High-purity unsaturated hyaluronic acid disaccharide for medical and cosmetic use, and preparation method and application thereof

Abstract

The application discloses a medical and cosmetic high-purity unsaturated hyaluronic acid disaccharide and a preparation method and application thereof, and belongs to the technical field of biomedical materials and saccharide biological manufacturing technology. The preparation method comprises the following steps: preparing low-heat-source high-activity hyaluronic acid cleavage enzyme, enzymolysis, and purifying the enzymolysis solution. The preparation of the low-heat-source high-activity hyaluronic acid cleavage enzyme comprises the following steps: activation, scale-up culture, fermentation, and purification of the hyaluronic acid cleavage enzyme. The scale-up culture is achieved by picking single colonies from the activated bacterial strains, inoculating into seed culture medium and oscillating culture to obtain a seed solution. The seed culture medium comprises calcium carbonate dispersion solution. The fermentation is achieved by inoculating the seed solution into fermentation culture medium to obtain a fermentation solution. The fermentation culture medium comprises starch complex. Through the preparation method, the generation of endotoxin can be controlled in fermentation, and the influence on the growth of bacterial strains and the enzyme expression amount is small. In the enzymolysis, high-efficiency and directional enzymolysis of hyaluronic acid can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and carbohydrate biomanufacturing technology, specifically relating to a method for preparing highly active hyaluronic acid lysase using a non-animal-derived fermentation system, obtaining low-endotoxin, high-purity unsaturated hyaluronic acid disaccharide through targeted enzymatic hydrolysis and pure physical purification, and the application of this disaccharide in medical aesthetic products. Background Technology

[0002] Hyaluronic acid (HA) is a linear acidic mucopolysaccharide formed by the alternating β-1,3 and β-1,4 glycosidic bonds of D-glucuronic acid and N-acetylglucosamine. It is widely found in skin, synovial fluid, vitreous humor, and the extracellular matrix. Due to its excellent moisturizing, lubricating, and biocompatibility, hyaluronic acid has been widely used in medical aesthetics, ophthalmology, orthopedics, and tissue engineering. In practical applications, hyaluronic acid of different molecular weights exhibits different biological functions. Specifically, high molecular weight hyaluronic acid mainly functions as a moisturizer and lubricant, while low molecular weight hyaluronic acid oligosaccharides, especially hyaluronic acid disaccharides, show distinct biological activities. Based on the biological activities exhibited by hyaluronic acid disaccharides, they have attracted widespread attention from researchers.

[0003] In the preparation of hyaluronic acid disaccharide, the main method employed is the degradation of hyaluronic acid. Various degradation methods are used, including acid hydrolysis, alkaline hydrolysis, oxidative degradation, ultrasonic degradation, and enzymatic hydrolysis. Enzymatic hydrolysis is favored due to its mild conditions and high specificity. Hyaluronic acid lyase specifically cleaves the β-1,4 glycosidic bond via a β-elimination reaction, generating a Δ4,5-unsaturated uronic acid structure at the non-reducing end. The resulting unsaturated oligosaccharide exhibits characteristic UV absorption at 232 nm, facilitating detection and quantification. When degradation reaches its limit, the product is unsaturated hyaluronic acid disaccharide (ΔDi-HA), which is the smallest fragment of hyaluronic acid obtainable after action by hyaluronic acid lyase.

[0004] Recent studies have revealed that it is precisely this non-reducing Δ4,5-unsaturated uronic acid structure that endows ΔDi-HA with a series of unique biological effects. Compared with chemically degraded saturated hyaluronic acid disaccharide, ΔDi-HA has a smaller molecular weight (only about 379 Da), significantly enhanced transdermal absorption, and exhibits highly efficient free radical scavenging, inhibition of inflammatory factor release, promotion of epidermal cell migration, and induction of tight junction protein expression. These properties make it show considerable promise in high-end medical aesthetic applications such as skin barrier repair, wound healing, and anti-aging. However, to truly bring ΔDi-HA to medical device-grade applications, several challenges remain in its preparation methods, as follows: First, regarding the fermentation of hyaluronic acid lyase, the fermentation process generally relies on traditional culture media containing animal-derived nitrogen sources such as animal peptone and beef extract. Although these media are inexpensive, they introduce a large amount of endotoxins and pyrogens from the source, putting great pressure on subsequent purification. Some researchers have tried to use non-animal-derived culture media, but due to the lack of certain nutrients in non-animal-derived media, problems such as slow cell growth and decreased enzyme expression levels occur during fermentation.

[0005] Secondly, regarding the enzymatic hydrolysis capacity of hyaluronic acid lyases, current methods generally suffer from insufficient hydrolysis ability. Chinese patent CN104293726A discloses a recombinant Bacillus subtilis strain that produces small-molecule hyaluronic acid. This patent utilizes hyaluronic acid synthase hasA derived from Streptococcus vesiculosus, integrated into Bacillus subtilis to achieve hyaluronic acid production. Simultaneously, overexpression of key genes in the hyaluronic acid synthesis pathway achieves high hyaluronic acid production in Bacillus subtilis. However, the small-molecule hyaluronic acid obtained using the aforementioned patent consists of HA10, HA8, HA6, and HA4, and is a mixture of different polymeric oligosaccharides. It fails to efficiently and directionally convert hyaluronic acid into the structurally singular ΔDi-HA. Furthermore, Chinese patent CN111004755A discloses a strain of Citrobacter T26 that specifically degrades hyaluronic acid, its hyaluronidase HylC, and its applications. The hyaluronidase HylC produced by this patent is obtained by fermentation of Citrobacter T26 and can specifically degrade hyaluronic acid. Its degradation mode is endo-degradation, and the final product is ΔDi-HA. However, as a Gram-negative bacterium, Citrobacter contains endotoxins in its cell wall, which leads to an increase in the endotoxin content in the product ΔDi-HA.

[0006] Third, from the perspective of endotoxin control, endotoxin is the core bottleneck restricting ΔDi-HA from reaching medical device applications. On the one hand, as analyzed above, endotoxins may exist in the cell walls of the microorganisms used in fermentation. On the other hand, endotoxins and other impurities may accumulate during enzymatic hydrolysis and concentration. When using hyaluronic acid raw materials for injection, according to pharmacopoeia standards, the endotoxin level should be controlled at <0.5 EU / mg. However, currently, the main method used is ethanol precipitation to purify unsaturated hyaluronic acid disaccharides. However, the ethanol precipitation method has limited ability to remove endotoxins, and it introduces residual organic solvents, increasing the burden of subsequent impurity removal and failing to meet the requirements of green manufacturing.

[0007] In summary, the industry urgently needs a comprehensive solution that covers the entire process from preparing hyaluronic acid lyase to targeted enzymatic hydrolysis by hyaluronic acid lyase, and then to the deep purification of unsaturated hyaluronic acid disaccharides. This solution would address the issues of slow cell growth and decreased enzyme expression during fermentation in non-animal-derived culture media, uneven hydrolysis products during hyaluronic acid lyase hydrolysis, and the easy introduction / accumulation of endotoxins and other impurities during fermentation, hydrolysis, and purification. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention aims to provide a high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics, its preparation method, and its application. The preparation method of the present invention can control the production of endotoxins during fermentation and has little impact on cell growth and enzyme expression. It can achieve efficient and targeted enzymatic hydrolysis of hyaluronic acid and obtain high-purity unsaturated hyaluronic acid disaccharide with low endotoxin content through physical means.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics includes preparing a low-pyrogen, high-activity hyaluronic acid lysin, enzymatic hydrolysis, and purification of the hydrolysate. The preparation of low-pyrogen, high-activity hyaluronic acid lyase includes: activation, large-scale culture, fermentation, and purification of hyaluronic acid lyase; The activation process involves streaking Bacillus subtilis onto a solid activation medium and activating it at 35-39°C for 20-30 hours to obtain the activated strain. The Bacillus subtilis strain described is the recombinant strain WB600 / pP43-abnA-HylP constructed according to the methods of Examples 1 and 2 in Chinese Patent CN117866931A, which describes an extracellular secreted hyaluronic acid lyase and its recombinant strain in application. The solid activated culture medium consists of: 9-11 g / L soybean peptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, with the remainder being water, and a pH of 6.9-7.2. The expanded culture involves picking a single colony from the activated strain and inoculating it into a seed culture medium. The culture is then shaken and cultured for 10-14 hours at a temperature of 35-39℃ and a shaking speed of 150-250 rpm to obtain a seed solution. In the aforementioned expanded culture, the amount of seed culture medium used is 40-60 mL; The seed culture medium consists of: 4-6 g / L glucose, 13-17 g / L soybean peptone, 4-6 g / L yeast extract, 3.7-4.3 g / L calcium carbonate dispersion, 1.8-2.2 g / L dipotassium hydrogen phosphate trihydrate, 0.4-0.6 g / L magnesium sulfate heptahydrate, with the balance being water, and a pH of 6.8-7.2. The calcium carbonate dispersion is prepared by mixing soluble starch and distilled water, stirring at 80-90℃ for 40-80 min, adding light calcium carbonate, stirring for 20-40 min, and cooling to room temperature to obtain the calcium carbonate dispersion. In the preparation of the calcium carbonate dispersion, the mass ratio of soluble starch, water, and light calcium carbonate is 9-11:280-320:45-55. The light calcium carbonate has a particle size of 1500-2500 mesh; The fermentation process involves inoculating the seed culture into the fermentation medium at an inoculation rate of 3.5-4.5%, stirring and fermenting at a temperature of 28-32℃ and a rotation speed of 400-600 rpm. During fermentation, the pH is controlled to 5.8-6.2 using ammonia water, and the glycerol concentration is monitored in real time. When the glycerol concentration drops below 10 g / L, the glycerol concentration is maintained at 5-15 g / L using a fed-batch culture medium. Fermentation is carried out for 28-30 hours to obtain the fermentation broth. In the fermentation process, the amount of fermentation medium used is 0.8-1.2L; The fermentation medium consists of: 60-70 g / L glycerol, 36-42 g / L soybean peptone, 11-15 g / L yeast extract, 13-17 g / L dipotassium hydrogen phosphate trihydrate, 5-7 g / L calcium carbonate dispersion, 1.8-2.2 g / L potassium dihydrogen phosphate, with the balance being water, and a pH of 5.8-6.2. The composition of the supplemental culture medium is: 310-340 g / L glycerol, 180-210 g / L soybean peptone, 60-70 g / L yeast extract, 35-45 g / L potassium dihydrogen phosphate, 12-18 g / L dipotassium hydrogen phosphate trihydrate, and 18-22 g / L starch complex. The method for preparing the calcium carbonate dispersion is the same as the method for preparing the calcium carbonate dispersion in the seed culture medium during the scaling-up culture step. The starch complex is prepared by mixing linear starch, L-tryptophan, and distilled water in a mass ratio of 8.5:0.9-1.1:140-180, stirring in a boiling water bath until transparent, adding anhydrous ethanol to a volume fraction of 35-45%, stirring and refluxing at 55-65℃ for 5-20 min, cooling to room temperature, centrifuging at 7000-9000 rpm for 15-25 min, taking the precipitate, and freeze-drying to obtain the starch complex. In the preparation of the starch complex, the method for preparing amylose is as follows: corn starch and hydrochloric acid aqueous solution are mixed and stirred at 33-37℃ for 3-5 days, filtered, and the filter cake is washed 4-6 times with deionized water and 2-4 times with anhydrous ethanol, and then dried at room temperature to obtain acid-hydrolyzed corn starch; all the above acid-hydrolyzed corn starch is mixed with water at a mass ratio of 1:8-10, and stirred and gelatinized in a boiling water bath for 20-40 minutes, naturally cooled to room temperature, centrifuged, and the supernatant is collected. The supernatant is frozen at -20℃ to -15℃ for 10-14 hours, thawed at room temperature, and when the temperature drops to -5℃ to 0℃, filtered, washed with cold water, and the filter cake is dried at room temperature to obtain amylose; wherein, the concentration of the hydrochloric acid aqueous solution is 2-2.5 mol / L; the mass ratio of corn starch to hydrochloric acid aqueous solution is 1:18-22; the centrifugation speed is 7000-9000 rpm, and the centrifugation time is 15-25 minutes; The ammonia water has a mass concentration of 20-28%; The purification of hyaluronic acid lyase involves the following steps: First, centrifuge the fermentation broth, collect the supernatant, filter the supernatant using a microfiltration membrane, collect the filtrate, add ammonium sulfate to 18-22%, stir for 20-40 minutes, add perlite to 9-11%, centrifuge a second time, collect the supernatant, add ammonium sulfate to 68-72%, stir for 20-40 minutes, let stand at 3-5°C for 1.5-2.5 hours, centrifuge a third time, collect the precipitate, dissolve it in a dissolving buffer to obtain a crude enzyme extract; concentrate the crude enzyme extract, change the medium, purify the ultrafiltered crude enzyme extract using nickel column affinity chromatography, and collect the elution peaks; In the purification of hyaluronic acid lysin, the filtration precision of the microfiltration membrane is 0.4-0.5 μm; The temperature for the first, second, and third centrifugation was 3-5℃, the centrifugation speed was 13000-17000 rpm, and the centrifugation time was 10-20 min. The dissolution buffer is a 45-55 mmol / L Na2HPO4-NaH2PO4 buffer solution with a pH of 5.5-6.5, and its volume is 9-11% of the fermentation supernatant volume; The concentration and liquid replacement process involves using an ultrafiltration membrane with a filtration accuracy of 8-12 kDa to ultrafilter the crude enzyme extract, concentrating it 4-6 times, and then replacing the liquid with NiA buffer. This concentration and liquid replacement process is repeated 3-5 times. The NiA buffer consists of: 20-30 mM phosphate buffer, 45-55 mM imidazole, 480-520 mM NaCl, and pH 7.3-7.5. The chromatography procedure in the nickel column affinity chromatography is as follows: equilibration, NiA buffer 4-6 CV; sample loading; rinsing, NiAbuffer 9-11 CV; elution, 50% NiB buffer, 4-6 CV; collection of elution peaks. The NiA buffer consists of: 20-30 mM phosphate buffer, 45-55 mM imidazole, 480-520 mM NaCl, and pH 7.3-7.5. The NiB buffer consists of: 20-30 mM phosphate buffer, 480-520 mM imidazole, 480-520 mM NaCl, and pH 7.3-7.5. The enzymatic hydrolysis involves adding purified hyaluronidase to a hyaluronic acid solution and stirring at 35-39°C for 9-11 hours to obtain the hydrolysate. In the enzymatic hydrolysis, the molecular weight of hyaluronic acid in the hyaluronic acid solution is 1000-2000 kDa; The concentration of the hyaluronic acid solution is 1.8-2.2 g / L; The concentration of hyaluronidase in the system is 100-200 U / mL; During enzymatic hydrolysis, hyaluronidase is added once every 0.5-2 hours; The purified enzymatic hydrolysate is prepared by inactivating the enzyme, filtering the enzymatic hydrolysate using a microfiltration membrane, taking the microfiltrate, performing primary ultrafiltration using an ultrafiltration membrane, taking the ultrafiltrate, performing secondary ultrafiltration using an ultrafiltration membrane, taking the concentrate, performing electrodialysis desalting, concentrating the desalted enzymatic hydrolysate under vacuum, and freeze-drying under vacuum to obtain unsaturated hyaluronic acid disaccharide powder. In the purified enzyme hydrolysate, the enzyme inactivation temperature is 75-85℃ and the time is 50-70 min; The filtration accuracy of the microfiltration membrane is 0.2-0.25 μm; In the primary ultrafiltration process, the ultrafiltration membrane has a molecular weight cutoff of 0.8-1.2 kDa. In the secondary ultrafiltration, the ultrafiltration membrane has a molecular weight cutoff of 300 Da; During the electrodialysis desalination process, the concentrate is introduced into the desalination chamber, pure water is introduced into the concentrated water chamber, and an aqueous sodium sulfate solution with a concentration of 8-12 g / L is introduced into the electrode water chamber. A voltage of 20-30 V is applied to the electrodes on both sides of the electrodialysis device. The operation is stopped when the conductivity of the desalination chamber drops to 100-200 μS / cm. The vacuum concentration is the process of vacuum concentrating to a solid content of 25-35%.

[0010] A high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics prepared by the aforementioned preparation method, wherein the endotoxin content of the high-purity unsaturated hyaluronic acid disaccharide is 0.08-0.1 EU / mg.

[0011] The application of a high-purity unsaturated hyaluronic acid disaccharide prepared by the aforementioned preparation method in the preparation of medical aesthetic products, wherein the medical aesthetic products have at least one of the following effects: skin barrier repair, wound healing, and anti-aging; The dosage forms of the medical aesthetic products include any one of the following: injection solution, lyophilized powder, gel, cream, spray, medical cold compress, microneedle, and facial mask.

[0012] Compared with the prior art, the present invention has the following advantages: (1) In terms of fermentation, this invention uses Gram-positive bacteria and a non-animal-derived low-pyrogen culture medium, which cuts off the introduction of animal-derived endotoxins at the source of fermentation. Combined with a multi-stage purification process, the endotoxin load of the enzyme solution is greatly reduced, and the enzyme activity of the purified hyaluronidase is 1.73 × 10⁻⁶. 7 U / mL, endotoxin content is 0.019 EU / mL; in terms of product uniformity, this invention selects highly specific hyaluronidase, thereby improving the enzymatic hydrolysate with high unsaturated hyaluronic acid disaccharide content; in terms of purification, this invention uses a combination of microfiltration-ultrafiltration-nanofiltration-electrodialysis, without introducing organic solvents and chemical precipitants throughout the process, avoiding the problem of chemical reagent residues. (2) Regarding the effect of endotoxin control, the present invention uses a dual strategy of controlling toxicity at the fermentation source and deep purification at the end to ensure that the endotoxin content of the final product can be stably lower than 0.5 EU / mg, and can reach as low as 0.088 EU / mg. (3) In fermentation, this invention uses calcium carbonate dispersion and starch complex. When preparing the calcium carbonate dispersion, the method involves mixing soluble starch with light calcium carbonate. Soluble starch can coat the surface of light calcium carbonate, improving its dispersibility. Furthermore, the interaction between soluble starch and calcium ions can regulate the release rate of calcium ions, thereby adjusting the effect of calcium ions on Bacillus subtilis. Utilizing the loading effect of light calcium carbonate and the promoting effect of calcium ions on enzyme production, the enzyme production of Bacillus subtilis is increased. When preparing the starch complex, The preparation method involves first preparing amylose, and then utilizing the interaction between amylose and L-tryptophan to prepare an amylose-tryptophan complex. Amylose has a stabilizing effect on hyaluronidase activity. Considering that soybean peptone has a lower tryptophan content compared to tryptophan, L-tryptophan was added as an extra supplement. Through the interaction between amylose and tryptophan, the release of tryptophan was controlled, thereby increasing the nitrogen content in the supplemental culture medium. This also avoids the inhibitory effect of excessive amino acid content on Bacillus subtilis enzyme production, further increasing the enzyme production of Bacillus subtilis. Detailed Implementation

[0013] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection.

[0014] In this embodiment, the method used to test the enzyme activity of hyaluronidase is as follows: 1. Preparation of hyaluronic acid solution substrate: Dissolve 2g of hyaluronic acid in 1L of 50mM citrate buffer to prepare a hyaluronic acid solution substrate with a concentration of 2g / L; 2. Test Method: The content of reduced ends produced by hyaluronic acid lyase degradation of hyaluronic acid was determined using the 3,5-dinitrosohydrate (DNS) colorimetric method. A mixture of hyaluronidase solution and hyaluronic acid substrate solution was used as the experimental group, while a mixture of inactivated hyaluronidase solution and hyaluronic acid substrate solution served as the control group. The reaction was carried out at 37℃ for 15 min, then DNS reagent was added, and the reaction was terminated by boiling in a water bath for 10 min. After cooling, the absorbance was measured at 540 nm using a microplate reader. Glucose solutions of different concentrations were prepared to establish a glucose standard curve. 3. Enzyme activity calculation method: The enzyme activity unit (1U) is defined as the amount of enzyme required to produce 1μg of glucose reduction equivalent per minute under the reaction conditions in step 2. During the test, 3 replicates are performed and the average value is taken.

[0015] Example 1 A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics, specifically comprising: 1. Preparation of low-pyrogen, high-activity hyaluronic acid lyase Following the methods described in Examples 1 and 2 of Chinese Patent CN117866931A, which discloses an extracellular secretory expression of hyaluronic acid lyase and its recombinant strain, a recombinant strain WB600 / pP43-abnA-HylP was constructed. Then, it was activated, cultured on a large scale, and fermented according to the following methods: (1) Activation: The recombinant strain WB600 / pP43-abnA-HylP, which was stored at -80℃, was streaked on a solid activation medium (the solid activation medium consisted of 10 g / L soybean peptone, 5 g / L yeast extract, 10 g / L sodium chloride, with the remainder being water and pH 7), and activated at 37℃ for 24 h to obtain the activated strain; (2) Expanded culture: Single colonies were picked from the activated strain and inoculated into 50 mL of seed culture medium (the components of the seed culture medium are: 5 g / L glucose, 15 g / L soybean peptone, 5 g / L yeast extract, 4 g / L calcium carbonate dispersion, 2 g / L dipotassium hydrogen phosphate trihydrate, 0.5 g / L magnesium sulfate heptahydrate, the remainder being water, and the pH is 7). The culture was shaken and cultured for 12 h at a temperature of 37℃ and a shaking speed of 200 rpm to obtain the seed liquid. The calcium carbonate dispersion is prepared by mixing 10g of soluble starch and 300g of distilled water, stirring at 85°C for 60min, adding 50g of light calcium carbonate, continuing to stir for 30min, and cooling to room temperature to obtain the calcium carbonate dispersion. The light calcium carbonate has a particle size of 2000 mesh; (3) Fermentation: The seed culture was inoculated into 1L of fermentation medium (the composition of the fermentation medium was: 65g / L glycerol, 39g / L soybean peptone, 13g / L yeast extract, 15g / L dipotassium hydrogen phosphate trihydrate, 6g / L calcium carbonate dispersion, 2g / L potassium dihydrogen phosphate, with the remainder being water and pH 6) at a rate of 30℃ and a stirring speed of 500rpm. During the fermentation process, the pH was controlled to 6 by adding 25% ammonia water. The glycerol concentration was monitored in real time during the fermentation process. Monitoring was conducted until the glycerol concentration dropped below 10 g / L. Then, fed-batch culture medium (composed of 325 g / L glycerol, 195 g / L soybean peptone, 65 g / L yeast extract, 40 g / L potassium dihydrogen phosphate, 15 g / L dipotassium hydrogen phosphate trihydrate, and 20 g / L starch complex) was added to maintain the glycerol concentration at 5-15 g / L. Enzyme activity in the fermentation system was monitored every hour to determine the peak enzyme production time. The tests showed that peak enzyme production was reached at 28 hours of fermentation, with an extracellular enzyme activity of 1.52 × 10⁻⁶. 5 U / mL; The preparation method of the calcium carbonate dispersion is the same as that of the calcium carbonate dispersion in (2); The preparation method of the starch complex is as follows: 50g of corn starch and 1000g of 2.2mol / L hydrochloric acid aqueous solution are mixed and stirred at 35℃ for 3 days. The mixture is then filtered, and the filter cake is washed 5 times with deionized water and 3 times with anhydrous ethanol. It is then air-dried at room temperature to obtain acid-hydrolyzed corn starch. All the acid-hydrolyzed corn starch is mixed with water at a mass ratio of 1:9, and the mixture is stirred and gelatinized in a boiling water bath for 30 minutes. After naturally cooling to room temperature, it is centrifuged at 8000rpm for 20 minutes. The supernatant is collected. The supernatant was frozen at -18℃ for 12 hours, thawed at room temperature, and filtered when cooled to -2℃. The filtered cake was then dried at room temperature to obtain amylose. Amylose was mixed with L-tryptophan and distilled water at a mass ratio of 8:1:160 and stirred in a boiling water bath until transparent. Anhydrous ethanol was added until the volume fraction of ethanol was 40%. The mixture was stirred and refluxed at 60℃ for 10 minutes, cooled to room temperature, and centrifuged at 8000 rpm for 20 minutes. The precipitate was collected, freeze-dried, and the starch complex was obtained. In summary, with 28 hours of fermentation as the fermentation endpoint, step (3) was repeated to obtain an enzyme activity of 1.52 × 10⁻⁶. 5 Fermentation broth with U / mL; (4) Purification of hyaluronic acid lyase: The fermentation broth was centrifuged at 4℃ and 15000rpm for 15min. The supernatant was collected (this supernatant was used as the fermentation supernatant). The supernatant was filtered through a microfiltration membrane with a filtration precision of 0.45μm. The filtrate was collected, and ammonium sulfate was added to 20% (w / v). The mixture was stirred for 30min. Perlite was added to 10% (w / v). The mixture was centrifuged at 4℃ and 15000rpm for 15min. The supernatant was collected, and ammonium sulfate was added to 70% (w / v). The mixture was stirred for 30min. The mixture was allowed to stand at 4℃ for 2h. The mixture was centrifuged at 4℃ and 15000rpm for 15min. The precipitate was collected and dissolved in 50mmol / L, pH 6 Na2HPO4-NaH2PO4 buffer (volume equal to 10% of the fermentation supernatant volume) to obtain the crude enzyme extract. The crude enzyme extract was ultrafiltered using an ultrafiltration membrane with a filtration accuracy of 10 kDa. After concentration by 5 times, the medium was changed (NiAbuffer was added to the original volume; the composition of NiA buffer was: 25 mM phosphate buffer, 50 mM imidazole, 500 mM NaCl, pH 7.4). The concentration and medium change were repeated as described above for a total of 4 times to obtain the ultrafiltered crude enzyme extract. The AKTA protein purification system was used to purify the ultrafiltration crude enzyme extract using nickel column affinity chromatography. The chromatography program was as follows: equilibration, NiA buffer 5CV; loading; rinsing, NiA buffer 10CV; elution, 50% NiB buffer, 5CV; collection of elution peaks. The NiA buffer consists of: 25 mM phosphate buffer, 50 mM imidazole, 500 mM NaCl, and pH 7.4. The NiB buffer consists of: 25 mM phosphate buffer, 500 mM imidazole, 500 mM NaCl, and pH 7.4. The elution peak was used as the purified hyaluronidase. The enzyme activity (using UV spectrophotometry) and endotoxin content (using Limulus amebocyte lysate (LAL) gel electrophoresis, detection limit ≥0.015 EU / mL) of the purified hyaluronidase were measured. The enzyme activity of the purified hyaluronidase was 1.73 × 10⁻⁶. 7 The endotoxin content was 0.019 EU / mL.

[0016] 2. Enzymatic hydrolysis Hyaluronic acid (molecular weight 1300kDa) was dissolved in distilled water to prepare a hyaluronic acid solution with a concentration of 2g / L. Purified hyaluronidase was added and its concentration in the system was controlled at 150U / mL. Hyaluronidase was added once every 1 hour. The solution was stirred at 37℃ for 10 hours to obtain the enzymatic hydrolysate. The content of unsaturated hyaluronic acid disaccharide in the enzymatic hydrolysate was detected by HPLC. The results showed that the content of unsaturated hyaluronic acid disaccharide accounted for 96.7% of the total content of hyaluronic acid oligosaccharides.

[0017] 3. Purify the enzyme hydrolysate The enzyme hydrolysate was inactivated by incubating at 80℃ for 60 min. The hydrolysate was then filtered using a microfiltration membrane with a filtration precision of 0.22 μm. The microfiltrate was then subjected to primary ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The ultrafiltrate was then subjected to secondary ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 300 Da. The concentrate was then subjected to electrodialysis desalination. During electrodialysis desalination, the concentrate was passed into the desalination chamber, pure water was passed into the concentration chamber, and a 10 g / L sodium sulfate aqueous solution was passed into the electrode water chamber. A voltage of 20 V was applied to both electrodes of the electrodialysis device. The operation was stopped when the conductivity of the desalination chamber dropped to 100 μS / cm. The desalted hydrolysate was then concentrated under vacuum to a solid content of 30%, and then freeze-dried under vacuum to obtain unsaturated hyaluronic acid disaccharide powder.

[0018] The purity of unsaturated hyaluronic acid disaccharide in the obtained hyaluronic acid disaccharide powder (HPLC method) and the endotoxin content (using the Limulus amebocyte lysate gel electrophoresis method, detection limit ≥0.015 EU / mL) were detected. The results showed that the purity of unsaturated hyaluronic acid disaccharide in the obtained hyaluronic acid disaccharide powder was 97.8%, and the endotoxin content was 0.088 EU / mg.

[0019] Example 2 A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics is as follows: The components of the seed culture medium in Example 1, 1. Preparation of low-pyrogen high-activity hyaluronic acid lyase and (2) expansion culture are changed to: 5 g / L glucose, 15 g / L soybean peptone, 5 g / L yeast extract, 3.7 g / L calcium carbonate dispersion, 2 g / L dipotassium hydrogen phosphate trihydrate, 0.5 g / L magnesium sulfate heptahydrate, with the remainder being water and the pH being 7; In Example 1, the components of 1. Preparation of low-pyrogen high-activity hyaluronic acid lyase and (3) Fermentation feeding medium were changed to: 325 g / L glycerol, 195 g / L soybean peptone, 65 g / L yeast extract, 40 g / L potassium dihydrogen phosphate, 15 g / L dipotassium hydrogen phosphate trihydrate, and 18 g / L starch complex. That is, based on Example 1, the amount of calcium carbonate dispersion and starch complex is increased, and the rest of the technical solution is the same as in Example 1.

[0020] In (3) determining the peak enzyme production time during fermentation, it was found that the peak enzyme production was reached at 30 hours of fermentation, with an extracellular enzyme activity of 1.45 × 10⁻⁶. 5 U / mL; This indicates that reducing the amount of calcium carbonate dispersion and starch complex will decrease the activity of hyaluronidase in the fermentation broth.

[0021] Example 3 A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics is as follows: The components of the seed culture medium in Example 1, 1. Preparation of low-pyrogen high-activity hyaluronic acid lyase and (2) expansion culture are changed to: 5 g / L glucose, 15 g / L soybean peptone, 5 g / L yeast extract, 4.3 g / L calcium carbonate dispersion, 2 g / L dipotassium hydrogen phosphate trihydrate, 0.5 g / L magnesium sulfate heptahydrate, with the remainder being water and the pH being 7; In Example 1, the components of 1. Preparation of low-pyrogen high-activity hyaluronic acid lyase and (3) Fermentation feeding medium were changed to: 325 g / L glycerol, 195 g / L soybean peptone, 65 g / L yeast extract, 40 g / L potassium dihydrogen phosphate, 15 g / L potassium dihydrogen phosphate trihydrate, and 22 g / L starch complex. That is, based on Example 1, the amount of calcium carbonate dispersion and starch complex is increased, and the rest of the technical solution is the same as in Example 1.

[0022] In (3) determining the peak enzyme production time during fermentation, it was found that the peak enzyme production was reached at 29 hours of fermentation, with an extracellular enzyme activity of 1.49 × 10⁻⁶. 5 U / mL; This indicates that increasing the amount of calcium carbonate dispersion and starch complex will reduce the activity of hyaluronidase in the fermentation broth.

[0023] Example 4 A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics is as follows: The components of the seed culture medium in Example 1, 1. Preparation of low-pyrogen high-activity hyaluronic acid lysin and (2) expansion culture are changed to: 5 g / L glucose, 15 g / L soybean peptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate trihydrate, 0.5 g / L magnesium sulfate heptahydrate, with the remainder being water and the pH being 7; (3) The composition of the fermentation medium during fermentation was changed to: 65 g / L glycerol, 39 g / L soybean peptone, 13 g / L yeast extract, 15 g / L dipotassium hydrogen phosphate trihydrate, 2 g / L potassium dihydrogen phosphate, with the remainder being water, and the pH being 6. That is, based on Example 1, the use of calcium carbonate dispersion is omitted, and the rest of the technical solutions are the same as in Example 1.

[0024] In (3) determining the peak enzyme production time during fermentation, it was found that the peak enzyme production was reached at 37 hours of fermentation, with an extracellular enzyme activity of 1.18 × 10⁻⁶. 5 U / mL; This indicates that omitting the use of calcium carbonate dispersion will reduce the activity of hyaluronidase in the fermentation broth.

[0025] Example 5 A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics is as follows: The components of the following in Example 1 are changed to: 1. Preparation of low-pyrogen high-activity hyaluronic acid lysin and (3) Fermentation feeding medium: 325g / L glycerol, 195g / L soybean peptone, 65g / L yeast extract, 40g / L potassium dihydrogen phosphate, and 15g / L dipotassium hydrogen phosphate trihydrate; That is, based on Example 1, the use of starch complex is omitted, and the rest of the technical solutions are the same as in Example 1.

[0026] In (3) determining the peak enzyme production time during fermentation, it was found that the peak enzyme production was reached at 35 hours of fermentation, with an extracellular enzyme activity of 1.26 × 10⁻⁶. 5 U / mL; This indicates that omitting the use of starch complex will reduce the activity of hyaluronidase in the fermentation broth.

[0027] Example 6 A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics is as follows: In Example 1, the soybean peptone in the solid activation culture medium, seed culture medium, fermentation culture medium, and fed culture medium used in the preparation of low-pyrogen, high-activity hyaluronic acid lysin is replaced with an equal mass of tryptone. Specifically, the composition of the solid activation culture medium is changed to: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, with the remainder being water, and a pH of 7; the composition of the seed culture medium is changed to: 5 g / L glucose, 15 g / L tryptone, 5 g / L yeast extract, 4 g / L calcium carbonate dispersion, 2 g / L dipotassium hydrogen phosphate trihydrate, 0. The initial composition of the fermentation medium was 5 g / L magnesium sulfate heptahydrate, with the remainder being water, and the pH was 7. The composition of the fermentation medium was changed to: 65 g / L glycerol, 39 g / L tryptone, 13 g / L yeast extract, 15 g / L dipotassium hydrogen phosphate trihydrate, 6 g / L calcium carbonate dispersion, 2 g / L potassium dihydrogen phosphate, with the remainder being water, and the pH was 6. The composition of the fed-batch medium was changed to: 325 g / L glycerol, 195 g / L tryptone, 65 g / L yeast extract, 40 g / L potassium dihydrogen phosphate, 15 g / L dipotassium hydrogen phosphate trihydrate, and 20 g / L starch complex.

[0028] The remaining technical solutions are the same as in Example 1.

[0029] In (3) determining the peak enzyme production time during fermentation, it was found that the peak enzyme production was reached at 27 hours of fermentation, with an extracellular enzyme activity of 1.65 × 10⁻⁶. 5 U / mL; Then, following the methods in 1. Preparing low-pyrogen, high-activity hyaluronic acid lysin and (4) purifying hyaluronic acid lysin, the purified hyaluronic acid enzyme was obtained. Then, following the methods in 2. Enzymatic hydrolysis and 3. Purification of the hydrolysate, hyaluronic acid disaccharide powder was obtained. The purity of unsaturated hyaluronic acid disaccharide in the obtained hyaluronic acid disaccharide powder (HPLC method) and the endotoxin content (using the Limulus amebocyte lysate gel electrophoresis method, detection limit ≥0.015 EU / mL) were detected. The results showed that the purity of unsaturated hyaluronic acid disaccharide in the obtained hyaluronic acid disaccharide powder was 97.3%, and the endotoxin content was 0.427 EU / mg.

[0030] This indicates that, in the culture medium, replacing soybean peptone with trypsin did not significantly increase enzyme production compared to Example 1, and it also significantly increased the endotoxin content in the obtained hyaluronic acid disaccharide powder. Analysis shows that there is a certain difference in enzyme production when trypsin and soybean peptone are used as nitrogen sources in the prior art. However, the difference in enzyme production between Example 1 and Example 6 is not significant, indicating that calcium carbonate dispersion and starch complex can shorten the difference in enzyme production when soybean peptone is used as a nitrogen source by increasing the enzyme production when soybean peptone is used as a nitrogen source.

[0031] Example 7 A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics, specifically: In Example 1, the soybean peptone in the solid activation culture medium, seed culture medium, fermentation culture medium, and fed culture medium used in the preparation of low-pyrogen, high-activity hyaluronic acid lysin is replaced with an equal mass of trypsin, and the use of calcium carbonate dispersion and starch complex is omitted. Specifically, the composition of the solid activation culture medium is changed to: 10 g / L trypsin, 5 g / L yeast extract, 10 g / L sodium chloride, with the remainder being water, and pH 7; the composition of the seed culture medium is changed to: 5 g / L glucose, 15 g / L trypsin, 5 g / L yeast extract, 2... The fermentation medium consisted of: 65 g / L dipotassium hydrogen phosphate trihydrate, 0.5 g / L magnesium sulfate heptahydrate, with the remainder being water, and pH 7. The fermentation medium was then modified to consist of: 65 g / L glycerol, 39 g / L tryptone, 13 g / L yeast extract, 15 g / L dipotassium hydrogen phosphate trihydrate, 2 g / L potassium dihydrogen phosphate, with the remainder being water, and pH 6. The fed-batch medium was modified to consist of: 325 g / L glycerol, 195 g / L tryptone, 65 g / L yeast extract, 40 g / L potassium dihydrogen phosphate, and 15 g / L dipotassium hydrogen phosphate trihydrate.

[0032] The remaining technical solutions are the same as in Example 1.

[0033] In (3) determining the peak enzyme production time during fermentation, it was found that the peak enzyme production was reached at 29 hours of fermentation, with an extracellular enzyme activity of 1.41 × 10⁻⁶. 5 U / mL; Then, following the methods in 1. Preparing low-pyrogen, high-activity hyaluronic acid lysin and (4) purifying hyaluronic acid lysin, the purified hyaluronic acid enzyme was obtained. Then, following the methods in 2. Enzymatic hydrolysis and 3. Purification of the hydrolysate, hyaluronic acid disaccharide powder was obtained. The purity of unsaturated hyaluronic acid disaccharide in the obtained hyaluronic acid disaccharide powder (HPLC method) and the endotoxin content (using the Limulus amebocyte lysate gel electrophoresis method, detection limit ≥0.015 EU / mL) were detected. The results showed that the purity of unsaturated hyaluronic acid disaccharide in the obtained hyaluronic acid disaccharide powder was 97.1%, and the endotoxin content was 0.525 EU / mg.

[0034] The results show that by using tryptone instead of soybean peptone in the culture medium and omitting the use of calcium carbonate dispersion and starch complex, the enzyme production decreased compared to Example 6, and the endotoxin content in the obtained hyaluronic acid disaccharide powder increased. This further demonstrates that calcium carbonate dispersion and starch complex can also increase the enzyme production when tryptone is used as a nitrogen source, and can also reduce the endotoxin content in the obtained hyaluronic acid disaccharide powder when tryptone is used as a nitrogen source.

[0035] Application example: The use of unsaturated hyaluronic acid disaccharide in skin repair compositions 1. Cell scratch assay The experiment used L929 cells, which were resuscitated and passaged 3 times, at a rate of 15 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 10 cells / well into 12-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2). Then, horizontal lines were marked on the bottom of the 12-well plates with a marker, the old culture medium was discarded, and the plates were washed with PBS. 500 μL of PBS was added to each well, and a 200 μL pipette tip was used to make a uniform scratch at a constant speed. The plates were then washed three times with PBS. 1 mL of PBS was added to each well, and a photograph was taken to record the initial scratch area. An aqueous solution of 5 mg / mL unsaturated hyaluronic acid disaccharide prepared in Example 1 and an aqueous solution of 5 mg / mL sodium hyaluronate (average molecular weight 300 kDa) were added to the 12-well plates, respectively. The control group used DMEM medium. The plates were incubated in a CO2 incubator (37°C, 5% CO2) for 48 h. The location where the photograph was taken at 0 h was identified, and the final scratch area was recorded. Cell migration rate was calculated using the following formula: Cell migration rate = (initial scratch area - final scratch area) / initial scratch area × 100%; The calculation results show that the cell migration rate corresponding to the unsaturated hyaluronic acid disaccharide aqueous solution is 89.8%, while the cell migration rate corresponding to the sodium hyaluronate (average molecular weight 300kDa) aqueous solution is 72.2%.

[0036] 2. Inhibitory effect on UV radiation-induced free radicals 2.2 g Na2HPO4, 0.3 g NaH2PO4 and 8.5 g NaCl were dissolved in 1000 mL to obtain a phosphate buffer solution with pH 7.2. Using this buffer solution as a solvent, 0.001% Rhodamine B solution (RhB), 0.5% sodium hyaluronate (HA-Na) aqueous solution (average molecular weight 300 kDa) and 0.5% aqueous solution of the HA-LYS composition prepared in Example 1 were prepared.

[0037] Take 1.2 mL of RhB solution, 0.4 mL of PBS solution, and 0.4 mL of HA-Na / HA-LYS aqueous solution (sample solution), mix well. Mix 1.2 mL of RhB solution, 0.4 mL of PBS solution, and 0.4 mL of 0.02 wt% Vc aqueous solution well as a positive control. Mix 1.2 mL of RhB solution, 0.4 mL of PBS solution, and 0.4 mL of distilled water well as a blank control. Examine under ultraviolet light (wavelength 254 nm, light intensity 5 mW / cm²). 2 Irradiation was performed under the following conditions. At 0 h and 1 h of irradiation, the absorbance (A0 and A1) at 555 nm was measured using a microplate reader. Each sample was tested in triplicate. The average change in absorbance at 0 h and 1 h (ΔA) was calculated. The inhibition rate of UV-induced free radicals was calculated using the following formula: The inhibition rate of UV-induced free radicals = (average change in absorbance of blank control - average change in absorbance of sample solution) / average change in absorbance of blank control × 100%; The calculation results show that the inhibition rate of UV-induced free radicals by the unsaturated hyaluronic acid disaccharide aqueous solution is 65.3%, while the inhibition rate of UV-induced free radicals by the sodium hyaluronate (average molecular weight 300kDa) aqueous solution is 21.8%.

[0038] The results above show that, compared with sodium hyaluronate with an average molecular weight of 300 kDa, unsaturated hyaluronic acid disaccharide has stronger skin repair and antioxidant capabilities, indicating that unsaturated hyaluronic acid disaccharide can be used to prepare medical aesthetic products with skin barrier repair, wound healing and anti-aging functions.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention. Any conventional adjustments and equivalent substitutions made by those skilled in the art based on these embodiments should be considered as falling within the protection scope of the present invention.

Claims

1. A method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics, characterized in that, This includes the preparation of a low-pyrogen, high-activity hyaluronic acid lysin, enzymatic hydrolysis, and purification of the hydrolysate; The preparation of low-pyrogen, high-activity hyaluronic acid lyase includes: activation, large-scale culture, fermentation, and purification of hyaluronic acid lyase; The expanded culture involves picking a single colony from the activated strain and inoculating it into a seed culture medium, then shaking and culturing it at a temperature of 35-39℃ and a shaking speed of 150-250 rpm to obtain a seed solution. The seed culture medium consists of: 4-6 g / L glucose, 13-17 g / L soybean peptone, 4-6 g / L yeast extract, 3.7-4.3 g / L calcium carbonate dispersion, 1.8-2.2 g / L dipotassium hydrogen phosphate trihydrate, 0.4-0.6 g / L magnesium sulfate heptahydrate, with the balance being water, and a pH of 6.8-7.

2. The fermentation process involves inoculating the seed culture into the fermentation medium at an inoculation rate of 3.5-4.5%, stirring and fermenting at a temperature of 28-32℃ and a rotation speed of 400-600 rpm, controlling the pH to 5.8-6.2 during fermentation, and monitoring the glycerol concentration in real time. When the glycerol concentration drops below 10 g / L, the glycerol concentration is maintained at 5-15 g / L by fed-batch culture medium, and fermentation is carried out for 28-30 hours to obtain the fermentation broth. The feed culture medium consists of: 310-340 g / L glycerol, 180-210 g / L soybean peptone, 60-70 g / L yeast extract, 35-45 g / L potassium dihydrogen phosphate, 12-18 g / L dipotassium hydrogen phosphate trihydrate, and 18-22 g / L starch complex.

2. The method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics according to claim 1, characterized in that, The activation process involves streaking Bacillus subtilis onto a solid activation medium and activating it at 35-39°C to obtain the activated strain. The Bacillus subtilis strain described is the recombinant strain WB600 / pP43-abnA-HylP constructed according to the methods of Examples 1 and 2 in Chinese Patent CN117866931A, which describes an extracellular secreted hyaluronic acid lyase and its recombinant strain in application. The solid activated culture medium consists of: 9-11 g / L soybean peptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, with the remainder being water, and a pH of 6.9-7.

2.

3. The method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics according to claim 1, characterized in that, In the aforementioned expanded culture, the amount of seed culture medium used is 40-60 mL; The calcium carbonate dispersion is prepared by mixing soluble starch and distilled water, stirring at 80-90°C, adding light calcium carbonate, stirring, and cooling to room temperature to obtain the calcium carbonate dispersion. In the preparation of the calcium carbonate dispersion, the mass ratio of soluble starch, water, and light calcium carbonate is 9-11:280-320:45-55. The light calcium carbonate has a particle size of 1500-2500 mesh.

4. The method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics according to claim 1, characterized in that, In the fermentation process, the amount of fermentation medium used is 0.8-1.2L; The fermentation medium consists of: 60-70 g / L glycerol, 36-42 g / L soybean peptone, 11-15 g / L yeast extract, 13-17 g / L dipotassium hydrogen phosphate trihydrate, 5-7 g / L calcium carbonate dispersion, 1.8-2.2 g / L potassium dihydrogen phosphate, with the balance being water, and a pH of 5.8-6.

2.

5. The method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics according to claim 1, characterized in that, The starch complex is prepared by mixing linear starch, L-tryptophan, and distilled water in a mass ratio of 7.5-8.5:0.9-1.1:140-180, stirring in a boiling water bath until transparent, adding ethanol to a volume fraction of 35-45%, stirring and refluxing at 55-65°C, cooling to room temperature, centrifuging, taking the precipitate, and freeze-drying to obtain the starch complex. In the preparation of the starch complex, the method for preparing amylose is as follows: corn starch and hydrochloric acid aqueous solution are mixed, stirred at 33-37℃, filtered, the filter cake is taken, washed, and dried at room temperature to obtain acid-hydrolyzed corn starch; all the above acid-hydrolyzed corn starch is mixed with water at a mass ratio of 1:8-10, gelatinized, cooled to room temperature, centrifuged, and the supernatant is taken. The supernatant is frozen at -20℃ to -15℃, thawed at room temperature, and when the temperature drops to -5℃ to 0℃, filtered, washed with cold water, the filter cake is taken, and dried at room temperature to obtain amylose; wherein, the concentration of the hydrochloric acid aqueous solution is 2-2.5 mol / L; the mass ratio of corn starch to hydrochloric acid aqueous solution is 1:18-22.

6. The method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics according to claim 1, characterized in that, The purification of hyaluronic acid lyase involves the following steps: First centrifugation of the fermentation broth, collection of the supernatant, filtration of the supernatant using a microfiltration membrane, collection of the filtrate, addition of ammonium sulfate to 18-22%, stirring for 20-40 minutes, addition of perlite to 9-11%, second centrifugation, collection of the supernatant, addition of ammonium sulfate to 68-72%, stirring for 20-40 minutes, standing at 3-5°C for 1.5-2.5 hours, third centrifugation, collection of the precipitate, dissolution using a dissolving buffer to obtain the crude enzyme extract. The crude enzyme extract was concentrated and the medium was changed. The ultrafiltration crude enzyme extract was purified by nickel column affinity chromatography, and the elution peak was collected.

7. The method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics according to claim 1, characterized in that, The enzymatic hydrolysis involves adding purified hyaluronidase to a hyaluronic acid solution and stirring at 35-39°C for 9-11 hours to obtain the hydrolysate. In the enzymatic hydrolysis, the molecular weight of hyaluronic acid in the hyaluronic acid solution is 1000-2000 kDa; The concentration of the hyaluronic acid solution is 1.8-2.2 g / L; The concentration of hyaluronidase in the system is 100-200 U / mL.

8. The method for preparing high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics according to claim 1, characterized in that, The purified enzymatic hydrolysate was subjected to enzyme inactivation, filtered using a microfiltration membrane, and the microfiltrate was subjected to primary ultrafiltration using an ultrafiltration membrane. The ultrafiltrate was then subjected to secondary ultrafiltration using an ultrafiltration membrane. The concentrate was then subjected to electrodialysis for desalting, and the desalted enzymatic hydrolysate was vacuum concentrated and then freeze-dried under vacuum to obtain unsaturated hyaluronic acid disaccharide powder.

9. A high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics prepared by the preparation method according to any one of claims 1-8, wherein the endotoxin content of the high-purity unsaturated hyaluronic acid disaccharide is 0.08-0.1 EU / mg.

10. The application of a high-purity unsaturated hyaluronic acid disaccharide for medical aesthetics prepared by the preparation method according to any one of claims 1-8 in the preparation of medical aesthetic products, wherein the medical aesthetic products have at least one of the following effects: skin barrier repair, wound healing, and anti-aging.