Method for producing composition containing n-acetylheparosan

Abstract

Provided is a method for producing a composition containing N-acetylheparosan, the method comprising: a step for (1) heating an aqueous composition containing N-acetylheparosan and microbial cells to 35-85°C under conditions in which the pH is 1.5-3.5, or (2) heating the aqueous composition to 65-85°C under conditions in which the pH is 3.5-5.5; and a step for removing at least a portion of the microbial cells from the aqueous composition by solid-liquid separation after the abovementioned step.

Description

Method for producing a composition containing N-acetylheparosan

[0001] The present invention relates to a method for producing a composition containing N-acetylheparosan.

[0002] N-acetylheparosan (hereinafter referred to as "heparosan") is a polysaccharide having a repeating disaccharide structure composed of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). Heparosan is known as a capsular polysaccharide produced by Escherichia coli K5 strain and Pasteurella multicida, among others.

[0003] Heparin is a type of sulfated polysaccharide with heparosan as its backbone and is widely used as an anticoagulant. Industrially, heparin extracted and purified from the intestinal mucosa of pigs has been mainly used. However, since a fatal accident occurred in 2008 due to the contamination of porcine-derived heparin with impurities, there is a need for the production of non-animal-derived heparin with controlled manufacturing processes and quality. As a method for producing non-animal-derived heparin, a method has been reported in which heparosan is deacetylated, isomerized, and sulfated using a combination of chemical and enzymatic methods to obtain heparin with anticoagulant activity. Therefore, heparosan is useful as a starting material when producing heparin. It is known that heparosan can be produced mainly by fermentation using microorganisms, and methods for producing heparosan using Escherichia coli Nissle 1917 strain, Escherichia coli BL21 strain, etc. have been reported (Non-Patent Literature 1, Patent Literature 2).

[0004] Heparosan produced by fermentation is obtained as an aqueous composition containing many insoluble components such as microbial cells, and therefore a solid-liquid separation process is necessary to make it usable in the production of heparin and other products. Known methods for solid-liquid separation of aqueous compositions containing heparosan include filtration (Patent Document 1) and centrifugation (Patent Document 2). Furthermore, a method has been reported to increase the recovery rate of heparosan by heating the aqueous composition containing heparosan at 80 to 121°C as a pretreatment for solid-liquid separation (Patent Document 3).

[0005] Furthermore, a method for purifying human milk oligosaccharides (HMOs) has been reported, which involves efficiently removing biomass from a culture medium containing HMOs by centrifuging it after pretreatment steps such as pH adjustment, dilution, and heating, thereby obtaining a clear supernatant containing HMOs (Patent Document 4).

[0006] Japanese Patent Publication No. 5-271305, International Publication No. 2015 / 050184, Japanese Patent No. 4084099, International Publication No. 2022 / 130324

[0007] Datta et al., “High density fermentation of probiotic E. coli Nissle 1917 towards heparosan production, characterization, and modification”, Applied Microbiology and Biotechnology (2021) 105:1051-1062

[0008] As a result of the inventors' investigation, it was found that the methods described in Patent Documents 1 to 3 have room for improvement in terms of the efficiency of removing E. coli, which is a microbial cell, from the culture medium. In view of the above circumstances, the present invention aims to provide a method for producing a heparosan-containing composition that can remove the above microbial cells from an aqueous composition containing heparosan and microbial cells with high efficiency.

[0009] The present inventors have found that the microbial cells in an aqueous composition containing heparosan and microbial cells can be removed with high efficiency by solid-liquid separation by (1) heating the composition at 35 to 85°C under conditions where the pH is 1.5 to 3.5, or (2) heating the composition at 65 to 85°C under conditions where the pH is 3.5 to 5.5.

[0010] This disclosure provides, for example, the inventions described in [1] to

[14] below: [1] A method for producing a composition containing N-acetylheparosan, comprising the steps of: (1) heating an aqueous composition containing N-acetylheparosan and microbial cells to 35 to 85°C under conditions where the pH is 1.5 to 3.5, or (2) heating it to 65 to 85°C under conditions where the pH is 3.5 to 5.5; and, after the above step, removing at least a portion of the microbial cells from the aqueous composition by solid-liquid separation. [2] The method according to [1], further comprising adding at least one acid selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, citric acid, acetic acid, propionic acid, and butyric acid to the aqueous composition to set the pH to the above conditions. [3] The method according to [1] or [2], wherein the aqueous composition is a culture medium. [4] The method according to any one of [1] to [3], further comprising the step of removing at least a portion of the microbial cells from the aqueous composition by the solid-liquid separation, followed by the step of desalting the aqueous composition using an ultrafiltration membrane, an ion exchange resin, or an ion exchange membrane. [5] The method according to [4], wherein the ultrafiltration membrane comprises at least one selected from the group consisting of porous ceramic, cellulose acetate, cellulose nitrate, regenerated cellulose, porous cellulose, polysulfone, polyacrylonitrile, polyamide, polyvinylidene fluoride, polytetrafluoroethylene, and polyimide as a material. [6] The method according to [4] or [5], wherein the ultrafiltration membrane comprises polysulfone or regenerated cellulose as a material. [7] The fractional molecular weight of the ultrafiltration membrane is 10 × 10 ３ The method according to any one of [4] to [6] below. [8] The method according to any one of [4] to [7] wherein the ultrafiltration membrane contains regenerated cellulose as a material. [9] The composition containing the above N-acetylheparosan produced is an aqueous composition having an N-acetylheparosan content of 1 to 30 g / L based on the above composition, an electrical conductivity of 20 mS / cm or less, and a pH of 0.5 to 3.0, wherein the N-acetylheparosan is 50 × 10 ３ ~300 x 10 ３The method according to any one of [4] to [8] having a weight average molecular weight.

[10] The composition containing the above-mentioned N-acetylheparosan to be produced has a content of the above-mentioned N-acetylheparosan of 1 to 30 g / L, an electrical conductivity of 3.0 mS / cm or less, and a pH of 2.0 to 4.0 based on the above composition, and is an aqueous composition, and the above-mentioned N-acetylheparosan is 50×10 ３ ~300×10 ３ The method according to [9] having a weight average molecular weight.

[11] 50×10 ３ ~300×10 ３ An aqueous composition containing N-acetylheparosan having a weight average molecular weight, having a content of the above-mentioned N-acetylheparosan of 1 to 30 g / L, an electrical conductivity of 20 mS / cm or less, and a pH of 0.5 to 5.0 based on the above aqueous composition.

[12] 50×10 ３ ~300×10 ３ An aqueous composition containing N-acetylheparosan having a weight average molecular weight, having a content of the above-mentioned N-acetylheparosan of 1 to 30 g / L, an electrical conductivity of 3.0 mS / cm or less, and a pH of 2.0 to 4.0 based on the above aqueous composition.

[13] A method for producing a composition containing N-acetylheparosan, comprising a step of heating an aqueous composition containing N-acetylheparosan and microbial cells (1) at 35 to 85°C under the condition that the pH is 1.5 to 3.5, or (2) at 65 to 85°C under the condition that the pH is 3.5 to 5.5.

[14] The method according to

[13] , further comprising a step of removing the microbial cells from the aqueous composition by solid-liquid separation after the above heating step.

[0011] According to the present invention, a method for producing a composition containing heparosan can be provided, which can efficiently remove the microbial cells from an aqueous composition containing heparosan and microbial cells.

[0012] Further, in the above production method, under the condition that the pH of the aqueous composition is 3.5 or less, microbial contamination of the aqueous composition can be suppressed.

[0013] Hereinafter, embodiments of the present disclosure will be described in detail.

[0014] A method for producing a composition containing heparosan according to this embodiment includes the steps of: (1) heating an aqueous composition containing heparosan and microbial cells to 35 to 85°C under conditions where the pH is 1.5 to 3.5, or (2) heating it to 65 to 85°C under conditions where the pH is 3.5 to 5.5 (hereinafter also referred to as the "heating step"), and after the above step, removing the microbial cells from the aqueous composition by solid-liquid separation (hereinafter also referred to as the "solid-liquid separation step").

[0015] In this specification, an aqueous composition containing heparosan and microbial cells before being subjected to the heating process may be referred to as the first composition, an aqueous composition containing heparosan and microbial cells after being subjected to the heating process but before being subjected to solid-liquid separation may be referred to as the second composition, and a composition containing heparosan after being subjected to the solid-liquid separation process may be referred to as the third composition.

[0016] (Aqueous composition containing heparosan and microbial cells: First composition) Heparosan is a polysaccharide consisting of a repeating structure of glucuronic acid and N-acetylglucosamine disaccharides, as shown in the general formula (1) below. In the general formula (1) below, "Ac-" represents an acetyl group.

[0017]

[0018] The first composition comprises heparosan and microbial cells. In this specification, the heparosan content refers to the value determined by gel filtration chromatography (GPC). The gel filtration chromatography is performed under the conditions described later.

[0019] The heparosan contained in the first composition is 250 × 10 ３ ~500 x 10 ３ It typically has a weight-average molecular weight of 150 × 10 ３ ~800 x 10 ３ It may also have a weight-average molecular weight. In this specification, weight-average molecular weight means the value determined by gel filtration chromatography (GPC). The gel filtration chromatography described above is performed under the conditions described later.

[0020] Gel filtration chromatography to determine the heparosan content and weight-average molecular weight of heparosan is performed under the following conditions: The columns are connected in the following order from the pump side: TSKgel guardcolumn PWH (inner diameter 7.5 mm × length 75 mm) and TSKgel G6000PW (inner diameter 7.5 mm × length 300 mm). The column temperature is 30°C. Detection is performed using a differential refractive index detector (RID) with a cell temperature of 30°C. The sample injection volume is 20 μL. A 0.1 mol / L aqueous solution of ammonium acetate is used as the mobile phase, with a flow rate of 0.6 mL / min. The chromatogram of the gel filtration chromatography is recorded using LabSolutions software (Shimadzu Corporation, version 6.117) and analyzed using the GPC reanalysis function of the same software. Here, when determining the heparosan content, a standard solution is used prepared by dissolving Heparin sodium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in purified water to a concentration of 4-5 g / L. When determining the weight-average molecular weight of heparosan, a calibration curve is created using molecular weight standard solutions prepared by dissolving the six types of standard compounds (P-20, P-50, P-100, P-200, P-400, and P-800) contained in STANDARD P-82 (manufactured by Shodex) in purified water to a concentration of 10 mg / mL each.

[0021] As for the microbial cells, those capable of producing heparosan are preferred. The microbial cells capable of producing heparosan are not particularly limited as long as they can produce heparosan, and may be microorganisms that originally have the ability to produce heparosan, or microorganisms that originally did not have the ability to produce heparosan into which the genes necessary for heparosan production have been introduced. Examples of microorganisms that originally have the ability to produce heparosan include Escherichia coli K5 strain (ATCC 23506), Escherichia coli Nissle 1917 strain (DSM 6601), and Pasteurella multocida type D. Microorganisms that do not originally possess heparosan-producing ability are preferably prokaryotes or yeast strains, more preferably prokaryotes belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Kluiveromyces, Trichosporon, Siwaniomyces, Pitia, or Candida, among which Escherichia bacteria are preferred, and Escherichia coli is more preferred. Examples of Escherichia coli include Escherichia coli W3110 strain (ATCC 27325), MG1655 strain (ATCC 47076), BL21 (DE3) strain, Escherichia coli W strain (ATCC 9637), and their derivative strains.The genes necessary for the production of heparosan include combinations of the kfiA, kfiB, kfiC, and kfiD genes, and combinations of the kfiA, tig, kfiC, and kfiD genes (Leroux, Melanie, et al. “Chaperone-assisted expression of KfiC glucuronyltransferase from Escherichia coli K5 leads to heparosan production in Escherichia coli BL21 in absence of the stabilisator KfiB.” Applied microbiology and biotechnology 100 (2016): 10355-10361.), and PmHS1 (Chen, Xiaofei, et al. “Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida, PmHS1.” Bioprocess and biosystems engineering 40 (2017): Examples include 675-681.), PmHS2 (Williams, Asher, et al. “Metabolic engineering of Bacillus megaterium for heparosan biosynthesis using Pasteurella multocida heparosan synthase, PmHS2.” Microbial cell factories 18 (2019): 1-13.). As for microbial cells with heparosan-producing ability, bacteria of the genus Escherichia are preferred, and Escherichia coli is more preferred.

[0022] The content of microbial cells in the first composition is not particularly limited, and may be such that the SSV (Suspended Solid Volume) of the first composition is 60-99%, preferably 75-95%, and more preferably 80-95%. According to the inventors' studies, the SSV can vary greatly depending on the pH and temperature history of the sample being measured, as well as the dilution state. Therefore, the SSV of the first composition mentioned above refers to the value measured in an unadjusted, unheated, and undiluted state.

[0023] In this specification, SSV means the percentage (%) of the volume occupied by the precipitated microbial solids (microbial pellets) relative to the total volume of the aqueous composition. The SSV of the first composition is measured by centrifuging the composition and visually measuring the total volume of the composition and the volume occupied by the microbial solids (microbial pellets). The centrifugation is performed under temperature conditions of 20 to 30°C, a centrifugal force of 4000 g at the radius of rotation, and a centrifugation time of 3 to 20 minutes.

[0024] The first composition is not particularly limited as long as it contains heparosan and microbial cells, but examples include a culture medium, a liquid sample derived from the culture medium, etc., with the culture medium being preferred. Examples of liquid samples derived from the culture medium include a dilution obtained by diluting the culture medium with deionized water, distilled water, etc. When diluting the culture medium, for example, the volume of the dilution can be 1.1 to 10 times, preferably 1.1 to 5 times, more preferably 1.1 to 3 times, and even more preferably 1.5 to 2.5 times, the volume of the culture medium before dilution.

[0025] The heparosan contained in the first composition may be heparosan produced by microbial cells. By culturing the microbial cells in a culture medium, an aqueous composition containing heparosan and microbial cells is obtained. This aqueous composition may be the culture medium described above as the first composition. This aqueous composition may be manufactured, for example, by the method described in Japanese Patent Application Publication No. 2024-60562.

[0026] The culture medium included in the culture solution as the first composition is not particularly limited as long as it can be used to cultivate microbial cells, and examples include, but is not limited to, LB medium (Luria-Bertani medium) and mineral medium (Carbohydrate Research, 2012, 360, 19-24). Furthermore, as the culture medium, for example, a medium containing components selected from a carbon source, nitrogen source, phosphorus source, sulfur source, and other various organic and inorganic components can be used as needed. The types and contents of the culture medium components may be appropriately determined by those skilled in the art.

[0027] Examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, fumaric acid, citric acid, succinic acid, and malic acid; alcohols such as glycerol, crude glycerol, and ethanol; and fatty acids. One carbon source may be used alone, or two or more carbon sources may be used in combination.

[0028] Examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; peptone; organic nitrogen sources such as yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. One nitrogen source may be used alone, or two or more nitrogen sources may be used in combination.

[0029] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. One type of phosphate source may be used alone, or two or more types of phosphate sources may be used in combination.

[0030] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. One sulfur source may be used alone, or two or more sulfur sources may be used in combination.

[0031] Other organic components include, for example, vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and peptones, casamino acids, yeast extracts, and soy protein hydrolysates containing these. These other organic components may be used individually or in combination of two or more.

[0032] Other inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; and trace metals such as iron, manganese, magnesium, and calcium. These other inorganic components may be used individually or in combination of two or more.

[0033] The above culture conditions can be set as appropriate. For example, aerobic culture may be performed by aeration culture, shaking culture, etc. The culture temperature may be, for example, 30 to 37°C. The culture period may be, for example, 16 to 72 hours. The pH of the culture medium is preferably 4.0 to 9.0, more preferably 5.0 to 8.0, and even more preferably 6.0 to 7.5. Furthermore, the culture may be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. Pre-culture may also be performed before the above culture. The conditions for pre-culture can also be set as appropriate. Pre-culture may be performed using the same or a different culture medium as the above culture, or it may be performed using plate medium.

[0034] The first composition may contain an antifoaming agent such as polyalkylene glycol, polyglycol, polyethylene glycol, polypropylene glycol, or polyvinyl alcohol. One antifoaming agent may be used alone, or two or more antifoaming agents may be used in combination. The content of the antifoaming agent in the first composition is not particularly limited, but may be 0.001 to 2% by volume relative to the solution of the first composition.

[0035] The first composition may be diluted with water. When diluting, for example, the volume of the diluted solution may be 1.1 to 10 times, preferably 1.1 to 5 times, more preferably 1.1 to 3 times, and even more preferably 1.5 to 2.5 times, the volume of the original composition.

[0036] (Heating step) In this specification, the heating step means heating the first composition (1) at 35 to 85°C under conditions where the pH is 1.5 to 3.5 (hereinafter also referred to as heating condition 1), or (2) at 65 to 85°C under conditions where the pH is 3.5 to 5.5 (hereinafter also referred to as heating condition 2).

[0037] Under heating condition 1, the pH is 3.5 or lower, but from the viewpoint of more efficiently removing microbial cells, it is preferable that the pH is 3.4 or lower, 3.3 or lower, 3.2 or lower, 3.0 or lower, 2.8 or lower, 2.5 or lower, or 2.3 or lower. Also, the pH is 1.5 or higher, but it may be 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, or 2.0 or higher. These upper and lower limits of pH can be freely combined, but examples of pH conditions include 1.6 to 3.4, 1.8 to 3.2, or 2.0 to 3.0. The heating temperature is 35°C or higher, but from the viewpoint of more efficiently removing microbial cells, it is preferable that the temperature is 35°C or higher, 37°C or higher, 38°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher. The heating temperature is 85°C or lower, but may be 84°C or lower, 83°C or lower, 82°C or lower, 81°C or lower, or 80°C or lower. These upper and lower limits of heating temperature can be freely combined, but examples of heating temperature conditions include 37-84°C, 38-82°C, 40-83°C, or 40-80°C. These pH ranges and heating temperature ranges can be freely combined. For example, under heating condition 1, heating can be performed at 37-84°C under conditions where the pH is 1.6-3.4, 38-82°C under conditions where the pH is 1.8-3.2, 38-83°C under conditions where the pH is 1.8-3.2, or 40-80°C under conditions where the pH is 2.0-3.0. Furthermore, under heating condition 1, (pH, heating temperature) = (1.5-3.5, 35-83), (1.5-3.5, 35-80), (1.5-3.5, 37-85), (1.5-3.5, 37-83), (1.5-3.5, 37-80), (1.5-3.5, 40-85), (1.5-3.5, 40-83), (1.5-3.5, 40-80), (1.5-3.2, 35-85), (1.5 ~3.2, 35-83), (1.5-3.2, 35-80), (1.5-3.2, 37-85), (1.5-3.2, 37-83), (1.5-3.2, 37-80), (1.5-3.2, 40-85), (1.5-3.2, 40-83), (1.5-3.2, 40-80), (1.5-3.0, 35-85), (1.5-3.0, 35-83), (1.5-3.0, 35-80),(1.5-3.0, 37-85), (1.5-3.0, 37-83), (1.5-3.0, 37-80), (1.5-3.0, 40-85), (1.5-3.0, 40-83), (1.5-3.0, 40-80), (1.8-3.5, 35-85), (1.8-3.5, 35-83), (1.8-3.5, 35-80), (1.8-3.5, 37-85), (1.8-3.5, 37-83), (1.8-3.5, 37-80), (1.8-3.5, 40-85), (1.8-3.5, 40-83), (1.8-3.5, 40-80) (1.8-3.2, 35-85), (1.8-3.2, 35-83), (1.8-3.2, 35-80), (1.8-3.2, 37-85), (1.8-3.2, 37-83), (1.8-3.2, 37-80), (1.8-3.2, 40-85), (1.8-3.2, 40-83), (1.8-3.2, 40-80), (1.8-3.0, 35-85), (1.8-3.0, 35-83), (1.8-3.0, 35-80), (1.8-3.0, 37-85), (1.8-3.0, 37-83), (1.8-3.0, 37-80) (1.8-3.0, 40-85), (1.8-3.0, 40-83), (1.8-3.0, 40-80), (2.0-3.5, 35-85), (2.0-3.5, 35-83), (2.0-3.5, 35-80), (2.0-3.5, 37-85), (2.0-3.5, 37-83), (2.0-3.5, 37-80), (2.0-3.5, 40-85), (2.0-3.5, 40-83), (2.0-3.5, 40-80), (2.0-3.2, 35-85), (2.0-3.2, 35-83), (2.0-3.2, 35-80) The following conditions can be adopted: (2.0-3.2, 37-85), (2.0-3.2, 37-83), (2.0-3.2, 37-80), (2.0-3.2, 40-85), (2.0-3.2, 40-83), (2.0-3.2, 40-80), (2.0-3.0, 35-85), (2.0-3.0, 35-83), (2.0-3.0, 35-80), (2.0-3.0, 37-85), (2.0-3.0, 37-83), (2.0-3.0, 37-80), (2.0-3.0, 40-85), or (2.0-3.0, 40-83). In this disclosure, "(pH, heating temperature) = (numerical range A, numerical range B)" meansThis means heating to a temperature range B (°C) under conditions where the pH is within the numerical range A.

[0038] Under heating condition 2, the pH is 5.5 or lower, but from the viewpoint of more efficiently removing microbial cells, it is preferable that the pH is 5.3 or lower, 5.2 or lower, 5.0 or lower, 4.8 or lower, 4.5 or lower, or 4.3 or lower. Also, the pH is 3.5 or higher, but it may be 3.6 or higher, 3.7 or higher, 3.8 or higher, 3.9 or higher, or 4.0 or higher. These upper and lower limits of pH can be freely combined, but conditions such as pH 3.6 to 5.4, 3.8 to 5.2, or 4.0 to 5.0 can be given. The heating temperature is 65°C or higher, but from the viewpoint of more efficiently removing microbial cells, it is preferable that the temperature is 67°C or higher, 68°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher. The heating temperature is 85°C or lower, but it may be 84°C or lower, 83°C or lower, 82°C or lower, 81°C or lower, or 80°C or lower. These upper and lower limits of heating temperature can be freely combined, but examples of heating temperatures include 67-84°C, 68-82°C, 70-83°C, or 70-80°C. These pH ranges and heating temperature ranges can be freely combined. For example, under heating condition 2, heating can be done at 67-84°C under conditions where the pH is 3.6-5.4, 68-82°C under conditions where the pH is 3.8-5.2, 68-83°C under conditions where the pH is 3.8-5.2, 70-80°C under conditions where the pH is 4.0, 70-80°C under conditions where the pH is 4.0, or 80°C under conditions where the pH is 5.0. Furthermore, under heating condition 2, (pH, heating temperature) = (3.5-5.5, 65-85), (3.5-5.5, 65-83), (3.5-5.5, 65-80), (3.5-5.5, 67-85), (3.5-5.5, 67-83), (3.5-5.5, 67-80), (3.5-5.5, 70-85), (3.5-5.5, 70-83), (3.5-5.5, 70-80), (3.5-5.2, 65- 85), (3.5-5.2, 65-83), (3.5-5.2, 65-80), (3.5-5.2, 67-85), (3.5-5.2, 67-83), (3.5-5.2, 67-80), (3.5-5.2, 70-85), (3.5-5.2, 70-83), (3.5-5.2, 70-80), (3.5-5.0, 65-85), (3.5-5.0, 65-83), (3.5-5.0, 65-80),(3.5-5.0, 67-85), (3.5-5.0, 67-83), (3.5-5.0, 67-80), (3.5-5.0, 70-85), (3.5-5.0, 70-83), (3.5-5.0, 70-80), (3.8-5.5, 65-85), (3.8-5.5, 65-83), (3.8-5.5, 65-80), (3.8-5.5, 67-85), (3.8-5.5, 67-83), (3.8-5.5, 67-80), (3.8-5.5, 70-85), (3.8-5.5, 70-83), (3.8-5.5, 70-80) (3.8-5.2, 65-85), (3.8-5.2, 65-83), (3.8-5.2, 65-80), (3.8-5.2, 67-85), (3.8-5.2, 67-83), (3.8-5.2, 67-80), (3.8-5.2, 70-85), (3.8-5.2, 70-83), (3.8-5.2, 70-80), (3.8-5.0, 65-85), (3.8-5.0, 65-83), (3.8-5.0, 65-80), (3.8-5.0, 67-85), (3.8-5.0, 67-83), (3.8-5.0, 67-80) (3.8-5.0, 70-85), (3.8-5.0, 70-83), (3.8-5.0, 70-80), (4.0-5.5, 65-85), (4.0-5.5, 65-83), (4.0-5.5, 65-80), (4.0-5.5, 67-85), (4.0-5.5, 67-83), (4.0-5.5, 67-80), (4.0-5.5, 70-85), (4.0-5.5, 70-83), (4.0-5.5, 70-80), (4.0-5.2, 65-85), (4.0-5.2, 65-83), (4.0-5.2, 65-80) The following conditions can be adopted: (4.0-5.2, 67-85), (4.0-5.2, 67-83), (4.0-5.2, 67-80), (4.0-5.2, 70-85), (4.0-5.2, 70-83), (4.0-5.2, 70-80), (4.0-5.0, 65-85), (4.0-5.0, 65-83), (4.0-5.0, 65-80), (4.0-5.0, 67-85), (4.0-5.0, 67-83), (4.0-5.0, 67-80), (4.0-5.0, 70-85), or (4.0-5.0, 70-83).

[0039] In this specification, the temperature for measuring pH is usually 10 to 40°C, preferably 15 to 35°C, and more preferably 20 to 30°C. In this specification, room temperature is defined as 20 to 30°C. Here, the pH at 20 to 30°C can be measured, for example, by heating the first composition to 20 to 30°C before heating and then measuring it using a known pH measurement method.

[0040] The pH of the first composition can be adjusted by adding an acid to the aqueous composition. The acid may be at least one acid selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, citric acid, acetic acid, propionic acid, and butyric acid, with sulfuric acid, hydrochloric acid, or citric acid being preferred, sulfuric acid or hydrochloric acid being more preferred, and sulfuric acid being most preferred. Another embodiment of the method for adjusting the pH of the first composition is H ＋ A proton-releasing immobilized support, such as a strongly acidic cation exchange resin that has been regenerated in the mold, can also be added.

[0041] In heating conditions 1 and 2, the duration of the heating temperature is preferably 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 7 hours or more, 10 hours or more, 13 hours or more, 15 hours or more, 17 hours or more, 20 hours or more, or 22 hours or more, from the viewpoint of more efficiently removing microbial cells. The heating time may also be 96 hours or less, 84 hours or less, 72 hours or less, 60 hours or less, or 48 hours or less. These upper and lower limits of heating time can be freely combined, but it is preferably 2 to 96 hours, 3 to 96 hours, 4 to 96 hours, 5 to 72 hours, 5 to 48 hours, or 10 to 48 hours. In heating conditions 1 and 2, when the duration of the heating temperature is more than 1 hour, the sedimentation of microbial cells is higher compared to when it is 1 hour or less (for example, 1 hour, 30 minutes, 5 minutes, etc.). This trend is particularly pronounced when the duration is 4 hours or longer, more pronounced when it is 5 hours or longer, even more pronounced when it is 10 hours or longer, and especially pronounced when it is 15 hours or longer. These heating times can be freely combined with the pH range and heating temperature range of heating conditions 1 or 2 described above.

[0042] Under heating condition 1, (pH, heating temperature, heating time) = (1.5-3.5, 35-85, 1-96), (1.5-3.5, 35-85, 1-48), (1.5-3.5, 35-85, 4-96), (1.5-3.5, 35-85, 4-48), (1.5-3.5, 35-80, 1-96), (1.5-3.5, 35-80, 1-48), (1.5-3.5, 35-80, 4-96), (1.5-3.5, 35-80, 4-48), (1.5-3.5, 40-85, 1-96), (1.5-3.5, 40-85, 1-48), (1.5-3.5, 40- 85, 4-96), (1.5-3.5, 40-85, 4-48), (1.5-3.5, 40-80, 1-96), (1.5-3.5, 40-80, 1-48), (1.5-3.5, 40-80, 4-96), (1.5-3.5, 40-80, 4-48), (1.5-3.0, 35-85, 1-96), (1.5-3.0, 35-85, 1-48), (1.5-3.0, 35-85, 4-96), (1.5-3.0, 35-85, 4-48), (1.5-3.0, 35-80, 1-96), (1.5-3.0, 35-80, 1-48), (1.5-3.0 , 35-80, 4-96), (1.5-3.0, 35-80, 4-48), (1.5-3.0, 40-85, 1-96), (1.5-3.0, 40-85, 1-48), (1.5-3.0, 40-85, 4-96), (1.5-3.0, 40-85, 4-48), (1.5-3.0, 40-80, 1-96), (1.5-3.0, 40-80, 1-48), (1.5-3.0, 40-80, 4-96), (1.5-3.0, 40-80, 4-48), (2.0-3.5, 35-85, 1-96), (2.0-3.5, 35-85, 1-48), (2.0 ~3.5, 35~85, 4~96), (2.0~3.5, 35~85, 4~48), (2.0~3.5, 35~80, 1~96), (2.0~3.5, 35~80, 1~48), (2.0~3.5, 35~80, 4~96), (2.0~3.5, 35~80, 4~48), (2.0~3.5, 40~85, 1~96), (2.0~3.5, 40~85, 1~48), (2.0~3.5, 40~85, 4~96), (2.0~3.5, 40~85, 4~48), (2.0~3.5, 40~80, 1~96), (2.0~3.5, 40~80, 1~48),(2.0-3.5, 40-80, 4-96), (2.0-3.5, 40-80, 4-48), (2.0-3.0, 35-85, 1-96), (2.0-3.0, 35-85, 1-48), (2.0-3.0, 35-85, 4-96), (2.0-3.0, 35-85, 4-48), (2.0-3.0, 35-80, 1-96), (2.0-3.0, 35-80, 1-48), (2.0-3.0, 35-80, 4-96), (2.0-3. The following conditions can be adopted: (0, 35-80, 4-48), (2.0-3.0, 40-85, 1-96), (2.0-3.0, 40-85, 1-48), (2.0-3.0, 40-85, 4-96), (2.0-3.0, 40-85, 4-48), (2.0-3.0, 40-80, 1-96), (2.0-3.0, 40-80, 1-48), (2.0-3.0, 40-80, 4-96), or (2.0-3.0, 40-80, 4-48). In this disclosure, "(pH, heating temperature, heating time) = (numerical range A, numerical range B, numerical range C)" means heating at numerical range B (°C) for numerical range C (hours) under the condition that pH is within numerical range A.

[0043] Under heating conditions 2, (pH, heating temperature, heating time) = (3.5-5.5, 65-85, 1-96), (3.5-5.5, 65-85, 1-48), (3.5-5.5, 65-85, 4-96), (3.5-5.5, 65-85, 4-48), (3.5-5.5, 65-80, 1-96), (3.5-5.5, 65-80, 1-48), (3.5-5.5, 65-80, 4-96), (3.5-5.5, 65-80, 4-48), (3.5-5.5, 70-85, 1-96), (3.5-5.5, 70-85, 1-48), (3.5-5.5, 70- 85, 4-96), (3.5-5.5, 70-85, 4-48), (3.5-5.5, 70-80, 1-96), (3.5-5.5, 70-80, 1-48), (3.5-5.5, 70-80, 4-96), (3.5-5.5, 70-80, 4-48), (3.5-5.0, 65-85, 1-96), (3.5-5.0, 65-85, 1-48), (3.5-5.0, 65-85, 4-96), (3.5-5.0, 65-85, 4-48), (3.5-5.0, 65-80, 1-96), (3.5-5.0, 65-80, 1-48), (3.5-5.0 , 65-80, 4-96), (3.5-5.0, 65-80, 4-48), (3.5-5.0, 70-85, 1-96), (3.5-5.0, 70-85, 1-48), (3.5-5.0, 70-85, 4-96), (3.5-5.0, 70-85, 4-48), (3.5-5.0, 70-80, 1-96), (3.5-5.0, 70-80, 1-48), (3.5-5.0, 70-80, 4-96), (3.5-5.0, 70-80, 4-48), (4.0-5.5, 65-85, 1-96), (4.0-5.5, 65-85, 1-48), (4.0 ~5.5, 65~85, 4~96), (4.0~5.5, 65~85, 4~48), (4.0~5.5, 65~80, 1~96), (4.0~5.5, 65~80, 1~48), (4.0~5.5, 65~80, 4~96), (4.0~5.5, 65~80, 4~48), (4.0~5.5, 70~85, 1~96), (4.0~5.5, 70~85, 1~48), (4.0~5.5, 70~85, 4~96), (4.0~5.5, 70~85, 4~48), (4.0~5.5, 70~80, 1~96), (4.0~5.5, 70~80, 1~48),(4.0-5.5, 70-80, 4-96), (4.0-5.5, 70-80, 4-48), (4.0-5.0, 65-85, 1-96), (4.0-5.0, 65-85, 1-48), (4.0-5.0, 65-85, 4-96), (4.0-5.0, 65-85, 4-48), (4.0-5.0, 65-80, 1-96), (4.0-5.0, 65-80, 1-48), (4.0-5.0, 65-80, 4-96), (4.0-5. The following conditions can be adopted: (0, 65-80, 4-48), (4.0-5.0, 70-85, 1-96), (4.0-5.0, 70-85, 1-48), (4.0-5.0, 70-85, 4-96), (4.0-5.0, 70-85, 4-48), (4.0-5.0, 70-80, 1-96), (4.0-5.0, 70-80, 1-48), (4.0-5.0, 70-80, 4-96), or (4.0-5.0, 70-80, 4-48).

[0044] The various conditions described above in the heating process are not limited by the solid-liquid separation method in the solid-liquid separation process described later. However, when centrifugal separation is used as the solid-liquid separation method, the heating conditions are particularly preferably pH 1.5 to 2.5 and 35 to 85°C, or pH 2.5 to 5.5 and 65 to 85°C. Furthermore, when membrane filtration is used as the solid-liquid separation method, there are no particular limitations, but the heating conditions are particularly preferably pH 1.5 to 3.5 and 45 to 75°C.

[0045] During the heating process, an antifoaming agent or the like may be added to the first composition. The addition of the antifoaming agent or the like to the composition may be performed before heating in the heating process. Examples of antifoaming agents include polyglycol, polyethylene glycol, polypropylene glycol, and polyvinyl alcohol. Furthermore, the composition may be stirred during the heating process.

[0046] By subjecting the first composition to a heating process, the sedimentation of the microbial cells is improved, and the second composition can be obtained.

[0047] (Aqueous composition containing heparosan and microbial cells: Second composition) By subjecting the first composition to a heating step, an aqueous composition containing heparosan and microbial cells (second composition) can be obtained in which the sedimentation properties of the microbial cells contained in the composition are improved.

[0048] Improved microbial sedimentation means that the microbial cells tend to settle easily, and when they settle, the volume of the microbial pellet tends to decrease.

[0049] The sedimentation properties of bacterial cells can be evaluated by the percentage (Suppened Solid Volume; hereinafter also referred to as "SSV%") of the volume occupied by the precipitated bacterial solids (bacterial pellets) relative to the volume of the composition. A small SSV% indicates that bacteria tend to precipitate easily, and when precipitated, the volume of the bacterial pellets tends to be small. A large SSV% indicates that bacteria do not precipitate easily in aqueous compositions, and when precipitated, the volume of the bacterial pellets tends to be large.

[0050] In this specification, it can be said that the sedimentation properties of the bacterial cells contained in the composition have improved when the SSV% of the second composition is smaller than the SSV% of the solution obtained by heating the first composition under the same conditions as the second composition except that the pH is 6.4 to 6.6 or 6.4 to 6.8 (hereinafter also referred to as the control solution). For example, it can be said that the sedimentation properties of the bacterial cells contained in the composition have improved by the above method when the value of (SSV%) of the second composition / (SSV%) of the control solution (hereinafter also referred to as the SSV ratio) is 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.85 times or less, 0.8 times or less, 0.7 times or less, 0.5 times or less, 0.3 times or less, or 0.1 times or less.

[0051] The second composition may be diluted with water. When diluting, for example, the volume of the diluted solution may be 1.1 to 10 times, preferably 1.1 to 5 times, more preferably 1.1 to 3 times, and even more preferably 1.5 to 2.5 times, the volume of the original composition.

[0052] (Solid-liquid separation step) In this specification, the solid-liquid separation step refers to the step of obtaining a third composition by removing at least a portion of microbial cells from the second composition by solid-liquid separation.

[0053] According to the method for producing a composition containing heparosan according to this embodiment, the sedimentation of microbial cells is greatly improved by the heating step, so that the microbial cells can be removed from the second composition with high efficiency by the solid-liquid separation step. High efficiency in removing microbial cells may mean that the microbial cells can be removed in a short time, that the microbial cells can be removed from culture broth that is highly viscous and cannot be obtained under neutral conditions or short heating time, that the microbial cells can be removed even under conditions on a kilogram scale which is a production level, or that the microbial cells can be removed under mild conditions such that the target fermentation product is not decomposed.

[0054] In this specification, "removing" microbial cells from an aqueous composition means "removing at least a portion" of the microbial cells from the aqueous composition. According to the method for producing a composition containing heparosan according to this embodiment, it is possible to remove almost all (e.g., 95% or more) or all of the microbial cells contained in the aqueous composition containing heparosan and microbial cells.

[0055] In the method for producing a composition containing heparosan according to this embodiment, the solid-liquid separation method is not particularly limited as long as it can remove microbial cells from the second composition, and examples include centrifugation, membrane filtration, etc., and these may be combined.

[0056] For centrifugation, for example, the temperature may be 4 to 80°C, preferably 10 to 50°C, and more preferably 20 to 30°C; the centrifugal force at the radius of rotation may be 500 to 500,000 g, preferably 1,000 to 100,000 g, and more preferably 1,000 to 5,000 g; and in the case of a batch system, the centrifugation time may be 10 seconds to 30 minutes, preferably 1 to 25 minutes, and more preferably 5 to 20 minutes. Furthermore, centrifugation may be performed once or two or more times.

[0057] Furthermore, centrifugation may be performed using, for example, a disk-type centrifuge, a decanter-type centrifuge, a basket-type centrifuge, etc. As a disk-type centrifuge, a nozzle-type centrifuge may be used. When a nozzle-type centrifuge is used, the supernatant and precipitate are discharged continuously during centrifugation, and the diameter of the nozzle from which the precipitate is discharged may be 0.1 to 1.2 mm, 0.2 to 1.1 mm, or 0.3 to 1.0 mm. In addition, the supply rate of the second composition to the nozzle-type centrifuge may be such that the residence time of the second composition in the nozzle-type centrifuge is the centrifugal time described above.

[0058] For example, when solid-liquid separation is performed by centrifugation, if the bacterial cells settle easily, they can settle in a short time, and the bacterial cells can settle with a small centrifugal force, so it can be said that solid-liquid separation from the second composition can be performed with high efficiency. In addition, the SSV% of the aqueous composition to be separated can be an indicator of the efficiency of solid-liquid separation, and a small SSV% indicates that solid-liquid separation can be performed with high efficiency, while a large SSV% indicates that solid-liquid separation is inefficient.

[0059] The membrane used for membrane filtration is not particularly limited as long as it can block and capture microbial cells and allow heparosan to pass through. For example, a membrane with a pore size of 0.2 to 10 μm or 0.3 to 1 μm may be used, and the molecular weight cutoff is 10 × 10 ３ ~1000 x 10 ３ or 20 x 10 ３ ~300 x 10 ３ A membrane may also be used.

[0060] The above-mentioned membrane may contain inorganic materials or organic polymers as materials. Examples of inorganic materials include porous ceramics, and examples of organic polymers include at least one selected from the group consisting of filter paper, cellulose acetate, cellulose nitrate, regenerated cellulose, porous cellulose, polysulfone, polyacrylonitrile, polyamide, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE) such as Teflon, and polyimide. These materials have excellent durability, such as heat resistance and chemical resistance.

[0061] Membrane filtration may be performed by pressure filtration. The pressure applied in pressure filtration may be 0.05 to 0.3 MPa or 0.07 to 0.2 MPa relative to atmospheric pressure. Pressure filtration may be performed until a sufficient filtrate is obtained, for example, from 30 seconds to 3 hours or from 1 minute to 30 minutes.

[0062] The membrane modules used in membrane filtration may be of the type such as stacked, spiral, tubular, or hollow fiber.

[0063] Membrane filtration may be performed by either cross-flow filtration or dead-end filtration, with dead-end filtration being more preferred. In cross-flow filtration, filtration may be performed using the pressure generated by a circulation pump, and in dead-end filtration, filtration may be performed using the pressure generated by a head difference, reduced pressure, etc.

[0064] When membrane filtration is used in the solid-liquid separation process, if the membrane filtration rate of the second composition is greater than that of the control solution obtained by heating the first composition under the same conditions as the second composition, except that the pH is 6.4 to 6.6, then the microbial cells can be removed with high efficiency from the aqueous composition containing heparosan and microbial cells. For example, by filtering the second composition using a membrane filter (pore size 0.1 μm to 0.45 μm, filter diameter 47 mm, material cellulose mixed ester) at room temperature and 0.1 MPa, it is preferable that the time required to obtain 1 g of filtrate is reduced by 10 minutes or more, and more preferably by 20 minutes or more, compared to filtering the control solution under the same conditions. Furthermore, for example, when the second composition is filtered using a membrane filter (pore size 0.1 μm to 0.45 μm, filter diameter 47 mm, material cellulose mixed ester) at room temperature and 0.1 MPa, if the average membrane filtration rate is 30 mins / g or less, 20 mins / g or less, or 10 mins / g or less, the above method can be said to be able to remove the microbial cells from the aqueous composition containing heparosan and microbial cells with high efficiency.

[0065] Furthermore, when solid-liquid separation is performed by membrane filtration, a smaller SSV% of the aqueous composition being separated allows for more efficient removal of bacterial cells, while a larger SSV% results in less efficient removal of bacterial cells. For example, according to Eiji Iritani, "New Developments in Solid-Liquid Separation Operations of Colloids," Grinding 61 (2017), there is a correlation between the sedimentation properties of fine particles contained in a suspension and the behavior of dead-end filtration. Combining this with the description in Eiji Iritani, "Development of Membrane Filtration Engineering in Particle-Liquid Separation," Membrane 36.5 (2011): 211-216, it is shown that the better the sedimentation properties of the solid components in the solution, the smaller the average cake resistance and the larger the average cake porosity, resulting in improved filterability. From these points, it can be said that in membrane filtration as well, a smaller SSV% allows for more efficient solid-liquid separation.

[0066] In the solid-liquid separation process, the sedimentation of microbial cells is greatly improved by the heating process described above, allowing for highly efficient removal of microbial cells from the second composition. The yield of heparosan in the solid-liquid separation may be 60-100%, preferably 70-99%, and more preferably 80-95%.

[0067] (Composition containing heparosan: Third composition) By subjecting the second composition to a solid-liquid separation step, a composition containing heparosan from which microbial cells have been removed (the third composition) can be obtained. This composition may be a heparosan-containing composition produced by the manufacturing method according to this embodiment.

[0068] The third composition contains heparosan, and the above composition may have a heparosan content of 1 to 30 g / L or 2 to 15 g / L based on the above composition.

[0069] The heparosan contained in the third composition is 20 x 10 ３ ~600 x 10 ３ , or 50 x 10 ３ ~500 x 10 ３ It may have a weight-average molecular weight of 100 × 10, and from the viewpoint of improving the efficiency when desalting using a membrane, ３ ~400 x 10３ , or 127 x 10 ３ ~300 x 10 ３ It is preferable that it has a weight-average molecular weight.

[0070] (Desalting step) The method for producing a composition containing heparosan according to this embodiment may further include a step of desalting the third composition using an ultrafiltration membrane, an ion exchange membrane, or an ion exchange resin after the solid-liquid separation step (desalting step). In this specification, desalting means removing at least a portion of the inorganic salts or low-molecular-weight organic salts contained in the target.

[0071] In this specification, a composition containing heparosan after being subjected to the desalting process may also be referred to as the fourth composition.

[0072] Before subjecting the third composition to the desalination process described above, it may be microfiltered. The membrane used for microfiltration is not particularly limited as long as it is a membrane that can allow heparosan to pass through, and for example, a membrane with a pore size of 0.1 to 10 μm may be used. The temperature conditions for the above membrane filtration (microfiltration) may be, for example, 4 to 80°C. If the third composition contains an antifoaming agent, it may have a cloud point of 50°C or lower. The antifoaming agent can be removed by heating to a temperature above the cloud point and performing microfiltration. The temperature conditions for the above membrane filtration (microfiltration) may be 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, or 75°C or higher, from the viewpoint of removing the antifoaming agent when the third composition contains one.

[0073] The above-mentioned microfiltration membrane can have residual contaminants removed by chemical washing after being subjected to the desalination process. The chemical washing method is not particularly limited as long as it can remove residual contaminants. For example, the membrane can be washed with water while supplying water to a circulation tank and allowing it to overflow for 20 to 120 minutes at room temperature. Next, a chemical solution can be added to the circulation tank and circulation filtration can be performed for 30 to 120 minutes at room temperature. After that, residual contaminants can be removed by washing with water for 15 to 120 minutes at room temperature while supplying water to the circulation tank. The module inlet pressure at this time is 60 kPa or less, and the module outlet pressure is 40 kPa or less. As the chemical solution, an aqueous solution containing 1 to 4% NaOH and 1000 to 4000 ppm NaClO can be used.

[0074] Before subjecting the third composition to the desalting step described above, heparosan may be purified by precipitation using an organic solvent. An example of a precipitation method using an organic solvent is to add an organic solvent or a mixture of an organic solvent and water to the third composition to precipitate heparosan. Examples of organic solvents include ethanol, propanol, methanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, etc., with ethanol or propanol being preferred. The amount of organic solvent added may be 2.5 to 3.5 parts by volume per 1 part by volume of the third composition.

[0075] Desalting using an ultrafiltration membrane is performed by blocking or capturing heparosan in the ultrafiltration membrane, allowing inorganic salts and low-molecular-weight organic salts to pass through. The above desalting may be performed, for example, by subjecting the third composition to ultrafiltration using an ultrafiltration membrane to remove inorganic salts and low-molecular-weight organic salts and obtain a concentrated heparosan solution, adding deionized water, purified water, etc. to the obtained concentrate, and then removing inorganic salts and low-molecular-weight organic salts and concentrating heparosan again by ultrafiltration using an ultrafiltration membrane, or by further repeating ultrafiltration and adding water such as deionized water or purified water. Desalting using an ultrafiltration membrane may be performed under conditions where the temperature of the target composition is between 4 and 80°C.

[0076] The molecular weight cutoff of the ultrafiltration membrane is 50 × 10 ３ Below, 30 x 10３ Below, 10 x 10 ３ Below, 7 x 10 ３ Below, 5 x 10 ３ The following, or 3 x 10 ３ The following is preferable: 1 × 10 ３ The above is 2 x 10 ３ The values ​​may be greater than or equal to these limits. These upper and lower limits can be combined in any way. If the molecular weight cutoff of the ultrafiltration membrane is within the above-mentioned range, low molecular weight compounds such as carbon sources, nitrogen sources, antifoaming agents, and heparosan by-products can also pass through the ultrafiltration membrane, in addition to inorganic salts and low molecular weight organic salts.

[0077] The ultrafiltration membrane may contain inorganic or organic polymers as materials. Examples of inorganic materials include porous ceramics, and examples of organic polymers include at least one selected from the group consisting of cellulose acetate, cellulose nitrate, regenerated cellulose, porous cellulose, polysulfone, polyacrylonitrile, polyamide, polyvinylidene fluoride, polytetrafluoroethylene such as Teflon, and polyimide. From the viewpoint of increasing the yield of heparosan in desalting, it is preferable that the ultrafiltration membrane contains regenerated cellulose as a material. When the ultrafiltration membrane contains regenerated cellulose as a material, the yield of heparosan in desalting can be 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%. In this case, the pH of the third composition is not particularly limited, but for example, the pH at 25°C may be 0.5 to 12.5.

[0078] Specifically, the ultrafiltration membranes used include Microza's AIP-0013D, ACP-0013D, ACP-0053D, AHP-0013D, SEP-0013, SAP-0013, SIP-0013, SLP-0053; Alfa Laval's RC10PE-3838 / 48, RC-10PE 2517 / 48; and Sartorius's Zartocon or Zartocube (molecular weight cutoff: 1 × 10⁻¹⁰). ３ , 2 x 10 ３ , 5 x 10 ３ , 10 x 10 ３ , 30 x 10 ３ , 50 x 10 ３), Merck's Pericon XL or Pericon 2 (fractionated molecular weight: 5 × 10 ３ , 8 x 10 ３ , 10 x 10 ３ , 30 x 10 ３ , 50 x 10 ３ ), Pericon 3 (fractionated molecular weight: 3 x 10) ３ , 5 x 10 ３ , 10 x 10 ３ , 30 x 10 ３ ), Delta membrane manufactured by Cytiva (fractionated molecular weight: 10 x 10 ３ , 30 x 10 ３ ), omega membrane (fractionated molecular weight: 1 x 10) ３ , 5 x 10 ３ , 10 x 10 ３ , 30 x 10 ３ , 50 x 10 ３ Examples include the following, with SEP-0013 or RC10PE-3838 / 48 being preferred.

[0079] Desalting using an ultrafiltration membrane is preferably carried out under conditions where the pH of the third composition at 25°C is 2.0 or higher, or 2.5 or higher, from the viewpoint of increasing the yield of heparosan in desalting. Desalting may also be carried out under conditions where the pH of the third composition at 25°C is 14 or lower, 13 or lower, or 12 or lower. These upper and lower limits can be arbitrarily combined. When desalting is carried out under conditions where the pH of the third composition at 25°C is 9 or higher, or 10 or higher, the yield of heparosan in desalting can be 90% or higher. In this case, the molecular weight cutoff of the ultrafiltration membrane is 10 × 10 ３ , 5 x 10 ３ The following, or 3 x 10 ３ The following is preferable, and the ultrafiltration membrane preferably contains polysulfone or regenerated cellulose as a material. The pH of the third composition at 25°C can be adjusted by adding sodium hydroxide or the like to the third composition. If the pH of the third composition at 25°C is adjusted to 9 or higher, or 10 or higher, precipitation may occur. In this case, the precipitate can be removed by vacuum filtration using filter paper with a pore size of 0.1 to 2 μm.

[0080] The membrane module used for desalting using an ultrafiltration membrane may be of the stacked, spiral, tubular, or hollow fiber type.

[0081] Membrane filtration may be performed by cross-flow filtration or by dead-end filtration. Cross-flow filtration is advantageous because it allows for membrane filtration while removing contaminants from the membrane through the flow of the target composition. In cross-flow filtration, filtration may be performed using the pressure generated by a circulation pump, while in dead-end filtration, filtration may be performed using the pressure generated by a head difference, reduced pressure, etc. Examples of dead-end filtration methods include pressurized filter presses and vacuum-operated continuous vacuum rotary filters. When performing dead-end filtration, pre-coating or body feeding with auxiliary agents may be performed as appropriate. Examples of filtration aids include diatomaceous earth, perlite, cellulose, carbon, acid clay, bentonite, and activated carbon.

[0082] The ultrafiltration membrane described above can be subjected to a desalination process and then washed with a chemical solution to remove residual contaminants. The method of chemical washing is not particularly limited as long as it can remove residual contaminants. For example, the membrane can be washed with water at 5 to 40°C for 10 to 120 minutes while overflowing, while supplying water to a circulation tank. Next, a chemical solution can be added to the circulation tank and circulating filtration can be performed at 10 to 55°C for 30 to 120 minutes. After that, residual contaminants can be removed by washing with water at 5 to 40°C for 30 to 120 minutes while supplying water to the circulation tank, or by washing with water until the electrical conductivity of the circulating liquid and permeate is undetectable. As the chemical solution, for example, an aqueous solution containing 2 to 4% NaOH and 1000 to 4000 ppm NaClO, or an aqueous NaOH solution with a pH of 10.5 to 11.5 can be used. Furthermore, if the amount of permeate does not recover sufficiently, UltraZeal 120 (manufactured by Ecolab), UltraZeal 620 (manufactured by Ecolab), and sodium dodecyl sulfate can be used to further enhance the washing.

[0083] Desalting using an ion exchange resin is performed by capturing inorganic salts or low-molecular-weight organic salts with the ion exchange resin and passing them through heparosan. Examples of ion exchange resins include strongly acidic cation exchange resins, combinations of strongly acidic cation exchange resins and strongly basic anion exchange resins, and combinations of strongly acidic cation exchange resins and weakly basic anion exchange resins. In desalting using an ion exchange resin, it is preferable to pass the third composition through a strongly acidic cation exchange resin, and more preferably to pass it through a strongly basic anion exchange resin or a weakly basic anion exchange resin after passing it through a strongly acidic cation exchange resin.

[0084] Examples of strongly acidic cation exchange resins include those having sulfonic acid groups as exchange groups, and whose resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[0085] The ionic form of the sulfonic acid group of the exchange group in the strongly acidic cation exchange resin is not particularly limited, for example, H ＋ Type, Na ＋ Type, K ＋ Type and NH ４ ＋ Examples include H ＋ It is preferable that it be a mold.

[0086] Examples of strongly acidic cation exchange resins include, specifically, Dow Chemical's DOWEX 88, DOWEX 88MB, DOWEX Monosphere 88, TG-Gel (also known as XUS40232-01), and DuPont's Amberlite (e.g., FPC16UPS Na, FPC88MB Na, FPC240H, CR3220 Ca, CR1310 Ca,Na, CR1360) Na, CR99K / 350, HPR1100Na, etc.), DuPont's Dowex Marathon C, Purolite's C100, C100E, C120E, C100x10, C100x16MBH, C145S, C150, C160, SGC650, Purolite's Purolite SST series (e.g., SSTC60, SSTC60H, SSTC80C, etc.), Mitsubishi Chemical's Diaion SK series (e.g., SK1B, SK1BH, SK1BL, SK1BLH, SKL10, SKT10L, SK104, SK110, SKT110, SKT110L, SK110L, SK112, SK112L, SK116, SKT20L, etc.), Mitsubishi Chemical's Diaion PK series (for example, PK208, PK208LH, PK212, PK212L, PK212LH, PK216, PK216L, PK216H, PK216LH, PK220, PK220L, PK228, PK228L, PK228LH, etc.), Mitsubishi Chemical's Diaion RCP series (e.g., RCP145H, RCP160M, etc.), Mitsubishi Chemical's Diaion HPK25, Mitsubishi Chemical's Diaion UBK series (e.g., UBK16, UBK14, UBK12, UBK10, UBK10H, UBK10HUP, UBK08, UBK08A, UBK08H, UBK08HUP, UBK04, UBK02, UBKN Examples include 1U, UBKN1UMB, UBK522M, UBK530, UBK530J, UBK530K, UBK535, UBK535J, UBK535K, UBK535L, UBK550, UBK555, etc.), Mitsubishi Chemical's Rewrite JC series (e.g., JC600, JC603, etc.), LANXESS's RevaChit S1668, and LANXESS's RevaChit MonoPlus series (e.g., S108, S108H, SP112, etc.).

[0087] Examples of strongly basic anion exchange resins include those having either a quaternary ammonium of type I, which has a trimethylammonium group or a triethylammonium group as an exchange group, or a type II, which has a dimethylethanolammonium group, and the resin matrix being porous, macroporous, gel, styrene, or acrylic.

[0088] The ionic form of the quaternary ammonium exchange group in a strongly basic anion exchange resin is not particularly limited, and for example, the hydroxide ion type (OH) － (Cl) － (SO type), sulfuric acid type (SO ４ ２－ (PO type), phosphate type (PO ４ ３－ type), nitric acid type (NO ３ － Examples include either the hydroxide ion type (OH) or a state in which an organic acid used as an eluent is bound, and － It is preferable that it be of the type.

[0089] Examples of strongly basic anion exchange resins include, specifically, Dow Chemical's 1x2, 1x4, 1x8, 22, MSA-2, and DuPont's Amberlite (e.g., HPR4700 Cl, HPR4700 OH, FPA400J Cl, IRA404J Cl, FPA420 OH, FPA900UPS Cl, HPR4580 Cl, SCAV4 Cl, FPA410J Cl, IRA411 Cl, FPA22UPS Cl, HPR4780 Cl, IRA400J Cl, IRA402BL Cl, IRA900J Cl, HPR4002 Cl, IRA410J Cl, IRA910CT Cl, HPR4010 Cl, HPR4100 Cl, HPR9200, Cl, HPR550, Cl, HPR550, OH, etc.), Purolite A400, A600, SGA550, A200, A300, A500, A501P, A502PS, A503, A510, A520E, A850, A860, A870, PFA520E, Purolite SST series (e.g., SSTA63, SSTA64), Mitsubishi Chemical Diaion PA series (e.g., PA3 06S, PA308, PA308L, PA312, PA312L, PA312LOH, PA312LTU, PA312LTUMB, PA316, PA316L, PA318L, PA318 LOH, PA408, PA412, PA412M, PA418, PA418L, PA418LL, PAF308L, HPA25L, HPA25M, HPA512L, HPA716, etc.), Mitsubishi Diaion NSA100, UMA130J from Chemical Co., Ltd., and Diaion SA series from Mitsubishi Chemical Corporation (e.g., SA10A, SA10AL, SA10ALLP, SA10AOH, SA10AP, SA10DL, SA11A, SA11AL, SA12A, SA12AL, SA12ALL, SA20A, SA20ALL, SA20ALLP, SA20AP, SA20AP2, S AF11AL, SANUPB, SAT10L, SAT20L, etc.), Mitsubishi Chemical's Diaion UBA series (e.g., UBA100, UBA100OH, UBA100OHUP, UBA120, UBA120A, UBA120OH, UBA120OHUP, UBA150, UBA200, etc.), Mitsubishi Chemical's Rewrite JA series (e.g., JA100, JA200,Examples include the JA400, JA420, JA450, etc., and the Lanxess LevaChit MonoPlus series (e.g., M500, M800, MP800, M600, MP600, etc.).

[0090] Examples of weakly basic anion exchange resins include those having primary to tertiary amino groups or polyamine groups as exchange groups, and whose resin matrix is ​​porous, macroporous, gel, styrene, or acrylic.

[0091] The ionic form of the amino group of the exchange group in a weakly basic anion exchange resin is not particularly limited, and for example, the hydroxide ion type (OH) － (Cl) － (SO type), sulfuric acid type (SO ４ ２－ (PO type), phosphate type (PO ４ ３－ type), nitric acid type (NO ３ － Examples include the hydroxide ion type (OH), the free form, and the state in which an organic acid used as an eluent is bound, and － It is preferable that it be in a molded or free form.

[0092] Examples of weakly basic anion exchange resins include the Dow Chemical Company's DowX Monosphere series (e.g., Monosphere 77) and DuPont's Amberlite (e.g., FPA95, FPA77UP, XE583GF, FPA53, HPR4780). Cl, IRA67, IRA96SB, IRA98, FPA95, FPA77UPS, FPA53, etc.), Purolite A100, A103S, A110, A111S, A133S, A830, A830W, A845, A847, Mitsubishi Chemical Diaion WA series (e.g., WA10, WA20, WA21J, WA30, WA30C, WA30LL, WA55, etc.), Mitsubishi Chemical Sepabees FPDA13, Mitsubishi Chemical Rewrite JA series (e.g., JA300, JA310, JA450, JA830, etc.), Lanxess Levatit MP62WS, Lanxess Levatit Monoplus MP64, Lanxess Levatit VP OC 1065 can be listed.

[0093] Desalting using ion exchange resins can be performed, for example, by packing a strongly acidic cation exchange resin and a strongly basic anion exchange resin or a weakly basic anion exchange resin into separate columns and passing the third composition through these columns.

[0094] In this disclosure, the flow rate when the third composition is passed through the ion exchange resin is defined by space velocity (the volume ratio of the solution passed through per hour when the volume of the stationary carrier is set to 1, hereinafter referred to as "SV").

[0095] When passing the third composition through the column, 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, and the temperature is 1 to 60°C, 1 to 45°C, or 1 to 30°C. The pH of the third composition may be 1 to 14 or 2 to 4.5. When desalting using an ion exchange resin is performed under these conditions, the yield of heparosan in desalting can be 80% or more.

[0096] Desalination using an ion exchange membrane can be carried out, for example, by electrodialysis. Electrodialysis is not particularly limited and can be carried out by conventional methods.

[0097] (Aqueous composition containing heparosan after the desalting process: Fourth composition) A composition containing desalted heparosan (the fourth composition) can be obtained by the desalting process. This composition may be a composition containing heparosan produced by the manufacturing method according to this embodiment.

[0098] The fourth composition contains heparosan, and the above composition may have a heparosan content of 1 to 30 g / L or 2 to 25 g / L based on the above composition.

[0099] The fourth composition may have an electrical conductivity of 20 mS / cm or less, 10 mS / cm or less, 5.0 mS / cm or less, 3.0 mS / cm or less, 2.5 mS / cm or less, 2.0 mS / cm or less, 1.5 mS / cm or less, or 1.0 mS / cm or less, and may also have an electrical conductivity of 0.05 mS / cm or more, 0.1 mS / cm or more, or 0.5 mS / cm or more. The upper and lower limits of these electrical conductivities can be freely combined. In the desalination step, by performing sufficient desalination, the fourth composition can have an electrical conductivity of 3.0 mS / cm or less.

[0100] In this specification, the electrical conductivity means the value measured by a compact electrical conductivity (EC) meter LAQUAtwin manufactured by Horiba, Ltd., a desktop pH / electrical conductivity meter F-74, a desktop electrical conductivity meter SD-72, etc.

[0101] The fourth composition may have a pH of 0.5 to 5.0 or 2.0 to 4.0.

[0102] The heparosan contained in the fourth composition is 50×10 ３ to 300×10 ３ 、100×10 ３ to 300×10 ３ 、100×10 ３ to 250×10 ３ 、or 150×10 ３ to 250×10 ３ and may have a weight average molecular weight.

[0103] The fourth composition may be an aqueous composition. Examples of the medium of the aqueous composition include water.

[0104] The fourth composition may further contain an antifoaming agent such as polyglycol, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, etc.

[0105] Examples are given below to explain the present invention in more detail, but the present invention is not limited only to these examples.

[0106] [Analysis of Heparosan] <Measurement of Heparosan Content> In the examples shown below, unless otherwise specified, the heparosan content in each aqueous composition was measured by gel filtration chromatography using the following method. The columns were connected in the following order from the pump side: TSKgel guardcolumn PWH (inner diameter 7.5 mm × length 75 mm) and TSKgel G6000PW (inner diameter 7.5 mm × length 300 mm), and the column temperature was set to 30°C. Detection was performed using a differential refractive index detector (RID), and the cell temperature was set to 30°C. The sample injection volume was 20 μL. A 0.1 mol / L aqueous solution of ammonium acetate was used as the mobile phase, and the flow rate was 0.6 mL / min. The chromatograms of the gel filtration chromatography were recorded using LabSolutions software (Shimadzu Corporation, version 6.117) and analyzed using the GPC reanalysis function of the same software. For the standard, we used heparin sodium reagent (Heparin Sodium, Special Grade, Lot. LEG5091, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in purified water at a concentration of 4-5 g / L. The water content of the above heparin sodium reagent was determined to be 15.35% by mass, as described later in <Measurement of Water Content of Heparin Sodium Reagent>. Therefore, the heparin sodium concentration of the standard solution, based on the powder weighing value of the heparin sodium reagent, was multiplied by 0.8465 to convert it to dry weight, and the heparosan concentration was calculated as the heparin sodium concentration from the area value of the major peak that eluted during the retention time of 17-22 minutes in the chromatogram obtained above.

[0107] <Measurement of Weight-Average Molecular Weight of Heparosan> In the examples shown below, unless otherwise specified, the weight-average molecular weight of heparosan in each aqueous composition was measured by gel filtration chromatography using the following method. The analysis by gel filtration chromatography was performed under the same conditions as in <Measurement of Heparosan Content> above, except that the weight-average molecular weight was determined using STANDARD P-82 (Shodex), a molecular weight standard, instead of determining the heparosan content using the standard solution described above. Calibration curves were created using molecular weight standard solutions prepared by dissolving the six types of standard (P-20, P-50, P-100, P-200, P-400, and P-800) contained in STANDARD P-82 in purified water at a concentration of 10 mg / mL, and the weight-average molecular weight was calculated.

[0108] <Measurement of Water Content of Heparin Sodium Reagent> The water content of the above heparin sodium reagent was measured using an automated water content analyzer AQV-2260S (manufactured by HIRANUMA Corporation) equipped with an AUTO SOLID EVAPORATOR EV-2010. The EV file settings, blank measurement, and sample measurement conditions were as follows, and the measurement was performed according to the instruction manual. [EV File] Step 1 Temperature 200°C, Time 0 min BG stabilization wait times: 60 Pre-purge mode: OFF Pre-purge wait time: 10 seconds Blank volume correction: OFF Sample density: 1.00000 g / cm³ Gas flow rate: 105-205 mL / min [Blank Measurement] Calculation formula: Automatic input of blank measurement value Wait time: 30 seconds Electrolytic current: MEDIUM S. Timer: 0 minutes Blank auto input: ON BG auto correction: ON Minimum electrolysis amount: 5 μg Electrolytic cell: Two chambers [Sample measurement] Waiting time: 30 seconds Electrolytic current: MEDIUM S. Timer: 0 minutes Moisture content unit: Auto BG auto correction: ON Auto interval: 0.0000 mg Minimum electrolysis amount: 5 μg Electrolytic cell: Two chambers

[0109] [Preparation of Culture Medium Containing Heparosan and Microbial Cells] In the following examples, a culture medium containing heparosan was prepared by introducing the heparosan synthesis gene group kfiA, kfiC, and kfiD, as well as the glmS gene involved in heparosan precursor supply, into an Escherichia coli strain, with reference to the description in Japanese Patent Publication No. 2024-60562. This Escherichia coli strain was cultured with reference to the culture conditions described in WO2021 / 201281A1 to obtain a culture medium containing heparosan and microbial cells.

[0110] [SSV% Measurement Method] In the examples shown below, unless otherwise specified, the SSV% was measured by the following method. First, the target composition was subjected to a spin test. For centrifugation in the spin test, an AX310 manufactured by Tommy Industries Co., Ltd. was used, and the target composition was placed on a TS-33 swing rotor and centrifugated at 25°C and 4,000 g for 10 minutes. After centrifugation, the percentage (%) of the volume occupied by the precipitated microbial solids (microbial pellets) relative to the total volume of the target composition (Suspended Solid Volume; hereinafter also referred to as "SSV%") was calculated.

[0111] [Example 1: Evaluation of the effect of adjusting the pH of the culture medium to 3 before or after heating on the SSV%] The culture medium obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above was divided into two parts, which were designated as Sample 1 and Sample 2. Sample 1 was heated to 70°C while stirring and held for 10 hours. After that, it was cooled to below 30°C and the pH at 25°C was adjusted to 3.0 by adding 96% sulfuric acid. On the other hand, the pH at 25°C of Sample 2 was adjusted to 3.0 by adding 96% sulfuric acid. After that, it was heated to 70°C while stirring and held for 10 hours and then cooled to below 30°C. The SSV% of Samples 1 and 2, which underwent heating and acidification, was calculated according to the method described in [SSV% measurement method] above.

[0112] As a result, sample 1, which was acidified after heating, had an SSV% of 88.2%, while sample 2, which was heated after acidification, had an SSV% of 61.8%, indicating that heating after acidification was effective in reducing the SSV%. Therefore, it was shown that heating a culture solution containing heparosan under acidic conditions such as pH 3 is effective in reducing the SSV%.

[0113] [Comparative Example 1: Evaluation of SSV% when culture medium is heated at 70°C and 122°C without pH adjustment] Sample 3 (unadjusted pH (pH 6.37 at 27°C)) and Sample 4 (unadjusted pH (pH 6.64 at 28.3°C)) were obtained using the method described in [Preparation of culture medium containing heparosan and microbial cells] above. Sample 3 was heated to 70°C while stirring and held for 24 hours, and Sample 4 was autoclaved at 122°C for 20 minutes, after which both were cooled to below 30°C. The SSV% of these samples was determined according to the [SSV% measurement method] above.

[0114] As a result, the SSV% of sample 3 was 85.0%, and the SSV% of sample 4 was 87.6%. These SSV%s were higher than those of sample 2. Therefore, it was shown that heating the culture medium to 70°C under acidic conditions such as pH 3 is more effective in improving the efficiency of solid-liquid separation than heating to 70°C or 122°C without adjusting the pH from pH 6.37 or pH 6.64.

[0115] [Example 2: Evaluation of the effect of adjusting the culture medium to pH 2.0 and then heating it at a temperature of 40-80°C on SSV%] Samples 9-13, which were culture media obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above, were each prepared by adding 96% by mass sulfuric acid to adjust the pH to 2.0 at room temperature. Next, 10 mL each of the unadjusted pH samples 5-8 and the pH-adjusted pH samples 9-13, obtained by the same method as above, were heated at 40, 50, 60, 70, or 80°C for 24 hours. After heating, each test solution was cooled to 30°C or below, and the SSV% was determined according to the [SSV% measurement method] above. The SSV% of samples 9-13 obtained were used as the SSV of the second composition, and the SSV% of samples 5-8, which were heated to each temperature without pH adjustment, were used as the SSV of the control solution. The results of calculating the SSV% of the second composition / SSV% of the control solution (SSV ratio) are shown in Table 1.

[0116] As shown in Table 1, the test solutions (Samples 9-13) prepared by heating a pH-adjusted solution at 40°C to 80°C showed lower SSV% values ​​at all temperature conditions compared to Samples 5-8, which were heated to the respective temperatures without pH adjustment. Furthermore, the SSV ratio was 0.90 or less in all cases. From these results, it was demonstrated that solid-liquid separation can be performed with high efficiency by heating a pH-adjusted solution at 40°C to 80°C.

[0117]

[0118] [Example 3 Evaluation of the effect of adjusting the culture medium to pH 3, 4, or 5 and then heating it at 80°C on SSV%] Samples 14, 15, and 16, which were culture mediums obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above, were each prepared by adding 96% by mass sulfuric acid to adjust the pH at 23-30°C to 3.0, 4.0, or 5.0. Next, 10 mL each of the unadjusted pH sample 8 and the pH-adjusted samples 14-16, obtained by the same method as the above culture, were heated to 80°C and incubated for 24 hours. After incubation, each test solution was cooled to 30°C or below, and the SSV% was determined according to the [SSV% measurement method] above. The SSV% of the obtained samples 14-16 was used as the SSV of the second composition, and the SSV% of sample 8 from [Example 2: Evaluation of the effect of adjusting the culture medium to pH 2.0 and then heating it at a temperature of 40-80°C], which was 91.3%, was used as the SSV of the control solution. The results of calculating the SSV of the second composition / SSV of the control solution (SSV ratio) are shown in Table 2.

[0119] As shown in Table 2, the SSV% was lower than that of Sample 8 obtained in Example 2 at all pH levels from 3.0 to 5.0. Furthermore, the SSV ratio was 0.90 or less in all cases. From the above, it was shown that solid-liquid separation can be performed with high efficiency by heating a solution adjusted to pH 3.0 to 5.0 at 80°C.

[0120]

[0121] [Example 4 Evaluation of the effect of heating the culture solution at 70°C after adjusting the pH to 3 or 4] Test solutions were obtained by adding 96% by mass sulfuric acid to samples 17 and 18, which were culture solutions obtained by the method described in [Preparation of culture solution containing heparosan and microbial cells] above, to adjust the pH at 23-30°C to 3.0 or 4.0. Next, 10 mL each of the test solutions of sample 7 (unadjusted pH) and the pH-adjusted samples 17 and 18, obtained by the same method as the culture described above, were heated to 70°C and kept warm for 24 hours. After warming, each test solution was cooled to 30°C or below, and the SSV% was determined according to the [SSV% measurement method] above. The SSV% of the obtained samples 17 and 18 were used as the SSV of the second composition, and the SSV% of sample 7 obtained in Example 2 was used as the SSV of the control solution. The results of calculating the SSV of the second composition / SSV of the control solution (SSV ratio) are shown in Table 3.

[0122] As shown in Table 3, the SSV% was lower at at least pH 3.0 or 4.0 compared to Sample 7 obtained in Example 2. Furthermore, the SSV ratio was 0.90 or less in all cases. From these results, it was demonstrated that solid-liquid separation can be performed with high efficiency by heating a solution adjusted to pH 3.0 or 4.0 at 70°C.

[0123]

[0124] [Example 5 Evaluation of the effect of the type of acid used to adjust the pH of the culture medium] Samples 21 to 27, which are culture medium obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above, were prepared by adding 35% by mass hydrochloric acid to adjust the pH at room temperature to 2.0 or 3.0, and by adding anhydrous citric acid powder under room temperature conditions to adjust the pH at room temperature to 4.0. Next, 10 mL each of the unadjusted pH samples 19, 20, and 8, and the pH-adjusted samples 21 to 27, obtained by the same method as the culture above, were heated to 80°C and kept warm for 5, 10, or 24 hours, respectively. After warming, each test solution was cooled to 30°C or below, and the SSV% was determined according to the [SSV% measurement method] above. Table 4 shows the results of calculating the SSV ratio of the second composition (SSV ratio) using the SSV% of the obtained samples 21-27 as the SSV of the second composition, and samples 8, 19, and 20, which were heated to each temperature without pH adjustment, as the SSV of the control solution.

[0125] As shown in Table 4, the SSV% was lower in the test solutions whose pH was adjusted with hydrochloric acid or citric acid compared to the test solutions whose pH was not adjusted, under all pH and incubation time conditions. Furthermore, the SSV ratio was 0.90 or less in all cases. From the above, it was shown that solid-liquid separation can be performed with high efficiency by adjusting the pH to acidity, regardless of the type of acid used.

[0126]

[0127] [Example 6: Evaluation of the effect of adjusting the culture medium to different pH levels and heating it on pressurized filtration] The culture medium obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above was divided into two. One was left unadjusted to a pH of 6.64 and heated to 80°C for 7 hours. The other was adjusted to a pH of 2.0 by adding 96% sulfuric acid at 25°C, and then heated to 70°C for 7 hours. After cooling each to below 30°C, a filtration test was performed using air pressure. Membrane filter (HAWP04700, pore size 0.45 μm, cellulose mixed ester, diameter φ47 mm, 17.3 cm) ２Eight mL of each test solution was placed in a cylindrical, sealed container equipped with a Merck filter, and filtered under a constant pressure of 0.1 MPa. The change in filtrate volume over time was measured. As a result, no filtrate was obtained even after 30 minutes of pressurization with the test solution heated without pH adjustment. On the other hand, with the test solution heated at pH 2.0, 1 mL of filtrate was obtained at 2, 4, 7, and 12 minutes, respectively. From the above, it was shown that solid-liquid separation by pressurized filtration can be performed with high efficiency by heating under acidic conditions such as pH 2.0.

[0128] [Example 7 Evaluation of the effect of heating the culture medium after adjusting its pH] Samples 28 to 31, which were culture media obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above, were prepared by adding 96% by mass sulfuric acid to adjust the pH at room temperature to 2.0 or 3.0. 10 mL of each test solution was heated to 50°C or 60°C and incubated for 4, 8, 16, or 48 hours. After incubation, sample 28 was subjected to MF-Millipore membrane (cellulose mixed ester, pore size 0.45 μm, diameter 47 mm (17.3 cm)). ２ Samples 29-31 were measured using an MF-Millipore membrane (cellulose mixed ester, hydrophilic, pore size 0.1 μm, diameter 47 mm, white, plain, VCWP04700, Merck) and the time (average filtration rate) to obtain the filtrate was measured by pressure filtration at 0.1 MPa. The results are shown in Table 5.

[0129] As shown in Table 5, filtrate could be obtained by pressure filtration even under conditions where the pH was 2.0 to 3.0 and the heating temperature was 50 to 60°C. Therefore, it was demonstrated that pressure filtration is feasible under pH and temperature conditions that result in a low SSV%.

[0130]

[0131] [Example 8 Evaluation of the effect of diluting the culture medium and heating it at various pH conditions on SSV%] Test solutions were obtained by adding 96% by mass sulfuric acid at room temperature to the culture medium obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above to adjust the pH at room temperature to 5.0, 4.0, 3.0, or 2.0 (Test solution group A), by diluting the above culture medium twice with deionized water after adjusting the pH (Test solution group B), by not adjusting the pH of the above culture medium (Test solution group C, pH 6.64 at room temperature), and by diluting the above culture medium twice with deionized water without adjusting the pH (Test solution group D, pH 6.64 at room temperature). 10 mL of each test solution was heated to 40, 50, 60, 70, or 80°C and kept warm for 24 hours. After maintaining temperature, test solutions A and C were diluted 2-fold with deionized water, and each test solution from test solutions A to D was cooled to 30°C or below. The SSV% was then determined according to the [SSV% measurement method] described above. The obtained SSV% (referred to as "SSV% (2-fold dilution)" in Tables 6 and 7) is shown in Tables 6 and 7. Furthermore, the SSV ratio was determined by using the SSV% of test solution A as the SSV of the second composition and the SSV% of test solution C as the SSV of the control solution. In addition, the SSV ratio was determined by using the SSV% of test solution B as the SSV of the second composition and the SSV% of the unadjusted pH test solution D as the SSV of the control solution. These results are shown in Table 8.

[0132] As shown in Tables 6-8, in the test solutions heated after adjusting the pH to 2-5 (test solution groups A and B), the SSV% was lower in all conditions compared to the unadjusted pH test solutions (test solution groups C and D) under the same conditions, both in terms of temperature and holding time. Furthermore, the SSV ratio of all test solutions heated after adjusting the pH to 2-5 was 0.90 or less. From the above, it was shown that, whether the solution was diluted after heating or heated after dilution, adjusting the pH to 2-5 and heating it allows for highly efficient solid-liquid separation.

[0133]

[0134]

[0135]

[0136] [Example 9 Desalination Using an Ultrafiltration Membrane (Molecular Weight Cutoff: 10 × 10 ３ ), Where the Material Using the Solution after Solid-Liquid Separation Is Regenerated Cellulose] 1010 L of the culture solution obtained by the method described in [Preparation of the Culture Solution Containing Heparan and Microbial Cells] above (weight-average molecular weight of heparan: 465 × 10 ３ ) was added with 24.9 kg of 35% by mass sulfuric acid, and the pH at 30°C was adjusted to 3.0. Then, 6 L of 20% (v / v) P-2000 (polyglycol, manufactured by Dow Chemical Japan) as an antifoaming agent was added, and the temperature was raised to 70°C and held for 10 hours (weight-average molecular weight of heparan: 205 × 10 ３ ). Thereafter, it was cooled until the temperature reached 30°C or lower, and deionized water was added to dilute it 1.86-fold. Then, using a nozzle-type centrifuge (DX203, manufactured by Alfa Laval) with a nozzle diameter of 0.5 mm, centrifugation was performed at a supply rate of 240 - 250 L / h and 8571 rpm (centrifugal sedimentation area: 900 m ２ ), and the supernatant was separated. Deionized water was added to the remaining concentrated cells to dilute them 2-fold, and then, using the above centrifuge, centrifugation was performed at a supply rate of 250 L / h and 8571 rpm (centrifugal sedimentation area: 900 m ２ ), and the supernatant was separated. When the supernatants obtained by the two centrifugations were combined, it was 2034 L (heparan content: 4.7 g / L), and the yield of heparan in the two centrifugations was 85%.

[0137] Next, the supernatant obtained by the above two centrifugations in the above solid-liquid separation step was subjected to a desalination step. As a result of microfiltration of the above supernatant using a microfiltration membrane (ULW-348, hollow fiber membrane: polyvinylidene fluoride, nominal pore diameter: 0.45 μm, manufactured by Asahi Kasei), 2006 L of a microfiltrate (heparan content: 4.6 g / L) was obtained. The yield of heparan in the above microfiltration was 98.5%. Among the above microfiltrate, 1803 L was subjected to an ultrafiltration membrane (RC10PE-3838 / 48, membrane material: regenerated cellulose, molecular weight cutoff: 10 × ３ , 4.8 m ２Using two Alfa Laval tubes, the solution was concentrated 3.67 times by volume. After that, five times the volume of deionized water was added to the concentrated liquid, and desalting was performed by ultrafiltration to obtain the desalted solution, which is the residue of the ultrafiltration. The desalting by ultrafiltration was carried out under conditions of maintaining a temperature of 12-17°C and a membrane differential pressure of 0.1 MPa. The desalted solution was 451 L (heparosan content: 18.5 g / L, weight-average molecular weight of heparosan: 207 × 10⁻⁶). ３ The yield of heparosan obtained in the above desalting was 99.3%. The yield loss to the filtrate side at this time was 0.7%. The electrical conductivity of the desalted solution was 1.6 mS / cm, and the pH of the desalted solution measured at 25°C was 3.2.

[0138] The desalination solution obtained in the above desalination process was subjected to cloud point measurement. Specifically, the desalination solution was cooled to 10°C and filtered through a 0.45 μm microfiltration membrane (HAWP04700, cellulose mixed ester, pore size 0.45 μm, filter diameter 47 mm, manufactured by Merck). The cloud point was then measured by visually observing the occurrence of turbidity while gradually increasing the temperature. As a result, the cloud point was found to be 25-26°C.

[0139] As described above, the yield in the desalting process was 99.3%, indicating that high-yield desalting is possible by using an ultrafiltration membrane containing regenerated cellulose as a material.

[0140] [Example 10: Solid-liquid separation and desalting under alkaline conditions using an ultrafiltration membrane with the solution after solid-liquid separation] 26.0 kg of 35% by mass sulfuric acid was added to 1000 L of the culture medium obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above, and the pH at 30°C was adjusted to 3.0. 1.5 L of 20% (v / v) P-2000 antifoaming agent (polyglycol, manufactured by Dow Chemical Japan) was added, and the temperature was raised to 70°C while stirring and held for 5 hours. After that, it was cooled to below 30°C, and the microbial cells were separated by centrifugation at 12,000 g for 10 minutes using a centrifuge. The obtained supernatant was filtered under reduced pressure using filter paper (No. 5C, pore size 1 μm, manufactured by Advantec) to obtain 740 L of supernatant (heparosan content: 9.1 g / L, heparosan mass: 6.8 kg). 249 L of the supernatant (heparosan content: 9.1 g / L, heparosan mass: 2.3 kg) was collected and 2.7 L of 48% by mass sodium hydroxide was added to adjust the pH to 10.0 at room temperature. The precipitate formed during pH adjustment was separated by vacuum filtration using filter paper (No. 5C, pore size 1 μm, manufactured by Advantec Co., Ltd.) to obtain 251 L of alkaline supernatant (heparosan content: 8.7 g / L, heparosan mass: 2.2 kg). The alkaline supernatant was then microfiltration using a microfiltration membrane (product name: ULP-143, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation) to obtain a fine filtrate.

[0141] Next, the obtained fine filtrate was subjected to a desalting process. Specifically, the fine filtrate was cooled to 15°C or below and subjected to an ultrafiltration membrane (product name: SEP-3013, hollow fiber membrane: polysulfone, nominal molecular weight cutoff: 3 × 10⁻⁶). ３ Diafiltration was performed using a product manufactured by Asahi Kasei Corporation. In the diafiltration, the fine filtrate was first concentrated to 1.8 times its volume to obtain a circulating concentrate. Next, 3.3 times its volume of deionized water was used on the obtained circulating concentrate, and the stationary addition of deionized water was repeated until the electrical conductivity of the circulating liquid was 3.0 mS / cm or less, thereby obtaining 170 L of desalinated solution (mass of heparosan: 2.1 kg).

[0142] [Example 11: Solid-liquid separation and desalting under acidic conditions using an ultrafiltration membrane with the solution after solid-liquid separation] 5.0 L of the culture medium obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above was mixed with 96% by mass sulfuric acid to adjust the pH to 3.0 at 25°C. The temperature was then raised to 70°C while stirring and maintained for 5 hours. After that, the temperature was cooled to below 30°C, and the microbial cells were separated by centrifugation at 12,000 g for 10 minutes using a centrifuge. The obtained supernatant was filtered under reduced pressure using filter paper (No. 5C, pore size 1 μm, Advantec Co., Ltd.) to obtain 2.7 L of supernatant (heparosan content: 11.4 g / L).

[0143] Next, the obtained supernatant was subjected to a desalting process. Specifically, 200 mL of the supernatant was taken and subjected to ultrafiltration (Microza UF, SEP-0013, nominal molecular weight cutoff: 3 × 10⁻¹⁰). ３ Hollow fiber membrane: polysulfone, effective membrane area: 0.017 m² ２ Desalination was performed by diafiltration using a (manufactured by Asahi Kasei Corporation) ultrafiltration membrane. In the diafiltration, the supernatant was first concentrated to 1.4 times its volume to obtain a circulating concentrate. Next, the obtained circulating concentrate was subjected to stationary hydration using 2.3 times its volume of deionized water to obtain 156 mL of desalination solution (heparosan content: 9.1 g / L, heparosan mass: 1.4 g). The yield of the above desalination was 62.5%. The weight-average molecular weight of heparosan was equal to the nominal fractionation molecular weight of the ultrafiltration membrane, 3 × 10⁻⁶. ３ A significant increase, 270 x 10 ３ However, during the desalting process described above, 21.4% of the heparosan contained in the 200 mL supernatant was lost by passing through the ultrafiltration membrane. In addition, 2.8% of the heparosan contained in the 200 mL supernatant was yield loss into the diafiltration apparatus, and 13.4% was yield loss of unknown cause.

[0144] Desalting was possible under both the alkaline conditions of Example 10 and the acidic conditions of Example 11. Therefore, it was shown that desalting using an ultrafiltration membrane is possible under both alkaline and acidic conditions.

[0145] [Example 12] An ultrafiltration membrane using solid-liquid separation and the solution after solid-liquid separation, where the material is regenerated cellulose (molecular weight cutoff: 10 × 10 ３ ) Desalting by the method described in [Preparation of culture medium containing heparosan and microbial cells] 1030 L of culture medium (weight-average molecular weight of heparosan: 478 × 10) ３ 25.5 kg of 35% by mass sulfuric acid was added to the mixture, and the pH at room temperature was adjusted to 2.98. 4 L of 20% (v / v) P-2000 defoaming agent (polyglycol, manufactured by Dow Chemical Japan) was added, and the mixture was heated to 70°C and held for 10 hours (weight-average molecular weight of heparosan: 197 × 10⁻⁶). ３ ). After that, it is cooled to below 30°C, and deionized water is added to dilute it 1.82 times. Using a nozzle-type centrifuge with a nozzle diameter of 0.5 mm (DX203, manufactured by Alfa Laval), the mixture is fed at a rate of 300 L / h and 8571 rpm (centrifugal sedimentation area: 900 m²). ２ The concentrated bacterial cells obtained were then diluted twice with deionized water, and then centrifuged using the above centrifuge at a feed rate of 240 L / h and 8571 rpm (centrifugal sedimentation area: 900 m²). ２ The mixture was centrifuged using a centrifuge. The supernatants obtained from these two centrifuges were combined to form 1906 L (heparosan content: 5.2 g / L), with a yield of 82.4%. Of the supernatant, 1700 L was microfiltered at 10°C or below using a microfiltration membrane (ULW-348, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation) to obtain 1664 L of microfiltrate (heparosan content: 5.4 g / L).

[0146] Next, the above-mentioned fine filtrate was subjected to a desalination process. Specifically, 1664 L of the fine filtrate was filtered through an ultrafiltration membrane (RC10PE-3838 / 48, membrane material: regenerated cellulose, molecular weight cutoff: 10 × 10). ３ , 4.8m ２Desalination was performed by diafiltration using one Alfa Laval (AVA) filtration system. In the diafiltration, the operating conditions were 10-15°C and the intermembrane pressure differential was 0.03 MPa. First, the fine filtrate was concentrated 3.3 times by volume to obtain a circulating concentrate. Next, the obtained circulating concentrate was subjected to stationary hydration using 5 times the volume of deionized water to obtain 487 L of desalination solution (heparosan content: 16.2 g / L, weight-average molecular weight of heparosan: 197 × 10⁻⁶). ３ The result obtained was 86.9% heparosan in the above desalting process. Since the permeate loss to the filtrate side was 0.6%, the remaining loss is estimated to be loss within the apparatus.

[0147] The desalination solution obtained in the above desalination process was subjected to cloud point measurement. Specifically, the desalination solution was cooled to 10°C and filtered through a 0.45 μm microfiltration membrane. The cloud point was then measured by visually observing the occurrence of turbidity while gradually increasing the temperature. As a result, the cloud point was found to be around 31°C. The final electrical conductivity of the desalination solution was 1.5 mS / cm, and the pH of the desalination solution measured at 25°C was 3.0.

[0148] As described above, the yield in the desalting process was 86.9%, indicating that high-yield desalting is possible by using an ultrafiltration membrane containing regenerated cellulose as a material.

[0149] [Example 13] An ultrafiltration membrane using a solution containing heparosan with different weight-average molecular weights after solid-liquid separation, where the material is regenerated cellulose (molecular weight cutoff: 10 × 10 ３ Desalting by ()) The supernatant from Example 12 was kept at 70°C for 14.8 hours, 29 hours, or 47.8 hours while being stirred in a water bath to allow the demolecular-weight reaction of heparosan to proceed. A portion of the supernatant was then collected and cooled to room temperature to stop the demolecular-weight reaction, thereby obtaining supernatants containing heparosan with different weight-average molecular weights. Each of the supernatants was subjected to microfiltration using a microfiltration membrane (Asahi Kasei Corporation, model: ULP-143, material: PVDF, nominal pore size: 0.45 μm) to obtain a fine filtrate containing heparosan with different weight-average molecular weights.

[0150] Next, the fine filtrates containing heparosan with different weight-average molecular weights were subjected to a desalting process. Specifically, each fine filtrate was subjected to an ultrafiltration membrane (RC10PE-2517 / 48, membrane material: regenerated cellulose, molecular weight cutoff: 10 × 10). ３ , 0.6m ２ Desalination was performed by diafiltration using one Alfa Laval (Alfa Laval) filter. The diafiltration was performed at room temperature of 15-30°C, maintaining a membrane differential pressure of 0.4 MPa. First, the fine filtrate was concentrated to 2.1-2.7 times its volume to obtain a circulating concentrate. Next, the obtained circulating concentrate was subjected to stationary hydration using 4 times its volume of deionized water to obtain each desalination solution. Table 9 shows the weight-average molecular weight of heparosan, the heparosan content, the volume of liquid subjected to or obtained by desalination, and the mass of heparosan in the fine filtrate and desalination solution of Example 13, Example 9, and Example 12, as well as the yield and permeate loss in desalination of Examples 9, 12, and 13. In Table 9, Examples 13-1, 13-2, and 13-3 correspond to the low molecular weight reaction in which the material was held at 70°C for 14.8 hours, 29 hours, and 47.8 hours, respectively. Furthermore, the molecular weight cutoff of the ultrafiltration membranes used for desalting in Examples 13, 9, and 12 was 10 × 10⁻⁶. ３ That is the case.

[0151] As shown in Table 9, the molecular weight cutoff is 10 × 10 ３ When using an ultrafiltration membrane, the weight-average molecular weight of heparosan in the microfiltrate should be at least 205 × 10 ３ ~197 x 10 ３ Desalting was possible within the specified range. Furthermore, the weight-average molecular weight of heparosan in the microfiltrate was 100 × 10⁶. ３ The above results demonstrate particularly low transmission loss and exceptionally high yield.

[0152]

[0153] [Example 14 Evaluation of the effect on the method of adding acid to adjust the pH of the culture medium] To 100 mL of the culture medium obtained by the method described in [Preparation of culture medium containing heparosan and microbial cells] above, H ＋ Regenerated Dowex Marathon C (manufactured by DuPont) was added to the mold, and the pH was adjusted to 3.0 at room temperature (23°C). The mixture was then stirred at 70°C for 10 hours to obtain the test solution. The amount of Marathon C required for pH adjustment was 8.2 g. The above test solution was subjected to a spin test, and the SSV% was determined to be 33%. The calculation of the SSV% by spin test was performed using the same procedure as in [Example 1: Evaluation of the effect of adjusting the pH to 3 before and after heating the culture medium on the SSV%]. After that, the above test solution was cooled to room temperature, and 50 mL of the above test solution was filtered under reduced pressure using filter paper (No. 5C, pore size 1 μm, manufactured by Advantec), and 13 mL of clarified filtrate was obtained in 7 minutes.

[0154] From the results above, H ＋ It was demonstrated that highly efficient solid-liquid separation can be achieved even when the pH is adjusted by adding recycled resins such as Dawex Marathon C to the mold.

[0155] [Example 15: Solid-liquid separation and desalting using ion exchange resin with the solution after solid-liquid separation] 1100 L of culture solution obtained by the method described in [Preparation of culture solution containing heparosan and microbial cells] above (weight-average molecular weight of heparosan: 422 × 10 ３ 23.6 kg of 35% by mass sulfuric acid was added to the mixture, and the pH at room temperature was adjusted to 3.01. 4 L of 20% (v / v) P-2000 defoaming agent (polyglycol, manufactured by Dow Chemical Japan) was added, and the mixture was heated to 70°C while stirring and held for 10 hours (weight-average molecular weight of heparosan: 194 × 10⁻¹⁶). ３ ). After that, it is cooled to below 30°C, and deionized water is added to dilute it 1.80 times. Using a nozzle-type centrifuge with a nozzle diameter of 0.6 mm (Y-55S, manufactured by Saito Centrifuge Industry Co., Ltd.), the mixture is fed at a rate of 1630 L / h and 8300 rpm (centrifugal sedimentation area: 10300 m²). ２The concentrated bacterial cells obtained were then diluted 3.0 times with deionized water, and then centrifuged using the above centrifuge at a feed rate of 1620 L / h and 8300 rpm (centrifugal sedimentation area: 10300 m²). ２ The mixture was centrifuged using a centrifuge. The supernatants obtained from these two centrifuges were combined to form 2672 L (heparosan content: 4.3 g / L), and the yield of heparosan from the two centrifuges was 82.2%. The entire supernatant was subjected to microfiltration at 40°C using a microfiltration membrane (ULW-348, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation) to obtain a microfiltrate of 3116 L (heparosan content: 3.6 g / L). The yield of heparosan from the above microfiltration was 97.0%.

[0156] Next, the above-mentioned precision filtrate was subjected to a desalination process using an ion exchange resin. Specifically, first, H ＋ 200 mL of recycled Dowex Marathon C (manufactured by DuPont) was packed into a 35 mm diameter glass column, and 3000 mL of the above-mentioned precision filtrate was added (heparosan content: 3.6 g / L, weight-average molecular weight of heparosan: 193 × 10⁻¹⁵). ３ The solution was passed through a column at room temperature at SV3.0 (flow rate: 600 mL / h) to obtain the filtrate. The pH changed from 2.99 in the fine filtrate before column passing to 1.38 after column passing, and the yield of heparosan in the column was 100%. Next, OH was added with NaOH. － Lewatit VP OC 1065 (manufactured by Lanxess), which has been regenerated in a mold, was packed into an 18 mm diameter glass column, and 365 mL of the above-mentioned perfusion of Marathon C (heparosan content: 3.6 g / L, weight-average molecular weight of heparosan: 177 × 10⁻¹⁵) was added. ３ The solution was passed through at room temperature at SV 1.0 (flow rate: 30 mL / h). When the electrical conductivity at the outlet of the ion exchange resin reached 0.1 mS / cm or higher, the recovery of the fraction containing heparosan was started, and the recovery was stopped when the electrical conductivity fell below 0.06 mS / cm during washing with deionized water. The obtained 435 mL of ion exchange resin filtration solution (pH 2.69) contained 2.5 g / L of heparosan, and the weight-average molecular weight of heparosan was 182 × 10⁶. ３The yield of desalting using Lewatit VP OC 1065 was 86.1%. The electrical conductivity of the ion exchange resin quenching solution was 0.51 mS / cm.

[0157] As described above, the desalting yield was high at 86.1%, and the electrical conductivity of the ion-exchange resin-passed liquid was sufficiently low at 0.51 mS / cm, demonstrating that desalting can be effectively achieved even when using ion-exchange resin.

[0158] [Example 16: Solid-liquid separation and desalting using an ultrafiltration membrane (molecular weight cutoff: 10 kDa) with regenerated cellulose as the material after solid-liquid separation and the resulting solution] 1102 L of culture solution obtained by the method described in [Preparation of culture solution containing heparosan and microbial cells] above (weight-average molecular weight of heparosan: 410 × 10 ３ 21.7 kg of 35% by mass sulfuric acid was added to the mixture, and the pH at room temperature was adjusted to 2.99. 6 L of 20% (v / v) P-2000 antifoaming agent (polyglycol, manufactured by Dow Chemical Japan) was added, the temperature was raised to 70°C and held for 10 hours, and then cooled to below 30°C. The resulting culture solution after the heating process (heparosan content: 11.7 g / L, weight-average molecular weight of heparosan: 197 × 10⁻⁶) ３ After diluting the mixture 1.8 times by adding deionized water, a nozzle-type centrifuge with a nozzle diameter of 0.6 mm (Y-55S, manufactured by Saito Centrifuge Industry Co., Ltd.) was used to centrifuge at a feed rate of 1810 L / h and 8300 rpm (centrifugal sedimentation area: 10300 m²). ２ The supernatant was separated by centrifugation. Deionized water was added to the remaining concentrated bacterial cells to dilute them 2.0 times, and then the above centrifuge was used to separate them at a feed rate of 1760 L / h and 8300 rpm (centrifugal sedimentation area: 10300 m²). ２ The mixture was centrifuged and the supernatant was separated. The supernatants obtained from the two centrifugations were combined to form 2146 L (heparosan content: 4.8 g / L), and the yield of heparosan from the two centrifugations was 80.0%.

[0159] Next, the supernatant obtained in the solid-liquid separation step described above was subjected to a desalting step. The supernatant was microfiltration at 38.0–45.8°C using a microfiltration membrane (ULW-348, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation), resulting in 2183 L of microfiltrate (heparosan content: 4.5 g / L). The yield of heparosan in the above microfiltration was 95.5%. Of the above microfiltrate, 2159 L was concentrated to 4.75 times its volume using two ultrafiltration membranes (RC10PE-3838 / 48, membrane material: regenerated cellulose, molecular weight cutoff: 10 kDa, 4.8 m2, manufactured by Alfa Laval), and then desalted by ultrafiltration with 5 times the volume of deionized water relative to the concentrated liquid volume to obtain a desalted solution. The desalination process by ultrafiltration described above was carried out under conditions of maintaining a temperature of 25.7–42.6°C and a membrane differential pressure of 0.4 MPa. The desalination solution was 477 L (heparosan content: 18.9 g / L, weight-average molecular weight of heparosan: 215 × 10⁻¹⁵). ３ The yield of heparosan obtained in the above desalting was 93.5%. The yield loss to the filtrate side at this time was 6.1%. The electrical conductivity of the above desalting solution was 0.09 mS / cm, and the pH of the above desalting solution measured at 20.0°C was 3.09. When the cloud point of the above desalting solution was measured in the same manner as in Example 12, no cloud point was observed in the range up to 60.3°C.

[0160] As described above, the yield in the desalination process was 93.5%, indicating that high-yield desalination is possible by using an ultrafiltration membrane containing regenerated cellulose as a material. Furthermore, the absence of a cloud point in the desalination solution suggests that the defoaming agent (polyglycol) was efficiently removed by microfiltration under conditions of 38.0–45.8°C.

[0161] [Example 17: Solid-liquid separation using ceramic membrane and desalting using ion exchange resin] 4 L of culture solution obtained by the method described in [Preparation of culture solution containing heparosan and microbial cells] above (weight-average molecular weight of heparosan: 414 × 10 ３96% by mass sulfuric acid was added to the culture medium to adjust the pH to 2.78 at room temperature. The culture medium was heated to 80°C and maintained for 24.5 hours, then cooled to below 30°C. 1.38 L of the culture medium after this heating process (heparosan content: 10.4 g / L, weight-average molecular weight of heparosan: 31.1 × 10⁻¹⁶) ３ ) is used in a ceramic membrane filter containing porous ceramic (length: 250 mm, pore size: 0.5 μm, area: 0.016 m²) ２ Solid-liquid separation was performed by cross-flow filtration using a NGK (North Angler) filter. Specifically, first, the culture medium was concentrated to 0.53 L under conditions of a temperature of 24-38°C and a membrane differential pressure of 0.17-0.20 MPa, and the concentrated filtrate (heparosan content: 9.45 g / L, weight-average molecular weight of heparosan: 31.4 × 10⁻¹⁴) was analyzed. ３ A concentrated filtrate was obtained. A portion of this concentrated filtrate was set aside for the desalination process described below. While maintaining the liquid level of the circulating fluid, 0.62 L of deionized water and 0.6 L of 1 mol / L NaCl aqueous solution were added, and cross-flow filtration was continued. The yield of heparosan that permeated the ceramic membrane filter was 94.1%.

[0162] Next, the concentrated filtrate separated for the desalination process was subjected to a desalination process using an ion exchange resin. First, a strongly acidic cation exchange resin, H ＋ A 30 mL bottle of DWEX Marathon C (DuPont) was packed into an 18 mm inner diameter glass column, and 165 mL of the concentrated filtrate was passed through it at room temperature and 1 SV. Deionized water was then passed through, and the flow was stopped when the electrical conductivity of the liquid at the column outlet fell below 0.5 mS / cm. The resulting decationized solution was 260 mL (heparosan content: 7.25 g / L, weight-average molecular weight of heparosan: 30.9 × 10⁶). ３ The pH of the concentrated filtrate before passing it through the ion exchange resin was 2.78, but it changed to 1.02 in the decation solution, and the yield of heparosan in the desalting process was 115.1%.

[0163] As described above, solid-liquid separation of the culture medium containing heparosan was possible by cross-flow filtration using a ceramic membrane filter containing porous ceramic. Furthermore, as described above, the yield in the desalting process was 115.1%, indicating that high-yield desalting is possible by using a strongly acidic cation exchange resin.

Claims

1. A method for producing a composition containing N-acetylheparosan, comprising the steps of: (1) heating an aqueous composition containing N-acetylheparosan and microbial cells to 35 to 85°C under conditions where the pH is 1.5 to 3.5, or (2) heating an aqueous composition containing N-acetylheparosan and microbial cells to 65 to 85°C under conditions where the pH is 3.5 to 5.5; and, after the first step, removing at least a portion of the microbial cells from the aqueous composition by solid-liquid separation.

2. The method according to claim 1, comprising adding at least one acid selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, citric acid, acetic acid, propionic acid, and butyric acid to the aqueous composition to set the pH to the conditions described above.

3. The method according to claim 1, wherein the aqueous composition is a culture medium.

4. The method according to claim 1, further comprising the step of removing at least a portion of the microbial cells from the aqueous composition by solid-liquid separation, followed by the step of desalting the aqueous composition using an ultrafiltration membrane, an ion exchange resin, or an ion exchange membrane.

5. The method according to claim 4, wherein the ultrafiltration membrane comprises at least one material selected from the group consisting of porous ceramic, cellulose acetate, cellulose nitrate, regenerated cellulose, porous cellulose, polysulfone, polyacrylonitrile, polyamide, polyvinylidene fluoride, polytetrafluoroethylene, and polyimide.

6. The method according to claim 5, wherein the ultrafiltration membrane comprises polysulfone or regenerated cellulose as a material.

7. The molecular weight cutoff of the ultrafiltration membrane is 10 × 10 ３ The method according to claim 6, which is as follows.

8. The method according to claim 7, wherein the ultrafiltration membrane contains regenerated cellulose as a material.

9. 50 x 10 ３ ~300 x 10 ３ An aqueous composition containing N-acetylheparosan having a weight-average molecular weight, wherein the aqueous composition has a content of N-acetylheparosan of 1 to 30 g / L based on the aqueous composition, an electrical conductivity of 20 mS / cm or less, and a pH of 0.5 to 5.

0.

10. The aqueous composition according to claim 9, having an electrical conductivity of 3.0 mS / cm or less and a pH of 2.0 to 4.0.

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