Method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure assisted enzymatic hydrolysis

Abstract

The present application relates to the field of biological medicine and food technology, and provides a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure assisted enzymatic hydrolysis. In the present application, sodium hyaluronate is dissolved in an acetic acid / sodium acetate buffer system, and a bovine testis-derived hyaluronidase is added under the condition of pH 4.5-5.5. The enzymatic hydrolysis is combined with 30-45 DEG C, 50-200 MPa static pressure and pulse pressure, and is cooperatively operated with two to three stages of tangential flow ultrafiltration external circulation separation. The molecular weight distribution is dynamically regulated through the segmentation interception and reflux strategy between the first and second membranes. The molecular weight interval target and flux are met, excessive shear and color rise are inhibited, and the product is obtained after enzyme inactivation, desalting and freeze-drying when the area ratio of 50-100 kDa is 55-95% or the distribution continuously changes by less than 5% twice. The product has a polydispersity index of 1.05-1.40, and the microimpurities and residues are strictly controlled. The product is suitable for cosmetics, functional food and biological medical materials.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of biomedical and food technology, specifically to a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure-assisted enzymatic hydrolysis. Background Technology

[0002] Hyaluronic acid, a polysaccharide with good biocompatibility and water retention, is widely used in cosmetics, functional foods, and biomedical materials. Different molecular weight ranges correspond to different application focuses. Among them, the low and medium molecular weight range often focuses on the coordination requirements 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 process throughput, while maintaining the distribution concentration and batch-to-batch consistency of the target molecular weight range. This provides a controllable basis for subsequent exfoliation, finished product stability, and user experience. On the other hand, process conditions have a significant impact on the integrity of the chain segments, color, and quality risks related to potential irritation. Therefore, under the premise of achieving large-scale continuous production and scale-up feasibility, establishing a process route that takes into account mild reactions, precise segmentation, and low impurity output has become the core demand of the industry to improve product performance consistency, processing adaptability, and cross-scenario applicability.

[0003] Currently, atmospheric pressure enzymatic hydrolysis or single-membrane fractionation pathways still have shortcomings in terms of distribution convergence speed, fine control of fractionation boundaries, and flux balance. These shortcomings mainly manifest in the difficulty of simultaneously meeting the requirements of high solids content and low viscosity while rapidly achieving the target range percentage. Furthermore, under long-term shear or high-shear conditions, excessive chain segmentation and color increase can easily occur, affecting product stability and user experience. In multi-stage membrane synergy, mismatched reflux strategies and transmembrane pressure window settings can also cause distribution oscillations and decreased batch-to-batch consistency. For example, Chinese patent CN117701598A discloses a novel enzymatic method for preparing hyaluronic acid of specific molecular weight using hyaluronidase, the hyaluronidase itself, and its applications. It emphasizes achieving specific distributions by optimizing the membrane's molecular weight cutoff range, but suffers from slow distribution regulation response, limited flux under high solids content conditions, and batch-to-batch consistency dependence on empirical adjustments. In addition, insufficient coupling between single-stage termination criteria and online monitoring can easily lead to deviations between the termination point and the target range percentage, further exacerbating subsequent purification load and quality fluctuations. Therefore, there is an urgent need to construct a more reproducible reaction-separation-reflux closed-loop control scheme. Summary of the Invention

[0004] (1) Technical problems to be solved

[0005] The purpose of this invention is to provide a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure-assisted enzymatic hydrolysis, which solves the coupling contradiction between high solid content and low viscosity and the proportion and consistency of the target molecular weight range, as well as the conflict between high pressure / high shear and chain segment integrity and low color / low irritation, thereby achieving scalable production and stable quality output.

[0006] (2) Technical solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure-assisted enzymatic hydrolysis includes:

[0009] S1 Raw Material Preparation: Dissolve sodium hyaluronate in a buffer system composed of acetic acid and sodium acetate, resulting in a solution with a mass-volume fraction of 0.3–1.5%. Adjust the pH to 4.5–5.5 and prepare the solution at a concentration of 20,000–80,000 U·g. -1 Sodium hyaluronate was added to hyaluronidase derived from bovine testis;

[0010] S2 high hydrostatic pressure enzymatic hydrolysis: The reaction is carried out at 30–45℃ and 50–200MPa hydrostatic pressure, with a cumulative reaction time of 1.0–5.0 h under pressure.

[0011] S3 Multistage Tangential Flow Ultrafiltration External Circulation Separation and Reflux: After the reaction solution is depressurized to atmospheric pressure, it undergoes external circulation separation through at least two stages of tangential flow ultrafiltration membrane arrays. The molecular weight cutoff of the first stage membrane is 110–300 kDa, and the molecular weight cutoff of the second stage membrane is 50–100 kDa. The feed solution that has passed through the first stage membrane and is retained by the second stage membrane is collected as the target product feed solution. A portion of the retentate from the first stage membrane is mixed with a portion of the permeate from the second stage membrane and refluxed to step S2 to continue the reaction. The remaining portion is discarded, and the solution is pressurized again to enter the next cycle.

[0012] S4 Termination and Purification: The reaction is terminated when the area ratio of the 50–100 kDa molecular weight range in the target product solution reaches 55–95% or the change in the molecular weight distribution of the product solution is less than 5% in two consecutive measurements. The pH of the product solution is adjusted to 6.5–7.5, followed by enzyme inactivation and desalting, and then freeze-drying to obtain the product.

[0013] Furthermore, the high hydrostatic pressure is applied in a pulsed manner, with 6–15 pressurization and depressurization cycles, and the time ratio of the pressurization segment to the depressurization segment is 1:0.2–2.

[0014] Furthermore, the static pressure is 80–150 MPa, the temperature is 35–42 °C, and the cumulative reaction time under pressure is 1.5–4.0 h.

[0015] Furthermore, the tangential flow ultrafiltration employs a three-stage membrane module, wherein the molecular weight cutoff of the first-stage membrane is 120–200 kDa, the molecular weight cutoff of the second-stage membrane is 50–100 kDa, and the molecular weight cutoff of the third-stage membrane is 8–20 kDa. The permeate from the third-stage membrane is discharged to remove small molecule impurities.

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

[0017] Furthermore, the single membrane cycle time is 5–30 min, and the total number of cycles is 2–10.

[0018] Furthermore, the hyaluronidase exists in an immobilized form and is packed in a replaceable basket core within a high hydrostatic chamber. The immobilization carrier is selected from at least one of cross-linked agarose gel, macroporous silica, cross-linked polystyrene microspheres, and magnetic nanoparticle composite carriers. The median particle size of the immobilized carrier is 100–200 μm, and its mechanical strength retention rate is 85–100% after 10–20 cycles under 50–200 MPa conditions.

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

[0020] Furthermore, in the method described, the amount of hyaluronidase used in step S1 is 30,000–60,000 U·g. -1 Sodium hyaluronate.

[0021] Furthermore, in the method described, when a three-stage membrane assembly is used in step S3, the retentate between the first-stage membrane and the second-stage membrane is used as the target product feed solution.

[0022] Furthermore, in the method described, 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⁻¹. -2 ·h -1 .

[0023] Furthermore, in the method described, the ratio of the return flow rate to the product flow rate in step S3 is 2–20.

[0024] Furthermore, in the method described, the single membrane cycle time in step S3 is 10–20 min, and the total number of cycles is 3–8.

[0025] Furthermore, in the method described, the immobilization carrier in step S2 is a cross-linked agarose gel or a magnetic nanoparticle composite carrier with a median particle size of 120–180 μm.

[0026] Furthermore, in the method described, the enzyme inactivation in step S4 is performed by heat treatment at a temperature of 70–90℃ for 10–20 min, and the desalting is performed by dialysis with a molecular weight cutoff of 3–10 kDa.

[0027] As a concept of this invention, an enzymatic hydrolysis-separation coupling design with high hydrostatic pressure pulsed pressurization at a mild temperature is employed to enhance the distribution and convergence efficiency and flux synergy of the process under high solids content conditions. A hydrostatic pressure of 50–200 MPa improves the conformational accessibility and local mass transfer of the substrate system, while avoiding the risk of excessive chain segment breakage due to continuous high shear. Two to three stages of tangential flow ultrafiltration external circulation achieve segmented enrichment of the target region between the first and second stages, and dynamically balances the distribution evolution and membrane surface load with a partial first-stage rejection and second-stage permeate reflux ratio, suppressing quality fluctuations caused by critical shear or concentration polarization. Termination is triggered by a percentage threshold or a threshold change between two measurements to reduce the probability of overprocessing, while also considering the product's low color, low irritation, and batch-to-batch consistency. This pathway improves convergence speed and scale-up adaptability while ensuring chain segment integrity, achieving a steady-state equilibrium between the target region percentage, rheological adaptability, and subsequent purification pressure, making it suitable for the stable supply of cosmetics, functional foods, and biomedical materials.

[0028] This invention also discloses a low molecular weight hyaluronic acid product, prepared using the above-described method. The product meets the following requirements: the area ratio of the 50–100 kDa molecular weight segment is 55–95%, the polydispersity index is 1.05–1.40, the area ratio of the molecular weight segment less than 10 kDa is 0–5%, the ash content is no more than 0.5%, the protein content is no more than 0.2%, the nucleic acid content is no more than 0.1%, the content of lead, arsenic, cadmium, and mercury is no more than 1 ppm each, the residual amounts of methanol and ethanol are no more than 10 ppm each, and the endotoxin content is no more than 0.5 units / mg. -1 The area ratio of the 50–100 kDa molecular weight range is 65–90%, and the polydispersity index is 1.08–1.30.

[0029] Furthermore, the low molecular weight hyaluronic acid is used 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℃ by 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 with 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 improve 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. Controlled polydispersity and micro-impurities: By triggering termination with a distribution threshold and combining enzyme inactivation and desalting, a polydispersity index of 1.05–1.40 is obtained. At the same time, quality indicators such as proteins, nucleic acids, heavy metals and residual solvents are limited to strict upper limits, supporting cross-scenario adaptation of cosmetics, functional foods and biomedical materials.

[0036] 4. Scale-up feasibility: The basket-style arrangement of the immobilized enzyme and the design of the cycle strength retention rate, combined with the segmented and reflux strategy of the external circulation membrane module, make the key window parameters reproducible, reduce quality fluctuations and energy consumption costs during the scale-up process, and improve industrial feasibility. Attached Figure Description

[0037] Figure 1 The pressure-temperature-time curve is the process trajectory of the pulsed high hydrostatic enzymatic hydrolysis device in Embodiment 1 of the present invention.

[0038] Figure 2 This is a transmission mode diagram of molecular weight distribution from a gel permeation chromatography-multi-angle laser light scattering / differential refractive index overlay in Example 1 of the present invention.

[0039] Figure 3 This is a box plot of the polydispersity index and a summary graph of the gel permeation chromatography-multi-angle laser light scattering detection results for Example 1 of the present invention.

[0040] Figure 4 This invention investigates the effect of static pressure on the proportion and yield of the 50-100 kDa molecular weight range.

[0041] Figure 5 This invention relates to the effect of sodium hyaluronate solution concentration on the proportion and viscosity of the 50-100 kDa molecular weight range.

[0042] Figure 6 This invention relates to the effect of pH value on the proportion of the 50-100 kDa molecular weight range and the polydispersity index.

[0043] Figure 7 This invention relates to the effect of the reflux flow rate / product flow rate ratio on the proportion and yield of the 50-100 kDa molecular weight range. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0045] Example 1

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

[0047] Step S1 Raw material preparation

[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 grade, purity ≥99.5%, commercially available) and sodium acetate (analytical grade, purity ≥99%, commercially available). In this embodiment, the total concentration of acetic acid and sodium acetate in the buffer system was 35 mmol·L⁻¹. -1 The molar ratio of acetic acid to sodium acetate was 1.1. A sodium hyaluronate solution with a mass-to-volume fraction of 0.8% was prepared, and then... -1 Acetic acid or 1 mol·L -1 Adjust the pH to 5.0 ± 0.1 with sodium hydroxide solution. (40,000 U·g) -1 The ratio of sodium hyaluronate to hyaluronidase derived from bovine testis (enzyme activity ≥ 50,000 U·mg) was added. -1 Egg white (lyophilized powder, commercially available) is thoroughly mixed to obtain an enzymatic hydrolysis reaction solution.

[0049] Step S2: High hydrostatic pressure enzymatic hydrolysis

[0050] The enzymatic hydrolysis solution obtained in step S1 was transferred to the reaction chamber of the high hydrostatic pressure treatment device (chamber volume 500 mL, sealed with PA / PE co-extruded membrane, air bubble venting). In this embodiment, the high hydrostatic pressure enzymatic hydrolysis was performed using a pulsed pressurization method. The reaction temperature was set at 38℃ (controlled by a jacketed circulating constant-temperature water bath), the static pressure was 100 MPa, the number of pulse cycles was 10, the time ratio of the pressurization segment to the depressurization segment was 1:1, the pressurization segment time in a single cycle was 10 min, the depressurization segment time was 10 min, and the cumulative reaction time under pressurization was 100 min (1.67 h). The pressurization medium was deionized water, and the pressurization rate was approximately 200 MPa·min. -1 Decompression rate ≤100 MPa·min -1 .

[0051] Step S3: Multi-stage tangential flow ultrafiltration external circulation separation and reflux

[0052] This embodiment uses a three-stage tangential flow ultrafiltration membrane module for external circulation separation. The membrane material is polyethersulfone (PES) hollow fiber membrane, and the membrane areas are 0.1 m² (first stage), 0.08 m² (second stage), and 0.05 m² (third stage).

[0053] After the reaction in step S2 is completed, the reaction solution, depressurized to atmospheric pressure, is subjected to tangential flow ultrafiltration separation. The specific operation is as follows:

[0054] (1) First-stage ultrafiltration: The reaction solution was pumped into the first-stage membrane module with a molecular weight cutoff of 150 kDa. The transmembrane pressure difference was set to 0.10 MPa and the membrane flux was 20 L·m -2 ·h -1 The circulation velocity is 1.5 m / s. -1 The temperature is controlled at 25±2℃. After collecting the retentate (high molecular weight component, >150 kDa) from the first-stage membrane, 70% of its volume is discarded, and the remaining 30% is returned to step S2 for the next cycle of high hydrostatic enzymatic hydrolysis. The permeate (molecular weight ≤150 kDa) from the first-stage membrane enters the second-stage ultrafiltration.

[0055] (2) Second-stage ultrafiltration: The permeate from the first-stage membrane is pumped into the second-stage membrane module with a molecular weight cutoff of 75 kDa. The transmembrane pressure difference is 0.10 MPa, and the membrane flux is 20 L·m. -2 ·h -1 The circulation velocity is 1.5 m / s. -1 The retentate from the second-stage membrane (molecular weight 75-150 kDa, containing the target product) is collected as the target product feed solution. 60% of the volume of the permeate from the second-stage membrane (molecular weight ≤75 kDa) is mixed with 30% of the volume of the retentate from the first-stage membrane and returned to step S2 for further enzymatic hydrolysis. The remaining 40% of the permeate enters the third-stage ultrafiltration.

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

[0057] In this embodiment, the ratio of reflux flow rate (total volume of feed liquid returned to S2) to product flow rate (volume of liquid retained by the second-stage membrane) is 10:1, the single membrane cycle time is 15 min, and the total number of cycles is 5.

[0058] Step S4 Termination and Purification

[0059] 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 72.5% and the change between two consecutive measurements (with an interval of one cycle) was less than 3%.

[0060] The target product solution was treated with 1 mol·L⁻¹ -1The pH was adjusted to 7.0 ± 0.2 with sodium hydroxide solution, followed by the following treatment:

[0061] (1) Enzyme inactivation: Heat the solution in an 80°C water bath for 15 min to inactivate residual enzyme activity, and then cool to room temperature.

[0062] (2) Desalination: Transfer the feed solution to a dialysis bag (regenerated cellulose membrane, commercially available) with a molecular weight cutoff of 5 kDa, and dialyze in deionized water for 48 h, changing the dialysate every 8 h (the dialysate volume is 20 times the feed solution volume) until the dialysate conductivity is <10 μS·cm -1 .

[0063] (3) Freeze-drying: The dialyzed solution was pre-frozen at -40℃ for 12 h, and then dried in a freeze dryer (initial drying stage: -30℃, vacuum degree <10 Pa, 24 h; desorption drying stage: 25℃, 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 embodiment employs a moderate parameter design, resulting in mild and stable reaction conditions and high process reliability. The pulsed pressurization uses a 1:1 equidistant cycle design, which facilitates the enzyme's recovery of its active conformation during the pressure release phase while preventing excessive substrate degradation. The three-stage membrane module, combined with a reasonable reflux strategy, ensures both the enrichment and purification of the target molecular weight range and maximizes raw material utilization through external circulation reflux. This embodiment is suitable for pilot-scale continuous production, with easily controllable process parameters, a narrow molecular weight distribution (polydispersity index of approximately 1.18), and a stable 70-75% proportion of the 50-100 kDa range, making it suitable as a cosmetic raw material (such as moisturizing serums and mask essences) or a functional food additive.

[0066] Example 2

[0067] This embodiment provides a method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure-assisted enzymatic hydrolysis, including the following steps:

[0068] Step S1 Raw material preparation

[0069] Sodium hyaluronate (molecular weight 1.2-2.0 MDa, purity ≥95%, food grade, commercially available) was dissolved in a buffer system composed of acetic acid (analytical grade, purity ≥99.5%, commercially available) and sodium acetate (analytical grade, purity ≥99%, commercially available). In this embodiment, the total concentration of acetic acid and sodium acetate in the buffer system was 25 mmol·L⁻¹. -1The molar ratio of acetic acid to sodium acetate was 0.8. A 0.5% (w / v) sodium hyaluronate solution was prepared using 1 mol·L⁻¹ solution. -1 Acetic acid or 1 mol·L -1 Adjust the pH to 4.7 ± 0.1 with sodium hydroxide solution. (30,000 U·g) -1 The ratio of sodium hyaluronate to hyaluronidase derived from bovine testis (enzyme activity ≥ 50,000 U·mg) was added. -1 Egg white (lyophilized powder, commercially available), stir and mix at room temperature for 10 min to obtain the enzymatic hydrolysis reaction solution.

[0070] Step S2: High hydrostatic pressure enzymatic hydrolysis

[0071] The enzymatic hydrolysis solution obtained in step S1 was transferred to the reaction chamber of the high hydrostatic pressure treatment device (chamber volume 500 mL, sealed with PA / PE co-extruded membrane, air bubble venting). In this embodiment, high hydrostatic pressure enzymatic hydrolysis was performed using a pulsed pressurization method. The reaction temperature was set at 35℃ (controlled by a jacketed circulating constant-temperature water bath), the static pressure was 80 MPa, the number of pulse cycles was 8, the time ratio of the pressurization segment to the depressurization segment was 1:0.5, the pressurization segment time in a single cycle was 10 min, the depressurization segment time was 5 min, and the cumulative reaction time under pressurization was 80 min (1.33 h). After the 4th and 8th cycles, the depressurization time was extended to 10 min each time to fully release protein conformational stress. The pressurization medium was deionized water, and the pressurization rate was approximately 180 MPa·min. -1 Decompression rate ≤90 MPa·min -1 .

[0072] Step S3: Multi-stage tangential flow ultrafiltration external circulation separation and reflux

[0073] This embodiment uses a three-stage tangential flow ultrafiltration membrane module for external circulation separation. The membrane material is polyethersulfone (PES) hollow fiber membrane, and the membrane areas are 0.12 m² (first stage), 0.09 m² (second stage), and 0.06 m² (third stage).

[0074] After the reaction in step S2 is completed, the reaction solution, depressurized to atmospheric pressure, is subjected to tangential flow ultrafiltration separation. The specific operation is as follows:

[0075] (1) First-stage ultrafiltration: The reaction solution was pumped into the first-stage membrane module with a molecular weight cutoff of 120 kDa. The transmembrane pressure difference was set to 0.08 MPa and the membrane flux was 15 L·m -2 ·h -1 The circulation velocity is 1.2 m / s. -1The temperature is controlled at 25±2℃. After collecting the retentate (high molecular weight component, >120 kDa) from the first-stage membrane, 65% of its volume is discarded, and the remaining 35% is returned to step S2 for the next cycle of high hydrostatic enzymatic hydrolysis. The permeate (molecular weight ≤120 kDa) from the first-stage membrane enters the second-stage ultrafiltration.

[0076] (2) Second-stage ultrafiltration: The permeate from the first-stage membrane is pumped into the second-stage membrane module with a molecular weight cutoff of 60 kDa. The transmembrane pressure difference is 0.08 MPa and the membrane flux is 15 L·m -2 ·h -1 The circulation velocity is 1.2 m / s. -1 The retentate from the second-stage membrane (molecular weight 60-120 kDa, containing the target product) is collected as the target product feed solution. 50% of the volume of the permeate from the second-stage membrane (molecular weight ≤60 kDa) is mixed with 35% of the volume of the retentate from the first-stage membrane and returned to step S2 for further enzymatic hydrolysis. The remaining 50% of the permeate enters the third-stage ultrafiltration.

[0077] (3) Third-stage ultrafiltration: Part of the permeate (50% by volume) from the second-stage membrane is pumped into the third-stage membrane module with a molecular weight cutoff of 8 kDa. The transmembrane pressure difference is 0.08 MPa and the membrane flux is 15 L·m -2 ·h -1 The retentate from the third-stage membrane (molecular weight 8-60 kDa) is returned and combined with the retentate from the second-stage membrane, while the permeate from the third-stage membrane (molecular weight < 8 kDa, consisting of small oligosaccharides, enzymes, and impurities) is discharged and discarded.

[0078] In this embodiment, the ratio of reflux flow rate (total volume of feed liquid returned to S2) to product flow rate (volume of liquid retained by the second-stage membrane) is 5:1, the single membrane cycle time is 12 min, and the total number of cycles is 4.

[0079] Step S4 Termination and Purification

[0080] 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 68.2% and the change between two consecutive measurements (with a one-cycle interval) was less than 4%.

[0081] The target product solution was treated with 1 mol·L⁻¹ -1 The pH was adjusted to 7.0 ± 0.2 with sodium hydroxide solution, followed by the following treatment:

[0082] (1) Enzyme inactivation: Heat the solution in a 75°C water bath for 12 min to inactivate residual enzyme activity, and then cool to room temperature.

[0083] (2) Desalination: Transfer the feed solution to a dialysis bag (regenerated cellulose membrane, commercially available) with a molecular weight cutoff of 5 kDa, and dialyze in deionized water for 44 h, changing the dialysate every 8 h (the dialysate volume is 18 times the feed solution volume) until the dialysate conductivity is <10 μS·cm -1 .

[0084] (3) Freeze-drying: The dialyzed solution was pre-frozen at -40℃ for 12 h, and then dried in a freeze dryer (initial drying stage: -30℃, vacuum degree <10 Pa, 24 h; desorption 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 initial sodium hyaluronate mass.

[0085] Features of Example 2

[0086] This embodiment employs a low-parameter design to verify the feasibility of the method under mild conditions. The low static pressure (80 MPa), temperature (35°C), and enzyme dosage (30,000 U·g) are key parameters. -1 This combination is suitable for pressure-sensitive enzyme preparations or applications requiring controlled degradation rates. The shorter cumulative pressurization time (1.33 h) and pulse asymmetry cycle (1:0.5) design reduce equipment energy consumption and extend the effective lifespan of the enzyme. The three-stage membrane module uses a lower molecular weight cutoff (120 kDa for the first stage and 8 kDa for the third stage), enabling more precise separation of the target molecular weight range. The 50-100 kDa segment consistently accounts for 66-70% of the product, with a polydispersity index of approximately 1.22 and lower levels of small molecule impurities (<3% of the <10 kDa segment). This embodiment is suitable for the preparation of small-batch, high-purity products, such as precursors for injectable medical-grade sodium hyaluronate or active ingredients in high-end cosmetics.

[0087] Example 3

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

[0089] Step S1 Raw material preparation

[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 grade, purity ≥99.5%, commercially available) and sodium acetate (analytical grade, purity ≥99%, commercially available). In this embodiment, the total concentration of acetic acid and sodium acetate in the buffer system was 42 mmol·L⁻¹. -1 The molar ratio of acetic acid to sodium acetate was 1.5. A 1.2% (w / v) sodium hyaluronate solution was prepared using 1 mol·L⁻¹ solution. -1 Acetic acid or 1 mol·L-1 The pH was adjusted to 5.3 ± 0.1 using sodium hydroxide solution. In this example, immobilized hyaluronidase was used, with cross-linked agarose gel (6% agarose, apparent density 1.15 g·mL) as the immobilization medium. -1 The pore size range is 10-40 nm (commercially available), and the median volumetric particle size is 150 μm (measured by a laser particle size analyzer). The mechanical strength retention rate of the immobilized carrier after 15 cycles at 50-200 MPa is 92% (assessed by carrier breakage rate). Immobilized enzymes were prepared using a covalent coupling method: 100 g of cross-linked agarose gel (containing water) was placed in phosphate buffer (pH 8.0, 50 mmol·L⁻¹). -1 The enzyme was activated with epichlorohydrin (10 mL) for 2 h, washed thoroughly, and then hyaluronidase solution (total enzyme activity 3 million U, dissolved in 20 mL of pH 7.5 phosphate buffer) was added. The mixture was slowly stirred at 4 °C for 16 h, achieving a coupling rate of approximately 75% and a retention rate of approximately 60% of the immobilized enzyme activity, resulting in an immobilized enzyme carrier (enzyme activity approximately 1.8 million U). The immobilized enzyme carrier was then loaded into a replaceable stainless steel mesh basket (100 μm mesh) within a high hydrostatic chamber, with a basket volume of 80 mL and an equivalent enzyme concentration of approximately 55,000 U·g. -1 Sodium hyaluronate.

[0091] Step S2: High hydrostatic pressure enzymatic hydrolysis

[0092] The sodium hyaluronate solution (without free enzyme) obtained in step S1 was pumped into the reaction chamber of a high hydrostatic treatment device containing an immobilized enzyme core (total chamber volume 600 mL, substrate solution volume 500 mL). In this embodiment, the high hydrostatic enzymatic hydrolysis was performed using a pulsed pressurization method. The reaction temperature was set at 42℃ (controlled by a jacketed circulating constant-temperature water bath), the hydrostatic pressure was 140 MPa, the number of pulse cycles was 12, the time ratio of the pressurization phase to the depressurization phase was 1:1.5, the pressurization phase time in a single cycle was 12 min, the depressurization phase time was 18 min, and the cumulative reaction time under pressurization was 144 min (2.4 h). The pressurization medium was deionized water, and the pressurization rate was approximately 220 MPa·min. -1 Decompression rate ≤110 MPa·min -1 In each depressurization section, the substrate is gently stirred by a built-in micro magnetic stirrer (100 rpm) to promote contact between the substrate and the immobilized enzyme.

[0093] Step S3: Multi-stage tangential flow ultrafiltration external circulation separation and reflux

[0094] This embodiment uses a three-stage tangential flow ultrafiltration membrane module for external circulation separation. The membrane material is polyethersulfone (PES) hollow fiber membrane, and the membrane areas are 0.15 m² (first stage), 0.12 m² (second stage), and 0.08 m² (third stage).

[0095] After the reaction in step S2 is completed, the reaction solution (excluding the immobilized enzyme core) is depressurized to atmospheric pressure and then subjected to tangential flow ultrafiltration. The specific operation is as follows:

[0096] (1) First-stage ultrafiltration: The reaction solution was pumped into the first-stage membrane module with a molecular weight cutoff of 180 kDa. The transmembrane pressure difference was set to 0.13 MPa and the membrane flux was 25 L·m -2 ·h -1 The circulation velocity is 1.8 m·s. -1 The temperature is controlled at 25±2℃. After collecting the retentate (high molecular weight component, >180 kDa) from the first-stage membrane, 75% of its volume is discarded, and the remaining 25% is returned to step S2 for the next cycle of high hydrostatic enzymatic hydrolysis (pumped back into the chamber containing the immobilized enzyme). The permeate (molecular weight ≤180 kDa) from the first-stage membrane enters the second-stage ultrafiltration.

[0097] (2) Second-stage ultrafiltration: The permeate from the first-stage membrane is pumped into the second-stage membrane module with a molecular weight cutoff of 85 kDa. The transmembrane pressure difference is 0.13 MPa, and the membrane flux is 25 L·m. -2 ·h -1 The circulation velocity is 1.8 m·s. -1 The retentate from the second-stage membrane (molecular weight 85-180 kDa, containing the target product) is collected as the target product feed solution. 70% of the volume of the permeate from the second-stage membrane (molecular weight ≤85 kDa) is mixed with 25% of the volume of the retentate from the first-stage membrane and returned to step S2 for further enzymatic hydrolysis. The remaining 30% of the permeate enters the third-stage ultrafiltration.

[0098] (3) Third-stage ultrafiltration: Part of the permeate (30% by volume) from the second-stage membrane is pumped into the third-stage membrane module with a molecular weight cutoff of 15 kDa. The transmembrane pressure difference is 0.13 MPa, and the membrane flux is 25 L·m. -2 ·h -1 The retentate from the third-stage membrane (molecular weight 15-85 kDa) is returned and combined with the retentate from the second-stage membrane, while the permeate from the third-stage membrane (molecular weight <15 kDa, consisting of small oligosaccharides and impurities, and containing no free enzymes) is discharged and discarded.

[0099] In this embodiment, the ratio of reflux flow rate (total volume of feed liquid returned to S2) to product flow rate (volume of liquid retained by the second-stage membrane) is 15:1, the single membrane cycle time is 18 min, and the total number of cycles 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] This embodiment employs a high-parameter design and introduces immobilized enzyme technology for the first time, validating the method's adaptability and industrialization potential under enhanced conditions. A higher sodium hyaluronate concentration (1.2%) increases single-batch production capacity. Higher temperatures (42°C), hydrostatic pressure (140 MPa), and pulse asymmetric cycles (1:1.5) accelerate the enzymatic hydrolysis reaction, shortening the total reaction time to approximately 2.4 hours of cumulative pressurization time (total reaction cycle approximately 6 hours including depressurization). The immobilized enzyme (cross-linked agarose gel carrier, median particle size 150 μm) retains 92% of its mechanical strength after 15 cycles at 50-200 MPa, demonstrating the carrier's excellent pressure resistance and allowing for reuse in 8-10 batches, significantly reducing enzyme costs. The three-stage membrane module employs a relatively high molecular weight cutoff (180 kDa for the first stage and 15 kDa for the third stage), combined with a high reflux ratio (15:1) and a long circulation time (18 min × 6 cycles), effectively enriching the target molecular weight range. The proportion of the 50-100 kDa range in the product is consistently maintained at 75-80%, with a polydispersity index of approximately 1.15 (narrower molecular weight distribution), and small molecule impurities <10 kDa account for <2%. This embodiment is suitable for large-scale continuous production. The immobilized enzyme system facilitates automated control and online monitoring, ensuring stable product quality. It is suitable as a raw material for medical devices (such as intra-articular injections and ophthalmic viscoelastic agents) or high-end cosmetics (such as anti-aging serums).

[0108] Example 4

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

[0110] Step S1 Raw material preparation

[0111] Sodium hyaluronate (molecular weight 1.5-2.2 MDa, purity ≥98%, pharmaceutical grade, commercially available) was dissolved in a buffer system composed of acetic acid (analytical grade, purity ≥99.5%, commercially available) and sodium acetate (analytical grade, purity ≥99%, commercially available). In this embodiment, the total concentration of acetic acid and sodium acetate in the buffer system was 48 mmol·L⁻¹. -1 The molar ratio of acetic acid to sodium acetate was 1.4. A 1.5% sodium hyaluronate solution (maximum concentration, first-order boundary verification) was prepared, and 1 mol·L⁻¹ was used. -1 Acetic acid or 1 mol·L -1The pH was adjusted to 5.5 ± 0.05 (maximum pH) using sodium hydroxide solution. In this embodiment, hyaluronidase was immobilized using a magnetic nanoparticle composite carrier. The carrier consisted of Fe3O4@SiO2 core-shell magnetic nanoparticles (core Fe3O4 particle size approximately 50 nm, SiO2 shell thickness approximately 15 nm, modified with aminosilanization, commercially available), with a median volumetric particle size of 180 μm (aggregate particle size, measured by laser particle size analyzer). After 18 cycles at 50-200 MPa, the mechanical strength retention rate was 88% (assessed by carrier breakage rate). The immobilized enzyme was prepared using a covalent coupling method: 120 g of the magnetic nanoparticle composite carrier was placed in phosphate buffer (50 mmol·L⁻¹) at pH 7.5. -1 The enzyme was activated with glutaraldehyde (8 mL, 2.5% v / v) for 1.5 h. After thorough washing, hyaluronidase solution (total enzyme activity 3.6 million U, dissolved in 25 mL pH 7.0 phosphate buffer) was added, and the enzyme was coupled by slow stirring at 4 °C for 18 h. The coupling rate was approximately 70%, and the immobilized enzyme activity retention rate was approximately 58%, yielding an immobilized enzyme carrier (enzyme activity approximately 2.1 million U). The immobilized enzyme carrier was then loaded into a replaceable stainless steel mesh basket (80 μm mesh, with an internal permanent magnet to enhance carrier fixation) within a high hydrostatic chamber. The basket volume was 100 mL, and the equivalent enzyme concentration was approximately 60,000 U·g. -1 Sodium hyaluronate.

[0112] Step S2: High hydrostatic pressure enzymatic hydrolysis

[0113] The sodium hyaluronate solution (without free enzyme) obtained in step S1 was pumped into the reaction chamber of a high hydrostatic treatment device containing an immobilized enzyme core (total chamber volume 700 mL, substrate solution volume 600 mL). In this embodiment, the high hydrostatic enzymatic hydrolysis was performed using a pulsed pressurization method. The reaction temperature was set at 40°C, the hydrostatic pressure at 150 MPa, and the number of pulse cycles was 6 (minimum number of cycles). The time ratio of the pressurization phase to the depressurization phase was 1:0.3. In a single cycle, the pressurization phase lasted 30 min, and the depressurization phase lasted 9 min. The cumulative reaction time under pressurization was 180 min (3.0 h). The pressurization medium was deionized water, and the pressurization rate was approximately 240 MPa·min. -1 Decompression rate ≤120 MPa·min -1 Within each depressurization section, an external electromagnetic stirrer generates a rotating magnetic field (frequency 5 Hz, magnetic field strength 0.1 T) to drive the magnetic carrier to rotate slightly, thereby enhancing the mass transfer efficiency between the substrate and the immobilized enzyme.

[0114] Step S3: Multi-stage tangential flow ultrafiltration external circulation separation and reflux

[0115] This embodiment uses a simplified two-stage tangential flow ultrafiltration membrane module for external circulation separation (to verify the boundary case where a three-stage membrane module can be simplified to a two-stage module). The membrane material is polyethersulfone (PES) hollow fiber membrane, and the membrane areas are 0.18 m² (first stage) and 0.14 m² (second stage).

[0116] After the reaction in step S2 is completed, the reaction solution, depressurized to atmospheric pressure (with the immobilized enzyme carrier rapidly separated and recovered to the core using an external magnetic separator, recovery rate >98%), is subjected to tangential flow ultrafiltration. The specific operation is as follows:

[0117] (1) First-stage ultrafiltration: The reaction solution was pumped into the first-stage membrane module with a molecular weight cutoff of 200 kDa. The transmembrane pressure difference was set to 0.15 MPa and the membrane flux was 30 L·m -2 ·h -1 The circulation velocity is 2.0 m / s. -1 The temperature is controlled at 25±2℃. After collecting the retentate (high molecular weight component, >200 kDa) from the first-stage membrane, 80% of its volume is discarded, and the remaining 20% ​​is returned to step S2 for the next cycle of high hydrostatic enzymatic hydrolysis (pumped back into the chamber containing the immobilized enzyme, which has already been pre-recovered into the basket via magnetic separation). The permeate from the first-stage membrane (molecular weight ≤200 kDa) enters the second-stage ultrafiltration.

[0118] (2) Second-stage ultrafiltration: The permeate from the first-stage membrane is pumped into the second-stage membrane module (minimum molecular weight cutoff) with a molecular weight cutoff of 50 kDa. The transmembrane pressure difference is 0.15 MPa, and the membrane flux is 30 L·m. -2 ·h -1 The circulation velocity is 2.0 m / s. -1 The retentate from the second-stage membrane (molecular weight 50-200 kDa, containing the target product) is collected as the target product feed solution. 80% of the volume of the permeate from the second-stage membrane (molecular weight ≤50 kDa, consisting of small oligosaccharides and impurities) is mixed with 20% of the volume of the retentate from the first-stage membrane and returned to step S2 for further enzymatic hydrolysis. The remaining 20% ​​of the permeate is discarded (simplified process, no third-stage ultrafiltration).

[0119] In this embodiment, the ratio of reflux flow rate (total volume of feed liquid returned to S2) to product flow rate (volume of liquid retained by the second-stage membrane) is 20:1, the single membrane cycle time is 20 min, and the total number of cycles is 8.

[0120] Step S4 Termination and Purification

[0121] 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 80.5% and the change between two consecutive measurements (with an interval of one cycle) was less than 2.5%.

[0122] The target product solution was treated with 1 mol·L⁻¹ -1 The pH was adjusted to 7.3 ± 0.2 with sodium hydroxide solution, followed by the following treatment (due to the use of rapidly separable magnetically immobilized enzymes, there were no free enzymes or carrier residues in the feed solution):

[0123] (1) Enzyme inactivation: Heat the solution in an 88°C water bath for 19 min to completely inactivate any trace amounts of enzyme activity that may dissociate, and then cool to room temperature.

[0124] (2) Desalination: Transfer the feed solution to a dialysis bag (regenerated cellulose membrane, commercially available) with a molecular weight cutoff of 9 kDa, and dialyze in deionized water for 52 h, changing the dialysate every 8 h (the dialysate volume is 25 times the feed solution volume) until the dialysate conductivity is <5 μS·cm -1 .

[0125] (3) Freeze-drying: The dialyzed solution was pre-frozen at -42℃ for 16 h, and then dried in a freeze dryer (initial drying stage: -32℃, vacuum degree <8 Pa, 28 h; desorption drying stage: 28℃, 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 (the yield is highest due to the high concentration of substrate and the efficient immobilized enzyme system).

[0126] Product quality inspection:

[0127] The low molecular weight hyaluronic acid product obtained in this embodiment was tested, and the following indicators were observed:

[0128] Area percentage of the 50-100 kDa molecular weight range: 80.5% (in line with the 55-95% range, and at the upper limit of the preferred 65-90%).

[0129] Polydispersity index: 1.12 (in line with the range of 1.05-1.40, and preferably in the range of 1.08-1.30)

[0130] Percentage of molecular weight segments less than 10 kDa: 1.8% (within the range of 0-5%)

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

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

[0133] Nucleic acid content (UV spectrophotometry, 260 nm): 0.05% (meets 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 each)

[0135] Organic solvent residue (GC method): Methanol < 2 ppm, Ethanol 5 ppm (both meet the requirement of ≤10 ppm each)

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

[0137] Features of Example 4:

[0138] This embodiment employs a combination of limiting parameters to systematically verify the feasibility of the method under boundary conditions and the stability of product quality. Several core parameters were taken as actual boundary values: sodium hyaluronate concentration (1.5%), pH value (5.5), and enzyme dosage (60,000 U·g). -1 ), static pressure (150 MPa), number of pulse cycles (6), molecular weight cutoff of the second-stage membrane (50 kDa), transmembrane pressure difference (0.15 MPa), membrane flux (30 L·m -2 ·h -1The synergistic effect of these boundary parameters—single membrane cycle time (20 min), total number of cycles (8 times)—fully demonstrates the industrial feasibility of the parameter range defined in the claims. This embodiment uses a magnetic nanoparticle composite carrier (median volumetric particle size 180 μm) to immobilize the enzyme, combined with pulsed high hydrostatic pressure (150 MPa × 6 cycles) and external rotating magnetic field-assisted mass transfer technology. High-efficiency enzymatic hydrolysis was achieved under high substrate concentration conditions (1.5%), with 80.5% of the product in the 50-100 kDa range (close to the preferred upper limit of 90%), a polydispersity index of only 1.12 (excellent uniformity), and extremely low levels of small molecule impurities (only 1.8% in the <10 kDa range). All quality indicators fully meet or exceed the standards of the low molecular weight hyaluronic acid product of this invention, especially with strict control of heavy metals and endotoxins, making it suitable for injectable medical sodium hyaluronate products (such as intra-articular injections and ophthalmic surgical adjuvants). The successful validation of the simplified two-stage membrane module demonstrates that, under specific process conditions, equipment complexity can be reduced. The high reflux ratio (20:1) and multiple cycles (8 times) strategy effectively improved feed utilization to 84% (maximum yield). The rapid separation characteristics of the magnetic carrier (external magnetic separator, recovery rate >98%) allow for continuous use of the immobilized enzyme for 12-15 batches, significantly reducing production costs and making it 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 industrial scale-up.

[0139] Comparative Example 1: It is basically the same as Example 1, except that the mass-volume fraction of sodium hyaluronate solution is 1.8%, while the amounts of other components and preparation conditions remain unchanged.

[0140] Comparative Example 2: It is basically the same as Example 1, except that the mass-volume fraction of sodium hyaluronate solution is 0.2%, while the amounts of other components and preparation conditions remain unchanged.

[0141] Comparative Example 3: Basically the same as Example 1, except that the pH was adjusted to 6.0, while the amounts of other components and preparation conditions remained unchanged.

[0142] Comparative Example 4: Basically the same as Example 1, except that the pH was adjusted to 4.0, while the amounts of other components and preparation conditions remained unchanged.

[0143] Comparative Example 5: Basically the same as Example 1, except that the enzyme dosage was 15000 U·g. -1 Sodium hyaluronate was added, while the amounts of other components and preparation conditions remained unchanged.

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

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

[0146] Comparative Example 8: Basically the same as Example 1, except that the reaction temperature is 28°C, while the amounts of other components and preparation conditions remain unchanged.

[0147] Comparative Example 9: Basically the same as Example 1, except that the reaction temperature is 48°C, while the amounts of other components and preparation conditions remain unchanged.

[0148] Comparative Example 10: Basically the same as Example 1, except that a continuous pressurization method was used (not a pulse pressurization method), and the reaction was carried out continuously at 100 MPa for 100 min. The amounts of other components and preparation conditions remained unchanged.

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

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

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

[0152] Performance testing:

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

[0154] Test Subjects: Low molecular weight hyaluronic acid products and samples from various examples and comparative examples. Test Objective: To determine the molecular weight distribution curve of the samples, calculate the area ratio of the 50-100 kDa molecular weight range, number-average molecular weight, weight-average molecular weight, and polydispersity index (PDI) to evaluate the homogeneity of the product's molecular weight and batch stability. Test Principle: High-performance liquid chromatography (HPLC) coupled with a multi-angle laser light scattering detector (MALLS) is used. After the sample is separated by the chromatographic column, MALLS directly determines the absolute molecular weight of each component based on the intensity of scattered light at different angles, while a differential refractive index (RI) detector determines the concentration. The combined use of these two techniques provides molecular weight distribution data without the need for standard calibration. Experimental Method: Weigh 5.0 mg of the sample and dissolve it in the mobile phase (0.15 mol·L⁻¹). -1 Prepare a solution of 1.0 mg / mL NaCl + 0.02% NaN3 aqueous solution. -1The solution was filtered through a 0.22 μm filter membrane and injected in 20 μL increments. The chromatographic column used was a TSKgel G5000PWXL + G4000PWXL + G3000PWXL tandem column (Tosoh, 300 × 7.8 mm), with a column temperature of 30℃ and a flow rate of 0.5 mL / min. -1 Isocratic elution was performed for 60 min. The MALLS detector (Wyatt miniDAWNTREOS) used a laser wavelength of 658 nm with detection angles of 42° / 90° / 138°, and the RI detector (Wyatt Optilab T-rEX) was used at 35°C. Data acquisition was performed using ASTRA software, and the molecular weight was calculated using the Zimm fitting model. The dn / dc value was taken as 0.153 mL·g. -1 Standard Basis: Refer to Appendix A of GB / T 26329-2010 "Sodium Hyaluronate" and ASTM D6474-12 standard. Key Parameter: Injection concentration 1.0 mg·mL -1 Flow rate 0.5 mL·min -1 The detection time was 60 min, and the data acquisition frequency was 1 Hz. Data processing: ASTRA software was used to automatically integrate the peak areas, calculate the percentage of the area in the 50-100 kDa range (%), the number-average molecular weight Mn (kDa), the weight-average molecular weight Mw (kDa), and PDI = Mw / Mn. Each sample was measured 3 times, and the average value ± standard deviation was taken.

[0155] Experimental Scheme 2: Viscosity Measurement

[0156] Test Subject: A 1% (w / v) aqueous solution of low molecular weight hyaluronic acid product. Test Objective: To determine the dynamic viscosity of the product's aqueous solution, evaluate its flowability and processing suitability, and verify the effectiveness of the high solids content, low viscosity technology. Test Principle: The shear stress of the fluid at a specific shear rate is measured using a rotational viscometer, based on Newton's law of viscosity. Calculate the dynamic viscosity (τ is the shear stress). (Shear rate). Experimental method: Accurately weigh 1.000 g of sample into a beaker, add deionized water to 100 mL, stir magnetically at 25℃ for 4 hours until completely dissolved (avoid air bubbles), transfer to a constant temperature water bath (25.0±0.1℃) for equilibration for 30 min. A Brookfield DV-II+ Pro rotational viscometer with an S61 rotor (cone-plate type) was used, with a sample volume of 0.5 mL and a shear rate set to 100 s⁻¹. -1 (Spindle speed 60 rpm), test time 60 s, read the viscosity value (mPa·s) after stabilization. Each sample was measured in triplicate, and the mean and standard deviation were calculated. Shear rates were also measured at 10, 50, 100, 200, and 500 s. -1Plot a rheological curve based on the viscosity to determine the fluid type (Newtonian or non-Newtonian). Standards: Refer to GB / T1632-1993 "Determination of Viscosity of Multicomponent Polymer Systems - Ubbelohde Viscometer Method" and ISO 3219-1993. Key parameters: Sample concentration 1.0% (w / v), test temperature 25℃, shear rate 100 s⁻¹. -1 Rotor S61. Data processing: Record 100 seconds. -1 Given the dynamic viscosity η (mPa·s) at 25℃, calculate the relative viscosity ηr = η / η0 (η0 is the viscosity of water at 25℃, which is 0.89 mPa·s). For each set of data, n=3, give the mean ± standard deviation.

[0157] Experimental scheme 3: Protein content determination

[0158] Test Subject: Low molecular weight hyaluronic acid product. Test Purpose: To determine the residual protein content in the product, evaluate purification effectiveness and biosafety (protein residue may cause allergic reactions). Test Principle: The BCA (diquinoline carboxylic acid) colorimetric method is used. Proteins react with Cu under alkaline conditions. 2+ Reduced to Cu + Cu + It chelates with BCA to form a purple complex with characteristic absorption at 562 nm; the absorbance is directly proportional to the protein concentration. Experimental method: Accurately weigh 50.0 mg of the sample into a 10 mL volumetric flask, add 0.5 mol·L⁻¹ -1 Dissolve the protein in 2 mL of NaOH solution (alkaline conditions promote protein dissolution), add water to the mark, mix well, and then take 200 μL of the sample solution into a microplate. Add 200 μL of BCA working solution (BCA reagent A: reagent B = 50:1), mix well, and incubate at 37℃ in the dark for 30 min. Measure the absorbance at 562 nm using a Bio-Rad iMark microplate reader. Simultaneously, prepare a bovine serum albumin (BSA) standard curve: prepare 0, 5, 10, 25, 50, 100, and 200 μg·mL⁻¹. -1 For a series of concentrations, the absorbance was measured using the same method, and a standard curve was plotted (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 Chapter 2.5.33. Key parameter: Sample concentration 5.0 mg·mL -1 BCA incubation was performed at 37℃ for 30 min, and the detection wavelength was 562 nm. Data processing: Sample protein concentration (μg·mL) was calculated based on the standard curve. -1The protein content (%) in the product is calculated as follows: (measured concentration × dilution factor × total volume) / (sample mass × 10000), n = 3. The mean ± standard deviation is given.

[0159] Experimental scheme 4: Nucleic acid content determination

[0160] Test Subject: Low molecular weight hyaluronic acid product. Test Purpose: To determine the residual nucleic acid (DNA / RNA) content in the product and evaluate the purification effect. Residual nucleic acid may affect product stability and safety. Test Principle: Nucleic acid molecules have a maximum ultraviolet absorption peak at 260 nm (π→π* transition of purine and pyrimidine bases). The absorbance follows the Lambert-Beer law with respect to nucleic acid concentration. By measuring A... 260 The nucleic acid content can be quantified. Experimental method: Accurately weigh 20.0 mg of sample into a 10 mL volumetric flask, add 0.01 mol·L⁻¹ -1 Dissolve the sample in 8 mL of NaOH solution, sonicate for 10 min to promote nucleic acid release, cool, and then add water to the mark. Filter through a 0.22 μm filter membrane. Take 1.0 mL of the filtrate in a quartz cuvette (1 cm path length) and measure the absorbance at 260 nm, 280 nm, and 320 nm (background correction) using a UV-Vis spectrophotometer (Thermo NanoDrop 2000c). Simultaneously, use 0.01 mol·L⁻¹... -1 NaOH solution was used as a blank control to determine A. 260 A 280 Value. Calculate A. 260 / A 280 Ratio (approximately 1.8-2.0 for pure nucleic acids; a low ratio suggests protein interference). Standard basis: Refer to GB 5009.268-2016 "National Food Safety Standard - Determination of Multiple Elements in Food" and USP 44 General Rules. <781> Nucleic acid assay. Key parameter: Sample concentration 2.0 mg / mL -1 Detection wavelengths: 260 nm / 280 nm / 320 nm, optical path length: 1 cm, blank control: 0.01 mol·L⁻¹ -1 NaOH. Data processing: Nucleic acid content (%) = (A 260 -A 320 ) × dilution factor × 50 μg·mL -1 (Nucleic acid extinction coefficient) / (sample concentration mg·mL) -1 ×1000), n=3, report the mean ± standard deviation and A 260 / A 280 ratio.

[0161] Experimental Scheme 5: Determination of Heavy Metal Content

[0162] Test Subject: Low molecular weight hyaluronic acid products. Test Purpose: To determine the content of four heavy metals—lead (Pb), arsenic (As), cadmium (Cd), and mercury (Hg)—in the product to ensure compliance with pharmaceutical-grade or food-grade safety standards (≤1 ppm each), thus guaranteeing product safety. Test Principle: Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is used. After microwave digestion, the sample is atomized and enters a plasma torch. The elements are ionized into ions, which are then separated by a mass analyzer according to their mass-to-charge ratio (m / z). The detector measures the signal intensity of each element ion, and quantification is achieved through a standard curve. Experimental Methods: Accurately weigh 0.5000 g of sample into a polytetrafluoroethylene digestion vessel, add 5 mL of HNO3 (analytical grade, ≥65%) and 2 mL of H2O2 (analytical grade, 30%), cover the vessel, and place it in a microwave digestion apparatus (CEM Mars 6). Proceed with the temperature program: 120℃ for 5 min, 150℃ for 5 min, and 180℃ for 20 min. After cooling, transfer to a 50 mL volumetric flask and dilute to volume with water. Simultaneously prepare a series of mixed standard solutions: 0, 0.5, 1.0, 5.0, 10.0, and 50.0 ppb of Pb, As, Cd, and Hg respectively (using 2% HNO3 as solvent), and plot a standard curve. ICP-MS (Agilent 7800) was used to 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" Method III and USP 44 General Rules. <233> Key parameters: Sample volume 0.5 g, digestion temperature 180℃, digestion time 20 min, final volume 50 mL, ICP-MS power 1550 W, carrier gas Ar flow rate 1.05 L·min -1 Data processing: Content (mg·kg) -1 = (measured concentration μg·L) -1 (×Volume at constant volume (L)×Dilution factor) / Sample mass (kg), n=3, report the mean ± standard deviation of each element.

[0163] Experimental scheme 6: Determination of endotoxin content

[0164] Test subject: Aqueous solution of low molecular weight hyaluronic acid product. Test purpose: To determine the endotoxin (bacterial lipopolysaccharide LPS) content of the product, ensuring it is ≤0.5 EU·mg. -1This test meets the safety requirements for injectable biological products. Test principle: A dynamic colorimetric method (Limulus amebocyte lysate (LAL) method) is used. Endotoxin catalyzes the activation of coagulase in the LAL reagent, causing the release of the substrate pNA (p-nitroaniline), producing a yellow product with an absorption peak at 405 nm. The rate of increase in absorbance is logarithmically linearly related to the endotoxin concentration. Experimental method: Accurately weigh 10.0 mg of the sample and use pyrogen-free water (LAL Reagent Water, <0.005 EU·mL) specifically for endotoxin detection. -1 Dissolve and dilute to 1.0 mg / mL -1 Equilibrate in a 37℃ water bath for 10 min. Prepare a series of endotoxin standards: 0.005, 0.05, 0.5, and 5.0 EU·mL. -1 (E. coli O55:B5 LPS, Charles River). In a pyrogen-free 96-well plate (pre-dried at 180°C for 4 h to remove pyrogens), add 100 μL of sample or standard and Limulus amebocyte lysate (Xiamen Bioendo, sensitivity 0.03 EU·mL) to each well. -1 100 μL was immediately incubated at 37°C using a Molecular Devices SpectraMax microplate reader. The absorbance at 405 nm was measured every 30 seconds, and data were recorded over 60 minutes. Standards based on: Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 1143, "Test Method for Bacterial Endotoxins" and USP 44 General Chapter. <85> Key parameter: Sample concentration 1.0 mg·mL -1 The sensitivity of the horseshoe crab reagent is 0.03 EU·mL. -1 The incubation temperature was 37℃, the detection time was 60 min, and the wavelength was 405 nm. Data processing: The reaction time (Onset Time) was calculated based on the slope of the standard curve, and the endotoxin concentration (EU·mL) was converted. -1 Then calculate the endotoxin content of the product (EU·mg). -1 (n=3), report the mean ± standard deviation.

[0165] Experimental Scheme 7: Colorimetric Measurement

[0166] Test subject: A 5% (w / v) aqueous solution of low molecular weight hyaluronic acid product. Test purpose: To determine the color (yellow index YI and brightness L) of the product solution. * This method evaluates the appearance quality of a product. Low color intensity indicates low degradation and high purity, which is beneficial for cosmetic applications. Testing principle: Using the CIE Lab color space system, the tristimulus values ​​X, Y, and Z of the sample are measured using a spectrophotometer. The luminance L (0 for black, 100 for white) and the chromaticity a along the yellow and blue axes are calculated. *The red-green axis chromaticity b and yellow index YI = 100 × (1.28X - 1.06Z) / Y. Experimental method: Accurately weigh 5.000 g of the sample into a 100 mL beaker, add deionized water to 100 mL, stir magnetically at 25℃ for 2 hours until completely dissolved, let stand for 30 min to remove air bubbles, and transfer to a quartz cuvette (10 mm optical path, four-sided transparent). Measure using a spectrophotometer (Konica Minolta CM-5) in transmission mode, with a light source of D65 (standard daylight), an angle of view of 10°, a measuring aperture of ϕ8 mm, and a measurement range of 400-700 nm. Using deionized water as a reference, record the L, a*, b*, X, Y, and Z values. Each sample was measured four times by rotating the cuvette 90°, and the average value was taken. Standard Basis: Refer to GB / T 3979-2008 "Methods for Measurement of Object Color" and ASTM E313-20 "Standard Practice for Calculating the Yellow Index of White or Near-White Opaque Materials". Key Parameters: Sample concentration 5% (w / v), optical path 10 mm, light source D65, viewing angle 10°, measurement wavelength range 400-700nm. Data Processing: Record L* (brightness), a*, b values ​​and calculate YI value, n=4, report the mean ± standard deviation; L>90 and YI<5 indicate a high-quality product (low color intensity).

[0167] Combination Figures 1 to 3 Quantitative proof can be provided: Figure 1 The pressure-temperature-time curves, across 10 independent batches, showed a peak pressure of 200 MPa, a pulse width of 30.0 ± 0.8 s, a period of 120 ± 2 s, a temperature rise ≤ 1.2 ℃ during the holding phase, and a steady-state temperature maintained at 35.0 ± 0.5 ℃. The pressure fluctuation coefficient (CV) within the same batch was 0.9%, and the inter-batch CV was 1.3%, with no abnormal overshoot or lag, indicating that the spatiotemporal parameters were controlled and reproducible. Correspondingly, Figure 2 The gel permeation chromatography-multi-angle laser light scattering / differential refractive index overlay showed a weight-average molecular weight (Mw) of 72.5 ± 1.8 kDa, a number-average molecular weight (Mn) of 61.9 ± 1.5 kDa, a main peak half-width (HW) of 0.21 ± 0.03 (volume fraction), a main peak percentage of ≥ 88%, a shoulder peak percentage of ≤ 8%, a peak position difference of ≤ 0.05 mL between multi-angle light scattering and differential refractive index at the same elution volume, a signal correlation coefficient (r) of ≥ 0.995, and a refractive index increment (dn / dc) calibrated to 0.153 mL / g. The calculated absolute molecular weight deviated from the external standard calibration value by ≤ 3%. Furthermore, Figure 3The polydispersity index (PDI) was 1.17 ± 0.04 (n = 10), the interquartile range (IQR) was 0.06, and the maximum outlier PDI was 1.25. Tracing back to the source, this corresponded to a single, minor temperature disturbance (+1.0 ℃, lasting approximately 2 min), which did not alter the overall statistical range. Simultaneously, the inter-batch RSD of Mw was 2.5%, and the inter-batch RSD after peak area normalization was 2.1%, consistent with the SEC-MALS summary parameters. The linear region of the fitted Zimm plot showed R² ≥ 0.992, and the recovery rate was 98.3 ± 1.2%. These data constitute a quantitative chain of evidence demonstrating process control (low CV, low drift), measurement reliability (consistent peak positions in both channels, high correlation, high recovery), and product consistency (low PDI, narrow distribution, low RSD). This proves that the proposed scheme meets engineering requirements in terms of rationality, reliability, and effectiveness, and can support the stable realization and scale-up reproduction of the target performance.

[0168] Figure 4 This invention investigates the effect of static pressure on the proportion and yield of the 50-100 kDa molecular weight range; basic parameters: sodium hyaluronate solution mass / volume fraction 0.8%, pH 5.0, enzyme dosage 40,000 U / g. -1 Sodium hyaluronate, reaction temperature 38℃, 10 pulse cycles, pressurization / depressurization time ratio 1:1, single-cycle pressurization time 10 min, first-stage membrane molecular weight cutoff 150 kDa, second-stage membrane molecular weight cutoff 75 kDa, transmembrane pressure difference 0.10 MPa, reflux flow rate / product flow rate ratio 10:1; variable parameters: static pressure 40-220 MPa; conclusion: the 50-100 kDa range in the 120-140 MPa range has the highest proportion and yield. Too low pressure leads to insufficient enzyme catalysis, and a decrease in the target range and yield. Too high pressure leads to excessive degradation and partial inactivation, and a decrease in the target range and yield. Figure 5 This invention investigates the effect of sodium hyaluronate solution concentration on the proportion and viscosity of the 50-100 kDa molecular weight range; basic parameters: pH 5.0, enzyme dosage 40,000 U·g. -1 Sodium hyaluronate, reaction temperature 38℃, static pressure 100 MPa, 10 pulse cycles, pressurization / depressurization time ratio 1:1, single-cycle pressurization time 10 min, first-stage membrane molecular weight cutoff 150 kDa, second-stage membrane molecular weight cutoff 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: the target fraction has the highest proportion and moderate viscosity at 1.0%-1.2% concentration. Too low a concentration results in insufficient substrate and a decreased proportion, while too high a concentration leads to a sharp increase in viscosity, limited contact, and a decreased proportion. Figure 6This invention investigates the effect of pH on the proportion of molecules in the 50-100 kDa molecular weight range and the polydispersity index; basic parameters: sodium hyaluronate solution mass / volume fraction 0.8%, enzyme dosage 40,000 U / g. -1 Sodium hyaluronate, reaction temperature 38℃, static pressure 100 MPa, 10 pulse cycles, pressurization / depressurization time ratio 1:1, single-cycle pressurization time 10 min, first-stage membrane molecular weight cutoff 150 kDa, second-stage membrane molecular weight cutoff 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: the target range of pH 5.0-5.3 has the best proportion and PDI, slightly acidic conditions enhance non-enzymatic degradation leading to a wider distribution, while slightly neutral conditions decrease enzyme activity leading to insufficient degradation. Figure 7 This study investigates the effect of the reflux flow rate / product flow rate ratio on the proportion and yield of the 50-100 kDa molecular weight range. Basic parameters: sodium hyaluronate solution mass / volume fraction 0.8%, pH 5.0, enzyme dosage 40,000 U / g. -1 Sodium hyaluronate, reaction temperature 38℃, static pressure 100 MPa, 10 pulse cycles, pressurization / depressurization time ratio 1:1, single-cycle pressurization time 10 min, first-stage membrane molecular weight cutoff 150 kDa, second-stage membrane molecular weight cutoff 75 kDa, transmembrane pressure difference 0.10 MPa; variable parameter: reflux flow rate / product flow rate ratio 0.5-35; conclusion: the target range of 15-25 has the highest proportion and yield. Too low a range results in insufficient reflux re-degradation, with a low proportion and yield, while too high a range results in small marginal benefits and increased process load, and the overall performance is not as good as the optimal range.

[0169] As can be seen from the performance summary of the examples and comparative examples in Tables 1 and 2, deviations from the optimal window in Example 1 all deteriorate the proportion, yield, and uniformity of the target 50–100 kDa segment: when the sodium hyaluronate concentration is too high (1.8%, Comparative Example 1), the system viscosity increases sharply, molecular entanglement intensifies, mass transfer is limited, and uneven enzymatic hydrolysis leads to a decrease in the proportion of the target segment; when it is too low (0.2%, Comparative Example 2), there is insufficient substrate, the effective collision frequency decreases, and the yield and the proportion of the target segment decrease simultaneously. Increasing the pH to 6.0 (Comparative Example 3) significantly inhibits enzyme activity and causes insufficient degradation, resulting in a worsening of the proportion and uniformity of the target segment; decreasing the pH to 4.0 (Comparative Example 4) enhances the acidic non-enzymatic pathway, partially inactivates the enzyme, broadens the molecular weight distribution, and increases the proportion of small molecules. The enzyme dosage was reduced to 15000 U·g. -1(Comparative Example 5) This results in insufficient catalytic sites, slower degradation kinetics, and a decrease in both target fragment enrichment and yield. A static pressure that is too low (40 MPa) (Comparative Example 6) leads to insufficient synergistic effects between enzymatic and pressure catalysis, inadequate chain fragmentation, and a decline in performance indicators; a pressure that is too high (220 MPa) (Comparative Example 7) causes excessive degradation and enzyme inactivation, resulting in a smaller molecular weight distribution, a lower proportion of the target fragment, and a decrease in yield. A temperature that is too low (28℃) (Comparative Example 8) reduces enzyme activity and mass transfer, slowing the reaction; a temperature that is too high (48℃) (Comparative Example 9) induces thermal inactivation of the enzyme and side reactions, both of which are detrimental to target fragment control. Using continuous pressurization instead of pulsed pressure (Comparative Example 10) weakens the selective shearing and mass transfer refresh effects of the pressure pulse, leading to a decrease in degradation selectivity and distribution control. Reducing the first-stage membrane cutoff to 80 kDa (Comparative Example 11) disrupts the molecular weight window management of the cascade separation, resulting in insufficient polymer recovery and re-degradation, escape or excessive retention of the target fragment, and impaired distribution and yield. When the reflux ratio drops to 0.5:1 (Comparative Example 12), there is essentially no effective reflux, and the polymer cannot be recycled and re-enzymatically hydrolyzed, resulting in a significant decrease in feed utilization and the proportion of the target segment. When the transmembrane pressure difference rises to 0.25 MPa (Comparative Example 13), concentration polarization and membrane compaction are exacerbated, the fractionation selectivity and flux stability deteriorate, and the distribution deviation is easily amplified, sacrificing the proportion of the target segment and the overall yield.

[0170] Table 1. Molecular weight distribution and rheological properties of the examples and comparative examples.

[0171] Table 2 summarizes the purity and safety performance of the examples and comparative examples.

[0172]

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing low molecular weight hyaluronic acid by high hydrostatic pressure-assisted enzymatic hydrolysis, characterized in that... ,include: S1 Raw Material Preparation: Dissolve sodium hyaluronate in a buffer system composed of acetic acid and sodium acetate, resulting in a solution with a mass-volume fraction of 0.3–1.5%. Adjust the pH to 4.5–5.5 and prepare the solution at a concentration of 20,000–80,000 U·g. -1 Sodium hyaluronate was added to hyaluronidase derived from bovine testis; S2 high hydrostatic pressure enzymatic hydrolysis: The reaction is carried out at 30–45℃ and 50–200MPa hydrostatic pressure, with a cumulative reaction time of 1.0–5.0 h under pressure. S3 Multistage Tangential Flow Ultrafiltration External Circulation Separation and Reflux: After the reaction solution is depressurized to atmospheric pressure, it undergoes external circulation separation through at least two stages of tangential flow ultrafiltration membrane arrays. The molecular weight cutoff of the first stage membrane is 110–300 kDa, and the molecular weight cutoff of the second stage membrane is 50–100 kDa. The feed solution that has passed through the first stage membrane and is retained by the second stage membrane is collected as the target product feed solution. A portion of the retentate from the first stage membrane is mixed with a portion of the permeate from the second stage membrane and refluxed to step S2 to continue the reaction. The remaining portion is discarded, and the solution is pressurized again to enter the next cycle. S4 Termination and Purification: The reaction is terminated when the area ratio of the 50-100kDa molecular weight range in the target product solution reaches 55-95% or the change in the molecular weight distribution of the product solution is less than 5% in two consecutive measurements. The pH of the product solution is adjusted to 6.5-7.5, and then enzyme inactivation and desalting are performed. The product is then freeze-dried. The high hydrostatic pressure is applied by 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. The transmembrane pressure difference of the tangential flow ultrafiltration is 0.03–0.20 MPa, and the membrane flux is 5–50 L·m⁻¹. -2 ·h -1 The ratio of return flow to product flow is 1–30.

2. The method according to claim 1, characterized in that... The static pressure is 80–150 MPa, the temperature is 35–42 °C, and the cumulative reaction time under pressure is 1.5–4.0 h.

3. The method according to claim 1, characterized in that... The tangential flow ultrafiltration uses a three-stage membrane module, wherein the molecular weight cutoff of the first stage membrane is 120–200 kDa, the molecular weight cutoff of the second stage membrane is 50–100 kDa, and the molecular weight cutoff of the third stage membrane is 8–20 kDa. The permeate from the third stage membrane is discharged to remove small molecule impurities.

4. The method according to claim 1, characterized in that... The single membrane cycle time is 5–30 min, and the total number of cycles is 2–10.

5. The method according to claim 1, characterized in that... The hyaluronidase exists in an immobilized form and is packed in a replaceable basket core within a high hydrostatic chamber. The immobilization carrier is selected from at least one of cross-linked agarose gel, macroporous silica, cross-linked polystyrene microspheres, and magnetic nanoparticle composite carriers. The median particle size of the immobilized carrier is 100–200 μm, and its mechanical strength retention rate is 85–100% after 10–20 cycles under 50–200 MPa conditions.

6. A low molecular weight hyaluronic acid product, characterized in that... The product is prepared by the method described in any one of claims 1-5, and the product satisfies the following conditions: the area ratio of the 50-100 kDa molecular weight segment is 55-95%, the polydispersity index is 1.05-1.40, the area ratio of the molecular weight segment less than 10 kDa 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 content of lead, arsenic, cadmium, and mercury is not more than 1 ppm each, the residual amount of methanol and ethanol is not more than 10 ppm each, and the endotoxin content is not more than 0.5 units / mg. -1 .

7. The product according to claim 6, characterized in that... The area ratio of the 50–100 kDa molecular weight range is 65–90%, and the polydispersity index is 1.08–1.

30.

8. The use of the low molecular weight hyaluronic acid product according to claim 6 or 7 in the preparation of cosmetics, functional foods or biomedical materials.

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