CN117164741A - Purification method and application of microbial polysaccharide hyaluronic acid - Google Patents
Purification method and application of microbial polysaccharide hyaluronic acid Download PDFInfo
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- CN117164741A CN117164741A CN202311010760.6A CN202311010760A CN117164741A CN 117164741 A CN117164741 A CN 117164741A CN 202311010760 A CN202311010760 A CN 202311010760A CN 117164741 A CN117164741 A CN 117164741A
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Abstract
The invention belongs to the technical field of polysaccharide preparation, and relates to a purification method and application of microbial polysaccharide hyaluronic acid. Adding alkali liquor into hyaluronic acid fermentation liquor for alkali regulation and viscosity reduction to enable OH in the fermentation liquor to be reduced in viscosity ‑ The concentration reaches 0.025 to 0.125mol/L; the viscosity of the hyaluronic acid fermentation liquor after alkali adjustment and viscosity reduction is further reduced to 40-75 mPa.s by mechanical shearing treatment, so that the viscosity of the hyaluronic acid fermentation liquor can be reduced by more than 99%; filtering the hyaluronic acid fermentation liquor subjected to high shear treatment, and sterilizing and removing impurities; adding acid into the filtered solution for neutralization, and then carrying out alcohol precipitation and drying to obtain the product. The invention can quickly reduce the viscosity of the hyaluronic acid fermentation liquor to reach the viscosity range for performing the subsequent operation and efficient sterilization and impurity removal, thereby breaking through the industrial bottleneck of sterilization and purification in the industrial production process of the microbial polysaccharide hyaluronic acid.
Description
Technical Field
The invention belongs to the technical field of polysaccharide preparation, and relates to a purification method and application of microbial polysaccharide hyaluronic acid.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The molecular structure of Hyaluronic Acid (HA) is linear macromolecular acidic mucopolysaccharide polymerized by disaccharide units of beta-1, 3-N-acetyl-D-glucosamine (UDP-GlcNAc) and beta-1, 4-D-glucuronic acid (UDP-GlcUA), and the molecular weight of the natural polysaccharide is 1.0-40 multiplied by 10 5 Da. It has good moisturizing effect, and can be used as raw material of cosmetics; meanwhile, the product is colorless and odorless, is easily dissolved in water, does not change the original properties of the product, and can be used as a food raw material.
At present, hyaluronic acid is mainly produced and obtained by adopting a microbial fermentation method. The downstream extraction and purification process is a key link for obtaining high-purity and high-quality HA products, and is a bottleneck for restricting most enterprises to produce high-standard HA. At present, the HA product extraction and purification process is usually realized by combining a plurality of extraction, separation and purification technologies, such as dilution viscosity reduction, filtration, sterilization, impurity removal, solvent precipitation, dilution viscosity reduction, membrane separation, sterilization, impurity removal, membrane concentration, solvent precipitation, dilution viscosity reduction, filtration, sterilization, impurity removal, quaternary ammonium salt precipitation and the like, but the inventor researches find that the extraction and purification technology inevitably presents a series of industrial problems of complex technological process, high solvent/quaternary ammonium salt consumption, high production cost and the like due to the difficult problem of high-viscosity treatment of the high-concentration hyaluronic acid fermentation liquor, and the stable production and scale expansion of the high-purity and high-quality HA product are restricted.
Disclosure of Invention
In order to solve the problem of high-viscosity treatment of hyaluronic acid fermentation liquor, the invention aims to provide a purification method and application of microbial polysaccharide hyaluronic acid, which can quickly reduce the viscosity to reach a viscosity range capable of performing efficient sterilization and impurity removal in subsequent operation, thereby breaking through the industrial bottleneck of sterilization and purification in the industrial production process of microbial polysaccharide hyaluronic acid.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
on the one hand, a purification method of microbial polysaccharide hyaluronic acid, which comprises adding alkali liquor into hyaluronic acid fermentation liquor for alkali regulation and viscosity reduction,OH in fermentation liquor - The concentration reaches 0.025 to 0.125mol/L; mechanically shearing the hyaluronic acid fermentation broth subjected to alkali adjustment and viscosity reduction to further reduce the viscosity to 40-75 mPa.s; filtering the hyaluronic acid fermentation liquor subjected to high shear treatment, and sterilizing and removing impurities; adding acid into the filtered solution for neutralization, and then carrying out alcohol precipitation and drying to obtain the product.
Because hyaluronic acid is water-soluble mucopolysaccharide, the hyaluronic acid fermentation broth is a high-viscosity non-Newtonian fluid, and in order to avoid the problems of complex process, high solvent/quaternary ammonium salt consumption and high production cost caused by dilution and viscosity reduction, the invention researches and discovers that alkali liquor is added into the high-viscosity hyaluronic acid fermentation broth to break hydrogen bonds formed between hyaluronic acid molecules and water molecules, so that the viscosity of the hyaluronic acid can be obviously reduced by changing the conformation of the hyaluronic acid in water.
Alkali liquor is added in the viscosity reduction process, so that the quality of a product is ensured, the alkali is required to be neutralized, new salt is introduced in the process, and in order to remove the introduced new salt, a common method is required to be concentrated, filtered and desalted. However, in the hyaluronic acid of the present invention, the solution viscosity after neutralization is extremely high, and it is difficult to perform the concentration operation, and thus it is difficult to realize the process of concentrating, filtering and desalting. However, the invention has less alkali liquid amount and OH in the viscosity reducing process - The concentration is 0.025-0.125 mol/L, thus the viscosity of the hyaluronic acid fermentation liquor can be obviously reduced, and the amount of new salt generated after neutralization is also less.
The purification method of the microbial polysaccharide hyaluronic acid is based on the process of producing the hyaluronic acid by a microbial fermentation method, so that on the other hand, the application of the purification method of the microbial polysaccharide hyaluronic acid in the production of the hyaluronic acid by the microbial fermentation method is provided.
The beneficial effects of the invention are as follows:
(1) According to the invention, on the premise of no dilution, the viscosity of the hyaluronic acid fermentation liquor is reduced by more than 99% only by an alkali adjusting and high-shear emulsifying technology, and the sterilization, impurity removal and purification of the hyaluronic acid fermentation liquor can be completed by utilizing controllable and simple filtering operation, so that the processing amount of materials and the production equipment cost in the subsequent extraction process are greatly reduced, and the low-cost and high-efficiency fractional extraction and purification of hyaluronic acid are realized.
(2) Compared with the prior art, the alcohol amount used for dehydration and separation of the product is reduced by 35% -50%, the energy is obviously saved, the consumption is reduced, and the technical method and the process equipment are simple to select and convenient to implement.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, 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 belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the problems of high viscosity and difficult treatment of hyaluronic acid fermentation broth, the bottleneck problem that the existing microbial polysaccharide hyaluronic acid separation and purification cost is high and the industrial development is restricted is caused, and the invention provides a purification method and application of the microbial polysaccharide hyaluronic acid.
In an exemplary embodiment of the invention, a purification method of microbial polysaccharide hyaluronic acid is provided, alkali liquor is added into hyaluronic acid fermentation liquor to regulate alkali and reduce viscosity, so thatOH in fermentation broths - The concentration reaches 0.025 to 0.125mol/L; mechanically shearing the hyaluronic acid fermentation broth subjected to alkali adjustment and viscosity reduction to further reduce the viscosity to 40-75 mPa.s; filtering the hyaluronic acid fermentation liquor subjected to high shear treatment, and sterilizing and removing impurities; adding acid into the filtered solution for neutralization, and then carrying out alcohol precipitation and drying to obtain the product.
In some embodiments, the hyaluronic acid content of the hyaluronic acid fermentation broth is 1.4-2.3% (w/v).
In some embodiments, the lye is slowly added to the hyaluronic acid broth. Avoiding the excessive addition of alkali liquor and affecting the subsequent alcohol precipitation treatment.
In some embodiments, the lye is sodium hydroxide solution or potassium hydroxide solution. The concentration of the alkali liquor is 5-10 mol/L.
In some embodiments, the mechanical shearing process is performed at a rate of 3000 to 5000r/min for a period of 5 to 10min. The viscosity reduction efficiency of the hyaluronic acid fermentation liquor is further accelerated under the condition.
In some embodiments, the filter aid is added during the filtration sterilization and decontamination process. The filter aid is one or more of diatomite, bentonite and perlite, preferably a mixture of diatomite, bentonite and perlite, and if the mixture of diatomite, bentonite and perlite is used as the filter aid, the weight ratio of the diatomite, bentonite and perlite is preferably 1:2:2-1:5:3. The addition amount of the filter aid is 0.5-1.2% (w/v) of the hyaluronic acid fermentation liquor. The filtration efficiency and the impurity removal efficiency can be improved by adding the filter aid.
In some embodiments, the pore size of the filter media is 500-1000 mesh (25-13 μm) during the filtration sterilization and decontamination process. The filtering equipment can be a vacuum belt filter or a plate filter and the like.
In some embodiments, the neutralization process is performed at a temperature of 10 to 40 ℃. Avoid damage to hyaluronic acid molecules caused by excessive temperature.
In some embodiments, the neutralization is to a pH of 6.0 to 7.0.
In some embodiments, the process of alcohol precipitation is: adding ethanol water solution into the neutralized feed liquid to fully precipitate hyaluronic acid. The volume concentration of the ethanol aqueous solution is 90-95 degrees (v/v). The addition amount of the ethanol aqueous solution is 1 to 1.3 times of the volume of the feed liquid.
In one or more embodiments, the precipitated hyaluronic acid is subjected to a dehydration wash with an aqueous ethanol solution. The method can obtain hyaluronic acid with low water content, and improve subsequent drying efficiency. The volume concentration of the ethanol aqueous solution is 90-95 degrees (v/v). The added volume of the ethanol aqueous solution is 1-2 times of the weight of the precipitated hyaluronic acid.
In some embodiments, the drying is performed at a temperature of 35 to 40 ℃. Can avoid the influence of the overhigh temperature on the quality of the hyaluronic acid. Preferably, vacuum drying is used. The drying efficiency is higher. The drying time is 2-5 hours.
In another embodiment of the invention, the invention provides an application of the purification method of the microbial polysaccharide hyaluronic acid in the production of hyaluronic acid by a microbial fermentation method.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
A purification method for low-cost and high-efficiency extraction of microbial polysaccharide hyaluronic acid comprises the following steps:
(1) Preliminary viscosity reduction of fermentation liquor
Slowly adding sodium hydroxide solution (5 mol/L) into hyaluronic acid fermentation broth with polysaccharide content of 1.4% (w/v) to obtain [ OH ] - ]The final concentration of (2) is 0.025mol/L, and the solution is uniform by continuously stirring, so that the viscosity of the fermentation liquor is obviously reduced.
(2) High shear rapid viscosity reduction
After alkali regulation and viscosity reduction, the viscosity of the hyaluronic acid alkali solution is rapidly reduced by adopting a high-shear emulsifying machine 3000r/min for 5min so as to facilitate subsequent filtration and sterilization, and the viscosity of the hyaluronic acid alkali solution after treatment is reduced from the initial viscosity of 8980 mPa.s to the viscosity of 40 mPa.s, and the viscosity reduction rate reaches 99.6%.
(3) Filtering, sterilizing and removing impurities
And (3) adding 0.8% (w/v) of filter aid into the hyaluronic acid fermentation liquor with the reduced viscosity in the step (2), and filtering to remove thalli and insoluble impurities to obtain a clear and transparent hyaluronic acid alkali solution. The filtering equipment is a vacuum belt filter, and the pore diameter of the filtering medium is 1000 meshes (13 mu m); the filter aid is a mixture of diatomite, bentonite and perlite (weight ratio is 1:5:3).
(4) Neutralization
Adding diluted hydrochloric acid into the hyaluronic acid-base solution from which the thalli and impurities are removed in the step (3) to neutralize until the pH value is 7.0, and controlling the temperature of the feed liquid below 40 ℃.
(5) Alcohol precipitation
Slowly adding 1.3 times of alcohol (95 DEG, v/v) with the volume of the feed liquid into the neutralized feed liquid in the step (4) to fully precipitate the hyaluronic acid, filtering and separating to obtain a water-containing hyaluronic acid product, adding 2 times of alcohol (v/w) with the volume of alcohol (95 DEG, v/v) into the product, further dehydrating and washing, and filtering and separating to obtain a low-water-content hyaluronic acid product.
(6) Drying
Drying the low-water content hyaluronic acid obtained in the step (5) for 2.5 hours at 40 ℃ by a vacuum dryer, crushing and screening to obtain a hyaluronic acid product, and determining that the glucuronic acid content of the obtained hyaluronic acid product is 44.5% (based on dry basis) and the molecular weight is 1.33+/-0.12 multiplied by 10 6 Da, the recovery rate of polysaccharide in fermentation liquor reaches 95.6 percent.
Example 2
A purification method for low-cost and high-efficiency extraction of microbial polysaccharide hyaluronic acid comprises the following steps:
(1) Preliminary viscosity reduction of fermentation liquor
Slowly adding potassium hydroxide solution (10 mol/L) into hyaluronic acid fermentation broth with polysaccharide content of 2.2% (w/v) to obtain [ OH ] - ]The final concentration of the fermentation liquor is 0.125mol/L, and the solution is even by continuous stirring, so that the viscosity of the fermentation liquor is obviously reduced.
(2) High shear rapid viscosity reduction
After alkali regulation and viscosity reduction, the viscosity of the hyaluronic acid alkali solution is rapidly reduced by adopting a high-shear emulsifying machine for 5000r/min to treat the hyaluronic acid alkali solution for 10min so as to facilitate subsequent filtration and sterilization, and the viscosity of the treated hyaluronic acid alkali solution is reduced from the initial viscosity of 17750 mPa.s to 72 mPa.s, and the viscosity reduction rate reaches 99.6%.
(3) Filtering, sterilizing and removing impurities
Adding 1.0% (w/v) of filter aid into the hyaluronic acid fermentation broth with the viscosity reduced in the step (2), filtering to remove thalli and insoluble impurities to obtain a clear and transparent hyaluronic acid alkali solution, wherein the filter of a plate filter of a filtering device is provided, the pore diameter of a filtering medium is 500 meshes (25 mu m), and the filter aid is a mixture of diatomite, bentonite and perlite (weight ratio is 1:2:2).
(4) Neutralization
Adding diluted hydrochloric acid into the hyaluronic acid-base solution from which the thalli and impurities are removed in the step (3) to neutralize until the pH value is 7.0, and controlling the temperature of the feed liquid below 40 ℃.
(5) Alcohol precipitation
Slowly adding 1-time volume of alcohol (95 DEG, v/v) into the neutralized feed liquid in the step (4) to fully precipitate the hyaluronic acid, filtering and separating to obtain a water-containing hyaluronic acid product, adding 1-time volume (v/w) of alcohol (95 DEG, v/v) into the product, further dehydrating and washing, and filtering and separating to obtain a low-water-content hyaluronic acid product.
(6) Drying
Drying the low-water-content hyaluronic acid obtained in the step (5) for 3.5 hours at 40 ℃ by a vacuum dryer, crushing and screening to obtain a hyaluronic acid product, and determining that the glucuronic acid content of the obtained hyaluronic acid product is 43.6% (based on dry basis) and the molecular weight is 1.31+/-0.09 multiplied by 10 6 Da, the recovery rate of polysaccharide in fermentation liquor reaches 96.3 percent.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for purifying hyaluronic acid from microbial polysaccharide is characterized by adding hyaluronic acid fermentation liquorAdding alkali liquor to regulate alkali and reduce viscosity, and adding OH in the fermentation liquor after alkali liquor - The concentration is 0.025-0.125 mol/L; mechanically shearing the hyaluronic acid fermentation broth subjected to alkali adjustment and viscosity reduction to further reduce the viscosity to 40-75 mPa.s; filtering, sterilizing and removing impurities from the hyaluronic acid fermentation liquor subjected to high-shear treatment and viscosity reduction; adding acid into the filtered solution for neutralization, and then carrying out alcohol precipitation and drying to obtain the product.
2. The method for purifying hyaluronic acid according to claim 1, wherein the hyaluronic acid content in the hyaluronic acid fermentation broth is 1.4-2.3%, w/v.
3. The method for purifying hyaluronic acid of microbial polysaccharide according to claim 1, wherein alkaline solution is slowly added into the hyaluronic acid fermentation broth;
or, the alkali liquor is sodium hydroxide solution or potassium hydroxide solution.
4. The method for purifying hyaluronic acid according to claim 1, wherein the mechanical shearing treatment is performed at a rate of 3000-5000 r/min for 5-10 min.
5. The method for purifying hyaluronic acid according to claim 1, wherein a filter aid is added during the filtration, sterilization and impurity removal process; preferably, the filter aid is one or more of diatomite, bentonite and perlite, and more preferably is a mixture of diatomite, bentonite and perlite; further preferably, the mixture of diatomite, bentonite and perlite is used as a filter aid, and the weight ratio of the diatomite, the bentonite and the perlite is preferably 1:2:2-1:5:3; preferably, the addition amount of the filter aid is 0.5-1.2% of the hyaluronic acid fermentation broth, w/v.
6. The method for purifying hyaluronic acid according to claim 1, wherein the pore size of the filter medium is 500-1000 mesh in the process of filtering, sterilizing and removing impurities.
7. The method for purifying hyaluronic acid according to claim 1, wherein the neutralization process is carried out at a temperature of 10 to 40 ℃;
or, the pH is neutralized to 6.0-7.0.
8. The method for purifying hyaluronic acid of microbial polysaccharide according to claim 1, wherein the alcohol precipitation process is as follows: adding ethanol water solution into the neutralized feed liquid to fully precipitate hyaluronic acid; preferably, the volume concentration of the ethanol water solution is 90-95 DEG, v/v; preferably, the addition amount of the ethanol water solution is 1 to 1.3 times of the volume of the feed liquid;
preferably, the precipitated hyaluronic acid is dehydrated and washed by ethanol water solution; preferably, the volume concentration of the ethanol water solution is 90-95 DEG, v/v; preferably, the added volume of the aqueous ethanol solution is 1 to 2 times the weight of the precipitated hyaluronic acid.
9. The method for purifying hyaluronic acid according to claim 1, wherein the temperature used for drying is 35-40 ℃; preferably vacuum drying is used; the drying time is preferably 2 to 5 hours.
10. Use of a purification method of a microbial polysaccharide hyaluronic acid according to any of claims 1-9 in the production of hyaluronic acid by microbial fermentation.
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