CN113493776A - Method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof - Google Patents
Method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof Download PDFInfo
- Publication number
- CN113493776A CN113493776A CN202110767111.5A CN202110767111A CN113493776A CN 113493776 A CN113493776 A CN 113493776A CN 202110767111 A CN202110767111 A CN 202110767111A CN 113493776 A CN113493776 A CN 113493776A
- Authority
- CN
- China
- Prior art keywords
- hyaluronic acid
- stage
- salt
- enzyme
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
A method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof comprises preparing crude enzyme solution, performing enzymolysis concentration, separating and extracting; performing enzymolysis concentration, mixing crude hyaluronic acid enzyme liquid and fermentation liquor in a membrane concentration reactor, controlling the enzymolysis temperature to be 35-45 ℃, stirring at 50-100rpm and pH of 7.1-8, performing enzymolysis for 1.8-2.2h, filtering by using a ceramic membrane, and performing continuous enzymolysis in the organic membrane concentration process to obtain final filtrate; the hyaluronic acid prepared by the invention has the molecular weight of 1000Da at the lowest, and the degradation range of 1KD-70KD, so that the molecular weight range prepared by the existing enzymolysis method is broken through, and especially the ultra-low molecular weight of oligosaccharide is realized; during extraction, a freeze drying technology is used, and the prepared hyaluronic acid or the salt thereof is far superior to products prepared by other processes in the aspects of solubility, bioactivity, molecular weight distribution, product color and the like.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof.
Background
Hyaluronic Acid (HA), also known as hyaluronic acid, is a glycosaminoglycan polysaccharide isolated from the vitreous humor of bovine eyes, and HAs unique molecular structure and physicochemical properties, so that the glycosaminoglycan polysaccharide HAs excellent moisture retention, viscoelasticity, biocompatibility and other characteristics, and is widely applied to the fields of cosmetics, health-care foods, medicines and the like. Hyaluronic acid is composed of a double-pond repeating unit formed by connecting D-glucuronic acid and N-acetylglucosamine through glycosidic bonds. HA is a linear single chain observed under an electron microscope, and is spread in a random coil shape in an aqueous solution. Each disaccharide unit in HA molecules contains a carboxyl, and can be dissociated into negative ions under physiological conditions, and the mutual repulsion of the negative ions at equal space distance enables the molecules to be in a loose state in aqueous solution, occupies a large amount of space, and can be combined with more than 1000 times of water per se. With the progress of research on HA in recent years, it HAs been found that HA plays an important role in processes such as tissue generation and repair, tumor invasion, and the like by acting on HA receptors in cells and cytoplasm to regulate cellular functions. Particularly, the low molecular hyaluronic acid has better permeability and absorption, and is widely applied to the fields of food, cosmetics and medicines.
In recent years, research and application of low molecular HA and HA oligosaccharides have been promoted, and research on degradation methods and preparation of degradation products thereof are increasing year by year at home and abroad. Currently, there are 3 major types of methods for HA degradation, physical degradation, chemical degradation, and biological degradation. Physical degradation methods generally employ mechanical, irradiation or sonication, but the extent of molecular weight reduction is limited. The chemical degradation causes environmental pollution and high energy consumption; secondly, the structure of the biomacromolecule is easily damaged, such as the breakage of glycosidic bonds on sugar chains, the hydrolysis of acetyl groups, the breakage of monosaccharide six-membered rings and the like, thereby influencing the biological activity of the substance; thirdly, the introduced small molecular substances are not easy to remove in the subsequent extraction process, and the quality of the product is influenced. The enzymolysis method is a mild and environment-friendly production technology which is generally accepted at present.
Patent CN102876748B discloses the production of hyaluronic acid by enzymatic hydrolysis, but its limitations are obvious, firstly, the reaction of finished hyaluronic acid or its salt with enzyme solution not only uses a large amount of organic solvent, increases carbon emission, and has low yield, but also continues to degrade on the basis of the finished hyaluronic acid product, and the production cycle continues to be prolonged, which is the main reason that the existing low molecular weight hyaluronic acid or its salt product is expensive and the cost is high. Secondly, the molecular weight range of the finished product prepared in the patent is 3kda-104kDa, narrow range, significantly limited applicability, and could not produce hyaluronic acid below 3 kDa.
Patent CN109897876B discloses the production of hyaluronic acid or its salts by enzymatic hydrolysis, with the following disadvantages: firstly, the molecular weight of hyaluronic acid or salt thereof is required to be 500-700 kda, the molecular weight of the prepared finished product is 1-60 kda, the enzymolysis process range is strictly limited, the application range is narrow, the number of pretreatment procedures is large, and the industrial feasibility is low; secondly, the finished product is prepared by the low-temperature spray drying technology in the extraction process, because the commercial finished product of the hyaluronic acid or the salt thereof is white or white-like powder, black spots are easily generated in the preparation process of a spray drying tower, and the hyaluronic acid with low molecular weight is easy to turn yellow after spray drying, so that the quality of the finished product is directly influenced, and the method is not suitable for the hyaluronic acid or the salt thereof with low molecular weight.
Patent CN108220364B discloses a method for preparing hyaluronic acid oligosaccharide and its salt with ultra-low molecular weight by combining solid-liquid two-phase enzymolysis and ultrafiltration, which has the following disadvantages besides the existing disadvantages: firstly, zirconia is introduced into an enzymolysis buffer solution, impurities in the substances have influence on the quality of a finished product, and the detection cost is increased in subsequent detection and analysis, no matter from the perspective of food safety factors or in the aspect of evolution analysis of a drug impurity spectrum; secondly, the time consumption is long in the process of converting a solid-liquid two-phase enzymolysis system into a liquid-liquid one-way system, and the polysaccharide feed liquid is analyzed from the enzymolysis condition and treated according to the method, so that the contamination is very easy to cause, and the irreparable loss is caused to the product quality; thirdly, activated carbon is needed for adsorption, and the method is very easy to cause the risk of carbon leakage and brings potential safety hazards to the quality of products.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof, so as to realize the following steps: the use of organic solvent is reduced, the yield is improved, the production period is shortened, the molecular weight of hyaluronic acid is reduced, and the problem of carbon leakage caused by adopting activated carbon for decolorization in the traditional method is avoided.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof comprises the steps of preparing a crude enzyme solution, performing enzymolysis and concentration, and separating and extracting.
The crude enzyme solution is prepared, and the enterobacter CGJ001 with the preservation number of CGMCC NO.18661 is cultured to prepare a bacterial solution containing hyaluronidase; then, centrifuging the prepared bacterial liquid containing hyaluronidase, and adding a PBS solution into the supernatant of the centrifugate in an equal volume to obtain a heavy suspension thallus; and (3) carrying out ultrasonic cell disruption on the resuspended thalli to prepare crude hyaluronic acid enzyme liquid, wherein the enzyme activity is 8100 and 8200U/ml.
The culture medium components per liter of the plate culture include: peptone 4.5-5.5g, yeast powder 4.5-5.5g, sodium hyaluronate 4.5-5.5g, K3PO4·3H2O 1.5-2.5g、MgSO4 ·7H20.4-0.6g of O, 1000mL of water and 19-21g of agar powder;
the pH value of the culture medium for plate culture is 5.5-6.5.
The seed culture medium comprises the following components: 0.9-1.1g/L hyaluronic acid, 0.9-1.1g/L K3PO4，0.09-0.11g/LMgSO40.9-1.1g/L peptone and 0.9-1.1g/L yeast powder;
the pH value of the seed culture medium is 5.5-6.5.
The fermentation medium comprises the following components: 0.9-1.1g/L hyaluronic acid, 0.9-1.1g/L K3PO4，0.09-0.11g/LMgSO40.9-1.1g/L peptone and 0.9-1.1g/L yeast powder;
the pH value of the fermentation medium is 5.5-6.5.
The PBS solution components per liter included: 2.4-2.6g of sodium dihydrogen phosphate, 0.9-1.1g of anhydrous disodium hydrogen phosphate, 8.1-8.3g of sodium chloride and 1000mL of water;
the pH value of the PBS solution is 6-6.5.
The parameters of the ultrasonic crusher are set as follows: the ultrasonic treatment time is 18-22min, the ultrasonic treatment is stopped for 4s and stopped for 6s, and the power is 290-310W.
And (2) performing enzymolysis concentration, namely mixing the crude hyaluronic acid enzyme solution and fermentation liquor in a membrane concentration reactor, controlling the enzymolysis temperature to be 35-45 ℃, controlling the stirring speed to be 50-100rpm and the pH to be 7.1-8, performing enzymolysis for 1.8-2.2h, filtering by using a ceramic membrane, removing impurities by filtering by using an organic membrane, continuing enzymolysis, performing filtration concentration by using the organic membrane at an interval of 25-35min, and continuing enzymolysis for 0.9-1.1h to obtain final filtrate.
And (2) performing enzymolysis concentration, namely mixing the crude hyaluronic acid enzyme solution and fermentation liquor in a membrane concentration reactor, controlling the enzymolysis temperature to be 35-45 ℃, controlling the stirring speed to be 90-100rpm, controlling the pH to be 7.1-8, performing enzymolysis for 1.8-2.2h, filtering by using a ceramic membrane, continuing enzymolysis for 3.5-4.5h until the solution is clear, then continuing enzymolysis after filtering by using an organic membrane, filtering and concentrating by using the organic membrane every 18-22min, and obtaining final filtrate after filtering by using the organic membrane until the solution is clear and transparent.
The volume ratio of the crude hyaluronic acid enzyme liquid to the fermentation liquid is 1: 100-1000.
Preferably, the volume ratio of the crude hyaluronic acid enzyme liquid to the fermentation liquid is 1: 100.
The fermentation liquid is one of hyaluronic acid fermentation liquid and sodium hyaluronate fermentation liquid.
The molecular weight of the hyaluronic acid in the hyaluronic acid fermentation liquor is 200-250KD, and the content of the hyaluronic acid is 10-15 g/L.
The filtration precision of the organic membrane is 50-100 KD.
Preferably, the organic membrane has a filtration precision of 50 KD.
The filtering precision of the ceramic membrane is 1KD-70 KD.
Preferably, the ceramic membrane has a filtration accuracy of 1 KD.
Performing separation and extraction, performing heat preservation treatment on the final filtrate at 85-95 ℃ for 8-12min, performing decolorization treatment by using a sodium hypochlorite solution, keeping the pH of the solution =3.5-4.5 in the decolorization process, adjusting the pH value to 6.5-7.5 after the decolorization treatment is finished, filtering the solution by using a liquid sterile filter, conveying the solution to a freeze dryer for freeze drying, and obtaining a hyaluronic acid product after the drying is finished.
The concentration of the sodium hypochlorite solution is 0.9-1.1 mg/L.
The filtration accuracy of the liquid sterile filter is 0.45 μm.
The freeze drying process is divided into a first stage, a second stage, a third stage, a fourth stage, a fifth stage, a sixth stage and a seventh stage.
Wherein, the second stage, the third stage, the fourth stage, the fifth stage, the sixth stage and the seventh stage are freeze-dried under the vacuum condition, and the vacuum degree is 25 Pa.
The temperature of the first stage is between 28 ℃ below zero and 32 ℃ below zero, and the time is 3.5 to 4.5 hours; the temperature of the second stage is between 18 ℃ below zero and 22 ℃ below zero, and the time is 1.5 to 2.5 hours; the temperature of the third stage is between 7 ℃ below zero and 3 ℃ below zero, and the time is 1.5 to 2.5 hours; the temperature of the stage four is between-2 ℃ and 2 ℃, and the time is 3.5 to 4.5 hours; the temperature of the stage five is 3-7 ℃, and the time is 1.5-2.5 h; the temperature of the sixth stage is 18-22 ℃, and the time is 1.5-2.5 h; the temperature of the stage seven is 28-32 ℃, and the time is 8-12 h.
The purity of the hyaluronic acid product is 98%.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof, the viscosity of the hyaluronic acid feed liquid is reduced by utilizing the reaction of the hyaluronic acid feed liquid and the enzyme liquid, the problem that membrane filtration equipment is not suitable for treating high-viscosity materials is solved, the concentration of a substrate is continuously improved by an organic membrane concentration technology, the hyaluronic acid or salt thereof is gradually degraded, and the molecular weight of the prepared hyaluronic acid is lower;
(2) the method for continuously preparing the enzyme-digested ultralow-molecular-weight hyaluronic acid or the salt thereof has no requirement on the molecular weight range of the hyaluronic acid or the salt thereof, reduces the production cost, and is simple and easy to implement;
(3) the method for continuously preparing the enzyme-digested ultralow-molecular-weight hyaluronic acid or the salt thereof, disclosed by the invention, combines the membrane filtration technology with the freeze drying technology, so that a large amount of organic solvent is saved, the quality of the prepared finished product is far superior to that of other technologies in aspects of color, solubility and the like, and the finished product is sterilized by moist heat in a phosphate system, so that the finished product is not turbid and does not change color;
(4) according to the method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof, disclosed by the invention, hyaluronidase is added into hyaluronic acid fermentation liquor, so that the viscosity of the feed liquid is reduced, the problem that membrane filtration equipment is not suitable for high-viscosity materials is effectively solved, and the problem that the high-molecular-weight viscosity of hyaluronic acid or salt thereof is high and the stability of the product is influenced by overlarge molecular weight distribution caused by local insufficient enzymolysis of the product due to easy caking in the dissolving process is avoided;
(5) according to the method for continuously preparing enzyme-digested ultralow molecular weight hyaluronic acid or salt thereof, the substrate concentration is continuously improved and the molecular weight of hyaluronic acid is continuously reduced by controlling the consistent conditions of membrane filtration and enzymolysis, so that the ultralow molecular weight is finally obtained, the lowest molecular weight can reach 1000Da, the degradation range can reach 1KD-70KD, the molecular weight range prepared by the existing enzymolysis method is broken through, and especially the ultralow molecular weight of oligosaccharide is realized;
(6) according to the method for continuously preparing enzyme-digested ultralow molecular weight hyaluronic acid or salt thereof, an extra hyaluronic acid finished product is not required to be added in the preparation process, and an ultralow molecular product is prepared through one-step degradation, so that the waste of repeated procedures in the preparation process is avoided, the cost is greatly saved, and the production period is shortened;
(7) the method for continuously preparing the enzyme-digested ultralow-molecular-weight hyaluronic acid or the salt thereof has the advantages that the membrane filtration process is adopted to replace the traditional alcohol precipitation process, the recovery rate is high, a large amount of solvents are saved, and the method is environment-friendly;
(8) according to the method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof, sodium hypochlorite is used for decoloring, and then the sodium hypochlorite is removed by utilizing the characteristic that the sodium hypochlorite is unstable under an alkaline condition, so that the problem of carbon leakage caused by decoloring by adopting activated carbon in the traditional method is avoided, and the method is more environment-friendly;
(9) the method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof uses a freeze-drying technology during extraction, the drying technology is more specific than other technologies, and the prepared hyaluronic acid or salt thereof is far superior to products prepared by other technologies in the aspects of solubility, bioactivity, molecular weight distribution, product color and the like.
Drawings
FIG. 1 shows the color of the solution obtained by dissolving the agent and subjecting it to high temperature treatment in example 1 and comparative example
The specific implementation mode is as follows:
while the invention has been described in terms of specific embodiments with a clear and complete understanding, it is to be understood that such embodiments are merely illustrative and not restrictive of the broad invention, and that the invention may be embodied or used in various other specific forms, which will become apparent to those skilled in the art from the description herein.
Example 1
A method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof comprises the following steps:
1. preparation of crude enzyme solution
(1) Carrying out plate culture on enterobacter CGJ001 with preservation number of CGMCC NO.18661 to obtain plate strains;
the culture medium components per liter of the plate culture include:
5g of peptone, 5g of yeast powder, 5g of sodium hyaluronate and K3PO4·3H2O 2g、MgSO4 ·7H20.5g of O, 1000mL of water and 20g of agar powder;
the pH value of the culture medium for plate culture is 6.
(2) Inoculating the flat strain into a sterilized seed culture medium, and culturing at 30 deg.C and 150rpm for 12h to obtain seed solution;
the seed cultureThe nutrient components comprise: 1g/L hyaluronic acid, 1g/L K3PO4，0.1g/LMgSO41g/L peptone and 1g/L yeast powder;
the pH value of the seed culture medium is 6.
(3) Inoculating the seed solution into a sterilized fermentation culture medium, and culturing for 12h at 30 ℃ and 150rpm to obtain a bacterial solution containing hyaluronidase;
the fermentation medium comprises the following components: 1g/L hyaluronic acid, 1g/L K3PO4，0.1g/LMgSO41g/L peptone and 1g/L yeast powder;
the pH value of the fermentation medium is 6.
(4) And centrifuging the prepared bacterial liquid containing the hyaluronidase for 10min at 6000rpm to obtain thalli.
(5) Discarding the supernatant of the centrifugate in the centrifugation process, and adding a PBS solution in the same volume to obtain the heavy suspension thallus;
the PBS solution components per liter included:
2.5g of sodium dihydrogen phosphate, 1.0g of anhydrous disodium hydrogen phosphate, 8.2g of sodium chloride and 1000mL of water;
the pH of the PBS solution was 6.2.
(6) And (3) carrying out ultrasonic cell disruption on the resuspended thalli for 20min to prepare crude hyaluronic acid enzyme liquid with the enzyme activity of 8139U/ml.
The parameters of the ultrasonic crusher are set as follows: the ultrasonic treatment time is 20min, the ultrasonic treatment time is 4s and the ultrasonic treatment time is 6s, and the power is 300W.
2. Concentrating by enzymolysis
Mixing 10ml of prepared crude hyaluronic acid enzyme liquid and 10L of hyaluronic acid fermentation liquor in a membrane concentration reactor, controlling the enzymolysis temperature at 40 ℃, the stirring speed at 50rpm and the pH value at 7.5, performing enzymolysis for 2 hours, filtering by using a 100KD ceramic membrane, removing impurities by filtering by using a 70KD organic membrane, continuing enzymolysis, performing filtration and concentration by using a 70KD organic membrane at intervals of 30min, and continuing enzymolysis for 1 hour to obtain final filtrate.
The molecular weight of hyaluronic acid in the hyaluronic acid fermentation liquor is 220KD, and the content of hyaluronic acid is 12 g/L.
3. Separating and extracting
And (3) preserving the temperature of the final filtrate at 90 ℃ for 10min, decoloring by using a sodium hypochlorite solution with the concentration of 1mg/L, keeping the pH =4 of the solution in the decoloring process, adjusting the pH value to 7 after the decoloring process is finished, filtering by using a 0.45-micrometer liquid sterile filter, conveying to a freeze dryer for freeze drying, and obtaining a 70kDa hyaluronic acid product with the purity of 98% after the drying is finished.
The freeze drying process is divided into a first stage, a second stage, a third stage, a fourth stage, a fifth stage, a sixth stage and a seventh stage.
Wherein, the second stage, the third stage, the fourth stage, the fifth stage, the sixth stage and the seventh stage are freeze-dried under the vacuum condition, and the vacuum degree is 25 Pa.
The temperature of the first stage is-30 ℃ and the time is 4 hours; the temperature of the second stage is-20 ℃ and the time is 2 hours; the temperature of the third stage is-5 ℃ and the time is 2 hours; the temperature of the stage four is 0 ℃, and the time is 4 hours; the temperature of the stage five is 5 ℃, and the time is 2 hours; the temperature of the sixth stage is 20 ℃, and the time is 2 hours; the temperature of the stage seven is 30 ℃ and the time is 10 h.
Example 2
Taking 100ml of the crude enzyme solution prepared in the embodiment 1, mixing 10L of hyaluronic acid fermentation liquor in a membrane concentration reactor, controlling the enzymolysis temperature at 40 ℃, the stirring speed at 100rpm, the pH value at 7.5, performing enzymolysis for 2h, filtering by a 50KD ceramic membrane, filtering until the solution is clear, continuing enzymolysis for 4h, then performing enzymolysis after filtering by a 1KD organic membrane, performing filtration and concentration by the 1KD organic membrane every 20min, and obtaining the final filtrate after filtering by the 1KD organic membrane until the solution is clear and transparent.
And (3) preserving the temperature of the final filtrate at 90 ℃ for 10min, decoloring by using a sodium hypochlorite solution with the concentration of 1mg/L, keeping the pH =4 of the solution in the decoloring process, adjusting the pH value to 7 after the decoloring process is finished, filtering by using a 0.45-micrometer liquid sterile filtering filter element, and freeze-drying by using a freeze dryer to obtain a hyaluronic acid product with the molecular weight of 1000D, wherein the purity of the hyaluronic acid product is 98%.
The freeze-drying procedure was the same as in example 1.
The molecular weight of hyaluronic acid in the hyaluronic acid fermentation liquor is 220KD, and the content of hyaluronic acid is 12 g/L.
Example 3
Taking example 1 and the control example, the competitors were dissolved in 100ml of PBS buffer solution according to the mass concentration of 2%, then the competitors were put into a 250ml triangular flask for sealing, and the solution was sterilized at 121 ℃ for 10min, and the color of the solution was observed and recorded, and the result is shown in FIG. 1.
The comparative example is a hyaluronic acid product with the molecular weight of 19KD prepared in example 1 in the preparation method of the preparation process of the low-molecular-weight sodium hyaluronate according to the publication number CN108484796B, and the competitive product is purchased from other companies on the market, has the molecular weight of 70KD and has the purity of 98%.
The components of the PBS buffer comprise: 0.027mol/L potassium chloride, 0.0147mol/L potassium dihydrogen phosphate, 1.37mol/L sodium chloride, and 0.077mol/L disodium hydrogen phosphate.
As shown in FIG. 1, the sample and the water sample in example 1 are dissolved in PBS buffer solution, and are still clear and transparent after being sterilized at high temperature, and the competitive products become turbid after being sterilized at high temperature, which shows that the product prepared by the invention meets the requirement that the turbid product is not generated after being sterilized at high temperature at the terminal after being dissolved in water.
Example 4
40 testers 20-40 years old are randomly selected, wherein 20 male and female testers are divided into experiment 1-4 groups according to 10 persons, then each sample in example 1, example 2, control example and competitive products is respectively coated on the cheek part of each tester, each sample is coated once a day and two drops are coated once, each sample is used for one month and then is scored, the score is 10 points, the scoring standard is shown in table 1, and the scoring result is shown in table 2.
TABLE 1
TABLE 2
As can be seen from Table 2, the skin feel effect of the samples prepared by the method is good after long-term use.
Claims (10)
1. A method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof is characterized by comprising the steps of preparing a crude enzyme solution, carrying out enzymolysis and concentration, and separating and extracting.
2. The method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or a salt thereof according to claim 1, wherein the enzymatic concentration is performed, crude hyaluronic acid enzyme solution and fermentation liquor are mixed in a membrane concentration reactor, the enzymatic hydrolysis temperature is controlled to be 35-45 ℃, the stirring speed is 90-100rpm, the pH is 7.1-8, after 1.8-2.2 hours of enzymatic hydrolysis, the crude hyaluronic acid enzyme solution is firstly filtered by using a ceramic membrane, the solution is filtered until the solution is clear, the enzymatic hydrolysis is continuously performed for 3.5-4.5 hours, then the enzymatic hydrolysis is continuously performed after the organic membrane is filtered, the organic membrane is filtered and concentrated every 18-22 minutes, and after the solution is filtered by the organic membrane until the solution is clear and transparent, the final filtrate is obtained.
3. The method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or a salt thereof according to claim 1, wherein the enzymatic hydrolysis concentration is performed, crude hyaluronic acid enzyme solution and fermentation liquor are mixed in a membrane concentration reactor, the enzymatic hydrolysis temperature is controlled to be 35-45 ℃, the stirring speed is 50-100rpm, the pH is 7.1-8, after 1.8-2.2 hours of enzymatic hydrolysis, a ceramic membrane is used for filtration, then the organic membrane is used for filtration and impurity removal, then the enzymatic hydrolysis is continued, the organic membrane is used for filtration and concentration every 25-35min, and the final filtrate is obtained after the enzymatic hydrolysis is continued for 0.9-1.1 hours.
4. The method for continuously preparing enzyme-cleaved ultra-low molecular weight hyaluronic acid or salt thereof according to any of claims 2 and 3, wherein the volume ratio of the crude hyaluronic acid enzyme solution to the fermentation broth is 1: 100-.
5. The method for continuously preparing enzymatically cut ultra-low molecular weight hyaluronic acid or a salt thereof according to any of claims 2 and 3, characterized in that the fermentation broth is one of a hyaluronic acid fermentation broth and a sodium hyaluronate fermentation broth.
6. The continuous production method of enzymatically cut ultra-low molecular weight hyaluronic acid or a salt thereof according to any of claims 2 and 3, characterized in that the filtration precision of the organic membrane is 50-100 KD; the filtering precision of the ceramic membrane is 1KD-70 KD.
7. The method for continuously preparing enzymatically-cleaved ultra-low molecular weight hyaluronic acid or a salt thereof as claimed in claim 5, wherein the molecular weight of hyaluronic acid in the hyaluronic acid fermentation broth is 200-250KD, and the content of hyaluronic acid is 10-15 g/L.
8. The method for continuously preparing enzyme-digested ultra-low molecular weight hyaluronic acid or a salt thereof according to claim 1, wherein the method comprises the steps of preparing a crude enzyme solution, and culturing enterobacter CGJ001 with the preservation number of CGMCC NO.18661 to obtain a hyaluronidase-containing solution; then, centrifuging the prepared bacterial liquid containing hyaluronidase, and adding a PBS solution into the supernatant of the centrifugate in an equal volume to obtain a heavy suspension thallus; and (3) carrying out ultrasonic cell disruption on the resuspended thalli to prepare crude hyaluronic acid enzyme liquid, wherein the enzyme activity is 8100 and 8200U/ml.
9. The method for continuously preparing enzyme-digested ultra-low molecular weight hyaluronic acid or a salt thereof according to claim 1, wherein the separation and extraction are performed, the final filtrate is subjected to heat preservation treatment at 85-95 ℃ for 8-12min, sodium hypochlorite solution is used for decoloring, the pH of the solution is kept between 3.5 and 4.5 during decoloring, the decoloring time is 1.8-2.2h, the pH value is adjusted to 6.5-7.5 after decoloring, the filtrate is filtered by a liquid sterile filter and then conveyed to a freeze dryer for freeze drying, and a hyaluronic acid product is obtained after drying.
10. The method for continuously preparing enzyme-cleaved ultra-low molecular weight hyaluronic acid or a salt thereof according to claim 9, wherein the freeze-drying process is divided into a first stage, a second stage, a third stage, a fourth stage, a fifth stage, a sixth stage and a seventh stage;
wherein, the second stage, the third stage, the fourth stage, the fifth stage, the sixth stage and the seventh stage are freeze-dried under the vacuum condition, and the vacuum degree is 25 Pa;
the temperature of the first stage is between 28 ℃ below zero and 32 ℃ below zero, and the time is 3.5 to 4.5 hours; the temperature of the second stage is between 18 ℃ below zero and 22 ℃ below zero, and the time is 1.5 to 2.5 hours; the temperature of the third stage is between 7 ℃ below zero and 3 ℃ below zero, and the time is 1.5 to 2.5 hours; the temperature of the stage four is between-2 ℃ and 2 ℃, and the time is 3.5 to 4.5 hours; the temperature of the stage five is 3-7 ℃, and the time is 1.5-2.5 h; the temperature of the sixth stage is 18-22 ℃, and the time is 1.5-2.5 h; the temperature of the stage seven is 28-32 ℃, and the time is 8-12 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110767111.5A CN113493776B (en) | 2021-07-07 | 2021-07-07 | Method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110767111.5A CN113493776B (en) | 2021-07-07 | 2021-07-07 | Method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113493776A true CN113493776A (en) | 2021-10-12 |
| CN113493776B CN113493776B (en) | 2022-06-14 |
Family
ID=77995850
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110767111.5A Active CN113493776B (en) | 2021-07-07 | 2021-07-07 | Method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113493776B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057909A (en) * | 2021-12-15 | 2022-02-18 | 沈阳博尔雅生物科技有限公司 | Oligomeric hyaluronic acid or salt thereof, and preparation method and application thereof |
| CN117535363A (en) * | 2023-12-27 | 2024-02-09 | 常州加源医药科技研发有限公司 | Production method of functional hyaluronic acid |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040016641A (en) * | 2002-08-19 | 2004-02-25 | 주식회사 코오롱 | Microorganism producing hyaluronic acid and method of producing hyalironic acid using thereof |
| CN101020724A (en) * | 2006-02-14 | 2007-08-22 | 镇江东方生物工程设备技术有限责任公司 | Process of preparing low molecular weight sodium hyaluronate |
| CN108220364A (en) * | 2016-12-09 | 2018-06-29 | 华熙福瑞达生物医药有限公司 | A kind of solid-liquid double-phase enzymolysis prepares Ultra-low molecular weight hyaluronic acid oligosaccharide and its method of salt with ultrafiltration combination |
| CN110923173A (en) * | 2019-12-26 | 2020-03-27 | 江南大学 | Enterobacter and application thereof |
| CN111040048A (en) * | 2019-12-21 | 2020-04-21 | 南京汉欣医药科技有限公司 | Ultra-low molecular weight hyaluronic acid and preparation method thereof |
| CN111893151A (en) * | 2020-08-21 | 2020-11-06 | 浙江绿创生物科技股份有限公司 | Method for continuously producing low molecular weight hyaluronic acid, low molecular weight hyaluronic acid obtained by method and application of low molecular weight hyaluronic acid |
| CN112662718A (en) * | 2021-01-13 | 2021-04-16 | 山东众山生物科技有限公司 | Preparation method of oligomeric sodium hyaluronate |
-
2021
- 2021-07-07 CN CN202110767111.5A patent/CN113493776B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040016641A (en) * | 2002-08-19 | 2004-02-25 | 주식회사 코오롱 | Microorganism producing hyaluronic acid and method of producing hyalironic acid using thereof |
| CN101020724A (en) * | 2006-02-14 | 2007-08-22 | 镇江东方生物工程设备技术有限责任公司 | Process of preparing low molecular weight sodium hyaluronate |
| CN108220364A (en) * | 2016-12-09 | 2018-06-29 | 华熙福瑞达生物医药有限公司 | A kind of solid-liquid double-phase enzymolysis prepares Ultra-low molecular weight hyaluronic acid oligosaccharide and its method of salt with ultrafiltration combination |
| CN111040048A (en) * | 2019-12-21 | 2020-04-21 | 南京汉欣医药科技有限公司 | Ultra-low molecular weight hyaluronic acid and preparation method thereof |
| CN110923173A (en) * | 2019-12-26 | 2020-03-27 | 江南大学 | Enterobacter and application thereof |
| CN111893151A (en) * | 2020-08-21 | 2020-11-06 | 浙江绿创生物科技股份有限公司 | Method for continuously producing low molecular weight hyaluronic acid, low molecular weight hyaluronic acid obtained by method and application of low molecular weight hyaluronic acid |
| CN112662718A (en) * | 2021-01-13 | 2021-04-16 | 山东众山生物科技有限公司 | Preparation method of oligomeric sodium hyaluronate |
Non-Patent Citations (1)
| Title |
|---|
| 鲍悦等: "陶瓷膜过滤在分离提取中国林蛙皮中透明质酸的应用研究", 《中国现代中药》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057909A (en) * | 2021-12-15 | 2022-02-18 | 沈阳博尔雅生物科技有限公司 | Oligomeric hyaluronic acid or salt thereof, and preparation method and application thereof |
| CN114057909B (en) * | 2021-12-15 | 2022-12-13 | 沈阳天贺新材料开发有限公司 | Oligomeric hyaluronic acid or salt thereof, and preparation method and application thereof |
| CN117535363A (en) * | 2023-12-27 | 2024-02-09 | 常州加源医药科技研发有限公司 | Production method of functional hyaluronic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113493776B (en) | 2022-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101512006B (en) | Efficient process for purification of high molecular weight hyaluronic acid | |
| CN113493776B (en) | Method for continuously preparing enzyme-digested ultralow-molecular-weight hyaluronic acid or salt thereof | |
| CN102876748B (en) | Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof | |
| CN103665371B (en) | A kind of method utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid | |
| JPS60251898A (en) | Preparation of hyaluronic acid by fermentation method | |
| CN102242165A (en) | Method for producing high molecular weight sodium hyaluronate through fermentation and culture medium utilized by same | |
| CN105695543B (en) | A kind of production method of surfactin | |
| EP0266578B1 (en) | Method of producing hyaluronic acid | |
| CN109897876A (en) | A method of preparing micromolecule hyaluronic acid or its salt | |
| US20140120584A1 (en) | Process for the co-production of chitin, its derivatives and polymers containing glucose, mannose and/or galactose, by the fermentation of the yeast pichia pastoris | |
| CN103805650A (en) | Fermentation production method for pigment-free pullulan | |
| CN110331178A (en) | A kind of enzyme cutting method prepares the method for micromolecule hyaluronic acid and gained micromolecule hyaluronic acid is applied with it | |
| CN107686854B (en) | Method for degrading and modifying schizophyllan by utilizing endoprotease produced by schizophyllum commune fermentation system | |
| CN111978421A (en) | Phellinus igniarius polysaccharide and preparation and application thereof | |
| CN108410926B (en) | Method for preparing and extracting high molecular weight hyaluronic acid | |
| Gummadi et al. | Production of extracellular water insoluble β-1, 3-glucan (curdlan) from Bacillus sp. SNC07 | |
| CN110592163A (en) | Production method for improving chromaticity of sodium hyaluronate | |
| CN110093389A (en) | The fermentation method for producing of instant xanthan gum | |
| CN113292613B (en) | Preparation method of D-glucosamine sulfate | |
| CN107557399B (en) | Method for simultaneously extracting beta-polymalic acid and pullulan from biological fermentation broth | |
| CN112358985B (en) | Pradazobium and application thereof in preparation of water-soluble beta-1, 3 glucan | |
| CN113563488A (en) | Preparation method of pharmaceutical-grade micromolecular marine organism polysaccharide | |
| CN107674854B (en) | Nitrogen-fixing sphingosine monad and application thereof in preparation of gellan gum | |
| WO2024131919A1 (en) | Paenibacillus and application thereof in preparation of tremella fuciformis oligosaccharide | |
| JPH0219393A (en) | N-acetylogalactosaminooligosaccahride and production thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221116 Address after: 273100 Economic Development Zone of Qufu High Speed Railway New Area, Jining City, Shandong Province Patentee after: Shandong focus furida biological Co.,Ltd. Patentee after: Shandong Baifu Furuida Pharmaceutical Co.,Ltd. Address before: 273100 Economic Development Zone of Qufu High Speed Railway New Area, Jining City, Shandong Province Patentee before: Shandong focus furida biological Co.,Ltd. |