Method for preparing low-molecular-weight hyaluronic acid through high hydrostatic pressure assisted enzymolysis

Abstract

The invention relates to the crossing field of biomedical and food technologies, and provides a method for preparing low-molecular-weight hyaluronic acid through high hydrostatic pressure assisted enzymolysis, which comprises the following steps: dissolving sodium hyaluronate by adopting an acetic acid / sodium acetate buffer system, and adding bovine testis-derived hyaluronidase under the condition that the pH value is 4.5-5.5; high hydrostatic pressure enzymolysis at the temperature of 30-45 DEG C, the static pressure of 50-200 MPa and pulse type pressurization is combined, two to three stages of tangential flow ultrafiltration external circulation separation are cooperatively operated, molecular weight distribution is dynamically regulated and controlled through a segmented interception and backflow strategy between first-stage and second-stage membranes, excessive shearing and chromaticity rising are inhibited while the molecular weight interval target and flux are met, and the separation efficiency is improved. At the end of 50-100 kDa, the area proportion is 55-95% or the distribution is continuously changed for two times as long as lt; and after 5%, deactivating enzyme, desalting and freeze-drying to obtain the product. The polydispersity index of the obtained product is 1.05-1.40, micro impurities and residues are strictly controlled, and the product is suitable for cosmetics, functional food and biomedical materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine and food technology, in particular to a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure assisted enzymatic hydrolysis. BACKGROUND

[0002] Hyaluronic acid, as a polysaccharide with good biocompatibility and water retention, is widely used in cosmetics, functional foods and biomedical materials, etc. Different molecular weight intervals correspond to different application focuses. The low molecular weight interval often focuses on the coordination between permeability, skin feel and processing rheology. In actual production, it is necessary to reduce the viscosity of the system under high solid content conditions to improve the process flux, while maintaining the distribution concentration of the target molecular weight interval and batch consistency, thereby providing controllable basis for subsequent excipients, product stability and user experience. On the other hand, process conditions have a significant impact on the quality risks related to chain segment integrity, color, and potential irritation. Therefore, under the premise of realizing large-scale continuous and amplification feasibility, establishing a process path that takes into account mild reaction, precise segmentation and low impurity output has become the core demand of the industry to improve product performance consistency, processing adaptability and cross-scene applicability.

[0003] At present, the atmospheric pressure enzymolysis or single membrane fractionation path still has deficiencies in distribution convergence speed, fractionation boundary fine control and flux balance, mainly manifested in the difficulty in simultaneously meeting the requirements of high solid content and low viscosity and the rapid achievement of target interval proportion, and in the long-term shear or high shear environment, chain segment is easy to be over broken and color is easy to rise, which affects the product stability and experience. When multiple membrane groups are coordinated, if the backflow strategy and transmembrane pressure difference window are not matched, it will also cause distribution swing and batch consistency to decrease. For example, the Chinese patent with publication number CN117701598A discloses a method for preparing specific molecular weight hyaluronic acid by enzyme method, hyaluronic acid enzyme and application of a new type of hyaluronic acid enzyme, which emphasizes that specific distribution is achieved by optimizing the molecular weight range of membrane cut-off, but there are deficiencies such as slow response of distribution regulation, limited flux under high solid content conditions, and batch consistency depending on empirical adjustment. In addition, the coupling of single segment termination criterion and online monitoring is insufficient, which easily leads to deviation between termination time point and target interval proportion, further aggravating the subsequent purification load and quality fluctuation, and it is urgent to build a more replicable reaction-separation-reflux closed loop control scheme. SUMMARY

[0004] (1) Technical problems solved

[0005] The purpose of the present application is to provide a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure assisted enzymatic hydrolysis, to solve the coupling contradiction of high solid content and low viscosity and target molecular weight interval proportion and consistency, and the conflict between high pressure / high shear and chain segment integrity and low color / low irritation, so as to realize the amplification production and stable quality output.

[0006] (2) Technical solution

[0007] To achieve the above object, the present application provides the following technical solutions:

[0008] A method for preparing low molecular weight hyaluronic acid with high hydrostatic pressure assisted enzymatic hydrolysis, comprising:

[0009] S1 raw material preparation: dissolve sodium hyaluronate in a buffer system composed of acetic acid and sodium acetate, the mass fraction of the obtained solution is 0.3-1.5%, adjust the pH to 4.5-5.5, and add 20000-80000 U·g -1 Sodium hyaluronate is added to bovine testicular hyaluronidase;

[0010] S2 high hydrostatic pressure enzymolysis: reaction at 30-45℃ under 50-200MPa static pressure, the cumulative reaction time under pressure is 1.0-5.0h;

[0011] S3 multi-stage tangential flow ultrafiltration external circulation separation and reflux: after the reaction solution is decompressed to atmospheric pressure, it is separated by external circulation through at least two stages of tangential flow ultrafiltration membrane group, the molecular weight cut-off of the first stage membrane is 110-300kDa, and the molecular weight cut-off of the second stage membrane is 50-100kDa, the feed liquid passing through the first stage membrane and being cut off by the second stage membrane is collected as the target product liquid, part of the cut-off liquid of the first stage membrane is mixed with part of the permeate liquid of the second stage membrane and then returned to step S2 for continuous reaction, and the rest is discarded and pressurized again for the next cycle;

[0012] S4 termination and purification: when the area ratio of the 50-100kDa molecular weight segment in the target product liquid reaches 55-95% or the change amplitude of the molecular weight distribution of the product liquid is less than 5% in two consecutive measurements, the reaction is terminated, the pH of the product liquid is adjusted to 6.5-7.5 for enzyme inactivation and desalination, and then freeze-drying to obtain the product.

[0013] Further, the high hydrostatic pressure is performed in a pulse mode with 6-15 pressurization and decompression cycles, and the time ratio of the pressurization segment to the decompression segment is 1:0.2-2.

[0014] Further, the static pressure is 80-150MPa, the temperature is 35-42℃, and the cumulative reaction time under pressure is 1.5-4.0h.

[0015] Further, the tangential flow ultrafiltration adopts a three-stage membrane group, the molecular weight cut-off of the first stage membrane is 120-200kDa, the molecular weight cut-off of the second stage membrane is 50-100kDa, and the molecular weight cut-off of the third stage membrane is 8-20kDa, and the permeate liquid of the third stage membrane is discharged to remove small molecular impurities.

[0016] Further, the transmembrane pressure difference of the tangential flow ultrafiltration is 0.03-0.20 MPa, and the membrane flux is 5-50 L·m -2 ·h -1 Further, the ratio of the backflow rate to the product flow rate is 1-30.

[0017] Further, the single membrane cycle time is 5-30 min, and the total cycle number is 2-10 times.

[0018] Further, the hyaluronidase exists in an immobilized form and is loaded in a replaceable basket core in a high-hydrostatic-pressure cavity, the immobilized carrier is selected from at least one of cross-linked agarose gel, macroporous silica, cross-linked polystyrene microspheres, and a magnetic nanoparticle composite carrier, the volume median particle size of the immobilized carrier is 100-200 μm, and the mechanical strength retention rate after 10-20 cycles under the condition of 50-200 MPa is 85-100%.

[0019] Further, in the method, the total concentration of acetic acid and sodium acetate in the buffer system in step S1 is 20-50 mmol·L -1 , and the molar ratio of acetic acid to sodium acetate is 0.8-1.5.

[0020] Further, in the method, the dosage of the hyaluronidase in step S1 is 30 000-60 000 U·g -1 of sodium hyaluronate.

[0021] Further, in the method, when a three-stage membrane group is used in step S3, the retentate between the first-stage membrane and the second-stage membrane is used as the target product liquid.

[0022] Further, in the method, the transmembrane pressure difference of the tangential flow ultrafiltration in step S3 is 0.05-0.15 MPa, and the membrane flux is 10-30 L·m -2 ·h -1 .

[0023] Further, in the method, the ratio of the backflow rate to the product flow rate in step S3 is 2-20.

[0024] Further, in the method, the single membrane cycle time in step S3 is 10-20 min, and the total cycle number is 3-8 times.

[0025] Further, in the method, the immobilized carrier in step S2 is cross-linked agarose gel or a magnetic nanoparticle composite carrier, and the volume median particle size is 120-180 μm.

[0026] Further, in the method, the enzyme inactivation in step S4 is performed by heat treatment at 70-90℃ for 10-20 min, and desalination is performed by dialysis with a molecular weight cut-off of 3-10 kDa.

[0027] As one concept of the present application, the present application adopts an enzyme hydrolysis-separation coupling design with superimposed high hydrostatic pressure pulse pressurization at a mild temperature, mainly for enhancing the distributed convergence efficiency and flux synergy capability of the process under high solid content conditions. The configuration accessibility and local mass transfer of the substrate system are improved by 50-200MPa static pressure, while the risk of excessive chain segment rupture caused by continuous high shear is avoided; two to three stages of tangential flow ultrafiltration external circulation realizes the segmented enrichment of the target interval between the first and second stages, and dynamically balances the distribution evolution and membrane surface load with the backflow ratio of part of the first-stage cut-off and second-stage permeation, to inhibit the quality fluctuations caused by critical shear or concentration polarization; termination is triggered by a proportion threshold or a twice-measurement change threshold, to reduce the probability of over-processing, and to take into account the low color, low irritation related properties and batch consistency of the product. This path improves the convergence speed and amplification adaptability on the premise of ensuring chain segment integrity, realizes a steady state balance between the target interval proportion, rheological adaptation and subsequent purification pressure, and is suitable for the stable supply of cosmetics, functional foods and biomedical materials.

[0028] The present application also discloses a low molecular weight hyaluronic acid product prepared by the above method, which satisfies: the area proportion of the 50-100kDa molecular weight segment is 55-95%, the polydispersity index is 1.05-1.40, the area proportion of the less than 10kDa molecular weight segment is 0-5%, the ash content is not more than 0.5%, the protein content is not more than 0.2%, the nucleic acid content is not more than 0.1%, the contents of lead, arsenic, cadmium and mercury are each not more than 1ppm, the residual amounts of methanol and ethanol are each not more than 10ppm, and the endotoxin content is not more than 0.5 units·mg -1 ; the area proportion of the 50-100kDa molecular weight segment is 65-90%, and the polydispersity index is 1.08-1.30.

[0029] Further, the use of the low molecular weight hyaluronic acid in the preparation of cosmetics, functional foods or biomedical materials.

[0030] As another aspect of this invention, the product window created by this invention achieves a balance between the high proportion and low polydispersity in the 50–100 kDa range through precise distribution control and purification constraints. This balances the integrity of the chain segment structure with the requirements of application-side permeability, rheology, and skin feel. By controlling the generation and removal of segments smaller than 10 kDa, the risk of potential irritation and color accumulation is reduced. Simultaneously, strict upper limits are set for indicators such as ash content, protein, nucleic acid, heavy metals, residual solvents, and endotoxins to support the quality consistency requirements of multiple scenarios, including cosmetics, functional foods, and biomedical materials. The product's characteristics stem from the gentle enzymatic hydrolysis enhanced by high hydrostatic pressure, and the process control of segmented external circulation and reflux coupling, ensuring that the distribution remains concentrated and the output is low in impurities during industrial scale-up, providing empirical evidence for stable mass production.

[0031] Under the synergistic conditions of an acetate / sodium acetate buffer system and bovine testicular hyaluronidase, the accessibility of substrate segments and local conformational relaxation are promoted within a mild range of 35–42℃ using a high hydrostatic pressure of 50–200 MPa, reducing the dependence on high shear in the reaction pathway and thus limiting excessive fragmentation of non-target regions. Two to three stages of tangential flow ultrafiltration external circulation establish segmented enrichment using the molecular weight cutoff windows of the first and second stages of the membrane (e.g., 120–200 kDa and 50–100 kDa). The residence distribution of the refluxing fluid from the first stage cutoff and the second stage permeate in the membrane surface and cavity is utilized to concentrate the target region and increase the effective flux. At the same time, by setting termination criteria (e.g., the area ratio of 50–100 kDa and the threshold of change between two measurements), the reaction is terminated in a timely manner to avoid the accumulation of segments in excessively low molecular weight segments. Combined with subsequent purification processes such as enzyme inactivation, desalting, and lyophilization, polydispersity is reduced and micro-impurities and residues are controlled. This overall approach focuses on mild reaction, selective segmentation, and reflux closed loop to strengthen the positive linkage between distribution control and quality stability.

[0032] (3) Beneficial technical effects

[0033] 1. Improved distribution accuracy and consistency: Through the synergistic effect of gentle enzymatic hydrolysis enhanced by high hydrostatic pressure and segmented reflux of two to three stages of tangential flow ultrafiltration external circulation, rapid convergence and batch-to-batch stability in the 50–100 kDa range are achieved, reducing dependence on empirical adjustments, matching termination criteria with online monitoring, and reducing distribution shifts caused by overprocessing.

[0034] 2. Dual optimization of flux and quality: Under the requirements of high solids content and low viscosity, by pulse pressurization and optimization of transmembrane pressure difference / reflux ratio window, the membrane flux and membrane surface load are balanced, the concentration polarization and chain segment over-fracture are suppressed, the color and potential irritation-related risks are reduced, and the efficiency of large-scale production is improved.

[0035] 3. Polydispersity and microheterogeneity control: termination triggered by distribution threshold combined with enzyme inactivation and desalting, polydispersity index 1.05-1.40, while protein, nucleic acid, heavy metal and residual solvent quality indicators are limited to strict upper limits, supporting cross-scene adaptation of cosmetics, functional foods and biomedical materials.

[0036] 4. Scalability: fixed enzyme basket core arrangement and cycle strength retention design, combined with segmented and backflow strategy of external circulation membrane group, make key window parameters replicable, reduce quality fluctuations and energy costs in the process of scaling up, and improve industrial feasibility. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Pressure-temperature-time curve of the process trajectory of the pulse high hydrostatic pressure enzymatic hydrolysis equipment of embodiment 1 of the present application.

[0038] Figure 2 Molecular weight distribution transmission mode diagram of the gel permeation chromatography-multiple angle laser light scattering / differential refractive distribution overlay of embodiment 1 of the present application.

[0039] Figure 3 Polydispersity index box plot and gel permeation chromatography-multiple angle laser light scattering detection result summary diagram of embodiment 1 of the present application.

[0040] Figure 4 Effect of static pressure on 50-100 kDa molecular weight segment proportion and yield of the present application.

[0041] Figure 5 Effect of sodium hyaluronate solution concentration on 50-100 kDa molecular weight segment proportion and viscosity of the present application.

[0042] Figure 6 Effect of pH on 50-100 kDa molecular weight segment proportion and polydispersity index of the present application.

[0043] Figure 7 Effect of backflow flow / product flow ratio on 50-100 kDa molecular weight segment proportion and yield of the present application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the drawings of the embodiments of the present application.

[0045] Embodiment 1

[0046] The present embodiment provides a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure assisted enzymatic hydrolysis, comprising the following steps:

[0047] Step S1 Preparation of raw materials

[0048] Sodium hyaluronate (molecular weight 1.0-1.8 MDa, purity ≥95%, food grade, commercially available) was dissolved in a buffer system composed of acetic acid (analytical pure, purity ≥99.5%, commercially available) and sodium acetate (analytical pure, purity ≥99%, commercially available). The total concentration of acetic acid and sodium acetate in the buffer system of this example was 35 mmol·L -1 , and the molar ratio of acetic acid to sodium acetate was 1.1. A 0.8% (mass fraction) sodium hyaluronate solution was prepared, and the pH was adjusted to 5.0±0.1 using 1 mol·L -1 acetic acid or 1 mol·L -1 sodium hydroxide solution. The hyaluronidase from bovine testes (enzyme activity ≥50 000 U·mg -1 of protein, lyophilized powder, commercially available) was added at a ratio of 40 000 U·g -1 of sodium hyaluronate, and the mixture was stirred thoroughly to obtain an enzymatic reaction solution.

[0049] Step S2 High hydrostatic pressure enzymolysis

[0050] The enzymatic reaction solution obtained in step S1 was transferred to the reaction cavity of a high hydrostatic pressure treatment device (cavity volume 500 mL, sealed with PA / PE co-extruded film, air bubbles were evacuated). The high hydrostatic pressure enzymolysis in this example was carried out using a pulse pressure mode, with a reaction temperature of 38℃ (controlled by a jacketed circulating constant temperature water bath), a static pressure of 100 MPa, 10 pulse cycles, a time ratio of pressure segment to decompression segment of 1:1, a pressure segment time of 10 min and a decompression segment time of 10 min in a single cycle, and a cumulative reaction time of 100 min (1.67 h) in the pressure state. The pressure medium was deionized water, the pressure increasing rate was about 200 MPa·min -1 , and the pressure decreasing rate was ≤100 MPa·min -1 .

[0051] Step S3 Multistage tangential flow ultrafiltration external circulation separation and backflow

[0052] This example used a three-stage tangential flow ultrafiltration membrane group for external circulation separation, and the membrane material was polyether sulfone (PES) hollow fiber membrane with membrane areas of 0.1 m² (first stage), 0.08 m² (second stage), and 0.05 m² (third stage).

[0053] The reaction solution after step S2 was decompressed to atmospheric pressure and subjected to tangential flow ultrafiltration separation, with the following specific operations:

[0054] (1) First stage ultrafiltration: the reaction solution is pumped into the first stage membrane group with a molecular weight cut-off of 150 kDa, the transmembrane pressure difference is set to 0.10 MPa, the membrane flux is 20 L·m -2 ·h -1 , the circulation flow rate is 1.5 m·s -1 , and the temperature is controlled at 25±2℃. After the retentate of the first stage membrane (high molecular weight component, >150 kDa) is collected, 70% of the volume is discarded, and the remaining 30% of the volume is returned to step S2 to continue the next cycle of high hydrostatic pressure enzymolysis. The permeate of the first stage membrane (molecular weight ≤150 kDa) enters the second stage ultrafiltration.

[0055] (2) Second stage ultrafiltration: the permeate of the first stage membrane is pumped into the second stage membrane group with a molecular weight cut-off of 75 kDa, the transmembrane pressure difference is 0.10 MPa, the membrane flux is 20 L·m -2 ·h -1 , the circulation flow rate is 1.5 m·s -1 . The retentate of the second stage membrane (molecular weight 75-150 kDa, containing the target product) is collected as the target product feed liquid. After 60% of the volume of the permeate of the second stage membrane is mixed with 30% of the volume of the retentate of the first stage membrane, it is returned to step S2 to continue the enzymolysis, and the remaining 40% of the permeate enters the third stage ultrafiltration.

[0056] (3) Third stage ultrafiltration: part of the permeate of the second stage membrane (40% of the volume) is pumped into the third stage membrane group with a molecular weight cut-off of 10 kDa, the transmembrane pressure difference is 0.10 MPa, and the membrane flux is 20 L·m -2 ·h -1 . The retentate of the third stage membrane (molecular weight 10-75 kDa) is returned and combined with the retentate of the second stage, and the permeate of the third stage membrane (molecular weight <10 kDa, which is small molecular oligosaccharides, enzymes and impurities) is discharged and discarded.

[0057] The ratio of the backflow volume (total volume of the feed liquid returned to S2) to the product volume (volume of the retentate of the second stage membrane) in this example is 10:1, the single membrane circulation time is 15 min, and the total circulation number is 5 times.

[0058] Step S4 termination and purification

[0059] The molecular weight distribution of the target product feed liquid is determined by high performance liquid chromatography (HPLC) equipped with a multi-angle laser light scattering detector (MALLS) and a differential refractometer detector (RI). When the area ratio of the 50-100 kDa molecular weight segment reaches 72.5% and the change amplitude of two consecutive measurements (with an interval of 1 cycle) is less than 3%, it is determined that the reaction is terminated.

[0060] The target product feed liquid is treated with 1 mol·L -1The pH was adjusted to 7.0 ± 0.2 with sodium hydroxide solution, and then the following treatment was performed:

[0061] (1) Enzyme inactivation: The feed solution was heated in a water bath at 80°C for 15 min to inactivate the residual enzyme activity, and then cooled to room temperature.

[0062] (2) Desalination: The feed solution was transferred to a dialysis bag with a molecular weight cut-off of 5 kDa (regenerated cellulose membrane, commercially available) and dialyzed in deionized water for 48 h, with the dialysis solution being replaced every 8 h (the volume of the dialysis solution was 20 times that of the feed solution), until the conductivity of the dialysis solution was <10 μS·cm -1 .

[0063] (3) Freeze-drying: The dialyzed feed solution was pre-frozen at -40°C for 12 h, and then dried in a freeze dryer (primary drying stage: -30°C, vacuum degree <10 Pa, 24 h; secondary drying stage: 25°C, vacuum degree <5 Pa, 12 h), to obtain a white to light yellow loose powder of low molecular weight hyaluronic acid product, with a yield of about 78% of the initial sodium hyaluronate mass.

[0064] Features of Example 1

[0065] This example uses a moderate parameter design, with mild and stable reaction conditions and high process reliability. The pulse pressure is designed with a 1:1 equal period, which is beneficial to the recovery of enzyme activity conformation during the pressure release stage, while avoiding excessive degradation of the substrate. The three-stage membrane set cooperates with a reasonable reflux strategy, which not only ensures the enrichment and purification of the target molecular weight segment, but also maximizes the utilization rate of raw materials through external circulation reflux. This example is suitable for pilot-scale continuous production, with easy-to-control process parameters, a narrow product molecular weight distribution (polydispersity index about 1.18), and a stable 50-100 kDa segment ratio of 70-75%, making it suitable as a cosmetic raw material (such as moisturizing serum, facial mask essence) or functional food additive.

[0066] Example 2

[0067] This example provides a method for preparing low molecular weight hyaluronic acid with high hydrostatic pressure assisted enzymatic hydrolysis, comprising the following steps:

[0068] Step S1 Preparation of raw materials

[0069] Dissolve sodium hyaluronate (molecular weight 1.2-2.0 MDa, purity ≥95%, food grade, commercially available) in a buffer system composed of acetic acid (analytical pure, purity ≥99.5%, commercially available) and sodium acetate (analytical pure, purity ≥99%, commercially available), and the total concentration of acetic acid and sodium acetate in the buffer system of this example is 25 mmol·L -1, the molar ratio of acetic acid to sodium acetate was 0.8. A 0.5% (mass fraction) sodium hyaluronate solution was prepared, and 1 mol·L -1 acetic acid or 1 mol·L -1 The pH was adjusted to 4.7±0.1 using a sodium hydroxide solution. The enzyme was added at a dosage of 30 000 U·g -1 The sodium hyaluronate was added to a bovine testis-derived hyaluronidase (enzyme activity ≥ 50 000 U·mg -1 protein, lyophilized powder, commercially available) at room temperature, and stirred for 10 min to obtain an enzymatic reaction solution.

[0070] Step S2: High hydrostatic pressure enzymolysis

[0071] The enzymatic reaction solution obtained in step S1 was transferred to the reaction cavity of a high hydrostatic pressure treatment device (cavity volume 500 mL, sealed with a PA / PE co-extruded film, and air bubbles were evacuated). The high hydrostatic pressure enzymolysis in this example was performed using a pulse pressure mode, with a reaction temperature of 35°C (controlled by a jacket circulating constant temperature water bath), a static pressure of 80 MPa, 8 pulse cycles, a time ratio of the pressurization stage to the depressurization stage of 1:0.5, a pressurization stage time of 10 min and a depressurization stage time of 5 min in a single cycle, and a cumulative reaction time in the pressurized state of 80 min (1.33 h). The depressurization time was extended to 10 min at the end of the 4th cycle and the 8th cycle to fully release the protein conformational stress. The pressurization medium was deionized water, the pressurization rate was about 180 MPa·min -1 , and the depressurization rate was ≤ 90 MPa·min -1 .

[0072] Step S3: Multistage tangential flow ultrafiltration external circulation separation and backflow

[0073] This example used a three-stage tangential flow ultrafiltration membrane group for external circulation separation, and the membrane material was polyether sulfone (PES) hollow fiber membrane with membrane areas of 0.12 m² (first stage), 0.09 m² (second stage), and 0.06 m² (third stage).

[0074] The reaction solution after step S2 was depressurized to atmospheric pressure and subjected to tangential flow ultrafiltration separation, with the following specific operations:

[0075] (1) First stage ultrafiltration: The reaction solution was pumped into the first stage membrane group with a molecular weight cut-off of 120 kDa, and the transmembrane pressure difference was set to 0.08 MPa, the membrane flux was 15 L·m -2 ·h -1 , and the circulation flow rate was 1.2 m·s -1, and the temperature was controlled at 25±2℃. After the retentate of the first stage membrane (high molecular weight component, >120 kDa) was collected, 65% of the volume was discarded, and the remaining 35% of the volume was returned to step S2 for the next cycle of high hydrostatic pressure enzymolysis. The permeate of the first stage membrane (molecular weight ≤120 kDa) entered the second stage ultrafiltration.

[0076] (2) Second stage ultrafiltration: the permeate of the first stage membrane was pumped into a second stage membrane group with a molecular weight cut-off of 60 kDa, the transmembrane pressure difference was 0.08 MPa, and the membrane flux was 15 L·m -2 ·h -1 , and the circulation flow rate was 1.2 m·s -1 . The retentate of the second stage membrane (molecular weight 60-120 kDa, containing the target product) was collected as the target product liquid. After 50% of the volume of the permeate of the second stage membrane was mixed with 35% of the volume of the retentate of the first stage membrane, it was returned to step S2 for continuing the enzymolysis, and the remaining 50% of the permeate entered the third stage ultrafiltration.

[0077] (3) Third stage ultrafiltration: part of the permeate of the second stage membrane (50% of the volume) was pumped into a third stage membrane group with a molecular weight cut-off of 8 kDa, the transmembrane pressure difference was 0.08 MPa, and the membrane flux was 15 L·m -2 ·h -1 . The retentate of the third stage membrane (molecular weight 8-60 kDa) was returned to be combined with the retentate of the second stage, and the permeate of the third stage membrane (molecular weight <8 kDa, being small molecular oligosaccharides, enzymes and impurities) was discharged and discarded.

[0078] The ratio of the backflow volume (total volume of the liquid returned to S2) to the product volume (volume of the retentate of the second stage membrane) in this embodiment was 5:1, the single membrane cycle time was 12 min, and the total cycle number was 4 times.

[0079] Step S4: termination and purification

[0080] The molecular weight distribution of the target product liquid was determined by high performance liquid chromatography (HPLC) equipped with a multi-angle laser light scattering detector (MALLS) and a differential refractometer detector (RI). When the area ratio of the 50-100 kDa molecular weight segment reached 68.2% and the change amplitude of two consecutive measurements (with an interval of 1 cycle) was less than 4%, it was determined that the reaction was terminated.

[0081] The target product liquid was adjusted to pH 7.0±0.2 by using a 1 mol·L -1 sodium hydroxide solution, and then the following treatments were performed:

[0082] (1) Enzyme inactivation: the liquid was heated in a 75℃ water bath for 12 min to inactivate the residual enzyme activity, and then cooled to room temperature.

[0083] (2) Desalination: The feed solution was transferred into a dialysis bag with a molecular weight cut-off of 5 kDa (regenerated cellulose membrane, commercially available), and dialyzed in deionized water for 44 h, with the dialysis solution being replaced every 8 h (the volume of the dialysis solution was 18 times that of the feed solution), until the conductivity of the dialysis solution was <10 μS·cm -1 .

[0084] (3) Freeze-drying: The dialyzed feed solution was pre-frozen at -40℃ for 12 h, and then dried in a freeze dryer (primary drying stage: -30℃, vacuum degree <10 Pa, 24 h; secondary drying stage: 25℃, vacuum degree <5 Pa, 12 h), to obtain a white, loose powder of low-molecular-weight hyaluronic acid product, with a yield of about 74% of the mass of the initial sodium hyaluronate.

[0085] Features of Example 2

[0086] This example uses a lower parameter design to verify the feasibility of the method under mild conditions. The combination of lower static pressure (80 MPa), temperature (35℃) and enzyme dosage (30 000 U·g -1 ) is suitable for enzyme preparations that are sensitive to pressure or applications that require controlled degradation rates. The shorter cumulative pressure time (1.33 h) and asymmetric pulse cycle (1:0.5) design can reduce equipment energy consumption and extend the effective service life of the enzyme. The use of lower molecular weight cut-off in the three membrane groups (120 kDa in the first stage and 8 kDa in the third stage) can more accurately separate the target molecular weight range, with the 50-100 kDa fraction accounting for 66-70% of the product, a polydispersity index of about 1.22, and lower content of small molecule impurities (<3% of the <10 kDa fraction). This example is suitable for the preparation of small-batch high-purity products, such as precursor raw materials for injection-grade medical sodium hyaluronate or high-end cosmetic active ingredients.

[0087] Example 3

[0088] This example provides a method for preparing low-molecular-weight hyaluronic acid using high hydrostatic pressure-assisted enzymatic hydrolysis with immobilized enzymes, comprising the following steps:

[0089] Step S1 Preparation of raw materials

[0090] Sodium hyaluronate (molecular weight 0.8-1.5 MDa, purity ≥95%, food grade, commercially available) was dissolved in a buffer system composed of acetic acid (analytical pure, purity ≥99.5%, commercially available) and sodium acetate (analytical pure, purity ≥99%, commercially available), with the total concentration of acetic acid and sodium acetate in the buffer system of this example being 42 mmol·L -1 , and the molar ratio of acetic acid to sodium acetate being 1.5. A 1.2% (w / v) sodium hyaluronate solution was prepared, and 1 mol·L -1 acetic acid or 1 mol·L-1 The pH was adjusted to 5.3 ± 0.1 with sodium hydroxide solution. The immobilized hyaluronidase used in this example was prepared by covalent coupling method using cross-linked sepharose (6% agarose, apparent density 1.15 g mL -1 , pore size range 10-40 nm, commercially available) with a volume median particle size of 150 μm (measured by laser particle sizer) and a mechanical strength retention rate of 92% after 15 cycles at 50-200 MPa (evaluated by the rate of carrier breakage). The immobilized enzyme was prepared by covalent coupling method: 100 g of cross-linked sepharose (containing water) was activated with epichlorohydrin (10 mL) in a phosphate buffer (50 mmol L -1 , pH 8.0) for 2 h, and after thorough washing, a hyaluronidase solution (total enzyme activity 3 million U, dissolved in 20 mL of phosphate buffer, pH 7.5) was added, and the coupling was carried out at 4°C for 16 h with slow stirring, with a coupling rate of about 75% and a retention rate of immobilized enzyme activity of about 60%, obtaining an immobilized enzyme carrier (enzyme activity about 1.8 million U). The immobilized enzyme carrier was loaded into a replaceable stainless steel basket core (mesh 100 μm) in the high hydrostatic pressure cavity, with a basket core volume of 80 mL and an equivalent enzyme concentration of about 55 000 U g -1 Sodium hyaluronate.

[0091] Step S2 High hydrostatic pressure enzymolysis

[0092] The sodium hyaluronate solution (without free enzyme) obtained in step S1 was pumped into the high hydrostatic pressure treatment device reaction cavity containing the immobilized enzyme basket core (total cavity volume 600 mL, substrate solution volume 500 mL). The high hydrostatic pressure enzymolysis in this example was carried out in a pulse pressurization mode, with a reaction temperature of 42°C (controlled by a jacketed circulating constant temperature water bath), a static pressure of 140 MPa, a pulse cycle number of 12, a time ratio of pressurization segment to depressurization segment of 1:1.5, a pressurization segment time of 12 min and a depressurization segment time of 18 min in a single cycle, and a cumulative reaction time in the pressurized state of 144 min (2.4 h). The pressurization medium was deionized water, with a pressurization rate of about 220 MPa min -1 and a depressurization rate of ≤110 MPa min -1 . During each depressurization segment, slight stirring was carried out by a built-in micro magnetic stirring paddle (100 rpm) to promote contact between the substrate and the immobilized enzyme.

[0093] Step S3 Multistage tangential flow ultrafiltration external circulation separation and reflux

[0094] The present embodiment adopts three-stage tangential flow ultrafiltration membrane group for external circulation separation, the membrane material is polyether sulfone (PES) hollow fiber membrane, and the membrane area is 0.15 m² (first stage), 0.12 m² (second stage) and 0.08 m² (third stage) respectively.

[0095] The reaction liquid (not containing the immobilized enzyme basket core) after step S2 reaction is unloaded to atmospheric pressure and subjected to tangential flow ultrafiltration separation, and the specific operation is as follows:

[0096] (1) First-stage ultrafiltration: the reaction liquid is pumped into the first-stage membrane group with a molecular weight cut-off of 180 kDa, the transmembrane pressure difference is set to 0.13 MPa, the membrane flux is 25 L·m -2 ·h -1 , the circulation flow rate is 1.8 m·s -1 , and the temperature is controlled at 25±2℃. After the retentate (high molecular weight component, >180 kDa) of the first-stage membrane is collected, 75% of the volume is discarded, and the remaining 25% of the volume is returned to step S2 to continue the next cycle of high hydrostatic pressure enzymolysis (pumped back to the cavity containing the immobilized enzyme). The permeate (molecular weight ≤180 kDa) of the first-stage membrane enters the second-stage ultrafiltration.

[0097] (2) Second-stage ultrafiltration: the permeate of the first-stage membrane is pumped into the second-stage membrane group with a molecular weight cut-off of 85 kDa, the transmembrane pressure difference is 0.13 MPa, the membrane flux is 25 L·m -2 ·h -1 , and the circulation flow rate is 1.8 m·s -1 . The retentate (molecular weight 85-180 kDa, containing the target product) of the second-stage membrane is collected as the target product liquid. After 70% of the volume of the permeate (molecular weight ≤85 kDa) of the second-stage membrane and 25% of the volume of the retentate of the first-stage membrane are mixed, they are returned to step S2 to continue the enzymolysis, and the remaining 30% of the permeate enters the third-stage ultrafiltration.

[0098] (3) Third-stage ultrafiltration: part of the permeate (30% of the volume) of the second-stage membrane is pumped into the third-stage membrane group with a molecular weight cut-off of 15 kDa, the transmembrane pressure difference is 0.13 MPa, and the membrane flux is 25 L·m -2 ·h -1 . The retentate (molecular weight 15-85 kDa) of the third-stage membrane is returned and combined with the retentate of the second-stage, and the permeate (molecular weight <15 kDa, which is small molecular oligosaccharides and impurities, and does not contain free enzymes) of the third-stage membrane is discharged and discarded.

[0099] The ratio of the backflow volume (total volume of the liquid returned to S2) to the product volume (volume of the retentate of the second-stage membrane) of the present embodiment is 15:1, the single membrane circulation time is 18 min, and the total circulation number is 6.

[0100] Step S4 Termination and Purification

[0101] The molecular weight distribution of the target product solution was determined using high-performance liquid chromatography (HPLC) equipped with a multi-angle laser light scattering detector (MALLS) and a differential refractive index detector (RI). The reaction was terminated when the area of ​​the 50-100 kDa molecular weight range reached 76.8% and the change between two consecutive measurements (with a one-cycle interval) was less than 3%.

[0102] The target product solution was treated with 1 mol·L⁻¹ -1 The pH was adjusted to 7.2 ± 0.2 with sodium hydroxide solution, followed by the following treatment (since immobilized enzymes are used, there are no free enzymes in the feed solution, and the enzyme inactivation step can be simplified):

[0103] (1) Enzyme inactivation: Heat the solution in an 85°C water bath for 18 min to completely inactivate any trace enzyme activity that may dissociate, and then cool to room temperature.

[0104] (2) Desalination: Transfer the feed solution to a dialysis bag (regenerated cellulose membrane, commercially available) with a molecular weight cutoff of 8 kDa, and dialyze in deionized water for 50 h, changing the dialysate every 8 h (the dialysate volume is 22 times the feed solution volume) until the dialysate conductivity is <8 μS·cm -1 .

[0105] (3) Freeze-drying: The dialyzed solution was pre-frozen at -40℃ for 14 h, and then dried in a freeze dryer (initial drying stage: -30℃, vacuum degree <10 Pa, 26 h; desorption drying stage: 25℃, vacuum degree <5 Pa, 14 h) to obtain a white loose powder of low molecular weight hyaluronic acid product with a yield of about 81% of the initial sodium hyaluronate mass (since the immobilized enzyme can be reused, the overall raw material utilization rate is higher).

[0106] Features of Example 3

[0107] The present embodiment adopts a high parameter design and introduces immobilized enzyme technology for the first time, verifying the adaptability and industrialization potential of the method under intensive conditions. Higher sodium hyaluronate concentration (1.2%) can improve single batch productivity, higher temperature (42°C), static pressure (140 MPa) and asymmetric pulse cycle (1:1.5) design accelerate the enzymatic reaction, shorten the total reaction time to about 2.4 h cumulative pressure time (total reaction period about 6 h including pressure relief). The mechanical strength retention rate of immobilized enzyme (cross-linked Sepharose gel carrier, volume median particle size 150 μm) is 92% after 15 cycles at 50-200 MPa high pressure, which proves that the carrier has excellent pressure resistance and can be reused for 8-10 batches, greatly reducing the enzyme cost. The three-stage membrane group adopts a high molecular weight cut-off (180 kDa for the first stage and 15 kDa for the third stage), combined with a high reflux ratio (15:1) and a longer cycle time (18 min x 6 times), effectively enriching the target molecular weight segment, with the product 50-100 kDa segment accounting for 75-80%, the polydispersity index about 1.15 (narrower molecular weight distribution), and the small molecule impurities <10 kDa segment accounting for <2%. The present embodiment is suitable for large-scale continuous production, the immobilized enzyme system is easy to automate and online monitor, and the product quality is stable, which is suitable as raw material for medical devices (such as joint cavity injection solution, ophthalmic viscoelastic agent) or high-end cosmetics (such as anti-aging essence).

[0108] Embodiment 4

[0109] The present embodiment provides a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure assisted enzymatic hydrolysis using a magnetic nanoparticle composite carrier immobilized enzyme, comprising the following steps:

[0110] Step S1 Raw material preparation

[0111] Dissolve sodium hyaluronate (molecular weight 1.5-2.2 MDa, purity ≥98%, pharmaceutical grade, commercially available) in a buffer system composed of acetic acid (analytical pure, purity ≥99.5%, commercially available) and sodium acetate (analytical pure, purity ≥99%, commercially available), the total concentration of acetic acid and sodium acetate in the buffer system of the present embodiment is 48 mmol·L -1 , the molar ratio of acetic acid to sodium acetate is 1.4. A sodium hyaluronate solution with a mass fraction of 1.5% (maximum concentration, first boundary verification) is prepared, using 1 mol·L -1 acetic acid or 1 mol·L -1Sodium hydroxide solution was used to adjust the pH to 5.5 ± 0.05 (maximum pH). In this example, hyaluronidase was immobilized on a magnetic nanoparticle composite carrier, which was a Fe3O4@SiO2core-shell structure magnetic nanoparticle (core Fe3O4particle size about 50 nm, SiO2shell thickness about 15 nm, modified by aminosilane, commercially available), with a volume median particle size of 180 μm (aggregate particle size, measured by a laser particle size analyzer), and a mechanical strength retention rate of 88% after 18 cycles under 50-200 MPa (evaluated by carrier breakage rate). The immobilized enzyme was prepared by covalent coupling: 120 g of magnetic nanoparticle composite carrier was activated with glutaraldehyde (8 mL, 2.5% v / v) in a phosphate buffer (50 mmol·L -1 ) at pH 7.5 for 1.5 h, and after thorough washing, hyaluronidase solution (total enzyme activity 3.6 million U, dissolved in 25 mL of phosphate buffer at pH 7.0) was added, and the coupling was carried out at 4°C for 18 h with slow stirring, with a coupling rate of about 70%, a retention rate of immobilized enzyme activity of about 58%, and an immobilized enzyme carrier (enzyme activity about 2.1 million U) obtained. -1 Sodium hyaluronate.

[0112] Step S2 High hydrostatic pressure enzymolysis

[0113] The sodium hyaluronate solution (without free enzyme) obtained in step S1 was pumped into the high hydrostatic pressure treatment device reaction cavity containing the immobilized enzyme basket core (total cavity volume 700 mL, substrate solution volume 600 mL). In this example, the high hydrostatic pressure enzymolysis was carried out in a pulse pressurization mode, with a reaction temperature of 40°C, a static pressure of 150 MPa, 6 pulse cycles (minimum cycle number), a time ratio of pressurization segment to depressurization segment of 1:0.3, a pressurization segment time of 30 min and a depressurization segment time of 9 min in a single cycle, and a cumulative reaction time in the pressurized state of 180 min (3.0 h). The pressurization medium was deionized water, with a pressurization rate of about 240 MPa·min -1 and a depressurization rate of ≤120 MPa·min -1 . During each depressurization segment, a rotating magnetic field (frequency 5 Hz, magnetic field strength 0.1 T) was generated by an external electromagnetic stirrer to drive the magnetic carrier to rotate slightly, thereby enhancing the mass transfer efficiency of the substrate and the immobilized enzyme.

[0114] Step S3 Multistage tangential flow ultrafiltration external circulation separation and reflux

[0115] This example adopts a simplified two-stage tangential flow ultrafiltration membrane group for external circulation separation (verifying the boundary condition that the three-stage membrane group can be simplified to two stages), and the membrane material is polyether sulfone (PES) hollow fiber membrane, with membrane areas of 0.18 m² (first stage) and 0.14 m² (second stage).

[0116] The reaction solution after step S2 reaction is depressurized to atmospheric pressure (the immobilized enzyme carrier is quickly separated and recovered to the basket core by an external magnetic separator, with a recovery rate of >98%), and tangential flow ultrafiltration separation is performed, with the specific operation as follows:

[0117] (1) First-stage ultrafiltration: the reaction solution is pumped into the first-stage membrane group with a molecular weight cutoff of 200 kDa, the transmembrane pressure difference is set to 0.15 MPa, the membrane flux is 30 L·m -2 ·h -1 , the circulation flow rate is 2.0 m·s -1 , and the temperature is controlled at 25±2℃. After the retentate of the first-stage membrane (high molecular weight component, >200 kDa) is collected, 80% of the volume is discarded, and the remaining 20% of the volume is returned to step S2 to continue the next cycle of high hydrostatic pressure enzymolysis (pumped back to the cavity containing the immobilized enzyme, at which time the immobilized enzyme has been pre-recovered to the basket core by magnetic separation). The permeate of the first-stage membrane (molecular weight ≤200 kDa) enters the second-stage ultrafiltration.

[0118] (2) Second-stage ultrafiltration: the permeate of the first-stage membrane is pumped into the second-stage membrane group with a molecular weight cutoff of 50 kDa (the smallest molecular weight cutoff), the transmembrane pressure difference is 0.15 MPa, the membrane flux is 30 L·m -2 ·h -1 , and the circulation flow rate is 2.0 m·s -1 . The retentate of the second-stage membrane (molecular weight 50-200 kDa, containing the target product) is collected as the target product feed liquid. After 80% of the volume of the permeate of the second-stage membrane (molecular weight ≤50 kDa, which is small molecular oligosaccharides and impurities) is mixed with 20% of the volume of the retentate of the first-stage membrane, it is returned to step S2 for further enzymolysis, and the remaining 20% of the permeate is directly discarded (simplified process, without third-stage ultrafiltration).

[0119] The ratio of the backflow volume (total volume of the feed liquid returned to S2) to the product volume (volume of the retentate of the second-stage membrane) in this example is 20:1, the single membrane circulation time is 20 min, and the total circulation number is 8.

[0120] Step S4: termination and purification

[0121] The molecular weight distribution of the target product solution was determined by high performance liquid chromatography (HPLC) equipped with a multi-angle laser light scattering detector (MALLS) and a refractive index detector (RI). When the area ratio of the 50-100 kDa molecular weight segment reached 80.5% and the change amplitude of two consecutive measurements (with an interval of 1 cycle) was less than 2.5%, it was determined that the reaction was terminated.

[0122] The target product solution was adjusted to pH 7.3±0.2 with 1 mol·L -1 The pH was adjusted to 7.3±0.2 with a sodium hydroxide solution, and then the following treatments were performed (since a rapidly separable magnetic immobilized enzyme was used, there were no free enzymes and carrier residues in the solution):

[0123] (1) Enzyme inactivation: The solution was heated in a 88°C water bath for 19 min to completely inactivate the extremely small amount of enzyme activity that may be dissociated, and cooled to room temperature.

[0124] (2) Desalination: The solution was transferred to a dialysis bag with a molecular weight cutoff of 9 kDa (regenerated cellulose membrane, commercially available) and dialyzed in deionized water for 52 h, with the dialysis solution being replaced every 8 h (the volume of the dialysis solution was 25 times that of the solution), until the conductivity of the dialysis solution was <5 μS·cm -1 .

[0125] (3) Freeze-drying: The dialyzed solution was pre-frozen at -42°C for 16 h, and then dried in a freeze dryer (primary drying stage: -32°C, vacuum degree <8 Pa, 28 h; secondary drying stage: 28°C, vacuum degree <3 Pa, 16 h), to obtain a white to slightly yellow loose powder of low molecular weight hyaluronic acid product, with a yield of about 84% of the initial sodium hyaluronate mass (due to the high concentration of substrate and the high efficiency of the immobilized enzyme system, the yield reached the highest).

[0126] Product quality inspection:

[0127] The low molecular weight hyaluronic acid product prepared in this example was tested, and the various indicators were as follows:

[0128] Area ratio of 50-100 kDa molecular weight segment: 80.5% (complying with the range of 55-95%, at the upper limit of the preferred range of 65-90%)

[0129] Polydispersity index: 1.12 (complying with the range of 1.05-1.40, at the range of 1.08-1.30)

[0130] Area ratio of less than 10 kDa molecular weight segment: 1.8% (complying with the range of 0-5%)

[0131] Ash content (sulfate method): 0.32% (complying with the requirement of ≤0.5%)

[0132] Protein content (BCA method): 0.12% (meet the requirement of ≤0.2%)

[0133] Nucleic acid content (ultraviolet spectrophotometry, 260 nm): 0.05% (meet the requirement of ≤0.1%)

[0134] Heavy metal content (ICP-MS method): lead 0.15 ppm, arsenic 0.08 ppm, cadmium 0.03 ppm, mercury <0.01 ppm (all meet the requirement of ≤1 ppm)

[0135] Organic solvent residue (GC method): methanol <2 ppm, ethanol 5 ppm (all meet the requirement of ≤10 ppm)

[0136] Endotoxin content (dynamic colorimetric method): 0.28 EU·mg -1 (meet the requirement of ≤0.5 EU·mg -1 )

[0137] Features of Example 4:

[0138] This example uses limit parameter combination design to systematically verify the implementability of the method under boundary conditions and the stability of product quality. A plurality of core parameters take actual boundary values: sodium hyaluronate concentration (1.5%), pH value (5.5), enzyme dosage (60 000 U·g -1 ), static pressure (150 MPa), pulse cycle number (6), second-stage membrane molecular weight cut-off (50 kDa), transmembrane pressure difference (0.15 MPa), membrane flux (30 L·m -2 ·h -1), single membrane cycle time (20 min), total cycle number (8 times), the synergistic work of these boundary parameters fully proves that the parameter range defined in the claims has industrial feasibility. In this embodiment, the magnetic nanoparticle composite carrier (volume median particle size 180 μm) immobilized enzyme is combined with pulse high hydrostatic pressure (150 MPa x 6 cycles) and external rotating magnetic field assisted mass transfer technology to realize high-efficiency enzymolysis under high-concentration substrate conditions (1.5%). The product 50-100 kDa segment accounts for 80.5% (close to the preferred upper limit of 90%), the polydispersity index is only 1.12 (excellent uniformity), the small molecule impurity is extremely low (<10 kDa segment is only 1.8%), and each quality index fully meets or is better than the standard of claim 8, especially the strict control of heavy metals and endotoxins, which is suitable for injection-grade medical sodium hyaluronate products (such as joint cavity injection, ophthalmic surgery adjuvant). The successful verification of the simplified two-stage membrane group shows that under certain process conditions, the equipment complexity can be reduced, and the high reflux ratio (20:1) and multiple cycle number (8 times) strategy effectively improves the raw material utilization rate to 84% (the highest yield). The rapid separation characteristics of the magnetic carrier (external magnetic separator, recovery rate >98%) enable the immobilized enzyme to be used continuously for 12-15 batches, greatly reducing the production cost and being suitable for large-scale continuous production. This embodiment fully demonstrates the robustness of the method under extreme process conditions and the controllability of product quality, providing important boundary data support for industrialization.

[0139] Comparative Example 1: Basically the same as Example 1, except that the mass fraction of the sodium hyaluronate solution is 1.8%, and the dosages and preparation conditions of other components remain unchanged.

[0140] Comparative Example 2: Basically the same as Example 1, except that the mass fraction of the sodium hyaluronate solution is 0.2%, and the dosages and preparation conditions of other components remain unchanged.

[0141] Comparative Example 3: Basically the same as Example 1, except that the pH is adjusted to 6.0, and the dosages and preparation conditions of other components remain unchanged.

[0142] Comparative Example 4: Basically the same as Example 1, except that the pH is adjusted to 4.0, and the dosages and preparation conditions of other components remain unchanged.

[0143] Comparative Example 5: Basically the same as Example 1, except that the enzyme dosage is 15000 U·g -1 sodium hyaluronate is added, and the dosages and preparation conditions of other components remain unchanged.

[0144] Comparative Example 6: Basically the same as Example 1, except that the static pressure is 40 MPa, and the dosages and preparation conditions of other components remain unchanged.

[0145] Comparative Example 7: Essentially the same as Example 1, except that the static pressure is 220 MPa, and the amounts of the other components and the preparation conditions are unchanged.

[0146] Comparative Example 8: Essentially the same as Example 1, except that the reaction temperature is 28℃, and the amounts of the other components and the preparation conditions are unchanged.

[0147] Comparative Example 9: Essentially the same as Example 1, except that the reaction temperature is 48℃, and the amounts of the other components and the preparation conditions are unchanged.

[0148] Comparative Example 10: Essentially the same as Example 1, except that a continuous pressure mode (not a pulse pressure mode) is used, and the reaction is continuously carried out at 100 MPa for 100 min, and the amounts of the other components and the preparation conditions are unchanged.

[0149] Comparative Example 11: Essentially the same as Example 1, except that the molecular weight cut-off of the first-stage membrane is 80 kDa, and the amounts of the other components and the preparation conditions are unchanged.

[0150] Comparative Example 12: Essentially the same as Example 1, except that the ratio of the backflow rate to the product rate is 0.5:1 (i.e., no effective backflow), and the amounts of the other components and the preparation conditions are unchanged.

[0151] Comparative Example 13: Essentially the same as Example 1, except that the transmembrane pressure difference is 0.25 MPa, and the amounts of the other components and the preparation conditions are unchanged.

[0152] Performance Test:

[0153] Experimental Scheme 1: Determination of Molecular Weight Distribution and Polydispersity Index

[0154] Test Object: Low-molecular-weight hyaluronic acid product and samples of each example and comparative example. Test Purpose: To determine the molecular weight distribution curve of the sample, calculate the area ratio of the 50-100 kDa molecular weight segment, the number-average molecular weight, the weight-average molecular weight, and the polydispersity index (PDI), in order to evaluate the uniformity of the molecular weight of the product and the batch stability. Test Principle: The high-performance liquid chromatography (HPLC) is combined with a multi-angle laser light scattering detector (MALLS), after the sample is separated by the chromatographic column, the MALLS directly determines the absolute molecular weight of each component according to the scattering light intensity at different angles, and the refractive index detector (RI) determines the concentration, and the combination of the two can obtain the molecular weight distribution data without the need for standard calibration. Experimental Method: 5.0 mg of the sample was dissolved in the mobile phase (0.15 mol·L -1 NaCl + 0.02% NaN3 aqueous solution) to make 1.0 mg·mL -1Solution, 0.22 μm filter membrane filtration 20 μL. Chromatographic column uses TSKgel G5000PWXL + G4000PWXL + G3000PWXL series column (Tosoh, 300x7.8 mm), column temperature 30℃, flow rate 0.5 mL·min -1 , isocratic elution 60 min. MALLS detector (Wyatt miniDAWNTREOS) laser wavelength 658 nm, detection angle 42° / 90° / 138°, RI detector (Wyatt Optilab T-rEX) temperature 35℃. Data collection with ASTRA software, using Zimm fitting model to calculate molecular weight, dn / dc value 0.153 mL·g -1 . Standard basis: refer to GB / T 26329-2010 "Sodium Hyaluronate" Appendix A and ASTM D6474-12 standard. Key parameters: sample concentration 1.0 mg·mL -1 , flow rate 0.5 mL·min -1 , detection time 60 min, data acquisition frequency 1 Hz. Data processing: using ASTRA software to automatically integrate the peak area, calculate the area ratio (%) of 50-100 kDa interval, number average molecular weight Mn (kDa), weight average molecular weight Mw (kDa) and PDI=Mw / Mn, each sample was determined 3 times to take the average value ± standard deviation.

[0155] Experimental scheme 2: viscosity determination

[0156] Test object: 1% (w / v) aqueous solution prepared from low molecular weight hyaluronic acid product. Test purpose: determine the dynamic viscosity of the product aqueous solution, evaluate its flowability and processing suitability, and verify the technical effect of high solid content and low viscosity. Test principle: use a rotary viscometer to measure the shear stress of the fluid under a certain shear rate, and calculate the dynamic viscosity according to the Newton viscosity law η=τ / γ̇ (τ is the shear stress, γ̇ is the shear rate). Experimental method: accurately weigh 1.000 g of sample in a beaker, add deionized water to 100 mL, 25℃ magnetic stirring for 4 hours to completely dissolve (avoiding air bubbles), transfer to a constant temperature water bath (25.0±0.1℃) and equilibrate for 30 min. Use Brookfield DV-II+ Pro rotary viscometer, with S61 rotor (cone and plate type), sample volume 0.5 mL, shear rate set to 100 s -1 (60 rpm), test time 60 s, read the stable viscosity value (mPa·s). Each sample was determined in triplicate, and the average value and standard deviation were calculated. Also determine the shear rate 10, 50, 100, 200, 500 s -1Rheological curve of viscosity vs. shear rate was plotted to determine the type of fluid (Newtonian or non-Newtonian). Standard basis: refer to GB / T 1632-1993 “Determination of viscosity of polymer multi-component systems - Ubbelohde viscometer method” and ISO 3219-1993. Key parameters: sample concentration 1.0% (w / v), test temperature 25°C, shear rate 100 s -1 , rotor S61. Data processing: record 100 s -1 , dynamic viscosity η (mPa-s) at 25°C, calculate relative viscosity ηr=η / η0 (η0 is the viscosity of water at 25°C 0.89 mPa-s), n=3 for each group of data, give the average value ± standard deviation.

[0157] Experimental scheme 3: protein content determination

[0158] Test object: low molecular weight hyaluronic acid product. Test purpose: determine the residual protein content in the product, evaluate the purification effect and biological safety (protein residues may cause allergic reactions). Test principle: BCA (bicinchoninic acid) colorimetric method, protein reduces Cu 2+ to Cu + under alkaline conditions, Cu + and BCA chelate to form a purple complex with characteristic absorption at 562 nm, and the absorbance is proportional to the protein concentration. Experimental method: accurately weigh 50.0 mg of sample into a 10 mL volumetric flask, add 0.5 mol-L -1 NaOH solution 2 mL for dissolution (alkaline conditions promote protein dissolution), add water to the mark for constant volume, mix well, and take 200 μL of sample solution into a microplate, add BCA working solution (BCA reagent A: reagent B=50:1) 200 μL, mix well, and incubate at 37°C for 30 min. The absorbance at 562 nm was measured on a microplate reader (Bio-Rad iMark). At the same time, prepare a bovine serum albumin (BSA) standard curve: prepare 0, 5, 10, 25, 50, 100, 200 μg-mL -1 series of concentrations, and determine the absorbance by the same method, and draw a standard curve (linear range 0-200 μg-mL -1 , R 2 ≥0.998). Standard basis: refer to GB 5009.5-2016 “National food safety standard Determination of protein in food” and EP 9.0 (European Pharmacopoeia) General 2.5.33. Key parameters: sample concentration 5.0 mg-mL -1 , BCA incubation temperature 37°C, time 30 min, detection wavelength 562 nm. Data processing: calculate the protein concentration (μg-mL -1), converted into the protein content in the product (%) = (measured concentration x dilution factor x total volume) / (sample mass x 10000), n = 3, average value ± standard deviation is given.

[0159] Experimental scheme 4: nucleic acid content determination

[0160] Test object: low molecular weight hyaluronic acid product. Test purpose: determine the residual nucleic acid (DNA / RNA) content in the product, evaluate the purification effect, and the residual nucleic acid may affect the stability and safety of the product. Test principle: nucleic acid molecules have a maximum ultraviolet absorption peak at 260 nm wavelength (π→π transition of purine and pyrimidine bases), the absorbance and nucleic acid concentration comply with the Lambert-Beer law, and the nucleic acid content can be quantified by measuring A * . 260 Experimental method: accurately weigh 20.0 mg of sample into a 10 mL volumetric flask, add 8 mL of 0.01 mol·L -1 NaOH solution to dissolve, ultrasonic treatment for 10 min to promote the release of nucleic acid, and then add water to constant volume after cooling, and filter through a 0.22 μm filter membrane. Take 1.0 mL of the filtrate in a quartz cuvette (light path 1 cm), and use an ultraviolet-visible spectrophotometer (Thermo NanoDrop 2000c) to measure the absorbance at 260 nm, 280 nm and 320 nm (background correction). At the same time, use 0.01 mol·L -1 NaOH solution as a blank control, and measure A 260 , A 280 values. Calculate A 260 / A 280 ratio (the ratio of pure nucleic acid is about 1.8-2.0, and a low ratio indicates protein interference). Standard basis: refer to GB 5009.268-2016 "National Food Safety Standard Determination of Multiple Elements in Food" and USP 44 General <781> Nucleic Acid Determination Method. Key parameters: sample concentration 2.0 mg·mL -1 , detection wavelength 260 nm / 280 nm / 320 nm, light path 1 cm, blank control 0.01 mol·L -1 NaOH. Data processing: nucleic acid content (%) = (A 260 -A 320 ) x dilution factor x 50 μg·mL -1 (nucleic acid extinction coefficient) / (sample concentration mg·mL -1 x 1000), n = 3, report average value ± standard deviation and A 260 / A 280 ratio.

[0161] Experimental scheme 5: heavy metal content determination

[0162] Test object: Low molecular weight hyaluronic acid product. Test purpose: To determine the content of lead (Pb), arsenic (As), cadmium (Cd), and mercury (Hg) in the product, to ensure compliance with pharmaceutical or food grade safety standards (each ≤1 ppm), and to ensure product safety. Test principle: Using inductively coupled plasma mass spectrometry (ICP-MS) technology, the sample is atomized into a plasma torch after microwave digestion, the elements are ionized into ions, separated by mass analyzer according to mass-to-charge ratio (m / z), and the signal intensity of each element ion is detected by the detector, and quantified by standard curve. Experimental method: Accurately weigh 0.5000 g of sample into a polytetrafluoroethylene digestion tank, add HNO3 (super pure, ≥65%) 5 mL, H2O2 (super pure, 30%) 2 mL, cover the tank cover and place it in a microwave digestion instrument (CEM Mars 6), and follow the program to heat: 120°C for 5 min, 150°C for 5 min, 180°C for 20 min, cool and transfer to a 50 mL volumetric flask, add water to constant volume. At the same time, prepare a series of mixed standard solution: Pb, As, Cd, Hg each 0, 0.5, 1.0, 5.0, 10.0, 50.0 ppb (2% HNO3 as solvent), draw the standard curve. Using ICP-MS (Agilent 7800) to detect, detect isotopes 208 Pb, 75 As, 111 Cd, 202 Hg, internal standard Rh, In correction. Standard basis: GB 5009.268-2016 "Determination of multiple elements in food" third method and USP 44 general <233>. Key parameters: sample size 0.5 g, digestion temperature 180°C, time 20 min, constant volume 50 mL, ICP-MS power 1550 W, carrier gas Ar flow 1.05 L·min -1 . Data processing: content (mg·kg -1 ) = (measured concentration μg·L -1 × constant volume L × dilution multiple) / sample mass kg, n = 3, report the average value ± standard deviation of each element.

[0163] Experimental scheme 6: Endotoxin content determination

[0164] Test object: Low molecular weight hyaluronic acid product aqueous solution. Test purpose: To determine the endotoxin (bacterial lipopolysaccharide LPS) content in the product, to ensure ≤0.5 EU·mg -1, to meet the safety requirements of injection-grade biological products. Test principle: using dynamic colorimetric method (limulus reagent method), endotoxin catalyzes the activation of coagulogen in limulus reagent, releases pNA (p-nitroaniline) substrate, produces yellow product with absorption peak at 405 nm, and the growth rate of absorbance is logarithmically linear with the concentration of endotoxin. Experimental method: accurately weigh 10.0 mg of sample, dissolve and dilute to 1.0 mg·mL -1 with endotoxin detection special pyrogen-free water (LAL Reagent Water, <0.005 EU·mL -1 ), and equilibrate in a 37℃ water bath for 10 min. Prepare endotoxin standard series: 0.005, 0.05, 0.5, 5.0 EU·mL -1 (Escherichia coli O55:B5 LPS, Charles River). In a pyrogen-free 96-well plate (previously 180℃ baked for 4 h to remove pyrogens), add 100 μL of sample or standard and 100 μL of limulus reagent (Xiamen Bioendo, sensitivity 0.03 EU·mL -1 ) per well, immediately incubate in a microplate reader (Molecular Devices SpectraMax) at 37℃, measure the absorbance at 405 nm every 30 seconds, and record the data within 60 min. Standard basis: Chinese Pharmacopoeia 2020 edition four chapters 1143 "Bacterial endotoxin test method" and USP 44 chapters <85>. Key parameters: sample concentration 1.0 mg·mL -1 , limulus reagent sensitivity 0.03 EU·mL -1 , incubation temperature 37℃, detection time 60 min, wavelength 405 nm. Data processing: calculate the reaction time (Onset Time) according to the slope of the standard curve, convert the endotoxin concentration (EU·mL -1 ), and then calculate the endotoxin content of the product (EU·mg -1 ), n=3, report the average value ± standard deviation.

[0165] Experimental scheme 7: colorimetric determination

[0166] Test object: 5% (w / v) aqueous solution of low molecular weight hyaluronic acid product. Test purpose: Determine the color of the product solution (yellow index YI and lightness L*), evaluate the appearance quality of the product, low color indicates low degree of degradation, high purity, which is beneficial for cosmetic application. Test principle: Using CIE Lab color space system, the three stimulus values X, Y, Z of the sample are determined by spectrophotometer, and the lightness L (0 for black, 100 for white), yellow-blue axis color a*, red-green axis color b and yellow index YI = 100 x (1.28X-1.06Z) / Y are calculated. Experimental method: Accurately weigh 5.000 g of sample into a 100 mL beaker, add deionized water to 100 mL, 25°C magnetic stirring for 2 hours to completely dissolve, stand for 30 min to degas bubbles, transfer to a quartz cuvette (light path 10 mm, four sides transmittance). Using spectrophotometer (Konica Minolta CM-5) in transmission mode, light source D65 (standard daylight), viewing angle 10°, measurement aperture ϕ8 mm, measurement range 400-700 nm. With deionized water as reference, record L, a*, b*, X, Y, Z values. Each sample is rotated 90° and measured 4 times, and the average value is taken. Standard basis: Refer to GB / T 3979-2008 "Method for measuring object color" and ASTM E313-20 "Standard practice for calculating yellow index of white or near-white opaque materials". Key parameters: sample concentration 5% (w / v), light path 10 mm, light source D65, viewing angle 10°, measurement wavelength range 400-700 nm. Data processing: Record L* (lightness), a*, b values and calculate YI value, n=4, report mean ± standard deviation; L>90 and YI<5 are high quality products (low color).

[0167] In combination Figures 1 to 3 , the quantitative conclusion can be given: Figure 1 The pressure-temperature-time curve of the product of formula (I) in 10 independent batches showed that the peak pressure was 200 MPa, the pulse width was 30.0±0.8 s, the cycle was 120±2 s, the holding pressure segment temperature rise was ≤1.2 ℃, the steady state segment temperature was maintained at 35.0±0.5 ℃, the pressure fluctuation coefficient CV was 0.9% within the same batch and 1.3% between batches, and there was no abnormal overshoot or lag, indicating that the space-time parameters were controlled and reproducible; corresponding to it, Figure 2The gel permeation chromatography-multiple angle laser light scattering / differential refractometry distribution superimposition shows that the weight average molecular weight Mw is 72.5±1.8 kDa, the number average molecular weight Mn is 61.9±1.5 kDa, the main peak half-peak width (in volume fraction) is 0.21±0.03, the main peak ratio is ≥ 88%, the shoulder peak ratio is ≤ 8%, the peak position difference of multiple angle light scattering and differential refractometry at the same elution volume is ≤ 0.05 mL, the signal correlation coefficient r is ≥ 0.995, the refractive index increment dn / dc is calibrated with 0.153 mL / g, the deviation of the calculated absolute molecular weight from the external calibration value is ≤ 3%; further, Figure 3 The polydispersity index PDI is 1.17±0.04 (n=10), the interquartile range IQR is 0.06, the maximum outlier PDI=1.25, which is traced back to a one-time slight temperature disturbance (+1.0 ℃, lasting about 2 min), but does not change the overall statistical interval; at the same time, the RSD of Mw between batches is 2.5%, and the RSD between batches after peak area normalization is 2.1%, which is consistent with the SEC-MALS summary parameters, the linear region R² of the fitted Zimm plot is ≥ 0.992, and the recovery rate is 98.3±1.2%. The above data constitute a quantitative evidence chain of process control (low CV, low drift), measurement credibility (consistent peak position of two channels, high correlation, high recovery) and product consistency (low PDI, narrow distribution, low RSD), which proves that the scheme meets the engineering requirements in terms of rationality, reliability and effectiveness, and can support the stable realization and scale-up of target performance.

[0168] Figure 4 Effect of static pressure on 50-100 kDa molecular weight segment ratio and yield of hyaluronic acid sodium; basic parameters: hyaluronic acid sodium solution mass fraction 0.8%, pH 5.0, enzyme dosage 40 000 U·g -1 Hyaluronic acid sodium, reaction temperature 38℃, pulse cycle number 10, pressure segment / decompression segment time ratio 1:1, single cycle pressure time 10 min, first stage membrane molecular weight cut-off 150 kDa, second stage membrane molecular weight cut-off 75 kDa, transmembrane pressure difference 0.10 MPa, reflux flow / product flow ratio 10:1; variable parameters: static pressure 40-220 MPa; conclusion: the 50-100 kDa segment ratio and yield are the highest in the range of 120-140 MPa, too low pressure leads to insufficient enzyme catalysis, target segment and yield decrease, and too high pressure leads to excessive degradation and partial inactivation, target segment and yield decrease. Figure 5 Effect of hyaluronic acid sodium solution concentration on 50-100 kDa molecular weight segment ratio and viscosity; basic parameters: pH 5.0, enzyme dosage 40 000 U·g -1Sodium hyaluronate, reaction temperature 38℃, static pressure 100 MPa, pulse cycle number 10, pressure segment / decompression segment time ratio 1:1, single cycle pressurization time 10 min, first stage membrane molecular weight cut-off 150 kDa, second stage membrane molecular weight cut-off 75 kDa, transmembrane pressure difference 0.10 MPa, reflux flow rate / product flow rate ratio 10:1; variable parameters: concentration 0.2%-1.8%(w / v); conclusion: when the concentration is 1.0%-1.2%, the target segment ratio is the highest and the viscosity is moderate, too low concentration of substrate is insufficient, the ratio decreases, and too high concentration of viscosity increases, contact is limited, and the ratio decreases. Figure 6 Influence of pH on 50-100 kDa molecular weight segment ratio and polydispersity index; basic parameters: sodium hyaluronate solution mass fraction 0.8%, enzyme dosage 40 000 U·g -1 Sodium hyaluronate, reaction temperature 38℃, static pressure 100 MPa, pulse cycle number 10, pressure segment / decompression segment time ratio 1:1, single cycle pressurization time 10 min, first stage membrane molecular weight cut-off 150 kDa, second stage membrane molecular weight cut-off 75 kDa, transmembrane pressure difference 0.10 MPa, reflux flow rate / product flow rate ratio 10:1; variable parameters: pH 4.0-6.0; conclusion: when the pH is 5.0-5.3, the target segment ratio and PDI are optimal, the non-enzymatic degradation is enhanced by the acidic pH, the distribution is widened, and the enzyme activity is decreased by the neutral pH, and the degradation is insufficient. Figure 7 Influence of reflux flow rate / product flow rate ratio on 50-100 kDa molecular weight segment ratio and yield; basic parameters: sodium hyaluronate solution mass fraction 0.8%, pH 5.0, enzyme dosage 40 000 U·g -1 Sodium hyaluronate, reaction temperature 38℃, static pressure 100 MPa, pulse cycle number 10, pressure segment / decompression segment time ratio 1:1, single cycle pressurization time 10 min, first stage membrane molecular weight cut-off 150 kDa, second stage membrane molecular weight cut-off 75 kDa, transmembrane pressure difference 0.10 MPa; variable parameters: reflux flow rate / product flow rate ratio 0.5-35; conclusion: when the reflux flow rate / product flow rate ratio is 15-25, the target segment ratio and yield are the highest, too low reflux re-degradation is insufficient, the ratio and yield are low, too high reflux has small marginal benefit and increases process load, and the comprehensive performance is not as good as the optimal interval.

[0169] From the performance summary of the examples and comparative examples in Table 1 and Table 2, it can be seen that deviating from the optimal window of Example 1 will cause the target 50-100 kDa segment proportion, yield and distribution uniformity to deteriorate: when the sodium hyaluronate concentration is too high at 1.8% (Comparative Example 1), the system viscosity increases sharply, the molecular entanglement is intensified, the mass transfer is limited, the enzyme hydrolysis is uneven, and the target segment proportion decreases; when it is too low at 0.2% (Comparative Example 2), the substrate is insufficient, the effective collision frequency is reduced, and the yield and target segment proportion decrease synchronously. The pH is raised to 6.0 (Comparative Example 3), which significantly inhibits the enzyme activity and causes insufficient degradation, and the target segment proportion and distribution uniformity are poor; the pH is lowered to 4.0 (Comparative Example 4), which enhances the acid non-enzyme pathway and partially inactivates the enzyme, and the molecular weight distribution is widened and the small molecule proportion increases. The enzyme dosage is lowered to 15000 U·g -1 (Corresponding Example 5) leads to insufficient catalytic sites and slow degradation kinetics, and the target segment enrichment and yield decrease. The static pressure is too low at 40 MPa (Comparative Example 6), which makes the enzyme and pressure synergistic effect insufficient, and the chain segment is not broken enough, and the indicators decrease; it is too high at 220 MPa (Comparative Example 7), which will cause excessive degradation and enzyme inactivation, and the molecular weight distribution is small, and the target segment proportion and yield decrease. The temperature is too low at 28℃ (Comparative Example 8), which reduces the enzyme activity and system mass transfer, and the reaction is slow; it is too high at 48℃ (Comparative Example 9), which induces enzyme thermal inactivation and side reactions, which are not conducive to the control of the target segment. Using continuous pressure instead of pulse (Comparative Example 10) will weaken the selective shearing and mass transfer refreshing effect of pressure pulse, leading to the decrease of degradation selectivity and distribution regulation ability. The first stage membrane cut-off is reduced to 80 kDa (Comparative Example 11), which destroys the molecular weight window management of cascade separation, and the high molecular is not enough for recycling and re-degradation, and the target segment escapes or is excessively intercepted, and the distribution and yield are damaged. The reflux ratio is reduced to 0.5:1 (Comparative Example 12), which is basically no effective reflux, and the high molecular cannot be recycled and re-enzymatic hydrolyzed, and the raw material utilization rate and target segment proportion decrease significantly. The transmembrane pressure difference is increased to 0.25 MPa (Comparative Example 13), which intensifies the concentration polarization and membrane compaction, and the classification selectivity and flux stability are poor, which is easy to enlarge the distribution deviation and sacrifice the target segment proportion and overall yield.

[0170] Table 1: Molecular weight distribution and rheological performance of examples and comparative examples

[0171] Table 2: Purity and safety performance of examples and comparative examples

[0172] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformation made under the concept of the present application, by using the content of the present application specification and drawings, should be covered in the protection scope of the claims of the present application.

Claims

1. A method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure assisted enzymatic hydrolysis, characterized by , including: S1 raw material preparation: sodium hyaluronate is dissolved in a buffer system composed of acetic acid and sodium acetate, the mass fraction of the obtained solution is 0.3-1.5%, the pH is adjusted to 4.5-5.5, and 20000-80000 U·g -1 Sodium hyaluronate is added to bovine testis-derived hyaluronidase; S2 high hydrostatic pressure enzymolysis: reaction at 30-45℃ under 50-200 MPa static pressure, cumulative reaction time under pressure state is 1.0-5.0 h; S3 multi-stage tangential flow ultrafiltration external circulation separation and reflux: after the reaction liquid is depressurized to normal pressure, it is separated by at least two stages of tangential flow ultrafiltration membrane group external circulation, wherein the molecular weight cut-off of the first stage membrane is 110-300 kDa, and the molecular weight cut-off of the second stage membrane is 50-100 kDa, the feed liquid passing through the first stage membrane and being cut off by the second stage membrane is collected as the target product liquid, part of the cut-off liquid of the first stage membrane is mixed with part of the permeate liquid of the second stage membrane and then refluxed to step S2 for continuous reaction, and the rest is discarded and pressurized again for the next cycle; S4 termination and purification: when the area ratio of the 50-100 kDa molecular weight segment in the target product liquid reaches 55-95% or the change amplitude of the molecular weight distribution of the product liquid is less than 5% in two consecutive measurements, the reaction is terminated, the product liquid is adjusted to pH 6.5-7.5 for enzyme inactivation and desalination, and then freeze-dried to obtain the product.

2. The method of claim 1, wherein , the high hydrostatic pressure is pulsed pressurization, with 6-15 pressurization and depressurization cycles, and the time ratio of the pressurization segment to the depressurization segment is 1:0.2-2.

3. The method of claim 1, wherein , the static pressure is 80-150 MPa, the temperature is 35-42℃, and the cumulative reaction time under pressure state is 1.5-4.0 h.

4. The method of claim 1, wherein , the tangential flow ultrafiltration uses a three-stage membrane group, wherein the molecular weight cut-off of the first stage membrane is 120-200 kDa, the molecular weight cut-off of the second stage membrane is 50-100 kDa, and the molecular weight cut-off of the third stage membrane is 8-20 kDa, and the permeate liquid of the third stage membrane is discharged to remove small molecular impurities.

5. The method of claim 1, wherein , the transmembrane pressure difference of the tangential flow ultrafiltration is 0.03-0.20 MPa, the membrane flux is 5-50 L·m -2 ·h -1 , the ratio of the backflow rate to the product flow rate is 1-30.

6. The method of claim 1, wherein , the single membrane circulation time is 5-30 min, and the total circulation number is 2-10 times.

7. The method of claim 1, wherein , the hyaluronidase exists in an immobilized form and is packed in a replaceable basket core in the high hydrostatic pressure cavity, the immobilized carrier is selected from at least one of cross-linked agarose gel, macroporous silica, cross-linked polystyrene microspheres, and magnetic nanoparticle composite carrier, the volume median particle size of the immobilized carrier is 100-200 μm, and the mechanical strength retention rate after 10-20 cycles under 50-200 MPa is 85-100%.

8. A low molecular weight hyaluronic acid product, characterized in that , the product prepared by the method of any one of claims 1-7, wherein the product satisfies: an area ratio of a molecular weight range of 50-100 kDa of 55-95%, a polydispersity index of 1.05-1.40, an area ratio of a molecular weight range of less than 10 kDa of 0-5%, an ash content of not more than 0.5%, a protein content of not more than 0.2%, a nucleic acid content of not more than 0.1%, a content of lead, arsenic, cadmium, and mercury each of not more than 1 ppm, a residual amount of methanol and ethanol each of not more than 10 ppm, and an endotoxin content of not more than 0.5 unit·mg -1 .

9. The product of claim 8, wherein , the area ratio of the 50-100 kDa molecular weight segment is 65-90%, and the polydispersity index is 1.08-1.

30.

10. Use of the low molecular weight hyaluronic acid according to claim 8 or 9 in the preparation of cosmetics, functional foods or biomedical materials.

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