Method for purifying microbial polysaccharide hyaluronic acid and application thereof

Abstract

The application belongs to the technical field of polysaccharide preparation and relates to a purification method and application of microbial polysaccharide hyaluronic acid. ‑ The viscosity of the hyaluronic acid fermentation liquor is reduced by more than 99% after the alkali-adjusted and viscosity-reduced hyaluronic acid fermentation liquor is subjected to mechanical shearing treatment, so that the viscosity is further reduced to 40-75 mPa s; the hyaluronic acid fermentation liquor after the high-shear treatment is filtered to remove bacteria and impurities; acid is added to the filtered solution for neutralization, and then alcohol precipitation and drying are performed, and the hyaluronic acid is obtained. The application can quickly reduce the viscosity of the hyaluronic acid fermentation liquor to reach the viscosity range for efficient bacteria removal and impurity removal in subsequent operations, thereby breaking through the industrial bottleneck of bacteria removal and purification in the industrialized production process of microbial polysaccharide hyaluronic acid.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide preparation technology, and relates to a purification method and application of microbial polysaccharide hyaluronic acid. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hyaluronic acid (HA) is a linear macromolecular acidic mucopolysaccharide composed of disaccharide units of β-1,3-N-acetyl-D-glucosamine (UDP-GlcNAc) and β-1,4-D-glucuronic acid (UDP-GlcUA). The molecular weight of the natural polysaccharide ranges from 1.0 to 40 × 10⁻⁶. 5 Da. It has good moisturizing properties and can be used as a raw material for cosmetics; at the same time, it is colorless and odorless, easily soluble in water, and does not change the original properties of the product, so it can be used as a food ingredient.

[0004] Currently, hyaluronic acid is mainly produced through microbial fermentation. Downstream extraction and purification processes are crucial for obtaining high-purity, high-quality HA products and represent a bottleneck for most companies producing high-standard HA. Currently, HA product extraction and purification processes often involve the combined use of several extraction, separation, and purification technologies, such as dilution and viscosity reduction-filtration for sterilization and impurity removal-solvent precipitation, dilution and viscosity reduction-membrane separation for sterilization and impurity removal-membrane concentration-solvent precipitation, and a combined process of dilution and viscosity reduction-filtration for sterilization and impurity removal-quaternary ammonium salt precipitation. However, the inventors have found that due to the challenge of handling the high viscosity of high-concentration hyaluronic acid fermentation broth, the aforementioned extraction and purification technologies inevitably present a series of industry problems, including complex processes, high solvent / quaternary ammonium salt consumption, and high production costs, thus hindering the stable production and large-scale expansion of high-purity, high-quality HA products. Summary of the Invention

[0005] To address the challenge of handling high-viscosity hyaluronic acid fermentation broth, this invention aims to provide a purification method and application for microbial polysaccharide hyaluronic acid. This method can rapidly reduce viscosity to a range suitable for efficient sterilization and impurity removal in subsequent operations, thereby overcoming the industrial bottleneck of sterilization and purification in the industrial production of microbial polysaccharide hyaluronic acid.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] On one hand, a method for purifying hyaluronic acid, a microbial polysaccharide, involves adding an alkaline solution to the hyaluronic acid fermentation broth to adjust the alkali and reduce viscosity, thereby reducing the OH- ions in the fermentation broth. -The concentration reaches 0.025–0.125 mol / L; the hyaluronic acid fermentation broth after alkali adjustment and viscosity reduction is subjected to mechanical shearing treatment to further reduce the viscosity to 40–75 mPa·s; the hyaluronic acid fermentation broth after high shear treatment is filtered to remove bacteria and impurities; acid is added to the filtered solution for neutralization, followed by alcohol precipitation and drying to obtain the final product.

[0008] Since hyaluronic acid is a water-soluble mucopolysaccharide, its fermentation broth is a high-viscosity non-Newtonian fluid. To avoid the problems of complex processes, high solvent / quaternary ammonium salt usage, and high production costs associated with dilution and viscosity reduction, this invention has found that adding alkali to the high-viscosity hyaluronic acid fermentation broth can break the hydrogen bonds formed between hyaluronic acid molecules and water molecules, thereby changing the conformation of hyaluronic acid in water and significantly reducing its viscosity. Therefore, this invention uses alkali adjustment for initial viscosity reduction, followed by mechanical shearing for further viscosity reduction, in order to facilitate subsequent sterilization and impurity removal.

[0009] In the viscosity reduction process, an alkaline solution is added. To ensure product quality, the alkali needs to be neutralized. However, this process introduces new salt, which typically requires concentration and filtration to remove. But for the hyaluronic acid of this invention, the neutralized solution has extremely high viscosity, making concentration difficult and thus hindering the concentration, filtration, and desalination process. However, this invention uses a smaller amount of alkaline solution added during viscosity reduction, resulting in a lower OH content. - A concentration of 0.025–0.125 mol / L can significantly reduce the viscosity of the hyaluronic acid fermentation broth, resulting in a smaller amount of new salt produced after neutralization. This invention has found that by adding a small amount of ethanol solution for subsequent alcohol precipitation, the small amount of new salt produced in the product can be removed. This not only eliminates the need for concentration and filtration desalting after neutralization, but also greatly reduces the amount of ethanol solution used in the alcohol precipitation process, thereby significantly reducing the purification cost of hyaluronic acid.

[0010] Since the purification method of the above-mentioned microbial polysaccharide hyaluronic acid provided by the present invention is based on the process of producing hyaluronic acid by microbial fermentation, on the other hand, the present invention provides an application of the above-mentioned purification method of microbial polysaccharide hyaluronic acid in the production of hyaluronic acid by microbial fermentation.

[0011] The beneficial effects of this invention are as follows:

[0012] (1) This invention reduces the viscosity of hyaluronic acid fermentation broth by more than 99% without dilution by adjusting the alkali and using high-shear emulsification technology. It can complete the sterilization and purification of hyaluronic acid fermentation broth by using a controllable and simple filtration operation, which greatly reduces the amount of material to be processed and the cost of production equipment in the subsequent extraction process, and achieves low-cost and high-efficiency extraction and purification of hyaluronic acid.

[0013] (2) Compared with the prior art, the amount of alcohol used for dehydration and separation of the product of the present invention is reduced by 35% to 50%, which significantly saves energy and reduces consumption. Moreover, the technical methods and process equipment of the present invention are simple to select and easy to implement. Detailed Implementation

[0014] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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.

[0015] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0016] Given the high viscosity and processing difficulties of hyaluronic acid fermentation broth, the high cost of separating and purifying microbial polysaccharide hyaluronic acid is a bottleneck restricting the development of the industry. This invention proposes a purification method and application for microbial polysaccharide hyaluronic acid.

[0017] A typical embodiment of the present invention provides a method for purifying microbial polysaccharide hyaluronic acid, which involves adding an alkaline solution to the hyaluronic acid fermentation broth to adjust the alkali and reduce viscosity, thereby reducing the OH- ions in the fermentation broth. - The concentration reaches 0.025–0.125 mol / L; the hyaluronic acid fermentation broth after alkali adjustment and viscosity reduction is subjected to mechanical shearing treatment to further reduce the viscosity to 40–75 mPa·s; the hyaluronic acid fermentation broth after high shear treatment is filtered to remove bacteria and impurities; acid is added to the filtered solution for neutralization, followed by alcohol precipitation and drying to obtain the final product.

[0018] In some embodiments, the hyaluronic acid content in the hyaluronic acid fermentation broth is 1.4–2.3% (w / v).

[0019] In some embodiments, an alkali solution is slowly added to the hyaluronic acid fermentation broth. Excessive alkali solution should be avoided, as it may affect subsequent alcohol precipitation.

[0020] In some embodiments, the alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution. The concentration of the alkaline solution is 5–10 mol / L.

[0021] In some embodiments, the mechanical shearing treatment rate is 3000–5000 r / min, and the time is 5–10 min. Under these conditions, it is beneficial to further accelerate the viscosity reduction efficiency of the hyaluronic acid fermentation broth.

[0022] In some embodiments, a filter aid is added during the filtration process to remove bacteria and impurities. The filter aid is one or more of diatomaceous earth, bentonite, and perlite, preferably a mixture of diatomaceous earth, bentonite, and perlite. If a mixture of diatomaceous earth, bentonite, and perlite is used as the filter aid, the weight ratio of diatomaceous earth, bentonite, and perlite is preferably 1:2:2 to 1:5:3. The amount of filter aid added is 0.5% to 1.2% (w / v) of the hyaluronic acid fermentation broth. Adding a filter aid can improve the filtration efficiency and impurity removal efficiency.

[0023] In some embodiments, during the filtration process for sterilization and impurity removal, the pore size of the filter medium is 500–1000 mesh (25–13 μm). The filtration equipment used can be a vacuum belt filter or a plate filter, etc.

[0024] In some embodiments, the neutralization process is carried out at 10–40°C. This is to avoid the damage to hyaluronic acid molecules caused by excessively high temperatures.

[0025] In some embodiments, the pH is neutralized to 6.0–7.0.

[0026] In some embodiments, the alcohol precipitation process involves adding an aqueous ethanol solution to the neutralized feed solution to allow hyaluronic acid to precipitate fully. The volume concentration of the aqueous ethanol solution is 90–95° (v / v). The amount of aqueous ethanol solution added is 1–1.3 times the volume of the feed solution.

[0027] In one or more embodiments, the precipitated hyaluronic acid is dehydrated and washed with an aqueous ethanol solution. This method yields hyaluronic acid with low water content, improving subsequent drying efficiency. The volume concentration of the aqueous ethanol solution is 90–95° (v / v). The volume of the aqueous ethanol solution added is 1–2 times the weight of the precipitated hyaluronic acid.

[0028] In some embodiments, the drying temperature is 35–40°C. This avoids excessively high temperatures from affecting the product quality of hyaluronic acid. Vacuum drying is preferred, as it offers higher drying efficiency. The drying time is 2–5 hours.

[0029] Another embodiment of the present invention provides an application of the above-mentioned purification method of microbial polysaccharide hyaluronic acid in the production of hyaluronic acid by microbial fermentation.

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0031] Example 1

[0032] A low-cost and efficient purification method for the extraction of microbial polysaccharide hyaluronic acid, comprising the following steps:

[0033] (1) Initial viscosity reduction of fermentation broth

[0034] Sodium hydroxide solution (5 mol / L) was slowly added to the hyaluronic acid fermentation broth with a polysaccharide content of 1.4% (w / v) to make [OH] - The final concentration of the solution was 0.025 mol / L. The solution was stirred continuously to make it homogeneous, and the viscosity of the fermentation broth was significantly reduced.

[0035] (2) High shear rapidly reduces viscosity

[0036] After adjusting the alkali and reducing the viscosity, the hyaluronic acid alkaline solution was treated with a high-shear emulsifier at 3000 r / min for 5 minutes to rapidly reduce its viscosity, so as to facilitate subsequent filtration and sterilization. After treatment, the viscosity of the hyaluronic acid alkaline solution was reduced from the initial 8980 mPa·s to 40 mPa·s, with a viscosity reduction rate of 99.6%.

[0037] (3) Filtration, sterilization and impurity removal

[0038] After adding 0.8% (w / v) of filter aid to the hyaluronic acid fermentation broth that reduced viscosity in step (2), the broth was filtered to remove the bacterial cells and insoluble impurities, resulting in a clear and transparent hyaluronic acid alkaline solution. The filtration equipment was a vacuum belt filter with a pore size of 1000 mesh (13 μm); the filter aid was a mixture of diatomaceous earth, bentonite, and perlite (weight ratio 1:5:3).

[0039] (4) Neutralization

[0040] Add dilute hydrochloric acid to the hyaluronic acid-base solution after removing bacteria and impurities in step (3) to neutralize to pH 7.0, and control the temperature of the solution to below 40℃.

[0041] (5) Alcohol precipitation

[0042] Slowly add 1.3 times the volume of alcohol (95°, v / v) to the neutralized liquid in step (4) to allow hyaluronic acid to fully precipitate. Filter and separate to obtain a water-containing hyaluronic acid product. Then add 2 times the volume (v / w) of alcohol (concentration 95°, v / v) to the product for further dehydration and washing, and then filter and separate to obtain a low-water-content hyaluronic acid product.

[0043] (6) Drying

[0044] The low-water-content hyaluronic acid obtained in step (5) was dried in a vacuum dryer at 40°C for 2.5 hours, and then pulverized and sieved to obtain the hyaluronic acid product. The glucuronic acid content of the obtained hyaluronic acid product was determined to be 44.5% (on a dry basis), and the molecular weight was 1.33 ± 0.12 × 10⁻⁶. 6 The polysaccharide recovery rate of the fermentation broth reached 95.6%.

[0045] Example 2

[0046] A low-cost and efficient purification method for the extraction of microbial polysaccharide hyaluronic acid, comprising the following steps:

[0047] (1) Initial viscosity reduction of fermentation broth

[0048] Slowly add potassium hydroxide solution (10 mol / L) to the hyaluronic acid fermentation broth with a polysaccharide content of 2.2% (w / v) to make [OH] - The final concentration of the solution was 0.125 mol / L. The solution was stirred continuously to make it homogeneous, and the viscosity of the fermentation broth was significantly reduced.

[0049] (2) High shear rapidly reduces viscosity

[0050] After adjusting the alkali and reducing the viscosity, the hyaluronic acid alkaline solution was treated with a high-shear emulsifier at 5000 r / min for 10 min to rapidly reduce its viscosity, so as to facilitate subsequent filtration and sterilization. After treatment, the viscosity of the hyaluronic acid alkaline solution was reduced from the initial 17750 mPa·s to 72 mPa·s, with a viscosity reduction rate of 99.6%.

[0051] (3) Filtration, sterilization and impurity removal

[0052] After adding 1.0% (w / v) of filter aid to the hyaluronic acid fermentation broth that has had its viscosity reduced in step (2), the bacterial cells and insoluble impurities are removed by filtration to obtain a clear and transparent hyaluronic acid alkaline solution. The filtration equipment is a plate filter with a pore size of 500 mesh (25 μm). The filter aid is a mixture of diatomaceous earth, bentonite and perlite (weight ratio of 1:2:2).

[0053] (4) Neutralization

[0054] Add dilute hydrochloric acid to the hyaluronic acid-base solution after removing bacteria and impurities in step (3) to neutralize to pH 7.0, and control the temperature of the solution to below 40℃.

[0055] (5) Alcohol precipitation

[0056] Slowly add 1 volume of alcohol (95°, v / v) to the neutralized liquid in step (4) to allow the hyaluronic acid to fully precipitate. Filter and separate to obtain a water-containing hyaluronic acid product. Then add 1 volume (v / w) of alcohol (concentration 95°, v / v) to the product for further dehydration and washing. Filter and separate to obtain a low-water-content hyaluronic acid product.

[0057] (6) Drying

[0058] The low-water-content hyaluronic acid obtained in step (5) was dried in a vacuum dryer at 40°C for 3.5 hours, and then pulverized and sieved to obtain the hyaluronic acid product. The glucuronic acid content of the obtained hyaluronic acid product was determined to be 43.6% (on a dry basis), and the molecular weight was 1.31 ± 0.09 × 10⁻⁶. 6 The polysaccharide recovery rate of the fermentation broth reached 96.3%.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for purifying microbial polysaccharide hyaluronic acid, characterized in that, Adding alkali to the hyaluronic acid fermentation broth to adjust the alkali content and reduce viscosity; the OH- ions in the fermentation broth after adding alkali... - The concentration is 0.025~0.125 mol / L; the hyaluronic acid fermentation broth after alkali adjustment and viscosity reduction is subjected to mechanical shear treatment to reduce the viscosity to 40~75 mPa·s; the hyaluronic acid fermentation broth after high shear treatment and viscosity reduction is filtered to remove bacteria and impurities; acid is added to the filtered solution for neutralization, followed by alcohol precipitation and drying to obtain the final product. The hyaluronic acid content in the fermentation broth was 1.4~2.3 w / v % %. Slowly add alkali solution to the hyaluronic acid fermentation broth; The neutralization process is carried out at 10~40 ℃.

2. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, The alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution.

3. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, The mechanical shearing process is performed at a rate of 3000~5000 r / min for 5~10 min.

4. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, A filter aid is added during the filtration process to remove bacteria and impurities; the filter aid is one or more of diatomaceous earth, bentonite, and perlite.

5. The purification method for microbial polysaccharide hyaluronic acid as described in claim 4, characterized in that, The filter aid is a mixture of diatomaceous earth, bentonite, and perlite.

6. The purification method for microbial polysaccharide hyaluronic acid as described in claim 5, characterized in that, A mixture of diatomaceous earth, bentonite, and perlite is used as a filter aid, with the weight ratio of diatomaceous earth, bentonite, and perlite being 1:2:2 to 1:5:

3.

7. The purification method for microbial polysaccharide hyaluronic acid as described in claim 4, characterized in that, The amount of the filter aid added is 0.5~1.2 w / v of the hyaluronic acid fermentation broth.

8. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, During the filtration process to remove bacteria and impurities, the pore size of the filter medium is 500~1000 mesh.

9. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, Neutralize to a pH of 6.0-7.

0.

10. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, The alcohol precipitation process is as follows: add an aqueous ethanol solution to the neutralized solution to allow hyaluronic acid to precipitate fully; the volume concentration of the aqueous ethanol solution is 90~95 v / v; the amount of aqueous ethanol solution added is 1~1.3 times the volume of the solution.

11. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, The precipitated hyaluronic acid was dehydrated and washed with an ethanol-water solution. The volume concentration of the ethanol-water solution is 90~95 v / v %; the volume of the ethanol-water solution added is 1~2 times the weight of the precipitated hyaluronic acid.

12. The purification method for microbial polysaccharide hyaluronic acid as described in claim 1, characterized in that, The drying temperature is 35~40℃; vacuum drying is used; and the drying time is 2~5 hours.

13. The application of the purification method of microbial polysaccharide hyaluronic acid according to any one of claims 1 to 12 in the production of hyaluronic acid by microbial fermentation.