Fermented product and method for producing fermented product

Abstract

The present invention relates to a fermented product containing a component X, a component Y, and a component Z, wherein the component X is a sialic acid or a sialylated oligosaccharide, the content of the component X per solid content of the fermented product is 80 mass% or more, the component Y is acetic acid, the ratio (component Y content / component X content) of the content of the component Y to the content of the component X in the fermented product is 0.010 or less on a mass basis, the component Z is at least one of a component Z1 and a component Z2, the component Z is a component other than the component X, the component Z1 is a neutral sugar, and the component Z2 is an acidic sugar.

Description

Fermented products and methods for producing fermented products

[0001] The present invention relates to a fermented product containing sialic acid or sialylated oligosaccharides, and a method for producing the fermented product.

[0002] Oligosaccharides contained in human breast milk (HMOs) have attracted attention as prebiotic materials and have been shown to be effective in the development of cognitive function, infection prevention, and improvement of the intestinal environment in infants (Non-Patent Document 1). Sialylated oligosaccharides are one of the important oligosaccharides contained in human breast milk and are believed to have physiological activities such as infection prevention against viruses and bacteria, and lactic acid bacteria proliferation activity. Sialylated oligosaccharides are also useful as raw materials or intermediates for products such as health foods, pharmaceuticals, and cosmetics. Sialic acid is also known as a component of breast milk. In particular, N-acetylneuraminic acid (NeuAc), a type of acidic amino sugar collectively referred to as sialic acid, is widely used as a raw material for pharmaceuticals such as anti-influenza drugs, as well as a component of foods, cosmetics, and cell culture media.

[0003] Various production methods for obtaining high-purity sialylated oligosaccharides have been investigated. For example, a method is known in which 6'-sialyllactose [O-(N-acetyl-α-neuraminosyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose] (hereinafter also referred to as "6SL") produced by chemical synthesis is spray-dried (Patent Document 1). Another method is known in which impurities are removed by crystallizing 6SL sodium salt (Patent Document 2). However, because the crystallization process requires the addition of organic solvents such as methanol, it cannot be ruled out that these solvents will remain in the final product, raising safety concerns. Therefore, in recent years, methods for purifying sialylated oligosaccharides produced by fermentation (Patent Documents 3 and 4) have become mainstream, and the final products obtained are used in foods and the like. However, it is difficult to remove the neutral sugars and organic acids generated during fermentation production, and it is known that a certain amount of these remain as impurities. Furthermore, components derived from fermentation bacteria that contaminate fermentation products also have physical properties similar to those of sialic acid or sialylated oligosaccharides, and it is therefore also necessary to remove such contaminants.

[0004] International Publication No. 2013 / 185780 International Publication No. 2017 / 086443 Japanese Patent Application Publication No. 2021-505170 Japanese Patent Application Publication No. 2020-531039

[0005] Int J Pediatr. 2019 Aug 4;2019:2390240.

[0006] To obtain a product containing a high concentration of sialylated oligosaccharides, it is necessary to reduce the amount of impurities, such as neutral sugars, acidic sugars other than sialylated oligosaccharides, and organic acids, in the final product. Chemical synthesis and crystallization are methods to prevent such impurities from contaminating the final product. However, these methods require the use of residual solvents, such as methanol, that fall into Class 1 to Class 3 as defined by the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Residual Solvents in Pharmaceuticals (Q3C). Strict control is required to prevent such residual solvents from contaminating the final product. Sialylated oligosaccharides, in particular, are often used in products for infants and young children, and therefore require high safety. Therefore, the chemically synthesized products and crystals produced by the above-mentioned methods pose safety challenges for the production of sialylated oligosaccharides.

[0007] Furthermore, in conventional methods for producing sialylated oligosaccharides by fermentation and purifying the fermentation product using ion exchange resins or the like, it is inevitable that neutral sugars such as lactose present during the production process will be contaminated into the final product. Furthermore, for example, acetic acid, which falls under ICH-Q3C Class 3 and is generated during fermentation production, will be contaminated into the final product as an impurity. Therefore, it has been desired to establish a production method that can reduce these impurities and achieve a sufficient recovery rate of sialic acid or sialylated oligosaccharides in industrial production.

[0008] Therefore, an object of the present invention is to provide a fermented product containing sialic acid or sialylated oligosaccharides and having a low impurity content, and a method for producing the same.

[0009] As a result of extensive research into the above-mentioned problems, the present inventors have found that a fermentation product containing a high content of sialic acid or sialylated oligosaccharides and few impurities can be produced by a method comprising treating solution A containing sialic acid or sialylated oligosaccharides with a strongly acidic cation exchange resin to obtain solution B, treating solution B with a weakly basic anion exchange resin to obtain solution C, adjusting the pH of solution C to obtain solution D, and treating solution D with a weakly basic anion exchange resin to obtain solution E, and have completed the present invention.

[0010] That is, the present invention is as follows: 1. A fermented product containing component X, component Y, and component Z, wherein component X is sialic acid or sialylated oligosaccharide, the content of component X relative to the solid content of the fermented product is 80% by mass or more, component Y is acetic acid, the ratio of the content of component Y to the content of component X in the fermented product (content of component Y / content of component X) is 0.010 or less by mass, component Z is at least one of component Z1 and component Z2, and component Z is a component other than component X, component Z1 is a neutral sugar, and component Z2 is an acidic sugar. 2. The fermented product according to item 1 above, wherein component X is N-acetylneuraminic acid. 3. The fermented product according to the above item 1, wherein the component X is any one compound selected from the group consisting of sialyllactose, sialyllact-N-biose (SLNB), sialyllact-N-acetylglucosamine (SLNac), sialyllact-N-tetraose (LST), and disialyllacto-N-tetraose (DSLNT). 4. The fermented product according to the above item 2, wherein the component Z2 is at least one compound selected from the group consisting of glucuronic acid, mannuronic acid, and galacturonic acid. 5. The fermented product according to the above item 3, wherein the component Z2 is at least one compound selected from the group consisting of N-acetylneuraminic acid, glucuronic acid, mannuronic acid, and galacturonic acid. 6. The fermented product according to the above item 3 or 5, further comprising a component W, wherein the component W is an isomer of component X and the component W is a substance different from the component Z2. 7. 7. The fermented product according to any one of items 1 to 6, further containing component V, wherein component V is a substance that is detected at RT 27.0 to 35.0 when the fermented product is measured by HPLC-CAD, and component V is a substance different from component Z2. 8. The fermented product according to item 4 or 5, wherein the ratio of the content of each compound of component Z2 to the content of component X in the fermented product (content of component Z2 / content of component X) is 0.02 or less by mass. 9. The fermented product according to any one of items 1 to 8, wherein component Z1 is at least one compound selected from the group consisting of glucose and lactose.10. The fermented product according to claim 9, wherein the ratio of the content of each compound of component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X) is 0.03 or less by mass. 11. The fermented product according to claim 6, wherein the ratio of the content of each compound of component W to the content of component X in the fermented product (content of component W / content of component X) is 0.0035 or less by mass. 12. The fermented product according to claim 7, wherein the ratio of the total content of component V to the content of component X in the fermented product (content of component V / content of component X) is 0.040 or less by mass. 13. 1. A method for producing a fermented product, comprising the following steps 1) to 4) in this order: 1) treating a solution A containing components X, Y, and Z with a strongly acidic cation exchange resin to obtain a solution B; 2) treating the solution B with a weakly basic anion exchange resin to obtain a solution C; 3) adjusting the pH of the solution C to obtain a solution D; and 4) treating the solution D with a weakly basic anion exchange resin to obtain a solution E, wherein the component X is sialic acid or a sialylated oligosaccharide, the component Y is acetic acid, the component Z is at least one of components Z1 and Z2, and the component Z is a component other than the component X, the component Z1 is a neutral sugar, and the component Z2 is an acidic sugar, and the ratio of the total mass of the component X in the solution E to the total mass of the component X in the solution A (total mass of component X in solution E / total mass of component X in solution A) is 0.5 or more, 14. A method for producing a fermented product, further comprising the following step 5): 5) powdering the solution E. The fermented product contains the components X, Y, and Z, the content of component X per solid content of the fermented product is 80 mass% or more, and the ratio of the content of component Y to the content of component X in the fermented product (component Y content / component X content) is 0.010 or less by mass.15. The method for producing a fermented product according to the above 13 or 14, wherein the ratio of the total mass of component Y in solution E to the total mass of component Y in solution A (total mass of component Y in solution E / total mass of component Y in solution A) is 0.5 or less. 16. The method for producing a fermented product according to any one of the above 13 to 15, wherein component X is N-acetylneuraminic acid. 17. The method for producing a fermented product according to any one of the above 13 to 15, wherein component X is any one compound selected from the group consisting of sialyllactose, sialyllact-N-biose (SLNB), sialyllact-N-acetylglucosamine (SLNac), sialyllact-N-tetraose (LST), and disialyllact-N-tetraose (DSLNT). 18. The method for producing a fermented product according to the above 16, wherein component Z2 is at least one compound selected from the group consisting of glucuronic acid, mannuronic acid, and galacturonic acid. 19. 20. A method for producing a fermented product according to claim 17, wherein component Z2 is at least one compound selected from the group consisting of N-acetylneuraminic acid, glucuronic acid, mannuronic acid, and galacturonic acid. 21. A method for producing a fermented product according to any one of claims 13 to 20, wherein the fermented product further contains component W, which is an isomer of component X and is a substance different from component Z2. 22. A method for producing a fermented product according to claim 18 or 19, wherein the fermented product further contains component V, which is a substance detected at RT 27.0 to 35.0 when the fermented product is measured by HPLC-CAD, and component V is a substance different from component Z2. 23. A method for producing a fermented product according to claim 18 or 19, wherein the ratio of the content of each compound of component Z2 to the content of component X in the fermented product (content of component Z2 / content of component X) is 0.02 or less by mass. 24. The method for producing a fermented product according to any one of items 13 to 22, wherein component Z1 is at least one compound selected from the group consisting of glucose and lactose. 25. The method for producing a fermented product according to item 23, wherein the ratio of the content of each compound of component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X) is 0.03 or less by mass.25. The method for producing a fermented product according to the above 13, wherein the step 2) comprises the following steps 2-1) to 2-3) in this order: 2-1) Passing solution B through a weakly basic anion exchange resin to remove at least a portion of the neutral impurities that do not adsorb to the weakly basic anion exchange resin; 2-2) Eluting at least a portion of the impurities remaining after step 2-1) from the weakly basic anion exchange resin; and 2-3) Separately eluting the acidic compounds including component X and the impurities remaining after step 2-2) from the weakly basic anion exchange resin. 26. The method for producing a fermented product according to the above 13, wherein the temperature at which the weakly basic anion exchange resin is passed in step 2-2) is 20°C or higher. 27. The method for producing a fermented product according to the above 25 or 26, wherein the temperature at which the weakly basic anion exchange resin is passed in step 2-3) is 20°C or lower. 28. 29. The method for producing a fermented product according to any one of 25 to 27 above, wherein the temperature at which the weakly basic anion exchange resin is passed through in 2-1) above is 15°C or lower. 29. The method for producing a fermented product according to 25 or 26 above, wherein the fermented product further contains component V, which is a substance detected at RT of 27.0 to 35.0 when the fermented product is measured by HPLC-CAD, and component V is a substance different from component Z2, and the ratio of the total content of component V to the content of component X in the fermented product (content of component V / content of component X) is 0.040 or lower by mass. 30. The method for producing a fermented product according to 25 or 27 above, wherein component W is an isomer of component X, and component W is a substance different from component Z2, and the ratio of the content of each compound of component W to the content of component X in the fermented product (content of component W / content of component X) is 0.0035 or lower by mass. 31. 26. The production method according to 25 above, wherein the temperature of the weakly basic anion exchange resin used in step 2-1) is 5 to 15°C, the temperature of the weakly basic anion exchange resin used in step 2-2) is 20 to 25°C, and the temperature of the weakly basic anion exchange resin used in step 2-3) is 8 to 20°C. 32. The production method according to 25 above, wherein the temperature of the weakly basic anion exchange resin used in step 2-2) is 20 to 25°C.33. A method for producing a fermented product according to 25 or 26 above, wherein the temperature for passing the weakly basic anion exchange resin in 2-3) above is 8 to 20°C. 34. A method for producing a fermented product according to any one of 25 to 27 above, wherein the temperature for passing the weakly basic anion exchange resin in 2-1) above is 5 to 15°C. 35. A method for producing a fermented product according to 13 above, wherein adjusting the pH of solution C in 3) above is a method of passing solution C through a cation exchange resin, and wherein the temperature for passing the cation exchange resin in 3) above is 20°C or lower. 36. A method for producing a fermented product according to 13 or 35 above, wherein the temperature for passing the weakly basic anion exchange resin in 4) above is 20°C or lower. 37. 32. The method for producing a fermented product according to claim 13 or 31, wherein adjusting the pH of the solution C in step 3) is a method of passing the solution C through a cation exchange resin, the temperature of the cation exchange resin in step 3) is 20°C or lower, and the temperature of the weakly basic anion exchange resin in step 4) is 20°C or lower.

[0011] The fermented product produced by the method for producing a fermented product according to one embodiment of the present invention has a high content of sialic acid or sialylated oligosaccharides and a low content of acetic acid per solid content of the fermented product. Furthermore, it is possible to produce a fermented product with less residual solvent than the crystallization process.

[0012] Fig. 1 is a graph showing the results of analyzing the composition of the eluate obtained in Comparative Example 1. Fig. 2 is a graph showing the results of analyzing the composition of the eluate obtained in Example 1. Fig. 3 is a graph showing the results of analyzing the composition of the eluate obtained in Comparative Example 2. Fig. 4 is a graph showing the results of analyzing the composition of the eluate obtained in Example 2.

[0013] The present invention will be described in detail below, but these are merely examples of preferred embodiments and are not intended to limit the scope of the present invention. The numerical range "to" includes the preceding and following numerical values. For example, "0% by mass to 100% by mass" means a range of 0% by mass or more and 100% by mass or less.

[0014] [1. Fermented Product] A fermented product according to one embodiment of the present invention is a fermented product containing component X, component Y, and component Z, wherein component X is sialic acid or sialylated oligosaccharide, the content of component X relative to the solid content of the fermented product is 80% by mass or more, component Y is acetic acid, the ratio of the content of component Y to the content of component X in the fermented product (component Y content / component X content) is 0.010 or less by mass, component Z is at least one of component Z1 and component Z2, and component Z is a component other than component X, and component Z1 is a neutral sugar and component Z2 is an acidic sugar.

[0015] A fermented product is a mixture of multiple compounds obtained by microbial fermentation. Examples of compounds that can be contained in the fermented product include the produced component X, a by-product compound of component X, an intermediate of component X, a decomposition product of component X or its intermediate, component Y, component Z, component W, component V, and medium components. The fermented product is a concept that encompasses solution E obtained in steps 1 to 4 described below, powder obtained in steps 1 to 5 described below, and powder obtained by powdering the solution obtained by concentrating, decolorizing, and / or filtering the solution obtained in steps 1 to 4 in step 5.

[0016] The solid content of the fermented product refers to the solid content remaining after volatile substances such as water have been removed from the fermented product, i.e., the evaporation residue, and may include non-volatile components such as sialic acid, sialylated oligosaccharides, and other impurities. Specifically, the solid content refers to, for example, the powder obtained by freeze-drying or spray-drying Solution E treated in steps 1 to 4 described below, after which water is removed and corrected using the method for analyzing the water content by Karl Fischer titration described below, or the evaporation residue remaining after evaporating the fermented product to dryness by heating, preferably the evaporation residue remaining after water is removed and corrected using the method for analyzing the water content by Karl Fischer titration described below. When the fermented product is a solution, the solid content refers to the solid content remaining after volatile substances such as water have been removed from the solution, preferably the evaporation residue remaining after water is removed and corrected using the method for analyzing the water content by Karl Fischer titration described below.

[0017] (Component X: Sialic Acid and Sialylated Oligosaccharides) The fermented product of one embodiment of the present invention contains, as component X, sialic acid or sialylated oligosaccharides.

[0018] Sialic acid is a general term for substances in which the amino group or hydroxy group of neuraminic acid is substituted. Examples of sialic acid include N-acetylneuraminic acid, N-glycolylneuraminic acid, glucuronic acid, N-acetylgalactosamine 6-sulfate, and mannose 6-phosphate, with N-acetylneuraminic acid being preferred.

[0019] Sialylated oligosaccharides are acidic oligosaccharides in which sialic acid is bound to an oligosaccharide, preferably sialic acid and lactose, or sialic acid and a neutral sugar. The neutral sugar is glucose, galactose, lactose, or sucrose, preferably lactose. Examples of sialylated oligosaccharides include sialyllactose, sialyllactose-N-biose (SLNB), sialyllactose-N-acetylglucosamine (SLNac), sialyllactose-N-tetraose (LST), disialyllactose-N-tetraose (DSLNT), 3-fucosyl-sialyllactose (F-SL), and fucosyl-LSTb (F-LSTb), with sialyllactose being preferred.

[0020] Sialyllactose is an acidic oligosaccharide in which sialic acid is bound to lactose. Examples of sialyllactose include 6'-sialyllactose [O-(N-acetyl-α-neuraminosyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose] (hereinafter also referred to as "6SL") and 3'-sialyllactose [O-(N-acetyl-α-neuraminosyl)-(2→3)-O-β-D-galactopyranosyl-(1→4)-D-glucose] (hereinafter also referred to as "3SL").

[0021] Sialyl lacto-N-biose is an acidic oligosaccharide in which sialic acid is bound to lacto-N-biose. Examples of sialyl lacto-N-biose include 6'-sialyllacto-N-biose (hereinafter also referred to as "6SLNB") and 3'-sialyllacto-N-biose (hereinafter also referred to as "3SLNB").

[0022] Sialyllacto-N-acetylglucosamine is an acidic oligosaccharide in which sialic acid is bound to lacto-N-acetylglucosamine. Examples of sialylacto-N-acetylglucosamine include 6'-sialylacto-N-acetylglucosamine (hereinafter also referred to as "6SLNAc") and 3'-sialylacto-N-acetylglucosamine (hereinafter also referred to as "3SLNAc").

[0023] Sialyl lacto-N-tetraose is an acidic oligosaccharide in which sialic acid is bound to lacto-N-tetraose or lacto-N-neotetraose. Examples of sialyllacto-N-tetraose include LST-a, LST-b, and LST-c.

[0024] Disialyl lacto-N-tetraose is an acidic oligosaccharide in which two molecules of sialic acid are bound to lacto-N-tetraose.

[0025] In one embodiment of the present invention, the content of component X relative to the solid content of the fermented product is 80% by mass or more, and in descending order of preference, is 82% by mass or more, 84% by mass or more, 85% by mass or more, 87% by mass or more, 88% by mass or more, 89% by mass or more, and 90% by mass or more. In this specification, the "content of component X" is synonymous with the "purity of component X."

[0026] When component X is sialic acid, the content of component X relative to the solid content of the fermented product of one embodiment of the present invention is 80% by mass or more, preferably 84% by mass or more, more preferably 87% by mass or more, more preferably 88% by mass or more, even more preferably 89% by mass or more, and most preferably 90% by mass or more. When component X is a sialylated oligosaccharide, the content of component X relative to the solid content of the fermented product of one embodiment of the present invention is 80% by mass or more, preferably 84% by mass or more, more preferably 87% by mass or more. In particular, when component X is at least one of 3SL and 6SL, the content of component X relative to the solid content of the fermented product of one embodiment of the present invention is preferably 87% by mass or more.

[0027] The sialic acid and sialylated oligosaccharide of component X may be contained in the fermented product as a sialic acid salt and a salt of sialylated oligosaccharide, respectively. In this specification, the content and amount of component X are calculated by converting the amount of sialic acid salt to the same mole of sialic acid, or by converting the amount of sialylated oligosaccharide salt to the same mole of sialylated oligosaccharide (hereinafter also referred to as the free form).

[0028] The content of component X per solid content of a fermented product means the proportion (purity) of component X in the solids, and can be calculated from the water content measured by Karl Fischer titration and the content of component X measured by a High Performance Liquid Chromatography-Charged Aerosol Detector (hereinafter also referred to as "HPLC-CAD"). More specific examples include the methods in the analytical examples and examples described below. When the fermented product is a solution, it can be determined by evaporating to dryness. The content of component X in the fermented product means the amount (concentration) of component X in the solution. Furthermore, when the fermented product is a solution, the content of component X can be calculated by subtracting the content of impurities in the fermented product from the content of component X in the fermented product (100% by mass). More specific examples include the methods in the analytical examples and examples described below.

[0029] The content of component X in the fermented product means the amount (concentration) of component X in a solution such as solution E described below, and can be measured and calculated by HPLC-CAD. More specifically, methods such as those in the analytical examples and examples described below can be mentioned.

[0030] (Component Y: Acetic Acid) The fermented product of one embodiment of the present invention further contains acetic acid as component Y. Acetic acid is an organic acid produced during microbial fermentation and is an acidic substance similar to component X. Therefore, if a process to increase the content of component X is performed during the microbial fermentation process, it may behave similarly to component X, resulting in an increase in the acetic acid content. Therefore, by performing a process to separate components X and Y, the content of acetic acid in the fermented product can be reduced and the content of component X can be increased. Such a process can provide a fermented product with sufficiently reduced impurities.

[0031] In particular, in one embodiment of the present invention, the ratio of the content of component Y to the content of component X in the fermented product (content of component Y / content of component X) is 0.010 or less by mass, preferably 0.008 or less, more preferably 0.006 or less, and most preferably 0.004 or less.

[0032] The sialic acid and sialylated oligosaccharide of component X may be contained in the fermented product as a sialic acid salt and a salt of sialylated oligosaccharide, respectively. Furthermore, the acetic acid of component Y may be contained in the fermented product as an acetate salt. The above (content of component Y / content of component X) is calculated using the value obtained by converting the amount of sialic acid salt to the same mole, the value obtained by converting the amount of sialylated oligosaccharide salt to the same mole, or the value obtained by converting the amount of acetate salt to the same mole of acetic acid.

[0033] As a method for calculating the ratio of the content of component Y to the content of component X in the fermented product (content of component Y / content of component X), the methods in the analytical examples and examples described below can be mentioned in detail.

[0034] (Component Z: Neutral sugar and acidic sugar) The fermented product of one embodiment of the present invention further contains at least one of component Z1 and component Z2 as component Z. Component Z is a compound different from component X. Component Z1 is a neutral sugar, and component Z2 is an acidic sugar.

[0035] (Component Z1: Neutral Sugars) Examples of neutral sugars contained as component Z1 include glucose, galactose, lactose, and sucrose used in fermentation production, as well as fructose, N-acetylmannosamine, and N-acetylglucosamine produced by the microorganisms used in fermentation production. In particular, glucose and sucrose added during the cultivation of the microorganisms used in fermentation production, and lactose added to the culture broth during the production of sialylated oligosaccharides, may have high contents at the end of fermentation. In particular, the content of component X in the fermentation product can be increased by removing glucose or lactose from the fermentation product.

[0036] In one embodiment of the fermented product of the present invention, the content of impurities such as neutral sugars is preferably low, and the ratio of the content of each compound of component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X) is preferably 0.03 or less, more preferably 0.01 or less, even more preferably 0.001 or less, and most preferably 0.0005 or less, on a mass basis.

[0037] "The ratio of the content of each compound of component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X) is preferably 0.03 or less by mass" means, for example, that when component Z1 is glucose only, the ratio of the glucose content to the component X content in the fermented product (content of glucose / content of component X) is preferably 0.03 or less. When component Z1 is lactose only, the ratio of the lactose content to the component X content in the fermented product (content of lactose / content of component X) is preferably 0.03 or less. Furthermore, when component Z1 is glucose and lactose, the ratio of the glucose content to the component X content in the fermented product (content of glucose / content of component X) is preferably 0.03 or less, and the ratio of the lactose content to the component X content in the fermented product (content of lactose / content of component X) is preferably 0.03 or less.

[0038] As a method for calculating the ratio of the content of each compound of component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X), the methods of the analytical examples and examples described below can be mentioned in detail.

[0039] (Component Z2: Acidic Sugars) Examples of acidic sugars contained as component Z2 include N-acetylneuraminic acid, N-glycolylneuraminic acid, glucuronic acid, N-acetylgalactosamine 6-sulfate, mannose 6-phosphate, and the like, which are produced by the microorganisms used in fermentation production, as well as N-acetylneuraminic acid and sialylated oligosaccharides other than component X to which N-acetylneuraminic acid is bound, which are produced during the production of sialylated oligosaccharides. Other examples of acidic sugars contained as component Z2 include mannuronic acid, galacturonic acid, and L-iduronic acid. Like component Y, these acidic sugars have similar physical properties, particularly acidity, to component X, and are therefore likely to be adsorbed to and eluted from a basic anion exchange resin together with component X. Therefore, when producing a fermentation product containing component X, component Z2 is likely to remain in the fermentation product as an impurity; however, by performing appropriate treatment, it is possible to increase the content of component X and reduce acidic sugars. In particular, it is preferable that the contents of glucuronic acid, mannuronic acid and galacturonic acid are low, and further, when component X is a sialylated oligosaccharide, it is preferable that the content of N-acetylneuraminic acid is low.

[0040] As described above, component Z, such as component Z2, is different from component X. Therefore, when a fermented product of one embodiment of the present invention contains N-acetylneuraminic acid as component X, N-acetylneuraminic acid is not a component Z, such as component Z2. On the other hand, for example, when a fermented product of one embodiment of the present invention contains a sialylated oligosaccharide such as sialyllactose and N-acetylneuraminic acid, if the sialylated oligosaccharide such as sialyllactose is component X, N-acetylneuraminic acid is component Z2.

[0041] The fermented product of one embodiment of the present invention preferably has a low content of impurities such as acidic sugars, and when component Z2 is at least one selected from the group consisting of glucuronic acid, mannuronic acid, galacturonic acid, and N-acetylneuraminic acid, the ratio of the content of each compound of component Z2 to the content of component X in the fermented product (content of component Z2 / content of component X) is preferably 0.02 or less, more preferably 0.01 or less, on a mass basis.

[0042] The phrase "the ratio of the content of each compound of component Z2 to the content of component X in the fermented product (content of component Z2 / content of component X) is preferably 0.02 or less on a mass basis" means, for example, that when component Z2 consists solely of N-acetylneuraminic acid, the ratio of the content of N-acetylneuraminic acid to the content of component X in the fermented product (content of N-acetylneuraminic acid / content of component X) is preferably 0.02 or less. When component Z2 consists solely of glucuronic acid, the ratio of the content of glucuronic acid to the content of component X in the fermented product (content of glucuronic acid / content of component X) is preferably 0.02 or less. Furthermore, when component Z2 is N-acetylneuraminic acid and glucuronic acid, the ratio of the content of N-acetylneuraminic acid to the content of component X in the fermented product (N-acetylneuraminic acid content / component X content) is preferably 0.02 or less, and the ratio of the content of glucuronic acid to the content of component X in the fermented product (glucuronic acid content / component X content) is preferably 0.02 or less.

[0043] As a method for calculating the ratio of the content of each compound of component Z2 to the content of component X in the fermented product (content of component Z2 / content of component X), the methods of the analytical examples and examples described below can be mentioned in detail.

[0044] (Component W: Isomer of Component X) When component X is a sialylated oligosaccharide, the isomer of component X contained as component W includes a compound in which the aldose monosaccharide unit at the reducing end of the sialylated oligosaccharide produced in the fermentation production and purification steps is converted to a ketose. Examples of compounds in which the aldose monosaccharide unit at the reducing end of a sialylated oligosaccharide is converted to a ketose include 3'-sialyllactulose and 6'-sialyllactulose. Component W is a substance different from component Z2.

[0045] The production of an isomer of component X is promoted under high-temperature storage conditions exceeding 20° C. Therefore, in producing a fermented product containing component X, in the step 2-3) of separately eluting an acidic compound group containing component X and impurities remaining after 2-2) from a weakly basic anion exchange resin, which will be described later, the temperature at which the liquid passes through the weakly basic anion exchange resin is set to 20° C. or lower, thereby suppressing the production of an isomer of component X and increasing the content of component X.

[0046] Furthermore, these isomers of component X can be produced under storage conditions at pH 4.8 or higher. For example, Figure 1 of WO 2020 / 148693 shows the amount of 6'-sialyllactulose formed in lyophilized 6SL samples with various pH values ​​during accelerated thermal stability testing. Because 6SL isomerizes and converts to 6'-sialyllactulose during storage, lowering the initial amount of sialylated oligosaccharide isomers in the ion exchange resin treatment can result in sialylated oligosaccharides that exhibit improved chemical stability and / or physical properties.

[0047] In one embodiment of the fermented product of the present invention, it is preferable that the content of impurities such as component W is low, and the ratio of the content of component W to the content of component X in the fermented product (content of component W / content of component X), by mass, can be, for example, 0.0080 or less, 0.0070 or less, 0.0060 or less, 0.0050 or less, 0.0040 or less, 0.0038 or less, 0.0035 or less, 0.0030 or less, 0.0025 or less, 0.0020 or less, or 0.0010 or less, preferably 0.0038 or less, more preferably 0.0035 or less, even more preferably 0.0030 or less, and most preferably 0.0025 or less. In other embodiments, the range is 0.00010 to 0.0080, 0.00010 to 0.0040, 0.000010 to 0.0035, 0.000010 to 0.0030, or 0.000010 to 0.0025.

[0048] As a method for calculating the ratio of the content of each compound of component W to the content of component X in the fermented product (content of component W / content of component X), the methods of the analytical examples and examples described below can be mentioned in detail.

[0049] (Component V: Substance detected at RT 27.0 to 35.0 when the fermented product is measured by HPLC-CAD) Component V is a general term for substances detected at RT 27.0 to 35.0 when the fermented product is measured by HPLC-CAD as in the analytical example described below, and contains one or more components. Component V is a substance different from component Z2. Specific examples include acidic sugars in which one or more sialic acids are bound to an oligosaccharide composed of 2 to 10 monosaccharides. When component X is 3'-sialyllactose or 6'-sialyllactose, examples of component V include sialyllact-N-tetraose (LST) and disialyllact-N-tetraose (DSLNT).

[0050] Like component Y, component V has similar physical properties to component X, particularly acidity, and is therefore likely to be adsorbed onto and eluted from the basic anion exchange resin together with component X.

[0051] Therefore, when producing a fermented product containing component X, component V is likely to remain in the fermented product as an impurity, but by carrying out appropriate treatment, the removal rate of component V can be improved and the content of component X in the fermented product can be increased.

[0052] A method for improving the removal rate of component V is to set the temperature of the weakly basic anion exchange resin to 20° C. or higher in the step 2-2) of eluting at least a portion of the impurities remaining after 2-1) from the weakly basic anion exchange resin, which will be described later. This allows component V, which has a weaker adsorption force to the weakly basic anion exchange resin than component X, to be separated.

[0053] Component V is a substance that can be produced by the microorganism used in fermentation production. As the composition of solution A before the first step, the ratio of the total mass of component V to the volume of the weakly basic ion exchange resin used in the second step [(content of component V per 1 L of solution A (g / L) × volume of solution A (L)) / volume of weakly basic ion exchange resin (L)] is usually 5 g / L or more, preferably 10 g / L or more, more preferably 15 g / L or more, even more preferably 17 g / L or more, still more preferably 20 g / L or more, and most preferably 25 g / L or more. When it is within the above range, the content of component X can be increased by using the method for improving the removal rate of component V.

[0054] In one embodiment of the present invention, the fermented product preferably has a low content of impurities such as component V, and the ratio of the total content of component V to the content of component X in the fermented product (content of component V / content of component X) is preferably 0.040 or less, more preferably 0.038 or less, even more preferably 0.036 or less, even more preferably 0.034 or less, and most preferably 0.032 or less, by mass. In another embodiment, the ratio is 0.0010 to 0.040, 0.0010 to 0.038, 0.0010 to 0.036, 0.0010 to 0.034, or 0.0010 to 0.032.

[0055] As a method for calculating the ratio of the total content of component V to the content of component X in the fermented product (content of component V / content of component X), the methods in the analytical examples and examples described below can be mentioned in detail.

[0056] When the removal rate of component V is improved, the content of component X per solid content of the fermentation product of one embodiment of the present invention is preferably 82% by mass or more, and the following preferred levels are 84% by mass or more, 85% by mass or more, 87% by mass or more, 88% by mass or more, 89% by mass or more, and 90% by mass or more.

[0057] (Other Components) In addition to the above-mentioned components, the fermented product of one embodiment of the present invention may contain inorganic ions or inorganic salts. Many inorganic ions are derived from components contained during fermentation production, but most of them are removed during the process of increasing the content of component X. Therefore, in addition to the inorganic ions that act as cations that serve as counter ions to component X contained in the fermented product of one embodiment of the present invention, very small amounts of inorganic ions, for example, 10 mg / kg or less, may be contained. In particular, since component X is an acidic substance, inorganic ions may be selected as counter ions to these acidic substances, for example, as monovalent cations, and inorganic salts may be contained in the fermented product of one embodiment of the present invention as monovalent cation salts. Note that examples of monovalent cations include hydrogen ions, sodium ions, potassium ions, ammonium ions, and lithium ions, but sodium ions or potassium ions are preferred from the perspective of use in foods, etc. Other inorganic ions include the inorganic anion Cl. － , P.O. ４ ３－ , S.O. ４ ２－ etc.

[0058] (Other Features of the Fermented Product) The method for producing a fermented product according to one embodiment of the present invention preferably does not include a crystallization process. For example, in the case of producing component X by chemical synthesis and purifying component X by crystallization, organic solvents such as ethanol, methanol, and toluene remain in the final product. On the other hand, the method for producing a fermented product according to one embodiment of the present invention, which will be described later, does not require the use of these organic solvents. Therefore, it can be said that the fermented product obtained by the production method according to one embodiment of the present invention is safer for the human body.

[0059] In one embodiment of the present invention, the fermented product preferably has a reduced content of compounds designated as "Class 3 solvents (solvents that should be regulated by GMP or other quality standards)" in the "Guidelines for Residual Solvents in Pharmaceuticals" of the International Council for Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) set out above, compared to the content of such compounds in fermented products obtained by conventional techniques. The ratio of the content of compounds designated as "Class 3 solvents (solvents that should be regulated by GMP or other quality standards)" in the fermented product of one embodiment of the present invention (Class 3 solvent content / target product content) is 0.010 or less, preferably 0.008 or less, and more preferably 0.006 or less, by mass. Examples of Class 3 solvents include acetic acid. As described above, the fermented product obtained by the production method of one embodiment of the present invention preferably has a low acetic acid content and is therefore safer for the human body than conventional fermented products.

[0060] When the fermented product of one embodiment of the present invention is a solution, the pH is preferably 1 to 7, more preferably 3 to 7, and even more preferably 3 to 5. When the fermented product of one embodiment of the present invention is a powder, the pH is preferably 3 to 5 when 1 g of the powder is dissolved in 0.01 L of water, 0.005 L of the powder is dispensed, and the solution is diluted 2-fold with the same amount of water (0.005 L).

[0061] [2. Method for Producing a Solution and a Fermented Product] The above [1. Fermented product] is a concept that encompasses solution E obtained by steps 1 to 4 described below, powder obtained by steps 1 to 5 described below, and powder obtained by powdering the solution obtained by steps 1 to 4 in step 5.

[0062] A method for producing a fermented product according to one embodiment of the present invention includes the following steps 1) to 4) in this order: 1) treating a solution A containing components X, Y, and Z with a strongly acidic cation exchange resin to obtain a solution B; 2) treating the solution B with a weakly basic anion exchange resin to obtain a solution C; 3) adjusting the pH of the solution C to obtain a solution D; and 4) treating the solution D with a weakly basic anion exchange resin to obtain a solution E.

[0063] wherein component X is sialic acid or a sialylated oligosaccharide, component Y is acetic acid, component Z is at least one of component Z1 and component Z2, and component Z is a component other than component X, component Z1 is a neutral sugar, and component Z2 is an acidic sugar, the ratio of the total mass of component X in solution E to the total mass of component X in solution A (total mass of component X in solution E / total mass of component X in solution A) is 0.5 or more, and the ratio of the content of component Y to the content of component X in solution E (content of component Y / content of component X) is 0.010 or less by mass.

[0064] The method for producing a fermented product according to one embodiment of the present invention may further include the following step 5): 5) powdering the solution E.

[0065] The fermented product obtained by the above production method preferably contains component X, component Y, and component Z, the content of component X relative to the solid content of the fermented product is 80 mass% or more, and the ratio of the content of component Y to the content of component X in the fermented product (content of component Y / content of component X) is 0.010 or less by mass.

[0066] The above production method can reduce the content of component Y and the recovery rate of component X, i.e., (total mass of component X in solution E / total mass of component X in solution A), is 0.5 or more, making it fully applicable to industrial production where the production amount of fermentation product is on the kilogram scale, for example. For efficient production of component X, a high recovery rate of component X is preferred, and the ratio of the total mass of component X in solution E to the total mass of component X in solution A (total mass of component X in solution E / total mass of component X in solution A) is preferably 0.5 or more, more preferably 0.6 or more, and even more preferably 0.7 or more.

[0067] The ratio of the total mass of component X in solution E to the total mass of component X in solution A (total mass of component X in solution E / total mass of component X in solution A) can be calculated by analysis using HPLC-CAD. More specifically, it can be calculated by the methods described in the analytical examples and examples below.

[0068] Furthermore, the ratio of the total mass of component Y in solution E to the total mass of component Y in solution A (total mass of component Y in solution E / total mass of component Y in solution A) is preferably 0.5 or less, more preferably 0.4 or less, and even more preferably 0.2 or less. When the ratio of the total mass of component Y in solution E to the total mass of component Y in solution A (total mass of component Y in solution E / total mass of component Y in solution A) is a value within the above range, it can be said that component Y is efficiently removed from the fermented product such as solution E.

[0069] The ratio of the total mass of component Y in solution E to the total mass of component Y in solution A (total mass of component Y in solution E / total mass of component Y in solution A) can be calculated by HPLC-UV. More specifically, it can be calculated by the methods described in the analytical examples and examples below.

[0070] The method for producing a fermented product according to one embodiment of the present invention may not include at least one of the steps of chemically synthesizing component X and crystallizing component X. By not including the steps of chemical synthesis or crystallization, the possibility of an organic solvent being contained in the final fermented product can be reduced.

[0071] In this specification, the above 1) will also be referred to as the first step, the above 2) as the second step, the above 3) as the third step, the above 4) as the fourth step, and the above 5) as the fifth step.

[0072] The fermented product of one embodiment of the present invention can be obtained by purifying a solution A containing components X, Y, and Z using a method including the following steps 1) to 5). This solution A can be produced by culturing a microorganism capable of producing component X.

[0073] Preferred examples of microorganisms capable of producing component X include microorganisms capable of producing sialic acid or sialylated oligosaccharides such as sialyllactose. Microorganisms capable of producing sialic acid or sialylated oligosaccharides are not particularly limited as long as they are capable of producing sialic acid or sialylated oligosaccharides, and may be microorganisms that inherently have the ability to produce sialic acid or sialylated oligosaccharides, or microorganisms into which genes necessary for the production of sialic acid or sialylated oligosaccharides have been introduced into microorganisms that do not inherently have the ability to produce sialic acid or sialylated oligosaccharides. Examples of microorganisms capable of producing sialic acid or sialylated oligosaccharides include recombinant microorganisms into which genes necessary for the production of sialic acid or sialylated oligosaccharides have been introduced.

[0074] Examples of the microorganism include bacteria such as Escherichia coli, Lactobacillus lactis, Corynebacterium glutamicum, Bacillus subtilis, and Pseudomonas putita, as well as bacteria such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. Examples of the Escherichia coli include the Escherichia coli W3110 strain (ATCC 27325), the Escherichia coli MG1655 strain (ATCC 47076), the Escherichia coli BL21 (DE3) strain, the Escherichia coli W strain (ATCC 9637), and derivatives thereof.

[0075] Examples of genes necessary for the production of sialic acid or sialylated oligosaccharides include a gene encoding glucosamine-6-phosphate synthase, a gene encoding glucosamine 6-phosphate N-acetyltransferase, a gene encoding N-acetylglucosamine 2-epimerase, a gene encoding UDP-N-acetylglucosamine 2-epimerase, a gene encoding N-acetylneuraminic acid synthase, and a CMP-N-acetylneuraminic acid synthetase. Examples of genes whose expression is preferably enhanced in a microorganism capable of producing sialic acid or sialylated oligosaccharides include a gene encoding glucosamine-6-phosphate synthase, a gene encoding phosphoenolpyruvate synthase, and a gene encoding pyruvate carboxylase. Examples of genes whose expression is preferably weakened or deleted in microorganisms capable of producing sialic acid or sialylated oligosaccharides include genes encoding β-galactosidase, genes encoding the nanRATEK enzyme group, genes encoding glucosamine-6-phosphate deaminase, and genes encoding PTS transporters such as manXYZ and nagE. Methods for producing microorganisms capable of producing sialic acid or sialylated oligosaccharides include those described in WO 2019 / 228993, JP 2021-505170 A, and JP 2020-531039 A.

[0076] (Preparation for the First Step) In the first step, a solution A containing components X, Y, and Z is used. Solution A may further contain at least one component selected from the group consisting of components Z1, Z2, W, and V. This solution A preferably contains a culture broth produced by a microorganism that produces component X. Here, before performing the first step, a step of separating solids containing microorganisms from the culture broth and recovering a culture supernatant containing component X may be performed. Methods for separating solids containing microorganisms from the culture broth include centrifugation, filtration, and cross-flow filtering. In this specification, the term "culture broth" refers to a microorganism, a medium, components X, Y, Z, etc. The term "culture supernatant" refers to a microorganism-containing solid separated from the culture broth.

[0077] The composition of solution A before the first step preferably includes, for example, sialyllactose as component X, the content of component X being 0.1 g / L to 100 g / L, N-acetylneuraminic acid as component Z2, the ratio of the content of component 2 to the content of component X (content of component Z2) / (content of component X) being 0.03 to 0.5 by mass, and acetic acid as component Y, the ratio of the content of component Y to the content of component X (content of component Y / content of component X) being 0.010 to 0.60 by mass.

[0078] (First Step) In the first step, solution A containing component X, component Y, and component Z is treated with a strongly acidic cation exchange resin to obtain solution B. That is, in the first step, solution A, for example, a culture supernatant obtained by microbial fermentation, is treated with a strongly acidic cation exchange resin, so that, for example, microbial impurities and positively charged contaminants are adsorbed onto the strongly acidic cation exchange resin and removed from solution A. Examples of positively charged contaminants include Na ＋ , K. ＋ , N.H. ４ ＋ Examples of impurities derived from microorganisms include nucleic acids, amino acids, and proteins.

[0079] The strong acid cation exchange resin is not particularly limited, and examples thereof include a strong acid cation exchange resin having a sulfonic acid group as an exchange group in the strong acid cation exchange resin. Examples of the matrix of the strong acid cation exchange resin include a porous type, a macroporous type, a gel type, a styrene type, and an acrylic type.

[0080] Specific examples of the strong acid cation exchange resin include the Diaion SK series (SK1B, SK1BH, SK1BL, SK1BLH, SKL10, SKT10L, SKT110L, SK110L, SK112L, SKT20L, SK104, SK110, SK112, SK116, etc.) manufactured by Mitsubishi Chemical Corporation, and the Diaion PK series (e.g., For example, PK208, PK212, PK216, PK220, PK228, PK208LH, PK212L, PK212LH, PK216L, PK216H, PK216LH, PK220L, PK228L, PK228LH, etc.), Mitsubishi Chemical Corporation's Diaion RCP series (for example, RCP145H, RCP160M, etc.), Mitsubishi Chemical Corporation's Diaion HPK25 , Mitsubishi Chemical Corporation's Diaion UBK series (e.g., UBK16, UBK14, UBK12, UBK10, UBK08, UBK04, UBK02, UBK10H, UBK10HUP, UBK08A, UBK08H, UBK08HUP, UBKN1U, UBKN1UMB, UBK522M, UBK530, UBK530J, UBK530K, UBK535, UBK53 5J, UBK535K, UBK535L, UBK550, UBK555, etc.), Mitsubishi Chemical Corporation's Relight JC series (e.g., JC600, JC603, etc.), Lanxess's Lewatit S1668, Lanxess's Lewatit Monoplus series (e.g., S108, S108H, SP112, etc.), Dow Chemical's DOWEX 88, DOWEX 88MB, DOWEX Examples of such a gel include Monosphere 88, TG-Gel (also known as XUS40232-01), Amberlite manufactured by DuPont (e.g., FPC16UPS Na, FPC88MB Na, FPC240H, CR3220 Ca, CR1310 Ca, Na, CR1360 Na, CR99K / 350, HPR1100Na, etc.), C100, C100E, C120E, C100x10, C100x16MBH, C145S, C150, C160, SGC650 manufactured by Purolite, and the Purolite SST series (e.g., SSTC60, SSTC60H, SSTC80C, etc.) manufactured by Purolite.

[0081] The strongly acidic cation exchange resin preferably has a sulfonic acid group as an exchange group, and the matrix of the strongly acidic cation exchange resin is more preferably a gel type. Specific examples include UBK04 and TG-Gel (also known as XUS40232-01). The ionic type of the sulfonic acid group of the exchange group in the strongly acidic cation exchange resin is not particularly limited, and examples thereof include H ＋ Type, Na ＋ Type, K ＋ Type, NH ４ ＋ H ＋ It is preferable that the mold is a mold.

[0082] The pH of solution A to be treated with a strongly acidic cation exchange resin, for example, a culture supernatant obtained by microbial fermentation, is preferably 1 to 7, more preferably 3 to 5. The concentration of component X in solution A is preferably 0.1 g / L to 100 g / L, more preferably 1 g / L to 70 g / L, and even more preferably 10 g / L to 50 g / L. When solution A, for example, a culture broth obtained by microbial fermentation, does not have the above-mentioned preferred pH or component X concentration, solution A can be pretreated. Examples of pretreatment include adjusting the pH and ultrafiltration of solution A.

[0083] In the first step, solutions that can be used to pass solution A through the strongly acidic cation exchange resin when treating it include deionized water, an eluent, and the like, in addition to solution A. Deionized water is used for the purpose of eluting substances that do not adsorb to the strongly acidic cation exchange resin to improve the recovery rate of component X. In this step, solution A and deionized water are preferably used as solutions to pass through the strongly acidic cation exchange resin when treating it.

[0084] In this specification, the flow rate when a solution is passed through an ion exchange resin is defined as the space velocity (the volume ratio of the solution passed through the column per hour when the resin volume of the ion exchange resin is taken as 1, hereinafter referred to as "SV"). As a flow rate condition when treating Solution A, for example, a culture broth obtained by microbial fermentation, with a strongly acidic cation exchange resin, the flow rate is preferably SV 0.1 to 5, more preferably SV 0.2 to 4, and even more preferably SV 0.5 to 2. As a flow rate condition, the temperature is preferably 3 to 30°C, more preferably 5 to 25°C, and even more preferably 8 to 20°C.

[0085] In this specification, the temperature at which the ion exchange resin passes through the column refers to the temperature at which at least one of the following temperatures is equilibrated to room temperature or the temperature of the chromatography chamber: the temperature of the solution introduced from the inlet of the column packed with the ion exchange resin, the temperature of the solution discharged from the outlet of the column, and the temperature of the equipment used in the experiment that includes the column packed with the ion exchange resin.

[0086] The liquid that has passed through the strongly acidic cation exchange resin, i.e., solution B, is recovered and used in the second step described below.

[0087] (Second Step) In the second step, the solution B is treated with a weakly basic anion exchange resin to obtain a solution C. That is, in the second step, the solution B obtained in the first step (the strongly acidic cation exchange resin in the first step) is treated with a weakly basic anion exchange resin to separate component X from the impurities described below. The second step preferably includes passing the solution B obtained in the first step through the weakly basic anion exchange resin, and more specifically, the second step preferably includes: 2-1) passing the solution B through the weakly basic anion exchange resin to remove at least a portion of the neutral impurities that do not adsorb to the weakly basic anion exchange resin; 2-2) eluting at least a portion of the impurities remaining after 2-1) from the weakly basic anion exchange resin; and 2-3) separately eluting the acidic compounds including component X and the impurities remaining after 2-2) from the weakly basic anion exchange resin.

[0088] An example of the neutral contaminant is the neutral sugar of component Z1.

[0089] The acidic compound group containing component X includes, in addition to component X, negatively charged impurities such as SO ４ ２－ , Cl － , P.O. ４ ３－ and the like, component Y, organic acids other than component Y such as formic acid and fatty acids, nucleic acids, amino acids, proteins, phospholipids, and the like.

[0090] The weakly basic anion exchange resin is not particularly limited, and examples thereof include ion exchange resins having primary to tertiary amino groups or polyamino groups as exchange groups in the weakly basic anion exchange resin. Examples of the matrix of the weakly basic anion exchange resin include porous, macroporous, gel, styrene, and acrylic types.

[0091] Specific examples of weakly basic anion exchange resins include Dowex (registered trademark) Monosphere series (e.g., Monosphere 77) manufactured by Dow Chemical Company, Purolite (registered trademark) A100, A103S, A110, A111S, A133S, A830W, A845, and A847 manufactured by Purolite Corporation, HPR4780, IRA67, IRA96SB, and IRA98 manufactured by Organo Corporation, and Amberlite (e.g., FPA77UP, XE583GF, FPA53, and HPR4780) manufactured by DuPont. Cl, IRA67, IRA96SB, IRA98FPA95, FPA96, FPA77UPS, XE583GF, FPA53, etc.), Mitsubishi Chemical Corporation's Diaion (registered trademark) WA series (e.g., WA10, WA20, WA21J, WA30, WA30C, WA30LL, WA55, etc.), Mitsubishi Chemical Corporation's Sepabeads FPDA13, Mitsubishi Chemical Corporation's Rewrite JA series (e.g., JA300, JA310, JA450, JA830, etc.), Lanxess AG's Lewatit MP62WS, and Lanxess AG's Lewatit Monoplus MP64.

[0092] As the weakly basic anion exchange resin, an ion exchange resin having a tertiary amino group as an exchange group in the weakly basic anion exchange resin is more preferable.Specific examples of the weakly basic anion exchange resin having a tertiary amino group as an exchange group in the weakly basic anion exchange resin include Purolite (registered trademark) A845 / 1967 and A830W manufactured by Purolite Corporation.

[0093] The ion type of the tertiary amino group of the exchange group in the weakly basic anion exchange resin is not particularly limited, and examples thereof include hydroxide ions (OH － type), chloride ions (Cl － type), or an organic acid having two or more carbon atoms, and － The organic acid having two or more carbon atoms is preferably a carboxylic acid having two or more carbon atoms. Examples of the organic acid having two or more carbon atoms include acetic acid, propionic acid, butyric acid, valeric acid, lactic acid, glycolic acid, pyruvic acid, gluconic acid, tartaric acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, malic acid, fumaric acid, maleic acid, citric acid, isocitric acid, and ascorbic acid. Among these, acetic acid, propionic acid, and butyric acid are preferred, and acetic acid and propionic acid are more preferred. The organic acid having two or more carbon atoms may be used alone or in combination. There is no particular upper limit on the number of carbon atoms of the organic acid, but from the viewpoint of solubility in water, it is preferably 8 or less carbon atoms, more preferably 6 or less carbon atoms, and even more preferably 3 or less carbon atoms. The organic acid having two or more carbon atoms may be a salt of an organic acid having two or more carbon atoms.

[0094] Solution B to be treated in the second step preferably has a pH of 0 to 7, more preferably a pH of 0.5 to 5, and even more preferably a pH of 1 to 3. The concentration of component X in solution B is preferably 0.1 g / L to 100 g / L, more preferably 1 g / L to 70 g / L, and even more preferably 10 g / L to 50 g / L. If solution B obtained in the first step does not have the above-mentioned preferred pH or concentration of component X, the pH of solution B can be adjusted or solution B can be subjected to pretreatment such as ultrafiltration before the second step.

[0095] In the second step, solutions that can be used for passing solution B through the weakly basic anion exchange resin when treating it with the weakly basic anion exchange resin include solution B, deionized water, an eluent, and the like. Deionized water is used for the purpose of eluting impurities that do not adsorb to the weakly basic anion exchange resin. The eluent is used in this step for the purpose of eluting substances that have adsorbed to the weakly basic anion exchange resin. In this step, solutions that can be used for passing solution B through the weakly basic anion exchange resin when treating it with the weakly basic anion exchange resin are preferably solution B, deionized water, and an eluent.

[0096] ((2-1) A step of passing the solution B obtained in the first step through a weakly basic anion exchange resin to remove at least a portion of the neutral impurities that are not adsorbed to the weakly basic anion exchange resin) As a condition for passing the solution B obtained in the first step through the weakly basic anion exchange resin, the flow rate is preferably SV0.1 to 5, more preferably SV0.2 to 4, and even more preferably SV0.5 to 2.

[0097] The temperature at which the liquid passes through the weakly basic anion exchange resin is preferably 3°C or higher, 5°C or higher, 8°C or higher, or 10°C or higher, and is preferably 30°C or lower, 25°C or lower, 20°C or lower, 15°C or lower, 14°C or lower, 13°C or lower, or 12°C or lower. These upper and lower limits can be combined arbitrarily. In another embodiment, the temperature is 3 to 30°C, more preferably 3 to 25°C, even more preferably 3 to 20°C, even more preferably 3 to 15°C, even more preferably 5 to 15°C, and most preferably 8 to 15°C.

[0098] By this step (2-1), at least a portion of the neutral impurities are eluted from the weakly basic anion exchange resin, and components other than the neutral impurities in solution B are adsorbed to the weakly basic anion exchange resin. The components other than the neutral impurities include component X.

[0099] ((2-2) Step of eluting at least a portion of the impurities remaining after 2-1) from the weakly basic anion exchange resin) To elute the impurities remaining after 2-1) from the weakly basic anion exchange resin, the eluent preferably contains at least one of water, ammonium hydroxide, sodium hydroxide, potassium hydroxide, etc., and more preferably contains water and sodium hydroxide.

[0100] The concentration of the basic substance, such as ammonium hydroxide, sodium hydroxide, or potassium hydroxide, in the eluent is preferably 0.0000 mol / L or more, and more preferably 0.0001 mol / L or more, 0.0005 mol / L or more, 0.001 mol / L or more, 0.002 mol / L or more, and 0.005 mol / L or more, and more preferably 0.5 mol / L or less, 0.1 mol / L, and 0.05 mol / L or less. These upper and lower limits can be combined arbitrarily. In another embodiment, the concentration is preferably 0.0000 to 0.5 mol / L, 0.0001 to 0.5 mol / L, 0.0005 to 0.5 mol / L, 0.001 to 0.5 mol / L, or 0.005 to 0.5 mol / L.

[0101] In this specification, the amount of solution passed through an ion exchange resin is expressed as resin volume (the volume ratio of the solution when the resin volume of the ion exchange resin is taken as 1, hereinafter referred to as "RV").

[0102] The flow rate of the eluent is preferably SV 0.1 to 5, more preferably SV 0.2 to 4, and even more preferably SV 0.5 to 2. The amount of the eluent is preferably 0.5 to 20 RV, more preferably 1 to 15 RV, even more preferably 2 to 12 RV, and most preferably 3 to 10 RV.

[0103] The temperature at which the weakly basic anion exchange resin is passed through is preferably 3°C or higher, in order of preference 5°C or higher, 8°C or higher, 20°C or higher, 25°C or higher, and 30°C or higher, and is preferably 60°C or lower, in order of preference 50°C or lower, 40°C or lower, 30°C or lower, and 25°C or lower. These upper and lower limits can be combined arbitrarily. The temperature at which the liquid is passed through is also preferably 3 to 60°C, more preferably 20 to 50°C, even more preferably 20 to 40°C, especially preferably 20 to 40°C, and most preferably 20 to 25°C.

[0104] In a more preferred embodiment, after passing water, a basic aqueous solution such as ammonium hydroxide, sodium hydroxide, potassium hydroxide or the like is passed through.

[0105] This step (2-2) washes away at least a portion of the impurities remaining in the weakly basic anion exchange resin after step (2-1). Furthermore, by setting the temperature within the above range, the content of component V in the final fermented product tends to be reduced and the content of component X tends to be increased.

[0106] ((2-3) Step of Separately Eluting the Acidic Compounds Containing Component X and the Impurities Remaining After 2-2) from the Weakly Basic Anion Exchange Resin) In order to separately elute the Acidic Compounds Containing Component X and the Impurities Remaining After 2-2) from the Weakly Basic Anion Exchange Resin, the eluent preferably contains at least one of water, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and the like, and more preferably contains sodium hydroxide.

[0107] The concentration of a basic substance such as ammonium hydroxide, sodium hydroxide, or potassium hydroxide in the eluent is preferably 0.001 mol / L or more, and more preferably 0.005 mol / L or more, 0.01 mol / L or more, 0.02 mol / L or more, and 0.03 mol / L or more, and more preferably 2.0 mol / L or less, 1.0 mol / L, and 0.5 mol / L or less. These upper and lower limits can be arbitrarily combined. In another embodiment, the concentration is preferably 0.001 to 2.0 mol / L, 0.005 to 1.0 mol / L, 0.001 to 1.0 mol / L, 0.001 to 0.5 mol / L, 0.01 to 0.5 mol / L, 0.02 to 0.5 mol / L, or 0.03 to 0.5 mol / L.

[0108] The flow rate of the eluent is preferably SV 0.1 to 5, more preferably SV 0.2 to 4, and even more preferably SV 0.5 to 2. The amount of the eluent is preferably 0.5 to 20 RV, more preferably 1 to 15 RV, even more preferably 2 to 12 RV, and most preferably 3 to 10 RV.

[0109] The temperature at which the weakly basic anion exchange resin is passed through is preferably 3°C or higher, more preferably 5°C or higher, even more preferably 8°C or higher, especially preferably 10°C or higher, and particularly preferably 15°C or higher, and is preferably 40°C or lower, more preferably 30°C or lower, even more preferably 25°C or lower, especially preferably 20°C or lower, and particularly preferably 15°C or lower. These upper and lower limits can be combined arbitrarily. The temperature at which the liquid is passed through is preferably 3 to 40°C, 3 to 30°C, 3 to 25°C, 3 to 20°C, 3 to 15°C, 5 to 20°C, 8 to 20°C, 10 to 20°C, or 15 to 20°C, in the following order of preference.

[0110] By setting the temperature at which the liquid passes through the weakly basic anion exchange resin within the above range, isomerization of aldose to ketose can be prevented or reduced, and the content of sialylated oligosaccharide isomers of component X can be reduced, tending to increase the content of component X.

[0111] Solution C obtained in the second step preferably has a pH of 5 to 12, more preferably a pH of 6 to 10, and even more preferably a pH of 7 to 9. The concentration of component X in solution C is preferably 0.1 g / L to 100 g / L, more preferably 1 g / L to 70 g / L, even more preferably 3 g / L to 50 g / L, and most preferably 5 g / L to 20 g / L.

[0112] (Third Step) In the third step, the pH of the above solution C is adjusted to obtain solution D. In other words, in the third step, the pH of solution C obtained in the second step, i.e., solution C eluted from the weakly basic anion exchange resin in the second step, is adjusted. Solution C obtained in the second step is basic due to ion exchange with the weakly basic anion exchange resin. Therefore, more specifically, solution C is adjusted to a pH of preferably 0 to 6, more preferably 1 to 5, and even more preferably 2 to 4 to obtain solution D. In this case, the pH adjustment in the third step is preferably performed without mixing with solution C separated in the second step. Because the pH adjustment in the third step improves the adsorption force to the weakly basic anion exchange resin, in the fourth step described below, the degree of separation between component X and other impurities can be improved by treating solution D with the weakly basic anion exchange resin.

[0113] The pH of solution C can be adjusted by any known method, including, for example, adding an inorganic acid, specifically hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, or the like, to solution C, or passing solution C through a cation exchange resin. The method of passing solution C through a cation exchange resin is preferred, and the method of passing solution C through a weakly acidic cation exchange resin is more preferred.

[0114] The cation exchange resin used in the third step is not particularly limited, and examples thereof include weakly acidic cation exchange resins having carboxylate groups as exchange groups. Examples of the base material of the cation exchange resin include porous, macroporous, gel, styrene, and acrylic types. Specific examples of cation exchange resins include WK10, WK10S, WK11, WT01S, WK40, WK60, and WK100 manufactured by Diaion Co., Ltd., Purolite (registered trademark) C104, C106, C107E, and C115E manufactured by Purolite Co., Ltd., Amberlite (e.g., FPC76J, FPC3500, etc.) manufactured by DuPont, Diaion WK series (e.g., WK10, WK100, WK10S, WK11, WK40, WK60, and WK60L, etc.) manufactured by Mitsubishi Chemical Corporation, Diaion WT01S manufactured by Mitsubishi Chemical Corporation, and Lewatit CNP80WS manufactured by Lanxess AG. The cation exchange resin used in the third step preferably has a carboxylate group as the exchange group in the cation exchange resin, and is more preferably a porous type cation exchange resin matrix. A specific example is Purolite (registered trademark) C115E manufactured by Purolite Co., Ltd. The ionic type of the carboxylate group of the exchange group in the cation exchange resin is not particularly limited, and examples thereof include H ＋ Type, Na ＋ Type, K ＋ Type, NH ４ ＋ H ＋ It is preferable that the mold is a mold.

[0115] The pH of solution D obtained in the third step is preferably 0 to 6, more preferably 1 to 5, and even more preferably 2 to 4. The concentration of component X in solution D is preferably 0.1 g / L to 100 g / L, more preferably 1 g / L to 70 g / L, even more preferably 3 g / L to 50 g / L, and most preferably 5 g / L to 20 g / L. If solution D obtained in the third step does not have the above-mentioned preferred pH or concentration of component X, solution D can be subjected to a treatment such as adjusting the pH after the third step.

[0116] In the third step, solutions that can be used to pass solution C through the cation exchange resin when treating it include solution C and deionized water. Deionized water is used for the purpose of eluting substances that do not adsorb to the acidic cation exchange resin to improve the recovery rate of component X. In this step, solution C and deionized water are preferably used as solutions to pass through the cation exchange resin when treating solution C through the cation exchange resin.

[0117] As a condition for passing solution C through the cation exchange resin, the flow rate is preferably SV 0.1 to 5, more preferably SV 0.2 to 4, and even more preferably SV 0.5 to 2. As a condition for passing solution C through the cation exchange resin, the temperature is preferably 30°C or lower, more preferably 25°C or lower, and even more preferably 20°C or lower. Specifically, the temperature is preferably 3 to 30°C, more preferably 5 to 25°C, and even more preferably 8 to 20°C. The liquid that has passed through the cation exchange resin, i.e., solution D, is recovered and used in the fourth step described below.

[0118] (Fourth Step) In the fourth step, the solution D is treated with a weakly basic anion exchange resin to obtain a solution E. That is, in the fourth step, the solution D obtained in the third step is treated with a weakly basic anion exchange resin to separate component X from acidic contaminants other than component X, thereby obtaining a solution E containing component X at a high concentration. Examples of acidic contaminants other than component X include component Y, organic acids other than component Y, and component Z.

[0119] The weakly basic anion exchange resin used in the fourth step can be any of those listed as the weakly basic anion exchange resins used in the second step. In particular, in the fourth step, the weakly basic anion exchange resin is preferably one in which the ion type of the tertiary amino group of the exchange group is in the free form.

[0120] Solution E obtained in the fourth step preferably has a pH of 5 to 12, more preferably a pH of 6 to 10, and even more preferably a pH of 7 to 9. The concentration of component X in solution E is preferably 0.1 g / L to 100 g / L, more preferably 1 g / L to 70 g / L, even more preferably 3 g / L to 50 g / L, and most preferably 5 g / L to 20 g / L. If solution E obtained in the fourth step does not have the above-mentioned preferred pH or concentration of component X, treatment such as adjustment of the pH of solution E can be performed after the fourth step. Alternatively, the pH of solution E may be adjusted to make it acidic.

[0121] In the fourth step, solutions that can be used to pass solution D through the weakly basic anion exchange resin when treating it with the weakly basic anion exchange resin include solution D, deionized water, an eluent, and the like. Deionized water is used for the purpose of eluting substances that do not adsorb to the weakly basic ion exchange resin to improve the recovery rate of component X. In this step, solution D and deionized water are preferably used as solutions to pass through the weakly basic anion exchange resin when treating solution D with the weakly basic anion exchange resin.

[0122] As conditions for passing Solution D through the weakly basic anion exchange resin, the flow rate is preferably SV 0.1 to 5, more preferably SV 0.2 to 4, and even more preferably SV 0.5 to 2.5. Under these conditions, the temperature is preferably 30°C or lower, more preferably 25°C or lower, and even more preferably 20°C or lower. Specifically, the temperature is preferably 3 to 30°C, more preferably 5 to 25°C, and even more preferably 8 to 20°C.

[0123] Furthermore, component X has a lower acidity than component Y and component Z, or a weaker adsorption force to the weakly basic anion exchange resin. When solution D is passed through a weakly basic anion exchange resin, component X tends to elute first, followed by components Y and Z. Therefore, as a fraction containing a large amount of component X and a small amount of components Y and Z, it is preferable to collect a fraction immediately after the inflection point of the electrical conductivity just before the end of the elution of component X, more preferably a fraction between the inflection point of the electrical conductivity just before the end of the elution of component X and 1.0 RV, even more preferably a fraction between the inflection point of the electrical conductivity just before the end of the elution of component X and 0.5 RV, and most preferably a fraction between the inflection point of the electrical conductivity just before the end of the elution of component X and 0.1 RV. The recovered solution is a fermented product containing component X at a high concentration. In this specification, the inflection point of electrical conductivity refers to a point at which the curve of a graph in which the horizontal axis represents the amount of liquid passed through a weakly basic anion exchange resin and the vertical axis represents electrical conductivity changes from convex (concave down) to concave (concave up), or vice versa.

[0124] (Fifth Step) In the fifth step, the solution E is powdered to obtain a fermented product. The solution E used in the fifth step may be a solution obtained by concentrating, decolorizing, and / or filtering the steps described below after the fourth step. Known methods can be used for powdering, such as freeze-drying and spray-drying, with spray-drying being preferred. It is also possible to use solution E or a solution obtained after concentration, decolorization, filtration, etc., as described below, as the fermented product without powdering the fermented product.

[0125] (Concentration, Decolorization, Filtration) After the fourth step, the solution E (fermented product) recovered in the fourth step may be concentrated, decolorized, and / or filtered. Known methods can be used for concentration, decolorization, and / or filtration. The order of concentration, decolorization, and filtration is not important.

[0126] (Concentration, Filtration) For example, there is mentioned a step of microfiltrating the solution E obtained in the fourth step using a membrane with a pore size of 0.2 to 1.2 μm, specifically a method of treating the solution E obtained in step 4 or a solution obtained by treating it with activated carbon using an MF membrane. Alternatively, the microfiltered solution may be concentrated under reduced pressure to a concentration of component X in the solution of 10 to 600 g / L, more preferably 100 to 550 g / L, and even more preferably 300 to 500 g / L.

[0127] (Decolorization) The concentrated solution may be subjected to a decolorization treatment by adding activated carbon. Specific examples of activated carbon used here include Carborafine, Strong Shirasagi, Purified Shirasagi, Special Shirasagi, Shirasagi A, Shirasagi C, Shirasagi ANOX-1, Shirasagi FAC-10, Shirasagi WP-H, Shirasagi DO-2, Shirasagi DO-5, Granular Shirasagi G2c, Granular Shirasagi WH2c, Granular Shirasagi W2c, Granular Shirasagi WH5c, Granular Shirasagi W5c, Granular Shirasagi LGK-100, Granular Shirasagi LGK-400, Granular Shirasagi KL, Granular Shirasagi LH2c, Spherical Shirasagi X8100H, Spherical Shirasagi XS8100H, Granular Shirasagi Sagi G2x, Granular Shirasagi G5x, Granular Shirasagi S2x, Granular Shirasagi WH2x, Granular Shirasagi X2M, Granular Shirasagi C2c, Granular Shirasagi C2x, Spherical Shirasagi X7000H, Spherical Shirasagi X7100H, Spherical Shirasagi XS7100H, Spherical Shirasagi X7000H-3, Spherical Shirasagi X71 00H-3, Spherical Shirasagi LGK-700, Spherical Shirasagi DX7-3, Shirasagi M, Shirasagi P, Granular Shirasagi GM2X, Seitz AKSJ, Taiko S, Taiko K, Taiko P, Taiko W, Taiko A, Taiko Y manufactured by Futamura Chemical Co., Ltd., and AltaPrint manufactured by Ingevity. 246, Altapyne Pitch, AquaGuard, AquaNuchar, Kraftsperse 1251, Nuchar, Nuchar ACS 310X-2L, Nuchar BAX 1100LD, Nuchar BAX 1500, Nuchar BAX 1700, Nuchar BAX LBE, Nuchar FuelSorb, Nuchar HCA LBE, Nuchar RGC Powder, Nuchar SA, Nuchar SA PCL, Nuchar SA-20, Nuchar SA-T, Nuchar SN, Nuchar Square HCA, Nuchar Standard HCA, Nuchar WV-A 1100 8x25, Nuchar WV-A 1100 8x35, Nuchar WV-A 1500 10x25, Nuchar WV-B 30 Granular, HR manufactured by Right Solution, GW-110, GW-120, GW-520, PW-100, PW-003, PW-210, PS-003, PS-250, PS-252, PG-100, PG-001, GG-770, GG-771, GG-772, GG-773 manufactured by Sun Frontier Chemical Co., Ltd.Examples include GG-775, GG-730, GG-731, GG-732, GG-733, GG-735, GD-120, GD-200, and PD-005, with Taiko Y being preferred.

[0128] Furthermore, the filtrate decolorized by the above process can be ultrafiltered by passing it through an ultrafiltration membrane (hereinafter also referred to as "UF membrane") (MWCO = 6000) capable of removing substances with a molecular weight of 6000 or more, for example, a UF membrane, to remove, for example, endotoxins, residual proteins, high molecular weight peptides, toxins, etc. from the decolorized filtrate. The ultrafiltered solution can also be concentrated under reduced pressure to a concentration of component X in the solution of 10 to 600 g / L, more preferably 100 to 550 g / L, and even more preferably 300 to 500 g / L.

[0129] (Fermented product obtained by the second step) The method for producing a fermented product may include the first step and the second step, but not the third step and the fourth step. The fermented product obtained by the second step includes the solution obtained by the first step and the second step, and a solution or powder obtained by concentrating, decolorizing, filtering, and / or powdering the solution by the above-mentioned methods.

[0130] Furthermore, the ratio of the content of component Z1 to the content of component X in the fermented product obtained by the second step (content of component Z1 / content of component X) is preferably 0.03 or less, more preferably 0.01 or less, and even more preferably 0.001 or less, by mass. Specific examples of neutral sugars of component Z1 include glucose and / or lactose.

[0131] Furthermore, the ratio of the content of component Z2 to the content of component X in the fermented product obtained by the second step (content of component Z2 / content of component X) is preferably 0.5 or less, more preferably 0.4 or less, by mass.

[0132] Other impurities contained in the fermented product obtained in the second step include inorganic anions such as Cl － , P.O. ４ ３－, S.O. ４ ２－ Examples of inorganic cations include sodium ions, potassium ions, and ammonium ions.

[0133] The fermented product obtained by the first to fifth steps described in [1. Fermented Product] of this specification has a higher content of component X and lower contents of component Y and component Z than the fermented products obtained by the first and second steps.

[0134] Preferred embodiments of the present invention will be described below, but the present invention is not limited to these. Unless otherwise specified, % indicates % by mass.

[0135] [Analysis Examples] In the Examples, the analysis and quantification of each component was carried out according to the following procedures.

[0136] Analysis Example 1-1: Measurement of the content and percentage content of sialic acid or sialylated oligosaccharide (component X) from solid content (Preparation of component X standard solution) The component X standard solution was prepared by precisely weighing out approximately 0.1 g of the following standard products, dissolving each in distilled water to adjust the concentration to 1 g / L, and then diluting with a diluent (a solution obtained by mixing HPLC-CAD mobile phase A and mobile phase B in a 6:4 volume ratio) to give component X concentrations of 0.04 g / L, 0.05 g / L, and 0.06 g / L. Standard product: 3SL·Na, manufactured by Kyowa Hakko Bio Co., Ltd., purity 99.2% (as 3SL·Na) Standard product: 6SL·Na, manufactured by Kyowa Hakko Bio Co., Ltd., purity 97.0% (as 6SL·Na)

[0137] (Preparation of sample solution) The fermented product was a solid such as a powder, and the sample solution was prepared by precisely weighing out about 0.5 g of the solid, dissolving it in distilled water, and diluting it with a diluent. Note that, when the peak area value of component X in the sample solution was a concentration outside the range of the calibration curve, the sample solution was appropriately adjusted with a diluent so that the concentration of component X was within the range of the calibration curve.

[0138] (Analysis of Component X) Measurement was performed under the following analytical conditions using the prepared sample solution and component X standard solution, and the peaks of component X (retention times: NeuAc 16.0 minutes, 3SL 21.4 minutes, 6SL 22.5 minutes) were detected. A calibration curve was created by the three-point calibration curve method from the peak area values ​​of component X in the component X standard solution, and the concentration of component X in the sample solution was quantified.

[0139] (Analysis conditions for component X) Component X was analyzed by HPLC-CAD under the following conditions. Analytical columns: Guard column: HILICpak VN-50G 4A, inner diameter 4.6 mm, length 10 mm, particle size 5 μm, Cat# F6711400 (Showa Denko K.K.) Column: HILICpak VN-50 4D, inner diameter 4.6 mm, length 150 mm, particle size 5 μm, Cat# F7630500 (Showa Denko K.K.) Column temperature: constant temperature around 60°C Mobile phase: Mobile phase A; acetonitrile, mobile phase B; 25 mM ammonium formate Mobile phase flow rate (flow rate): 1.0 mL / min Detector: Charged Aerosol Detector (CAD) CAD drying temperature: 35°C

[0140] The gradient was performed under the conditions shown in Table 1 below.

[0141]

[0142] (Calculation of the content of component X from solid matter) The slope of the calibration curve calculated from the peak area value of component X in the obtained component X standard solution was C, the intercept was D, the moisture value (mass%) of the sample calculated by the Karl Fischer titration method in Analysis Example 7 was E, the concentration of the fermented product in the sample solution was F (g / L), and the peak area value of the sample solution was G. The content of component X was calculated using the following formula (1a): Content of component X (mass%) = (C × G + D) ÷ (F × (100 - E)) × 100 × 100 ... formula (1a)

[0143] Analysis Example 1-2: Calculation of the Content of Component X from a Solution (Calculation of the Content of Other Impurities Detected by HPLC) Analysis was performed under the same conditions as in Analysis Example 1-1 using a component X standard solution and a sample solution containing 1 g / L of component X, and the amount of other impurities detected by HPLC was quantified using the single-point calibration curve method from the component X peak area value of the component X standard solution. Here, other impurities detected by HPLC refer to components detected by HPLC other than component X, component Y, component Z, component W, and component V. The sample solution containing 1 g / L of component X was prepared based on the content of component X determined in Analysis Example 1-1. The peak area value of the component X standard solution was defined as R, and the peak area value of the other impurities detected by HPLC in the sample solution was defined as S. The content of other impurities detected by HPLC in the sample was calculated using the following formula (13a): Content of other impurities detected by HPLC in the sample solution (g / L) = 0.05 × (R / S) Equation (13a)

[0144] (Calculation of the content of other impurities detected by HPLC in the fermented product / the content of component X) Here, the content of other impurities detected by HPLC in the sample solution calculated by formula (13a) is the content of other impurities detected by HPLC in a sample solution with a component X content of 1 g / L, and therefore the content of other impurities detected by HPLC relative to the content of component X in the fermented product was calculated by the following formula (13b). Content of other impurities detected by HPLC / content of component X=(content of other impurities detected by HPLC in the sample (g / L) / content of component X in the sample solution (g / L)...formula (13b)

[0145] (Calculation of the sum of (content of other impurities detected by HPLC in the fermentation product / content of component X)) The total number of impurities detected in the sample is n, and the sum of the ratios of the contents of each impurity to the content of component X is U. ２ was calculated by the following formula (13c): In the following formula (13c), U is the ratio of the content of each impurity detected by HPLC to the content of component X.

[0146]

[0147] (Calculation of the content of component X from the solution) The ratio of the content of acetic acid to the content of component X (specifically, sialyllactose) detected from the sample is Y, and the ratio of the content of neutral sugars (specifically, lactose) to the content of component X is Z. １ The ratio of the content of the acidic sugar (specifically, NeuAc) to the content of the component X is Z. ２ , the ratio of the content of component W to the content of component X is W, the ratio of the content of component V to the content of component X is V, and the ratio of the content of inorganic metal to the content of component X is M. １ The moisture value when the solution is solidified using SD etc. is U ３ , the sum of the ratios of the contents of each impurity to the content of component X in the sample solution is I １ , the sum of the ratios of the contents of each impurity to the content of component X in the sample solution on a wet basis is I ２ , the purity of component X in the wet weight sample solution is X １ , the purity of component X in the sample solution is X ２ In this case, the purity of component X in the sample solution can be calculated using the following formulas (14a) to (14d): The sum of the ratios of the content of each impurity to the content of component X in the sample solution (I1) = Y + Z １ +Z ２ +W+V+U ２ +M １ ...Equation (14a) The sum of the ratios of the contents of each impurity to the content of component X in the wet weight sample solution (I ２ ) = I １ × (100-U ３ ) / 100 ... Equation (14b) The purity of component X in the wet weight sample solution (X １ ) = 100 - (I ２ ×100)-U ３ ...Equation (14c) Content of component X in the sample solution (X ２ ) = X １ ×100 / (100-U ３ ) ...Formula (14d)

[0148] (Calculation of the content of component X) When the fermented product was a solid such as a powder, the content of component X was calculated using the following formula (1b). Note that the definitions of C, G, and D in formula (1b) are the same as those of C, G, and D in formula (1a). Content of component X (g / g) = (C × G + D) ÷ F formula (1b)

[0149] Furthermore, when the fermented product is a solution and the content of component X is calculated using the fermented product as a sample solution without evaporating it to dryness, the fermented product is analyzed using the sample solution as is, and the peak area value of the obtained sample solution is designated as G'. If the sample solution is diluted to calculate G, the dilution factor is designated as y, and the content (g / L) of component X in the fermented product is calculated using formula (1c). Note that the definitions of C and D in formula (1c) are the same as those of C and D in formula (1a). Content (g / L) of component X = (C × G' + D) × y Formula (1c)

[0150] <Analysis Example 2: Measurement of Acetic Acid (Component Y) Content> (Preparation of Acetic Acid Standard Solution) The acetic acid standard solution was prepared by precisely weighing out approximately 0.14 g of sodium acetate (Fujifilm Wako Pure Chemical Industries, Ltd., product number 198-15965) as a standard and dissolving it in water to give a solution with an acetic acid concentration of approximately 0.1 g / L.

[0151] (Analysis of Acetic Acid (Component Y)) Using a 0.1 g / L acetic acid standard solution and a sample solution containing approximately 10 g / L of component X, analysis was performed under the following analytical conditions. The acetic acid peak (retention time: acetic acid 34.7 minutes) was detected, and component Y (i.e., acetic acid) was quantified using the single-point calibration curve method from the peak area value of the standard. The sample solution containing approximately 10 g / L of component X was prepared based on the content of component X calculated in Analysis Example 1-1.

[0152] (Analysis conditions for acetic acid (component Y)) The quantitative determination of acetic acid was carried out by HPLC-UV under the following conditions. Guard column: SUGAR SH-G, inner diameter 6.0 mm, length 50 mm, particle size 10 μm, P / N: F6700080 (Showa Denko K.K.) Column: SUGAR SH1011, inner diameter 8.0 mm, length 300 mm, particle size 6 μm, two columns connected, P / N: F6378100 (Showa Denko K.K.) Column temperature: constant temperature around 60°C Mobile phase: 5 mM H ２ SO ４ Mobile phase flow rate: 0.6 mL / min Detector: UV-visible absorption detector (SPD-20A, manufactured by Shimadzu Corporation) Detection wavelength: 210 nm Detection limit: 0.650 mg / L Quantitation limit: 1.97 mg / L

[0153] (Calculation of the Content of Component Y) The peak area value of the acetic acid standard solution is A, and the peak area value of the sample solution is B. The acetic acid concentration in the sample solution was calculated using the following formula (2a): Acetic acid content in sample solution (g / L) = 0.1 × (B / A) Formula (2a)

[0154] (Calculation of Content of Component Y / Content of Component X in Fermented Product) Here, the content of acetic acid in the sample solution calculated by formula (1) is the content of component Y in a sample solution in which the content of component X is 10 g / L, and therefore the content of component Y relative to the content of component X in the fermented product (content of component Y / content of component X) was calculated by the following formula (2b): Content of component Y / content of component X=content of acetic acid in sample solution (g / L) / content of component X in sample solution (g / L) ... formula (2b)

[0155] Analysis Example 3: Analysis of lactose (component Z1) (Preparation of lactose standard solution) Lactose monohydrate (FUJIFILM Wako Pure Chemical Corporation, 128-00095) was used as a standard for quantitative analysis of lactose (component Z1) by HPLC. The lactose standard solution was prepared by precisely weighing approximately 0.11 g of the lactose standard and dissolving it in distilled water so that the lactose content was 0.025 g / L.

[0156] (Calculation of the content of component Z1) Analysis was carried out under the following conditions using a lactose standard solution and a sample solution with a component X content of approximately 1 g / L, and component Z1 was quantified using the single-point calibration curve method from the peak area value of lactose in the lactose standard solution. The sample solution with a component X content of approximately 1 g / L was prepared based on the content of component X calculated in Analysis Example 1-1. The lactose content in the sample solution was calculated using the following formula (3a), where the peak area value of the lactose standard solution is J and the peak area value of the sample solution is K. Z1 (lactose) content in the sample solution (g / L) = 0.025 × (K / J) ... formula (3a)

[0157] (Conditions for Analyzing Lactose (Z1)) Lactose was quantified by HPLC-PAD under the following conditions. Guard column: Dionex CarboPac PA1 IC Standard Bore Guard Column, inner diameter 4 mm, length 50 mm, particle size 10 μm, Cat# 043096 (Thermo Fisher Scientific) Column: Dionex CarboPac PA1 IC Standard Bore Analytical Column, inner diameter 4 mm, length 250 mm, particle size 10 μm, Cat# 035391 (Thermo Fisher Scientific) Column temperature: constant temperature around 30°C Mobile phase: Mobile phase A: distilled water, mobile phase B: 0.5 mol / L NaOH, mobile phase C: 0.28 M Na acetate + 0.03 M NaOH Mobile phase flow rate: 1.0 mL / min Detector: Pulsed amperometric detector (PAD) Detection limit: 0.451 mg / L Quantitation limit: 1.37 mg / L

[0158] The gradient was performed under the conditions shown in Table 2 below.

[0159]

[0160] (Calculation of the content of component Z1 / content of component X in the fermented product) Here, the content of Z1 in the sample solution calculated by formula (3a) is the content of component Z1 in a sample solution in which the content of component X is 1 g / L, and therefore the content of lactose (component Z1) relative to the content of component X in the fermented product was calculated by the following formula (3b): Content of component Z1 / content of component X=(content of lactose (component Z1) in the sample solution) / (content of component X in the sample solution)...formula (3b)

[0161] In addition, when component Z1 is glucose, the content of component Z1 / content of component X in the fermented product can be calculated in the same manner as in the above-mentioned Analysis Example 3.

[0162] Analysis Example 4: Analysis of NeuAc (component Z2) (Preparation of NeuAc standard solution) N-acetylneuraminic acid dihydrate (MA167834, manufactured by Biosynth Carbosynth) was used as a standard for quantitative analysis of NeuAc (component Z2) by HPLC. The NeuAc standard solution was prepared by precisely weighing out approximately 0.11 g of the NeuAc standard and dissolving it in distilled water to prepare a 1 g / L solution, followed by dilution with a diluent (a 6:4 mixture of HPLC-CAD mobile phase A and mobile phase B) to prepare a NeuAc concentration of 0.04 g / L.

[0163] (Calculation of the Content of Component Z2) Analysis was performed under the same conditions as in Analysis Example 1-1 using a NeuAc standard solution and a sample solution containing 1 g / L of component X, and the amount of component Z2 was determined by a single-point calibration curve method from the NeuAc peak area value of the NeuAc standard solution. The sample solution containing 1 g / L of component X was prepared based on the content of component X determined in Analysis Example 1-1. The peak area value of the NeuAc standard was taken as L, and the NeuAc peak area value of the sample solution was taken as M, and the NeuAc content in the sample was calculated by the following formula (4a): Content (g / L) of Z2 (NeuAc) in the sample solution = 0.04 × (M / L) ... formula (4a)

[0164] (Calculation of Content of Component Z2 / Content of Component X in Fermented Product) Here, the content of Z2 in the sample solution calculated by formula (4a) is the content of component Z2 in a sample solution with a component X content of 1 g / L, and therefore the content of NeuAc (component Z2) relative to the content of component X in the fermented product was calculated by the following formula (4b): Content of component Z2 / Content of component X=(Content of NeuAc (component Z2) in sample (g / L) / Content of component X in sample solution (g / L)...formula (4b)

[0165] Analysis Example 5: Analysis of isomer of component X (component W) (Calculation of the content of component W) Analysis was performed under the same conditions as in Analysis Example 1-1 using a component X standard solution and a sample solution containing 1 g / L of component X, and the amount of component W was quantified by the single-point calibration method from the component X peak area value of the component X standard solution. The sample solution containing 1 g / L of component X was prepared based on the content of component X determined in Analysis Example 1-1. The component X peak area value of the component X standard solution was set to N, and the peak area value of component W in the sample solution was set to O, and the content of component W in the sample was calculated using the following formula (11a): Content (g / L) of component W (isomer of component X) in the sample solution = 0.05 × (O / N) ... formula (11a)

[0166] (Calculation of Content of Component W / Content of Component X in Fermented Product) Here, the content of W in the sample solution calculated by formula (11a) is the content of component W in a sample solution in which the content of component X is 1 g / L, and therefore the content of the isomer of component X (component W) relative to the content of component X in the fermented product was calculated by the following formula (11b): Content of component W in fermented product / Content of component X=(content of isomer of component X (component W) in sample (g / L) / content of component X in sample solution (g / L)...formula (11b)

[0167] Analysis Example 6: Analysis of Component V (Calculation of the Content of Component V) Analysis was performed under the same conditions as in Analysis Example 1-1 using a component X standard solution and a sample solution containing 1 g / L of component X, and the amount of component V was quantified using the single-point calibration curve method from the peak area value of component X in the standard solution. The sample solution containing 1 g / L of component X was prepared based on the content of component X determined in Analysis Example 1-1. The peak area value of component X in the standard solution was P, and the peak area value of component V in the sample solution was Q, and the content of component V in the sample was calculated using the following formula (12a): Content of component V in sample solution (g / L) = 0.05 × (P / Q) ... formula (12a)

[0168] (Calculation of Content of Component V / Content of Component X in Fermented Product) Here, the content of component V in the sample solution calculated by formula (12a) is the content of component V in a sample solution in which the content of component X is 1 g / L, and therefore the content of component V relative to the content of component X in the fermented product was calculated by the following formula (12b): Content of component V in fermented product / Content of component X=Content of component V in sample (g / L) / Content of component X in sample solution (g / L) Formula (12b)

[0169] Analysis Example 7: Measurement of water content in fermented product The water content (% by mass, the water content value of E in formula (1a)) in the fermented product was analyzed by Karl Fischer titration under the following conditions: Test method: JP17<2.48> (Water content determination method of the Japanese Pharmacopoeia, 17th Edition) Amount added: 0.1 g of powder Titrant: Aquamicron (registered trademark) Titrant SS-Z 3 mg (Karl Fischer reagent SS-Z) Dehydrating solvent: Aquamicron (registered trademark) Dehydrating solvent ML (general use) Dissolution time: up to 5 min

[0170] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples as long as it does not depart from the gist of the invention. In this specification, 3SL is 3-sialyllactose, and 3'-sialyllactose sodium salt, which is the sodium salt of 3SL, is also referred to as 3SL·Na. In this specification, 6SL is 6-sialyllactose, and 6'-sialyllactose sodium salt, which is the sodium salt of 6SL, is also referred to as 6SL·Na.

[0171] Test Example 1: Production of a fermented product with a high content of 3'-sialyllactose (3SL) Preparation Example 1 Fermentation production of 3SL The culture broth was prepared by fermentation production of 3SL using recombinant Escherichia coli according to the methods described in International Publication No. 2015 / 037698 and Japanese Patent Application Laid-Open No. 2008-5794. The culture broth was then heat-treated at 70°C for 40 minutes to inactivate the cells, and the pH was adjusted to 4.3±0.3 by adding sulfuric acid. Subsequently, the culture broth was passed through a cross-flow filter (0.1 μm filter) to separate the inactivated cells (biomass) from the fermentation liquid, and a culture supernatant (referred to as Solution A) was obtained. Solution A was obtained for two lots, Lot 1 and Lot 2. Table 3 shows the composition of the resulting Solution A.

[0172] Comparative Example 1 Purification of 3SL by First and Second Steps In Comparative Example 1, 0.355 L of Solution A of Lot 1 at 10°C obtained in Preparation Example 1 above was used for the ion exchange chromatography in the first and second steps.

[0173] In the first step, H ＋0.11 L of TG-Gel (also known as XUS 40232.01 (manufactured by The Dow Chemical Company)), a type of strongly acidic cation exchange resin, was used, and solution A was passed through the TG-gel at an SV of 0.86, resulting in the adsorption of cationic impurities in solution A onto the strongly acidic cation exchange resin, thereby obtaining solution B from which the cationic impurities had been removed. In this specification, the "treated solution" refers to a solution that is treated in steps 1 to 5 to obtain a fermented product containing a high proportion of component X. Furthermore, in Examples 1 to 4 and Comparative Examples 1 and 2, unless otherwise specified, RV and SV are the volume ratios of the solution when the resin volume of the second tower A845 / 1967 is taken as 1.

[0174] Next, the above solution B is added to the resin in the second step. － The solution was treated with 0.142 L of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin. First, solution B at 10°C was passed through the ion exchange resin at an SV of 0.83 to adsorb the anionic impurities and 3SL of the solution B onto the weakly basic anion exchange resin. Subsequently, 3.2 RV of deionized water at 25°C was passed through the resin at an SV of 0.83 to remove neutral substances that did not adsorb to the weakly basic anion exchange resin. Subsequently, the anionic impurities adsorbed on the weakly basic anion exchange resin were removed by passing 2.3 RV of a 0.005 mol / L aqueous NaOH solution at 25°C through the resin at an SV of 0.83. Thereafter, a 0.035 mol / L aqueous NaOH solution at 10°C was passed through the resin at an SV of 0.83, and the 3SL adsorbed on the weakly basic anion exchange resin was eluted as a sodium salt. The solution was collected at an end point of 4.65 RV in Figure 1, yielding 0.66 L of eluate (solution C). During the elution, the eluate was sampled every 0.155 RV, and the composition of the eluate was analyzed.

[0175] The results of analyzing the composition of this eluate are shown in Figure 1. In Figure 1, the horizontal axis shows the volume of the liquid passed through the column in the second step as "volume of liquid passed through the column in the second column," and the vertical axis shows the concentration of each component and electrical conductivity. Electrical conductivity is shown as Cond.

[0176] In Table 3, the composition of Solution C obtained in the second step is shown in the column for Comparative Example 1. The composition of Solution C obtained in the second step was calculated by accumulating the values ​​from the line graph in Figure 1 obtained by analyzing the eluate fractionated in the treatment of the second step. Note that 3SL is also contained in the eluate as 3SL·Na, but the content and percentage of 3SL shown in Table 3 are values ​​converted to 3SL (free form).

[0177] Example 1 Purification of 3SL Through First to Fourth Steps In Example 1, 0.355 L of solution A from lot 2 at 10°C obtained in Preparation Example 1 above was used to carry out the treatments in steps 1 to 4. The first and second steps were carried out in the same manner as in Comparative Example 1 above, except that in the second step, 2.9 RV of deionized water at 25°C was passed through at an SV of 0.83, and then 2.8 RV of a 0.005 mol / L aqueous NaOH solution at 25°C was passed through at an SV of 0.83, and solution C obtained in the second step was passed through in the third step. Solution A from lot 2 obtained in Preparation Example 1 was treated to obtain solution C.

[0178] The solution C obtained in the second step was passed through Purolite® C115E and Purolite® A845 / 1967 successively in the third and fourth steps as follows: In the third step, the solution C obtained in the second step was passed through Purolite® C115E and Purolite® A845 / 1967 in a continuous manner while being cooled to 10° C. ＋ The solution was passed through 0.08 L of a weakly acidic cation exchange resin Purolite (registered trademark) C115E (manufactured by Purolite Co., Ltd.) of the type 2, and the pH of the solution was adjusted to 2 to 4, to obtain a solution D. The solution C was passed through Purolite (registered trademark) C115E at an SV of 1.5, assuming that the resin volume of the third column Purolite (registered trademark) C115E was 1. In the fourth step, the solution D obtained in the third step was cooled to 10°C while OH was added. －The eluate was passed through 0.051 L of Purolite® A845 / 1967 (manufactured by Purolite Corporation), a weakly basic anion exchange resin, to remove related substances, specifically acidic impurities, and the solution was collected at an end point of 5.8 RV in Figure 2, yielding 0.82 L of eluate (Solution E). Solution D was prepared by passing Purolite® A845 / 1967 at an SV of 2.3 relative to the resin volume of the fourth column, Purolite® A845 / 1967, and fractions of the eluate were collected every 0.148 RV, and the eluate composition was analyzed.

[0179] The results of analyzing the composition of this solution E are shown in Figure 2. In Figure 2, the horizontal axis represents the amount of liquid passed through the column in the fourth step, and the vertical axis represents the content of each component and electrical conductivity. Electrical conductivity is shown as Cond. In addition, the composition of solution E obtained in the fourth step is shown in the column for Example 1 in Table 3.

[0180] In Table 3, the value of 3SL (g / L) is the value calculated by formula (1c) in the above-mentioned Analysis Example 1-1, the value of NeuAc (g / L) is the value calculated by formula (4a) in the above-mentioned Analysis Example 4, and the value of acetic acid (g / L) is the value calculated by formula (2a) in the above-mentioned Analysis Example 2.

[0181] In Table 3, the "yield" for Example 1 is a value calculated using the following formula (5a), and the "yield" for Comparative Example 1 is a value calculated using the following formula (5b): {3SL concentration of solution E (g / L) × liquid volume (L)} / {3SL concentration of solution A (g / L) × liquid volume (L)} ... formula (5a) {3SL concentration of solution C (g / L) × liquid volume (L)} / {3SL concentration of solution A (g / L) × liquid volume (L)} ... formula (5b)

[0182] The "yield" in the examples is a value corresponding to the "ratio of the total mass of component X in solution E to the total mass of component X in solution A (total mass of component X in solution E / total mass of component X in solution A)" in this specification.

[0183] In Table 3, the "residual rate" of acetic acid in Example 1 is a value calculated by the following formula (9a), and the "residual rate" of acetic acid in Comparative Example 1 is a value calculated by the following formula (9a): {Acetic acid concentration of solution E (g / L) × liquid volume (L)} / {Acetic acid concentration of solution A (g / L) × liquid volume (L)} ... formula (9a) {Acetic acid concentration of solution C (g / L) × liquid volume (L)} / {Acetic acid concentration of solution A (g / L) × liquid volume (L)} ... formula (9b)

[0184] The "residual rate" of acetic acid in Example 1 is a value corresponding to "the ratio of the total mass of component Y in solution E to the total mass of component Y in solution A (total mass of component Y in solution E / total mass of component Y in solution A)" in this specification.

[0185]

[0186] 1 shows that in Comparative Example 1, acetic acid is eluted after the volume of liquid passing through the second column reaches about 3 RV, and that acetic acid is eluted while 3SL is eluted. On the other hand, FIG. 2 shows that in Example 1, the amount of acetic acid eluted while 3SL is eluted is suppressed compared to FIG. 1.

[0187] As shown in Table 3, in solution E obtained in Example 1 (i.e., the fermented product of the present invention), the ratio (content of component Z2 (NeuAc) / content of component X (3SL)) ("Ratio of NeuAc to 3SL" in Table 3) was less than 0.02. Furthermore, the ratio (content of component Y (acetic acid) / content of component X (3SL)) ("Ratio of acetic acid to 3SL" in Table 3) was less than 0.010. Meanwhile, in solution C obtained in Comparative Example 1, the ratio (content of component Z2 (NeuAc) / content of component X (3SL)) was greater than 0.02. Furthermore, the ratio (content of component Y (acetic acid) / content of component X (3SL)) ("Ratio of acetic acid to 3SL" in Table 3) was greater than 0.010.

[0188] Test Example 2: Production of a fermented product with a high content of 6'-sialyllactose (6SL) Preparation Example 2 Fermentative production of 6SL The culture broth was prepared by fermentation production of 6SL using recombinant Escherichia coli according to the methods described in International Publication No. 2015 / 037698 and Japanese Patent Application Laid-Open No. 2008-5794. The cells were inactivated by heat treatment at 70°C for 40 minutes, and the pH was adjusted to 4.3±0.3 by adding sulfuric acid. Subsequently, the inactivated cells (biomass) were separated from the fermentation broth using a cross-flow filter (0.1 μm filter) to obtain a culture supernatant (solution A). The resulting solution A is shown in Table 4.

[0189] [Comparative Example 2] <Purification of 6SL by the first and second steps> In Comparative Example 2, 51.9 kL of industrial-scale solution A obtained in the above [Preparation Example 2] was used in the first and second steps of ion exchange chromatography.

[0190] In the first step, H ＋ 17 kL of a strongly acidic cation exchange resin (TG-Gel) of this type was used, and solution A at 8.0°C was passed through the TG-gel at an SV of 1.0, causing the cationic impurities in solution A to be adsorbed onto the strongly acidic cation exchange resin, thereby obtaining solution B from which the cationic impurities had been removed.

[0191] Next, the resin in the second step was treated with the same OH as in Example 1 and Comparative Example 1. －22 kL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, was used. Solution B was passed through the weakly basic anion exchange resin at 11.1°C at an SV of 0.73 to adsorb the anionic impurities and 6SL in Solution B onto the weakly basic anion exchange resin. Subsequently, 3.6 RV of deionized water at 10.0°C was passed through the resin at an SV of 0.81, and neutral substances not adsorbed to the weakly basic anion exchange resin were removed by washing. Furthermore, the anionic impurities adsorbed to the weakly basic anion exchange resin were removed by passing 2.6 RV of a 0.005 mol / L aqueous NaOH solution at 10.0°C through the resin at an SV of 0.82. Subsequently, a 0.035 mol / L aqueous NaOH solution at 10.0°C was passed through the resin at an SV of 0.62, and the 6SL adsorbed on the weakly basic anion exchange resin was eluted as a sodium salt. The eluate was collected at 0.1-0.2 RV intervals, and the composition of each eluate was analyzed. The solution with an end point of 2.73 RV (Figure 3) was collected and combined into a single solution, yielding 60.0 kL of eluate (Solution C). During the elution, eluate was collected every 15 minutes, and the composition of the eluate was analyzed.

[0192] The results of analyzing the composition of the collected eluate are shown in Figure 3. In Figure 3, the horizontal axis indicates the volume of the liquid passed through the column in the second step as "volume of liquid passed through the column in the second column," and the vertical axis indicates the concentration of each component and the electrical conductivity. The electrical conductivity is indicated as Cond.

[0193] In Table 4, the composition of the eluate obtained in the second step is shown in the column for Comparative Example 2. The composition of Solution C in Comparative Example 2 in Table 4 was calculated by accumulating the values ​​from the line graph in Figure 3 obtained by analyzing the composition of the eluate fractionated in the treatment of the second step. Note that 6SL is also contained in the eluate as 6SL·Na, but the content and percentage of 6SL shown in Table 4 are values ​​converted to 6SL (free form).

[0194] [Example 2] <Purification of 6SL through Steps 1 to 4> In Example 2, the eluate from Step 2 (Solution C) obtained in [Comparative Example 2] was passed through Step 3, and the eluate from Step 3 (Solution D) was passed through Step 4, thereby carrying out the treatments of Steps 1 to 4.

[0195] In the third and fourth steps, the eluate obtained in the second step was passed through Purolite® C115E and Purolite® A845 / 1967 successively as follows: In the third step, the eluate obtained in the second step (solution C) was passed through H ＋ Solution D was obtained by passing it through 12 kL of a weakly acidic cation exchange resin Purolite (registered trademark) C115E (manufactured by Purolite Co., Ltd.) of the type 2, adjusted to a pH of 2 to 4. Solution C was passed through Purolite (registered trademark) C115E at an SV of 1.4, assuming that the resin volume of the third column Purolite (registered trademark) C115E was 1. In the fourth step, solution D obtained in the third step was cooled to 8.0°C while OH was added. － The eluate was passed through 9.5 kL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, to remove related substances, specifically acidic impurities, and the solution was collected at an end point of 2.69 RV in Figure 4, yielding 54.0 kL of eluate (Solution E). Note that, when the resin volume of the fourth column, Purolite® A845 / 1967, was taken as 1, Solution D was passed through Purolite® A845 / 1967 at an SV of 1.6, and the eluate was collected every 15 minutes and analyzed for composition.

[0196] The results of analyzing the composition of this solution E are shown in Figure 4. In Figure 4, the horizontal axis shows the amount of liquid passed through the column in the fourth step as "amount of liquid passed through the column at the fourth column," and the vertical axis shows the content of each component and electrical conductivity. Electrical conductivity is shown as Cond. In addition, the composition of solution E obtained in the fourth step is shown in the column for Example 2 in Table 4.

[0197] In Table 4, the amount of 6SL (g / L) is the value calculated by formula (1c) in Analysis Example 1-1 above, the amount of NeuAc (g / L) is the value calculated by formula (4a) in Analysis Example 4 above, and the amount of acetic acid (g / L) is the value calculated by formula (2a) in Analysis Example 2 above.

[0198] The "yield" and "residual rate" of acetic acid in Table 4 are values ​​calculated in the same manner as the "yield" and "residual rate" of acetic acid in Table 3, except that 3SL is replaced with 6SL.

[0199]

[0200] 3 shows that in Comparative Example 2, acetic acid was eluted immediately after the start of the second step and continued to be eluted while 6SL was being eluted. On the other hand, FIG. 4 shows that in Example 2, the amount of acetic acid eluted while 6SL was being eluted was reduced compared to Comparative Example 2.

[0201] As shown in Table 4, in solution E obtained in Example 2 (i.e., the fermented product of the present invention), the (content of component Z2 (NeuAc) / content of component X (6SL)) ("Ratio of NeuAc to 6SL" in Table 4) was less than 0.02. Also, the (content of component Y (acetic acid) / content of component X (6SL)) ("Ratio of acetic acid to 6SL" in Table 4) was less than 0.010. On the other hand, in solution C obtained in Comparative Example 2, the (content of component Z2 (NeuAc) / content of component X (3SL)) was greater than 0.02, and the (content of component Y (acetic acid) / content of component X (3SL)) ("Ratio of acetic acid to 3SL" in Table 4) was greater than 0.010.

[0202] [Example 3] <Purification of 3SL and Production of Powder Containing 3SL> Fermentation production of 3SL was carried out using recombinant Escherichia coli according to the methods described in International Publication No. 2015 / 037698 and Japanese Patent Application Laid-Open No. 2008-5794. The cells were inactivated by heat treatment at 70 ° C. for 40 minutes, and sulfuric acid was added to adjust the pH to 4.3 ± 0.3, yielding 78.7 kL of culture broth having the composition listed in sample "A. Broth" in Tables 5 and 6. Subsequently, the inactivated cells (biomass) were separated from the fermentation broth using a cross-flow filter (0.1 μm filter), yielding a total of 128 kL of culture supernatant (Solution A). The 3SL concentration, NeuAc [ratio to 3SL], and acetic acid [ratio to 3SL] of this Solution A are as listed in Tables 5 and 6.

[0203] The resulting solution A was then divided into two portions, and the first to fourth steps were carried out in the same manner as in Examples 1 and 2, as follows.

[0204] The first portion of the obtained solution A (64.1 kL) and the second portion (63.9 kL) were used separately in the first and second steps, respectively.

[0205] In the first step, a strong acid cation exchange resin, H ＋ 17 kL of TG-Gel (manufactured by The Dow Chemical Company) was used, and solution A at 8.0°C was passed through the first strongly acidic cation exchange resin at an SV of 0.95 relative to the TG-gel, and solution A at 8.0°C was passed through the second strongly acidic cation exchange resin at an SV of 1.0 relative to the TG-gel, thereby allowing the cationic impurities in solution A to be adsorbed onto the strongly acidic cation exchange resin, and two solutions B were obtained from solution A from which the cationic impurities had been removed.

[0206] Next, the second step resin is OH －23 kL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, was used. For each solution B, in order to adsorb the anionic impurities and 3 SL in solution B onto the weakly basic anion exchange resin, solution B at 8.9°C was passed through the first weakly basic anion exchange resin at an SV of 0.72, and solution B at 8.7°C was passed through the second weakly basic anion exchange resin at an SV of 0.74. Subsequently, the first weakly basic anion exchange resin was washed with 3.1 RV of deionized water at 10.0°C, and the second weakly basic anion exchange resin was washed with 3.1 RV of deionized water at 10.0°C, both at an SV of 0.78. Neutral substances not adsorbed to the weakly basic anion exchange resin were removed from each weakly basic anion exchange resin. The anionic impurities adsorbed to the weakly basic anion exchange resin were then removed by passing 2.7 RV of 0.005 mol / L NaOH aqueous solution at 10.0 ° C. through the first weakly basic anion exchange resin, and 2.6 RV of 0.005 mol / L NaOH aqueous solution at 10.0 ° C. through the second weakly basic anion exchange resin, each at an SV of 0.78. The first weakly basic anion exchange resin was then passed through 0.035 mol / L NaOH aqueous solution at 10.0 ° C., and the second weakly basic anion exchange resin was passed through 0.035 mol / L NaOH aqueous solution at 10.0 ° C., each at an SV of 0.77. The 3SL adsorbed to the weakly basic anion exchange resin was eluted as a sodium salt, yielding two solutions C.

[0207] Then, in the third step, the two solutions C obtained in the second step are mixed with H ＋ Each solution was passed through 11 kL of a weakly acidic cation exchange resin, Purolite (registered trademark) C115E (manufactured by Purolite Corporation), to obtain two solutions D, each adjusted to a pH range of 2 to 4. Solution C was passed through Purolite (registered trademark) C115E at an SV of 1.6 relative to the resin volume of the third column, Purolite (registered trademark) C115E, while being cooled to 11.1°C and 11.0°C, respectively.

[0208] Then, in the fourth step, the two solutions D obtained in the third step were cooled to 8.0°C while OH was added. － The solution was passed through 10 kL of Purolite® A845 / 1967 (manufactured by Purolite Corporation), a weakly basic anion exchange resin, to obtain 85.0 kL of eluate (Solution E) in the first division and 80.5 kL of eluate (Solution E) in the second division. Note that, when the resin volume of the Purolite® A845 / 1967 in the fourth column is taken as 1, Solution D was passed through Purolite® A845 / 1967 at an SV of 1.8.

[0209] The two solutions E obtained in the fourth step were each adjusted to a pH of 5.5 to 6.1 by adding 0.5 mol / L hydrochloric acid. Then, each pH-adjusted solution E was passed through a 1.2 μm line filter (MF membrane) and a 0.45 μm line filter (MF membrane), in that order, to obtain two treated solutions. Each treated solution was then concentrated until the SL·Na concentration reached 400 g / L, and the two concentrated solutions were mixed to obtain one concentrated solution.

[0210] Next, activated carbon (Taiko (registered trademark) Y) manufactured by Futamura Chemical Co., Ltd. was added to this concentrate in order to remove colored components and the like from the concentrate by adsorption onto the activated carbon. Then, the pH of the concentrate to which activated carbon had been added was adjusted to 5.3-6.2 by adding 0.5 mol / L of hydrochloric acid immediately before proceeding to the filtration process. This pH-adjusted concentrate was passed through a 0.45 μm line filter (MF membrane) in the filtration process to remove fine activated carbon and foreign matter, and then passed through a UF membrane (6000 Da cutoff) to remove endotoxins and the like from the concentrate. The permeate obtained by passing through the UF membrane was then concentrated to an SL·Na concentration of approximately 400 g / L, and passed through a 0.2 μm line filter to obtain a sterilized treated liquid (liquid fermentation product).

[0211] Then, in the fifth step, the sterilized treatment solution was spray-dried using a spray dryer to obtain a spray-dried powder (SD) as a fermentation product. As a result, 2,281 kg of powder was obtained, as shown in the sample "SD" column in Tables 5 and 6, with an SL purity of 88% by mass, a NeuAc content of 0.00552 relative to 3SL, and an acetic acid content of 0.000473 relative to 3SL.

[0212] Tables 5 and 6 show the results of analyzing the compositions of Solution A, Solution E, and SD obtained in Example 3. In Tables 5 and 6, 3SL [g / L] is the value calculated using formula (1c) in Analysis Example 1-1 above, NeuAc [g / L] is the value calculated using formula (4a) in Analysis Example 4 above, and acetic acid [g / L] is the value calculated using formula (2a) in Analysis Example 2 above.

[0213] The "yield" in Table 5 is a value calculated using the following formula (10): {3SL concentration of solution E (g / L) × liquid volume (L)} / {3SL concentration of solution A (g / L) × liquid volume (L)} Formula (10)

[0214] The "3SL purity" in Table 5 is a value corresponding to the "content of component X per solid content of the fermented product" in this specification. The "3SL purity" was calculated by the method described in formula (1a) of Analysis Example 1-1 above.

[0215] The [ratio to 3SL] of acetic acid in Table 5 corresponds to the "ratio of the content of component Y to the content of component X in solution E (content of component Y / content of component X)" by mass in this specification when the sample is solution E, and corresponds to the "ratio of the content of component Y to the content of component X in the fermented product (content of component Y / content of component X)" by mass in this specification when the sample is SD. This value was calculated using formula (2b) in Analysis Example 2 above.

[0216] The [ratio to 3SL] of NeuAc in Table 6 corresponds to the mass-based "ratio of the content of each compound of component Z2 to the content of component X in the fermentation product (content of component Z2 / content of component X)" in this specification. This value was calculated using formula (4b) in Analysis Example 4 above.

[0217] The "residual rate" of acetic acid in Table 5 was calculated in the same manner as the "residual rate" of acetic acid in Example 1.

[0218] In this powder, the neutral sugars Z1, glucose and lactose, were present in a ratio of 3SL (the ratio of the content of each compound of component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X)), which was below the detection limit.

[0219]

[0220]

[0221] [Example 4] <Purification of 6SL and Production of Powder Containing 6SL> Fermentation production of 6SL was carried out using recombinant Escherichia coli according to the methods described in International Publication No. 2015 / 037698 and Japanese Patent Application Laid-Open No. 2008-5794. The cells were inactivated by heat treatment at 70 ° C. for 40 minutes, and sulfuric acid was added to adjust the pH to 4.3 ± 0.3, yielding 83.4 kL of culture broth having the composition listed in sample "A. Broth" in Tables 7 and 8. Subsequently, the inactivated cells (biomass) were separated from the fermentation broth using a cross-flow filter (0.1 μm filter), yielding a total of 121 kL of culture supernatant (Solution A). The 6SL concentration, NeuAc [ratio to 6SL], and acetic acid [ratio to 6SL] of this Solution A are as listed in Tables 7 and 8.

[0222] The resulting solution A was then divided into two portions, and the first to fourth steps were carried out in the same manner as in Examples 1 and 2 as follows.

[0223] The resulting first portion of Solution A (60.8 kL) and second portion (60.0 kL) were used in the first and second steps, respectively.

[0224] In the first step, H ＋17 kL of TG-Gel (manufactured by The Dow Chemical Company), a type of strongly acidic cation exchange resin, was used. Solution A at 8.0°C was passed through the first portion of the strongly acidic cation exchange resin at an SV of 1.0 relative to the TG-gel, and solution A at 8.0°C was passed through the second portion of the strongly acidic cation exchange resin at an SV of 1.0 relative to the TG-gel, thereby allowing the cationic impurities to be adsorbed onto the strongly acidic cation exchange resin, and two solutions B were obtained from solution A from which the cationic impurities had been removed.

[0225] Next, the second step resin is OH － 22 kL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, was used. To adsorb anionic impurities and 6SL onto the weakly basic anion exchange resin, each solution B was passed through the weakly basic anion exchange resin at 12.3°C (SV 0.77) for the first portion and at 14.0°C (SV 0.77) for the second portion. The weakly basic anion exchange resin was then washed with 3.1 RV of deionized water at 10.0°C (SV 0.79) for the first portion and 3.6 RV of deionized water at 10.0°C (SV 0.70) for the second portion, respectively. Neutral substances not adsorbed to the weakly basic anion exchange resin were removed from each weakly basic anion exchange resin. The anionic impurities adsorbed to the weakly basic anion exchange resin were then removed by passing 3.1 RV of 0.005 mol / L NaOH aqueous solution at 10.0 ° C through the first weakly basic anion exchange resin, and 2.9 RV of 0.005 mol / L NaOH aqueous solution at 10.0 ° C through the second weakly basic anion exchange resin at SV 0.82 and SV 0.70, respectively. Then, 0.035 mol / L NaOH aqueous solution at 10.0 ° C through the first weakly basic anion exchange resin, and 0.035 mol / L NaOH aqueous solution at 10.0 ° C through the second weakly basic anion exchange resin at SV 0.81 and SV 0.70, respectively. The 6SL adsorbed on the weakly basic anion exchange resin was eluted as a sodium salt, yielding two solutions C.

[0226] Then, in the third step, the two solutions C obtained in the second step are mixed with H ＋ Each solution was passed through 11.5 kL of a weakly acidic cation exchange resin, Purolite (registered trademark) C115E (manufactured by Purolite Corporation), to obtain two solutions D, each adjusted to a pH range of 2 to 4. Solution C was passed through Purolite (registered trademark) C115E at SVs of 1.5 and 1.4, respectively, while being cooled to 11.1°C and 15.2°C, respectively, where the resin volume of the third column, Purolite (registered trademark) C115E, was taken as 1.

[0227] Then, in the fourth step, the two solutions D obtained in the third step were cooled to 8.0°C while OH was added. － The solution was passed through 9.5 kL of Purolite® A845 / 1967 (manufactured by Purolite Corporation), a weakly basic anion exchange resin, to obtain 108 kL of eluate (Solution E) in the first portion and 80.6 kL of eluate (Solution E) in the second portion. Note that, when the resin volume of the Purolite® A845 / 1967 in the fourth column was taken as 1, Solution D was passed through Purolite® A845 / 1967 at SVs of 1.9 and 1.7, respectively.

[0228] The two solutions E obtained in the fourth step were adjusted to a pH of 5.5 to 6.1 by adding 0.5 mol / L hydrochloric acid. Then, each pH-adjusted solution E was passed through a 1.2 μm line filter (MF membrane) and a 0.45 μm line filter (MF membrane), respectively, in this order, to obtain two treated solutions. Next, each treated solution was concentrated until the SL·Na concentration in the treated solution reached 400 g / L, and the two concentrated solutions were mixed to obtain one concentrated solution.

[0229] Next, activated carbon (Taiko (registered trademark) Y) manufactured by Futamura Chemical Co., Ltd. was added to this concentrate in order to remove colored components and the like from the concentrate by adsorption onto the activated carbon. Then, the pH of the concentrate to which activated carbon had been added was adjusted to 5.3-6.2 by adding 0.5 mol / L of hydrochloric acid immediately before proceeding to the filtration process. This pH-adjusted concentrate was passed through a 0.45 μm line filter (MF membrane) in the filtration process to remove fine activated carbon and foreign matter, and then passed through a UF membrane (6000 Da cutoff) to remove endotoxins and the like from the concentrate. The permeate obtained by passing through the UF membrane was then concentrated to an SL·Na concentration of approximately 400 g / L, and passed through a 0.2 μm line filter to obtain a sterilized treated liquid (liquid fermentation product).

[0230] Then, in the fifth step, the sterilized treatment solution was spray-dried using a spray dryer to obtain a spray-dried powder (SD) as a fermentation product. As a result, 2,099 kg of powder was obtained, as shown in the sample "SD" column in Tables 7 and 8, with an SL purity of 88% by mass, a NeuAc content of 0.0170 relative to 6SL, and an acetic acid content of 0.00318 relative to 6SL.

[0231] Tables 7 and 8 show the results of analyzing the compositions of Solution A, Solution E, and SD obtained in Example 4. The various parameters shown in Tables 7 and 8 were calculated in the same manner as the various parameters shown in Tables 5 and 6, except that 3SL was replaced with 6SL. In this powder, the neutral sugars Z1, glucose and lactose, were below the detection limit in terms of the ratio to 6SL (the ratio of the content of each compound in component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X)).

[0232]

[0233]

[0234] [Preparation Example 3] <Fermentation production of 3SL> A culture broth was prepared by fermentation production of 3SL using recombinant Escherichia coli according to the methods described in International Publication No. 2015 / 037698 and Japanese Patent Application Laid-Open No. 2008-5794. The cells were inactivated by heat treatment at 70°C for 40 minutes, and the pH was adjusted to 4.3±0.3 by adding sulfuric acid. Subsequently, the inactivated cells (biomass) were separated from the fermentation liquid using a cross-flow filter (0.1 µm filter), and a culture liquid (solution A) was obtained.

[0235] Example 5 Purification of 3SL by First to Fourth Steps In Example 5, 0.468 L of solution A obtained in Preparation Example 3 above was used to carry out the treatments of the first to fourth steps.

[0236] In the first step, H ＋ 126 mL of TG-Gel (manufactured by The Dow Chemical Company), a type of strongly acidic cation exchange resin, was used, and solution A at 15.0°C was passed through the TG-gel at an SV of 1.0, causing the cationic impurities in solution A to be adsorbed onto the strongly acidic cation exchange resin, thereby obtaining solution B from which the cationic impurities had been removed.

[0237] Next, the second step resin is OH － 140 mL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, was used. To adsorb the anionic impurities in Solution B and 3SL onto this weakly basic anion exchange resin, Solution B at 15.0°C was passed through the weakly basic anion exchange resin at an SV of 0.9. Subsequently, 3.5 RV of deionized water at 15.0°C was passed through at an SV of 1.0, and neutral substances that did not adsorb onto the weakly basic anion exchange resin were removed from the weakly basic anion exchange resin. The anionic impurities adsorbed onto the weakly basic anion exchange resin were then removed by passing a 0.005 mol / L aqueous NaOH solution at 15.0°C through at 3.0 RV and SV of 1.0. Thereafter, a 0.035 mol / L aqueous NaOH solution at 15.0° C. was passed through at an SV of 1.0, and the 3SL adsorbed on the weakly basic anion exchange resin was eluted as a sodium salt, thereby obtaining solution C.

[0238] Then, in the third step, the solution C obtained in the second step is ＋ The solution was passed through 84 mL of a weakly acidic cation exchange resin, Purolite (registered trademark) C115E (manufactured by Purolite Corporation), to obtain Solution D, the pH of which was adjusted to a range of 2 to 4. Solution C was passed through Purolite (registered trademark) C115E at an SV of 1.6 while being cooled to 15.0°C, assuming that the resin volume of the third column, Purolite (registered trademark) C115E, was 1.

[0239] Then, in the fourth step, the solution D obtained in the third step was cooled to 15.0°C while OH was added. － The solution was passed through 74 mL of Purolite (registered trademark) A845 / 1967 (manufactured by Purolite Corporation), a weakly basic anion exchange resin, to obtain 1.1050 L of eluate (Solution E). Solution D was passed through Purolite (registered trademark) A845 / 1967 at an SV of 1.9 relative to the resin volume of the fourth column, Purolite (registered trademark) A845 / 1967, and the eluate was collected every 0.6 RV to analyze its composition.

[0240] The composition of Solution E obtained in the fourth step was analyzed, and the results are shown in the column for Example 5 in Tables 10 and 11.

[0241] [Example 6] <Purification of 3SL by the First to Fourth Steps> In Example 6, the first to fourth steps were carried out using 0.468 L of Solution A obtained in the above [Preparation Example 3].

[0242] In the first step, H ＋ 126 mL of TG-Gel (manufactured by The Dow Chemical Company), a type of strongly acidic cation exchange resin, was used, and solution A at 10.0°C was passed through the TG-gel at an SV of 1.0, causing the cationic impurities in solution A to be adsorbed onto the strongly acidic cation exchange resin, thereby obtaining solution B from which the cationic impurities had been removed.

[0243] Next, the second step resin is OH －140 mL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, was used. To adsorb the anionic impurities in Solution B and 3SL onto this weakly basic anion exchange resin, Solution B at 10.0°C was passed through the weakly basic anion exchange resin at an SV of 0.9. Subsequently, 3.5 RV of deionized water at 25.0°C was passed through at an SV of 1.0, and neutral substances that did not adsorb onto the weakly basic anion exchange resin were removed from the weakly basic anion exchange resin. The anionic impurities adsorbed onto the weakly basic anion exchange resin were then removed by passing a 0.005 mol / L aqueous NaOH solution at 25.0°C through at 3.0 RV and SV of 1.0. Thereafter, a 0.035 mol / L aqueous NaOH solution at 25.0° C. was passed through at an SV of 1.0, and the 3SL adsorbed on the weakly basic anion exchange resin was eluted as a sodium salt, thereby obtaining solution C.

[0244] Then, in the third step, the solution C obtained in the second step is ＋ The solution was passed through 84 mL of a weakly acidic cation exchange resin, Purolite (registered trademark) C115E (manufactured by Purolite Corporation), to obtain Solution D, the pH of which was adjusted to a range of 2 to 4. Note that Solution C was passed through Purolite (registered trademark) C115E at 25.0°C and an SV of 1.6, assuming that the resin volume of the third column, Purolite (registered trademark) C115E, was 1.

[0245] Then, in the fourth step, the solution D obtained in the third step was subjected to OH treatment at 25.0°C. － The solution was passed through 74 mL of Purolite (registered trademark) A845 / 1967 (manufactured by Purolite Corporation), a weakly basic anion exchange resin, to obtain 1.095 L of eluate (Solution E). Solution D was passed through Purolite (registered trademark) A845 / 1967 at an SV of 1.9 relative to the resin volume of the fourth column, Purolite (registered trademark) A845 / 1967, and the eluate was collected every 0.6 RV to analyze its composition.

[0246] The composition of Solution E obtained in the fourth step was analyzed, and the results are shown in the Example 6 column of Tables 10 and 11.

[0247] Example 7 Purification of 3SL by First to Fourth Steps In Example 7, 0.468 L of solution A obtained in Preparation Example 3 above was used to carry out the treatments of the first to fourth steps.

[0248] In the first step, H ＋ 126 mL of TG-Gel (manufactured by The Dow Chemical Company), a type of strongly acidic cation exchange resin, was used, and solution A at 10.0°C was passed through the TG-gel at an SV of 1.0, causing the cationic impurities in solution A to be adsorbed onto the strongly acidic cation exchange resin, thereby obtaining solution B from which the cationic impurities had been removed.

[0249] Next, the second step resin is OH － 140 mL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, was used. To adsorb the anionic impurities in Solution B and 3SL onto this weakly basic anion exchange resin, Solution B at 10.0°C was passed through the weakly basic anion exchange resin at an SV of 0.9. Subsequently, 3.5 RV of deionized water at 30.0°C was passed through at an SV of 1.0 to wash the resin, thereby removing neutral substances that did not adsorb onto the weakly basic anion exchange resin. Subsequently, the anionic impurities adsorbed onto the weakly basic anion exchange resin were removed by passing a 0.005 mol / L aqueous NaOH solution at 30.0°C through the resin at 3.0 RV and an SV of 1.0. Thereafter, a 0.035 mol / L aqueous NaOH solution at 30.0° C. was passed through at an SV of 1.0, and the 3SL adsorbed on the weakly basic anion exchange resin was eluted as a sodium salt, thereby obtaining solution C.

[0250] Then, in the third step, the solution C obtained in the second step is ＋ The solution was passed through 84 mL of a weakly acidic cation exchange resin, Purolite (registered trademark) C115E (manufactured by Purolite Corporation), to obtain Solution D, the pH of which was adjusted to a range of 2 to 4. Note that Solution C was passed through Purolite (registered trademark) C115E at 30.0°C and an SV of 1.6, assuming that the resin volume of the third column, Purolite (registered trademark) C115E, was 1.

[0251] Then, in the fourth step, the solution D obtained in the third step was heated at 10.0°C with OH. － The solution was passed through 74 mL of Purolite (registered trademark) A845 / 1967 (manufactured by Purolite Corporation), a weakly basic anion exchange resin, to obtain 1.080 L of eluate (Solution E). Solution D was passed through Purolite (registered trademark) A845 / 1967 at an SV of 1.9 relative to the resin volume of the fourth column, Purolite (registered trademark) A845 / 1967, and the eluate was collected every 0.6 RV to analyze its composition.

[0252] The composition of Solution E obtained in the fourth step was analyzed, and the results are shown in the column for Example 7 in Tables 10 and 11.

[0253] [Example 8] <Purification of 3SL by the first to fourth steps> In Example 8, the first to fourth steps were carried out using 0.468 L of solution A obtained in the above [Preparation Example 3].

[0254] In the first step, H ＋ 126 mL of TG-Gel (manufactured by The Dow Chemical Company), a type of strongly acidic cation exchange resin, was used, and solution A at 10.0°C was passed through the TG-gel at an SV of 1.0, causing the cationic impurities in solution A to be adsorbed onto the strongly acidic cation exchange resin, thereby obtaining solution B from which the cationic impurities had been removed.

[0255] Next, the second step resin is OH －140 mL of Purolite® A845 / 1967 (Purolite Corporation), a weakly basic anion exchange resin, was used. To adsorb the anionic impurities in Solution B and 3SL onto this weakly basic anion exchange resin, Solution B at 25.0°C was passed through the weakly basic anion exchange resin at an SV of 0.9. Subsequently, 3.5 RV of deionized water at 25.0°C was passed through at an SV of 1.0 to wash the resin, thereby removing neutral substances that did not adsorb onto the weakly basic anion exchange resin. Subsequently, the anionic impurities adsorbed onto the weakly basic anion exchange resin were removed by passing a 0.005 mol / L aqueous NaOH solution at 25.0°C through the resin at 3.0 RV and an SV of 1.0. Thereafter, a 0.035 mol / L aqueous NaOH solution at 15.0° C. was passed through at an SV of 1.0, and the 3SL adsorbed on the weakly basic anion exchange resin was eluted as a sodium salt, thereby obtaining solution C.

[0256] Then, in the third step, the solution C obtained in the second step is ＋ The solution was passed through 84 mL of a weakly acidic cation exchange resin, Purolite (registered trademark) C115E (manufactured by Purolite Corporation), to obtain Solution D, the pH of which was adjusted to a range of 2 to 4. Solution C was passed through Purolite (registered trademark) C115E at an SV of 1.6 while being cooled to 15.0°C, assuming that the resin volume of the third column, Purolite (registered trademark) C115E, was 1.

[0257] Then, in the fourth step, the solution D obtained in the third step was cooled to 15.0°C while OH was added. － The solution was passed through 74 mL of Purolite (registered trademark) A845 / 1967 (manufactured by Purolite Corporation), a weakly basic anion exchange resin, to obtain 1.150 L of eluate (Solution E). Solution D was passed through Purolite (registered trademark) A845 / 1967 at an SV of 1.9 relative to the resin volume of the fourth column, Purolite (registered trademark) A845 / 1967, and the eluate was collected every 0.6 RV to analyze its composition.

[0258] The composition of Solution E obtained in the fourth step was analyzed, and the results are shown in the column for Example 8 in Tables 10 and 11.

[0259] Table 9 shows the liquid passing temperatures in the first to fourth steps of Examples 5 to 8.

[0260] In Table 10, 3SL [g / L] is the value calculated using formula (1c) in Analysis Example 1-1 above, NeuAc [g / L] is the value calculated using formula (4a) in Analysis Example 4 above, 3'-sialyllactulose [g / L] is the value calculated using formula (11a) in Analysis Example 5 above, and Component V [g / L] is the value calculated using formula (12a) in Analysis Example 6 above.

[0261] The various parameters shown in Table 10 were calculated using the same method as the various parameters shown in Table 3. The various parameters shown in Table 11 were calculated using the same method as the various parameters shown in Table 6.

[0262] In Table 13, acetic acid [ratio to 3SL] is the value calculated by formula (2b) in Analysis Example 2 above, lactose [ratio to 3SL] is the value calculated by formula (3b) in Analysis Example 3 above, NeuAc [ratio to 3SL] is the value calculated by formula (4b) in Analysis Example 4 above, 3'-sialyllactulose [ratio to 3SL] is the value calculated by formula (11b) in Analysis Example 5 above, impurities [ratio to 3SL] detected at RT 27.0 to RT 35.0 are the values ​​calculated by formula (12b) in Analysis Example 6 above, and the impurities [ratio to 3SL] detected by HPLC are the values ​​calculated by formula (13b) in Analysis Example 6 above. is the value calculated by the formula (13b) of the above Analysis Example 1-2, and the total value of other impurities detected by HPLC [ratio to 3SL] is the value calculated by the formula (13c) of the above Analysis Example 1-2, and the total impurities and sodium [ratio to 3SL] are values ​​calculated by the formula (14a) of the above Analysis Example 1-2, and the total impurities and sodium on a wet basis are values ​​calculated by the formula (14b) of the above Analysis Example 1-2, and the 3SL content on a wet basis is the value calculated by the formula (14c) of the above Analysis Example 1-2, and the 3SL content is the value calculated by the formula (14d) of the above Analysis Example 1-2. As shown in Table 13, the 3SL contents of Examples 5 to 8 were all 80% by mass or more. Here, the "3SL content" in Table 13 means the "content of component X per solid content of the fermented product" when component X is 3SL.

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] Thus, it was found that by processing a solution containing sialyllactose through steps 1 to 4, a solution E containing a high concentration of sialyllactose and a low concentration of acetic acid was obtained. Furthermore, it was found that by powdering this solution in step 5, a powdered fermented product containing a high concentration of sialyllactose and a low concentration of acetic acid was obtained. Such fermented products with a low concentration of acetic acid can be said to be highly safe for the human body.

[0269] It is clear that a person skilled in the art can conceive of various modifications and alterations within the scope of the claims, and it is understood that these also naturally fall within the technical scope of the present invention. Furthermore, the components in the above-described embodiments may be combined in any manner without departing from the spirit of the invention.

[0270] This application is based on an international application (PCT / JP2023 / 044738) filed on December 13, 2023, the contents of which are incorporated herein by reference.

Claims

1. A fermented product containing components X, Y and Z, wherein component X is sialic acid or sialylated oligosaccharide, the content of component X in the solid content of the fermented product is 80% by mass or more, component Y is acetic acid, the ratio of the content of component Y to the content of component X in the fermented product (content of component Y / content of component X) is 0.010 or less by mass, component Z is at least one of components Z1 and Z2, and component Z is a component other than component X, component Z1 is a neutral sugar, and component Z2 is an acidic sugar.

2. The fermentation product according to claim 1, wherein the component X is N-acetylneuraminic acid.

3. The fermentation product according to claim 1, wherein the component X is any one compound selected from the group consisting of sialyllactose, sialyllact-N-biose (SLNB), sialyllact-N-acetylglucosamine (SLNac), sialyllact-N-tetraose (LST) and disialyllact-N-tetraose (DSLNT).

4. The fermentation product according to claim 2, wherein the component Z2 is at least one compound selected from the group consisting of glucuronic acid, mannuronic acid and galacturonic acid.

5. The fermentation product according to claim 3, wherein the component Z2 is at least one compound selected from the group consisting of N-acetylneuraminic acid, glucuronic acid, mannuronic acid and galacturonic acid.

6. The fermentation product according to claim 3 or 5, further comprising a component W, said component W being an isomer of component X, said component W being a substance different from said component Z2.

7. The fermentation product according to any one of claims 1 to 6, further comprising a component V, said component V being a substance detected at RT 27.0 to 35.0 when said fermentation product is measured by HPLC-CAD, and said component V being a substance different from said component Z2.

8. The fermented product according to claim 4 or 5, wherein the ratio of the content of each compound of component Z2 to the content of component X in the fermented product (content of component Z2 / content of component X) is 0.02 or less on a mass basis.

9. The fermentation product according to any one of claims 1 to 8, wherein the component Z1 is at least one compound selected from the group consisting of glucose and lactose.

10. The fermented product according to claim 9, wherein the ratio of the content of each compound of component Z1 to the content of component X in the fermented product (content of component Z1 / content of component X) is 0.03 or less on a mass basis.

11. The fermented product according to claim 6, wherein the ratio of the content of each compound of component W to the content of component X in the fermented product (content of component W / content of component X) is 0.0035 or less on a mass basis.

12. The fermented product according to claim 7, wherein the ratio of the total content of the component V to the content of the component X in the fermented product (content of component V / content of component X) is 0.040 or less on a mass basis.

13. A method for producing a fermented product, comprising the following steps 1) to 4) in this order: 1) treating solution A containing components X, Y, and Z with a strongly acidic cation exchange resin to obtain solution B; 2) treating solution B with a weakly basic anion exchange resin to obtain solution C; 3) adjusting the pH of solution C to obtain solution D; and 4) treating solution D with a weakly basic anion exchange resin to obtain solution E, wherein component X is sialic acid or sialylated oligosaccharide, component Y is acetic acid, component Z is at least one of components Z1 and Z2, and component Z is a component other than component X, component Z1 is a neutral sugar, and component Z2 is an acidic sugar, and the ratio of the total mass of component X in solution E to the total mass of component X in solution A (total mass of component X in solution E / total mass of component X in solution A) is 0.5 or more, A method for producing a fermented product, wherein a ratio of a content of the component Y to a content of the component X in the solution E (content of component Y / content of component X) is 0.010 or less on a mass basis.

14. A method for producing a fermented product, further comprising the following 5): 5) powderizing the solution E; the fermented product contains the component X, the component Y and the component Z; the content of the component X per solid content of the fermented product is 80 mass% or more; and the ratio of the content of the component Y to the content of the component X in the fermented product (content of component Y / content of component X) is 0.010 or less by mass. The method for producing a fermented product according to claim 13.

15. A method for producing a fermented product described in claim 13 or 14, wherein the ratio of the total mass of component Y in solution E to the total mass of component Y in solution A (total mass of component Y in solution E / total mass of component Y in solution A) is 0.5 or less.

16. The method for producing a fermentation product according to any one of claims 13 to 15, wherein the component X is N-acetylneuraminic acid.

17. The method for producing a fermentation product according to any one of claims 13 to 15, wherein the component X is any one compound selected from the group consisting of sialyllactose, sialyllact-N-biose (SLNB), sialyllact-N-acetylglucosamine (SLNac), sialyllact-N-tetraose (LST) and disialyllact-N-tetraose (DSLNT).

18. The method for producing a fermentation product according to claim 16, wherein the component Z2 is at least one compound selected from the group consisting of glucuronic acid, mannuronic acid and galacturonic acid.

19. The method for producing a fermentation product according to claim 17, wherein the component Z2 is at least one compound selected from the group consisting of N-acetylneuraminic acid, glucuronic acid, mannuronic acid and galacturonic acid.

20. The method for producing a fermentation product according to claim 17 or 19, wherein the fermentation product further contains a component W, which is an isomer of component X and is a substance different from component Z2.

21. A method for producing a fermented product according to any one of claims 13 to 20, wherein the fermented product further contains a component V, the component V being a substance that is detected at RT 27.0 to 35.0 when the fermented product is measured by HPLC-CAD, and the component V being a substance different from the component Z2.

22. A method for producing a fermented product according to claim 18 or 19, wherein the ratio of the content of each compound of component Z2 to the content of component X in the fermented product (content of component Z2 / content of component X) is 0.02 or less on a mass basis.

23. A method for producing a fermentation product according to any one of claims 13 to 22, wherein the component Z1 is at least one compound selected from the group consisting of glucose and lactose.

24. The method for producing a fermented product according to claim 23, wherein the ratio of the content of each compound of the component Z1 to the content of the component X in the fermented product (content of component Z1 / content of component X) is 0.03 or less on a mass basis.

25. The method for producing a fermented product according to claim 13, wherein the step 2) comprises the following steps 2-1) to 2-3) in this order: 2-1) Passing solution B through a weakly basic anion exchange resin to remove at least a portion of neutral impurities that are not adsorbed to the weakly basic anion exchange resin; 2-2) Eluting at least a portion of the impurities remaining after 2-1) from the weakly basic anion exchange resin; and 2-3) Eluting the acidic compound group including component X and the impurities remaining after 2-2) separately from the weakly basic anion exchange resin.

26. The method for producing a fermented product according to claim 25, wherein the temperature at which the weakly basic anion exchange resin is passed through in 2-2) is 20°C or higher.

27. A method for producing a fermented product according to claim 25 or 26, wherein the temperature at which the weakly basic anion exchange resin is passed through in 2-3) is 20°C or lower.

28. A method for producing a fermented product described in any one of claims 25 to 27, wherein the temperature at which the weakly basic anion exchange resin is passed in 2-1) is 15°C or lower.

29. The method according to claim 25 or 26, wherein the fermented product further contains a component V, the component V being a substance that is detected at RT 27.0 to 35.0 when the fermented product is measured by HPLC-CAD, the component V being a substance different from the component Z2, and the ratio of the total content of the component V to the content of the component X in the fermented product (content of component V / content of component X) is 0.040 or less by mass.

30. The method according to claim 25 or 27, wherein the fermented product further contains component W, which is an isomer of component X and is a substance different from component Z2, and the ratio of the content of each compound of component W to the content of component X in the fermented product (content of component W / content of component X) is 0.0035 or less by mass.

31. The production method according to claim 25, wherein the temperature of the weakly basic anion exchange resin in step 2-1) is 5 to 15°C, the temperature of the weakly basic anion exchange resin in step 2-2) is 20 to 25°C, and the temperature of the weakly basic anion exchange resin in step 2-3) is 8 to 20°C.

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