Method for preparing colanic acid or salt thereof

Abstract

Provided is the large-scale preparation of a high-purity hydrolyzed colanic acid or a salt thereof. The method comprises: 1) adding a solution containing a colanic acid or a salt thereof to a colanic acid degradation enzyme comprising an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity thereto; and 2) performing an enzyme termination reaction, wherein the termination in step 2) is achieved by means of 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, absolute ethanol, methanol, EDTA, trichloroacetic acid and sodium carbonate, and preferably, activated carbon is used to achieve the termination in step 2), so as to obtain the hydrolyzed colanic acid or a salt thereof. The molecular structure of the hydrolyzed sodium colanate prepared by the provided method features acetylation and pyruvic acid modification. Moreover, the preparation conditions are mild, the production cost is low, continuous production can be achieved, and industrial scale-up is easily implemented. The prepared hydrolyzed sodium colanate has the characteristics of high purity and low endotoxin residue, and has potential and broad application value in fields such as cosmetics, medical devices, and oligosaccharide drugs.

Description

A method for preparing colacid or its salt

[0001] Related applications

[0002] This disclosure claims priority to Chinese application CN202411683922.7, filed on November 22, 2024, the entire contents of which are expressly incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biotechnology, and in particular relates to a method for large-scale purification and preparation of high-purity hydrolyzed colacid or its salt. Background Technology

[0004] Microbial extracellular polysaccharides are biopolymers produced by bacteria, fungi, cyanobacteria, and other microorganisms during metabolism, providing protection for these microorganisms. Under natural conditions, polysaccharides coating the surface of bacteria play a crucial role in their survival and growth in competitive environments. Kolanic acid is one such microbial extracellular polysaccharide. In solid form, it is a white, fibrous, amorphous heteropolysaccharide primarily composed of D-glucuronic acid, D-glucose, D-galactose, and L-fucose in a 1:2:2:1 ratio. It also contains various chemically modified side chains, including O-acetyl and pyruvate, and is a high-molecular-weight polymeric polysaccharide with an average molecular weight exceeding 10 million Daltons.

[0005] Microbial extracellular polysaccharides possess unique physical and rheological properties and are widely used in the food industry as stabilizers, thickeners, gelling agents, and emulsifiers. Recent studies have revealed significant biological activities of bacterial polysaccharides, including antitumor, antiviral, immunostimulatory, anti-inflammatory, antioxidant, and antimicrobial effects, attracting considerable attention. In 2017, the journal *Cell* first reported that kolanic acid induced mitochondrial fragmentation and extended the lifespan of *C. elegans*. Due to its porous cellulose structure and numerous hydrophilic groups on the colloidal surface, kolanic acid is a natural hydrogel with excellent hydrating capabilities and a soft texture, making it a promising candidate for the future cosmetics and healthcare markets. Furthermore, as a unique biopolymer, kolanic acid possesses special biological characteristics and physiological parameters, offering broad research and application prospects for lifespan extension and anti-aging.

[0006] Literature studies show that the bioactivity of polysaccharides depends on their molecular weight, with polysaccharides in different molecular weight ranges exhibiting different physiological functions. For example, high molecular weight hyaluronic acid has good viscoelasticity, moisturizing properties, anti-inflammatory effects, and lubrication, and can be used in the cosmetics industry, as a viscoelastic agent in ophthalmic surgery, and for intra-articular injection therapy. Medium molecular weight hyaluronic acid has good moisturizing, lubricating, and drug-releasing effects, and can be widely used in cosmetics, eye drops, skin burn healing, and postoperative anti-adhesion. Low molecular weight hyaluronic acid and oligomeric hyaluronic acid exhibit very strong bioactivity, including antioxidant, anti-inflammatory, collagen-promoting, and wound-healing activities. Most importantly, due to their small molecular size, they can penetrate the stratum corneum of the skin to exert their effects, and can be widely used in cosmetics.

[0007] Sodium coralamate, biosynthesized using *E. coli*, has a relatively large molecular weight, approximately 5-10 million Daltons. This high-molecular-weight polysaccharide has applications in cosmetics and health foods. However, there is currently no research on production processes for preparing hydrolyzed coralamates, especially sodium coralamate, with specific molecular weights. This significantly limits the application scenarios and efficacy of sodium coralamate. Therefore, developing a large-scale production process for finished coralamate or its salts (1×10⁻⁶) is crucial. 3 Da-2.0×10 6 The production process of Da is of great significance.

[0008] To address the aforementioned problems, this invention provides a method for the continuous, large-scale production of finished colacid or its salts. The colacid or salts prepared by the method of this invention have intact structures (including acetylation and pyruvate modification), high purity, and low endotoxin residues. Furthermore, the preparation conditions are mild and efficient, with low production costs, enabling continuous production and facilitating industrial-scale scaling. Summary of the Invention

[0009] This invention discloses a method for the continuous large-scale production of hydrolyzed colacid or its salts. Specifically, this disclosure provides a method for preparing hydrolyzed colacid or its salts, comprising the following steps: 1) adding a solution containing colacid or its salts to a colacid-degrading enzyme containing an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity with it; and 2) terminating the enzyme reaction: terminating the reaction in step 2) by means of 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 using activated carbon to terminate the reaction in step 2); thereby obtaining hydrolyzed colacid or its salts.

[0010] According to some embodiments of this disclosure, in the aforementioned method, the collamerate is sodium collamerate.

[0011] According to some embodiments of this disclosure, in the aforementioned method, in step 1), the weight-average molecular weight of the colacid or its salt is greater than 2 × 10⁻⁶. 6 Da.

[0012] In some preferred embodiments, in the aforementioned method, in step 1), the amino acid sequence of the ketamine degrading enzyme is as shown in SEQ ID NO: 1.

[0013] According to some embodiments of this disclosure, in the aforementioned method, in step 1), the final concentration of the colacid-degrading enzyme is between about 0.01 mg / L and 10 mg / L.

[0014] According to some embodiments of this 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.

[0015] According to some embodiments of this disclosure, in the aforementioned method, in step 1), the reaction time is about 1 hour to about 20 hours.

[0016] According to some embodiments of this 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.

[0017] According to some embodiments of this disclosure, the above method further includes steps 3) filtration and purification; 4) concentration and desalting; and 5) drying.

[0018] According to some embodiments of this disclosure, the above method further includes 6) multiple enzymatic hydrolysis.

[0019] According to some embodiments of this 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%.

[0020] According to some embodiments of this disclosure, in step 3), the pH is adjusted to approximately 3.0 to approximately 6.5 before filtration. In some preferred embodiments, the pH is adjusted to approximately 3.0 to approximately 6.0 before filtration.

[0021] According to some embodiments of this disclosure, in step 5), the drying is achieved by spray drying, freeze drying, or vacuum drying.

[0022] According to some embodiments of this disclosure, in the aforementioned method, the weight-average molecular weight of the hydrolyzed colaic acid or its salt is in the range of about 1 × 10⁻⁶. 3 Da - about 3×10 6In some preferred embodiments, the hydrolyzed colaic acid or its salt has a weight-average molecular weight range of about 1 × 10⁻⁶. 3 Da - about 2×10 6 Da.

[0023] According to some embodiments of this disclosure, in the aforementioned method, the hydrolyzed colaic acid or its salt contains acetylation and / or pyruvate modification.

[0024] According to some embodiments of this disclosure, in the aforementioned method, the acetylation modification rate in the hydrolyzed colaic acid or its salt is about 80-100%.

[0025] According to some embodiments of this disclosure, in the aforementioned method, the endotoxin residue in the hydrolyzed colacid or its salt is less than about 0.5 EU / mg, preferably less than about 0.1 EU / mg.

[0026] According to some embodiments of this disclosure, in the aforementioned method, the residual protein in the hydrolyzed colaic acid or its salt is less than about 0.1%, preferably less than about 0.07%.

[0027] According to some embodiments of this disclosure, in the aforementioned method, the heavy metal residue in the hydrolyzed colaic acid or its salt is less than about 10 mg / kg.

[0028] According to some embodiments of this disclosure, in the aforementioned method, the hydrolyzed colacid or its salt is colacid hexasaccharide or its salt, and / or colacid dodecanose or its salt.

[0029] Another aspect of this disclosure provides hydrolyzed colaic acid or its salt obtained according to the aforementioned method.

[0030] According to some embodiments of this disclosure, the hydrolyzed colaic acid or its salt has a weight-average molecular weight range of about 1 × 10⁻⁶. 3 Da - about 3×10 6 In some preferred embodiments, the hydrolyzed colaic acid or its salt has a weight-average molecular weight range of about 1 × 10⁻⁶. 3 Da - about 2×10 6 Da.

[0031] According to some embodiments of this disclosure, the hydrolyzed colaic acid or its salt contains acetylation and / or pyruvate modification.

[0032] According to some embodiments of this disclosure, the acetylation modification rate in the hydrolyzed colaic acid or its salt is about 80-100%.

[0033] According to some embodiments of this disclosure, the endotoxin residue in the hydrolyzed colacid or its salt is less than about 0.5 EU / mg, preferably less than about 0.1 EU / mg.

[0034] According to some embodiments of this disclosure, the residual protein in the hydrolyzed colaic acid or its salt is less than about 0.1%, preferably less than about 0.07%.

[0035] According to some embodiments of this disclosure, the heavy metal residue in the hydrolyzed colaic acid or its salt is less than about 10 mg / kg.

[0036] According to some embodiments of this disclosure, the hydrolyzed colacid or its salt is colacid hexasaccharide or its salt, and / or colacid dodecanose or its salt.

[0037] Effects of the invention:

[0038] This invention utilizes colacid-degrading enzymes to catalyze the hydrolysis and purification of hydrolyzed colacid or its salts, which has a direct objective. Firstly, the high-purity hydrolyzed colacid or its salts obtained by this invention have a weight-average molecular weight covering the smallest unit of colacid or its salts (1.1 × 10⁻⁶). 3 Da) to 2.0×10 6 The product contains acetylation and pyruvate modification, laying the foundation for academic research on the structure and structure-activity relationship of colacid or its salts; secondly, the reaction conditions of this invention are extremely simple, requiring no instruments or equipment, and can be carried out under mild conditions; finally, the product prepared by this 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. Attached Figure Description

[0039] Figure 1 shows a molecular weight of 1.2 × 10⁻⁶. 6 Da, 7.6×10 5 Da, 4.2×10 5 High-performance liquid chromatography (HPLC) chromatogram of Da's sodium collamate product.

[0040] Figure 2 shows the high performance liquid chromatogram of the hydrolyzed sodium collamate oligosaccharide composition.

[0041] Figure 3 shows the high performance liquid chromatogram of hydrolyzed colarate sodium hexasaccharide.

[0042] Figure 4 shows the high performance liquid chromatogram of hydrolyzed sodium collamate dodecanose.

[0043] Figure 5 shows the mass spectrum of hydrolyzed sodium colarate hexasaccharide.

[0044] Figure 6 shows the mass spectrum of hydrolyzed sodium colarate dodecanose.

[0045] Figure 7 shows the NMR spectrum of the hydrolyzed sodium collamerate oligosaccharide composition. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of this invention. The raw materials used in the embodiments are all commercially available.

[0047] definition

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0049] All numerical designations used in this document, such as pH, temperature, time, concentration, content, and molecular weight, including ranges, are approximate values ​​and are varied (+) 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 "approximately," although not always explicitly stated.

[0050] The terms “about” and “approximately” include quantities within ±10% of the relevant numerical value.

[0051] As those skilled in the art will understand, for any and all purposes, particularly in providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficiently descriptive, and the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc.

[0052] In this article, "hydrolyzed colaic acid or its salt" refers to the product obtained after the degradation reaction, with a weight-average molecular weight in the range of approximately 1 × 10⁻⁶. 3 Da - about 5×10 6 Preferably, the hydrolyzed colaic acid or its salt has a weight-average molecular weight range of about 1 × 10⁻⁶. 3 Da - about 2×10 6 It should be noted that the weight-average molecular weight of the reactant colaic acid or its salt is greater than that of the hydrolyzed colaic acid or its salt.

[0053] When "oligosaccharide composition" or "oligosaccharide single component" is used, it only indicates whether the product at a specific molecular weight is separated. Both refer to hydrolyzed colaic acid or its salt prepared according to the technical solution of this invention. The former means that a product with a specific molecular weight can be identified but not separated, while the latter means that a product with a specific molecular weight is separated. In this document, "oligosaccharide" means a weight-average molecular weight of less than 1 × 10⁻⁶. 4 Da's colacid or its salts, such as colacid sodium hexasaccharide or colacid sodium dodecasaccharide.

[0054] When used herein, the term "solution" is a broad term that is not limited to the traditional concept and refers to a composition. A "solution" can be an aqueous or non-aqueous mixture, for example, it can include homogeneous or heterogeneous mixtures of polymers and water, and it can also include forms such as suspensions, colloids, atomized liquids, gels, ointments, creams or lotions, and it can also include fermentation broths.

[0055] In this invention, "colamine salt" refers to the salt formed by the reaction of colacid with any alkaline substance. Depending on the specific type of cation in the colamine salt, the colamine salt can be sodium colamine, potassium colamine, magnesium colamine, aluminum colamine, calcium colamine, ammonium colamine, etc. Colamine salts may also contain more than one cation, such as sodium or potassium colamine. One aspect of this disclosure provides a method for preparing hydrolyzed colacid or its salt, comprising the following steps: 1) adding a solution containing colacid or its salt to a colacid-degrading enzyme containing an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity with it; and 2) terminating the enzyme reaction: terminating the reaction in step 2) by means of 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 using activated carbon to terminate the reaction in step 2); thereby obtaining hydrolyzed colacid or its salt.

[0056] According to some embodiments of this disclosure, in the aforementioned method, in step 1), the weight-average molecular weight of the colacid or its salt is greater than 1 × 10⁻⁶. 4 In some preferred embodiments, the weight-average molecular weight range of colacid or its salts is preferably in the range of about 1 × 10⁻⁶. 4 Da - about 6×10 6 Between Da, preferably around 5×10 5 Da - about 6×10 6 Between Da. For example, the weight-average molecular weight of colaic acid or its salts can be any value within the range or any range, and the weight-average molecular weight can be approximately 1 × 10⁻⁶. 4 Da, 2×10 4 Da, 3×10 4 Da, 4×10 4Yes, 5×10 4 Yes, 6×10 4 Yes, 7×10 4 Yes, 8×10 4 Yes, 9×10 4 Yes, 1×10 5 Yes, 2×10 5 Yes, 3×10 5 Yes, 4×10 5 Yes, 5×10 5 Yes, 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 6Da, 4.3×10 6 Da, 4.4×10 6 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, 5×10 6 Da, 5.1×10 6 Da, 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.

[0057] In this disclosure, the colacid-degrading enzyme comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity with it. For example, the colacid-degrading enzyme comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with SEQ ID NO: 1. In some preferred embodiments, the amino acid sequence of the colacid-degrading enzyme is as shown in SEQ ID NO: 1.

[0058] In some embodiments, in step 1), the colarate is sodium colarate.

[0059] In some embodiments, in the aforementioned method, the final concentration of the colacid-degrading enzyme in step 1) is between approximately 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, 0.13 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, and 0.26 mg / L. , 0.27mg / L, 0.28mg / L, 0.29mg / L, 0.30mg / L, 0.31mg / L, 0.32mg / L, 0.33mg / L, 0.34mg / L, 0.35mg / L, 0.36mg / L, 0.37mg / L, 0.38mg / L, 0.39mg / L , 0.40mg / L, 0.41mg / L, 0.42mg / L, 0.43mg / L, 0.44mg / L, 0.45mg / L, 0.46mg / L, 0.47mg / L, 0.48mg / L, 0.49mg / L, 0.50mg / L, 0.51mg / L, 0.52mg / L, 0.53mg / L, 0.54mg / L, 0.55mg / L, 0.56mg / L, 0.57mg / L, 0.58mg / L, 0.59mg / L, 0.60mg / L, 0.61mg / L, 0.62mg / L, 0.63mg / L, 0.64mg / L, 0.65mg / L, 0.66mg / L, 0.67mg / L, 0.68mg / L, 0.69mg / L, 0.70mg / L, 0.71mg / L, 0.72mg / L, 0.73mg / L, 0.74mg / L, 0.75mg / L, 0.76mg / L, 0.77mg / L, 0.78mg / L, 0 .79mg / L, 0.80mg / L, 0.81mg / L, 0.82mg / L, 0.83mg / L, 0.84mg / L, 0.85mg / L, 0.86mg / L, 0.87mg / L, 0.88mg / L, 0.89mg / L, 0.90mg / L, 0.91mg / L, 0 .92mg / L, 0.93mg / L, 0.94mg / L, 0.95mg / L, 0.96mg / L, 0.97mg / L, 0.98mg / L, 0.99mg / L, 1.00mg / L, 1.01mg / L, 1.02mg / L, 1.03mg / L, 1.04mg / L, 1.0.5 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 1.19mg / L,1.20mg / L,1.21mg / L,1.22mg / L,1.23mg / L,1.24mg / L,1.25mg / L ,1.26mg / L,1.27mg / L,1.28mg / L,1.29mg / L,1.30mg / L,1.31mg / L,1.32mg / L 1.33mg / L, 1.34mg / L, 1.35mg / L, 1.36mg / L, 1.37mg / L, 1.38mg / L, 1.39mg / L, 1.40mg / L, 1.41mg / L, 1.42mg / L, 1.43mg / L, 1.44mg / L, 1.45mg / L, 1.46mg / L,1.47mg / L,1.48mg / L,1.49mg / L,1.50mg / L,1.51mg / L,1.52mg / L,1.53mg / L,1.54mg / L,1.55mg / L,1.56mg / L,1.57mg / L,1.58mg / L,1.59mg / L,1.60mg / 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 g / L,1.75mg / L,1.76mg / L,1.77mg / L,1.78mg / L,1.79mg / L,1.80mg / L,1.81 mg / L,1.82mg / L,1.83mg / L,1.84mg / L,1.85mg / L,1.86mg / L,1.87mg / L,1.88 mg / L,1.89mg / L,1.90mg / L,1.91mg / L,1.92mg / L,1.93mg / L,1.94mg / L,1.9 5mg / L,1.96mg / L,1.97mg / L,1.98mg / L,1.99mg / L,2.00mg / L,2.01mg / L,2.0 2mg / L, 2.03mg / L, 2.04mg / L, 2.05mg / L, 2.06mg / L, 2.07mg / L, 2.08mg / L, 2.09mg / L, 2.10mg / L, 2.11mg / L, 2.12mg / L, 2.13mg / L, 2.14mg / L, 2.15mg / L, 2.16mg / L,2.17mg / L,2.18mg / L,2.19mg / L,2.20mg / L,2.21mg / L,2.22mg / L,2.23mg / L,2.24mg / L,2.25mg / L,2.26mg / L,2.27mg / L,2.28mg / L,2.29mg / L, 2.30mg / L,2.31mg / L,2.32mg / L,2.33mg / L,2.34mg / L,2.35mg / L,2.36mg / L,2.37mg / L,2.38mg / L,2.39mg / L,2.40mg / L,2.41mg / L,2.42mg / L,2.43mg / L ,2.44mg / L,2.45mg / L,2.46mg / L,2.47mg / L,2.48mg / L,2.49mg / L,2.50mg / L,2.51mg / L,2.52mg / L,2.53mg / L,2.54mg / L,2.55mg / L,2.56mg / L,2.57mg / L,2.58mg / L,2.59mg / L,2.60mg / L,2.61mg / L,2.62mg / L,2.63mg / L,2.64mg / L,2.65mg / L,2.66mg / L,2.67mg / L,2.68mg / L,2.69mg / L,2.70mg / L,2.71mg / L, 2.72mg / L, 2.73mg / L, 2.74mg / L, 2.75mg / L, 2.76mg / L, 2.77mg / L, 2.78mg / L, 2.79mg / L, 2.80mg / L, 2.81mg / L, 2.82mg / L, 2.83mg / L, 2.84mg / L, 2.85mg / L, 2.86mg / L, 2.87mg / L, 2.88mg / L, 2.89mg / L, 2.90mg / L, 2.91mg / L, 2.92mg / L, 2.93mg / L, 2.94mg / L, 2.95mg / L, 2.96mg / L, 2.97mg / L, 2.98mg / L, 2.99 mg / L,3.00mg / L,3.01mg / L,3.02mg / L,3.03mg / L,3.04mg / L,3.05mg / L,3.06mg / L,3.07mg / L,3.08mg / L,3.09mg / L,3.10mg / L,3.11mg / L,3.12mg / L,3.1 3mg / L,3.14mg / L,3.15mg / L,3.16mg / L,3.17mg / L,3.18mg / L,3.19mg / L,3.20mg / L,3.21mg / L,3.22mg / L,3.23mg / L,3.24mg / L,3.25mg / L,3.26mg / L.27mg / L, 3.28mg / L, 3.29mg / L, 3.30mg / L, 3.31mg / L, 3.32mg / L, 3.33mg / L, 3.34mg / L, 3.35mg / L, 3.36mg / L, 3.37mg / L, 3.38mg / L, 3.39mg / L, 3.40mg / L 3.41mg / L,3.42mg / L,3.43mg / L,3.44mg / L,3.45mg / L,3.46mg / L,3.47mg / L ,3.48mg / L,3.49mg / L,3.50mg / L,3.51mg / L,3.52mg / L,3.53mg / L,3.54mg / L 3.55mg / L, 3.56mg / L, 3.57mg / L, 3.58mg / L, 3.59mg / L, 3.60mg / L, 3.61mg / L, 3.62mg / L, 3.63mg / L, 3.64mg / L, 3.65mg / L, 3.66mg / L, 3.67mg / L, 3.68mg / L,3.69mg / L,3.70mg / L,3.71mg / L,3.72mg / L,3.73mg / L,3.74mg / L,3.75mg / L,3.76mg / L,3.77mg / L,3.78mg / L,3.79mg / L,3.80mg / L,3.81mg / L,3.82mg / 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 g / L,3.97mg / L,3.98mg / L,3.99mg / L,4.00mg / L,4.01mg / L,4.02mg / L,4.03 mg / L,4.04mg / L,4.05mg / L,4.06mg / L,4.07mg / L,4.08mg / L,4.09mg / L,4.10 mg / L,4.11mg / L,4.12mg / L,4.13mg / L,4.14mg / L,4.15mg / L,4.16mg / L,4.1 7mg / L,4.18mg / L,4.19mg / L,4.20mg / L,4.21mg / L,4.22mg / L,4.23mg / L,4.2 4mg / L, 4.25mg / L, 4.26mg / L, 4.27mg / L, 4.28mg / L, 4.29mg / L, 4.30mg / L, 4.31mg / L, 4.32mg / L, 4.33mg / L, 4.34mg / L, 4.35mg / L, 4.36mg / L, 4.37mg / L, 4.38mg / L,4.39mg / L,4.40mg / L,4.41mg / L,4.42mg / L,4.43mg / L,4.44mg / L,4.45mg / L,4.46mg / L,4.47mg / L,4.48mg / L,4.49mg / L,4.50mg / L,4.51mg / L 4.52mg / L,4.53mg / L,4.54mg / L,4.55mg / L,4.56mg / L,4.57mg / L,4.58mg / L,4.59mg / L,4.60mg / L,4.61mg / L,4.62mg / L,4.63mg / L,4.64mg / L,4.65mg / L ,4.66mg / L,4.67mg / L,4.68mg / L,4.69mg / L,4.70mg / L,4.71mg / L,4.72mg / L,4.73mg / L,4.74mg / L,4.75mg / L,4.76mg / L,4.77mg / L,4.78mg / L,4.79mg / L,4.80mg / L,4.81mg / L,4.82mg / L,4.83mg / L,4.84mg / L,4.85mg / L,4.86mg / L,4.87mg / L,4.88mg / L,4.89mg / L,4.90mg / L,4.91mg / L,4.92mg / L,4.93mg / L, 4.94mg / L, 4.95mg / L, 4.96mg / L, 4.97mg / L, 4.98mg / L, 4.99mg / L, 5.00mg / L, 5.01mg / L, 5.02mg / L, 5.03mg / L, 5.04mg / L, 5.05mg / L, 5.06mg / L, 5.07mg / L, 5.08mg / L, 5.09mg / L, 5.10mg / L, 5.11mg / L, 5.12mg / L, 5.13mg / L, 5.14mg / L, 5.15mg / L, 5.16mg / L, 5.17mg / L, 5.18mg / L, 5.19mg / L, 5.20mg / L, 5.21 mg / L,5.22mg / L,5.23mg / L,5.24mg / L,5.25mg / L,5.26mg / L,5.27mg / L,5.28mg / L,5.29mg / L,5.30mg / L,5.31mg / L,5.32mg / L,5.33mg / L,5.34mg / L,5.3 5mg / L, 5.36mg / L, 5.37mg / L, 5.38mg / L, 5.39mg / L, 5.40mg / L, 5.41mg / L, 5.42mg / L, 5.43mg / L, 5.44mg / L, 5.45mg / L, 5.46mg / L, 5.47mg / L, 5.48mg / L.49mg / L,5.50mg / L,5.51mg / L,5.52mg / L,5.53mg / L,5.54mg / L,5.55mg / L,5 .56mg / L,5.57mg / L,5.58mg / L,5.59mg / L,5.60mg / L,5.61mg / L,5.62mg / L, 5.63mg / L,5.64mg / L,5.65mg / L,5.66mg / L,5.67mg / L,5.68mg / L,5.69mg / L ,5.70mg / L,5.71mg / L,5.72mg / L,5.73mg / L,5.74mg / L,5.75mg / L,5.76mg / L ,5.77mg / L,5.78mg / L,5.79mg / L,5.80mg / L,5.81mg / L,5.82mg / L,5.83mg / L,5.84mg / L,5.85mg / L,5.86mg / L,5.87mg / L,5.88mg / L,5.89mg / L,5.90mg / L,5.91mg / L,5.92mg / L,5.93mg / L,5.94mg / L,5.95mg / L,5.96mg / L,5.97mg / L,5.98mg / L,5.99mg / L,6.00mg / L,6.01mg / L,6.02mg / L,6.03mg / L,6.04mg / 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 g / L,6.19mg / L,6.20mg / L,6.21mg / L,6.22mg / L,6.23mg / L,6.24mg / L,6.25 mg / L,6.26mg / L,6.27mg / L,6.28mg / L,6.29mg / L,6.30mg / L,6.31mg / 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, 6.4 6mg / L,6.47mg / L,6.48mg / L,6.49mg / L,6.50mg / L,6.51mg / L,6.52mg / L,6. 53mg / L,6.54mg / L,6.55mg / L,6.56mg / L,6.57mg / L,6.58mg / L,6.59mg / L,6.60mg / L,6.61mg / L,6.62mg / L,6.63mg / L,6.64mg / L,6.65mg / L,6.66mg / L,6 .67mg / L,6.68mg / L,6.69mg / L,6.70mg / L,6.71mg / L,6.72mg / L,6.73mg / L, 6.74mg / L,6.75mg / L,6.76mg / L,6.77mg / L,6.78mg / L,6.79mg / L,6.80mg / L ,6.81mg / L,6.82mg / L,6.83mg / L,6.84mg / L,6.85mg / L,6.86mg / L,6.87mg / L ,6.88mg / L,6.89mg / L,6.90mg / L,6.91mg / L,6.92mg / L,6.93mg / L,6.94mg / L,6.95mg / L,6.96mg / L,6.97mg / L,6.98mg / L,6.99mg / L,7.00mg / L,7.01mg / 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, 7.16mg / L, 7.17mg / L, 7.18mg / L, 7.19mg / L, 7.20mg / L, 7.21mg / L, 7.22mg / L, 7.23mg / L, 7.24mg / L, 7.25mg / L, 7.26mg / L, 7.27mg / L, 7.28mg / L, 7.29mg / L g / L,7.30mg / L,7.31mg / L,7.32mg / L,7.33mg / L,7.34mg / L,7.35mg / L,7.36 mg / L,7.37mg / L,7.38mg / L,7.39mg / L,7.40mg / L,7.41mg / L,7.42mg / L,7.43 mg / L,7.44mg / L,7.45mg / L,7.46mg / L,7.47mg / L,7.48mg / L,7.49mg / L,7.5 0mg / L,7.51mg / L,7.52mg / L,7.53mg / L,7.54mg / L,7.55mg / L,7.56mg / L,7.5 7mg / L, 7.58mg / L, 7.59mg / L, 7.60mg / L, 7.61mg / L, 7.62mg / L, 7.63mg / L, 7.64mg / L, 7.65mg / L, 7.66mg / L, 7.67mg / L, 7.68mg / L, 7.69mg / L, 7.70mg / L, 7.71mg / L, 7.72mg / L, 7.73mg / L, 7.74mg / L, 7.75mg / L, 7.76mg / L, 7.77mg / L, 7.78mg / L, 7.79mg / L, 7.80mg / L, 7.81mg / L, 7.82mg / L, 7.83mg / L, 7.84mg / L, 7.85mg / L, 7.86mg / L, 7.87mg / L, 7.88mg / L, 7.89mg / L, 7.90mg / L, 7.91mg / L, 7.92mg / L, 7.93mg / L, 7.94mg / L, 7.95mg / L, 7.96mg / L, 7.97mg / L, 7.98mg / L ,7.99mg / L,8.00mg / L,8.01mg / L,8.02mg / L,8.03mg / L,8.04mg / L,8.05mg / L,8.06mg / L,8.07mg / L,8.08mg / L,8.09mg / L,8.10mg / L,8.11mg / L,8.12mg / L L, 8.13mg / L, 8.14mg / L, 8.15mg / L, 8.16mg / L, 8.17mg / L, 8.18mg / L, 8.19mg / L, 8.20mg / L, 8.21mg / L, 8.22mg / L, 8.23mg / L, 8.24mg / L, 8.25mg / L, 8.26mg / L, 8.27mg / L, 8.28mg / L, 8.29mg / L, 8.30mg / L, 8.31mg / L, 8.32mg / L, 8.33mg / L, 8.34mg / L, 8.35mg / L, 8.36mg / L, 8.37mg / L, 8.38mg / L, 8.39mg / L, 8.40mg / L, 8.41mg / L, 8.42mg / L, 8.43mg / L, 8.44mg / L, 8.45mg / L, 8.46mg / L, 8.47mg / L, 8.48mg / L, 8.49mg / L, 8.50mg / L, 8.51mg / L, 8.52mg / L, 8.53mg / L, 8.54 mg / L,8.55mg / L,8.56mg / L,8.57mg / L,8.58mg / L,8.59mg / L,8.60mg / L,8.61mg / L,8.62mg / L,8.63mg / L,8.64mg / L,8.65mg / L,8.66mg / L,8.67mg / L,8.6 8mg / L, 8.69mg / L, 8.70mg / L, 8.71mg / L, 8.72mg / L, 8.73mg / L, 8.74mg / L, 8.75mg / L, 8.76mg / L, 8.77mg / L, 8.78mg / L, 8.79mg / L, 8.80mg / L, 8.81mg / L.82mg / L, 8.83mg / L, 8.84mg / L, 8.85mg / L, 8.86mg / L, 8.87mg / L, 8.88mg / L, 8.89mg / L, 8.90mg / L, 8.91mg / L, 8.92mg / L, 8.93mg / L, 8.94mg / L, 8.95mg / L, 8.96mg / L, 8.97mg / L, 8.98mg / L, 8.99mg / L, 9.00mg / L, 9.01mg / L, 9.02mg / L, 9.03mg / L, 9.04mg / L, 9.05mg / L, 9.06mg / L, 9.07mg / L, 9.08mg / L, 9.09mg / L ,9.10mg / L,9.11mg / L,9.12mg / L,9.13mg / L,9.14mg / L,9.15mg / L,9.16mg / L,9.17mg / L,9.18mg / L,9.19mg / L,9.20mg / L,9.21mg / L,9.22mg / L,9.23mg / L L,9.24mg / L,9.25mg / L,9.26mg / L,9.27mg / L,9.28mg / L,9.29mg / L,9.30mg / L,9.31mg / L,9.32mg / L,9.33mg / L,9.34mg / L,9.35mg / L,9.36mg / L,9.37mg / L, 9.38mg / L, 9.39mg / L, 9.40mg / L, 9.41mg / L, 9.42mg / L, 9.43mg / L, 9.44mg / L, 9.45mg / L, 9.46mg / L, 9.47mg / L, 9.48mg / L, 9.49mg / L, 9.50mg / L, 9.51mg / L, 9.52mg / L, 9.53mg / L, 9.54mg / L, 9.55mg / L, 9.56mg / L, 9.57mg / L, 9.58mg / L, 9.59mg / L, 9.60mg / L, 9.61mg / L, 9.62mg / L, 9.63mg / L, 9.64mg / L, 9.65 mg / L,9.66mg / L,9.67mg / L,9.68mg / L,9.69mg / L,9.70mg / L,9.71mg / L,9.72mg / L,9.73mg / L,9.74mg / L,9.75mg / L,9.76mg / L,9.77mg / L,9.78mg / L,9.7 9mg / L, 9.80mg / L, 9.81mg / L, 9.82mg / L, 9.83mg / L, 9.84mg / L, 9.85mg / L, 9.86mg / L, 9.87mg / L, 9.88mg / L, 9.89mg / L, 9.90mg / L, 9.91mg / L, 9.92mg / L.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.

[0060] In some embodiments, in the aforementioned method, the reaction temperature in step 1) is between about 10°C and about 70°C, preferably between about 20°C and 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.

[0061] In some embodiments, in the aforementioned method, the reaction time in step 1) is about 1-20 hours, for example 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours.

[0062] 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.

[0063] In some embodiments, the above method further includes steps 3) filtration and purification; 4) concentration and desalting; and 5) drying.

[0064] In some embodiments, the above method further includes 6) multiple enzymatic hydrolysis, such as secondary enzymatic hydrolysis, tertiary enzymatic hydrolysis, etc.

[0065] 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 10 g of activated carbon added to 1 L of colacid solution. 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, activated carbon content of 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%, or 0.18 wt%. , 0.19wt%, 0.2wt%, 0.21wt%, 0.22wt%, 0.23wt%, 0.24wt%, 0.25wt%, 0. 26wt%, 0.27wt%, 0.28wt%, 0.29wt%, 0.3wt%, 0.31wt%, 0.32wt%, 0.33w t%, 0.34wt%, 0.35wt%, 0.36wt%, 0.37wt%, 0.38wt%, 0.39wt%, 0.4wt%, 0.41wt%, 0.42wt%, 0.43wt%, 0.44wt%, 0.45wt%, 0.46wt%, 0.47wt%, 0. 48wt%, 0.49wt%, 0.5wt%, 0.51wt%, 0.52wt%, 0.53wt%, 0.54wt%, 0.55w t%, 0.56wt%, 0.57wt%, 0.58wt%, 0.59wt%, 0.6wt%, 0.61wt%, 0.62wt%, 0.63wt%, 0.64wt%, 0.65wt%, 0.66wt%, 0.67wt%, 0.68wt%, 0.69wt%, 0. 7wt%, 0.71wt%, 0.72wt%, 0.73wt%, 0.74wt%, 0.75wt%, 0.76wt%, 0.77w t%, 0.78wt%, 0.79wt%, 0.8wt%, 0.81wt%, 0.82wt%, 0.83wt%, 0.84wt%, 0.85wt%, 0.86wt%, 0.87wt%, 0.88wt%, 0.89wt%, 0.9wt%, 0.91wt%, 0.9 2wt%, 0.93wt%, 0.94wt%, 0.95wt%, 0.96wt%, 0.97wt%, 0.98wt%, 0.99w t%, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt%, 2.5wt%, 2.6wt%, 2.7wt%, 2.8wt%, 2.9wt%, 3wt%, 3.1wt%, 3.2wt%, 3.3wt%, 3.4wt%, 3.5wt%, 3.6wt%, 3.7wt%, 3.8wt%, 3.9wt%, 4wt%, 4.1wt%, 4.2wt%, 4.3wt%, 4.4wt%, 4.5wt%, 4.6wt%, 4.7wt%, 4.8wt%, 4.9wt%, or 5wt%.

[0066] In some embodiments, in step 3), the pH is adjusted to approximately 3.0-6.5 before filtration; in some preferred embodiments, the pH is adjusted to approximately 3.0-approximately 6.5 before filtration; and in some preferred embodiments, the pH is adjusted to approximately 3.0-approximately 6.0 before filtration. For example, the pH can be adjusted to approximately 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.

[0067] In some embodiments, in step 5), the drying is achieved by spray drying, freeze drying, or vacuum drying.

[0068] In some embodiments, in the foregoing method, the weight-average molecular weight of the hydrolyzed colaic acid or its salt is in the range of about 1 × 10⁻⁶. 3 Da - about 3×10 6 In some preferred embodiments, the hydrolyzed colaic acid or its salt has a weight-average molecular weight range of about 1 × 10⁻⁶. 3 Da - about 2×10 6 Da. The weight-average molecular weight of the hydrolyzed colaic acid or its salt can be any value within a range or any range, for example, 1 × 10⁻⁶. 3 Da, 2×10 3 Da, 3×10 3 Da, 4×10 3 Da, 5×10 3 Da, 6×10 3 Da, 7×10 3 Da, 8×10 3 Da, 9×10 3 Da, 1×10 4 Da, 2×10 4 Da, 3×104 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, 4.3×10 5 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, 4.9×105 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 5 Yes, 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, 9.5×105 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.

[0069] In some embodiments, the hydrolyzed colaic acid or its salt in the foregoing method comprises acetylation and / or pyruvate modification.

[0070] In some embodiments, in the foregoing method, the acetylation modification rate in the hydrolyzed colaic acid or its salt is about 80% to 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%.

[0071] In some embodiments, the endotoxin residue in the hydrolyzed colacid or its salt is less than about 0.5 EU / mg, and in some preferred embodiments, the endotoxin residue is less than about 0.1 EU / mg.

[0072] In some embodiments, in the foregoing method, the residual protein in the hydrolyzed colaic 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%, or about 0.01%. In some preferred embodiments, the residual protein 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%.

[0073] In some embodiments, in the foregoing method, the heavy metal residue in the hydrolyzed colaic 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, or about 1 mg / kg.

[0074] In some embodiments, the hydrolyzed colacid or its salt in the foregoing method may also be colacid hexasaccharide or its salt, and / or colacid dodecanoic acid or its salt. For example, the hydrolyzed colacid or its salt includes, but is not limited to, compositions comprising colacid hexasaccharide or its salt, and colacid dodecanoic acid or its salt; or colacid hexasaccharide or its salt, or colacid dodecanoic acid or its salt, which can be isolated separately.

[0075] Another aspect of this disclosure provides hydrolyzed colaic acid or its salt prepared according to the foregoing method.

[0076] In some embodiments, the weight-average molecular weight of the hydrolyzed colaic acid or its salt prepared according to the foregoing method is in the range of about 1 × 10⁻⁶. 3 Da - about 5×10 6 In some preferred embodiments, the hydrolyzed colaic acid or its salt has a weight-average molecular weight range of about 1 × 10⁻⁶. 3 Da - about 2×10 6 In some preferred embodiments, the hydrolyzed colaic acid or its salt has a weight-average molecular weight in the range of about 1 × 10⁻⁶. 3 Da-about 1.5×10 6 Da. The weight-average molecular weight of the hydrolyzed colaic acid or its salt can be any value within a range or any range, for example, 1 × 10⁻⁶. 3 Da, 2×10 3 Da, 3×10 3 Da, 4×10 3 Da, 5×10 3 Da, 6×10 3 Da, 7×10 3 Da, 8×10 3 Da, 9×10 3 Da, 1×104 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, 4.3×10 5 Yes, 4.4×10 5 Yes, 4.5×10 5 Yes, 4.6×10 5 Yes, 4.7×10 5Yes, 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 5 Yes, 8.9×10 5 Yes, 9×10 5 Yes, 9.1×10 5 Yes, 9.2×10 5 Yes, 9.3×10 5Yes, 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, 4.4×10 6 Yes, 4.5×10 6 Yes, 4.6×10 6 Yes, 4.7×10 6 Yes, 4.8×10 6 Yes, 4.9×106 Da, or 5×10 6 Da.

[0077] In some embodiments, the hydrolyzed colacid or its salt prepared according to the foregoing method comprises acetylation and / or pyruvate modification.

[0078] In some embodiments, the acetylation modification rate in the hydrolyzed colaic acid or its salt prepared according to the foregoing method is about 80% to 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%.

[0079] In some embodiments, the endotoxin residue in the hydrolyzed colacid or its salt prepared according to the foregoing method is less than about 0.5 EU / mg, and in some preferred embodiments, the endotoxin residue is less than about 0.1 EU / mg.

[0080] In some embodiments, the residual protein content in the hydrolyzed colaic acid or its salt prepared according to the foregoing 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 residual protein content 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%.

[0081] In some embodiments, the heavy metal residue in the hydrolyzed colaic acid or its salt prepared according to the foregoing 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.

[0082] In some embodiments, the hydrolyzed colacid or its salt prepared according to the foregoing method may also be colacid hexasaccharide or its salt, and / or colacid dodecanoate or its salt. For example, the hydrolyzed colacid or its salt includes, but is not limited to, compositions comprising colacid hexasaccharide or its salt, and colacid dodecanoate or its salt; or colacid hexasaccharide or its salt, or colacid dodecanoate or its salt, which can be isolated separately.

[0083] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0084] The colacid fermentation broth used in this invention was provided by Shenzhen Baiyin Biotechnology Co., Ltd. using an engineered strain of Escherichia coli (refer to Chinese invention patent application CN115287314A), and the colacid degrading enzyme was provided by Shenzhen Baiyin Biotechnology Co., Ltd. using an engineered strain of Escherichia coli (refer to Chinese invention patent application CN118685474A) for fermentation and purification.

[0085] Test methods

[0086] Protein content was determined using the Lorry method, heavy metal residues were determined using ICP-MS, endotoxins were determined using the Limulus Amebocyte Lysate (LAL) gel electrophoresis, and acetylation was quantified by comparing the CH3 peak of pyruvate in the H-chromosome spectrum.

[0087] Methods for analyzing the purity and weight-average molecular weight of hydrolyzed sodium colarate: The instrument used was an Agilent 1260II with a differential detector. The chromatographic column was either a PolySep-GFC-P6000 tandem with a PolySep-GFC-P4000 or a PolySep-GFC-P4000 tandem with a PolySep-GFC-P2000. The mobile phase was 20 mM sodium chloride, the flow rate was 0.6 mL / min, and the isocratic cycle lasted for 50 min.

[0088] Example

[0089] Example 1-1

[0090] A certain amount of Coriander fermentation broth was taken, flocculant was added, and the pH was adjusted to alkaline. After dilution and standing, the solution was sterilized and then adsorbed with an adsorbent under acidic conditions. After filtration, anhydrous ethanol was added to precipitate the precipitate. 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 approximately 2:8 to 4:6). Subsequently, it was dehydrated with anhydrous ethanol and finally dried to obtain 1.0 kg of a product with a weight average molecular weight of 5.0 × 10⁻⁶. 6 Da's crude pure sodium collamate powder has an acetylation modification rate of 99.7%.

[0091] Take 5 portions of the above-prepared material with a molecular weight of 5.0 × 10⁻⁶. 6 0.01 kg of sodium colarate powder was added to 1 L of pure water and stirred until dissolved, maintaining the water temperature at 50°C. After complete dissolution, the temperature was lowered to 25°C, and 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 0.2 mg / L, and the mixture was stirred for 1 hour. After the reaction, the five samples were treated as follows:

[0092] (1) Activated carbon termination: Add activated carbon (0.5% wt) and adjust the pH to 4.5 for 1 h, then collect the filtrate by plate and frame filtration;

[0093] (2) High temperature termination: Heat to 75℃ for 1 hour, then cool to room temperature;

[0094] (3) Termination with anhydrous ethanol: Add 2L of anhydrous ethanol and treat for 1h;

[0095] (4) Proteinase termination: Add proteinase K at a final concentration of 125 mg / L and treat for 1 h;

[0096] (5) Do not do anything.

[0097] Add 0.2 kg of sodium chloride powder to each of the five solutions and stir to dissolve. Then, filter the solution through a 0.65 μm Nenz filter. Next, precipitate the solution with 20 L of anhydrous ethanol. Collect the precipitate and wash it three times with a mixed solution of sodium chloride and ethanol of the same concentration. Then, dehydrate the precipitate three times with anhydrous ethanol. Finally, dry the precipitate under vacuum to obtain the hydrolyzed sodium collamerate powders F1-F5 shown in Table 1 below.

[0098] The data in the table show that activated carbon treatment yields superior results compared to other termination methods. Failure to terminate enzyme activity leads to further molecular weight reduction during subsequent treatments; high-temperature treatment easily causes acetylation loss; ethanol treatment poses a hazard when used in non-specific containers; protease treatment is not ideal for termination and introduces proteins; while activated carbon can simultaneously adsorb impurities and enzymes, resulting in a significant termination effect and convenient treatment. Therefore, using activated carbon as the termination method is more ideal.

[0099] Table 1. Effects of different termination methods

[0100] Examples 1-2

[0101] Take one portion of the sample prepared in Example 1-1 with a molecular weight of 5.0 × 10⁻⁶. 6 1.0 kg of sodium colarate powder was dissolved in 100 L of pure water under a controlled temperature of 50 °C. After complete dissolution, the solution was cooled to 25 °C, and 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 1.0 mg / L, and the mixture was stirred for 3 hours. After the reaction was complete, activated carbon (0.25% wt) was added to the solution, and the pH was adjusted to 6.0. The solution was then subjected to plate and frame filtration to collect the filtrate. The filtrate was then finely filtered through a 0.65 μm Capillary filter, replaced with purified water, and finally spray-dried to obtain a weight-average molecular weight of 1.2 × 10⁻⁶. 4 Da's hydrolyzed sodium colarate product.

[0102] Comparative Example 1

[0103] Take one portion of the sample prepared in Example 1-1 with a molecular weight of 5.0 × 10⁻⁶. 60.1 kg of sodium collamerate 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 lowered to 25 °C, and the pH was adjusted to 8.0 with sodium phosphate. The temperature was then raised to 134 °C and reacted for 1 hour. After the reaction was complete, activated carbon (0.25% wt) was added to the solution, and the pH was adjusted to 6.0. The solution was then subjected to plate and frame filtration to collect the filtrate. The filtrate was then finely filtered through a 0.65 μm Lennox filter, replaced with purified water, and finally spray-dried to obtain a weight-average molecular weight of 1.1 × 10⁻⁶. 4 Da's hydrolyzed sodium colarate product.

[0104] As can be seen from Examples 1-2 and Comparative Example 1, high temperature can also achieve the purpose of degrading colacid, but it will affect the acetylation modification of sodium colacid hydrolysis; while the enzymatic hydrolysis conditions are mild and the acetylation modification is completely preserved after the reaction.

[0105] Table 2. Results of product determination for sodium colarate, a raw material of the same molecular weight, degraded by different degradation methods.

[0106] It should be noted that "finished product yield" is the percentage of the final product relative to the amount of the reaction substrate (e.g., powder, fermentation broth, etc.), for example, 5.0 × 10⁻⁶. 6 If 1.0 kg of sodium dacronate powder is used and 0.701 kg of the final product is collected, the yield is 70.1%.

[0107] Example 2

[0108] Take 5 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, maintaining the water temperature at 50°C. After complete dissolution, the solution was cooled to 25°C, and 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 final concentrations of 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 complete, activated carbon (0.25% wt) was added to the solution, and the pH was adjusted to 6.0. The solution was then subjected to plate and frame filtration to collect the filtrate. The filtrate was finely filtered using a 0.65 μm Nennis filter. 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 products F1'-F5'. The data are shown in Table 3 below.

[0109] The results showed that products with molecular weight, protein residue, degree of acetylation modification, and yield were all within the expected range 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.

[0110] Table 3. Results of product determination for the degradation of sodium collamerate of the same molecular weight starting material with different enzyme amounts and reaction times.

[0111] Example 3

[0112] Take 8 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, maintaining the water temperature at 50°C. After complete dissolution, the temperature was lowered to 25°C, and 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, 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, maintaining the molecular weight at 0.6 × 10⁻⁶. 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 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 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.

[0113] 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.

[0114] Table 4. Results of product determination for the degradation of sodium colarate, a raw material of the same molecular weight, under different pH conditions.

[0115] Example 4

[0116] Take 9 portions of the sample prepared in Example 1-1 with a molecular weight of 5.0 × 10⁻⁶. 60.1 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 terminated 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 the sodium collamate 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.

[0117] The results showed that acetylation modification was easily lost at temperatures above 70℃.

[0118] Table 5. Results of product degradation of sodium colarate of the same molecular weight under different temperature conditions.

[0119] Example 5: Preparation of sodium collamerate products with high weight-average molecular weight range

[0120] Take 3 portions of the sample prepared in Example 1-1 with a molecular weight of 5.0 × 10⁻⁶. 6 1.0 kg of sodium colarate powder was added to 100 L of pure water and stirred until dissolved, with the water temperature controlled at 50 °C. After complete dissolution, the temperature was lowered to 25 °C, and 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 final concentrations of 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, and the pH was adjusted to 6.0. The filtrate was then collected by plate and frame filtration. The filtrate was finely filtered through a 0.65 μm Nenberg filter, and then 200 L of anhydrous ethanol was added while stirring to hydrolyze the sodium colarate 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 a weight-average molecular weight of 1.2 × 10⁻⁶. 6 Da, 7.6×10 5 Da, 4.2×10 5 The finished sodium collamerate powder products of Da (E1-E3). Its high performance liquid chromatogram is shown in Figure 1.

[0121] It should be noted that different purification methods used in the hydrolysis of colacid or its salts can yield products with different molecular weights. For example, for hydrolyzed sodium colacid with a weight-average molecular weight, the molecular weight is greater than 5.0 × 10⁻⁶. 5 For Da, alcohol precipitation followed by drying can be used to obtain the product in a more time-efficient manner; however, for values ​​below 5.0 × 10⁻⁶, the product can be obtained more efficiently. 5 Da's products show better results with pure water than with alcohol precipitation.

[0122] Example 6: Preparation of sodium colarate products with low weight-average molecular weight range

[0123] Take 5 portions of the sample prepared in Example 1-1 with a molecular weight of 5.0 × 10⁻⁶. 6 1.0 kg of sodium colarate powder from Da was added to 100 L of pure water and stirred until dissolved, with the water temperature controlled at 50 °C. After complete dissolution, the temperature was lowered to 25 °C, and 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 final concentrations of 0.3 mg / L, 0.5 mg / L, 1.0 mg / L, 2.5 mg / L, and 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 to the solution, and the pH was adjusted to 6.0. The filtrate was then collected by plate and frame filtration. The filtrate was finely filtered through a 0.65 μm Nenz filter, and then the solution was replaced with purified water. Finally, the solution was spray-dried to obtain the hydrolyzed sodium colarate product (E4-E8).

[0124] Table 6. Determination results of different molecular weights of hydrolyzed sodium colarate or hydrolyzed sodium colarate oligosaccharides

[0125] Example 7: Preparation of sodium collamylate oligosaccharide composition

[0126] Example 7-1

[0127] The molecular weight of the sample prepared in Example 1-1 was 5.0 × 10⁻⁶. 61.0 kg of sodium collamate powder was added to 100 L of pure water and stirred until dissolved, with the water temperature controlled at 40 °C. After complete dissolution, the temperature was lowered to 20 °C, and the pH was adjusted to 6.5 with sodium phosphate. Then, collamate degrading enzyme (SEQ ID NO: 1) was added to the sodium collamate 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, and the pH was adjusted to 4.5. The filtrate was then collected by plate and frame filtration. The filtrate was concentrated 20 times by nanofiltration, and then replaced with purified water. Finally, it was spray-dried to obtain a hydrolyzed sodium collamate oligosaccharide composition containing sodium collamate hexasaccharide (molecular weight 1126.34) and sodium collamate dodecanose (molecular weight 2234.67). Its high performance liquid chromatogram is shown in Figure 2, and its nuclear magnetic resonance spectrum is shown in Figure 7.

[0128] Example 7-2

[0129] The molecular weight of the sample prepared in Example 1-1 was 5.0 × 10⁻⁶. 6 1.0 kg of sodium colarate powder was added to 100 L of pure water and stirred until dissolved, with the water temperature controlled at 60 °C. After complete dissolution, the temperature was lowered to 50 °C, and the pH was adjusted to 5.0 with sodium phosphate. Then, colarate degrading enzyme (SEQ ID NO: 1) was added to the sodium colarate 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, and the pH was adjusted to 3.0. The filtrate was then collected by plate and frame filtration. The filtrate was concentrated 20 times by nanofiltration, and then replaced with purified water. Finally, the solution was spray-dried to obtain a hydrolyzed sodium colarate oligosaccharide composition containing sodium colarate hexasaccharide (molecular weight 1126.34) and sodium colarate dodecanose (molecular weight 2234.67).

[0130] Example 7-3

[0131] The molecular weight of the sample prepared in Example 1-1 was 5.0 × 10⁻⁶. 6 1.0 kg of sodium collamate powder was added to 100 L of pure water and stirred until dissolved, with the water temperature controlled at 50 °C. After complete dissolution, the temperature was lowered to 35 °C, and the pH was adjusted to 8.0 with sodium phosphate. Then, collamate degrading enzyme (SEQ ID NO: 1) was added to the sodium collamate 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, and the pH was adjusted to 6.0. The filtrate was then collected by plate and frame filtration. The filtrate was concentrated 20 times by nanofiltration, and then replaced with purified water. Finally, the solution was spray-dried to obtain a hydrolyzed sodium collamate oligosaccharide composition containing sodium collamate hexasaccharide (molecular weight 1126.34) and sodium collamate dodecanose (molecular weight 2234.67).

[0132] Example 8: Preparation of sodium colarate hexasaccharide and sodium colarate dodecanose

[0133] Example 8-1

[0134] The molecular weight of the sample prepared in Example 1-1 was 5.0 × 10⁻⁶. 6 1.0 kg of sodium colarate powder was added to 100 L of pure water and stirred until dissolved, maintaining the water temperature at 40 °C. After complete dissolution, the temperature was lowered to 20 °C, and the pH was adjusted to 6.5 with sodium phosphate. Then, colarate-degrading enzyme (SEQ ID NO: 1) was added to the sodium colarate 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, and the pH was adjusted to 4.5. The filtrate was then collected by plate and frame filtration. The filtrate was subjected to Q anion exchange chromatography, eluted with 10 mM sodium chloride at pH 8.5, and eluent fraction one was collected; then eluted with 50 mM sodium chloride at pH 8.5, and eluent fraction two was collected. The eluted fractions one and two were concentrated 20-fold by nanofiltration, then the liquid was replaced with purified water, and finally spray-dried to obtain hydrolyzed colarate sodium hexasaccharide (molecular weight 1126.34) and hydrolyzed colarate sodium dodecanose (molecular weight 2234.67). Their high-performance liquid chromatograms are shown in Figures 3 and 4, and their mass spectra are shown in Figures 5 and 6.

[0135] Example 8-2

[0136] The molecular weight of the sample prepared in Example 1-1 was 5.0 × 10⁻⁶. 6 1.0 kg of sodium colarate powder was added to 100 L of pure water and stirred until dissolved, maintaining the water temperature at 60 °C. After complete dissolution, the temperature was lowered to 50 °C, and the pH was adjusted to 5.0 with sodium phosphate. Then, colarate-degrading enzyme (SEQ ID NO: 1) was added to the sodium colarate 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, and the pH was adjusted to 3.0. The solution was then subjected to plate and frame filtration to collect the filtrate. The filtrate was subjected to Q anion exchange chromatography, eluted with 10 mM sodium chloride at pH 9.0, and the first eluent fraction was collected. Then, it was eluted with 50 mM sodium chloride at pH 9.0, and the second eluent fraction was collected. The eluted fractions 1 and 2 were concentrated 20 times by nanofiltration, then replaced with purified water, and finally spray-dried to obtain hydrolyzed colarate sodium hexasaccharide (molecular weight 1126.34) and hydrolyzed colarate sodium dodecanose (molecular weight 2234.67).

[0137] Example 8-3

[0138] The molecular weight of the sample prepared in Example 1-1 was 5.0 × 10⁻⁶. 61.0 kg of sodium colarate powder was added to 100 L of pure water and stirred until dissolved, maintaining the water temperature at 50 °C. After complete dissolution, the temperature was lowered to 35 °C, and 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 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, and the pH was adjusted to 6.0. The solution was then subjected to plate and frame filtration to collect the filtrate. The filtrate was subjected to Q anion exchange chromatography, eluted with 10 mM sodium chloride at pH 8.0, and the first eluent fraction was collected. Then, it was eluted with 50 mM sodium chloride at pH 8.0, and the second eluent fraction was collected. The eluted fractions 1 and 2 were concentrated 20 times by nanofiltration, then replaced with purified water, and finally spray-dried to obtain hydrolyzed colarate sodium hexasaccharide (molecular weight 1126.34) and hydrolyzed colarate sodium dodecanose (molecular weight 2234.67).

[0139] Table 7. Determination results of hydrolyzed sodium collamate oligosaccharide compositions or single-component finished products

[0140] Example 9: Preparation of sodium collamerate powder or oligosaccharide composition / single component product from fermentation broth

[0141] Example 9-1

[0142] 100 L of colacid fermentation broth was taken and the temperature was controlled at 30 °C. Colacid-degrading enzyme (SEQ ID NO: 1) was added to a final concentration of 0.01 mg / L, and the reaction was carried out for 16 h. After the reaction, the mixture was sterilized by plate and frame filtration. Activated carbon (2% wt) was added to the filtrate, and the pH was adjusted to 5.0. After 1 h of treatment, the mixture was filtered again by plate and frame filtration, followed by filtration through a 0.65 μm Nenz filter. The precipitate was precipitated with two volumes of anhydrous ethanol. The precipitate was collected and washed three times with a mixed solution of sodium chloride and ethanol of the same concentration. Then, it was dehydrated three times with anhydrous ethanol and finally dried under vacuum to obtain a weight-average molecular weight of 1.4 × 10⁻⁶. 6 Da's crude pure sodium collamerate powder.

[0143] Example 9-2

[0144] Take 100 L of colacid fermentation broth, control the temperature at 30℃, add colacid-degrading enzyme (SEQ ID NO: 1) to a final concentration of 0.2 mg / L, and react for 2 h. After the reaction, sterilize by plate and frame filtration, add activated carbon (2% wt) to the filtrate, adjust the pH to 5.0, treat for 1 h, and filter again by plate and frame filtration. Then filter through a 0.65 μm Nenz filter, precipitate with 2 volumes of anhydrous ethanol, collect the precipitate, wash three times with a mixed solution of sodium chloride and ethanol of the same concentration, dehydrate three times with anhydrous ethanol, and finally vacuum dry to obtain a weight-average molecular weight of 7.1 × 10⁻⁶. 5Da's crude pure sodium collamerate powder.

[0145] Example 9-3

[0146] Take 100 L of colacid fermentation broth, control the temperature at 30℃, add colacid-degrading enzyme (SEQ ID NO: 1) to a final concentration of 0.01 mg / L, and react for 16 h. After the reaction, sterilize by plate and frame filtration, add activated carbon (2% wt) to the filtrate, adjust the pH to 5.0, treat for 1 h, and filter again by plate and frame filtration. Then filter through a 0.65 μm Capstone filter and collect the filtrate. Concentrate the filtrate using a commercially available 500 kD hollow fiber membrane and replace the solution with pure water. After the solution replacement, adjust the pH to 6.5 with sodium phosphate, then add colacid-degrading enzyme to a final concentration of 0.5 mg / L, and stir to react for 2 h. After the secondary reaction, activated carbon (2% wt) was added to the colacid solution, the pH was adjusted to 5.0, and the solution was treated for 1 hour. The solution was then filtered again by plate and frame filter press. The filtrate was concentrated by nanofiltration 20 times, and then purified water was used to replace the solution. Finally, the solution was spray-dried to obtain a hydrolyzed colacid sodium oligosaccharide composition containing colacid sodium hexasaccharide (molecular weight 1126.34) and colacid sodium dodecanose (molecular weight 2234.67).

[0147] It should be noted that the fermentation broth can be directly enzymatically hydrolyzed into products of the target molecular weight, including low molecular weight products. However, the fermentation broth contains many small molecule impurities, and the cost of purifying small molecule colacid or its salts from it is high. Therefore, in Examples 9-3 and 9-4, a second enzymatic hydrolysis was performed after separating medium and large molecule colacid or its salts from the broth.

[0148] Example 9-4

[0149] Take 100 L of colacid fermentation broth, control the temperature at 30℃, add colacid-degrading enzyme (SEQ ID NO: 1) to a final concentration of 0.01 mg / L, and react for 16 h. After the reaction, sterilize by plate and frame filtration, add activated carbon (2% wt) to the filtrate, adjust the pH to 5.0 and treat for 1 h, then filter again by plate and frame filtration, followed by filtration through a 0.65 μm Capstone filter, and collect the filtrate. Concentrate the filtrate using a 500 kD hollow fiber membrane and replace the liquid with pure water. After the liquid replacement, adjust the pH to 6.5 with sodium phosphate, then add colacid-degrading enzyme to a final concentration of 0.5 mg / L, and stir to react for 2 h.

[0150] After the secondary reaction, activated carbon (2% wt) was added to the colacid solution, the pH was adjusted to 5.0, and the solution was treated for 1 hour. The solution was then filtered again using a plate and frame filter press. The filtrate was subjected to Q anion exchange chromatography, eluted with 10 mM sodium chloride at pH 9.0, and fraction one was collected. Fraction two was then eluted with 50 mM sodium chloride at pH 9.0. Fractions one and two were concentrated 20-fold by nanofiltration, then replaced with purified water, and finally spray-dried to obtain hydrolyzed colacid sodium hexasaccharide (molecular weight 1126.34) and hydrolyzed colacid sodium dodecanose (molecular weight 2234.67).

[0151] Table 8. Results of determination of the final product composition prepared by enzymatic hydrolysis of fermentation broth for sodium hydrolysate.

[0152] Table 9. Results of the preparation of hydrolyzed sodium colarate hexasaccharide and dodecanose by enzymatic hydrolysis of fermentation broth

[0153] Table 10. Sequence Information

[0154] By incorporating via reference

[0155] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0156] Equivalence

[0157] This invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases, and not as limiting of the invention described herein. Consequently, the scope of the invention is defined by the appended claims rather than by the foregoing description, and is intended to be encompassed by all variations within the equivalent meaning and scope of the claims.

Claims

1. A method for preparing hydrolyzed colaic acid or its salt, comprising the following steps: 1) Adding a solution containing colacid or its salt to a colacid-degrading enzyme comprising an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% sequence identity with it; and 2) Enzyme termination reaction: The 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 activated carbon is used to achieve the termination in step 2). This leads to the hydrolysis of colacid or its salt.

2. The method of claim 1, comprising any one or more of the following: (i) The collamerate is sodium collamerate, and / or (ii) In step 1), the weight-average molecular weight of the colacid or its salt is greater than 1 × 10⁻⁶. 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 concentration of the colacid-degrading enzyme is between approximately 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 approximately 1 hour to approximately 20 hours, 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 step 3) filtration purification; 4) concentration desalting; and 5) drying.

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

5. The method according to any one of claims 1-4, 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 3-5, wherein, In step 3), the pH is adjusted to about 3.0 to about 6.5 before filtration, preferably about 3.0 to about 6.

0.

7. The method according to any one of claims 3-6, 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 1-7, wherein, The hydrolyzed colaic acid or its salt has a weight-average molecular weight range of approximately 1 × 10⁻⁶. 3 Da to approximately 3 × 10 6 Preferably, the hydrolyzed colaic acid or its salt has a weight-average molecular weight range of about 1 × 10⁻⁶. 3 Da to approximately 2 × 10 6 Da.

9. The method according to any one of claims 1-8, wherein, The hydrolyzed colaic acid or its salt contains acetylation and / or pyruvate modification.

10. The method according to any one of claims 1-9, wherein, The hydrolyzed colaic acid or its salt The acetylation modification rate is greater than 80%, preferably greater than 90%; and / or Endotoxin residue less than about 0.5 EU / mg, preferably less than about 0.1 EU / mg; and / or Protein residue is less than about 0.1%, preferably less than about 0.07%; and / or Heavy metal residue is less than approximately 10 mg / kg.

11. The method according to any one of claims 1-10, wherein, The hydrolyzed colacid or its salt is colacid hexasaccharide or its salt, and / or colacid dodecanose or its salt.

12. Hydrolyzed colacid or its salt prepared by the method according to any one of claims 1-11.

Images