A process for the preparation of carbenoxolide or a salt thereof

Abstract

The present application belongs to the field of biotechnology, and relates to a method for large-scale preparation of high-purity hydrolyzed colanic acid or a salt thereof. The method comprises: 1) adding a solution containing colanic acid or a salt thereof into 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 termination agent, a boiling water bath and / or an ice bath, wherein the termination agent is selected from one or more of activated carbon, anhydrous ethanol, methanol, EDTA, trichloroacetic acid and sodium carbonate, preferably termination in step 2) is achieved by using activated carbon; thereby obtaining hydrolyzed colanic acid or a salt thereof. The hydrolyzed sodium colanic acid prepared by the method provided in the present application has a molecular structure with acetylation and pyruvic acid modification, and the preparation conditions are mild, the production cost is low, continuous production can be realized, and industrial amplification is easy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for large-scale purification and preparation of high-purity hydrolyzed colanic acid or a salt thereof. BACKGROUND

[0002] Microbial exopolysaccharide is a biological polymer produced by bacteria, fungi, blue algae and other microorganisms in the metabolic process. Under natural conditions, the polysaccharide coated on the surface of bacteria plays an important role in the survival and growth of bacteria in a competitive environment. Colanic acid is one of the microbial exopolysaccharides, and the solid is a white fibrous amorphous structure heteropolysaccharide mainly composed of D-glucuronic acid, D-glucose, D-galactose and L-fucose in a ratio of 1:2:2:1, and also contains O-acetyl and pyruvic acid and other chemical modification structures on the side chain. It is a high-molecular-weight polysaccharide with an average molecular weight of more than 10 million daltons.

[0003] Microbial exopolysaccharide has unique physical and rheological properties and is widely used as a stabilizer, thickener, gelling agent and emulsifier in the food industry. In recent years, research has found that bacterial polysaccharide has shown significant biological activity in anti-tumor, anti-virus, immune stimulation, anti-inflammatory activity, antioxidant effect and antimicrobial effect, thus attracting more attention. In 2017, it was first reported in the journal cell that colanic acid causes mitochondrial fragmentation and has the effect of prolonging the life of Caenorhabditis elegans. Due to its porous cellulose structure and a large number of hydrophilic groups on the colloid surface, colanic acid is a natural hydrogel with excellent water-retaining capacity and soft texture, and is a good candidate product for the future cosmetics and healthcare market. In addition, as a unique active biopolymer, colanic acid has special biological characteristics and physiological parameters, and has a wide research and application prospect in prolonging life and anti-aging.

[0004] Literature research shows that the biological activity of polysaccharide depends on the size of its 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 applied to the cosmetics industry, viscoelastic agents for eye surgery and intra-articular injection therapy. Medium-molecular-weight hyaluronic acid has good moisturizing, lubricating and drug 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 exhibit very strong biological activity, with antioxidant, anti-inflammatory, collagen production promoting, wound healing promoting and other biological activity. What is particularly important is that due to its small molecular size, it can penetrate into the stratum corneum of the skin to play a role, and can be widely applied to cosmetics.

[0005] The molecular weight of sodium colanic acid biosynthesized by E. coli is large, about 5-10 million Daltons. Such a large molecular weight polysaccharide has certain applications in the fields of cosmetics, health foods, etc. However, there is currently no production process for preparing hydrolyzed colanic acid salt with a specific molecular weight, especially sodium colanic acid, which greatly limits the application scenarios and efficacy of sodium colanic acid. Therefore, it is of great significance to develop a production process for large-scale preparation of finished colanic acid or salt (1 x 10 3 Da-2.0 x 10 6 Da).

[0006] To solve the above problems, the present application provides a method for continuous large-scale production of finished colanic acid or salt. The method provided by the present application has the advantages of complete structure (containing acetylation and pyruvic acid modification), high purity, low endotoxin residue, mild and efficient preparation conditions, low production cost, continuous production, and easy industrialization. SUMMARY

[0007] The present application discloses a method for continuous large-scale production of hydrolyzed colanic acid or salt thereof. Specifically, the present disclosure provides a method for preparing hydrolyzed colanic acid or salt thereof, which comprises the following steps: 1) adding a solution containing colanic acid or 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: termination in step 2) is achieved by a termination agent, a boiling water bath and / or an ice bath, wherein the termination agent 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 salt thereof.

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

[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 salt thereof is greater than 2 x 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 reaction final concentration of the colanic acid degrading enzyme is between about 0.01 mg / L-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 aforementioned method further comprises steps 3) filtration and purification; 4) concentration and desalination; and 5) drying.

[0016] According to some embodiments of the present disclosure, the aforementioned 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% to 1 wt%, and more preferably about 0.01 wt% to 5 wt%.

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

[0019] According to some embodiments of the present disclosure, in step 5), the drying is achieved by spray drying or freeze drying, 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 salt thereof ranges from about 1 x 10 3 Da to about 3 x 10 6 Da. In some preferred embodiments, the weight average molecular weight of the hydrolyzed colanic acid or salt thereof ranges from about 1 x 10 3 Da to about 2 x 10 6 Da.

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

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

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

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

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

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

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

[0028] According to some embodiments of the present disclosure, the hydrolyzed colanic acid or salt thereof has a weight average molecular weight ranging from about 1 x 10 3 Da to about 3 x 10 6 Da. In some preferred embodiments, the hydrolyzed colanic acid or salt thereof has a weight average molecular weight ranging from about 1 x 10 3 Da to about 2 x 10 6 Da.

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

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

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

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

[0033] According to some embodiments of the present disclosure, the hydrolyzed colanic acid or salt thereof has less than about 10 mg / kg of heavy metal residue.

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

[0035] Effects of the present disclosure:

[0036] The present application has the direct purpose of using the acid-degrading enzyme to catalyze the hydrolysis and purify the refined hydrolyzed colanic acid or its salt. First, the high-purity hydrolyzed colanic acid or its salt obtained by the present application has a weight average molecular weight ranging from 1.1 x 10 3 Da to 2.0 x 10 6 Da, and the finished product contains acetylation and pyruvic acid modification, which lays a foundation for the academic research on the structure and structure-activity relationship of colanic acid or its salt; second, the reaction condition of the present application is extremely simple, and no instrument equipment is required, and the reaction can be carried out under mild conditions; finally, the product prepared by the present application 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 DRAWINGS

[0037] Figure 1 The high-performance liquid chromatogram of the finished product of sodium colanic acid with a molecular weight of 1.2 x 10 6 Da, 7.6 x 10 5 Da and 4.2 x 10 5 Da.

[0038] Figure 2 The high-performance liquid chromatogram of the hydrolyzed sodium colanic acid oligosaccharide composition.

[0039] Figure 3 The high-performance liquid chromatogram of the hydrolyzed sodium colanic acid hexose.

[0040] Figure 4 The high-performance liquid chromatogram of the hydrolyzed sodium colanic acid dodecahexose.

[0041] Figure 5 The mass spectrum of the hydrolyzed sodium colanic acid hexose.

[0042] Figure 6 The mass spectrum of the hydrolyzed sodium colanic acid dodecahexose.

[0043] Figure 7 The nuclear magnetic resonance spectrum of the hydrolyzed sodium colanic acid oligosaccharide composition. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application. A number of modifications and improvements made on the basis of the inventive concept by those skilled in the art are within the scope of protection of the present application. The raw materials used in the examples can be obtained by commercial means.

[0045] Definitions

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0047] All numerical designations, e.g., pH, temperature, time, concentration, potency, and molecular weight, as used herein are approximations unless otherwise indicated. It will be understood that, although the numerical designations are expressed in whole numbers, the numerical designations can be approximations and can vary by + / - 0.1 or 1.0, as appropriate. It will be understood that the numerical designations can be preceded by the word "about" unless otherwise indicated.

[0048] The terms "about" and "approximately" include amounts within ±10% of the stated value.

[0049] As will be understood by those of ordinary skill in the art, all ranges disclosed herein are also intended to encompass any and all possible sub-ranges and combinations thereof, for any and all purposes. Any listed range can be easily recognized as sufficiently describing and supporting the disclosure, and 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 easily broken down into three ranges of equal size, e.g., a range of 1-10 can be broken down into 1-4, 5-6, and 7-10.

[0050] As used herein, "hydrolyzed colanic acid or a salt thereof" means the product obtained after the degradation reaction, which has a weight average molecular weight ranging from about 1 x 10 3 Da to about 5 x 10 6 Da, preferably, the weight average molecular weight of the hydrolyzed colanic acid or a salt thereof ranges from about 1 x 10 3 Da to about 2 x 10 6 Da. It is noted that the weight average molecular weight of the reactant colanic acid or a salt thereof is greater than the weight average molecular weight of the hydrolyzed colanic acid or a salt thereof.

[0051] As used herein, "oligosaccharide composition" and "oligosaccharide single component" are used only to reflect whether the product at a specific molecular weight is isolated or not, and both refer to the hydrolyzed colanic acid or a salt thereof prepared according to the technical solution of the present application, the former means that the product at a specific molecular weight can be identified but not isolated, and the latter means that the product at a specific molecular weight is isolated alone. As used herein, "oligosaccharide" means colanic acid or a salt thereof having a weight average molecular weight less than 1 x 10 4 Da, such as sodium colanic acid hexaose, sodium colanic acid dodecaose.

[0052] As used herein, the term "solution" is a broad term that is intended to have its ordinary meaning unless otherwise limited in the context by its use. A "solution" can be an aqueous or non-aqueous mixture, for example, it can include a homogeneous or heterogeneous mixture of a polymer with water, it can also include a suspension, a colloid, an emulsion, a gel, a paste, an ointment, a cream, or the like, it can also include a fermentation broth.

[0053] In the present disclosure, "colanic acid salt" refers to a salt formed by the reaction of colanic acid with any basic substance. Depending on the specific type of cation in the colanic acid salt, the colanic acid salt can be sodium colanic acid, potassium colanic acid, magnesium colanic acid, aluminum colanic acid, calcium colanic acid, ammonium colanic acid, etc. The colanic acid salt can also contain more than one cation, for example, sodium potassium colanic acid. One aspect of the present disclosure provides a method for preparing hydrolyzed colanic acid or a salt thereof, comprising the steps of: 1) adding a solution containing colanic acid or a salt thereof to a colanic acid-degrading enzyme comprising an amino acid sequence as set forth in SEQ ID NO: 1 or having at least 90% sequence identity thereto; and 2) terminating the enzyme reaction: termination in step 2) is achieved by a terminating agent, a boiling water bath, and / or an ice bath, wherein the terminating agent is selected from one or more of activated carbon, anhydrous ethanol, methanol, EDTA, trichloroacetic acid, and sodium carbonate, preferably termination in step 2) is achieved using activated carbon; thereby obtaining hydrolyzed colanic acid or a salt thereof.

[0054] 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 a salt thereof is greater than 1 x 10 4 Da. In some preferred embodiments, the weight average molecular weight of the colanic acid or a salt thereof is preferably in the range of about 1 x 10 4 Da to about 6 x 10 6 Da. In some preferred embodiments, the weight average molecular weight of the colanic acid or a salt thereof is preferably in the range of about 1 x 10 5 Da to about 6 x 10 6 Da. In some preferred embodiments, the weight average molecular weight of the colanic acid or a salt thereof is preferably in the range of about 1 x 10 4 Da, 2 x 10 4 Da, 3 x 10 4 Da, 4 x 10 4 Da, 5 x 10 4 Da, 6 x 10 4 Da, 7 x 10 4 Da, 8 x 10 4 Da, 9 x 10 4 Da, 1 x 10 5 Da, 2 x 10 5 Da, 3 x 10 5 Da, 4 x 10 5 Da, 5 x 10 5Da, 6 x 10 5 Da, 7 x 10 5 Da, 8 x 10 5 Da, 9 x 10 5 Da, 1 x 10 6 Da, 1.1 x 10 6 Da, 1.2 x 10 6 Da, 1.3 x 10 6 Da, 1.4 x 10 6 Da, 1.5 x 10 6 Da, 1.6 x 10 6 Da, 1.7 x 10 6 Da, 1.8 x 10 6 Da, 1.9 x 10 6 Da, 2 x 10 6 Da, 2.1 x 10 6 Da, 2.2 x 10 6 Da, 2.3 x 10 6 Da, 2.4 x 10 6 Da, 2.5 x 10 6 Da, 2.6 x 10 6 Da, 2.7 x 10 6 Da, 2.8 x 10 6 Da, 2.9 x 10 6 Da, 3 x 10 6 Da, 3.1 x 10 6 Da, 3.2 x 10 6 Da, 3.3 x 10 6 Da, 3.4 x 10 6 Da, 3.5 x 10 6 Da, 3.6 x 10 6 Da, 3.7 x 10 6 Da, 3.8 x 10 6 Da, 3.9 x 10 6 Da, 4 x 10 6 Da, 4.1 x 10 6 Da, 4.2 x 10 6 Da, 4.3 x 10 6 Da, 4.4 x 10 6 Da, 4.5 x 10 6 Da, 4.6 x 10 6 Da, 4.7 x 10 6 Da, 4.8 x 10 6 Da, 4.9 x 10 6 Da, 5 x 10 6 Da, 5.1 x 10 6Da, 5.2 x 10 6 Da, 5.3 x 10 6 Da, 5.4 x 10 6 Da, 5.5 x 10 6 Da, 5.6 x 10 6 Da, 5.7 x 10 6 Da, 5.8 x 10 6 Da, 5.9 x 10 6 Da, or 6 x 10 6 Da.

[0055] In the present disclosure, the colanic acid-degrading enzyme comprises an amino acid sequence as set forth 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 99% 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 set forth in SEQ ID NO: 1.

[0056] In some embodiments, in step 1), the colanic acid salt is sodium colanic acid.

[0057] In some embodiments, in the foregoing method, the reaction final concentration of the said acid-degrading enzyme in step 1) is between about 0.01 mg / L-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, 0.13 mg / 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, 0.28 mg / 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, 0.43 mg / 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, 0.58 mg / 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, 0.88 mg / 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, 1.03 mg / 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, 1.18 mg / 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, 1.33 mg / 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, 1.48 mg / 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, 1.63 mg / 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, 1.78 mg / 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, 1.93 mg / 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, 2.08 mg / 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, 2.23 mg / 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, 2.38 mg / 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, 2.53 mg / 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, 2.68 mg / 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, 2.83 mg / 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, 2.98 mg / 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, 3.13 mg / 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, 3.28 mg / 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, 3.43 mg / 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, 3.58 mg / 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, 3.73 mg / 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, 3.88 mg / 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, 4.03 mg / 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, 4.18 mg / 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, 4.33 mg / 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, 4.48 mg / 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, 4.63 mg / 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, 4.78 mg / 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, 4.93 mg / 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, 5.08 mg / 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, 5.23 mg / 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, 5.38 mg / 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, 5.53 mg / 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, 5.68 mg / 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, 5.83 mg / 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, 5.98 mg / 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, 6.13 mg / 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, 6.28 mg / 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, 6.43 mg / 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, 6.58 mg / 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, 6.73 mg / 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, 6.88 mg / 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, 7.03 mg / 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, 7.18 mg / 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, 7.33 mg / 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, 7.48 mg / 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, 7.63 mg / 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, 7.78 mg / 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, 7.93 mg / 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, 8.08 mg / 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, 8.23 mg / 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, 8.38 mg / 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, 8.53 mg / 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, 8.68 mg / 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, 8.83 mg / 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, 8.98 mg / 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, 9.13 mg / 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, 9.28 mg / 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, 9.43 mg / 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, 9.58 mg / 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, 9.73 mg / 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, 9.88 mg / 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.

[0058] In some embodiments, in the aforementioned method, the reaction temperature in step 1) is between about 10 °C to 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.

[0059] 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.

[0060] In some embodiments, in the aforementioned method, the pH of the reaction solution in step 1) is about 3 to about 9, for example, 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.

[0061] In some embodiments, the aforementioned method further comprises steps 3) filtration and purification; 4) concentration and desalination; and 5) drying.

[0062] In some embodiments, the aforementioned method further comprises 6) multiple enzymatic hydrolysis, for example, twice enzymatic hydrolysis, thrice enzymatic hydrolysis, etc.

[0063] 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 colacid solution; for example, 1 wt% means 1 L of colacid solution plus 10 g of activated carbon. In some preferred embodiments, the activated carbon content is 0.005 wt%-1 wt%, and 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%. 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%, 0.96 wt%, 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%, 2.5 wt%, 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%, 3.5 wt%, 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%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, or 5 wt%.

[0064] In some embodiments, in step 3) the pH is adjusted to about 3.0 to about 6.5 prior to filtration; in some preferred embodiments, the pH is adjusted to about 3.0 to about 6.5 prior to filtration, and in some preferred embodiments, the pH is adjusted to about 3.0 to about 6.0 prior to 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.

[0065] In some embodiments, in step 5) the drying is achieved by spray drying or freeze drying, vacuum drying.

[0066] In some embodiments, in the foregoing methods, the hydrolyzed colanic acid or salt thereof has a weight average molecular weight ranging from about 1 x 10 3 Da to about 3 x 10 6 Da. In some preferred embodiments, the hydrolyzed colanic acid or salt thereof has a weight average molecular weight ranging from about 1 x 10 3 Da to about 2 x 10 6 Da. The weight average molecular weight of the hydrolyzed colanic acid or salt thereof can be any value or any range within the ranges, for example, 1 x 10 3 Da, 2 x 10 3 Da, 3 x 10 3 Da, 4 x 10 3 Da, 5 x 103 Da, 6 x 10 3 Da, 7 x 10 3 Da, 8 x 10 3 Da, 9 x 10 3 Da, 1 x 10 4 Da, 2 x 10 4 Da, 3 x 10 4 Da, 4 x 10 4 Da, 5 x 10 4 Da, 6 x 10 4 Da, 7 x 10 4 Da, 8 x 10 4 Da, 9 x 10 4 Da, 1.0 x 10 5 Da, 1.1 x 10 5 Da, 1.2 x 10 5 Da, 1.3 x 10 5 Da, 1.4 x 10 5 Da, 1.5 x 10 5 Da, 1.6 x 10 5 Da, 1.7 x 10 5 Da, 1.8 x 10 5 Da, 1.9 x 10 5 Da, 2 x 10 5 Da, 2.1 x 10 5 Da, 2.2 x 10 5 Da, 2.3 x 10 5 Da, 2.4 x 10 5 Da, 2.5 x 10 5 Da, 2.6 x 10 5 Da, 2.7 x 10 5 Da, 2.8 x 10 5 Da, 2.9 x 10 5 Da, 3 x 10 5 Da, 3.1 x 10 5 Da, 3.2 x 10 5 Da, 3.3 x 10 5 Da, 3.4 x 10 5 Da, 3.5 x 10 5 Da, 3.6 x 10 5 Da, 3.7 x 10 5 Da, 3.8 x 10 5 Da, 3.9 x 10 5 Da, 4 x 10 5 Da, 4.1 x 10 5 Da, 4.2 x 10 5 Da, 4.3 x 105 Da, 4.4 x 10 5 Da, 4.5 x 10 5 Da, 4.6 x 10 5 Da, 4.7 x 10 5 Da, 4.8 x 10 5 Da, 4.9 x 10 5 Da, 5 x 10 5 Da, 5.1 x 10 5 Da, 5.2 x 10 5 Da, 5.3 x 10 5 Da, 5.4 x 10 5 Da, 5.5 x 10 5 Da, 5.6 x 10 5 Da, 5.7 x 10 5 Da, 5.8 x 10 5 Da, 5.9 x 10 5 Da, 6 x 10 5 Da, 6.1 x 10 5 Da, 6.2 x 10 5 Da, 6.3 x 10 5 Da, 6.4 x 10 5 Da, 6.5 x 10 5 Da, 6.6 x 10 5 Da, 6.7 x 10 5 Da, 6.8 x 10 5 Da, 6.9 x 10 5 Da, 7 x 10 5 Da, 7.1 x 10 5 Da, 7.2 x 10 5 Da, 7.3 x 10 5 Da, 7.4 x 10 5 Da, 7.5 x 10 5 Da, 7.6 x 10 5 Da, 7.7 x 10 5 Da, 7.8 x 10 5 Da, 7.9 x 10 5 Da, 8 x 10 5 Da, 8.1 x 10 5 Da, 8.2 x 10 5 Da, 8.3 x 10 5 Da, 8.4 x 10 5 Da, 8.5 x 10 5 Da, 8.6 x 10 5 Da, 8.7 x 10 5 Da, 8.8 x 10 5Da, 8.9 x 10 5 Da, 9 x 10 5 Da, 9.1 x 10 5 Da, 9.2 x 10 5 Da, 9.3 x 10 5 Da, 9.4 x 10 5 Da, 9.5 x 10 5 Da, 9.6 x 10 5 Da, 9.7 x 10 5 Da, 9.8 x 10 5 Da, 9.9 x 10 5 Da, 1.0 x 10 6 Da, 1.1 x 10 6 Da, 1.2 x 10 6 Da, 1.3 x 10 6 Da, 1.4 x 10 6 Da, 1.5 x 10 6 Da, 1.6 x 10 6 Da, 1.7 x 10 6 Da, 1.8 x 10 6 Da, 1.9 x 10 6 Da, 2 x 10 6 Da, 2.1 x 10 6 Da, 2.2 x 10 6 Da, 2.3 x 10 6 Da, 2.4 x 10 6 Da, 2.5 x 10 6 Da, 2.6 x 10 6 Da, 2.7 x 10 6 Da, 2.8 x 10 6 Da, 2.9 x 10 6 Da, or 3 x 10 6 Da.

[0067] In some embodiments, the foregoing methods, the hydrolyzed colanic acid or salt thereof comprises acetylation and / or pyruvic acid modification.

[0068] In some embodiments, the foregoing methods, the hydrolyzed colanic acid or salt thereof comprises acetylation and / or pyruvic acid modification.

[0069] In some embodiments, the foregoing method, the hydrolyzed colanic acid or salt thereof has less than about 0.5 EU / mg of endotoxin residual, and in some preferred embodiments, less than about 0.1 EU / mg of endotoxin residual.

[0070] In some embodiments, the foregoing method, the hydrolyzed colanic acid or salt thereof has 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 % of protein residual, and in some preferred embodiments, less than about 0.07 %, about 0.06 %, about 0.05 %, about 0.04 %, about 0.03 %, about 0.02 %, or about 0.01 % of protein residual.

[0071] In some embodiments, the foregoing method, the hydrolyzed colanic acid or salt thereof has 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 of heavy metal residual.

[0072] In some embodiments, the foregoing method, the hydrolyzed colanic acid or salt thereof can also be a colanic acid hexaose or salt thereof, and / or a colanic acid dodecaose or salt thereof. For example, the hydrolyzed colanic acid or salt thereof includes, but is not limited to, a composition comprising a combination of a colanic acid hexaose or salt thereof, and a colanic acid dodecaose or salt thereof; or a colanic acid hexaose or salt thereof, or a colanic acid dodecaose or salt thereof, that is isolated individually.

[0073] Another aspect of the present disclosure provides a hydrolyzed colanic acid or salt thereof prepared according to the foregoing method.

[0074] In some embodiments, the hydrolyzed colanic acid or salt thereof prepared according to the foregoing method has a weight average molecular weight ranging from about 1 x 10 3 Da to about 5 x 10 6 Da. In some preferred embodiments, the hydrolyzed colanic acid or salt thereof has a weight average molecular weight ranging from about 1 x 10 3 Da to about 2 x 10 6 Da. In some more preferred embodiments, the hydrolyzed colanic acid or salt thereof has a weight average molecular weight ranging from about 1 x 10 3 Da to about 1.5 x 10 6 Da. The weight average molecular weight of the hydrolyzed colanic acid or salt thereof can be any value or any range within the range, for example, 1 x 10 3 Da, 2 x 10 3 Da, 3 x 10 3 Da, 4 x 10 3 Da, 5 x 103 Da, 6 x 10 3 Da, 7 x 10 3 Da, 8 x 10 3 Da, 9 x 10 3 Da, 1 x 10 4 Da, 2 x 10 4 Da, 3 x 10 4 Da, 4 x 10 4 Da, 5 x 10 4 Da, 6 x 10 4 Da, 7 x 10 4 Da, 8 x 10 4 Da, 9 x 10 4 Da, 1.0 x 10 5 Da, 1.1 x 10 5 Da, 1.2 x 10 5 Da, 1.3 x 10 5 Da, 1.4 x 10 5 Da, 1.5 x 10 5 Da, 1.6 x 10 5 Da, 1.7 x 10 5 Da, 1.8 x 10 5 Da, 1.9 x 10 5 Da, 2 x 10 5 Da, 2.1 x 10 5 Da, 2.2 x 10 5 Da, 2.3 x 10 5 Da, 2.4 x 10 5 Da, 2.5 x 10 5 Da, 2.6 x 10 5 Da, 2.7 x 10 5 Da, 2.8 x 10 5 Da, 2.9 x 10 5 Da, 3 x 10 5 Da, 3.1 x 10 5 Da, 3.2 x 10 5 Da, 3.3 x 10 5 Da, 3.4 x 10 5 Da, 3.5 x 10 5 Da, 3.6 x 10 5 Da, 3.7 x 10 5 Da, 3.8 x 10 5 Da, 3.9 x 10 5 Da, 4 x 10 5 Da, 4.1 x 10 5 Da, 4.2 x 10 5 Da, 4.3 x 105 Da, 4.4 x 10 5 Da, 4.5 x 10 5 Da, 4.6 x 10 5 Da, 4.7 x 10 5 Da, 4.8 x 10 5 Da, 4.9 x 10 5 Da, 5 x 10 5 Da, 5.1 x 10 5 Da, 5.2 x 10 5 Da, 5.3 x 10 5 Da, 5.4 x 10 5 Da, 5.5 x 10 5 Da, 5.6 x 10 5 Da, 5.7 x 10 5 Da, 5.8 x 10 5 Da, 5.9 x 10 5 Da, 6 x 10 5 Da, 6.1 x 10 5 Da, 6.2 x 10 5 Da, 6.3 x 10 5 Da, 6.4 x 10 5 Da, 6.5 x 10 5 Da, 6.6 x 10 5 Da, 6.7 x 10 5 Da, 6.8 x 10 5 Da, 6.9 x 10 5 Da, 7 x 10 5 Da, 7.1 x 10 5 Da, 7.2 x 10 5 Da, 7.3 x 10 5 Da, 7.4 x 10 5 Da, 7.5 x 10 5 Da, 7.6 x 10 5 Da, 7.7 x 10 5 Da, 7.8 x 10 5 Da, 7.9 x 10 5 Da, 8 x 10 5 Da, 8.1 x 10 5 Da, 8.2 x 10 5 Da, 8.3 x 10 5 Da, 8.4 x 10 5 Da, 8.5 x 10 5 Da, 8.6 x 10 5 Da, 8.7 x 10 5 Da, 8.8 x 10 5Da, 8.9 x 10 5 Da, 9 x 10 5 Da, 9.1 x 10 5 Da, 9.2 x 10 5 Da, 9.3 x 10 5 Da, 9.4 x 10 5 Da, 9.5 x 10 5 Da, 9.6 x 10 5 Da, 9.7 x 10 5 Da, 9.8 x 10 5 Da, 9.9 x 10 5 Da, 1.0 x 10 6 Da, 1.1 x 10 6 Da, 1.2 x 10 6 Da, 1.3 x 10 6 Da, 1.4 x 10 6 Da, 1.5 x 10 6 Da, 1.6 x 10 6 Da, 1.7 x 10 6 Da, 1.8 x 10 6 Da, 1.9 x 10 6 Da, 2 x 10 6 Da, 2.1 x 10 6 Da, 2.2 x 10 6 Da, 2.3 x 10 6 Da, 2.4 x 10 6 Da, 2.5 x 10 6 Da, 2.6 x 10 6 Da, 2.7 x 10 6 Da, 2.8 x 10 6 Da, 2.9 x 10 6 Da, 3 x 10 6 Da, 3.1 x 10 6 Da, 3.2 x 10 6 Da, 3.3 x 10 6 Da, 3.4 x 10 6 Da, 3.5 x 10 6 Da, 3.6 x 10 6 Da, 3.7 x 10 6 Da, 3.8 x 10 6 Da, 3.9 x 10 6 Da, 4 x 10 6 Da, 4.1 x 10 6 Da, 4.2 x 10 6 Da, 4.3 x 10 6 Da, 4.4 x 106 Da, 4.5 x 10 6 Da, 4.6 x 10 6 Da, 4.7 x 10 6 Da, 4.8 x 10 6 Da, 4.9 x 10 6 Da, or 5 x 10 6 Da.

[0075] In some embodiments, the hydrolyzed colanic acid or salt thereof prepared according to the foregoing methods comprises acetylation and / or pyruvic acid modification.

[0076] In some embodiments, the hydrolyzed colanic acid or salt thereof prepared according to the foregoing methods comprises an acetylation modification rate of about 80-100%, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0077] In some embodiments, the hydrolyzed colanic acid or salt thereof prepared according to the foregoing methods comprises less than about 0.5 EU / mg of endotoxin residual, and in some preferred embodiments, less than about 0.1 EU / mg of endotoxin residual.

[0078] In some embodiments, the hydrolyzed colanic acid or salt thereof prepared according to the foregoing methods comprises 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% of protein residual, and in some preferred embodiments, less than about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, or about 0.01% of protein residual.

[0079] In some embodiments, the hydrolyzed colanic acid or salt thereof prepared according to the foregoing methods comprises 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 of heavy metal residual.

[0080] In some embodiments, the hydrolyzed colanic acid or salt thereof prepared according to the foregoing methods can also be a colanic acid hexaose or salt thereof, and / or a colanic acid dodecaose or salt thereof. For example, the hydrolyzed colanic acid or salt thereof includes, but is not limited to, a composition comprising a combination of a colanic acid hexaose or salt thereof, and a colanic acid dodecaose or salt thereof; or a colanic acid hexaose or salt thereof, or a colanic acid dodecaose or salt thereof, that is isolated separately.

[0081] The specific implementation of the present application is described in detail below in combination with specific examples.

[0082] The colanic acid fermentation liquor used in the present application is provided by fermentation of an engineered strain of Escherichia coli constructed by Shenzhen Boyin Biotechnology Co., Ltd. (see Chinese Invention Patent Application CN115287314A), and the colanic acid degrading enzyme is provided by fermentation and purification of an engineered strain of Escherichia coli constructed by Shenzhen Boyin Biotechnology Co., Ltd. (see Chinese Invention Patent Application CN118685474A).

[0083] Test method

[0084] The protein content was determined by the Lorry method, the heavy metal residue was determined by ICP-MS, the endotoxin was determined by the limulus reagent gel method, and the acetylation quantification was obtained by comparing the CH3 peak integral of the H spectrum of pyruvic acid.

[0085] The purity and weight average molecular weight analysis method of hydrolyzed colanic acid sodium: the instrument is Agilent 1260II, the differential detector, and the 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, the flow rate is 0.6 mL / min, and the isocratic 50 min.

[0086] Examples

[0087] Example 1-1

[0088] A certain amount of colanic acid fermentation liquor was taken, a flocculating agent was added, the pH value was adjusted to alkaline, and after dilution and standing, the bacteria were removed, and then the solution was filtered under acidic conditions using an adsorbent, precipitated by adding anhydrous ethanol, and the precipitate was collected, washed repeatedly with a mixed solution of sodium chloride and ethanol (the volume ratio of sodium chloride to ethanol is about 2:8 to 4:6), then dehydrated with anhydrous ethanol, and finally dried to obtain 1.0 kg of colanic acid sodium with a weight average molecular weight of 5.0×10 6 The crude and pure colanic acid sodium powder of Da has an acetylation modification rate of 99.7%.

[0089] Five portions of 0.01 kg of colanic acid sodium powder with a molecular weight of 5.0×10 6 Da were each added to 1 L of pure water and stirred to dissolve, and the water temperature was controlled at 50°C. After complete dissolution, the temperature was lowered to 25°C, the pH was adjusted to 8.0 with sodium phosphate salt, then colanic acid degrading enzyme (SEQ ID NO: 1) was added to the colanic acid sodium solution to a final concentration of 0.2 mg / L, and the mixture was stirred for 1 h. After the reaction was completed, the five samples were treated as follows:

[0090] (1) Activated carbon termination: add activated carbon (0.5% wt) and adjust pH to 4.5 for 1 h, then collect filtrate by plate and frame pressure filtration;

[0091] (2) High temperature termination: heat to 75 °C for 1 h, then decrease to room temperature;

[0092] (3) Anhydrous ethanol termination: add 2 L of anhydrous ethanol for 1 h;

[0093] (4) Proteinase termination: add proteinase K with a final concentration of 125 mg / L for 1 h;

[0094] (5) Do nothing.

[0095] To 5 portions of the solution, add 0.2 kg of sodium chloride powder respectively, stir to dissolve, and then perform fine filtration with a 0.65 μm capsule filter. Then perform alcohol precipitation with 20 L of anhydrous ethanol, collect the precipitate, and repeat washing with a mixed solution of the same concentration of sodium chloride and ethanol for 3 times, and then dehydrate with anhydrous ethanol for 3 times. Finally, vacuum dry to obtain hydrolyzed sodium colforsin powders F1-F5 as shown in Table 1.

[0096] The data in the table show that the result of activated carbon treatment is superior to that of other termination methods. Non-termination of enzyme activity can lead to further reduction of molecular weight in the subsequent process; high temperature treatment is easy to cause acetylation to fall off; ethanol treatment is easy to cause danger when used in a non-specific container; proteinase treatment is not ideal and introduces protein; and activated carbon can simultaneously adsorb impurities and enzymes, so the termination effect is obvious and the treatment is convenient. Therefore, activated carbon is more ideal as a termination method.

[0097] Table 1 Effects of different termination methods

[0098]

[0099] Example 1-2

[0100] Take 1 portion of the sodium colforsin powder with a molecular weight of 5.0 x 10 6 Da prepared in Example 1-1, add it to 100 L of pure water respectively, stir to dissolve, and control the water temperature at 50 °C. After fully dissolving, decrease the temperature to 25 °C, adjust the pH to 8.0 with sodium phosphate salt, then add colforsin degrading enzyme (SEQ ID NO: 1) to the sodium colforsin solution to make the final concentration 1.0 mg / L, and stir for 3 h. After the reaction is completed, add activated carbon (0.25% wt) to the solution, adjust the pH to 6.0, and then collect the filtrate by plate and frame pressure filtration. The filtrate is fine filtered with a 0.65 μm capsule filter, then exchanged with purified water, and finally spray dried to obtain a hydrolyzed sodium colforsin product with a weight average molecular weight of 1.2 x 10 4 Da.

[0101] Comparative Example 1

[0102] Take 1 part of the molecular weight of 5.0 x 10 6 Da sodium colaurum powder 0.1 kg was added to 10 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After complete dissolution, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 with sodium phosphate salt, and then the temperature was raised to 134 °C for 1 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 plate and frame pressure filtration was performed to collect the filtrate. The filtrate was refined with a 0.65 μm Cellophane filter, then exchanged with purified water, and finally spray dried to obtain a final product of hydrolyzed sodium colaurum with a weight average molecular weight of 1.1 x 10 4 Da sodium colaurum powder 0.1 kg was added to 10 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After complete dissolution, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 with sodium phosphate salt, and then the temperature was raised to 134 °C for 1 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 plate and frame pressure filtration was performed to collect the filtrate. The filtrate was refined with a 0.65 μm Cellophane filter, then exchanged with purified water, and finally spray dried to obtain a final product of hydrolyzed sodium colaurum with a weight average molecular weight of 1.1 x 10

[0103] As can be seen from Example 1-2 and Comparative Example 1, high temperature can also achieve the purpose of degrading colaurum, but will affect the acetylation modification of hydrolyzed sodium colaurum; and enzymatic degradation is mild, and the acetylation modification is completely retained after the reaction.

[0104] Table 2 Product determination results of degrading the same molecular weight raw material sodium colaurum by different degradation methods

[0105]

[0106] It should be noted that the "product yield" is the percentage of the amount of the final product relative to the reaction substrate (such as powder, fermentation broth, etc.), for example, take 5.0 x 10 6 Da sodium colaurum powder 0.1 kg was added to 10 L of pure water and stirred to dissolve, and the water temperature was controlled at 50 °C. After complete dissolution, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 with sodium phosphate salt, and then the temperature was raised to 134 °C for 1 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 plate and frame pressure filtration was performed to collect the filtrate. The filtrate was refined with a 0.65 μm Cellophane filter, then exchanged with purified water, and finally spray dried to obtain a final product of hydrolyzed sodium colaurum with a weight average molecular weight of 1.1 x 10

[0107] Example 2

[0108] Take 5 parts of the molecular weight of 5.0 x 10 6Da's sodium colforsate powder 0.1 kg was added into 10 L pure water respectively to be stirred and dissolved, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 by sodium phosphate salt, then sodium colforsate degrading enzyme (SEQ ID NO: 1) was added into the sodium colforsate solution to make its final concentration 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L respectively, and stirred to react for 20 h, 10 h, 4 h, 2 h, 1 h respectively. After the reaction was completed, activated carbon (0.25% wt) was added into the solution to adjust the pH to 6.0, then plate and frame pressure filtration was carried out to collect the filtrate. The filtrate was refined by 0.65 μm Carius filter, then 20 L anhydrous ethanol was added while stirring to make the hydrolyzed sodium colforsate precipitate, the precipitate was collected and washed with the same concentration of sodium chloride and ethanol mixed solution for 3 times, and anhydrous ethanol for 3 times, and finally vacuum dried to obtain the finished product F1'-F5' of hydrolyzed sodium colforsate, the data are shown in Table 3 below.

[0109] The results show that when the enzyme final concentration is in the range of 0.01 mg / L-0.2 mg / L and the reaction time is in the range of 1 h-20 h, the molecular weight, protein residue, acetylation modification degree and product yield of the product obtained are all in the expected range.

[0110] Table 3 Product determination results of degrading the same molecular weight raw material sodium colforsate under different enzyme amounts and reaction times

[0111]

[0112] Example 3

[0113] 8 portions of the sodium colforsate with a molecular weight of 5.0 x 10 6 Da's sodium colforsate powder 0.1 kg was added into 10 L pure water respectively to be stirred and dissolved, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 by sodium phosphate salt, then sodium colforsate degrading enzyme (SEQ ID NO: 1) was added into the sodium colforsate solution to make its final concentration 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L respectively, and stirred to react for 20 h, 10 h, 4 h, 2 h, 1 h respectively. After the reaction was completed, activated carbon (0.25% wt) was added into the solution to adjust the pH to 6.0, then plate and frame pressure filtration was carried out to collect the filtrate. The filtrate was refined by 0.65 μm Carius filter, then 20 L anhydrous ethanol was added while stirring to make the hydrolyzed sodium colforsate precipitate, the precipitate was collected and washed with the same concentration of sodium chloride and ethanol mixed solution for 3 times, and anhydrous ethanol for 3 times, and finally vacuum dried to obtain the finished product F1'-F5' of hydrolyzed sodium colforsate, the data are shown in Table 3 below. 6 ~1.0×10 6The reaction was stopped within the range of Da. After the reaction was completed, activated carbon (0.25% wt) was added to the solution to adjust the pH to 6.0, and then plate and frame filtration was performed to collect the filtrate. The filtrate was finely filtered through a 0.65 μm Nenner filter, and then 20 L of anhydrous ethanol was added while stirring to precipitate the hydrolyzed sodium colarate. The precipitate was collected and washed three times with a mixed solution of sodium chloride and ethanol of the same concentration, and dehydrated three times with anhydrous ethanol. Finally, it was vacuum dried to obtain the hydrolyzed sodium colarate product (F1''-F8''), and the data are shown in Table 4 below.

[0114] The data in the table show that acetylation is lost at pH 10, while the desired product can be obtained at pH 3-9. Among these conditions, the yield is slightly lower at pH 3-4, and the reaction is slower and the molecular weight is higher at pH 8-9. The enzymatic hydrolysis at pH 5-7 shows excellent performance in terms of product molecular weight, protein residue, endotoxin residue, heavy metals, acetylation modification and pyruvate modification, and product yield.

[0115] Table 4. Results of product determination for degradation of sodium colarate of the same molecular weight under different pH conditions.

[0116]

[0117] Example 4

[0118] Take 9 portions of the sample prepared in Example 1-1 with a molecular weight of 5.0 × 10⁻⁶. 6 0.1 kg of sodium colarate powder was added to 10 L of pure water and stirred until dissolved, with the water temperature controlled at 50 °C. After complete dissolution, the temperature was adjusted to 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C, respectively. The pH was adjusted to 8.0 with sodium phosphate. Then, colarate-degrading enzyme (SEQ ID NO: 1) was added to the sodium colarate solution to a final concentration of 0.1 mg / L, and the reaction was stirred, controlling the molecular weight at 0.6 × 10⁻⁶. 6 ~1.0×10 6 The reaction was stopped within the range of Da. After the reaction was completed, activated carbon (0.25% wt) was added to the solution to adjust the pH to 6.0, and then plate and frame filtration was performed to collect the filtrate. The filtrate was finely filtered through a 0.65 μm Nenz filter, and then 20 L of anhydrous ethanol was added while stirring to hydrolyze sodium collamate to precipitate. The precipitate was collected and washed three times with a mixed solution of sodium chloride and ethanol of the same concentration, and dehydrated three times with anhydrous ethanol. Finally, it was vacuum dried to obtain the finished sodium collamate (F1'''-F9'''), and the data are shown in Table 5 below.

[0119] The results showed that acetylation modification was easily lost at temperatures above 70 °C.

[0120] Table 5. Results of product determination of sodium colforsate with the same molecular weight of raw material under different temperature conditions

[0121]

[0122] Example 5: Preparation of sodium colforsate product with high range of weight average molecular weight

[0123] Take 3 parts of the sodium colforsate powder with a molecular weight of 5.0 x 10 6 Da were respectively added into 100 L of pure water for stirring and dissolving, and the water temperature was controlled at 50 °C. After sufficient dissolution, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 by sodium phosphate salt, and then the sodium colforsate solution was added with colforsate degrading enzyme (SEQ ID NO: 1) to make the final concentration 0.1 mg / L, 0.2 mg / L, and 0.4 mg / L respectively, and stirred for 1.0 h. After the reaction was completed, activated carbon (0.25% wt) was added to the solution to adjust the pH to 6.0, and then plate and frame pressure filtration was performed to collect the filtrate. The filtrate was refined by 0.65 μm Cellophane filter, and then 200 L of anhydrous ethanol was added while stirring to precipitate the hydrolyzed sodium colforsate, which was collected and washed repeatedly 3 times with a mixed solution of sodium chloride and ethanol with the same concentration, and dehydrated 3 times with anhydrous ethanol. Finally, vacuum drying was performed to obtain sodium colforsate powder products (E1-E3) with a weight average molecular weight of 1.2 x 10 6 Da, 7.6 x 10 5 Da, 4.2 x 10 5 Da. The high performance liquid chromatogram of the sodium colforsate powder product (E1-E3) is shown in FIG. 1. Figure 1

[0124] It should be noted that different purification methods used in the process of hydrolyzed colforsate or its salt can obtain products with different molecular weights. For example, for the hydrolyzed sodium colforsate with a weight average molecular weight, when the molecular weight is greater than 5.0 x 10 5 Da, the precipitation and drying method can be used to obtain the product in a more time-saving manner; and for the product with a molecular weight less than 5.0 x 10 5 Da, the effect of pure water is better than that of alcohol precipitation.

[0125] Example 6: Preparation of sodium colforsate product with low range of weight average molecular weight

[0126] Take 5 parts of the sodium colforsate powder with a molecular weight of 5.0 x 10 6 ​Da's sodium colfosceinate powder 1.0 kg was added into 100 L purified water to be stirred and dissolved, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 by sodium phosphate salt, then the sodium colfosceinate solution was added with colfosceinate degrading enzyme (SEQ ID NO: 1) to make the final concentration 0.3 mg / L, 0.5 mg / L, 1.0 mg / L, 2.5 mg / L, 5 mg / L respectively, and the reaction was stirred for 2.0 h. After the reaction was completed, activated carbon (0.25% wt) was added into the solution to adjust the pH to 6.0, then the plate and frame filter press was used to collect the filtrate. The filtrate was refined by 0.65 μm capsule filter, then the purified water was used for liquid exchange, and finally the spray drying was performed to obtain the hydrolyzed sodium colfosceinate product (E4-E8).

[0127] Table 6 Determination results of hydrolyzed sodium colfosceinate or hydrolyzed sodium colfosceinate oligosaccharide with different molecular weights

[0128]

[0129] Example 7: Preparation of sodium colfosceinate oligosaccharide composition

[0130] Example 7-1

[0131] The sodium colfosceinate with a molecular weight of 5.0 x 10 6 Da's sodium colfosceinate powder 1.0 kg was added into 100 L purified water to be stirred and dissolved, and the water temperature was controlled at 50 °C. After being fully dissolved, the temperature was lowered to 25 °C, the pH was adjusted to 8.0 by sodium phosphate salt, then the sodium colfosceinate solution was added with colfosceinate degrading enzyme (SEQ ID NO: 1) to make the final concentration 0.3 mg / L, 0.5 mg / L, 1.0 mg / L, 2.5 mg / L, 5 mg / L respectively, and the reaction was stirred for 2.0 h. After the reaction was completed, activated carbon (0.25% wt) was added into the solution to adjust the pH to 6.0, then the plate and frame filter press was used to collect the filtrate. The filtrate was refined by 0.65 μm capsule filter, then the purified water was used for liquid exchange, and finally the spray drying was performed to obtain the hydrolyzed sodium colfosceinate product (E4-E8). Figure 2 The HPLC chromatogram thereof is shown in Figure 7 The NMR spectrum thereof is shown in

[0132] Example 7-2

[0133] The sodium colfosceinate with a molecular weight of 5.0 x 10 6Da sodium colanic acid powder 1.0 kg was added to 100 L purified water and stirred to dissolve, controlling the water temperature at 60 °C. After complete dissolution, the temperature was lowered to 50 °C, the pH was adjusted to 5.0 with sodium phosphate salt, then sodium colanic acid degrading enzyme (SEQ ID NO: 1) was added to the sodium colanic acid solution to a final concentration of 10 mg / L, and 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, then plate and frame pressure filtration was performed to collect the filtrate. The filtrate was concentrated 20-fold by nanofiltration, then exchanged with purified water, and finally spray-dried to obtain a hydrolyzed sodium colanic acid oligosaccharide composition product containing sodium colanic acid hexasaccharide (molecular weight 1126.34) and sodium colanic acid dodecasaccharide (molecular weight 2234.67).

[0134] Example 7-3

[0135] Example 1-1 was taken as the starting material, and the molecular weight of the sodium colanic acid was adjusted to 5.0 x 10 6 Da sodium colanic acid powder 1.0 kg was added to 100 L purified water and stirred to dissolve, controlling the water temperature at 60 °C. After complete dissolution, the temperature was lowered to 50 °C, the pH was adjusted to 5.0 with sodium phosphate salt, then sodium colanic acid degrading enzyme (SEQ ID NO: 1) was added to the sodium colanic acid solution to a final concentration of 10 mg / L, and 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, then plate and frame pressure filtration was performed to collect the filtrate. The filtrate was concentrated 20-fold by nanofiltration, then exchanged with purified water, and finally spray-dried to obtain a hydrolyzed sodium colanic acid oligosaccharide composition product containing sodium colanic acid hexasaccharide (molecular weight 1126.34) and sodium colanic acid dodecasaccharide (molecular weight 2234.67).

[0136] Example 8: Preparation of sodium colanic acid hexasaccharide and sodium colanic acid dodecasaccharide

[0137] Example 8-1

[0138] Example 1-1 was taken as the starting material, and the molecular weight of the sodium colanic acid was adjusted to 5.0 x 10 6Da sodium colanic acid powder 1.0 kg was added to 100 L of pure water and stirred to dissolve, with the water temperature controlled at 40 °C. After complete dissolution, the temperature was lowered to 20 °C, the pH was adjusted to 6.5 with sodium phosphate, and then colanic acid-degrading enzyme (SEQ ID NO: 1) was added to the sodium colanic acid solution to a final concentration of 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 plate-and-frame pressure filtration was performed to collect the filtrate. The filtrate was subjected to Q anion chromatography, eluted with 10 mM sodium chloride at pH 8.5, and the elution fraction one was collected; then it was eluted with 50 mM sodium chloride at pH 8.5 and the elution fraction two was collected. The elution and collection fractions one and two were separately concentrated 20 times by nanofiltration, then exchanged 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). The high-performance liquid chromatogram thereof is shown in Figure 3 、 Figure 4 , and the mass spectrum thereof is shown in Figure 5 、 Figure 6 .

[0139] Example 8-2

[0140] The sodium colanic acid powder with a molecular weight of 5.0 x 10 6 Da was added to 100 L of pure water and stirred to dissolve, with the water temperature controlled at 60 °C. After complete dissolution, the temperature was lowered to 50 °C, the pH was adjusted to 5.0 with sodium phosphate, and then colanic acid-degrading enzyme (SEQ ID NO: 1) was added to the sodium colanic acid solution to a final concentration of 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 plate-and-frame pressure filtration was performed to collect the filtrate. The filtrate was subjected to Q anion chromatography, eluted with 10 mM sodium chloride at pH 9.0, and the elution fraction one was collected; then it was eluted with 50 mM sodium chloride at pH 9.0 and the elution fraction two was collected. The elution and collection fractions one and two were separately concentrated 20 times by nanofiltration, then exchanged 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).

[0141] Example 8-3

[0142] The sodium colanic acid powder with a molecular weight of 5.0 x 10 6Da sodium colanic acid powder 1.0 kg was added to 100 L of pure water and stirred to dissolve, with the water temperature controlled at 50 °C. After complete dissolution, the temperature was lowered to 35 °C, the pH was adjusted to 8.0 with sodium phosphate, and then sodium colanic acid degrading enzyme (SEQ ID NO: 1) was added to the sodium colanic acid solution to a final concentration of 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 plate and frame pressure filtration was performed to collect the filtrate. The filtrate was subjected to Q anion chromatography, eluted with 10 mM sodium chloride at pH 8.0, and the elution fraction one was collected; then eluted with 50 mM sodium chloride at pH 8.0 and the elution fraction two was collected. The elution and collection fractions one and two were separately subjected to nanofiltration concentration 20 times, then exchanged 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).

[0143] Table 7. Hydrolyzed sodium colanic acid oligosaccharide composition or single component product assay results

[0144]

[0145] Example 9: Preparation of sodium colanic acid powder product or oligosaccharide composition product / single component product in fermentation broth

[0146] Example 9-1

[0147] Take 100 L of colanic acid fermentation broth, control the temperature at 30 °C, add sodium colanic acid degrading enzyme (SEQ ID NO: 1) to a final concentration of 0.01 mg / L, and react for 16 h. After the reaction is completed, remove bacteria by plate and frame pressure filtration, add activated carbon (2% wt) to the filtrate, adjust the pH to 5.0, and treat for 1 h before again performing plate and frame pressure filtration, then filter with a 0.65 μm Cellophane filter, alcohol precipitate with 2 volumes of anhydrous ethanol, collect the precipitate, wash repeatedly 3 times with a mixed solution of the same concentration of sodium chloride and ethanol, dehydrate 3 times with anhydrous ethanol, and finally vacuum dry to obtain sodium colanic acid powder with a weight average molecular weight of 1.4 x 10 6 Da crude and purified sodium colanic acid powder.

[0148] Example 9-2

[0149] Take the colanic acid fermentation broth 100 L, control the temperature at 30 ℃, add colanic acid degrading enzyme (SEQ ID NO: 1) to make its final concentration 0.2 mg / L, react for 2 h. After the reaction is completed, the bacteria are removed by plate and frame filter pressing, activated carbon (2% wt) is added to the filtrate, the pH is adjusted to 5.0, treated for 1 h, then plate and frame filter pressing again, then filtered with 0.65 μm capsule filter, alcohol precipitated with 2 volumes of anhydrous ethanol, the precipitate is collected and washed with a mixed solution of sodium chloride and ethanol of the same concentration for 3 times, then dehydrated with anhydrous ethanol for 3 times, finally vacuum dried to obtain a sodium colanic acid oligosaccharide composition product containing sodium colanic acid hexasaccharide (molecular weight 1126.34) and sodium colanic acid dodecasaccharide (molecular weight 2234.67). 5 Da's crude and pure sodium colanic acid powder.

[0150] Example 9-3

[0151] Take the colanic acid fermentation broth 100 L, control the temperature at 30 ℃, add colanic acid degrading enzyme (SEQ ID NO: 1) to make its final concentration 0.2 mg / L, react for 2 h. After the reaction is completed, the bacteria are removed by plate and frame filter pressing, activated carbon (2% wt) is added to the filtrate, the pH is adjusted to 5.0, treated for 1 h, then plate and frame filter pressing again, then filtered with 0.65 μm capsule filter, collect the filtrate. The filtrate is concentrated and replaced with pure water using a commercially available 500kD hollow fiber membrane, the pH is adjusted to 6.5 with sodium phosphate salt after the replacement is completed, then colanic acid degrading enzyme is added to make its final concentration 0.5 mg / L, stirred for 2 h. After the second reaction is completed, activated carbon (2% wt) is added to the colanic acid solution, the pH is adjusted to 5.0, treated for 1 h, then plate and frame filter pressing again, the filtrate is concentrated 20 times by nanofiltration, then replaced with purified water, finally spray dried to obtain a hydrolyzed sodium colanic acid oligosaccharide composition product containing sodium colanic acid hexasaccharide (molecular weight 1126.34) and sodium colanic acid dodecasaccharide (molecular weight 2234.67).

[0152] It should be noted that the fermentation broth can be directly enzymatically degraded into products of target molecular weight, including low molecular weight products, but there are many small molecule impurities in the fermentation broth, and the cost of purifying small molecule colanic acid or its salt therefrom is high, therefore, the present example 9-3 and example 9-4 adopt one-time enzymatic degradation to separate out medium and large molecular colanic acid or its salt, and then perform secondary enzymatic degradation.

[0153] Example 9-4

[0154] 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 its final concentration 0.01 mg / L, and react for 16 h. After the reaction is completed, remove bacteria by plate and frame pressure filtration, add activated carbon (2% wt) to the filtrate, adjust the pH to 5.0, and treat for 1 h before filtering again by plate and frame pressure filtration, then filter with a 0.65 μm capsule filter, and collect the filtrate. Concentrate the filtrate to pure water with a 500 kD hollow fiber membrane, adjust the pH to 6.5 with sodium phosphate salt after the liquid change is completed, then add colanic acid degrading enzyme to make its final concentration 0.5 mg / L, and stir for 2 h of reaction.

[0155] After the secondary reaction is completed, add activated carbon (2% wt) to the colanic acid solution, adjust the pH to 5.0, and treat for 1 h before filtering again by plate and frame pressure filtration, and then perform Q anion chromatography on the filtrate, elute with 10 mM sodium chloride at pH 9.0, and collect elution component one; then elute with 50 mM sodium chloride at pH 9.0 and collect elution component two. Concentrate the elution collected components one and two by nanofiltration by 20 times, then change the liquid with purified water, and finally spray dry to obtain hydrolyzed colanic acid sodium hexose (molecular weight 1126.34) and hydrolyzed colanic acid sodium dodecaose (molecular weight 2234.67).

[0156] Table 8. Determination results of hydrolyzed colanic acid sodium end product composition prepared by fermentation broth enzymolysis

[0157]

[0158] Table 9. Determination results of hydrolyzed colanic acid sodium hexose and dodecaose prepared by fermentation broth enzymolysis

[0159]

[0160] Table 10. Sequence information

[0161]

Claims

1. A method of preparing hydrolyzed colanic acid or a salt thereof, comprising the steps of: 1) adding a colanic acid-degrading enzyme having an amino acid sequence as set forth in SEQ ID NO: 1 to a solution containing colanic acid or a salt thereof; and 2) terminating the enzyme reaction; 3) filtration purification; further obtaining hydrolyzed colanic acid or a salt thereof, wherein in step 1), the final concentration of the colanic acid-degrading enzyme is between 0.01 mg / L and 10 mg / L, the reaction temperature is between 10 °C and 70 °C, the reaction solution pH is 3-9, and the termination in step 2) is achieved using activated carbon, and the activated carbon content is 0.01 wt% or more, and in step 3), the pH is adjusted to 3.0-6.0 before filtration.

2. The method of claim 1, wherein any one or more of the following are included: (i) the colanic acid salt is sodium colanic acid, and / or (ii) in step 1), the weight average molecular weight of the colanic acid or salt thereof is greater than 1 x 10 4 Da, and / or (iii) in step 1), the reaction temperature is between 20 °C and 50 °C, and / or (iv) in step 1), the reaction time is 1 h-20 h, and / or (v) in step 1), the reaction solution pH is 5-8.

3. The method of claim 2, wherein, In step 1), the weight average molecular weight of the carboxylic acid or salt thereof is greater than 5 x 10 5 Da.

4. The method of claim 2, wherein, In step 1), the weight average molecular weight of the carboxylic acid or salt thereof is greater than 2 x 10 6 Da.

5. The method of claim 1, wherein, The method further comprises steps 4) concentration and desalination; and 5) drying.

6. The method of claim 1, wherein, The method further comprises 6) multiple enzymatic hydrolysis.

7. The method of any one of claims 1-6, wherein, The activated carbon content is 0.01 wt%-5 wt%.

8. The method of claim 5, wherein, In step 5), the drying is achieved by spray drying or freeze drying, vacuum drying.

9. The method of any one of claims 1-6, wherein, The weight average molecular weight of the hydrolyzed colanic acid or salt thereof ranges from 1 x 10 3 Da to 3 x 10 6 Da.

10. The method of claim 9, wherein, The weight average molecular weight of the hydrolyzed colanic acid or salt thereof ranges from 1 x 10 3 Da to 2 x 10 6 Da.

11. The method of any one of claims 1-6, wherein, The hydrolyzed colanic acid or a salt thereof comprises acetylation and / or pyruvate modification.

12. The method of any one of claims 1-6, wherein, In the hydrolyzed colanic acid or a salt thereof the acetylation modification rate is greater than 80%; and / or the endotoxin residue is less than 0.5 EU / mg; and / or the protein residue is less than 0.1%; and / or the heavy metal residue is less than 10 mg / kg.

13. The method of claim 12, wherein, In the hydrolyzed colanic acid or a salt thereof, the acetylation modification rate is greater than 90%.

14. The method of claim 12, wherein, In the hydrolyzed colanic acid or a salt thereof, the endotoxin residue is less than 0.1 EU / mg.

15. The method of claim 12, wherein, In the hydrolyzed colanic acid or a salt thereof, the protein residue is less than 0.07%.

16. The method of any one of claims 1-6, wherein, The hydrolyzed colanic acid or a salt thereof is colanic acid hexasaccharide or a salt thereof, and / or colanic acid dodecasaccharide or a salt thereof.

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