A method for separation and purification of oligosaccharides and its application

Abstract

The present invention discloses a separation and purification method for oligosaccharides and its application, which combines enzymatic hydrolysis, chemical precipitation and membrane methods. The method has mild conditions, simple process, short time consumption, and the generated precipitate can be used in fertilizers and other fields. And the method does not affect the yield of the target oligosaccharide.

Description

Technical Field

[0001] The invention relates to a highly efficient preparation method of a series of sialylated lacto-N-tetraoses and also provides a separation and purification method of oligosaccharides. Background Art

[0002] Oligosaccharides, also known as oligosaccharides, refer to a type of sugar that is a linear or branched chain of 2 to 10 monosaccharides connected by glycosidic bonds. Human milk oligosaccharides (HMOs), one of the typical oligosaccharides, are the third largest solid substance in breast milk after lipids and lactose, with a concentration of up to 9-24 g / L. It has broad market prospects in the food industry, especially in infant formula milk powder. However, due to the complex process of separating and purifying human milk oligosaccharides from mammalian breast milk and the low yield, most oligosaccharides are now obtained based on chemical methods or enzymatic methods, or cell factory fermentation. The chemical method requires rigorous design, including protective groups, selection of catalysts, and appropriate reaction conditions. Enzymatic methods and cell factory fermentation are usually carried out under mild conditions, without the need for certain complex steps. Oligosaccharide synthesis is usually achieved in vivo or in vitro through the action of various enzymes, which usually include various glycosyltransferases, glycoside hydrolases, and phosphorylases. The role of glycosyltransferases is to efficiently and specifically catalyze the transfer of sugar groups from sugar nucleotide donors to receptors. However, since sugar nucleotides are expensive and the generated nucleotide phosphates usually cause the glycosylation pathway to be subject to very obvious feedback inhibition, a sugar nucleotide regeneration system needs to be added to this type of system. In particular, in the sugar nucleotide regeneration system for enzymatic synthesis of oligosaccharides, a large amount of phosphate will be produced, and phosphate will be added to the fermentation medium for the production of human milk oligosaccharides by cell factory fermentation. Based on this, there will be a large amount of phosphate multivalent anions in the fermentation broth or reaction liquid, and the presence of these phosphates poses a huge challenge to the separation of oligosaccharides.

[0003] As for the current chemical synthesis, enzymatic synthesis and fermentation-based preparation methods, it is actually difficult to achieve large-scale preparation of oligosaccharides, especially HMOs products, with sufficient quality for food applications. The reason is that chemical synthesis requires several toxic chemicals, which may contaminate the final product. However, enzymatic methods and fermentation-based methods tend to produce complex mixtures of oligosaccharides, so that the desired product is contaminated by starting materials such as lactose, as well as intermediates, unwanted by-products (e.g., by-products derived from the side activities of certain glycosyltransferases) and substrates such as various monosaccharides and polypeptides. Specifically, human milk oligosaccharides are synthesized from scratch using glycosyltransferases in genetically engineered bacteria or enzymatic biosynthesis processes. For example, the synthesis of human milk oligosaccharides containing 5 sugar units means that it is necessary to start from disaccharides and synthesize trisaccharides, tetrasaccharides and pentasaccharides in sequence under the action of glycosyltransferases. In this biosynthesis process, a compound containing the target oligosaccharide will be obtained, that is, a reaction mixture or a cell fermentation broth, which contains a small amount of residual substrates and products of the intermediate process (non-target oligosaccharides), as well as some inorganic ions. Moreover, the structure and physicochemical properties of these non-target oligosaccharides are often very similar to those of the target oligosaccharides, and the molecular weight of the oligosaccharides obtained in adjacent reaction processes differs by only one sugar unit. In addition, it is worth mentioning that genetically engineered fermentation broths, especially those containing recombinant microorganisms (bacteria or eukaryotic microorganisms, such as Pasteurella), are much more complex than milk-derived product streams. For example, the composition of whey is ~94% water, 4-5% lactose, 0.5-1% protein, and only a few identified minerals (such as calcium, potassium and phosphorus), as well as some vitamins, which are simple matrices that are concentrated and demineralized only in dairy streams. In contrast, the matrix of the sugar solution obtained from the recombinant microbial fermentation process is very complex. First, the recombinant biomass is separated and inactivated according to the regulatory requirements, and the clarified liquid obtained is an uncertain matrix containing different salts and ions, and also contains heavy metals and trace elements. Secondly, the challenge of such liquids is to remove cell debris, membrane fragments such as lipids, proteins, and molecules derived from microbial cell metabolism. Therefore, the recovery of oligosaccharides produced by recombinant processing aids (such as genetically modified bacteria) is more challenging compared to whey and dairy streams because the contaminants therein are very diverse in terms of molecular weight, charged molecules (singly and multiply charged), and colored molecules.

[0004] Based on the above, it is not difficult to find that the main reason why the bioenzymatic method and the fermentation method tend to produce a complex mixture of oligosaccharides is that, in addition to the expected target oligosaccharides, there are at least the following undesirable substances in the bioenzymatic reaction liquid or the fermentation broth of the fermentation method:

[0005] 1) Oligosaccharide product analogs; the product analogs include compounds with a molecular weight lower than the molecular weight of the oligosaccharide to be purified or the sialylated oligosaccharide, and the compounds include the carbohydrate substrates required for fermentation or enzymatic reaction and various levels of oligosaccharide intermediates generated by various glycosyltransferases; for example, in the enzymatic synthesis of LST-a, residues of four substrates, lactose, N-acetylneuraminic acid (sialic acid), N-acetylglucosamine, and galactose, as well as residues of two intermediate products, LNTⅡ (lactose-N-triose) and LNT (lactose-N-tetraose).

[0006] 2) Remove polyvalent anions; Typical polyvalent anions include phosphate ions and sulfate ions. In the fermentation system for oligosaccharide generation, the polyvalent anions, especially phosphate ions and sulfate ions, are derived from the fermentation medium; in the enzymatic reaction system, the polyvalent anions mainly refer to phosphate ions. It should also be emphasized that for cost considerations, most systems for enzymatic synthesis of oligosaccharides require the addition of a sugar nucleotide regeneration system, while phosphate is added to the fermentation medium for the production of human milk oligosaccharides by cell factory fermentation. Based on this, a large amount of phosphate will be produced at the end of the above two types of synthesis systems, and the presence of these phosphates poses a huge challenge to the separation of oligosaccharides. Based on this, the concentration / content of phosphate ions / polyvalent anions in the reaction solution is generally not less than 150mM, and the ratio of acid ions / polyvalent anions in enzymatic synthesis is not less than 70%.

[0007] 3) Soluble protein: after the fermentation or enzymatic reaction is completed, soluble protein still exists in the solution after centrifugation; soluble protein in fermentation mainly refers to soluble protein secreted outside the cell during the fermentation process and hydrolases or lyases added to remove product analogs during purification. Soluble protein in enzymatic reaction system mainly refers to enzyme solution added as catalyst in the enzymatic reaction system and hydrolases or lyases added to remove product analogs.

[0008] 4) Cations: The cations include those added to the fermentation medium or the raw materials in the enzymatic reaction system in the form of sodium salts, as well as pH regulators added during the process and trace amounts of metal ions remaining from the precipitation reaction during the purification process. Typical cations include sodium ions, magnesium ions, potassium ions, etc.

[0009] 5) Anions, including monovalent anions added to the fermentation medium or the raw materials in the enzymatic reaction system in the form of chloride ion salts, as well as pH adjusters added during the process and trace amounts of anions remaining from the precipitation reaction during the purification process. Typical anions include chloride ions, phosphate ions, sulfate ions, etc.

[0010] Therefore, how to effectively and scalably separate the target oligosaccharides also has an important impact on the commercialization process of HMOs products. The existing methods for purifying each oligosaccharide from an oligosaccharide mixture are technically complex, difficult to scale up, and not economical for food applications. At present, when purifying human milk oligosaccharides from these phosphate-containing bioenzymatic reaction solutions and fermentation broths, as described in Patent Document 1, most of them use anion exchange resins to adsorb phosphate (CN201980036628.8). On the one hand, ion exchange resins have good removal capabilities for inorganic ions. However, if the phosphate concentration is very high, a large amount of resin is required for adsorption in industrial production, which means that the regeneration of the resin requires a large amount of regeneration agent and produces a huge amount of wastewater.

[0011] Patent document 1: CN201980036628.8, a simple method for purifying sialyllactose. Summary of the invention

[0012] 1. Problem to be solved

[0013] In a first aspect, one of the objects of the present invention is to provide a separation and purification method for purifying oligosaccharides produced by microbial fermentation or enzymatic reaction, which method can effectively achieve the separation of target oligosaccharides, is suitable for commercial scale or industrial scale, and can produce products with a purity that makes the products suitable for human consumption;

[0014] A second object of the present invention is to provide a method for separation and purification of oligosaccharides, which can effectively and environmentally friendly achieve the removal of high-concentration multivalent anions, especially phosphate ions, which method is economical, effective and environmentally friendly, suitable for commercial or industrial scale, and can produce a product with a purity that makes the product suitable for human consumption;

[0015] In a second aspect, the present invention aims to provide an oligosaccharide obtained according to the first aspect;

[0016] In a third aspect, the present invention aims to provide the oligosaccharide obtained according to the first aspect, or the use of the oligosaccharide according to the second aspect.

[0017] 2. Technical solution

[0018] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0019] [1] According to the first aspect, the present invention provides a method for separating and purifying oligosaccharides, the method comprising the following steps:

[0020] The fermentation liquid or the reaction liquid is separated and purified, and the treatment comprises the following steps:

[0021] a) removing product analogs;

[0022] b) removing polyvalent anions;

[0023] c) Remove soluble proteins, cations and anions.

[0024] [1.1] According to any embodiment of the first aspect of the present invention,

[0025] In a), the fermentation liquid or the reaction liquid is treated with a hydrolase to remove the product analogs; or

[0026] In a), the fermentation liquid or the reaction liquid is treated with a lyase to remove the product analog; or

[0027] In a), the fermentation liquid or reaction liquid is treated with a hydrolase or a lyase to remove the product analogs.

[0028] [1.2] According to the method for separating and purifying oligosaccharides of any embodiment of the first aspect of the present invention, the hydrolase is a glycoside hydrolase, and the final concentration of the hydrolase is 1 to 50 U / mL or 500 to 5000 ALU / L (for example, β-galactosidase, final concentration); the lyase is N-acetylneuraminic acid aldolase or sialic acid lyase; the final concentration of the lyase is 1 to 50 U / mL.

[0029] [1.3] According to the method for separating and purifying oligosaccharides in any embodiment of the first aspect of the present invention, in b), the fermentation liquid or the reaction liquid is treated with metal ions to remove the multivalent anions;

[0030] The added concentration of the metal ions is 5-1000 mM; preferably 100-500 mM, and more preferably 100-400 mM.

[0031] [1.4] According to the method for separating and purifying oligosaccharides in any embodiment of the first aspect of the present invention, in b), the fermentation liquid or the reaction liquid is treated with metal ions to remove the multivalent anions;

[0032] The added concentration of the metal ions is 5-1000 mM; preferably 100-500 mM, and more preferably 100-400 mM.

[0033] [1.5] According to the method for separating and purifying oligosaccharides in any embodiment of the first aspect of the present invention, the metal ions include any one or more of calcium ions, nickel ions, cobalt ions, zinc ions, copper ions, lead ions, manganese ions, magnesium ions, aluminum ions, iron ions, barium ions, and silver ions;

[0034] The metal ion is added in the form of a solution containing a salt of the metal ion;

[0035] The concentration of the salt solution is the concentration of a saturated solution corresponding to the metal salt solution.

[0036] [1.6] A method for separating and purifying oligosaccharides according to any embodiment of the first aspect of the present invention,

[0037] The pH of the fermentation liquid or reaction liquid after treatment b) is set to 6-12, preferably 7-10, and more preferably 8-9.

[0038] [1.7] A method for separating and purifying oligosaccharides according to any embodiment of the first aspect of the present invention,

[0039] In c), the fermentation liquid or the reaction liquid is ultrafiltered;

[0040] Preferably, ultrafiltration is performed to remove soluble proteins with a molecular weight ≥20 kDa from the fermentation broth or reaction liquid, more preferably, ultrafiltration is performed to remove soluble proteins with a molecular weight ≥10 kDa from the fermentation broth or reaction liquid, and most preferably, ultrafiltration is performed to remove soluble proteins with a molecular weight ≥4 kDa from the fermentation broth or reaction liquid.

[0041] [1.8] A method for separating and purifying oligosaccharides according to any embodiment of the first aspect of the present invention,

[0042] c), wherein the removal of cations from the fermentation broth or reaction broth is carried out by cation exchange chromatography;

[0043] Preferably, the stationary phase of the cation exchange chromatography is a cation exchange resin, more preferably a strongly acidic cation exchange resin;

[0044] c), wherein the removal of anions from the fermentation broth or reaction broth is performed by anion exchange chromatography;

[0045] Preferably, the stationary phase of the anion exchange chromatography is an anion exchange resin, more preferably a weakly basic anion exchange resin.

[0046] [1.9] A method for separating and purifying oligosaccharides according to any embodiment of the first aspect of the present invention,

[0047] Also includes one or more processing steps selected from the following:

[0048] Ⅰ) performing solid-liquid separation treatment on the fermentation liquid or reaction liquid after the treatment in b);

[0049] The solid-liquid separation treatment method includes filtration, microfiltration or centrifugation;

[0050] II) filtering the fermentation liquid or reaction liquid after the treatment in c), wherein the filtering method is preferably nanofiltration;

[0051] Nanofiltration concentration using nanofiltration membranes can remove residual pH regulator ions and monosaccharide products after enzymatic hydrolysis. The molecular weight cutoff of the nanofiltration membrane should be between the molecular weight of the target oligosaccharide and the molecular weight of the impurities.

[0052] Preferably, the molecular weight cut-off of the nanofiltration membrane is less than 500 Daltons. More preferably, the molecular weight cut-off of the nanofiltration membrane is 100 to 300 Daltons.

[0053] [1.10] A method for separating and purifying oligosaccharides according to any embodiment of the first aspect of the present invention,

[0054] Also includes one or more processing steps selected from the following:

[0055] III) decolorizing the fermentation liquid or reaction liquid after treatment in II); preferably, decolorizing with activated carbon;

[0056] IV) The fermentation liquid or reaction liquid treated by III) is subjected to crystallization, alcohol precipitation, freeze-drying or spray-drying.

[0057] [1.11] A method for separating and purifying oligosaccharides according to any embodiment of the first aspect of the present invention,

[0058] The oligosaccharides include neutral human milk oligosaccharides and acidic human milk oligosaccharides (sialylated human milk oligosaccharides);

[0059] Including lactosyl-N-tetraose (LNT), lactosyl-N-neotetraose (LNnT), lactose-N-difucosylhexaose I (LNDFHⅠ), lactose-N-difucosylhexaose II (LNDFHⅡ), lactose-N-hexaose (LNH), lactose-N-neohexose (LNnH), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 6'-sialyllactosamine (6'-SLN), 3'-sialyllactosamine (3'-SLN), 3'-sialyllactose-3-fucosyllactose (3'S3FL), disialyl lactose (DSL), disialyl lacto-N-tetraose (DSLNT), sialyllactose-N-tetraose a (LST a), sialyllactose-N-tetraose b (LST b), sialyllactose-N-tetraose c (LST c), fucosyl disialyl lactose-N-hexasaccharide I (FDS-LNH-I), fucosyl disialyl lactose-N-hexasaccharide II (FDS-LNH-II), etc., but are not limited to these oligosaccharides.

[0060] [2] According to the second aspect, the present invention provides a preparation containing oligosaccharides, wherein the oligosaccharides are purified by any of the above methods; and the oligosaccharides are present in the preparation containing oligosaccharides at a purity of ≥90% (mass).

[0061] [3] According to a third aspect, the present invention provides use of any of the above-described preparations for the manufacture of a nutritional composition, preferably an infant formula.

[0062] Beneficial Effects

[0063] 1. The method provided by the present invention is simple and easy to control, the whole process conditions are mild, and the equipment used is simple, which is conducive to the industrial production of target oligosaccharides;

[0064] 2. The method provided by the present invention can effectively remove product analogs produced during the fermentation process or residual in the enzymatic reaction, increase the difference in physical and chemical properties between these substances and the target oligosaccharides, reduce the difficulty of separation and purification of the target oligosaccharides, and effectively avoid the use of complex and expensive separation processes such as size-dependent gel filtration chromatography and chromatographic separation. In addition, the hydrolase or lyase used in the present invention will not have an enzymatic effect on human milk oligosaccharides, so it does not affect the yield of the product.

[0065] 3. The method provided by the present invention can effectively remove water-soluble phosphate ions, and the generated phosphate precipitate particles are large, and the subsequent filtration speed is fast and the time is short. It has been confirmed by experiments that the final phosphate content in the solution is less than 10mmol / L through the method provided by the present invention. And the phosphate precipitate produced by the present invention will not have an adsorption effect on human milk oligosaccharides, so it does not affect the yield of the product. And the present invention generates a phosphate precipitate through reaction, and the precipitate can be used in the field of building materials or agriculture, which is conducive to the recycling of waste.

[0066] 4. The present invention combines enzymatic hydrolysis, chemical precipitation, ion exchange resin and nanofiltration membrane to make the content of impurity ions in the final product very low, greatly reducing the use of ion exchange resin and the amount of wastewater generated in the separation process.

[0067] 5. The purity of the final product obtained in the present invention can reach more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : The structure of 6'-sialyllactose;

[0069] Figure 2 :Enzymatic synthesis of 6'-sialyllactose

[0070] Figure 3 : Schematic diagram of the separation and purification method of target oligosaccharides;

[0071] Figure 4 : HPLC chart of 6'-sialyllactose at the end of reaction;

[0072] Figure 5 : HPLC chart after adding N-acetylneuraminic acid aldolase to the 6'-sialyllactose reaction solution for 3 hours;

[0073] Figure 6 : HPLC chart after adding β-galactosidase to the 6'-sialyllactose reaction solution and reacting for 3 hours;

[0074] Figure 7 : HPLC chart of purified 6'-sialyllactose;

[0075] Figure 8 : LC-MS image of purified 6'-sialyllactose;

[0076] Fig. 9 :Multi-enzyme catalytic synthesis route of LNT;

[0077] Fig.10 : HPLC chart of purified LNT. DETAILED DESCRIPTION

[0078] The present disclosure may be more easily understood by reference to the following description in conjunction with the accompanying drawings and examples, all of which constitute a part of the present disclosure. It should be understood that the present disclosure is not limited to the specific products, methods, conditions or parameters described and / or shown herein. Further, the terms used herein are only used for the purpose of describing specific embodiments by way of example and are not intended to be limiting unless otherwise stated.

[0079] It should also be understood that, for the sake of clarity, certain features of the present disclosure may be described herein in the context of separate embodiments, but may also be provided in combination with each other in a single embodiment. That is, unless clearly incompatible or specifically not included, each separate embodiment is considered to be combinable with any other embodiment, and the combination is considered to represent another different embodiment. Conversely, for the sake of simplicity, the various features of the present disclosure described in the context of a single embodiment may also be provided individually or in any sub-combination. Finally, although a particular embodiment may be described as part of a series of steps or part of a more general structure, each step or substructure itself may also be considered to be an independent embodiment.

[0080] Unless otherwise indicated, it should be understood that each individual element in the same statement and each combination of individual elements in the statement will be interpreted as a different embodiment. For example, a list of embodiments expressed as "A, B, or C" should be interpreted to include embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0081] As used herein, the singular forms of the articles "a," "an," and "the" also include the corresponding plural references, and a reference to a specific value includes at least that specific value unless the context clearly dictates otherwise. Thus, for example, a reference to "a substance" is a reference to at least one of that substance and equivalents thereof.

[0082] As used herein, the terms "comprise, include, contain, have", when preceding the description of a step in a method, mean that the method encompasses one or more steps beyond those explicitly described, and that these additional one or more steps may be performed before, between, and / or after the described steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of a, b, c, and x, and a method of steps x, a, b, and c.

[0083] In addition, when preceding the recitation of steps in a method, the term "comprising" does not (although it may) require sequential performance of the listed steps unless the context clearly indicates otherwise. For example, a method comprising steps a, b, and c encompasses the following illustrated order: a method in which the steps are performed in the order of steps a, c, and b, the order of steps a, b, and c, the order of steps b, a, and c, the order of steps b, c, and a, the order of steps c, b, and a, and the order of steps c, a, and b.

[0084] Generally, the use of the term "about" indicates an approximate value that can vary according to the desired properties obtained by the disclosed subject matter, and will be interpreted in a context-dependent manner based on function. Therefore, a person of ordinary skill in the art will be able to interpret a certain degree of difference on a case-by-case basis. In some cases, the number of important figures used when expressing a particular value can be a representative technique for determining the difference allowed by the term "about". In other cases, a gradient in a series of values ​​can be used to determine the range of differences allowed by the term "about". Further, all ranges in the present disclosure are inclusive and combinable, and the reference to the value described in the range includes each value within the range. Unless otherwise indicated, all numbers used herein to represent the amount of ingredients, properties such as molecular weight, reaction conditions, etc. should be understood to be modified by the term "about" in all instances. Therefore, unless otherwise indicated, the numerical parameters herein are approximate values ​​that can vary according to the desired properties attempted to be obtained by the present invention. At the very least, and without limiting the application of equivalent declarations to the scope of the claims, the number of reported significant figures should at least be considered and each numerical parameter should be understood by applying ordinary rounding techniques. While the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain standard deviations necessarily resulting from errors found in the numerical testing measurements.

[0085] The terms including ordinal numbers such as "first" and "second" can be used to explain various components or fluids, but these components and fluids are not limited by these terms. Therefore, without departing from the teachings of the present disclosure, these terms are only used to distinguish the component / fluid from another component / fluid.

[0086] When items are described by using the conjunction terms "... and / or...," etc., the description should be understood to include any one of the associated listed items and all combinations of one or more thereof.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terms used herein and / or include any and all combinations of one or more of the associated listed items.

[0088] As used herein, "oligosaccharide" refers to the synthesis of one or more enzymes in the glycosyltransferase, glycosidase, and sugar nucleotide regeneration system. Based on this, the biosynthesis of the oligosaccharide includes genetic engineering bacteria fermentation and bioenzymatic synthesis.

[0089] The separation and purification process of oligosaccharides provided by the present invention can be applied to purify oligosaccharides produced by fermentation of genetically engineered bacteria. Cells capable of producing the desired oligosaccharides are cultured under conditions that allow the cells to produce the desired oligosaccharides. Suitable cells for producing the desired oligosaccharides include bacteria, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or yeast, such as Saccharomyces cerevisiae and Pichia pastoris.

[0090] Cells can be genetically engineered to produce desired oligosaccharides, such as human milk oligosaccharides (genetically unmodified precursor cells are unable to produce the desired oligosaccharides. For example, E. coli is a preferred host for metabolic engineering and has been used for the fermentation of HMOs, including neutral HMOs as well as sialylated HMOs. However, other host strains, such as yeast (e.g., Saccharomyces cerevisiae), Corynebacterium glutamicum, Bacillus species, can also be engineered to produce oligosaccharides.

[0091] The oligosaccharides include human milk oligosaccharides. HMOs can be divided into neutral human milk oligosaccharides (neutral HMOs) and acidic human milk oligosaccharides, namely sialylated human milk oligosaccharides (sialylated HMOs). The HMOs include lactosyl-N-tetraose (LNT), lactosyl-N-neotetraose (LNnT), lactose-N-difucohexaose I (LNDFHI), lactose-N-difucohexaose II (LNDFHI), lactose-N-hexaose (LNH), lactose-N-neohexose (LNnH), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 6'-sialyllactosamine (6'-SLN), 3'-sialyllactosamine (3'-SLN), 3'-sialyllactose-3-fucosyllactose (3'S3FL), disialyllactose (DSL), disialyllacto-N-tetraose (DSLNT), sialyllactose-N-tetraose a (LST a), sialyllactose-N-tetraose b (LST b), sialyllactose-N-tetraose c (LST c), fucosyl disialyl lactose-N-hexasaccharide I (FDS-LNH-I), fucosyl disialyl lactose-N-hexasaccharide II (FDS-LNH-II), etc., but are not limited to these oligosaccharides.

[0092] Among them, for the production of the desired sialylated HMOs, the bacterial or yeast host strain usually needs to start from glucose or glycerol, and synthesize sialic acid by heterologously expressing enzymes related to sialic acid synthesis in the cell. At the same time, lactose is also needed as a raw material, and the chain is extended under the action of a specific heterologously expressed glycosyltransferase, and finally, sialylated HMO is synthesized under the action of the heterologously expressed glycosyltransferase. In this process, the host cell needs to be modified to improve the excretion of the product. At the same time, some intermediates, such as the generated or added sialic acid, the intermediates generated by the substrate lactose during the sugar chain extension process, etc., will be secreted into the culture medium, becoming product analogs that need to be removed from the target product during purification.

[0093] The term "desired" with respect to (sialylated / neutral) oligosaccharides refers to oligosaccharides that should be produced by or are expected to be produced by a cell. Desired oligosaccharides are oligosaccharides to be purified by the process disclosed herein. As used herein, the term "desired" with respect to sialylated oligosaccharides is also used to distinguish sialylated oligosaccharides to be produced from other sialylated oligosaccharides that a cell may unintentionally produce.

[0094] The separation and purification process of oligosaccharides provided by the present invention can be applied to purify oligosaccharides synthesized by bioenzymatic methods. The desired oligosaccharides are obtained by one or more in vitro enzymatic reactions, and can be purified from the reaction mixture by subjecting the reaction mixture (or cell-free fermentation broth, clarified lysate) to the purification process described herein at the end of the biocatalytic reaction. It should be understood that the purification of oligosaccharides from the reaction mixture of in vitro biocatalysis does not require the removal of biomass from the reaction mixture.

[0095] The cell-free fermentation broth, clarified lysate or reaction mixture contains the desired oligosaccharides and a large amount of impurities and unwanted components, including other oligosaccharides other than the desired oligosaccharides, monovalent salts, divalent salts, amino acids, polypeptides, proteins, nucleic acids, residual monosaccharides from the reaction, residual substrate lactose from the reaction, and product analogs such as intermediates that may be generated during the reaction.

[0096] The oligosaccharides include human milk oligosaccharides. HMOs can be divided into neutral human milk oligosaccharides (neutral HMOs) and acidic human milk oligosaccharides, namely sialylated human milk oligosaccharides (sialylated HMOs). The HMOs include lactosyl-N-tetraose (LNT), lactosyl-N-neotetraose (LNnT), lactose-N-difucohexaose I (LNDFHI), lactose-N-difucohexaose II (LNDFHI), lactose-N-hexaose (LNH), lactose-N-neohexose (LNnH), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 6'-sialyllactosamine (6'-SLN), 3'-sialyllactosamine (3'-SLN), 3'-sialyllactose-3-fucosyllactose (3'S3FL), disialyllactose (DSL), disialyllacto-N-tetraose (DSLNT), sialyllactose-N-tetraose a (LST a), sialyllactose-N-tetraose b (LST b), sialyllactose-N-tetraose c (LST c), fucosyl disialyl lactose-N-hexasaccharide I (FDS-LNH-I), fucosyl disialyl lactose-N-hexasaccharide II (FDS-LNH-II), etc., but not limited to these oligosaccharides. And the aforementioned HMOs can be synthesized by bioenzymatic method.

[0097] Based on this, according to the first aspect, a method for separating and purifying oligosaccharides, the method separates and purifies the fermentation liquid or the reaction liquid, comprising the following steps:

[0098] a) removing product analogs;

[0099] b) removing polyvalent anions;

[0100] c) Remove soluble proteins, cations and anions.

[0101] As described herein, the "oligosaccharide product analogs" mainly include compounds with a molecular weight lower than the molecular weight of the oligosaccharide to be purified or the sialylated oligosaccharide, and the compounds include the carbohydrate substrates required for fermentation or enzymatic reactions and various levels of oligosaccharide intermediates generated by the action of various glycosyltransferases; for example, in the enzymatic synthesis of LST-a, residues of four substrates, lactose, N-acetylneuraminic acid (sialic acid), N-acetylglucosamine, and galactose, as well as residues of two intermediates, LNTⅡ (lactose-N-triose) and LNT (lactose-N-tetraose).

[0102] As described herein, "polyvalent anions" mainly include phosphate ions and sulfate ions. In the fermentation system for oligosaccharide generation, the polyvalent anions, especially phosphate ions and sulfate ions, are derived from the fermentation medium; in the enzymatic reaction system, the polyvalent anions mainly refer to phosphate ions. And for cost considerations, most systems for enzymatic synthesis of oligosaccharides require the addition of a sugar nucleotide regeneration system, while phosphates are added to the fermentation medium for the production of human milk oligosaccharides by fermentation in cell factories. Based on this, a large amount of phosphates will also be produced in the above two types of synthesis systems, and the presence of these phosphates poses a huge challenge to the separation of oligosaccharides. Based on this, the concentration / content of phosphate ions / polyvalent anions in the reaction solution is generally not less than 150mM.

[0103] As mentioned herein, "soluble protein" mainly refers to the soluble protein that remains in the solution after centrifugation at the end of the fermentation or enzymatic reaction; wherein the soluble protein in the fermentation method mainly refers to the soluble protein secreted outside the cell during the fermentation process and the hydrolase or lyase added in the purification to remove the product analogs. The soluble protein in the enzymatic reaction system mainly refers to the enzyme solution added as a catalyst in the enzymatic reaction system and the hydrolase or lyase added in order to remove the product analogs.

[0104] The "cations" mentioned herein include cations added to the fermentation medium or the raw materials in the enzymatic reaction system in the form of sodium salts, as well as pH adjusters added during the process and trace amounts of metal ions remaining from precipitation reactions during the purification process. Typical cations include sodium ions, magnesium ions, potassium ions, etc.

[0105] As used herein, "anions" include monovalent anions added to the fermentation medium or the raw materials in the enzymatic reaction system in the form of chloride ion salts, as well as pH regulators added during the process and trace amounts of anions remaining from precipitation reactions during the purification process. Typical anions include chloride ions, phosphate ions, sulfate ions, etc.

[0106] In one embodiment of the first aspect of the present invention, the operation of removing the product analogs includes treating the fermentation liquid or the reaction liquid with an enzyme solution. In the step a) of producing the product analogs during the enzymatic fermentation process or the residual product analogs during the enzymatic reaction, the enzyme solution includes or only has a hydrolase. Preferably, the hydrolase is used to hydrolyze the intermediate by-products, and the hydrolase is further preferably a glycoside hydrolase (referred to as glycosidase) capable of hydrolyzing the substrate or the intermediate by-products.

[0107] As described herein, "glycoside hydrolase" refers to a glycosidase that can hydrolyze product analogs produced during the fermentation process or residual in the enzymatic reaction; the glycoside hydrolase includes galactosidase, fucosidase, sialidase, N-acetylglucosaminidase or N-acetylglucosaminidase, etc. Preferably, the glycoside hydrolase includes β-galactosidase, α-fucosidase, α-sialidase, β-N-acetylglucosaminidase or β-N-acetylglucosaminidase. Further preferably, the glycoside hydrolase includes β-1,3 galactosidase, α-1,2 / 1,3 / 1,4 fucosidase, α-2,3 / 2,6 sialidase, β-1,3 / 1,6N-acetylglucosaminidase or β-N-acetylglucosaminidase. Most preferably, the glycoside hydrolase refers to an exoglycosidase that is specific for the non-reducing end of a product analog produced during a fermentation process or remaining in an enzymatic reaction, including β-1,3 galactosidase, α-1,2 / 1,3 / 1,4 fucosidase, α-2,3 / 2,6 sialidase, β-1,3 / 1,6 N-acetylglucosaminidase or β-N-acetylglucosaminidase.

[0108] Further, in some embodiments, in the step a) of producing the product analogs in the enzymatic fermentation process or the residual product in the enzymatic reaction, the fermentation liquid or the reaction liquid is treated with an enzyme solution, wherein the enzyme solution includes or contains only hydrolases and lyases. Preferably, the hydrolases are used to hydrolyze the intermediate byproducts, and more preferably, the lyases are used to degrade the substrates.

[0109] As described herein, the term "lyase" refers to a lyase that can lyse product analogs produced during the fermentation process or residual in the enzymatic reaction. After the genetically engineered bacteria fermentation liquid or the enzymatic reaction liquid is treated with glycoside hydrolase, the residual substrate and product analogs are all monosaccharide structures, including glucose, N-acetylglucosamine, galactose, sialic acid, fucose, etc. Monosaccharides other than sialic acid can be separated from the product by a nanofiltration membrane, and sialic acid is close to the sialylated HMOs in properties, molecular structure and molecular weight, resulting in it being unable to be separated from the product by a nanofiltration method. Therefore, the present invention further processes sialic acid using a lyase in order to obtain a higher product purity. The lyase is further preferably N-acetylneuraminic acid aldolase and / or sialyl lyase.

[0110] Further, in some embodiments, the addition amount (final concentration) of the hydrolase is 500-5000 ALU / L, preferably 1000-3000 ALU / L, more preferably 1500-2500 ALU / L; the final concentration of the hydrolase is 1-50 U / mL or 500-5000 ALU / L (purchased β-galactosidase can be used in subsequent embodiments);

[0111] The amount of the lytic enzyme added (final concentration) is 1 to 50 U / mL, preferably 1 to 30 U / mL, and more preferably 5 to 10 U / mL.

[0112] In the first aspect of the present invention, in one embodiment, in b), the operation of removing the polyvalent anions comprises: treating the fermentation broth or reaction broth with metal ions to remove the polyvalent anions; the added metal ions will combine with the polyvalent anions to form a salt precipitate with extremely low solubility, so in some embodiments, it is necessary to remove the precipitate by centrifugation. In other and / or alternative embodiments, the precipitate is separated from the fermentation broth or reaction broth by filtration. Suitable filtration methods for separating the precipitate from the fermentation broth include microfiltration and ultrafiltration.

[0113] Microfiltration itself is a physical filtration process in which a fluid containing particles is passed through a membrane with a specific pore size to separate the particles from the fluid. As used herein, the term "microfiltration" refers to a physical filtration process in which precipitated or soluble proteins are separated from a fermentation broth.

[0114] Ultrafiltration is a variety of membrane filtration and is not fundamentally different. In ultrafiltration, forces such as pressure or concentration gradients cause separation across a semipermeable membrane. High molecular weight cells, suspended solids, precipitates or soluble proteins and solutes are retained in the so-called retentate, while water and low molecular weight solutes such as the desired sialylated oligosaccharides pass through the membrane into the permeate (filtrate).

[0115] Further, in another embodiment, the added concentration of the metal ion is 5-1000mM; preferably 100-500mM, and more preferably 100-400mM. Usually, the metal ion is selected from one or more of calcium ion, nickel ion, cobalt ion, zinc ion, copper ion, lead ion, manganese ion, magnesium ion, aluminum ion, iron ion, barium ion, and silver ion; and, usually, the metal ion is added in the form of a solution containing a salt of the metal ion. Further, in another embodiment, the metal ion is added in the form of a solution containing a salt of the metal ion; the concentration of the salt solution is the concentration of the saturated solution corresponding to the metal salt solution. Further preferably, in some embodiments, the pH of the fermentation broth or reaction liquid after step b) treatment is 6-12, preferably 7-10, and more preferably 8-9.

[0116] In some preferred embodiments, during the operation of removing the polyvalent anions, the optimal pH of the liquid environment needs to be maintained at 6-12, preferably 7-10, and more preferably 8-9; based on the consideration of minimizing the use of impurity ions that are difficult to remove, it is preferred to adjust the pH value by adding an alkali solution containing sodium hydroxide or potassium hydroxide. Moreover, if the concentration of the alkali solution is too high, it will actually lead to product degradation, and if it is too low, the adjustment speed will be too slow. Therefore, the concentration of the alkali solution is generally 3-6 mol / L.

[0117] Further, in some preferred embodiments, during the operation of removing the polyvalent anions, the liquid environment needs to be kept at a certain temperature, and the temperature described herein will affect the solubility of the precipitate and the stability of the product. At a high temperature, the stability of the product is poor, and degradation is prone to occur, and the solubility of the phosphate precipitate is high. At a low temperature, energy consumption increases. Therefore, considering comprehensively, the temperature should be 0-45°C, preferably 5-30°C, and the optimal temperature is 10-20°C.

[0118] In one embodiment of the first aspect of the present invention, the method for separating and purifying oligosaccharides, the operation of removing the soluble protein includes ultrafiltration. In some preferred embodiments, in c), the fermentation broth or reaction liquid is ultrafiltered to remove soluble proteins with a molecular weight of ≥20 kDa from the fermentation broth or reaction liquid. In some preferred embodiments, in c), the fermentation broth or reaction liquid is ultrafiltered to remove soluble proteins with a molecular weight of ≥10 kDa from the fermentation broth or reaction liquid; in some preferred embodiments, in c), the fermentation broth or reaction liquid is ultrafiltered to remove soluble proteins with a molecular weight of ≥4 kDa from the fermentation broth or reaction liquid.

[0119] In one embodiment of the first aspect of the present invention, the method for separating and purifying oligosaccharides comprises a cation removal operation to remove positively charged compounds from a cell-free fermentation broth, clarified lysate or reaction mixture.

[0120] Therefore, in some embodiments, in step c), the removal of cations from the fermentation broth or reaction liquid is performed by cation exchange chromatography, the stationary phase of the cation exchange chromatography is a cation exchange resin, more preferably a strongly acidic cation exchange resin, and the cation exchange resin treatment is performed to remove positively charged compounds from the cell-free fermentation broth, clarified lysate or reaction mixture.

[0121] In some preferred embodiments, the cation exchange resin is a strong acid cation exchange resin; available strong acid cation exchange resins include H type or Na type, preferably H type resin. Schematically, suitable cation exchange resins for removing positively charged compounds include strong acid cation exchange resins and weak acid cation exchange resins such as D113; the strong acid cation exchange resins are particularly strong acid styrene cation exchange resins, such as: 0017 strong acid styrene cation exchange resin, 732 strong acid cation exchange resin, IR-120 (or AmberliteIR-120, CAS No. 9002-23-7) cation exchange resin, 001×7 strong acid styrene cation exchange, 732 cation resin, lewatit-100 (such as: German Lanxess ion exchange resin Lewatit MonoPlus S100), Diaion SK-1 (such as: Japanese Mitsubishi cation exchange resin DIAION SK-1).

[0122] In some preferred embodiments, during the operation of removing the cations, an alkali solution is used to adjust the pH of the solution to 5-9, preferably 6-7.

[0123] In one embodiment of the first aspect of the present invention, the method for separating and purifying oligosaccharides comprises an anion removal operation to remove negatively charged compounds from a cell-free fermentation broth, clarified lysate or reaction mixture.

[0124] Therefore, in some embodiments, in step c), the removal of anions from the fermentation broth or reaction liquid is performed by anion exchange chromatography; the stationary phase of the anion exchange chromatography is an anion exchange resin, more preferably a weakly basic anion exchange resin.

[0125] In some preferred embodiments, the available weakly basic anion exchange resin can be OH type or Cl type, preferably OH type resin. Schematically, suitable anion exchange resins include D201, 201*7, Amberlite IRA-400 (CAS No.: 9002-24-8), lewatit-M 500 (for example: German Bayer Lanxess resin MonoPlus M500), Diaion SA-10A (for example: Mitsubishi Chemical anion exchange resin gel type), and other strong basic anion resins, as well as D301 macroporous weakly basic styrene type I anion exchange resin, D113, Amberlite-200 (CAS No.: 12626-25-4), lewatit-MP 500 (for example: German Bayer Lanxess resin MonoPlus MP 500), Diaion PA308 (for example: Mitsubishi Chemical anion exchange resin porous type) and other weakly basic anion exchange resins.

[0126] In some preferred embodiments, during the operation of removing the anions, an acid solution is simultaneously used to adjust the pH of the solution to 5-9, preferably 6-8.

[0127] In one embodiment of the first aspect of the present invention, after the fermentation broth or reaction liquid is subjected to the product analogs and polyvalent anion removal operation, the method for separating and purifying oligosaccharides further comprises: step I) subjecting the fermentation broth or reaction liquid after the treatment in step b) to solid-liquid separation; the solid-liquid separation treatment comprises filtration, microfiltration or centrifugation.

[0128] In one embodiment of the first aspect of the present invention, after removing product analogs, multivalent anions, soluble proteins, cations, anions, etc. from the fermentation broth or reaction liquid, the method for separating and purifying oligosaccharides generally further comprises:

[0129] Step II), filtering the fermentation liquid or reaction liquid after treatment in c), wherein the filtering method is preferably nanofiltration;

[0130] The enzymatic substrate or by-product is ultrafiltered after removing the precipitate, and the desalted solution is subjected to nanofiltration. This step can further reduce and remove some ions contained in the solution, small molecules formed after the hydrolase or lyase treatment, and further reduce the impurity content in the final product.

[0131] In some embodiments, the filtration treatment method is preferably nanofiltration; the solution 6 is concentrated by nanofiltration using a nanofiltration membrane, and residual pH regulator ions and monosaccharide products after enzymatic hydrolysis can be removed at the same time. The molecular weight cutoff of the nanofiltration membrane should be between the molecular weight of the target oligosaccharide and the molecular weight of the impurity;

[0132] The molecular weight cutoff of the nanofiltration membrane should be between the molecular weight of the target oligosaccharide and the molecular weight of the impurity. Preferably, the molecular weight cutoff of the nanofiltration membrane is less than 500 Daltons. More preferably, the molecular weight cutoff of the nanofiltration membrane is 100 to 300 Daltons.

[0133] In one embodiment of the first aspect of the present invention, the method for separating and purifying oligosaccharides further comprises one or more processing steps selected from the following:

[0134] Decolorization treatment; and crystallization, or alcohol precipitation, or freeze drying, or spray drying treatment.

[0135] In some embodiments, the method comprises step III), wherein the fermentation broth or reaction broth treated by II) is subjected to decolorization treatment; preferably, the decolorization treatment is performed using activated carbon. Further, in some embodiments, the method comprises step IV), wherein the fermentation broth or reaction broth treated by III) is subjected to crystallization, alcohol precipitation, freeze drying, or spray drying.

[0136] In one embodiment of the first aspect of the present invention, the method for separating and purifying oligosaccharides provided by the present invention comprises the following steps:

[0137] (1) quantifying the amount of each product analogue produced during the fermentation process or remaining in the enzymatic reaction, adding an appropriate amount of enzyme solution to the fermentation liquid or the enzymatic reaction liquid to remove the product analogue produced during the fermentation process or remaining in the enzymatic reaction, and obtaining a liquid 1 containing the target oligosaccharide;

[0138] (2) Accurately determine the content of phosphate ions in the solution, and remove phosphate ions in the fermentation broth or enzymatic reaction solution by adding metal ions to the solution at a molar ratio of 1:1. Maintain the pH value of the solution at 8-9, wait for the precipitation of precipitates, and obtain solution 2 containing human milk oligosaccharides and precipitates;

[0139] (3) After the precipitate no longer precipitates, remove the precipitate from the solution by filtering, microfiltration or centrifugation to obtain a solution 3 containing human milk oligosaccharides;

[0140] (4) removing soluble protein in solution 3 by ultrafiltration to obtain solution 4 containing human milk oligosaccharides;

[0141] (5) removing cations in solution 4 to obtain solution 5 containing oligosaccharides;

[0142] (6) removing anions from solution 5 to obtain solution 6 containing oligosaccharides;

[0143] (7) Solution 6 is subjected to nanofiltration to obtain solution 7 containing high-purity target oligosaccharides.

[0144] (8) subjecting solution 7 to an activated carbon decolorization treatment to obtain a concentrated solution 8 containing high-purity oligosaccharides;

[0145] (9) Solution 8 is treated to obtain high-purity oligosaccharide crystals or powder.

[0146] In one embodiment of the first aspect of the invention, the purification process provides the desired oligosaccharides in a preparation, wherein the purity of the desired oligosaccharides is ≥80%, ≥85%, ≥90%, ≥95%. The process provides a preparation of sialylated oligosaccharides, wherein the purity of the oligosaccharides is suitable for food and feed applications.

[0147] Based on the above, according to the method for separating and purifying oligosaccharides of any embodiment of the first aspect of the present invention, the oligosaccharides include neutral human milk oligosaccharides and acidic human milk oligosaccharides (sialylated human milk oligosaccharides); including lactosyl-N-tetraose (LNT), lactosyl-N-neotetraose (LNnT), lactose-N-difucosylhexaose I (LNDFHⅠ), lactose-N-difucosylhexaose II (LNDFHⅡ), lactose-N-hexaose (LNH) , lactose-N-neohexose (LNnH), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 6'-sialyllactosamine (6'-SLN), 3'-sialyllactosamine (3'-SLN), 3'-sialyl-3-fucosyllactose (3'S3FL), disialyl lactose (DSL), disialyl lacto-N-tetraose (DSLNT), sialyl lactose-N-tetraose a (LST a), sialyl lactose-N-tetraose b (LST b), sialyl lactose-N-tetraose c (LST c), fucosyl disialyl lactose-N-hexasaccharide Ⅰ (FDS-LNH-Ⅰ), fucosyl disialyl lactose-N-hexasaccharide Ⅱ (FDS-LNH-Ⅱ), etc., but are not limited to these oligosaccharides.

[0148] According to an embodiment of the second aspect of the present invention, there is provided a preparation containing oligosaccharides, wherein the oligosaccharides are purified by any of the above methods.

[0149] In one embodiment of the second aspect of the present invention, the oligosaccharide is present in the formulation at a purity of ≥80% by mass. In some embodiments, the oligosaccharide is present in the formulation at a purity of ≥85% by mass. Further, in some preferred embodiments, the oligosaccharide is present in the formulation at a purity of ≥90% by mass. Further, in some preferred embodiments, the oligosaccharide is present in the formulation at a purity of ≥95% by mass.

[0150] The process provides preparations of sialylated oligosaccharides wherein the oligosaccharides are of a purity suitable for food and feed applications.

[0151] According to an embodiment of the third aspect of the present invention, there is provided a use of any of the above-described formulations for the manufacture of a nutritional composition, preferably an infant formula. The components of an exemplary infant formula include the following:

[0152] Table 1 Ingredient information of infant formula

[0153] Components Per 100g / component g energy 1800~2100KJ protein 10～20g Fat 10～30g carbohydrate 50～70g Of which lactose 42～58g Galacto-oligosaccharides 1～3g HMO 3.5~5.1g / L 2'FL' 1.8~2.4g / L 3FL 0.5~0.6g / L LNT 0.85~1.15g / L LqCy 0~0.2g / L 3'SL 0.05~0.28g / L 6'SL 0.12~0.4g / L Salt 0.4～0.6g

[0154] The present invention is further described below in conjunction with more specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The essential features and remarkable effects of the present invention can be reflected from the following examples, and the described embodiments are part of the embodiments of the present invention, rather than all the embodiments, and therefore, they do not limit the present invention in any way, and those skilled in the art make some non-essential improvements and adjustments based on the content of the present invention, which all belong to the protection scope of the present invention.

[0155] Example 1

[0156] An enzyme reaction system is used to generate an enzyme reaction solution containing 6'-sialyllactose (6'SL);

[0157] See also Figure 2 The following is a roadmap for the enzymatic synthesis of 6'SL. Reaction system composition and conditions:

[0158] 100ml reaction system, containing:

[0159] 80 mM sialic acid, 100 mM lactose, 80 mM ATP, Tris-HCl (pH 8.5, 100 mM), 60 mM MgCl2,

[0160] The concentrations of NmCSS (CMP-sialic acid synthetase, Yu H, et al., Bioorganic & Medicinal Chemistry, 2004, 12, 6427–6435), Pd26ST (α-2,6-sialyltransferase, Sun M, et al., Biotechnol Lett. 2008, 30, 671–676), and PPA (pyrophosphatase, Li L, et al., Org. Lett., 2013, 15, 5528-5530) were all 2 μM;

[0161] React at 37°C and 140 rpm for 48 hr.

[0162] The conversion rate of the reaction can reach 92%, and the final 6'SL production is 73.6mM, the lactose residue is 9.03g / L, and the sialic acid residue is 1.98g / L. In the multi-enzyme catalytic synthesis, due to the addition of PPA, 2 molecules of phosphate are generated for every molecule of product generated. The residual substrate and product analogs of the reaction are lactose and sialic acid.

[0163] Example 2

[0164] Purify 6'-sialyllactose from the enzymatic reaction solution of Example 1, as follows Figure 3 As shown:

[0165] This laboratory first used an enzymatic reaction system to generate 6'-sialyllactose, such as Figure 4 The reaction is completed. After the reaction is completed, 10U / mL (final concentration) of N-acetylneuraminic acid aldolase (expressed in this laboratory) is directly added to the reaction system. N-acetylneuraminic acid aldolase is a cleavage enzyme that can effectively degrade the residual N-acetylneuraminic acid (sialic acid) in the solution after the reaction into sodium pyruvate and N-acetylglucosamine. This step corresponds to Figure 5 After the reaction was carried out at 37°C for 3 hours, the pH value of the solution was adjusted to 5.0, the reaction temperature was adjusted to 55°C, and 2500 ALU / L (final concentration) of β-galactosidase was added for 3 hours. This step corresponds to Figure 6 β-galactosidase is a glycosidase that can effectively decompose the lactose remaining in the solution after the reaction into galactose and glucose. The β-galactosidase used in this experiment was purchased from Sino Biotechnology Development Jiangsu Co., Ltd. and is a white powder. Its temperature range: 5℃~65℃, the optimal temperature is 55℃~60℃. The effective pH range is 3.0~8.0, and the optimal pH is 4.0~5.5. After the reaction is completed, the reaction system is cooled to 20℃, and then the content of phosphate ions in the solution is accurately determined to be 160mM, and equimolar magnesium chloride is added to the solution. Then adjust the pH to 8.5 and wait for precipitation. After the precipitate no longer precipitates, adjust the pH value of the solution to 8.5 again. After the precipitate no longer precipitates, centrifuge at 10000rpm to remove the precipitate to obtain a 6'-sialyllactose solution containing more than 50g / L. The final concentration of phosphate in the solution was determined to be 9mM.

[0166] pass Figure 5 and Figure 6 The results showed that N-acetylneuraminic acid aldolase can effectively degrade the sialic acid substrates remaining in the solution, but has no degradation effect on the sialic acid groups attached to the 6'-sialyllactose molecule. Similarly, β-galactosidase can only selectively hydrolyze lactose, but has no hydrolysis effect on the Gal(β1-4)Glu at the non-reducing end of 6'-sialyllactose.

[0167] The protein in the enzymatic reaction system is retained by an ultrafiltration membrane (molecular weight cutoff of 5000 Daltons).

[0168] The permeate passes through the strong acid cation exchange resin and the weak base anion exchange resin in turn. The filler of the strong acid cation exchange resin is 001*7. The pretreatment method is: the treatment is carried out in the order of acid-base-acid, and the sample loading speed is one time the bed volume flows through per hour. The loading solution is five times the bed volume. The filler of the weak base anion exchange resin is D301. The pretreatment method is: the treatment is carried out in the order of base-acid-base, and the sample loading speed is one time the bed volume flows through per hour. The loading solution is five times the bed volume.

[0169] The flow-through solution that has passed through the strong acid cation exchange resin and the weak base anion exchange resin in sequence is subjected to nanofiltration, and the nanofiltration membrane uses Dow's NF270-4040. This step is mainly used to concentrate the target product, and can also remove trace amounts of small molecular products and pH regulator ions after hydrolysis or degradation in the reaction system. Nanofiltration finally concentrates the solution to a concentration of 6'-sialyllactose of more than 100g / L.

[0170] The nanofiltration retentate was then decolorized by activated carbon.

[0171] The 6'-sialyllactose powder was obtained by freeze drying using a freeze dryer.

[0172] This Example 2 is also applicable to sialylated oligosaccharides synthesized by other multi-enzyme catalysis than Example 1.

[0173] Example 3

[0174] The purity of 6'-sialyllactose obtained in Example 2 was determined by HPLC. Figure 7 shown.

[0175] The purity of the sample was determined by HPLC, chromatographic column: XAmide, 100A (4.6×250 mm, 5 μm) (ACCHROM). B-acetonitrile, C-100 mM ammonium formate aqueous solution (pH 3.2). Elution program: 0-20 minutes, 80% B-20% C to 65% B-35% C. Flow rate 1 mL / min.

[0176] The 6'-sialyllactose product after freeze-drying was configured to a concentration of 5 g / L for HPLC analysis. According to the external standard method calculation, the purity of the 6'-sialyllactose powder obtained after freeze-drying by the freeze dryer can reach 96.3%.

[0177] Example 4

[0178] The ash content of 6'-sialyllactose obtained in Example 2 was measured.

[0179] Weigh 1g of freeze-dried sample into a crucible, heat it with a low fire to fully carbonize the sample until it is smokeless, then place it in a muffle furnace and burn it at 550±25℃ for 4h. Cool it to about 200℃, take it out, put it in a desiccator and cool it for 30min to constant weight, and calculate the ash content (weight percentage g / 100g, hereinafter referred to as %).

[0180] After measurement and calculation, the ash content of the 6'-sialyllactose powder obtained after freeze-drying is 5.2%.

[0181] Example 5

[0182] LC-MS identified the structure of 6'-sialyllactose, such as Figure 8 shown.

[0183] The freeze-dried 6'-sialyllactose product was configured to a concentration of 0.5 g / L for LC-MS analysis.

[0184] LC method: Chromatographic column: Amide (2.1×150 mm, 2.5 μm) (Waters). B-acetonitrile, C-100 mM ammonium formate aqueous solution (pH 3.2). Elution program: 0-40 minutes, 80% B-20% C to 65% B-35% C. Flow rate: 0.3 mL / min.

[0185] Mass spectrometry method: ion source voltage: 3.5KV, source temperature: 150°C, collision voltage 37V, scanning range 200~1000m / z

[0186] Example 6

[0187] like Fig. 9 As shown, an enzymatic reaction system is used to generate an enzymatic reaction solution containing LNT (neutral HMOs).

[0188] Reaction system composition and conditions: 100 ml reaction system, 50 mM galactose, 50 mM lactose, 50 mM N-acetylglucosamine, 50 mM UTP, 50 mM ATP, Tris-HCl (pH 8.0, 100 mM), 20 mM MgCl2, 0.5 mg / mL NahK (Li Y, et al, Molecules, 2011, 16, 6396-6407), 0.5 mg / mL GlmU (Chen Y, et al, Chem. Commun., 2011, 47, 10815-10817), 0.5 mg / mL LgtA (Li Y, et al, Bioorganic & Medicinal Chemistry, 2016, 24, 1696-1705) and 0.5 mg / mL PPA (Li L, et al. al., Org. Lett., 2013, 15, 5528-5530), 0.5 mg / mL GalK (Chen X, et al, J. Am. Chem. Soc. 2001, 123, 2081-2082), 0.5 mg / mL BLUSP (Muthana MM, et al. al, Chem. Commun., 2012, 48, 2728-2730), 0.5 mg / mL WbgO (Liu The conversion rate of the reaction can reach 79%, and the final LNT production is 27.95g / L, with residual lactose of 1.08g / L, residual galactose of 1.34g / L, residual N-acetylglucosamine of 4.29g / L, and residual intermediate LNTⅡ of 4.72g / L.

[0189] In the enzyme-catalyzed synthesis, due to the addition of PPA, 4 molecular weights of phosphate are generated for every molecule of product generated. The residual substrate and product analogs of the reaction are lactose, N-acetylglucosamine, galactose and LNTⅡ.

[0190] Example 7

[0191] LNT was purified from the enzymatic reaction solution of Example 6.

[0192] After the reaction is completed, 5U / mL (final concentration) of β-N-acetylhexosaminidase (expressed in this laboratory) is added to the solution. After reacting at 37°C for 5h, the pH value of the solution is adjusted to 5.0. After adjusting the reaction temperature to 55°C, 2500ALU / L (final concentration) of β-galactosidase is added to react for 3h. Then the temperature is lowered to 20°C, and the content of phosphate ions in the solution is accurately determined to be 179mM. An equimolar saturated solution of magnesium chloride is added to the solution. Then the pH is adjusted to 8.5 and the precipitation is waited for. After the precipitate is no longer precipitated, the pH value of the solution is adjusted to 8.5 again. After the precipitate is no longer precipitated, the precipitate is removed by centrifugation at 10000rpm to obtain a LNT solution containing more than 27.95g / L. The final concentration of phosphate in the solution was determined to be 6.3mM.

[0193] The protein in the enzymatic reaction system is retained by an ultrafiltration membrane (molecular weight cutoff of 5000 Daltons).

[0194] The permeate passes through the strong acid cation exchange resin and the weak base anion exchange resin in turn. The filler of the strong acid cation exchange resin is 001*7. The pretreatment method is: the treatment is carried out in the order of acid-base-acid, and the sample loading speed is one time the bed volume flows through per hour. The loading solution is five times the bed volume. The filler of the weak base anion exchange resin is D301. The pretreatment method is: the treatment is carried out in the order of base-acid-base, and the sample loading speed is one time the bed volume flows through per hour. The loading solution is five times the bed volume.

[0195] The flow-through liquid that has passed through the strong acid cation exchange resin and the weak base anion exchange resin is subjected to nanofiltration, and the nanofiltration membrane is NF270-4040 from Dow. This step is mainly used to concentrate the target product, and can also remove trace amounts of small molecular products and pH regulator ions after hydrolysis or degradation in the reaction system. Nanofiltration finally concentrates the solution to a LNT concentration of more than 100g / L.

[0196] The nanofiltration retentate was then decolorized by activated carbon.

[0197] The powder of LNT was obtained by freeze drying.

[0198] This Example 7 is also applicable to other neutral oligosaccharides synthesized by multi-enzyme catalysis other than Example 6.

[0199] Example 8

[0200] The purity of the LNT obtained in Example 7 was determined by HPLC to be 95.1%, and the ash content was 3.8%.

[0201] The purity of the sample was determined by HPLC, chromatographic column: Sugar-Pak Column, 10μm, 6.5mm×300mm, 1 / pk (Waters). The mobile phase was pure water, column temperature was 80℃, flow rate was 0.5mL / min, injection volume was 10ul, detector was RID detector, and external standard method was used for quantification.

[0202] The results are as follows Fig.10 shown.

[0203] Example 9

[0204] This embodiment provides a preparation containing oligosaccharides, wherein the oligosaccharides are prepared and purified by the methods described in the above embodiments 1 and 2.

[0205] The oligosaccharides are present in the preparation in a purity of ≥ 95% by mass.

[0206] At the same time, this embodiment provides the use of the preparation for infant formula. The components of the infant formula include the following:

[0207] Table 2 Composition information of infant formula

[0208]

[0209]

[0210] Comparative Example 1

[0211] In this comparative example, 6'-sialyllactose was purified from the enzymatic reaction solution of Example 1, except that:

[0212] Only β-galactosidase is added to the enzymatic reaction solution, and N-acetylneuraminic acid aldolase is not added;

[0213] The rest is the same as Example 2.

[0214] The purity of the 6'-sialyllactose purified in this comparative example was determined by HPLC to be 91.9%, and the ash content was 5.21%.

[0215] Comparative Example 2

[0216] In this comparative example, 6'-sialyllactose was purified from the enzymatic reaction solution of Example 1, except that:

[0217] After adding enzyme solution to the enzymatic reaction solution, the enzymatic reaction solution is not treated with magnesium chloride, and then the soluble protein, cations and anions are directly removed; and the loading volume of the loading solution is still five times the bed volume during the treatment process. The rest is the same as Example 2.

[0218] The purity of the 6'-sialyllactose purified in this comparative example was determined by HPLC to be 78.13%, and the ash content was 24.61%.

[0219] Comparative Example 3

[0220] In this comparative example, 6'-sialyllactose was purified from the enzymatic reaction solution of Example 1, except that:

[0221] After adding enzyme solution to the enzymatic reaction solution for treatment, the phosphate ion content in the solution was accurately measured to be 160 mM, and then the phosphate ions were removed by using resin instead of adding magnesium chloride to the solution to remove the phosphate ions.

[0222] The rest is the same as Example 2.

[0223] In fact, in order to reduce the concentration of phosphate ions in the resin flow-through liquid to the same concentration as that in the resin flow-through liquid in Example 2, it is necessary to repeatedly use D301 (the loading solution is five times the volume of the bed, the same as in Example 2) resin to remove phosphate ions 19 times. Then nanofiltration is performed to obtain a 6'-sialyllactose solution containing more than 100g / L. Comparing Example 2 and Comparative Example 3, a traditional resin is used to remove phosphate, replacing the metal salt precipitation in the present invention. In Comparative Example 3, the amount of wastewater generated in this step is 19 times that of the embodiment. Using the treatment of 1t of enzymatic reaction liquid as an example, the amount of wastewater generated in the removal process of polyvalent anions using the technical scheme of Example 2 (i.e., the present invention) is 2.8t, while the amount of wastewater generated in the removal process of polyvalent anions in Comparative Example 3 is 53.2t.

[0224] The purity of 6'-sialyllactose obtained by purification in this comparative example was determined by HPLC to be 96.4%.

Claims

1. A method for separating and purifying oligosaccharides, characterized in that: The fermentation liquid at the end of the oligosaccharide preparation reaction by the fermentation method or the reaction liquid at the end of the oligosaccharide preparation reaction by the bioenzyme method is separated and purified. The oligosaccharide includes acidic human milk oligosaccharide, and the acidic human milk oligosaccharide includes one or more of 3'-sialyllactose, 6'-sialyllactose, 6'-sialyllactosamine, and 3'-sialyllactosamine; The process comprises the following steps: a) Removal of product analogs; The product analogs include: compounds having a molecular weight lower than that of the oligosaccharide to be purified; wherein the compounds include sugars and sialic acid; Treating the fermentation broth or reaction liquid with a hydrolase and a lyase to remove the product analog; the final concentration of the hydrolase is 500-5000 ALU / L, and the final concentration of the lyase is 1-50 U / mL; The hydrolase is a glycoside hydrolase; The lyase is N-acetylneuraminic acid aldolase; b) removing polyvalent anions; The multivalent anions include phosphate ions; c) removal of soluble proteins; Remove cations; Remove anions.

2. The method for separation and purification of oligosaccharides according to claim 1, characterized in that: In b), the fermentation liquid or the reaction liquid is treated with metal ions to remove the multivalent anions; The added concentration of the metal ions is 5-1000 mM.

3. The method for separation and purification of oligosaccharides according to claim 2, characterized in that: In b), the fermentation liquid or the reaction liquid is treated with metal ions to remove the multivalent anions; The added concentration of the metal ions is 100-500 mM.

4. The method for separation and purification of oligosaccharides according to claim 2, characterized in that: In b), the fermentation liquid or the reaction liquid is treated with metal ions to remove the multivalent anions; The added concentration of the metal ions is 100-400 mM.

5. The method for separation and purification of oligosaccharides according to claim 2, characterized in that: The metal ions include any one or more of calcium ions, nickel ions, cobalt ions, zinc ions, copper ions, lead ions, manganese ions, magnesium ions, aluminum ions, iron ions, barium ions, and silver ions; The metal ion is added in the form of a solution containing a salt of the metal ion; The concentration of the salt solution is the concentration of a saturated solution corresponding to the metal salt solution.

6. The method for separation and purification of oligosaccharides according to claim 2, characterized in that: The pH of the fermentation liquid or reaction liquid after treatment in b) is set at 6-12.

7. The method for separation and purification of oligosaccharides according to claim 2, characterized in that: The pH of the fermentation liquid or reaction liquid after treatment in b) is set at 7-10.

8. The method for separation and purification of oligosaccharides according to claim 2, characterized in that: The pH of the fermentation liquid or reaction liquid after treatment in b) is set at 8-9.

9. The method for separating and purifying oligosaccharides according to any one of claims 2 to 8, characterized in that: In the above c), the fermentation broth or reaction liquid is subjected to ultrafiltration to remove soluble proteins with a molecular weight of ≥4 kDa from the fermentation broth or reaction liquid.

10. The method for separation and purification of oligosaccharides according to claim 9, characterized in that: In the above c), the fermentation broth or reaction liquid is subjected to ultrafiltration to remove soluble proteins with a molecular weight of ≥10 kDa from the fermentation broth or reaction liquid.

11. The method for separation and purification of oligosaccharides according to claim 9, characterized in that: In the above c), the fermentation broth or reaction liquid is subjected to ultrafiltration to remove soluble proteins with a molecular weight of ≥20 kDa from the fermentation broth or reaction liquid.

12. The method for separating and purifying oligosaccharides according to any one of claims 2 to 8, characterized in that: c), wherein the removal of cations from the fermentation broth or reaction broth is carried out by cation exchange chromatography; Wherein, the stationary phase of the cation exchange chromatography is a cation exchange resin.

13. The method for separating and purifying oligosaccharides according to any one of claims 2 to 8, characterized in that: c), wherein the removal of cations from the fermentation broth or reaction broth is carried out by cation exchange chromatography; Wherein, the stationary phase of the cation exchange chromatography is a strongly acidic cation exchange resin.

14. The method for separating and purifying oligosaccharides according to any one of claims 2 to 8, characterized in that: c), wherein the removal of anions from the fermentation broth or reaction broth is performed by anion exchange chromatography; Wherein, the stationary phase of the anion exchange chromatography is anion exchange resin.

15. The method for separating and purifying oligosaccharides according to any one of claims 2 to 8, characterized in that: c), wherein the removal of anions from the fermentation broth or reaction broth is performed by anion exchange chromatography; Wherein, the stationary phase of the anion exchange chromatography is a weakly basic anion exchange resin.

16. The method for separation and purification of oligosaccharides according to claim 12, characterized in that: Also includes one or more processing steps selected from the following: Ⅰ) performing solid-liquid separation on the fermentation liquid or reaction liquid after the treatment in b); The solid-liquid separation treatment method includes filtration, microfiltration or centrifugation; II) filtering the fermentation liquid or reaction liquid after the treatment in c); Nanofiltration membrane is used for nanofiltration concentration to remove residual pH regulator ions and monosaccharide products after enzymatic hydrolysis. The molecular weight cutoff of the nanofiltration membrane is between the molecular weight of the target oligosaccharide and the molecular weight of the impurities.

17. The method for separation and purification of oligosaccharides according to claim 16, characterized in that: The filtration method is nanofiltration, and the molecular weight cutoff of the nanofiltration membrane is less than 500 Daltons.

18. The method for separation and purification of oligosaccharides according to claim 17, characterized in that: The molecular weight cut-off of the nanofiltration membrane is selected to be 100-300 Daltons.

19. The method for separating and purifying oligosaccharides according to any one of claims 16 to 18, characterized in that: Also includes one or more processing steps selected from the following: III) decolorizing the fermentation liquid or reaction liquid after the treatment of II); IV) crystallizing, or alcohol precipitation, or freeze-drying, or spray drying the fermentation liquid or reaction liquid after the treatment of III).

20. The method for separation and purification of oligosaccharides according to claim 19, characterized in that: Decolorization is carried out using activated carbon.

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