Method for preparing corac acid or salt thereof

Abstract

The invention belongs to the technical field of biology, and relates to a method for large-scale preparation of high-purity hydrolyzed corac acid or a salt thereof. The method comprises the following steps: 1) adding a solution containing corac acid or a salt thereof into a corac acid degrading enzyme containing an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity with the SEQ ID NO: 1; terminating in the step 2) is achieved through a terminating agent, a boiling water bath and / or an ice bath, the terminating agent is selected from one or more of activated carbon, absolute ethyl alcohol, methyl alcohol, EDTA, trichloroacetic acid and sodium carbonate, and preferably, the activated carbon is used for achieving terminating in the step 2); and further obtaining the hydrolyzed corac acid or the salt thereof. The molecular structure of the hydrolyzed sodium currate prepared by the method provided by the invention has acetylation and pyruvic acid modification, the preparation condition is mild, the production cost is low, continuous production can be realized, and industrial amplification is easy.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing high-purity hydrolyzed colanic acid or its salt by large-scale purification. Background Art

[0002] Microbial extracellular polysaccharides are biopolymers produced by bacteria, fungi, cyanobacteria and other microorganisms during metabolism that have a protective effect on microorganisms. Under natural conditions, polysaccharides coated on the surface of bacteria play an important role in the survival and growth of bacteria in a competitive environment. Colanic acid is one of the microbial extracellular polysaccharides. The solid is a white fibrous amorphous structured heteropolysaccharide, mainly composed of D-glucuronic acid, D-glucose, D-galactose and L-fucose, with a composition ratio of 1:2:2:1. It also contains chemically modified structures on various side chains such as O-acetyl and pyruvic acid. It is a high molecular weight polymer polysaccharide with an average molecular weight of more than 10 million Daltons.

[0003] Microbial extracellular polysaccharides have unique physical and rheological properties and are widely used as stabilizers, thickeners, gelling agents and emulsifiers in the food industry. In recent years, studies have found that bacterial polysaccharides have shown significant biological activities in anti-tumor, anti-viral, immunostimulatory, anti-inflammatory, antioxidant, and antimicrobial effects, and have therefore attracted more attention. In 2017, it was first reported in the journal Cell that colanic acid caused mitochondrial fragmentation and had the effect of extending the lifespan of Caenorhabditis elegans. Due to its porous cellulose structure and a large number of hydrophilic groups on the surface of the colloid, colanic acid is a natural hydrogel with excellent hydration ability and soft texture, making it a good candidate product for the future cosmetics and healthcare markets. In addition, as a unique active biopolymer, colanic acid has special biological properties and physiological parameters, and has broad research and application prospects in extending lifespan and anti-aging.

[0004] Literature research shows that the biological activity of polysaccharides depends on their molecular weight, and polysaccharides in different molecular weight ranges exhibit different physiological functions. For example, high molecular weight hyaluronic acid has good viscoelasticity, moisturizing, anti-inflammatory, lubricating and other functions, and can be used in the cosmetics industry, ophthalmic surgery viscoelastic agents and intra-articular injection therapy. Medium molecular weight hyaluronic acid has good moisturizing, lubricating and drug sustained release effects, and can be widely used in cosmetics, eye drops, skin burn healing and postoperative adhesion prevention. Low molecular weight hyaluronic acid and oligomeric hyaluronic acid show very strong biological activity, with antioxidant, anti-inflammatory, collagen production promotion, wound healing and other biological activities. More importantly, due to its small molecular size, it can penetrate into the stratum corneum of the skin to exert its effects and can be widely used in cosmetics.

[0005] Sodium colanate synthesized by Escherichia coli has a large molecular weight of about 5 to 10 million Daltons. This large molecular weight polysaccharide has certain applications in cosmetics, health foods and other fields. However, there is currently no research on the production process of preparing hydrolyzed colanate salts of specific molecular weight, especially sodium colanate, which greatly limits the application scenarios and efficacy of sodium colanate. Therefore, it is necessary to develop a large-scale preparation method for finished colanate acid or salt (1×10 3 Da-2.0×10 6 Da)'s production process is of great significance.

[0006] In order to solve the above problems, the present invention provides a method for continuous large-scale production of finished colanic acid or salt. The finished colanic acid or salt prepared by the method provided by the present invention has a complete structure (including acetylation and pyruvic acid modification), high purity, low endotoxin residue, mild and efficient preparation conditions, low production cost, can realize continuous production, and is easy to scale up for industrial production. Summary of the invention

[0007] The present invention discloses a method for continuous large-scale production of hydrolyzed colanic acid or its salt. Specifically, the present invention provides a method for preparing hydrolyzed colanic acid or its salt, which comprises the following steps: 1) adding a solution containing colanic acid or its salt to a colanic acid degrading enzyme comprising an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity therewith; and 2) enzyme termination reaction: the termination in step 2) is achieved by a terminator, a boiling water bath and / or an ice bath, wherein the terminator is selected from one or more of activated carbon, anhydrous ethanol, methanol, EDTA, trichloroacetic acid and sodium carbonate, preferably the termination in step 2) is achieved by using activated carbon; thereby obtaining hydrolyzed colanic acid or its salt.

[0008] According to some embodiments of the present disclosure, in the aforementioned method, the colanate salt is sodium colanate.

[0009] According to some embodiments of the present disclosure, in the aforementioned method, in step 1), the weight average molecular weight of the colanic acid or its salt is greater than 2×10 6 Da.

[0010] In some preferred embodiments, in the aforementioned method, in step 1), the amino acid sequence of the colanic acid degrading enzyme is as shown in SEQ ID NO: 1.

[0011] According to some embodiments of the present disclosure, in the aforementioned method, in step 1), the final reaction concentration of the colanic acid degrading enzyme is between about 0.01 mg / L and 10 mg / L.

[0012] According to some embodiments of the present disclosure, in the aforementioned method, in step 1), the reaction temperature is between about 10 ° C. and about 70 ° C. In some preferred embodiments, the reaction temperature is between about 20 ° C. and about 50 ° C.

[0013] According to some embodiments of the present disclosure, in the aforementioned method, in step 1), the reaction time is about 1 h to about 20 h.

[0014] According to some embodiments of the present disclosure, in the aforementioned method, in step 1), the pH of the reaction solution is about 3 to about 9. In some preferred embodiments, the pH is about 5 to about 8.

[0015] According to some embodiments of the present disclosure, the method further comprises the steps of 3) filtration and purification; 4) concentration and desalination; and 5) drying.

[0016] According to some embodiments of the present disclosure, the above method further comprises 6) multiple enzymatic hydrolysis.

[0017] According to some embodiments of the present disclosure, in the aforementioned method, the activated carbon content is about 0.005 wt% or more, preferably 0.005 wt%-1 wt%, and more preferably about 0.01 wt%-5 wt%.

[0018] According to some embodiments of the present disclosure, in step 3), the pH is adjusted to about 3.0-about 6.5 before filtration. In some preferred embodiments, the pH is adjusted to about 3.0-about 6.0 before filtration.

[0019] According to some embodiments of the present disclosure, in step 5), the drying is achieved by spray drying, freeze drying or vacuum drying.

[0020] According to some embodiments of the present disclosure, in the aforementioned method, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 3×10 6 In some preferred embodiments, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 2×10 6 Da.

[0021] According to some embodiments of the present disclosure, in the aforementioned method, the hydrolyzed colanic acid or its salt comprises acetylation and / or pyruvic acid modification.

[0022] According to some embodiments of the present disclosure, in the aforementioned method, the acetylation modification rate of the hydrolyzed colanic acid or its salt is about 80-100%.

[0023] According to some embodiments of the present disclosure, in the aforementioned method, the residual endotoxin in the hydrolyzed colanic acid or its salt is less than about 0.5 EU / mg, preferably less than about 0.1 EU / mg.

[0024] According to some embodiments of the present disclosure, in the aforementioned method, the protein residue in the hydrolyzed colanic acid or its salt is less than about 0.1%, preferably less than about 0.07%.

[0025] According to some embodiments of the present disclosure, in the aforementioned method, the residual heavy metal in the hydrolyzed colanic acid or its salt is less than about 10 mg / kg.

[0026] According to some embodiments of the present disclosure, in the aforementioned method, the hydrolyzed colanic acid or its salt is colanic acid hexasaccharide or its salt, and / or colanic acid dodecasaccharide or its salt.

[0027] Another aspect of the present disclosure also provides hydrolyzed colanic acid or a salt thereof obtained according to the aforementioned method.

[0028] According to some embodiments of the present disclosure, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 3×10 6 In some preferred embodiments, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 2×10 6 Da.

[0029] According to some embodiments of the present disclosure, the hydrolyzed colanic acid or its salt comprises acetylation and / or pyruvic acid modification.

[0030] According to some embodiments of the present disclosure, the acetylation modification rate of the hydrolyzed colanic acid or its salt is about 80-100%.

[0031] According to some embodiments of the present disclosure, the residual endotoxin in the hydrolyzed colanic acid or its salt is less than about 0.5 EU / mg, preferably less than about 0.1 EU / mg.

[0032] According to some embodiments of the present disclosure, the protein residue in the hydrolyzed colanic acid or its salt is less than about 0.1%, preferably less than about 0.07%.

[0033] According to some embodiments of the present disclosure, the residual heavy metal in the hydrolyzed colanic acid or its salt is less than about 10 mg / kg.

[0034] According to some embodiments of the present disclosure, the hydrolyzed colanic acid or its salt is colanic acid hexasaccharide or its salt, and / or colanic acid dodecasaccharide or its salt.

[0035] Effects of the present invention: The present invention uses colanic acid degrading enzyme to catalyze hydrolysis and purify hydrolyzed colanic acid or its salt, which has a direct purpose. First, the weight average molecular weight of the high-purity hydrolyzed colanic acid or its salt obtained by the present invention covers the minimum unit of colanic acid or its salt (1.1×10 3 Da) to 2.0×10 6 The finished product contains acetylation and pyruvic acid modification, which lays a foundation for the academic community to study the structure and structure-activity relationship of colanic acid or its salts; secondly, the reaction conditions of the present invention are extremely simple, and there is no requirement for instruments and equipment, and it can be implemented under mild conditions; finally, the product prepared by the present invention has low protein residue, low heavy metal residue and low endotoxin, and has potential and wide application value in the fields of cosmetics, medical devices and oligosaccharide drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The molecular weight is 1.2×10 6 Da, 7.6×10 5 Da, 4.2×10 5 HPLC chromatogram of Da's sodium colanate product.

[0037] Figure 2 The figure is a high performance liquid chromatogram of the hydrolyzed sodium colanate oligosaccharide composition.

[0038] Figure 3 This is the HPLC chromatogram of hydrolyzed sodium colanate hexasaccharide.

[0039] Figure 4 This is the HPLC chromatogram of hydrolyzed sodium colanate dodecasaccharide.

[0040] Figure 5 This is the mass spectrum of hydrolyzed sodium colanate hexasaccharide.

[0041] Figure 6 This is the mass spectrum of hydrolyzed sodium colanate dodecasaccharide.

[0042] Figure 7 The figure is the NMR spectrum of the hydrolyzed sodium colanate oligosaccharide composition. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. For those skilled in the art, on the basis of this invention and creation concept, some modifications and improvements made belong to the protection scope of the present invention. The raw materials used in the embodiments can all be obtained through commercial channels.

[0044] definition Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] All numerical designations used herein, such as pH values, temperatures, times, concentrations, amounts and molecular weights, including ranges, are approximate and vary (+) or (-) in increments of 0.1 or 1.0 where appropriate. It will be understood that all numerical designations may be preceded by the term "about", although not always explicitly stated.

[0046] The terms "about" and "approximately" include amounts within ±10% of the associated numerical value.

[0047] As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily identified as fully described, and the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc.

[0048] As used herein, "hydrolyzed colanic acid or its salt" means the product obtained after the degradation reaction, and its weight average molecular weight ranges from about 1×10 3 Da - about 5×10 6 Da, preferably, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 2×10 6 It should be noted that the weight average molecular weight of the reactant colanic acid or its salt is greater than the weight average molecular weight of the hydrolyzed colanic acid or its salt.

[0049] When "oligosaccharide composition" or "oligosaccharide single component" is used, it only reflects whether the product with a specific molecular weight is separated separately. They are all hydrolyzed colanic acid or its salt prepared by the technical scheme of the present invention. The former means that the product with a specific molecular weight can be identified but not separated, and the latter means that the product with a specific molecular weight is separated separately. When used in this article, "oligosaccharide" means a product with a weight average molecular weight of less than 1×10 4 Da of colanic acid or its salt, for example, sodium colanate hexasaccharide, sodium colanate dodecasaccharide.

[0050] As used herein, the term "solution" is a broad term, which is not limited to the traditional concept and means a composition. The "solution" may be an aqueous or non-aqueous mixture, for example, it may include a homogeneous or heterogeneous mixture of a polymer and water, it may also include a suspension, a colloid, a nebulized liquid, a gel, an ointment, a cream or the like, and it may also include a fermentation broth.

[0051] In the present invention, "colanate" refers to a salt substance formed by the reaction of colanic acid with any alkaline substance. Depending on the specific type of cation in the colanate, the colanate may be sodium colanate, potassium colanate, magnesium colanate, aluminum colanate, calcium colanate, ammonium colanate, etc. Colanate may also contain more than one cation, such as sodium potassium colanate. One aspect of the present disclosure provides a method for preparing hydrolyzed colanic acid or its salt, comprising the following steps: 1) adding a solution containing colanic acid or its salt to a colanic acid degrading enzyme comprising an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity thereto; and 2) enzyme termination reaction: termination in step 2) is achieved by a terminator, a boiling water bath and / or an ice bath, wherein the terminator is selected from one or more of activated carbon, anhydrous ethanol, methanol, EDTA, trichloroacetic acid and sodium carbonate, preferably activated carbon is used to achieve termination in step 2); thereby obtaining hydrolyzed colanic acid or its salt.

[0052] According to some embodiments of the present disclosure, in the aforementioned method, in step 1), the weight average molecular weight of the colanic acid or its salt is greater than 1×10 4 In some preferred embodiments, the weight average molecular weight of colanic acid or its salt is preferably in the range of about 1×10 4 Da - about 6×10 6 Da, preferably about 5×10 5 Da - about 6×10 6 For example, the weight average molecular weight range of colanic acid or its salt can be any value or any range within the range, and the weight average molecular weight can be about 1×10 4 Da, 2×10 4 Da, 3×10 4 Da, 4×10 4 Da, 5×10 4 Da, 6×10 4 Da, 7×10 4 Da, 8×10 4 Da, 9×10 4 Da, 1×10 5 Da, 2×10 5 Da, 3×10 5 Da, 4×10 5 Da, 5×10 5Yes, 6×10 5 Yes, 7×10 5 Yes, 8×10 5 Yes, 9×10 5 Yes, 1×10 6 Yes, 1.1×10 6 Yes, 1.2×10 6 Yes, 1.3×10 6 Yes, 1.4×10 6 Yes, 1.5×10 6 Yes, 1.6×10 6 Yes, 1.7×10 6 Yes, 1.8×10 6 Yes, 1.9×10 6 Yes, 2×10 6 Yes, 2.1×10 6 Yes, 2.2×10 6 Yes, 2.3×10 6 Yes, 2.4×10 6 Yes, 2.5×10 6 Yes, 2.6×10 6 Yes, 2.7×10 6 Yes, 2.8×10 6 Yes, 2.9×10 6 Yes, 3×10 6 Yes, 3.1×10 6 Yes, 3.2×10 6 Yes, 3.3×10 6 Yes, 3.4×10 6 Yes, 3.5×10 6 Yes, 3.6×10 6 Yes, 3.7×10 6 Yes, 3.8×10 6 Yes, 3.9×10 6 Yes, 4×10 6 Yes, 4.1×10 6 Yes, 4.2×10 6 Yes, 4.3×10 6 Yes, 4.4×10 6 Yes, 4.5×10 6 Yes, 4.6×10 6 Yes, 4.7×10 6 Yes, 4.8×10 6 Yes, 4.9×10 6 Yes, 5×10 6 Yes, 5.1×10 6Da, 5.2×10 6 Da, 5.3×10 6 Da, 5.4×10 6 Da, 5.5×10 6 Da, 5.6×10 6 Da, 5.7×10 6 Da, 5.8×10 6 Da, 5.9×10 6 Da or 6×10 6 Da.

[0053] In the present disclosure, the colanic acid degrading enzyme comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity thereto. For example, the colanic acid degrading enzyme comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity to SEQ ID NO: 1. In some preferred embodiments, the amino acid sequence of the colanic acid degrading enzyme is as shown in SEQ ID NO: 1.

[0054] In some embodiments, in step 1), the colanate salt is sodium colanate.

[0055] In some embodiments, in the aforementioned method, the final reaction concentration of the colanic acid degrading enzyme in step 1) is between about 0.01 mg / L and 10 mg / L. For example, 0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L, 0.10 mg / L, 0.11 mg / L, 0.12 mg / L, 0.13mg / L, 0.14 mg / L, 0.15 mg / L, 0.16 mg / L, 0.17 mg / L, 0.18 mg / L, 0.19 mg / L, 0.20 mg / L, 0.21 mg / L, 0.22 mg / L, 0.23 mg / L, 0.24 mg / L, 0.25 mg / L, 0.26 mg / L, 0.27 mg / L, 0.28mg / L, 0.29 mg / L, 0.30 mg / L, 0.31 mg / L, 0.32 mg / L, 0.33 mg / L, 0.34 mg / L, 0.35 mg / L, 0.36 mg / L, 0.37 mg / L, 0.38 mg / L, 0.39 mg / L, 0.40 mg / L, 0.41 mg / L, 0.42 mg / L, 0.43mg / L, 0.44 mg / L, 0.45 mg / L, 0.46 mg / L, 0.47 mg / L, 0.48 mg / L, 0.49 mg / L, 0.50 mg / L, 0.51 mg / L, 0.52 mg / L, 0.53 mg / L, 0.54 mg / L, 0.55 mg / L, 0.56 mg / L, 0.57 mg / L, 0.58mg / L, 0.59 mg / L, 0.60 mg / L, 0.61 mg / L, 0.62 mg / L, 0.63 mg / L, 0.64 mg / L, 0.65 mg / L, 0.66 mg / L, 0.67 mg / L, 0.68 mg / L, 0.69 mg / L, 0.70 mg / L, 0.71 mg / L, 0.72 mg / L, 0.73 mg / L, 0.74 mg / L, 0.75 mg / L, 0.76 mg / L, 0.77 mg / L, 0.78 mg / L, 0.79 mg / L, 0.80 mg / L, 0.81 mg / L, 0.82 mg / L, 0.83 mg / L, 0.84 mg / L, 0.85 mg / L, 0.86 mg / L, 0.87 mg / L, 0.88mg / L, 0.89 mg / L, 0.90 mg / L, 0.91 mg / L, 0.92 mg / L, 0.93 mg / L, 0.94 mg / L, 0.95 mg / L,0.96 mg / L,0.97 mg / L,0.98 mg / L,0.99 mg / L,1.00 mg / L,1.01 mg / L,1.02 mg / L,1.03mg / L,1.04 mg / L,1.05 mg / L,1.06 mg / L,1.07 mg / L,1.08 mg / L,1.09 mg / L,1.10 mg / L,1.11 mg / L,1.12 mg / L,1.13 mg / L,1.14 mg / L,1.15 mg / L,1.16 mg / L,1.17 mg / L,1.18mg / L,1.19 mg / L,1.20 mg / L,1.21 mg / L,1.22 mg / L,1.23 mg / L,1.24 mg / L,1.25 mg / L,1.26 mg / L,1.27 mg / L,1.28 mg / L,1.29 mg / L,1.30 mg / L,1.31 mg / L,1.32 mg / L,1.33mg / L,1.34 mg / L,1.35 mg / L,1.36 mg / L,1.37 mg / L,1.38 mg / L,1.39 mg / L,1.40 mg / L,1.41 mg / L,1.42 mg / L,1.43 mg / L,1.44 mg / L,1.45 mg / L,1.46 mg / L,1.47 mg / L,1.48mg / L,1.49 mg / L,1.50 mg / L,1.51 mg / L,1.52 mg / L,1.53 mg / L,1.54 mg / L,1.55 mg / L,1.56 mg / L,1.57 mg / L,1.58 mg / L,1.59 mg / L,1.60 mg / L,1.61 mg / L,1.62 mg / L,1.63mg / L,1.64 mg / L,1.65 mg / L,1.66 mg / L,1.67 mg / L,1.68 mg / L,1.69 mg / L,1.70 mg / L,1.71 mg / L,1.72 mg / L,1.73 mg / L,1.74 mg / L,1.75 mg / L,1.76 mg / L,1.77 mg / L,1.78mg / L,1.79 mg / L,1.80 mg / L,1.81 mg / L,1.82 mg / L,1.83 mg / L,1.84 mg / L,1.85 mg / L,1.86 mg / L,1.87 mg / L,1.88 mg / L,1.89 mg / L,1.90 mg / L,1.91 mg / L,1.92 mg / L,1.93mg / L,1.94 mg / L,1.95 mg / L,1.96 mg / L,1.97 mg / L,1.98 mg / L,1.99 mg / L,2.00 mg / L,2.01 mg / L,2.02 mg / L,2.03 mg / L,2.04 mg / L,2.05 mg / L,2.06 mg / L,2.07 mg / L,2.08mg / L,2.09 mg / L,2.10 mg / L,2.11 mg / L,2.12 mg / L,2.13 mg / L,2.14 mg / L,2.15 mg / L,2.16 mg / L,2.17 mg / L,2.18 mg / L,2.19 mg / L,2.20 mg / L,2.21 mg / L,2.22 mg / L,2.23mg / L,2.24 mg / L,2.25 mg / L,2.26 mg / L,2.27 mg / L,2.28 mg / L,2.29 mg / L,2.30 mg / L,2.31 mg / L,2.32 mg / L,2.33 mg / L,2.34 mg / L,2.35 mg / L,2.36 mg / L,2.37 mg / L,2.38mg / L,2.39 mg / L,2.40 mg / L,2.41 mg / L,2.42 mg / L,2.43 mg / L,2.44 mg / L,2.45 mg / L,2.46 mg / L,2.47 mg / L,2.48 mg / L,2.49 mg / L,2.50 mg / L,2.51 mg / L,2.52 mg / L,2.53mg / L,2.54 mg / L,2.55 mg / L,2.56 mg / L,2.57 mg / L,2.58 mg / L,2.59 mg / L,2.60 mg / L,2.61 mg / L,2.62 mg / L,2.63 mg / L,2.64 mg / L,2.65 mg / L,2.66 mg / L,2.67 mg / L,2.68mg / L,2.69 mg / L,2.70 mg / L,2.71 mg / L,2.72 mg / L,2.73 mg / L,2.74 mg / L,2.75 mg / L,2.76 mg / L,2.77 mg / L,2.78 mg / L,2.79 mg / L,2.80 mg / L,2.81 mg / L,2.82 mg / L,2.83mg / L,2.84 mg / L,2.85 mg / L,2.86 mg / L,2.87 mg / L,2.88 mg / L,2.89 mg / L,2.90 mg / L,2.91 mg / L,2.92 mg / L,2.93 mg / L,2.94 mg / L,2.95 mg / L,2.96 mg / L,2.97 mg / L,2.98mg / L,2.99 mg / L,3.00 mg / L,3.01 mg / L,3.02 mg / L,3.03 mg / L,3.04 mg / L,3.05 mg / L,3.06 mg / L,3.07 mg / L,3.08 mg / L,3.09 mg / L,3.10 mg / L,3.11 mg / L,3.12 mg / L,3.13mg / L,3.14 mg / L,3.15 mg / L,3.16 mg / L,3.17 mg / L,3.18 mg / L,3.19 mg / L,3.20 mg / L,3.21 mg / L,3.22 mg / L,3.23 mg / L,3.24 mg / L,3.25 mg / L,3.26 mg / L,3.27 mg / L,3.28mg / L,3.29 mg / L,3.30 mg / L,3.31 mg / L,3.32 mg / L,3.33 mg / L,3.34 mg / L,3.35 mg / L,3.36 mg / L,3.37 mg / L,3.38 mg / L,3.39 mg / L,3.40 mg / L,3.41 mg / L,3.42 mg / L,3.43mg / L,3.44 mg / L,3.45 mg / L,3.46 mg / L,3.47 mg / L,3.48 mg / L,3.49 mg / L,3.50 mg / L,3.51 mg / L,3.52 mg / L,3.53 mg / L,3.54 mg / L,3.55 mg / L,3.56 mg / L,3.57 mg / L,3.58mg / L,3.59 mg / L,3.60 mg / L,3.61 mg / L,3.62 mg / L,3.63 mg / L,3.64 mg / L,3.65 mg / L,3.66 mg / L,3.67 mg / L,3.68 mg / L,3.69 mg / L,3.70 mg / L,3.71 mg / L,3.72 mg / L,3.73mg / L,3.74 mg / L,3.75 mg / L,3.76 mg / L,3.77 mg / L,3.78 mg / L,3.79 mg / L,3.80 mg / L,3.81 mg / L,3.82 mg / L,3.83 mg / L,3.84 mg / L,3.85 mg / L,3.86 mg / L,3.87 mg / L,3.88mg / L,3.89 mg / L,3.90 mg / L,3.91 mg / L,3.92 mg / L,3.93 mg / L,3.94 mg / L,3.95 mg / L,3.96 mg / L,3.97 mg / L,3.98 mg / L,3.99 mg / L,4.00 mg / L,4.01 mg / L,4.02 mg / L,4.03mg / L,4.04 mg / L,4.05 mg / L,4.06 mg / L,4.07 mg / L,4.08 mg / L,4.09 mg / L,4.10 mg / L,4.11 mg / L,4.12 mg / L,4.13 mg / L,4.14 mg / L,4.15 mg / L,4.16 mg / L,4.17 mg / L,4.18mg / L,4.19 mg / L,4.20 mg / L,4.21 mg / L,4.22 mg / L,4.23 mg / L,4.24 mg / L,4.25 mg / L,4.26 mg / L,4.27 mg / L,4.28 mg / L,4.29 mg / L,4.30 mg / L,4.31 mg / L,4.32 mg / L,4.33mg / L,4.34 mg / L,4.35 mg / L,4.36 mg / L,4.37 mg / L,4.38 mg / L,4.39 mg / L,4.40 mg / L,4.41 mg / L,4.42 mg / L,4.43 mg / L,4.44 mg / L,4.45 mg / L,4.46 mg / L,4.47 mg / L,4.48mg / L,4.49 mg / L,4.50 mg / L,4.51 mg / L,4.52 mg / L,4.53 mg / L,4.54 mg / L,4.55 mg / L,4.56 mg / L,4.57 mg / L,4.58 mg / L,4.59 mg / L,4.60 mg / L,4.61 mg / L,4.62 mg / L,4.63mg / L,4.64 mg / L,4.65 mg / L,4.66 mg / L,4.67 mg / L,4.68 mg / L,4.69 mg / L,4.70 mg / L,4.71 mg / L,4.72 mg / L,4.73 mg / L,4.74 mg / L,4.75 mg / L,4.76 mg / L,4.77 mg / L,4.78mg / L,4.79 mg / L,4.80 mg / L,4.81 mg / L,4.82 mg / L,4.83 mg / L,4.84 mg / L,4.85 mg / L,4.86 mg / L,4.87 mg / L,4.88 mg / L,4.89 mg / L,4.90 mg / L,4.91 mg / L,4.92 mg / L,4.93mg / L,4.94 mg / L,4.95 mg / L,4.96 mg / L,4.97 mg / L,4.98 mg / L,4.99 mg / L,5.00 mg / L,5.01 mg / L,5.02 mg / L,5.03 mg / L,5.04 mg / L,5.05 mg / L,5.06 mg / L,5.07 mg / L,5.08mg / L,5.09 mg / L,5.10 mg / L,5.11 mg / L,5.12 mg / L,5.13 mg / L,5.14 mg / L,5.15 mg / L,5.16 mg / L,5.17 mg / L,5.18 mg / L,5.19 mg / L,5.20 mg / L,5.21 mg / L,5.22 mg / L,5.23mg / L,5.24 mg / L,5.25 mg / L,5.26 mg / L,5.27 mg / L,5.28 mg / L,5.29 mg / L,5.30 mg / L,5.31 mg / L,5.32 mg / L,5.33 mg / L,5.34 mg / L,5.35 mg / L,5.36 mg / L,5.37 mg / L,5.38mg / L,5.39 mg / L,5.40 mg / L,5.41 mg / L,5.42 mg / L,5.43 mg / L,5.44 mg / L,5.45 mg / L,5.46 mg / L,5.47 mg / L,5.48 mg / L,5.49 mg / L,5.50 mg / L,5.51 mg / L,5.52 mg / L,5.53mg / L,5.54 mg / L,5.55 mg / L,5.56 mg / L,5.57 mg / L,5.58 mg / L,5.59 mg / L,5.60 mg / L,5.61 mg / L,5.62 mg / L,5.63 mg / L,5.64 mg / L,5.65 mg / L,5.66 mg / L,5.67 mg / L,5.68mg / L,5.69 mg / L,5.70 mg / L,5.71 mg / L,5.72 mg / L,5.73 mg / L,5.74 mg / L,5.75 mg / L,5.76 mg / L,5.77 mg / L,5.78 mg / L,5.79 mg / L,5.80 mg / L,5.81 mg / L,5.82 mg / L,5.83mg / L,5.84 mg / L,5.85 mg / L,5.86 mg / L,5.87 mg / L,5.88 mg / L,5.89 mg / L,5.90 mg / L,5.91 mg / L,5.92 mg / L,5.93 mg / L,5.94 mg / L,5.95 mg / L,5.96 mg / L,5.97 mg / L,5.98mg / L,5.99 mg / L,6.00 mg / L,6.01 mg / L,6.02 mg / L,6.03 mg / L,6.04 mg / L,6.05 mg / L,6.06 mg / L,6.07 mg / L,6.08 mg / L,6.09 mg / L,6.10 mg / L,6.11 mg / L,6.12 mg / L,6.13mg / L,6.14 mg / L,6.15 mg / L,6.16 mg / L,6.17 mg / L,6.18 mg / L,6.19 mg / L,6.20 mg / L,6.21 mg / L,6.22 mg / L,6.23 mg / L,6.24 mg / L,6.25 mg / L,6.26 mg / L,6.27 mg / L,6.28mg / L,6.29 mg / L,6.30 mg / L,6.31 mg / L,6.32 mg / L,6.33 mg / L,6.34 mg / L,6.35 mg / L,6.36 mg / L,6.37 mg / L,6.38 mg / L,6.39 mg / L,6.40 mg / L,6.41 mg / L,6.42 mg / L,6.43mg / L,6.44 mg / L,6.45 mg / L,6.46 mg / L,6.47 mg / L,6.48 mg / L,6.49 mg / L,6.50 mg / L,6.51 mg / L,6.52 mg / L,6.53 mg / L,6.54 mg / L,6.55 mg / L,6.56 mg / L,6.57 mg / L,6.58mg / L,6.59 mg / L,6.60 mg / L,6.61 mg / L,6.62 mg / L,6.63 mg / L,6.64 mg / L,6.65 mg / L,6.66 mg / L,6.67 mg / L,6.68 mg / L,6.69 mg / L,6.70 mg / L,6.71 mg / L,6.72 mg / L,6.73mg / L,6.74 mg / L,6.75 mg / L,6.76 mg / L,6.77 mg / L,6.78 mg / L,6.79 mg / L,6.80 mg / L,6.81 mg / L,6.82 mg / L,6.83 mg / L,6.84 mg / L,6.85 mg / L,6.86 mg / L,6.87 mg / L,6.88mg / L,6.89 mg / L,6.90 mg / L,6.91 mg / L,6.92 mg / L,6.93 mg / L,6.94 mg / L,6.95 mg / L,6.96 mg / L,6.97 mg / L,6.98 mg / L,6.99 mg / L,7.00 mg / L,7.01 mg / L,7.02 mg / L,7.03mg / L,7.04 mg / L,7.05 mg / L,7.06 mg / L,7.07 mg / L,7.08 mg / L,7.09 mg / L,7.10 mg / L,7.11 mg / L,7.12 mg / L,7.13 mg / L,7.14 mg / L,7.15 mg / L,7.16 mg / L,7.17 mg / L,7.18mg / L,7.19 mg / L,7.20 mg / L,7.21 mg / L,7.22 mg / L,7.23 mg / L,7.24 mg / L,7.25 mg / L,7.26 mg / L,7.27 mg / L,7.28 mg / L,7.29 mg / L,7.30 mg / L,7.31 mg / L,7.32 mg / L,7.33mg / L,7.34 mg / L,7.35 mg / L,7.36 mg / L,7.37 mg / L,7.38 mg / L,7.39 mg / L,7.40 mg / L,7.41 mg / L,7.42 mg / L,7.43 mg / L,7.44 mg / L,7.45 mg / L,7.46 mg / L,7.47 mg / L,7.48mg / L,7.49 mg / L,7.50 mg / L,7.51 mg / L,7.52 mg / L,7.53 mg / L,7.54 mg / L,7.55 mg / L,7.56 mg / L,7.57 mg / L,7.58 mg / L,7.59 mg / L,7.60 mg / L,7.61 mg / L,7.62 mg / L,7.63mg / L,7.64 mg / L,7.65 mg / L,7.66 mg / L,7.67 mg / L,7.68 mg / L,7.69 mg / L,7.70 mg / L,7.71 mg / L,7.72 mg / L,7.73 mg / L,7.74 mg / L,7.75 mg / L,7.76 mg / L,7.77 mg / L,7.78mg / L,7.79 mg / L,7.80 mg / L,7.81 mg / L,7.82 mg / L,7.83 mg / L,7.84 mg / L,7.85 mg / L,7.86 mg / L,7.87 mg / L,7.88 mg / L,7.89 mg / L,7.90 mg / L,7.91 mg / L,7.92 mg / L,7.93mg / L,7.94 mg / L,7.95 mg / L,7.96 mg / L,7.97 mg / L,7.98 mg / L,7.99 mg / L,8.00 mg / L,8.01 mg / L,8.02 mg / L,8.03 mg / L,8.04 mg / L,8.05 mg / L,8.06 mg / L,8.07 mg / L,8.08mg / L,8.09 mg / L,8.10 mg / L,8.11 mg / L,8.12 mg / L,8.13 mg / L,8.14 mg / L,8.15 mg / L,8.16 mg / L,8.17 mg / L,8.18 mg / L,8.19 mg / L,8.20 mg / L,8.21 mg / L,8.22 mg / L,8.23mg / L,8.24 mg / L,8.25 mg / L,8.26 mg / L,8.27 mg / L,8.28 mg / L,8.29 mg / L,8.30 mg / L,8.31 mg / L,8.32 mg / L,8.33 mg / L,8.34 mg / L,8.35 mg / L,8.36 mg / L,8.37 mg / L,8.38mg / L,8.39 mg / L,8.40 mg / L,8.41 mg / L,8.42 mg / L,8.43 mg / L,8.44 mg / L,8.45 mg / L,8.46 mg / L,8.47 mg / L,8.48 mg / L,8.49 mg / L,8.50 mg / L,8.51 mg / L,8.52 mg / L,8.53mg / L,8.54 mg / L,8.55 mg / L,8.56 mg / L,8.57 mg / L,8.58 mg / L,8.59 mg / L,8.60 mg / L,8.61 mg / L,8.62 mg / L,8.63 mg / L,8.64 mg / L,8.65 mg / L,8.66 mg / L,8.67 mg / L,8.68mg / L,8.69 mg / L,8.70 mg / L,8.71 mg / L,8.72 mg / L,8.73 mg / L,8.74 mg / L,8.75 mg / L,8.76 mg / L,8.77 mg / L,8.78 mg / L,8.79 mg / L,8.80 mg / L,8.81 mg / L,8.82 mg / L,8.83mg / L,8.84 mg / L,8.85 mg / L,8.86 mg / L,8.87 mg / L,8.88 mg / L,8.89 mg / L,8.90 mg / L,8.91 mg / L,8.92 mg / L,8.93 mg / L,8.94 mg / L,8.95 mg / L,8.96 mg / L,8.97 mg / L,8.98mg / L,8.99 mg / L,9.00 mg / L,9.01 mg / L,9.02 mg / L,9.03 mg / L,9.04 mg / L,9.05 mg / L,9.06 mg / L,9.07 mg / L,9.08 mg / L,9.09 mg / L,9.10 mg / L,9.11 mg / L,9.12 mg / L,9.13mg / L,9.14 mg / L,9.15 mg / L,9.16 mg / L,9.17 mg / L,9.18 mg / L,9.19 mg / L,9.20 mg / L,9.21 mg / L,9.22 mg / L,9.23 mg / L,9.24 mg / L,9.25 mg / L,9.26 mg / L,9.27 mg / L,9.28mg / L,9.29 mg / L,9.30 mg / L,9.31 mg / L,9.32 mg / L,9.33 mg / L,9.34 mg / L,9.35 mg / L,9.36 mg / L,9.37 mg / L,9.38 mg / L,9.39 mg / L,9.40 mg / L,9.41 mg / L,9.42 mg / L,9.43mg / L,9.44 mg / L,9.45 mg / L,9.46 mg / L,9.47 mg / L,9.48 mg / L,9.49 mg / L,9.50 mg / L,9.51 mg / L,9.52 mg / L,9.53 mg / L,9.54 mg / L,9.55 mg / L,9.56 mg / L,9.57 mg / L,9.58mg / L,9.59 mg / L,9.60 mg / L,9.61 mg / L,9.62 mg / L,9.63 mg / L,9.64 mg / L,9.65 mg / L,9.66 mg / L,9.67 mg / L,9.68 mg / L,9.69 mg / L,9.70 mg / L,9.71 mg / L,9.72 mg / L,9.73mg / L,9.74 mg / L,9.75 mg / L,9.76 mg / L,9.77 mg / L,9.78 mg / L,9.79 mg / L,9.80 mg / L,9.81 mg / L,9.82 mg / L,9.83 mg / L,9.84 mg / L,9.85 mg / L,9.86 mg / L,9.87 mg / L,9.88mg / L,9.89 mg / L,9.90 mg / L,9.91 mg / L,9.92 mg / L,9.93 mg / L,9.94 mg / L,9.95 mg / L, 9.96 mg / L, 9.97 mg / L, 9.98 mg / L, 9.99 mg / L or 10.00 mg / L. .

[0056] In some embodiments, in the aforementioned method, the reaction temperature in step 1) is between about 10 °C and about 70 °C, preferably, the reaction temperature is between about 20-50 °C, for example, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C or 50 °C.

[0057] In some embodiments, in the aforementioned method, the reaction time in step 1) is about 1-20 h, for example, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h or 20 h.

[0058] In some embodiments, in the aforementioned method, the pH of the reaction solution in step 1) is about 3 to about 9, such as 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9. Preferably, the pH of the reaction solution is about 5 to about 8.

[0059] In some embodiments, the above method further comprises the steps of 3) filtration purification; 4) concentration and desalination; and 5) drying.

[0060] In some embodiments, the above method further comprises 6) multiple enzymatic hydrolysis, such as secondary enzymatic hydrolysis, tertiary enzymatic hydrolysis, etc.

[0061] In other embodiments, the activated carbon content is about 0.005 wt % or more. The activated carbon content is the mass relative to the volume of the colanic acid solution, for example, 1 wt % means 1 L of colanic acid solution plus 10 g of activated carbon. In some preferred embodiments, the activated carbon content is 0.005 wt%-1 wt%. In some more preferred embodiments, the activated carbon content is about 0.01 wt%-5 wt%, which can be any value within this range, for example, the activated carbon content is about 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, 0.15 wt%, 0.16 wt%, 0.17 wt%, 0.18 wt%, 0.19 wt%, 0.2 wt%, 0.21 wt%, 0.22 wt%, 0.23 wt%, 0.24 wt%, 0.25 wt%, 0.26 wt%, 0.27 wt%, 0.28wt%, 0.29 wt%, 0.3 wt%, 0.31 wt%, 0.32 wt%, 0.33 wt%, 0.34 wt%, 0.35 wt%, 0.36 wt%, 0.37 wt%, 0.38 wt%, 0.39 wt%, 0.4 wt%, 0.41 wt%, 0.42 wt%, 0.43 wt%, 0.44 wt%, 0.45wt%, 0.46 wt%, 0.47 wt%, 0.48 wt%, 0.49 wt%, 0.5 wt%, 0.51 wt%, 0.52 wt%, 0.53 wt%, 0.54 wt%, 0.55 wt%, 0.56 wt%, 0.57 wt%, 0.58 wt%, 0.59 wt%, 0.6 wt%, 0.61 wt%, 0.62wt%, 0.63 wt%, 0.64 wt%, 0.65 wt%, 0.66 wt%, 0.67 wt%, 0.68 wt%, 0.69 wt%, 0.7 wt%, 0.71 wt%, 0.72 wt%, 0.73 wt%, 0.74 wt%, 0.75 wt%, 0.76 wt%, 0.77 wt%, 0.78 wt%, 0.79wt%, 0.8 wt%, 0.81 wt%, 0.82 wt%, 0.83 wt%, 0.84 wt%, 0.85 wt%, 0.86 wt%, 0.87 wt%, 0.88 wt%, 0.89 wt%, 0.9 wt%, 0.91 wt%, 0.92 wt%, 0.93 wt%, 0.94 wt%, 0.95 wt%, 0.96wt%, 0.97 wt%, 0.98 wt%, 0.99 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5wt%, 4.6wt%, 4.7wt%, 4.8wt%, 4.9wt% or 5wt%. .

[0062] In some embodiments, in step 3), the pH is adjusted to about 3.0-6.5 before filtration; in some preferred embodiments, the pH is adjusted to about 3.0-about 6.5 before filtration, and in some preferred embodiments, the pH is adjusted to about 3.0-about 6.0 before filtration. For example, the pH can be adjusted to about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5.

[0063] In some embodiments, in step 5), the drying is achieved by spray drying, freeze drying or vacuum drying.

[0064] In some embodiments, in the aforementioned method, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 3×10 6 In some preferred embodiments, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 2×10 6 The weight average molecular weight of the hydrolyzed colanic acid or its salt can be any value or any range within the range, for example, 1×10 3 Da, 2×10 3 Da, 3×10 3 Da, 4×10 3 Da, 5×103 Yes, 6×10 3 Yes, 7×10 3 Yes, 8×10 3 Yes, 9×10 3 Yes, 1×10 4 Yes, 2×10 4 Yes, 3×10 4 Yes, 4×10 4 Yes, 5×10 4 Yes, 6×10 4 Yes, 7×10 4 Yes, 8×10 4 Yes, 9×10 4 Yes, 1.0×10 5 Yes, 1.1×10 5 Yes, 1.2×10 5 Yes, 1.3×10 5 Yes, 1.4×10 5 Yes, 1.5×10 5 Yes, 1.6×10 5 Yes, 1.7×10 5 Yes, 1.8×10 5 Yes, 1.9×10 5 Yes, 2×10 5 Yes, 2.1×10 5 Yes, 2.2×10 5 Yes, 2.3×10 5 Yes, 2.4×10 5 Yes, 2.5×10 5 Yes, 2.6×10 5 Yes, 2.7×10 5 Yes, 2.8×10 5 Yes, 2.9×10 5 Yes, 3×10 5 Yes, 3.1×10 5 Yes, 3.2×10 5 Yes, 3.3×10 5 Yes, 3.4×10 5 Yes, 3.5×10 5 Yes, 3.6×10 5 Yes, 3.7×10 5 Yes, 3.8×10 5 Yes, 3.9×10 5 Yes, 4×10 5 Yes, 4.1×10 5 Yes, 4.2×10 5 Yes, 4.3×105 Yes, 4.4×10 5 Yes, 4.5×10 5 Yes, 4.6×10 5 Yes, 4.7×10 5 Yes, 4.8×10 5 Yes, 4.9×10 5 Yes, 5×10 5 Yes, 5.1×10 5 Yes, 5.2×10 5 Yes, 5.3×10 5 Yes, 5.4×10 5 Yes, 5.5×10 5 Yes, 5.6×10 5 Yes, 5.7×10 5 Yes, 5.8×10 5 Yes, 5.9×10 5 Yes, 6×10 5 Yes, 6.1×10 5 Yes, 6.2×10 5 Yes, 6.3×10 5 Yes, 6.4×10 5 Yes, 6.5×10 5 Yes, 6.6×10 5 Yes, 6.7×10 5 Yes, 6.8×10 5 Yes, 6.9×10 5 Yes, 7×10 5 Yes, 7.1×10 5 Yes, 7.2×10 5 Yes, 7.3×10 5 Yes, 7.4×10 5 Yes, 7.5×10 5 Yes, 7.6×10 5 Yes, 7.7×10 5 Yes, 7.8×10 5 Yes, 7.9×10 5 Yes, 8×10 5 Yes, 8.1×10 5 Yes, 8.2×10 5 Yes, 8.3×10 5 Yes, 8.4×10 5 Yes, 8.5×10 5 Yes, 8.6×10 5 Yes, 8.7×10 5 Yes, 8.8×10 5Da, 8.9×10 5 Da, 9×10 5 Da, 9.1×10 5 Da, 9.2×10 5 Da, 9.3×10 5 Da, 9.4×10 5 Da, 9.5×10 5 Da, 9.6×10 5 Da, 9.7×10 5 Da, 9.8×10 5 Da, 9.9×10 5 Da, 1.0×10 6 Da, 1.1×10 6 Da, 1.2×10 6 Da, 1.3×10 6 Da, 1.4×10 6 Da, 1.5×10 6 Da, 1.6×10 6 Da, 1.7×10 6 Da, 1.8×10 6 Da, 1.9×10 6 Da, 2×10 6 Da, 2.1×10 6 Da, 2.2×10 6 Da, 2.3×10 6 Da, 2.4×10 6 Da, 2.5×10 6 Da, 2.6×10 6 Da, 2.7×10 6 Da, 2.8×10 6 Da, 2.9×10 6 Da or 3×10 6 Da.

[0065] In some embodiments, in the aforementioned methods, the hydrolyzed colanic acid or its salt comprises acetylation and / or pyruvic acid modification.

[0066] In some embodiments, in the aforementioned method, the acetylation modification rate of the hydrolyzed colanic acid or its salt is about 80-100%, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0067] In some embodiments, in the aforementioned method, the residual endotoxin in the hydrolyzed colanic acid or its salt is less than about 0.5 EU / mg. In some preferred embodiments, the residual endotoxin is less than about 0.1 EU / mg.

[0068] In some embodiments, in the aforementioned method, the protein residue in the hydrolyzed colanic acid or its salt is less than about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%, and in some preferred embodiments, the protein residue is less than about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, or about 0.01%.

[0069] In some embodiments, in the aforementioned method, the heavy metal residue in the hydrolyzed colanic acid or its salt is less than about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg.

[0070] In some embodiments, in the aforementioned method, the hydrolyzed colanic acid or its salt may also be colanic acid hexasaccharide or its salt, and / or colanic acid dodecasaccharide or its salt. For example, the hydrolyzed colanic acid or its salt includes but is not limited to a composition comprising colanic acid hexasaccharide or its salt, and colanic acid dodecasaccharide or its salt; or colanic acid hexasaccharide or its salt, or colanic acid dodecasaccharide or its salt that can be separated separately.

[0071] Another aspect of the present disclosure provides hydrolyzed colanic acid or a salt thereof prepared according to the aforementioned method.

[0072] In some embodiments, the weight average molecular weight of the hydrolyzed colanic acid or its salt prepared according to the aforementioned method is in the range of about 1×10 3 Da - about 5×10 6 In some preferred embodiments, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da - about 2×10 6 In some more preferred embodiments, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da-about 1.5×10 6 The weight average molecular weight of the hydrolyzed colanic acid or its salt can be any value or any range within the range, for example, 1×10 3 Da, 2×10 3 Da, 3×10 3 Da, 4×10 3 Da, 5×103 Yes, 6×10 3 Yes, 7×10 3 Yes, 8×10 3 Yes, 9×10 3 Yes, 1×10 4 Yes, 2×10 4 Yes, 3×10 4 Yes, 4×10 4 Yes, 5×10 4 Yes, 6×10 4 Yes, 7×10 4 Yes, 8×10 4 Yes, 9×10 4 Yes, 1.0×10 5 Yes, 1.1×10 5 Yes, 1.2×10 5 Yes, 1.3×10 5 Yes, 1.4×10 5 Yes, 1.5×10 5 Yes, 1.6×10 5 Yes, 1.7×10 5 Yes, 1.8×10 5 Yes, 1.9×10 5 Yes, 2×10 5 Yes, 2.1×10 5 Yes, 2.2×10 5 Yes, 2.3×10 5 Yes, 2.4×10 5 Yes, 2.5×10 5 Yes, 2.6×10 5 Yes, 2.7×10 5 Yes, 2.8×10 5 Yes, 2.9×10 5 Yes, 3×10 5 Yes, 3.1×10 5 Yes, 3.2×10 5 Yes, 3.3×10 5 Yes, 3.4×10 5 Yes, 3.5×10 5 Yes, 3.6×10 5 Yes, 3.7×10 5 Yes, 3.8×10 5 Yes, 3.9×10 5 Yes, 4×10 5 Yes, 4.1×10 5 Yes, 4.2×10 5 Yes, 4.3×105 Yes, 4.4×10 5 Yes, 4.5×10 5 Yes, 4.6×10 5 Yes, 4.7×10 5 Yes, 4.8×10 5 Yes, 4.9×10 5 Yes, 5×10 5 Yes, 5.1×10 5 Yes, 5.2×10 5 Yes, 5.3×10 5 Yes, 5.4×10 5 Yes, 5.5×10 5 Yes, 5.6×10 5 Yes, 5.7×10 5 Yes, 5.8×10 5 Yes, 5.9×10 5 Yes, 6×10 5 Yes, 6.1×10 5 Yes, 6.2×10 5 Yes, 6.3×10 5 Yes, 6.4×10 5 Yes, 6.5×10 5 Yes, 6.6×10 5 Yes, 6.7×10 5 Yes, 6.8×10 5 Yes, 6.9×10 5 Yes, 7×10 5 Yes, 7.1×10 5 Yes, 7.2×10 5 Yes, 7.3×10 5 Yes, 7.4×10 5 Yes, 7.5×10 5 Yes, 7.6×10 5 Yes, 7.7×10 5 Yes, 7.8×10 5 Yes, 7.9×10 5 Yes, 8×10 5 Yes, 8.1×10 5 Yes, 8.2×10 5 Yes, 8.3×10 5 Yes, 8.4×10 5 Yes, 8.5×10 5 Yes, 8.6×10 5 Yes, 8.7×10 5 Yes, 8.8×10 5Yes, 8.9×10 5 Yes, 9×10 5 Yes, 9.1×10 5 Yes, 9.2×10 5 Yes, 9.3×10 5 Yes, 9.4×10 5 Yes, 9.5×10 5 Yes, 9.6×10 5 Yes, 9.7×10 5 Yes, 9.8×10 5 Yes, 9.9×10 5 Yes, 1.0×10 6 Yes, 1.1×10 6 Yes, 1.2×10 6 Yes, 1.3×10 6 Yes, 1.4×10 6 Yes, 1.5×10 6 Yes, 1.6×10 6 Yes, 1.7×10 6 Yes, 1.8×10 6 Yes, 1.9×10 6 Yes, 2×10 6 Yes, 2.1×10 6 Yes, 2.2×10 6 Yes, 2.3×10 6 Yes, 2.4×10 6 Yes, 2.5×10 6 Yes, 2.6×10 6 Yes, 2.7×10 6 Yes, 2.8×10 6 Yes, 2.9×10 6 Yes, 3×10 6 Yes, 3.1×10 6 Yes, 3.2×10 6 Yes, 3.3×10 6 Yes, 3.4×10 6 Yes, 3.5×10 6 Yes, 3.6×10 6 Yes, 3.7×10 6 Yes, 3.8×10 6 Yes, 3.9×10 6 Yes, 4×10 6 Yes, 4.1×10 6 Yes, 4.2×10 6 Yes, 4.3×10 6 Yes, 4.4×106 Da, 4.5×10 6 Da, 4.6×10 6 Da, 4.7×10 6 Da, 4.8×10 6 Da, 4.9×10 6 Da, or 5×10 6 Da.

[0073] In some embodiments, the hydrolyzed colanic acid or its salt prepared according to the aforementioned method comprises acetylation and / or pyruvic acid modification.

[0074] In some embodiments, the acetylation modification rate of the hydrolyzed colanic acid or its salt prepared according to the aforementioned method is about 80-100%, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0075] In some embodiments, the hydrolyzed colanic acid or its salt prepared according to the aforementioned method has a residual endotoxin of less than about 0.5 EU / mg. In some preferred embodiments, the residual endotoxin is less than about 0.1 EU / mg.

[0076] In some embodiments, the protein residue in the hydrolyzed colanic acid or its salt prepared according to the aforementioned method is less than about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, or about 0.01%. In some preferred embodiments, the protein residue is less than about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, or about 0.01%.

[0077] In some embodiments, the residual heavy metal content in the hydrolyzed colanic acid or its salt prepared according to the aforementioned method is less than about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, or about 1 mg / kg.

[0078] In some embodiments, the hydrolyzed colanic acid or its salt prepared according to the above method may also be colanic acid hexasaccharide or its salt, and / or colanic acid dodecasaccharide or its salt. For example, the hydrolyzed colanic acid or its salt includes but is not limited to a composition comprising colanic acid hexasaccharide or its salt, and colanic acid dodecasaccharide or its salt; or colanic acid hexasaccharide or its salt, or colanic acid dodecasaccharide or its salt that can be separated separately.

[0079] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0080] The colanic acid fermentation broth used in the present invention is provided by Shenzhen Boyin Biological Co., Ltd. using an engineering strain constructed by Escherichia coli (refer to Chinese invention patent application CN115287314A), and the colanic acid degrading enzyme is provided by Shenzhen Boyin Biological Co., Ltd. using an engineering strain constructed by Escherichia coli (refer to Chinese invention patent application CN118685474A) and fermented and purified.

[0081] Test Method Protein content was determined by the Lorry method, heavy metal residues were determined by ICP-MS, endotoxins were determined by the Limulus amebocyte lysate gel method, and acetylation was quantified by the CH 3 Peak integration comparison.

[0082] Purity and weight-average molecular weight analysis method of hydrolyzed sodium colanate: The instrument is Agilent 1260II, differential detector, chromatographic column is PolySep-GFC-P6000 in series with PolySep-GFC-P4000 or PolySep-GFC-P4000 in series with PolySep-GFC-P2000. The mobile phase is selected as: 20 mM sodium chloride, flow rate is 0.6 mL / min, isocratic for 50 min.

[0083] Example Example 1-1 A certain amount of colanic acid fermentation broth was taken, a flocculant was added, the pH value was adjusted to an alkaline solution, diluted and allowed to stand, sterilized, adsorbed with an adsorbent under acidic conditions and filtered, anhydrous ethanol was added for precipitation, the precipitate was collected, and repeatedly washed with a mixed solution of sodium chloride and ethanol (the volume ratio of sodium chloride to ethanol was about 2:8~4:6), then dehydrated with anhydrous ethanol, and finally dried to obtain 1.0 kg of a 5.0×10 6 The crude pure sodium colanate powder of Da has an acetylation modification rate of 99.7%.

[0084] Take 5 portions of the above prepared solution with a molecular weight of 5.0×10 6 0.01 kg of sodium colanate powder of Da was added to 1 L of pure water and stirred to dissolve. The water temperature was controlled at 50 °C. After fully dissolved, the temperature was lowered to 25 °C, and the pH was adjusted to 8.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 0.2 mg / L, and the reaction was stirred for 1 h. After the reaction, the five samples were treated as follows: (1) Activated carbon termination: Add activated carbon (0.5%wt) and adjust the pH to 4.5 for 1h, then collect the filtrate by plate and frame filtration; (2) High temperature termination: heating to 75 °C for 1 h, then cooling to room temperature; (3) Termination with anhydrous ethanol: Add 2 L of anhydrous ethanol and treat for 1 h; (4) Protease termination: add proteinase K at a final concentration of 125 mg / L and treat for 1 h; (5) No action is taken.

[0085] 0.2 kg of sodium chloride powder was added to each of the five solutions, stirred and dissolved, and then finely filtered with a 0.65 μm filter. Then, 20 L of anhydrous ethanol was used for alcohol precipitation, and the precipitate was collected and repeatedly washed with a mixed solution of sodium chloride and ethanol of the same concentration for three times, and then dehydrated with anhydrous ethanol for three times, and finally vacuum dried to obtain hydrolyzed sodium colanate powders F1-F5 as shown in Table 1 below.

[0086] The data in the table show that the results of activated carbon treatment are superior to those of other termination methods. Failure to terminate the enzyme activity will lead to further reduction of molecular weight in the subsequent treatment process; high temperature treatment is prone to acetylation shedding, and ethanol treatment is prone to danger when used in non-specific containers; protease treatment termination is not ideal and introduces protein; while activated carbon can adsorb impurities and enzymes at the same time, with obvious termination effect and convenient treatment, so it is more ideal to use activated carbon as a termination method.

[0087] Table 1 Effects of different termination methods

[0088] Example 1-2 Take 1 portion of the sample prepared in Example 1-1 with a molecular weight of 5.0×10 6 1.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, and the pH was adjusted to 8.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 1.0 mg / L, and the reaction was stirred for 3 h. After the reaction was completed, activated carbon (0.25% wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filter pressing. The filtrate was finely filtered with a 0.65 μm capsule filter, and then the solution was replaced with purified water. Finally, it was spray dried to obtain a weight average molecular weight of 1.2×10 4 Da finished product of hydrolyzed sodium colanate.

[0089] Comparative Example 1 Take 1 portion of the sample prepared in Example 1-1 with a molecular weight of 5.0×10 60.1 kg of sodium colanate powder of Da was added to 10 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, and the pH was adjusted to 8.0 with sodium phosphate, and then the temperature was raised to 134 °C for reaction for 1 h. After the reaction, activated carbon (0.25% wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filtration. The filtrate was finely filtered with a 0.65 μm capsule filter, and then the liquid was replaced with purified water, and finally spray dried to obtain a weight average molecular weight of 1.1×10 4 Da finished product of hydrolyzed sodium colanate.

[0090] From Examples 1-2 and Comparative Example 1, it can be seen that high temperature can also achieve the purpose of degrading colanic acid, but it will affect the acetylation modification of hydrolyzed sodium colanate; while the enzymatic hydrolysis conditions are mild, and the acetylation modification is completely retained after the reaction.

[0091] Table 2 Determination results of products of sodium colanate with the same molecular weight after degradation by different degradation methods

[0092] It should be noted that the “yield of finished product” is the percentage of the amount of the final product relative to the amount of the reaction substrate (such as powder, fermentation broth, etc.). For example, 5.0×10 6 Da sodium colanate powder 1.0 kg, final product yield 0.701 kg, the yield is 70.1%.

[0093] Example 2 Take 5 portions of the product prepared in Example 1-1 with a molecular weight of 5.0×10 6 0.1 kg of sodium colanate powder of Da was added to 10 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, and the pH was adjusted to 8.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentrations 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, and 0.2 mg / L, respectively, and stirred for 20 h, 10 h, 4 h, 2 h, and 1 h, respectively. After the reaction was completed, activated carbon (0.25% wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filtration. The filtrate was finely filtered with a 0.65 μm bladder filter, and then 20 L of anhydrous ethanol was added while stirring to precipitate the hydrolyzed sodium colanate. The precipitate was collected and washed three times with a mixed solution of sodium chloride and ethanol of the same concentration, dehydrated with anhydrous ethanol three times, and finally vacuum dried to obtain the hydrolyzed sodium colanate finished products F1'-F5'. The data are shown in Table 3 below.

[0094] The results showed that when the final enzyme concentration was in the range of 0.01 mg / L-0.2 mg / L and the reaction time was in the range of 1 h-20 h, products with molecular weight, protein residue, acetylation modification degree and finished product yield within the expected range could be obtained.

[0095] Table 3 Determination results of products of sodium colanate with the same molecular weight after degradation at different enzyme dosages and reaction times

[0096] Example 3 Take 8 portions of the 5.0×10 6 0.1 kg of sodium colanate powder of Da was added to 10 L pure water and stirred to dissolve. The water temperature was controlled at 50 °C. After fully dissolved, the temperature was lowered to 25 °C. The pH was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 0.1 mg / L. The reaction was stirred and the molecular weight was controlled at 0.6×10 6 ~1.0×10 6 The reaction was terminated within the range of Da. After the reaction was completed, activated carbon (0.25%wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filtration. The filtrate was finely filtered with a 0.65μm capsule filter, and then 20 L of anhydrous ethanol was added while stirring to precipitate the hydrolyzed sodium colanate. The precipitate was collected and washed repeatedly with a mixed solution of sodium chloride and ethanol of the same concentration for 3 times, dehydrated with anhydrous ethanol for 3 times, and finally vacuum dried to obtain the finished product of hydrolyzed sodium colanate (F1''-F8''), the data are shown in Table 4 below.

[0097] From the data in the table, we can see that acetylation is lost under pH 10, and the desired product can be obtained under pH 3-9. Among them, the yield is slightly lower under pH 3-4, the reaction is slow and the molecular weight is high under pH 8-9, and the enzymatic reaction pH 5-7 is excellent in terms of product molecular weight, protein residue, endotoxin residue, heavy metals, acetylation modification and pyruvic acid modification, and finished product yield.

[0098] Table 4 Determination results of products of sodium colanate with the same molecular weight under different pH conditions

[0099] Example 4 Take 9 portions of the 5.0×10 60.1 kg of sodium colanate powder of Da was added to 10 L pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was adjusted to 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C, and the pH was adjusted to 8.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 0.1 mg / L, and the reaction was stirred to control the molecular weight at 0.6×10 6 ~1.0×10 6 The reaction was terminated within the range of Da. After the reaction was completed, activated carbon (0.25%wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filtration. The filtrate was finely filtered with a 0.65 μm capsule filter, and then 20 L of anhydrous ethanol was added while stirring to hydrolyze the sodium colanate precipitate. The precipitate was collected and washed repeatedly with a mixed solution of sodium chloride and ethanol of the same concentration for 3 times, dehydrated with anhydrous ethanol for 3 times, and finally vacuum dried to obtain the finished sodium colanate (F1'''-F9'''), the data are shown in Table 5 below.

[0100] The results showed that acetylation modification was easily lost when the temperature was higher than 70 ℃.

[0101] Table 5 Determination results of products of sodium colanate with the same molecular weight under different temperature conditions

[0102] Example 5: Preparation of Sodium Colanate Products with High Weight Average Molecular Weight Range Take 3 portions of the product prepared in Example 1-1 with a molecular weight of 5.0×10 6 1.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 ℃. After being fully dissolved, the temperature was lowered to 25 ℃, and the pH was adjusted to 8.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentrations 0.1 mg / L, 0.2 mg / L, and 0.4 mg / L, respectively, and the reaction was stirred for 1.0 h. After the reaction was completed, activated carbon (0.25% wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filter pressing. The filtrate was finely filtered with a 0.65 μm capsule filter, and then 200 L of anhydrous ethanol was added while stirring to hydrolyze the sodium colanate precipitate. The collected precipitate was repeatedly washed with a mixed solution of sodium chloride and ethanol at the same concentration for 3 times, dehydrated with anhydrous ethanol for 3 times, and finally vacuum dried to obtain 1.2×10 6 Da, 7.6×10 5 Da, 4.2×10 5 Da's sodium colanate powder products (E1-E3). Its HPLC chromatogram is as follows Figure 1 shown.

[0103] It should be noted that different purification methods are used in the process of hydrolyzing colanic acid or its salts to obtain products with different molecular weights. For example, for the hydrolyzed sodium colanate with a weight average molecular weight greater than 5.0×10 5 When Da is less than 5.0×10 5 Da's products are better precipitated with pure water than with alcohol.

[0104] Example 6: Preparation of Sodium Colanate Product in Low Weight Average Molecular Weight Range Take 5 portions of the product prepared in Example 1-1 with a molecular weight of 5.0×10 6 1.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, and the pH was adjusted to 8.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentrations 0.3 mg / L, 0.5 mg / L, 1.0 mg / L, 2.5 mg / L, and 5 mg / L, respectively, and stirred for 2.0 h. After the reaction was completed, activated carbon (0.25% wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filter pressing. The filtrate was finely filtered with a 0.65 μm capsule filter, and then the solution was replaced with purified water, and finally spray dried to obtain the hydrolyzed sodium colanate products (E4-E8).

[0105] Table 6 Determination results of hydrolyzed sodium colanate or hydrolyzed sodium colanate oligosaccharides with different molecular weights

[0106] Example 7: Preparation of sodium colanate oligosaccharide composition Example 7-1 Take the molecular weight of 5.0×10 61.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 40 °C. After being fully dissolved, the temperature was lowered to 20 °C, and the pH was adjusted to 6.5 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make its final concentration 2.5 mg / L, and the reaction was stirred for 4 h. After the reaction was completed, activated carbon (0.5% wt) was added to the solution, and the pH was adjusted to 4.5, and then the filtrate was collected by plate and frame filtration. The filtrate was concentrated 20 times by nanofiltration, and then the liquid was replaced with purified water. Finally, it was spray-dried to obtain the finished product of the hydrolyzed sodium colanate oligosaccharide composition containing sodium colanate hexasaccharide (molecular weight 1126.34) and sodium colanate dodecasaccharide (molecular weight 2234.67). Its high performance liquid chromatogram is shown as follows Figure 2 Its NMR spectrum is shown in Figure 7 shown.

[0107] Example 7-2 Take the molecular weight of 5.0×10 6 1.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 60 ° C. After being fully dissolved, the temperature was lowered to 50 ° C, the pH was adjusted to 5.0 by sodium phosphate, and then colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 10 mg / L, and the reaction was stirred for 1 h. After the reaction was completed, activated carbon (0.01% wt) was added to the solution, the pH was adjusted to 3.0, and then the filtrate was collected by plate and frame filter pressing. The filtrate was concentrated 20 times by nanofiltration, and then the liquid was replaced with purified water, and finally spray dried to obtain a finished product of a hydrolyzed sodium colanate oligosaccharide composition containing sodium colanate hexasaccharide (molecular weight 1126.34) and sodium colanate dodecasaccharide (molecular weight 2234.67).

[0108] Example 7-3 Take the molecular weight of 5.0×10 6 1.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 35 °C, the pH was adjusted to 8.0 by sodium phosphate, and then colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 5.0 mg / L, and the reaction was stirred for 2.5 hours. After the reaction was completed, activated carbon (0.25% wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filter pressing. The filtrate was concentrated 20 times by nanofiltration, and then the liquid was replaced with purified water, and finally spray dried to obtain a finished product of a hydrolyzed sodium colanate oligosaccharide composition containing sodium colanate hexasaccharide (molecular weight 1126.34) and sodium colanate dodecasaccharide (molecular weight 2234.67).

[0109] Example 8: Preparation of sodium colanate hexasaccharide and sodium colanate dodecasaccharide Example 8-1 Take the molecular weight of 5.0×10 6 1.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 40 °C. After being fully dissolved, the temperature was lowered to 20 °C, and the pH was adjusted to 6.5 by sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 2.5 mg / L, and the reaction was stirred for 4 h. After the reaction was completed, activated carbon (0.5%wt) was added to the solution, the pH was adjusted to 4.5, and then the filtrate was collected by plate and frame filter pressing. The filtrate was subjected to Q anion chromatography, eluted with 10 mM sodium chloride at pH 8.5, and eluted component one was collected; then eluted with 50 mM sodium chloride at pH 8.5 and eluted component two was collected. The eluted fractions 1 and 2 were concentrated by nanofiltration 20 times, then replaced with purified water, and finally spray-dried to obtain hydrolyzed sodium colanate hexasaccharide (molecular weight 1126.34) and hydrolyzed sodium colanate dodecasaccharide (molecular weight 2234.67). The HPLC chromatogram is shown in Figure 3 , Figure 4 Its mass spectrum is shown in Figure 5 , Figure 6 shown.

[0110] Example 8-2 Take the molecular weight of 5.0×10 6 1.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 60 °C. After being fully dissolved, the temperature was lowered to 50 °C, and the pH was adjusted to 5.0 with sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 10 mg / L, and the reaction was stirred for 1 h. After the reaction was completed, activated carbon (0.01% wt) was added to the solution, the pH was adjusted to 3.0, and then the filtrate was collected by plate and frame filter pressing. The filtrate was subjected to Q anion chromatography, eluted with 10 mM sodium chloride at pH 9.0, and eluted component one was collected; then eluted with 50 mM sodium chloride at pH 9.0 and eluted component two was collected. The eluted collected fractions one and two were concentrated 20 times by nanofiltration, then the liquid was replaced with purified water, and finally spray-dried to obtain hydrolyzed sodium colanate hexasaccharide (molecular weight 1126.34) and hydrolyzed sodium colanate dodecasaccharide (molecular weight 2234.67).

[0111] Example 8-3 Take the molecular weight of 5.0×10 61.0 kg of sodium colanate powder of Da was added to 100 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 35 °C, and the pH was adjusted to 8.0 by sodium phosphate. Then, colanate degrading enzyme (SEQ ID NO: 1) was added to the sodium colanate solution to make the final concentration 5.0 mg / L, and the reaction was stirred for 2.5 h. After the reaction was completed, activated carbon (0.25%wt) was added to the solution, the pH was adjusted to 6.0, and then the filtrate was collected by plate and frame filter pressing. The filtrate was subjected to Q anion chromatography, eluted with 10 mM sodium chloride at pH 8.0, and eluted component one was collected; then eluted with 50 mM sodium chloride at pH 8.0 and eluted component two was collected. The eluted collected fractions one and two were concentrated 20 times by nanofiltration, then the liquid was replaced with purified water, and finally spray-dried to obtain hydrolyzed sodium colanate hexasaccharide (molecular weight 1126.34) and hydrolyzed sodium colanate dodecasaccharide (molecular weight 2234.67).

[0112] Table 7. Test results of hydrolyzed sodium colanate oligosaccharide composition or single component finished product

[0113] Example 9: Preparation of sodium colanate powder product or oligosaccharide composition product / single component product in fermentation broth Example 9-1 100 L of colanic acid fermentation broth was taken, the temperature was controlled at 30 °C, colanic acid degrading enzyme (SEQ ID NO: 1) was added to make the final concentration 0.01 mg / L, and the reaction was carried out for 16 h. After the reaction was completed, the filtrate was sterilized by plate and frame filter pressing, activated carbon (2% wt) was added to the filtrate, the pH was adjusted to 5.0, and the filtrate was filtered again by plate and frame filter pressing for 1 h, and then filtered with a 0.65 μm bladder filter, precipitated with 2 volumes of anhydrous ethanol, and the precipitate was collected and washed repeatedly with a mixed solution of sodium chloride and ethanol at the same concentration for 3 times, and then dehydrated with anhydrous ethanol for 3 times, and finally vacuum dried to obtain a weight average molecular weight of 1.4×10 6 Da of crude pure sodium colanate powder.

[0114] Example 9-2 100 L of colanic acid fermentation broth was taken, the temperature was controlled at 30 °C, colanic acid degrading enzyme (SEQ ID NO: 1) was added to make the final concentration 0.2 mg / L, and the reaction was carried out for 2 h. After the reaction was completed, the filtrate was sterilized by plate and frame filter pressing, activated carbon (2% wt) was added to the filtrate, the pH was adjusted to 5.0, and the filtrate was filtered again by plate and frame filter pressing for 1 h, and then filtered with a 0.65 μm bladder filter, precipitated with 2 volumes of anhydrous ethanol, and the precipitate was collected and washed repeatedly with a mixed solution of sodium chloride and ethanol at the same concentration for 3 times, and then dehydrated with anhydrous ethanol for 3 times, and finally vacuum dried to obtain a weight average molecular weight of 7.1×10 5 Da of crude pure sodium colanate powder.

[0115] Example 9-3 Take 100 L of colanic acid fermentation broth, control the temperature at 30 °C, add colanic acid degrading enzyme (SEQ ID NO: 1) to make the final concentration 0.01 mg / L, and react for 16 h. After the reaction is completed, sterilize by plate and frame filter pressing, add activated carbon (2% wt) to the filtrate, adjust the pH to 5.0 for 1 h, and then filter again by plate and frame filter pressing, and then filter with a 0.65 μm capsule filter to collect the filtrate. The filtrate is concentrated with a commercially available 500kD hollow fiber membrane and replaced with pure water. After the replacement, adjust the pH to 6.5 with sodium phosphate, then add colanic acid degrading enzyme to make the final concentration 0.5 mg / L, and stir for 2 h. After the secondary reaction, activated carbon (2%wt) was added to the colanic acid solution, and the pH was adjusted to 5.0 and treated for 1 h before plate and frame filter pressing again. The filtrate was concentrated 20 times by nanofiltration, and then replaced with purified water. Finally, it was spray-dried to obtain a finished product of the hydrolyzed sodium colanic acid oligosaccharide composition containing sodium colanic acid hexasaccharide (molecular weight 1126.34) and sodium colanic acid dodecasaccharide (molecular weight 2234.67).

[0116] It should be noted that the fermentation broth can be directly hydrolyzed into products of target molecular weight, including low molecular weight products. However, there are many small molecular impurities in the fermentation broth, and the cost of purifying small molecular colanic acid or its salt therefrom is high. Therefore, in Examples 9-3 and 9-4, a primary hydrolysis was performed to separate medium and large molecular colanic acid or its salt, and then a secondary hydrolysis was performed.

[0117] Example 9-4 Take 100L of colanic acid fermentation broth, control the temperature at 30℃, add colanic acid degrading enzyme (SEQ ID NO: 1) to make the final concentration 0.01 mg / L, and react for 16 hours. After the reaction is completed, sterilize by plate and frame filter pressing, add activated carbon (2% wt) to the filtrate, adjust the pH to 5.0, treat for 1 hour, and then filter again by plate and frame filter pressing, and then filter with 0.65 μm capsule filter to collect the filtrate. The filtrate is concentrated with 500 kD hollow fiber membrane and replaced with pure water. After the replacement, adjust the pH to 6.5 with sodium phosphate, then add colanic acid degrading enzyme to make the final concentration 0.5 mg / L, and stir for 2 hours.

[0118] After the secondary reaction, activated carbon (2% wt) was added to the colanic acid solution, and the pH was adjusted to 5.0 for 1 h before plate and frame filter pressing again. The filtrate was subjected to Q anion chromatography, eluted with 10 mM sodium chloride at pH 9.0, and eluted component 1 was collected; then eluted with 50 mM sodium chloride at pH 9.0 and eluted component 2 was collected. The eluted and collected components 1 and 2 were concentrated 20 times by nanofiltration, and then the solution was replaced with purified water, and finally spray dried to obtain hydrolyzed sodium colanic acid hexasaccharide (molecular weight 1126.34) and hydrolyzed sodium colanic acid dodecasaccharide (molecular weight 2234.67).

[0119] Table 8. Results of the preparation of hydrolyzed sodium colanate finished product composition by enzymatic hydrolysis of fermentation broth

[0120] Table 9. Determination results of the hydrolyzed sodium colanate hexasaccharide and dodecasaccharide prepared by enzymatic hydrolysis of fermentation broth

[0121] Table 10. Sequence information

Claims

1. A method for preparing hydrolyzed colanic acid or its salt, comprising the following steps: 1) adding a solution containing colanic acid or a salt thereof to a colanic acid degrading enzyme comprising an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity thereto; and 2) Enzyme termination reaction: The termination in step 2) is achieved by using a terminator, a boiling water bath and / or an ice bath, wherein the terminator is selected from one or more of activated carbon, anhydrous ethanol, methanol, EDTA, trichloroacetic acid and sodium carbonate, preferably activated carbon is used to achieve the termination in step 2); Then, hydrolyzed colanic acid or its salt is obtained.

2. The method according to claim 1, comprising any one or more of the following: (i) the colanate is sodium colanate, and / or (ii) In step 1), the weight average molecular weight of the colanic acid or its salt is greater than 1×10 4 Da, preferably greater than 5×10 5 Da, more preferably greater than 2×10 6 Da, and / or (iii) in step 1), the final reaction concentration of the colanic acid degrading enzyme is between about 0.01 mg / L and 10 mg / L, and / or (iv) in step 1), the reaction temperature is between about 10 °C and about 70 °C, preferably between about 20 °C and about 50 °C, and / or (v) in step 1), the reaction time is about 1 h to about 20 h, and / or (vi) In step 1), the pH of the reaction solution is about 3 to about 9, preferably about 5 to about 8.

3. The method according to claim 1 or 2, wherein the method further comprises the steps of 3) purification by filtration; 4) concentration and desalination; and 5) drying.

4. The method according to any one of claims 1 to 3, wherein the method further comprises 6) multiple enzymatic hydrolysis.

5. The method according to any one of claims 1 to 4, wherein: The activated carbon content is about 0.005 wt% or more, preferably 0.005 wt%-1 wt%, and more preferably about 0.01 wt%-5 wt%.

6. The method according to any one of claims 3 to 5, wherein: In step 3), the pH is adjusted to about 3.0 to about 6.5, preferably about 3.0 to about 6.0, before filtration.

7. The method according to any one of claims 3 to 6, wherein: In step 5), the drying is achieved by spray drying, freeze drying or vacuum drying.

8. The method according to any one of claims 1 to 7, wherein: The weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da is about 3×10 6 Da, preferably, the weight average molecular weight of the hydrolyzed colanic acid or its salt is in the range of about 1×10 3 Da is about 2×10 6 Da.

9. The method according to any one of claims 1 to 8, wherein: The hydrolyzed colanic acid or its salt comprises acetylation and / or pyruvic acid modification.

10. The method according to any one of claims 1 to 9, wherein: The hydrolyzed colanic acid or its salt The acetylation modification rate is greater than 80%, preferably greater than 90%; and / or Residual endotoxin is less than about 0.5 EU / mg, preferably less than about 0.1 EU / mg; and / or The protein residue is less than about 0.1%, preferably less than about 0.07%; and / or The heavy metal residue is less than about 10 mg / kg.

11. The method according to any one of claims 1 to 10, wherein: The hydrolyzed colanic acid or its salt is colanic acid hexasaccharide or its salt, and / or colanic acid dodecasaccharide or its salt.

12. The hydrolyzed colanic acid or its salt prepared by the method according to any one of claims 1 to 11.

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