In the present invention, the term "sialylated oligosaccharide" refers to a sugar polymer containing at least two monosaccharide units, at least one of which is a sialyl (N-acetylneuramidyl) moiety. Sialyl oligosaccharides may have a linear or branched structure, which comprises monosaccharide units linked to each other by interglycosidic bonds. Sialyl oligosaccharides are sialylated human milk oligosaccharides.

[0052] The term "sialylated human milk oligosaccharide", also known as "acidic human milk oligosaccharide (acidic HMO)", refers to a complex carbohydrate found in human breast milk, comprising a core structure of a lactose unit at the reducing end, which may be extended by one or more β-N-acetyl-lactosamine units and / or one or more β-lactosyl-N-diosyl units, and which is substituted by α-N-acetyl-neuraminic acid (sialic acid) moieties and optionally by L-fucopyranosyl moieties. In this regard, it has at least one sialic acid residue in its structure. Sialyl human milk oligosaccharides include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), LSTa, fucosyl-LSTa (FLSTa), LSTb, fucosyl-LSTb (FLST b), LSTc, fucosyl-LSTc (FLST c), sialic acid-LNH (SLNH), sialic acid-lacto-N-hexaose (SLNH), sialic acid-lacto-N-neohexose I (SLNH-I), sialic acid-lacto-N-neohexose II (SLNH-II) and disialyl-lacto-N-tetraose (DS-LNT).

[0053] The term "sialyllactose" is preferably 3'-SL or 6'-SL.

[0054] The term "aqueous solution containing sialylated oligosaccharides" preferably refers to the raw material for the ion exchange resin treatment of the present invention, which is an optional pretreatment containing sialylated oligosaccharides. The aqueous medium containing sialylated oligosaccharides can be directly subjected to the ion exchange resin treatment of the present invention, or can be pretreated by one or more of the steps described above before being subjected to the ion exchange resin treatment. Through the pretreatment, the aqueous medium is partially purified, and the content of impurities such as bacteria and proteins is reduced or eliminated.

[0055] In some specific embodiments of the present invention, a method for purifying sialyllactose from a fermentation broth is provided, and the technical route is as follows: fermentation broth → ceramic membrane filtration to remove bacteria → collection of clear liquid → protein removal → decolorization → collection of clear liquid → nanofiltration → collection of retentate → treatment with a strong alkaline anion exchange resin to remove impurities such as sialic acid → treatment with an acidic cation exchange resin → treatment with a chloride-type weakly alkaline anion exchange resin to remove impurities such as lactose and salt → elution with 0.03~0.05M sodium chloride or sodium acetate solution → collection of eluate → nanofiltration desalination → drying (freeze drying or spray drying).

[0056] In other specific embodiments of the present invention, a method for purifying sialyllactose from a fermentation broth is provided, and the technical route is as follows: fermentation broth → ceramic membrane filtration to remove bacteria → collection of clear liquid → protein removal → decolorization → collection of clear liquid → nanofiltration → collection of retentate → treatment with acidic cation exchange resin → treatment with chloride-type weakly basic anion exchange resin to remove impurities such as sialic acid, lactose, and salt → elution with 0.03~0.05M sodium chloride or sodium acetate solution → collection of eluate → nanofiltration desalination → drying (freeze drying or spray drying).

[0057] The above-mentioned method for purifying sialyllactose from fermentation broth specifically comprises the following steps: (1) Filtering the fermentation liquid through a ceramic membrane, adding water to filter out the clear liquid, and obtaining a ceramic membrane filtrate; (2) Raise the temperature of the ceramic membrane filtrate to 80-85°C, inactivate the filtrate and precipitate the protein, and keep the temperature for 25-35 minutes to obtain the inactivated product; (3) Cool the inactivated material to 40-45°C, add activated carbon, stir, and filter to obtain a decolorized solution; (4) Filter the decolorized liquid through a 2-4 kDa ultrafiltration membrane, add water to filter out the clear liquid, and obtain an ultrafiltration membrane filtrate; (5) further filtering the ultrafiltration membrane filtrate of step (4) using a nanofiltration membrane to remove impurities such as salt to obtain a nanofiltration retentate, wherein the concentration of sialyllactose in the nanofiltration retentate is controlled at 30-50 g / L; (6) The nanofiltration retentate is first treated with a strong alkaline anion exchange resin, and the clear liquid is collected; the clear liquid is then treated with an acidic cation exchange resin, and the clear liquid is collected; the clear liquid is then treated with a chloride-type weakly alkaline anion exchange resin, and the sialyllactose is adsorbed on the chloride-type weakly alkaline anion exchange resin, and eluted with sodium chloride or sodium acetate, and the eluate is collected. The pH of the eluate is 6-7 (Cl type); (7) filtering the eluate containing step (6) using a nanofiltration membrane to remove the eluent, and collecting the retentate containing sialyllactose; (8) Drying: Drying the retentate containing sialyllactose from step (7) to obtain a sialyllactose product.

[0058] The sialyllactose content of the sialyllactose product obtained by the purification method is greater than 95%, and the product yield is greater than 80%.

[0059] In the present invention, the non-limiting examples of weakly basic anion exchange resins include LX-360, D354-FD, Dowex 66, Amberlite FPA53, Lewatit S6368 A, Lewatit S4268, Lewatit S5528, Lewatit S6368A (Lanxess AG. Cologne, DE), Dowex AG 1x2 (Mesh 200-400), Dowex 1x8 (Mesh 100-200), Purolite Chromalite CGA 100x4 (Purolite GmbH, Ratingen, DE), Dow Amberlite FPA51 (Dow Chemicals, M1, USA) and other different brands or models. Chlorine-based weakly basic anion exchange resins can be obtained by modifying the above-mentioned weakly basic anion exchange resins with chloride ions.

[0060] Taking LX-360 as an example, the following methods can be used to modify the chloride ion: 1. Load the resin into the exchanger and backwash the resin with clean water until the effluent is clear. 2. Pass twice the volume of the resin into 8-10% NaCl solution, soak the resin in the salt solution for about 24 hours, and then rinse with clean water until the effluent water is colorless and odorless. 3. First pass twice the volume of the resin into about 4% HCl, first let one times the volume of the resin directly pass through the column, then soak the resin with another times the volume of the resin for 4-8h, and wash with clean water until the pH is about 3. Then pass twice the volume of the resin into about 4% NaOH, first let one times the volume of the resin directly pass through the column, then soak the resin with another times the volume of the resin for 4-8h, and wash with clean water until the pH is about 10. Finally, 2 to 3 times the volume of the resin 4% sodium chloride is passed through at a rate of 2 to 4 BV / h to obtain a chlorine-type weakly basic anion exchange resin, which is used in the following examples. This transformation method is a conventional resin modification method in the art. Those skilled in the art can make adaptive adjustments according to the specific resin conditions without making too many restrictions.

[0061] In the present invention, non-limiting examples of acidic cation exchange resins include: Amberlite™ IR100, Amberlite™ IR120, Amberlite™ FPC22, Dowex TM50WX, Finex™ CS16GC, Finex™ CS13GC, Finex™ CS12GC, Finex™ CS11GC, Lewatit™ S, Diaion™ SK, Diaion™ UBK, Amberjet™ 1000, Amberjet™ 1200, Dowex™ 88, etc.

[0062] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] The nanofiltration retentate containing sialyllactose used in the following examples was prepared by the following method: (1) Separation of fermentation broth and extraction of clear liquid: The fermentation broth produced by microbial fermentation to produce sialyllactose (the content of sialyllactose in the fermentation broth is about 30 g / L) is filtered through a 30 nm ceramic membrane, and process water is added to filter out the clear liquid to obtain the ceramic membrane filtrate.

[0064] (2) Inactivation by heating: The ceramic membrane filtrate is heated to 80°C to inactivate it and precipitate the protein. The temperature is kept for 30 minutes to obtain the inactivated product.

[0065] (3) Decolorization and filtration: Cool the inactivated material to 40-45°C, add 4% activated carbon and stir for 30 minutes, then filter to obtain a decolorized solution.

[0066] (4) Ultrafiltration: The decolorized liquid is filtered through a 3 kDa ultrafiltration membrane, and process water is added to filter the clear liquid to obtain the ultrafiltration membrane filtrate.

[0067] (5) Nanofiltration: The ultrafiltration membrane filtrate of step (4) is further filtered using a nanofiltration membrane to remove impurities such as salt, thereby obtaining a nanofiltration retentate containing sialyllactose (i.e., an aqueous solution containing sialyllactose). The concentration of sialyllactose in the nanofiltration retentate is controlled at 30-50 g / L and the pH is 6-7.

[0068] Example 1 This embodiment provides a method for purifying sialyllactose, which comprises the following steps: The nanofiltration retentate containing 3'-sialyllactose was passed through H +The supernatant is collected by adding a strong acid cation exchange resin (LX-150) to a chloride-type weakly basic anion exchange resin (obtained by modifying the weakly basic anion exchange resin LX-360 with chloride ions). At this time, sialyllactose will be adsorbed onto the chloride-type weakly basic anion exchange resin, while lactose will directly flow through the chloride-type weakly basic anion exchange resin with the supernatant; then, 0.05 mol / L sodium chloride is used for elution to obtain an eluate containing sialyllactose.

[0069] The content of sialyllactose in the eluate was detected, and the percentage of sialic acid loss was calculated based on the content of sialyllactose in the nanofiltration retentate and the content of sialyllactose in the eluate. The results showed that the percentage of sialyllactose loss during the ion exchange resin treatment was only 2.2%.

[0070] The eluate containing sialyllactose is subjected to nanofiltration and vacuum drying to obtain a sialyllactose finished product, wherein the content of sialyllactose is 95.5% and the comprehensive yield is 85.3%.

[0071] Example 2 This embodiment provides a method for purifying sialyllactose, which comprises the following steps: The nanofiltration retentate containing 3'-sialyllactose is first passed through a strong basic anion exchange resin (WSD260). SA in the material will be adsorbed on the resin, while SL will pass through directly without being adsorbed, and the clear liquid is collected. The clear liquid collected by the strong basic anion exchange resin is then passed through H + The supernatant is collected by adding a strong acid cation exchange resin (LX-150) to a chloride-type weakly basic anion exchange resin (obtained by modifying the weakly basic anion exchange resin LX-360 with chloride ions). At this time, sialyllactose will be adsorbed onto the chloride-type weakly basic anion exchange resin, while lactose will directly flow through the chloride-type weakly basic anion exchange resin with the supernatant; then, 0.05 mol / L sodium chloride is used for elution to obtain an eluate containing sialyllactose.

[0072] The content of sialyllactose in the eluate was detected, and the percentage of sialic acid loss was calculated based on the content of sialyllactose in the nanofiltration retentate and the content of sialyllactose in the eluate. The results showed that the percentage of sialyllactose loss during the ion exchange resin treatment was only 4.5%.

[0073] The eluate containing sialyllactose is subjected to nanofiltration and vacuum drying to obtain a sialyllactose finished product, wherein the content of sialyllactose is 96.2% and the yield is 82.4%.

[0074] Example 3 This embodiment provides a method for purifying sialyllactose, which comprises the following steps: The nanofiltration retentate containing sialyllactose was passed through H + The supernatant is collected by adding a strong acid cation exchange resin (LX-150) to a chloride-type weakly basic anion exchange resin (obtained by modifying the weakly basic anion exchange resin LX-360 with chloride ions). At this time, sialyllactose will be adsorbed onto the chloride-type weakly basic anion exchange resin, while lactose will directly flow through the chloride-type weakly basic anion exchange resin with the supernatant; then, 0.03 mol / L sodium acetate is used for elution to obtain an eluate containing sialyllactose.

[0075] The content of sialyllactose in the eluate was detected, and the percentage of sialic acid loss was calculated based on the content of sialyllactose in the nanofiltration retentate and the content of sialyllactose in the eluate. The results showed that the percentage of sialyllactose loss during the ion exchange resin treatment was only 2.8%.

[0076] The eluate containing sialyllactose is subjected to nanofiltration and vacuum drying to obtain a sialyllactose finished product, wherein the sialyllactose content is 95.8% and the yield is 84.6%.

[0077] Comparative Example 1 This comparative example provides a method for purifying sialyllactose, which differs from the method in Example 1 only in that the chloride-type weakly basic anion exchange resin (obtained by modifying the weakly basic anion exchange resin LX-360 with chloride ions) is replaced by LX-360 (weakly basic anion exchange resin not modified with chloride ions (OH - )).

[0078] The content of sialyllactose in the eluate was detected, and the percentage of sialic acid loss was calculated based on the content of sialyllactose in the nanofiltration retentate and the content of sialyllactose in the eluate. The results showed that the percentage of sialyllactose loss during the ion exchange resin treatment was 14.5%.

[0079] The eluate containing sialyllactose is subjected to nanofiltration and vacuum drying to obtain a sialyllactose finished product, wherein the content of sialyllactose is 82.5% and the yield is 65.4%.

[0080] Comparative Example 2 This comparative example provides a method for purifying sialyllactose, which differs from the method in Example 1 only in that the chloride-type weakly basic anion exchange resin (obtained by modifying the weakly basic anion exchange resin LX-360 with chloride ions) is replaced with a strongly basic anion exchange resin WSD260.

[0081] The content of sialyllactose in the eluate was detected, and the percentage of sialic acid loss was calculated based on the content of sialyllactose in the nanofiltration retentate and the content of sialyllactose in the eluate. The results showed that the percentage of sialyllactose loss during the ion exchange resin treatment was 8.3%, indicating that the adsorption capacity of the strong basic anion exchange resin for sialyllactose was significantly worse than that of the chloride-type weak basic anion exchange resin.

[0082] The eluate containing sialyllactose is subjected to nanofiltration and vacuum drying to obtain a sialyllactose finished product, wherein the content of sialyllactose is 81.3% and the yield is 68.3%.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying sialylated oligosaccharides, characterized in that: The method comprises: treating a solution containing sialylated oligosaccharides with an ion exchange resin; The ion exchange resin treatment includes: using a chloride-type weakly basic anion exchange resin for treatment, so that sialylated oligosaccharides are adsorbed on the chloride-type weakly basic anion exchange resin, and after elution, collecting the eluate containing sialylated oligosaccharides.

2. The method according to claim 1, characterized in that The ion exchange resin treatment also includes treatment with an acidic cation exchange resin; Preferably, the acidic cation exchange resin is H + Type strong acid cation exchange resin.

3. The method according to claim 1 or 2, characterized in that: The ion exchange resin treatment comprises: treating the solution containing sialylated oligosaccharides with an acidic cation exchange resin first, and then treating the solution with a chloride-type weakly basic anion exchange resin; Alternatively, the solution containing sialylated oligosaccharides is first treated with a strongly basic anion exchange resin, then with an acidic cation exchange resin, and then with a chloride-type weakly basic anion exchange resin.

4. The method according to any one of claims 1 to 3, characterized in that In the treatment process of the chloride-type weakly basic anion exchange resin, the eluent used for elution is a neutral salt and / or a weakly acidic salt.

5. The method according to claim 4, characterized in that The concentration of the eluent is 0.01-0.05M; Preferably, the eluent is a sodium salt.

6. The method according to claim 4 or 5, characterized in that: After the treatment with the chloride-type weakly basic anion exchange resin, the step of removing the eluent is also included; Preferably, nanofiltration is used to remove the eluent.

7. The method according to any one of claims 1 to 6, characterized in that: The solution containing sialylated oligosaccharides is an aqueous solution containing sialylated oligosaccharides; Preferably, the pH of the aqueous solution is 6-7.

8. The method according to claim 7, characterized in that The solution containing sialylated oligosaccharides is obtained by subjecting a fermentation broth or an enzymatic reaction solution containing sialylated oligosaccharides to one or more pretreatment steps; Preferably, the pretreatment comprises one or more selected from centrifugation, ultrafiltration, concentration, desalting and decolorization.

9. The method according to claim 7 or 8, characterized in that: The method for preparing the solution containing sialylated oligosaccharides comprises: removing bacteria and protein from the fermentation liquid containing sialylated oligosaccharides, performing decolorization treatment, and concentrating and desalting the clear liquid obtained by decolorization.

10. The method according to any one of claims 1 to 9, characterized in that: The sialylated oligosaccharide comprises at least one sialic acid group; Preferably, the sialylated oligosaccharide is sialylated human milk oligosaccharide; more preferably, it is sialyllactose.