Plant biological reaction composite stock solution and preparation process thereof

Abstract

This invention belongs to the field of plant polysaccharide complex stock solution technology, and provides a plant bioreaction complex stock solution and its preparation process. This invention constructs a complex nano-complex by aldehyde-modified oligosaccharide of snow lotus and a collagen-hyaluronic acid conjugated intermediate, and combines it with N-acetylglucosamine, yeast β-glucan, sodium chloride and citrate buffer system to achieve coordinated improvement in dispersion stability, low irritation, processing fluidity and moisture retention. It solves the problem of balancing stability and mildness, fluidity and moisture retention in the complex system of plant polysaccharides, collagen and hyaluronic acid. It is suitable for biomaterial stock solutions, functional stock solutions and related complex preparations.

Description

Technical Field

[0001] This invention relates to the field of plant polysaccharide complex stock solution technology, specifically to a plant bioreaction complex stock solution and its preparation process. Background Technology

[0002] Plant-derived active polysaccharides, collagen-related intermediates, and hyaluronic acid-related components have attracted significant attention in biomaterials, functional feedstocks, and composite aqueous systems. These systems typically require uniform dispersion at high water content, mild usability, suitable flowability, and sustained moisture retention. Furthermore, they need to maintain stable appearance and controllable particle size during preparation, filtration, filling, and storage. As composite functional feedstocks evolve from single-channel moisture retention to multi-channel regulation, the system not only needs to provide a hydrophilic network, compliant application characteristics, and basic osmotic pressure regulation, but also needs to consider the degree of intermediate reactivity, ionic strength, buffering environment, and low-viscosity processing window. If the composite system lacks coordination among intermolecular interactions, particle size distribution, and solids content, it will be difficult to simultaneously meet the requirements of production operability, shelf stability, and mild usability. Therefore, constructing a composite feedstock with synergistic matching of plant oligosaccharides, collagen-hyaluronic acid conjugated intermediates, and mild aqueous excipients is of great significance for improving the application adaptability of composite active systems.

[0003] Existing plant polysaccharide, collagen, and hyaluronic acid complex systems typically rely on increasing intermolecular interaction strength, enhancing colloidal networks, or adjusting the ionic environment to improve dispersion stability. However, these methods can easily lead to excessively high system reactivity, increased residual control pressure, or a higher risk of irritation. Conversely, reducing reactivity, decreasing solids contribution, or enhancing purification can easily loosen the complex structure and weaken dispersion stability. On the other hand, to reduce processing viscosity and improve filtration and filling convenience, existing systems often employ low-molecular-weight or low-viscosity treatments. However, these treatments may reduce the contribution of hydrophilic water-retaining structures; while increasing the content of water-retaining components can increase viscosity and the risk of aggregation. Chinese patent CN114699335A discloses a stable collagen-hyaluronic acid miscible system and its preparation method. It obtains a miscible system by modifying hyaluronic acid with cationic cellulose and then mixing it with a collagen solution. However, this type of solution mainly focuses on regulating the miscibility of collagen and hyaluronic acid, and does not systematically design the multi-parameter coupling between plant aldehyde oligosaccharides, collagen-hyaluronic acid conjugated intermediates, nanocomplex particle size, and mild buffer solution. Summary of the Invention

[0004] The purpose of this invention is to provide a plant bioreaction complex stock solution and its preparation process, which solves the problem that it is difficult to simultaneously achieve stability, mildness, processing fluidity and moisture retention in the current plant polysaccharide, collagen and hyaluronic acid complex system.

[0005] This invention achieves a balance between the need for reaction stabilization and low irritation by matching the ratio of aldehyde-modified oligosaccharide and collagen-hyaluronic acid conjugated intermediates and reducing stabilization. Furthermore, it mitigates the adverse effects of single enhancement or single low viscosity treatment by leveraging the synergistic regulation of flowability and moisture retention contribution of nano-complex particle size, salt flushing, and hydrophilic small molecules.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A plant bioreaction complex stock solution, comprising the following components: excluding purified water, the remaining components are calculated based on the total solids of the plant bioreaction complex stock solution. The content of A1-derived plant oligopolysaccharide intermediates is 15-45 wt%, and the A1-derived plant oligopolysaccharide intermediates are aldehyde-based oligosaccharides of Saussurea involucrata obtained by oxidative oligomerization of Saussurea involucrata culture powder. The content of B1-derived collagen-hyaluronic acid intermediate is 5-25 wt%, and the B1-derived collagen-hyaluronic acid intermediate is a conjugated intermediate obtained by coupling hydrolyzed collagen or gelatin with sodium hyaluronate. The content of N-acetylglucosamine is 0.1-10 wt%; The content of yeast β-glucan is 0.1-10 wt%; The sodium chloride content is 0.1-1.5 wt%; The total content of one or both of citric acid and trisodium citrate dihydrate is 0.05-1.0 wt%; based on the total mass of the plant bioreaction compound stock solution, The remaining solids are the balance, which include coexisting solids from the extraction and grading process of snow lotus culture powder, the purification process of A1-derived plant oligosaccharide intermediates, the purification process of B1-derived collagen-hyaluronic acid intermediates, and the adjustment process of the citrate buffer system. The remainder is purified water.

[0007] Furthermore, the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate are subjected to aldehyde-amino condensation and reduction stabilization at a solids mass ratio of 4:1-1:4 to form a composite nanocomplex. The composite nanocomplex has a dynamic light scattering Z-average particle size of 80-300 nm in the plant bioreaction composite stock solution, the pH value of the plant bioreaction composite stock solution is 4.2-6.2, and the solids content of the plant bioreaction composite stock solution is 1-15 wt%.

[0008] Furthermore, the A1-derived plant oligosaccharide intermediate is prepared through the following steps: A1. Raw material preparation: By weight, the raw materials include 100 parts by weight of snow lotus culture powder, 3-30 parts by weight of sodium periodate, 1-10 parts by weight of glycerol, and 1000-3000 parts by weight of purified water; A2. Extraction and grading: Disperse snow lotus culture powder in purified water and extract for 1-4 hours at a temperature of 45-65℃. After filtration, pass the filtrate through a membrane with a molecular weight cutoff of 50kDa and collect the permeate. Then pass the permeate through a membrane with a molecular weight cutoff of 1kDa and collect the retentate as the oligosaccharide enrichment portion. A3. Oxidative oligomerization: Adjust the pH of the oligosaccharide enrichment obtained in step A2 to 4.0-7.0, and react it with sodium periodate at 10-30℃ under light-protected conditions for 0.5-4 hours; A4. Termination and Post-processing: Glycerol was added to terminate the reaction, followed by dialysis or ultrafiltration to remove salt and concentration, to obtain the A1-derived plant oligosaccharide intermediate with an aldehyde content of 0.20-1.50 mmol / g and a weight-average molecular weight of 1-20 kDa based on dry basis.

[0009] Furthermore, the B1-derived collagen-hyaluronic acid intermediate is prepared through the following steps: B1. Raw material preparation: By weight, the raw materials include 10-40 parts sodium hyaluronate, 20-80 parts hydrolyzed collagen or gelatin, 2-20 parts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-15 parts N-hydroxysuccinimide, and 500-3000 parts buffer solution composed of citric acid and trisodium citrate dihydrate. B2. Dissolution and activation: Sodium hyaluronate is dissolved in the buffer solution with a pH of 4.5-6.0, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially at 10-25°C, and the mixture is activated for 0.5-2 hours. B3. Coupling reaction: Add hydrolyzed collagen or gelatin and react at 15-35℃ for 2-12 hours; B4. Purification: Remove unreacted small molecule reagents by ultrafiltration or dialysis, then concentrate or freeze-dry to obtain the B1-derived collagen-hyaluronic acid intermediate with a binding rate of 5-30 wt% based on hyaluronic acid and a residual amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride not exceeding 100 mg / kg.

[0010] Furthermore, the composite nanocomplex is prepared through the following steps: C1. The A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate are mixed in a solid mass ratio of 4:1 to 1:4 in a buffer system with a pH of 4.8-6.2 composed of citric acid and trisodium citrate dihydrate; C2. The condensation reaction of aldehyde and amino groups is carried out at 15-30℃ for 1-6 hours; C3. Based on 100 parts by weight of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate, add 0.1-5 parts by weight of sodium borohydride, reduce and stabilize at 0-10°C for 0.2-2 hours, and remove borates and small molecule salts by dialysis or ultrafiltration. C4. The composite nanocomplex with an average particle size of 80-300 nm is obtained by high-pressure homogenization treatment at 40-90 MPa 2-5 times.

[0011] Furthermore, based on the total solids of the plant bioreaction complex stock solution, the content of the A1-derived plant oligosaccharide intermediate is 20-40 wt%, the content of the B1-derived collagen-hyaluronic acid intermediate is 8-20 wt%, the content of the N-acetylglucosamine is 0.5-5 wt%, and the content of the yeast β-glucan is 0.1-5 wt%. The A1-derived plant oligosaccharide intermediate has an aldehyde content of 0.20-1.00 mmol / g on a dry basis and a weight-average molecular weight of 2-10 kDa; the sodium hyaluronate has an average molecular weight of 5-300 kDa. When the protein component in the B1-derived collagen-hyaluronic acid intermediate is hydrolyzed collagen, its average molecular weight is 0.5-10 kDa; when the protein component in the B1-derived collagen-hyaluronic acid intermediate is gelatin, its Bloom strength is 100-300 g; the snow lotus culture powder is derived from the callus culture of Tian Shan snow lotus, and its dried product contains not less than 7 wt% total flavonoids and not less than 20 wt% protein.

[0012] As a concept of this invention, a composite nanocomplex is designed to form a complex of aldehyde-derived oligosaccharide from *Saussurea involucrata* and a collagen-hyaluronic acid conjugated intermediate. This design aims to achieve a synergistic balance between dispersion stability and low irritation. Existing plant polysaccharide and collagen-hyaluronic acid composite systems typically enhance intermolecular reactions or network constraints to improve dispersion stability; however, excessively strong reactions can increase residue control and irritation risks. Conversely, to improve mildness, reactivity is often reduced or purification is intensified, leading to insufficient complexation and decreased dispersion stability. This invention utilizes aldehyde-amino group condensation, reduction stabilization, buffered pH, and particle size control to provide controllable reaction sites for the A1-derived plant oligosaccharide intermediate and a flexible hydrophilic framework for the B1-derived collagen-hyaluronic acid intermediate. Furthermore, N-acetylglucosamine, yeast β-glucan, and salts regulate the aqueous microenvironment, thereby reducing the adverse effects of single stabilization or single low-irritation treatments.

[0013] This invention also discloses a method for preparing a plant bioreaction complex stock solution, comprising the following steps: S1. Provide the prepared A1-derived plant oligosaccharide intermediate; S2. Provide the prepared B1-derived collagen-hyaluronic acid intermediate; S3. The A1-derived plant oligosaccharide intermediate provided in step S1 and the B1-derived collagen-hyaluronic acid intermediate provided in step S2 are mixed in a buffer system with a pH of 4.8-6.2 composed of citric acid and trisodium citrate dihydrate at a solid mass ratio of 4:1-1:4. The aldehyde and amino groups are condensed, and the mixture is then reduced and stabilized with sodium borohydride and homogenized under high pressure to obtain a composite nanocomplex with an average particle size of 80-300 nm. S4. Add the composite nano-complex obtained in step S3, N-acetylglucosamine, yeast β-glucan, and sodium chloride to purified water. Based on the total solids of the plant bioreaction composite stock solution obtained in step S6, the amount of N-acetylglucosamine added is 0.1-10 wt%, the amount of yeast β-glucan added is 0.1-10 wt%, and the amount of sodium chloride added is 0.1-1.5 wt%. S5. Adjust the pH of the system obtained in step S4 to 4.2-6.2 using one or both of citric acid and trisodium citrate dihydrate; S6. The system obtained in step S5 is filtered using a combination of 200-1000 mesh filtration and 0.45μm terminal filtration, or it is filled in a low-bacterial environment where the microbial limit test results meet the requirements for release of low-bacterial stock solution, and purified water is added to bring the solid content of the obtained stock solution to 1-15wt% to obtain the plant bioreaction composite stock solution.

[0014] Further, in step S3, the solid mass ratio of the A1-derived plant oligosaccharide intermediate to the B1-derived collagen-hyaluronic acid intermediate is 2:1-1:2; the amount of sodium borohydride added is 0.2-2 wt% of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate; the reduction stabilization temperature is 0-5℃; the high-pressure homogenization pressure is 50-80 MPa, the homogenization times are 3-4 times, and the average particle size of the resulting composite nanocomplex is 100-250 nm.

[0015] Furthermore, in step S4, based on the total solids of the plant bioreaction complex stock solution obtained in step S6, the amount of N-acetylglucosamine added is 0.5-5 wt%, the amount of yeast β-glucan added is 0.1-5 wt%, and the amount of sodium chloride added is 0.3-0.9 wt%.

[0016] Furthermore, in step S6, the filtration process involves first performing a 200-1000 mesh pre-filtration, followed by a 0.45μm terminal filtration.

[0017] Furthermore, in step S6, the obtained plant bioreaction compound stock solution is filled into single-use bottles, cans, or bags.

[0018] Furthermore, the membrane separation in step A2 is carried out in a sequential fractionation manner. The filtrate obtained by hot water extraction and filtration of snow lotus culture powder is first passed through a membrane with a molecular weight cutoff of 50 kDa, and the permeate is collected. Then, the permeate is passed through a membrane with a molecular weight cutoff of 1 kDa, and the retentate is collected as the oligosaccharide enrichment part. The oligosaccharide enrichment part enters step A3 for oxidative oligomerization.

[0019] Furthermore, the oligosaccharide enrichment obtained in step A2 was tested for solids content, polysaccharide content, protein content, and molecular weight distribution. The polysaccharide content was determined and recorded using the phenol-sulfuric acid method, the protein content was determined and recorded using the BCA method, and the molecular weight distribution was determined and recorded using the GPC / SEC method. The obtained test data were used for the feed amount control in step A3 and the intermediate quality control in step A4.

[0020] Further, in step A3, before adding sodium periodate, the pH of the oligosaccharide enrichment obtained in step A2 is adjusted to 4.0-7.0 and kept at 10-30°C in the dark. After adding sodium periodate, the reaction is kept in the dark for 0.5-4 hours. In step A4, after adding glycerol to terminate the reaction, iodate, unreacted sodium periodate and glycerol are removed by dialysis or ultrafiltration. The resulting A1-derived plant oligosaccharide intermediate enters step C1.

[0021] Furthermore, the aldehyde content of A1-derived plant oligosaccharide intermediates was determined using dry basis samples. The quantitative data of aldehydes were obtained by hydroxylamine hydrochloride titration or DNPH method, and the quantitative data of aldehydes were converted to mmol / g. The weight-average molecular weight was determined using the same batch of A1-derived plant oligosaccharide intermediates. The molecular weight distribution and weight-average molecular weight were determined and recorded by GPC / SEC method.

[0022] Furthermore, the snow lotus culture powder is subjected to batch testing before feeding in step A1. The dry powder is used as the test object, and the total flavonoid content, protein content, moisture content and total polysaccharide content are recorded. The tested snow lotus culture powder is fed into step A1 in 100 parts by mass.

[0023] Further, in step B2, after dissolving sodium hyaluronate in a buffer solution with a pH of 4.5-6.0 composed of citric acid and trisodium citrate dihydrate, a condensing agent and an activator are added sequentially according to the following feeding relationship: 10-40 parts by weight of sodium hyaluronate, 2-20 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1-15 parts by weight of N-hydroxysuccinimide. After activation at 10-25°C for 0.5-2 hours, the process proceeds to step B3.

[0024] Further, in step B3, hydrolyzed collagen or gelatin is added to the activation system obtained in step B2, so that the feeding ratio of sodium hyaluronate, hydrolyzed collagen or gelatin and buffer is maintained at 10-40 parts by mass, 20-80 parts by mass and 500-3000 parts by mass, and the reaction is carried out at 15-35℃ for 2-12 hours; in step B4, unreacted small molecule reagents are removed by ultrafiltration or dialysis, and then concentrated or lyophilized to obtain the B1-derived collagen-hyaluronic acid intermediate.

[0025] Furthermore, before adding sodium hyaluronate in step B1, the weight-average molecular weight was determined and recorded using the GPC / SEC method. Before adding hydrolyzed collagen, the weight-average molecular weight was determined and recorded using the GPC / SEC method. Before adding gelatin, the Bloom strength was recorded. The batch data of the above raw materials were stored in correspondence with the binding rate data of the B1-derived collagen-hyaluronic acid intermediate obtained in step B4, and entered into step C1 along with the B1-derived collagen-hyaluronic acid intermediate.

[0026] Furthermore, the binding rate of the B1-derived collagen-hyaluronic acid intermediate was measured using the purified B1-derived collagen-hyaluronic acid intermediate as the test sample. After separating free hydrolyzed collagen or free gelatin from the test sample, the mass of sodium hyaluronate, the mass of the purified sample, and the amount of protein components bound were recorded, and the binding rate was calculated using hyaluronic acid. The residual data of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were determined and recorded using HPLC on the same test sample.

[0027] Further, in step C1, the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate are added to a buffer system with a pH of 4.8-6.2 composed of citric acid and trisodium citrate dihydrate at a solid mass ratio of 4:1-1:4. After the aldehyde and amino groups are condensed at 15-30℃ for 1-6 hours, the temperature is lowered to 0-10℃ to enter the reduction and stabilization step.

[0028] Further, in step C3, based on 100 parts by mass of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate, 0.1-5 parts by mass of sodium borohydride are added in batches to the reaction system obtained in step C2 at 0-10℃ and reduced and stabilized for 0.2-2 hours. After the reduction and stabilization is completed, the system is dialyzed or ultrafiltered to remove borate and small molecule salts, and the resulting composite nanocomplex dispersion system enters step C4.

[0029] Furthermore, samples were taken from the reaction system before and after reduction stabilization in step C3. The changes in free amino content were determined and recorded using the TNBS method, and the changes in aldehyde content were determined and recorded using the hydroxylamine hydrochloride titration method or the DNPH method. The data on the changes in free amino content and aldehyde content were used as quality control data for the condensation of aldehyde and amino groups and the degree of reduction stabilization.

[0030] Furthermore, the Z-average particle size and PDI of the composite nanocomplex dispersion system obtained in step C4 were determined and recorded by dynamic light scattering method. The test sample was a plant bioreaction composite stock solution with a pH of 4.2-6.2 and a solid content of 1-15 wt%. The recorded data were used to release composite nanocomplex dispersion systems with an average particle size of 80-300 nm.

[0031] Further, in step S4, the composite nano-complex obtained in step S3 is mixed with N-acetylglucosamine, yeast β-glucan, and sodium chloride. Based on the total solids of the obtained plant bioreaction composite stock solution, the amount of N-acetylglucosamine added is 0.1-10 wt%, the amount of yeast β-glucan added is 0.1-10 wt%, and the amount of sodium chloride added is 0.1-1.5 wt%. After mixing, the pH of the system is adjusted to 4.2-6.2 using one or both of citric acid and trisodium citrate dihydrate, and purified water is added to make the solids content of the obtained stock solution 1-15 wt%.

[0032] Furthermore, in step S6, the pH-adjusted system is first pre-filtered through a 200-1000 mesh filter medium, and then terminally filtered through a 0.45μm filter medium. The filtered stock solution is then filled into single-use bottles, cans, or bags. After filling, the packaging specifications, solid content before and after filtration, average particle size before and after filtration, and microbial limit test data are recorded.

[0033] As another aspect of this invention, it employs a stepwise preparation process of A1-derived plant oligosaccharide intermediates and B1-derived collagen-hyaluronic acid intermediates, followed by condensation-reduction stabilization and high-pressure homogenization. This is primarily used to achieve, fix, or amplify the aforementioned synergistic effects. Existing processes, if directly compounding plant polysaccharides, collagen components, and hyaluronic acid salts, are prone to aggregation due to uneven molecular weight distribution, uncontrollable reaction sites, or localized viscosity increases; excessively reducing the reaction intensity makes it difficult to form a stable dispersion. This invention first obtains A1-derived plant oligosaccharide intermediates with controllable aldehyde content through extraction, fractionation, and oxidative oligomerization. Then, it obtains B1-derived collagen-hyaluronic acid intermediates by coupling sodium hyaluronate with hydrolyzed collagen or gelatin. Subsequently, it undergoes condensation, reduction stabilization, and high-pressure homogenization in a buffer system according to the solids mass ratio. This ensures that the reaction degree, particle size, and final stock solution pH of the intermediates are controllable, reducing localized over-reaction and viscosity loss through process sequence.

[0034] The A1-derived plant oligosaccharide intermediate primarily addresses the issues of dispersion and controllable reaction sites after the plant-derived active components have been reduced to low molecular weight. Excessive content or overly strong aldehyde reactions may increase system constraint and pressure on mildness control. The B1-derived collagen-hyaluronic acid intermediate mainly provides the collagen-related framework and the hyaluronic acid hydrophilic network; excessive content may increase viscosity, aggregation, and filtration burden. If the two are simply mixed, the results tend to favor one end over the other in terms of stability and flowability. This invention utilizes aldehyde-amino condensation, reduction stabilization, pH buffering, solids mass ratio control, and high-pressure homogenization to ensure that the reactivity of the aldehyde-derived oligosaccharide is absorbed by the collagen-hyaluronic acid conjugated framework. Furthermore, the viscosity contribution of the collagen-hyaluronic acid network is regulated by oligomerization, particle size control, and small-molecule hydrophilic components, thus achieving a balance between dispersion stability, low irritation, processing flowability, and moisturizing properties.

[0035] Beneficial technical effects 1. By preparing snow lotus culture powder into an A1-derived plant oligopolysaccharide intermediate with controllable aldehyde content and weight-average molecular weight, the plant-derived polysaccharide has more suitable reaction sites and oligomeric dispersion in the aqueous phase, reducing the viscosity increase and uneven dispersion when using high molecular weight plant polysaccharides directly, which is conducive to the formation of a filterable and fillable composite stock solution.

[0036] 2. By coupling hydrolyzed collagen or gelatin with sodium hyaluronate to form B1-derived collagen-hyaluronic acid intermediates, the collagen-related structures and the hydrophilic skeleton of hyaluronic acid are pre-conjugated. Then, the intermediates are condensed with A1-derived plant oligosaccharide intermediates, which helps to reduce the risk of aggregation caused by simple blending and improve the stability of aqueous dispersion.

[0037] 3. Through continuous processing of aldehyde-amino condensation, sodium borohydride reduction stabilization, and high-pressure homogenization, a coordinated window is formed between particle size, pH, and solid content in the composite nanocomplex. This avoids both the mild stress caused by simply increasing the degree of reaction and the loose dispersion structure caused by insufficient reaction, thereby improving the overall feasibility of the original solution system.

[0038] 4. By combining N-acetylglucosamine, yeast β-glucan, sodium chloride, and a buffer system of citric acid and trisodium citrate dihydrate, the balance between water retention contribution, hydrophilic water-holding contribution, ionic strength, and pH stability can be adjusted, so that the stock solution maintains good processing adaptability under different solid contents and packaging forms, and is suitable for biomaterial stock solutions, functional stock solutions, and related compound preparations. Attached Figure Description

[0039] Figure 1 The following are the DLS particle size distribution diagrams for Example 1, Comparative Example 7, and Comparative Example 11.

[0040] Figure 2 The cumulative particle size distribution of DLS in Example 1, Comparative Example 7, and Comparative Example 11 is shown.

[0041] Figure 3 The graph shows the storage variation of Z-average average particle size for Examples 1, 7, and 11.

[0042] Figure 4 The diagram shows the changes in PDI storage in Example 1, Comparative Example 7, and Comparative Example 11.

[0043] Figure 5 This is a graph showing the stage-wise changes in aldehyde content for Example 1, Comparative Example 1, and Comparative Example 11.

[0044] Figure 6 This is a graph showing the stage-wise changes in free amino content in Example 1, Comparative Example 1, and Comparative Example 11.

[0045] Figure 7 The graph shows the changes in condensation reduction consumption rate for Example 1, Comparative Example 1, and Comparative Example 11.

[0046] Figure 8 The graph shows the apparent viscosity-shear rate changes for Example 1, Comparative Example 3, and Comparative Example 7.

[0047] Figure 9 The graph shows the difference in filtered solids for Example 1, Comparative Example 3, and Comparative Example 7.

[0048] Figure 10 The diagram shows the dose-response pattern of HaCaT cell viability in Example 1, Comparative Example 6, and Comparative Example 8.

[0049] Figure 11The above are in vitro tissue model viability-dose response diagrams for Example 1, Comparative Example 6, and Comparative Example 8.

[0050] Figure 12 The graph shows the relationship between moisture residue and apparent viscosity for Examples 1, 9, and 10.

[0051] Figure 13 The graph shows the relationship between moisture residue and apparent viscosity for Example 1, Comparative Example 9, and Comparative Example 10.

[0052] Figure 14 This is a macroscopic optical photograph of the plant bioreaction complex stock solution from Example 1.

[0053] Figure 15 Here is a scanning electron microscope image of the composite nanocomplex stock solution from Example 1; Figure 15 a is a low-magnification scanning electron microscope image of the composite nanocomplex stock solution of Example 1; Figure 15 b and Figure 15 c is a medium-to-high magnification scanning electron microscope image of the composite nanocomplex stock solution of Example 1; Figure 15 d is a cross-sectional or partially magnified scanning electron microscope image of the original liquid of the composite nanocomplex in Example 1.

[0054] Figure 16 The image shows the transmission electron microscopy characterization of the composite nanocomplex in Example 1. Figure 16 a is a bright-field transmission electron microscope image of the composite nanocomplex of Example 1; Figure 16 b is a magnified transmission electron microscope image of the composite nanocomplex of Example 1; Figure 16 c is a high-resolution transmission electron microscope image of the composite nanocomplex of Example 1; Figure 16 d is the selected area electron diffraction pattern of the composite nanocomplex of Example 1. Detailed Implementation

[0055] 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.

[0056] Example 1 Overall production scale and product form This embodiment prepares 1000g of plant bioreaction complex stock solution. The product is a light yellow, transparent to microemulsified aqueous stock solution, packaged in single-use bottles, 5g per bottle. The stock solution in this embodiment has a solids content of 1.00wt%, a pH of 4.20, and a Z-average particle size of 80nm for the composite nanocomplex. Based on the total solids of the stock solution, this embodiment contains 15wt% A1-derived plant oligosaccharide intermediate, 5wt% B1-derived collagen-hyaluronic acid intermediate, 0.1wt% N-acetylglucosamine, 0.1wt% yeast β-glucan, 0.1wt% sodium chloride, and 0.05wt% citric acid and trisodium citrate dihydrate. Purified water is added to bring the total volume to 1000g.

[0057] Raw materials, components or material specifications The snow lotus culture powder in this embodiment is derived from the dried powder of callus culture of Tian Shan snow lotus. Before feeding, the total flavonoid content was 7.0 wt%, the protein content was 20.0 wt%, the moisture content was 6.5 wt%, and the total polysaccharide content was 32.0 wt%. Sodium periodate was analytical grade with a content ≥99.0%; glycerol was analytical grade with a content ≥99.0%; sodium hyaluronate was a commercially available pharmaceutical grade powder with a weight-average molecular weight of 5 kDa determined by GPC / SEC method; hydrolyzed collagen was a commercially available pharmaceutical grade powder with a weight-average molecular weight of 0.5 kDa determined by GPC / SEC method; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were analytical grade; N-acetylglucosamine, yeast β-glucan, sodium chloride, citric acid, and trisodium citrate dihydrate were all commercially available pharmaceutical grade or analytical grade raw materials.

[0058] A1. Raw material preparation Weigh out 100.0g of snow lotus culture powder, 3.0g of sodium periodate, 1.0g of glycerol, and 1000.0g of purified water. Add the snow lotus culture powder to the purified water and mechanically stir at 300rpm for 20min to ensure uniform dispersion of the powder. The system is kept under normal pressure and air atmosphere, with an ambient temperature of 25℃ and a relative humidity of 50%.

[0059] A2. Extraction and Grading The dispersion was heated to 45°C at a rate of 2°C / min and extracted at this temperature for 1 hour with a stirring speed of 300 rpm. After extraction, the mixture was filtered through a 200-mesh filter cloth. The filtrate was first passed through a membrane with a molecular weight cutoff of 50 kDa, and the permeate was collected. Then, the permeate was passed through a membrane with a molecular weight cutoff of 1 kDa, and the retentate was collected as the oligosaccharide enrichment fraction. The oligosaccharide enrichment fraction was found to have a solids content of 1.2 wt% by drying loss method, a polysaccharide content of 45.0 wt% (dry basis) by phenol-sulfuric acid method, and a protein content of 8.0 wt% (dry basis) by BCA method.

[0060] A3. Oxidative oligomerization The oligosaccharide-enriched fraction obtained in step A2 was adjusted to pH 4.0 with citric acid and trisodium citrate dihydrate, and incubated at 10°C in the dark for 15 min. 3.0 g of sodium periodate was prepared as an aqueous solution and added dropwise to the oligosaccharide-enriched fraction over 20 min. During the addition, the mixture was kept at 10°C, in the dark, and stirred at 300 rpm. After the addition was complete, the reaction was continued in the dark for 0.5 h. The reaction endpoint was determined by the stabilization of free periodate consumption as measured by sampling.

[0061] A4. Termination and Post-processing Add 1.0 g of glycerol to the system obtained in step A3 in one step, and continue stirring at 10 °C for 10 min to terminate the oxidation reaction. After termination, dialyze the system in purified water for 24 h using a dialysis bag with a molecular weight cutoff of 1 kDa, changing the dialysate every 6 h. Then, concentrate the solution by ultrafiltration until the solids content is 2.0 wt%, obtaining the A1-derived plant oligosaccharide intermediate. The aldehyde content of the A1-derived plant oligosaccharide intermediate in this example was determined to be 0.20 mmol / g by hydroxylamine hydrochloride titration, and the weight-average molecular weight was determined to be 1.0 kDa by GPC / SEC method.

[0062] B1. Raw material preparation Weigh out 10.0 g of sodium hyaluronate, 20.0 g of hydrolyzed collagen, 2.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1.0 g of N-hydroxysuccinimide, and 500.0 g of pH 4.5 buffer solution composed of citric acid and trisodium citrate dihydrate. Filter the prepared buffer solution through a 0.45 μm filter membrane for later use.

[0063] B2. Dissolution and Activation 10.0 g of sodium hyaluronate was slowly added to 500.0 g of pH 4.5 buffer solution and dissolved for 2 h under mechanical stirring at 10 °C and 250 rpm to obtain a sodium hyaluronate solution. Subsequently, 2.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.0 g of N-hydroxysuccinimide were added sequentially at 5 min intervals. The activation temperature was maintained at 10 °C and the activation time was 0.5 h.

[0064] B3. Coupling reaction 20.0g of hydrolyzed collagen was added to the activation system obtained in step B2 in three batches. After each batch was added, the mixture was stirred at 250 rpm for 10 min. After all the collagen was added, the reaction was carried out at 15°C for 2 h. The reaction was carried out under normal pressure and air atmosphere, and the pH was maintained at 4.5-4.7. The reaction endpoint was determined by the stabilization of the system viscosity and the cessation of significant changes in the degree of activation of free carboxyl groups.

[0065] B4. Purification The system obtained in step B3 was subjected to cyclic ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The membrane was washed with purified water until the conductivity of the permeate was below 50 μS / cm, and then concentrated to a solids content of 3.0 wt% to obtain the B1-derived collagen-hyaluronic acid intermediate. After separation of free hydrolyzed collagen, the binding rate, calculated as hyaluronic acid, was 5.0 wt%. HPLC analysis revealed that the residual 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 100 mg / kg, and the residual N-hydroxysuccinimide was 45 mg / kg.

[0066] C1. Composite nanocomplex mixture The A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate were added to a pH 4.8 buffer system composed of citric acid and trisodium citrate dihydrate at a solids mass ratio of 3:1. The solids content of the A1-derived plant oligosaccharide intermediate was 1.50 g, and the solids content of the B1-derived collagen-hyaluronic acid intermediate was 0.50 g. The order of addition was as follows: first, the A1-derived plant oligosaccharide intermediate dispersion was added, and then the B1-derived collagen-hyaluronic acid intermediate dispersion was added dropwise over 30 min. The stirring speed was 400 rpm.

[0067] C2. Condensation reaction The system obtained in step C1 was stirred at 15°C for 1 hour, maintaining pH 4.8, and the reaction was carried out in the dark. The reaction endpoint was determined by the decrease and stabilization of the free amino group content as measured by the TNBS method and the decrease and stabilization of the aldehyde group content as measured by the hydroxylamine hydrochloride titration method.

[0068] C3. Reduction Stabilization The system obtained in step C2 was cooled to 0°C. Based on 100 parts by weight of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate, 0.1 parts by weight of sodium borohydride were added in two batches, with a 5-minute interval between each batch. During the addition, the mixture was stirred at 0°C and 300 rpm. The reduction stabilization time was 0.2 h. After completion, borates and small molecule salts were removed by ultrafiltration. The process was terminated when the conductivity of the permeate was below 80 μS / cm.

[0069] C4. High-pressure homogenization The composite nanocomplex dispersion system obtained in step C3 was homogenized twice under high pressure at 40 MPa, with the feed temperature maintained at 10 °C, and cooled for 5 min after each homogenization. After homogenization, the Z-average particle size was measured by dynamic light scattering, and the PDI was 0.19.

[0070] S4. Preparation of stock solution Based on 100 parts by weight of the solid composite nanocomplex obtained in step C4, add 0.5 parts by weight of N-acetylglucosamine, 0.5 parts by weight of yeast β-glucan, and 0.5 parts by weight of sodium chloride. First, dissolve N-acetylglucosamine in a portion of purified water, then add the yeast β-glucan dispersion, followed by the sodium chloride aqueous solution, and finally add the composite nanocomplex dispersion system. Stir at 300 rpm for 30 min at 25°C.

[0071] S5. pH Adjustment The pH of the system obtained in step S4 was adjusted to 4.20 using an aqueous solution of citric acid and trisodium citrate dihydrate, while maintaining stirring at 25°C and 300 rpm during the adjustment process. A pH fluctuation of no more than 0.05 within 10 minutes was considered the completion criterion.

[0072] S6. Filtration and Filling The system obtained in step S5 was first pre-filtered through a 200-mesh filter medium, and then terminally filtered through a 0.45μm filter medium, maintaining a filtration pressure of 0.08MPa. After filtration, purified water was added to bring the total volume to 1000g, so that the solid content of the stock solution was 1.00wt%. The stock solution was then filled into single-use bottles (5g per bottle) under low-bacterial conditions, sealed after filling, and the packaging specifications were recorded.

[0073] Quality testing methods and results Three samples of the stock solution were taken for testing in this embodiment. The pH was measured using a calibrated pH meter, and the result was 4.20±0.03; the solid content was determined by drying at 105℃ to constant weight, and the result was 1.00±0.04wt%; the average particle size of the composite nanocomplex was determined by dynamic light scattering method, and the sample was diluted to a transparent state before testing, with a Z-average particle size of 80±3nm and a PDI of 0.19±0.02; the aldehyde content of the A1-derived plant oligosaccharide intermediate was 0.20±0.02mmol / g; the binding rate of the B1-derived collagen-hyaluronic acid intermediate was 5.0±0.4wt%; the difference in solid content before and after filtration was 0.03wt%, and the change in average particle size after filtration was 3nm.

[0074] Features and application scenarios of this embodiment This embodiment adopts a relatively conservative low-ratio scheme. The solid content, composite nanocomplex particle size, pH and the amount of main excipients added are all under mild processing conditions. It is suitable for biomaterial stock solutions or functional stock solutions with high requirements for flowability, filtration and single-use packaging convenience. It is also suitable for demonstrating the feasibility and application adaptability of preparing composite nanocomplexes under a low solid content system.

[0075] Example 2 Overall production scale and product form This embodiment prepares 2000g of plant bioreaction complex stock solution. The product is a light yellow, homogeneous aqueous stock solution, packaged in single-use bags, 10g per bag. The stock solution in this embodiment has a solids content of 15.00wt%, a pH of 6.20, and a Z-average particle size of 300nm for the composite nanocomplex. Based on the total solids of the stock solution, this embodiment contains 45wt% A1-derived plant oligosaccharide intermediate, 25wt% B1-derived collagen-hyaluronic acid intermediate, 10wt% N-acetylglucosamine, 10wt% yeast β-glucan, 1.5wt% sodium chloride, and a total of 1.0wt% citric acid and trisodium citrate dihydrate. Purified water is added to bring the total to 2000g.

[0076] Raw materials, components or material specifications The snow lotus culture powder in this embodiment was derived from the dried powder of callus culture of *Saussurea involucrata* from the Tian Shan Mountains. Before feeding, the total flavonoid content was 8.5 wt%, the protein content was 24.0 wt%, the moisture content was 5.8 wt%, and the total polysaccharide content was 36.0 wt%. Sodium periodate, glycerol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were all analytical grade. Sodium hyaluronate was a commercially available pharmaceutical grade powder with a weight-average molecular weight of 300 kDa determined by GPC / SEC. Gelatin was a commercially available pharmaceutical grade powder with a Bloom strength of 300 g. N-acetylglucosamine, yeast β-glucan, sodium chloride, citric acid, and trisodium citrate dihydrate were all commercially available pharmaceutical grade or analytical grade raw materials.

[0077] A1. Raw material preparation Weigh out 100.0g of snow lotus culture powder, 30.0g of sodium periodate, 10.0g of glycerol, and 3000.0g of purified water. Add the snow lotus culture powder to the purified water and mechanically stir at 500rpm for 30min to ensure uniform dispersion of the powder. The system is kept under normal pressure and air atmosphere, with an ambient temperature of 25℃ and a relative humidity of 50%.

[0078] A2. Extraction and Grading The dispersion was heated to 65°C at a rate of 2°C / min and extracted at this temperature for 4 hours with a stirring speed of 500 rpm. After extraction, the mixture was filtered through a 200-mesh filter cloth. The filtrate was first passed through a membrane with a molecular weight cutoff of 50 kDa, and the permeate was collected. Then, the permeate was passed through a membrane with a molecular weight cutoff of 1 kDa, and the retentate was collected as the oligosaccharide enrichment fraction. The oligosaccharide enrichment fraction was found to have a solids content of 2.5 wt% by drying loss method, a polysaccharide content of 52.0 wt% (dry basis) by phenol-sulfuric acid method, and a protein content of 7.5 wt% (dry basis) by BCA method.

[0079] A3. Oxidative oligomerization The oligosaccharide-enriched fraction obtained in step A2 was adjusted to pH 7.0 with citric acid and trisodium citrate dihydrate, and incubated at 30°C in the dark for 15 min. 30.0 g of sodium periodate was prepared as an aqueous solution and added dropwise to the oligosaccharide-enriched fraction over 60 min. During the addition, the temperature was maintained at 30°C, in the dark, and the mixture was stirred at 500 rpm. After the addition was complete, the reaction was continued in the dark for 4 h. The reaction endpoint was determined by the stabilization of the aldehyde group formation rate as measured by sampling.

[0080] A4. Termination and Post-processing 10.0 g of glycerol was added to the system obtained in step A3 in one step, and the oxidation reaction was terminated by stirring at 30 °C for 20 min. The terminated system was washed with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa until the conductivity of the permeate was below 80 μS / cm, and then concentrated to a solids content of 5.0 wt% to obtain the A1-derived plant oligopolysaccharide intermediate. The aldehyde content of the A1-derived plant oligopolysaccharide intermediate in this example was determined to be 1.50 mmol / g by DNPH method, and the weight-average molecular weight was determined to be 20 kDa by GPC / SEC method.

[0081] B1. Raw material preparation Weigh out 40.0 g of sodium hyaluronate, 80.0 g of gelatin, 20.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 15.0 g of N-hydroxysuccinimide, and 3000.0 g of pH 6.0 buffer solution composed of citric acid and trisodium citrate dihydrate. Filter the prepared buffer solution through a 0.45 μm filter membrane for later use.

[0082] B2. Dissolution and Activation 40.0 g of sodium hyaluronate was slowly added to 3000.0 g of pH 6.0 buffer solution and dissolved for 6 h under mechanical stirring at 25 °C and 400 rpm to obtain a sodium hyaluronate solution. Subsequently, 20.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 15.0 g of N-hydroxysuccinimide were added sequentially at 10 min intervals. The activation temperature was maintained at 25 °C, and the activation time was 2 h.

[0083] B3. Coupling reaction 80.0g of gelatin was added to the activation system obtained in step B2 in four batches. After each batch was added, the mixture was stirred at 400rpm for 15min. After all the gelatin was added, the mixture was reacted at 35℃ for 12h. The reaction was carried out under normal pressure and air atmosphere, and the pH was maintained between 5.8 and 6.0. The reaction endpoint was determined by sampling and testing the viscosity and binding rate of the system until they tended to stabilize.

[0084] B4. Purification The system obtained in step B3 was subjected to dialysis to remove unreacted small molecule reagents. The dialysis medium was purified water, and the dialysis time was 36 hours, with the dialysis water changed every 6 hours. After dialysis, the B1-derived collagen-hyaluronic acid intermediate was obtained by lyophilization. After separation of free gelatin, the binding rate, calculated based on hyaluronic acid, was 30.0 wt%. HPLC analysis revealed that the residual 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 50 mg / kg, and the residual N-hydroxysuccinimide was 35 mg / kg.

[0085] C1. Composite nanocomplex mixture The A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate were added to a pH 6.2 buffer system composed of citric acid and trisodium citrate dihydrate at a solids mass ratio of 1.8:1. The order of addition was as follows: first, the B1-derived collagen-hyaluronic acid intermediate dispersion was added, followed by the A1-derived plant oligosaccharide intermediate dispersion over 60 minutes, with the stirring speed at 500 rpm.

[0086] C2. Condensation reaction The system obtained in step C1 was stirred at 30°C for 6 hours, maintaining a pH of 6.2, and the reaction was carried out in the dark. The reaction endpoint was determined by the stabilization of the changes in free amino content (measured by TNBS method) and aldehyde content (measured by DNPH method).

[0087] C3. Reduction Stabilization The system obtained in step C2 was cooled to 10°C. Based on 100 parts by weight of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate, 5 parts by weight of sodium borohydride were added in 5 batches, with an 8-minute interval between each batch. The mixture was stirred at 10°C and 400 rpm during the addition process. The reduction stabilization time was 2 hours. After completion, borates and small molecule salts were removed by ultrafiltration. The process was terminated when the conductivity of the permeate was below 100 μS / cm.

[0088] C4. High-pressure homogenization The composite nanocomplex dispersion system obtained in step C3 was homogenized five times under high pressure at 90 MPa, with the feed temperature maintained at 15 °C, and cooled 8 min after each homogenization. After homogenization, the Z-average particle size was measured using dynamic light scattering, and the PDI was 0.23.

[0089] S4. Preparation of stock solution Based on 100 parts by weight of the solid composite nanocomplex obtained in step C4, add 10 parts by weight of N-acetylglucosamine, 10 parts by weight of yeast β-glucan, and 1.5 parts by weight of sodium chloride. First, dissolve N-acetylglucosamine in a portion of purified water, then add sodium chloride aqueous solution, followed by yeast β-glucan dispersion, and finally add the composite nanocomplex dispersion system. Stir at 500 rpm for 60 min at 25°C.

[0090] S5. pH Adjustment The pH of the system obtained in step S4 was adjusted to 6.20 using an aqueous solution of citric acid and trisodium citrate dihydrate, while maintaining a stirring speed of 500 rpm at 25°C during the adjustment process. A pH fluctuation of no more than 0.05 within 10 minutes was considered the completion criterion.

[0091] S6. Filtration and Filling The system obtained in step S5 was processed using a low-sterile filling method. Before filling, it was pre-filtered through a 500-mesh filter medium at a pressure of 0.10 MPa. After filtration, purified water was added to bring the total volume to 2000 g, resulting in a solid content of 15.00 wt%. The stock solution was filled into single-use bags, 10 g per bag, and sealed after filling, with the packaging specifications recorded.

[0092] Quality testing methods and results Three samples of the stock solution were taken for testing in this embodiment. The pH was measured using a calibrated pH meter, and the result was 6.20±0.04; the solid content was determined by drying at 105℃ to constant weight, and the result was 15.00±0.15wt%; the average particle size of the composite nanocomplex was determined by dynamic light scattering, and the Z-average average particle size was 300±9nm, and the PDI was 0.23±0.03; the aldehyde content of the A1-derived plant oligosaccharide intermediate was 1.50±0.05mmol / g; the binding rate of the B1-derived collagen-hyaluronic acid intermediate was 30.0±1.2wt%; the difference in solid content before and after filtration was 0.08wt%, and the change in average particle size after filtration was 7nm.

[0093] Features and application scenarios of this embodiment This embodiment adopts an optimized scheme with a relatively high loading. The addition amounts of A1-derived plant oligosaccharide intermediate, B1-derived collagen-hyaluronic acid intermediate, N-acetylglucosamine, and yeast β-glucan are all in a relatively high ratio range. The moisture retention contribution is suitable for compound functional stock solutions with high solids content, strong moisture retention contribution, and requiring bagged low-sterile filling.

[0094] Example 3 Overall production scale and product form This embodiment prepares 1500g of plant bioreaction complex stock solution. The product is a light yellow, homogeneous aqueous stock solution, packaged in single-use containers, 20g per container. The stock solution in this embodiment has a solids content of 8.00wt%, a pH of 4.80, and a Z-average particle size of 100nm for the composite nanocomplex. Based on the total solids of the stock solution, this embodiment contains 20wt% A1-derived plant oligosaccharide intermediate, 8wt% B1-derived collagen-hyaluronic acid intermediate, 5wt% N-acetylglucosamine, 5wt% yeast β-glucan, 0.3wt% sodium chloride, and a total of 0.4wt% citric acid and trisodium citrate dihydrate. Purified water is added to bring the total to 1500g.

[0095] Raw materials, components or material specifications The snow lotus culture powder in this embodiment is derived from the dried powder of callus culture of Tian Shan snow lotus. Before feeding, the total flavonoid content was 7.8 wt%, the protein content was 22.0 wt%, the moisture content was 6.0 wt%, and the total polysaccharide content was 34.5 wt%. Sodium hyaluronate was a commercially available pharmaceutical-grade powder with a weight-average molecular weight of 120 kDa as determined by GPC / SEC. Hydrolyzed collagen was a commercially available pharmaceutical-grade powder with a weight-average molecular weight of 10 kDa as determined by GPC / SEC. All other raw materials were commercially available pharmaceutical-grade or analytical grade raw materials.

[0096] A1. Raw material preparation Weigh out 100.0g of snow lotus culture powder, 15.0g of sodium periodate, 5.0g of glycerol, and 2000.0g of purified water. Add the snow lotus culture powder to the purified water and mechanically stir at 400rpm for 25min to ensure uniform dispersion of the powder. The system is kept under normal pressure and air atmosphere, with an ambient temperature of 25℃ and a relative humidity of 50%.

[0097] A2. Extraction and Grading The dispersion was heated to 55℃ at a rate of 2℃ / min and extracted at 55℃ for 2.5 h with stirring at 400 rpm. After extraction, the mixture was filtered through a 400-mesh filter cloth. The filtrate was first passed through a membrane with a molecular weight cutoff of 50 kDa, and the permeate was collected. Then, the permeate was passed through a membrane with a molecular weight cutoff of 1 kDa, and the retentate was collected as the oligosaccharide enrichment fraction. The oligosaccharide enrichment fraction was found to have a solids content of 1.8 wt% by drying loss method, a polysaccharide content of 48.5 wt% on a dry basis by phenol-sulfuric acid method, and a protein content of 7.8 wt% on a dry basis by BCA method.

[0098] A3. Oxidative oligomerization The oligosaccharide-enriched fraction obtained in step A2 was adjusted to pH 5.5 with citric acid and trisodium citrate dihydrate, and incubated at 20°C in the dark for 15 min. 15.0 g of sodium periodate was prepared as an aqueous solution and added dropwise to the oligosaccharide-enriched fraction over 40 min. During the addition, the temperature was maintained at 20°C, in the dark, and the mixture was stirred at 400 rpm. After the addition was complete, the reaction was continued in the dark for 2 h. The reaction endpoint was determined by titration with hydroxylamine hydrochloride, where the aldehyde content reached the target range and tended to stabilize.

[0099] A4. Termination and Post-processing Add 5.0 g of glycerol to the system obtained in step A3 in one step, and continue stirring at 20 °C for 15 min to terminate the oxidation reaction. After termination, remove iodate, unreacted sodium periodate, and glycerol by ultrafiltration. Wash and filter until the conductivity of the permeate is below 80 μS / cm, then concentrate to a solids content of 4.0 wt% to obtain the A1-derived plant oligopolysaccharide intermediate. The aldehyde content of the A1-derived plant oligopolysaccharide intermediate in this example was determined to be 1.00 mmol / g by hydroxylamine hydrochloride titration, and the weight-average molecular weight was determined to be 10 kDa by GPC / SEC method.

[0100] B1. Raw material preparation Weigh out 25.0 g of sodium hyaluronate, 50.0 g of hydrolyzed collagen, 10.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 8.0 g of N-hydroxysuccinimide, and 1500.0 g of pH 5.2 buffer solution composed of citric acid and trisodium citrate dihydrate. Filter the prepared buffer solution through a 0.45 μm filter membrane for later use.

[0101] B2. Dissolution and Activation 25.0 g of sodium hyaluronate was slowly added to 1500.0 g of pH 5.2 buffer solution and dissolved for 4 h under mechanical stirring at 18 °C and 350 rpm to obtain a sodium hyaluronate solution. Subsequently, 10.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 8.0 g of N-hydroxysuccinimide were added sequentially at 8-min intervals. The activation temperature was maintained at 18 °C, and the activation time was 1 h.

[0102] B3. Coupling reaction 50.0g of hydrolyzed collagen was added to the activation system obtained in step B2 in four batches. After each batch was added, the mixture was stirred at 350rpm for 10min. After all the collagen was added, the reaction was carried out at 25℃ for 6h. The reaction was carried out under normal pressure and air atmosphere, and the pH was maintained at 5.1-5.3. The reaction endpoint was determined by the stability of the binding rate through sampling.

[0103] B4. Purification The system obtained in step B3 was subjected to ultrafiltration to remove unreacted small molecule reagents, and then concentrated to a solid content of 5.0 wt% to obtain the B1-derived collagen-hyaluronic acid intermediate. After separation of free hydrolyzed collagen, the binding rate was calculated to be 18.0 wt% based on hyaluronic acid. HPLC analysis revealed that the residual 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 40 mg / kg, and the residual N-hydroxysuccinimide was 25 mg / kg.

[0104] C1. Composite nanocomplex mixture This embodiment employs a combined approach of two batches of composite nanocomplex dispersion systems. In the first batch, A1-derived plant oligosaccharide intermediates and B1-derived collagen-hyaluronic acid intermediates were added to a pH 4.8 buffer system at a solids mass ratio of 4:1. In the second batch, A1-derived plant oligosaccharide intermediates and B1-derived collagen-hyaluronic acid intermediates were added to the pH 4.8 buffer system at a solids mass ratio of 1:1. Both batches of dispersion systems were added in the order of first adding the A1-derived plant oligosaccharide intermediate dispersion, followed by the B1-derived collagen-hyaluronic acid intermediate dispersion, with a stirring speed of 400 rpm.

[0105] C2. Condensation reaction The two batches of systems obtained in step C1 were stirred and reacted at 20°C for 3 h, maintaining pH 4.8, and the reaction was carried out in the dark. The reaction endpoint was determined by the stabilization of the free amino group content as measured by the TNBS method and the stabilization of the aldehyde group content as measured by the hydroxylamine hydrochloride titration method.

[0106] C3. Reduction Stabilization Both batches of the system obtained in step C2 were cooled to 5°C. Based on 100 parts by weight of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate, 0.2 parts by weight of sodium borohydride were added in three batches, with a 5-minute interval between each batch. During the addition, the mixture was stirred at 5°C and 350 rpm. The reduction stabilization time was 1 hour. After completion, borates and small molecule salts were removed by ultrafiltration. The process was terminated when the conductivity of the permeate was below 80 μS / cm.

[0107] C4. High-pressure homogenization The two batches of composite nanocomplex dispersions obtained in step C3 were combined and homogenized three times under high pressure at 50 MPa, with the feed temperature maintained at 12 °C. Cooling was performed 6 minutes after each homogenization. Dynamic light scattering was used for analysis after homogenization, and the Z-average particle size was 100 nm, with a PDI of 0.20.

[0108] S4. Preparation of stock solution Based on 100 parts by weight of the solid composite nanocomplex obtained in step C4, add 5 parts by weight of N-acetylglucosamine, 5 parts by weight of yeast β-glucan, and 0.3 parts by weight of sodium chloride. First, dissolve N-acetylglucosamine in a portion of purified water, then add the yeast β-glucan dispersion, followed by the sodium chloride aqueous solution, and finally add the composite nanocomplex dispersion system. Stir at 400 rpm for 45 min at 25°C.

[0109] S5. pH Adjustment The pH of the system obtained in step S4 was adjusted to 4.80 using an aqueous solution of citric acid and trisodium citrate dihydrate, while maintaining stirring at 25°C and 400 rpm during the adjustment process. A pH fluctuation of no more than 0.05 within 10 minutes was considered the completion criterion.

[0110] S6. Filtration and Filling The system obtained in step S5 was first pre-filtered through a 600-mesh filter medium, and then terminally filtered through a 0.45μm filter medium, maintaining a filtration pressure of 0.09MPa. After filtration, purified water was added to bring the total volume to 1500g, resulting in a solids content of 8.00wt% in the stock solution. The stock solution was then filled into single-use containers, 20g per container, sealed after filling, and the packaging specifications were recorded.

[0111] Quality testing methods and results Three samples of the stock solution were taken for testing in this embodiment. The pH was measured using a calibrated pH meter, and the result was 4.80±0.03; the solid content was determined by drying at 105℃ to constant weight, and the result was 8.00±0.09wt%; the average particle size of the composite nanocomplex was determined by dynamic light scattering, and the Z-average average particle size was 100±4nm, and the PDI was 0.20±0.02; the aldehyde content of the A1-derived plant oligosaccharide intermediate was 1.00±0.04mmol / g; the binding rate of the B1-derived collagen-hyaluronic acid intermediate was 18.0±0.8wt%; the difference in solid content before and after filtration was 0.05wt%, and the change in average particle size after filtration was 4nm.

[0112] Features and application scenarios of this embodiment This embodiment employs process conditions with moderate solids content and a preference for low-temperature reduction stabilization. It obtains a smaller particle size stock solution by merging two batches of composite nanocomplex dispersion systems, making it suitable for composite stock solutions that balance filtration and filling, usability, and high contribution of hydrophilic components.

[0113] Example 4 Overall production scale and product form This embodiment prepares 1200g of plant bioreaction complex stock solution. The product is a light yellow, homogeneous aqueous stock solution, packaged in single-use bags, 8g per bag. The stock solution in this embodiment has a solids content of 12.00wt%, a pH of 6.00, and an average Z-average particle size of 250nm for the composite nanocomplex. Based on the total solids of the stock solution, this embodiment contains 40wt% A1-derived plant oligosaccharide intermediate, 20wt% B1-derived collagen-hyaluronic acid intermediate, 0.5wt% N-acetylglucosamine, 0.1wt% yeast β-glucan, 0.9wt% sodium chloride, and 0.5wt% citric acid and trisodium citrate dihydrate. Purified water is added to bring the total to 1200g.

[0114] Raw materials, components or material specifications The snow lotus culture powder in this embodiment is derived from the dried powder of callus culture of Tian Shan snow lotus. Before feeding, the total flavonoid content was 8.0 wt%, the protein content was 23.0 wt%, the moisture content was 5.9 wt%, and the total polysaccharide content was 35.0 wt%. Sodium hyaluronate was a commercially available pharmaceutical-grade powder with a weight-average molecular weight of 80 kDa as determined by GPC / SEC method; gelatin was a commercially available pharmaceutical-grade powder with a Bloom strength of 100 g; other raw materials were all commercially available pharmaceutical-grade or analytical grade raw materials.

[0115] A1. Raw material preparation Weigh out 100.0g of snow lotus culture powder, 20.0g of sodium periodate, 8.0g of glycerol, and 2500.0g of purified water. Add the snow lotus culture powder to the purified water and mechanically stir at 450rpm for 25min to ensure uniform dispersion of the powder. The system is kept under normal pressure and air atmosphere, with an ambient temperature of 25℃ and a relative humidity of 50%.

[0116] A2. Extraction and Grading The dispersion was heated to 60℃ at a rate of 2℃ / min and extracted at 60℃ for 3 hours with a stirring speed of 450 rpm. After extraction, the mixture was filtered through a 1000-mesh filter. The filtrate was first passed through a membrane with a molecular weight cutoff of 50 kDa, and the permeate was collected. Then, the permeate was passed through a membrane with a molecular weight cutoff of 1 kDa, and the retentate was collected as the oligosaccharide enrichment fraction. The oligosaccharide enrichment fraction was found to have a solids content of 2.0 wt% by drying loss method, a polysaccharide content of 50.0 wt% (dry basis) by phenol-sulfuric acid method, and a protein content of 7.6 wt% (dry basis) by BCA method.

[0117] A3. Oxidative oligomerization The oligosaccharide-enriched fraction obtained in step A2 was adjusted to pH 6.0 with citric acid and trisodium citrate dihydrate, and incubated at 25°C in the dark for 15 min. 20.0 g of sodium periodate was prepared as an aqueous solution and added dropwise to the oligosaccharide-enriched fraction over 50 min. During the addition, the temperature was maintained at 25°C, in the dark, and the mixture was stirred at 450 rpm. After the addition was complete, the reaction was continued in the dark for 3 h. The reaction endpoint was determined by the DNPH method, where the aldehyde content reached the target range and tended to stabilize.

[0118] A4. Termination and Post-processing Add 8.0 g of glycerol to the system obtained in step A3 in one step, and continue stirring at 25 °C for 15 min to terminate the oxidation reaction. After termination, remove iodate, unreacted sodium periodate, and glycerol by a combination of dialysis and ultrafiltration. Wash and filter until the conductivity of the permeate is below 80 μS / cm, then concentrate to a solids content of 4.5 wt% to obtain the A1-derived plant oligosaccharide intermediate. The aldehyde content of the A1-derived plant oligosaccharide intermediate in this example was determined to be 1.00 mmol / g by DNPH method, and the weight-average molecular weight was determined to be 2.0 kDa by GPC / SEC method.

[0119] B1. Raw material preparation Weigh out 30.0 g of sodium hyaluronate, 60.0 g of gelatin, 12.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 10.0 g of N-hydroxysuccinimide, and 2000.0 g of pH 5.8 buffer solution composed of citric acid and trisodium citrate dihydrate. Filter the prepared buffer solution through a 0.45 μm filter membrane for later use.

[0120] B2. Dissolution and Activation 30.0 g of sodium hyaluronate was slowly added to 2000.0 g of pH 5.8 buffer solution and dissolved for 5 h under mechanical stirring at 20 °C and 400 rpm to obtain a sodium hyaluronate solution. Subsequently, 12.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10.0 g of N-hydroxysuccinimide were added sequentially at 8-min intervals. The activation temperature was maintained at 20 °C, and the activation time was 1.5 h.

[0121] B3. Coupling reaction 60.0g of gelatin was added to the activation system obtained in step B2 in three batches. After each batch was added, the mixture was stirred at 400rpm for 15min. After all the gelatin was added, the reaction was carried out at 30℃ for 8h. The reaction was carried out under normal pressure and air atmosphere, and the pH was maintained at 5.7-5.9. The reaction endpoint was determined by the stability of the system viscosity and binding rate through sampling.

[0122] B4. Purification The system obtained in step B3 was subjected to ultrafiltration to remove unreacted small molecule reagents, and then concentrated to a solid content of 5.5 wt% to obtain the B1-derived collagen-hyaluronic acid intermediate. After separation of free gelatin, the binding rate based on hyaluronic acid was calculated to be 5.0 wt%. HPLC analysis revealed that the residual 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 60 mg / kg, and the residual N-hydroxysuccinimide was 30 mg / kg.

[0123] C1. Composite nanocomplex mixture This embodiment employs a three-batch composite nanocomplex dispersion system. In the first batch, A1-derived plant oligosaccharide intermediate and B1-derived collagen-hyaluronic acid intermediate were added to a pH 6.2 buffer system at a solids mass ratio of 1:4, with A1 solids comprising 1.00 parts by mass and B1 solids comprising 4.00 parts by mass. In the second batch, A1-derived plant oligosaccharide intermediate and B1-derived collagen-hyaluronic acid intermediate were added to a pH 6.2 buffer system at a solids mass ratio of 1:2, with A1 solids comprising 3.57 parts by mass and B1 solids comprising 7.14 parts by mass. In the third batch, A1-derived plant oligosaccharide intermediate and B1-derived collagen-hyaluronic acid intermediate were added to a pH 6.2 buffer system at a solids mass ratio of 4:1, with A1 solids comprising 35.43 parts by mass and B1 solids comprising 8.86 parts by mass. All three batches of dispersion systems were prepared by first adding the B1-derived collagen-hyaluronic acid intermediate dispersion, followed by the A1-derived plant oligosaccharide intermediate dispersion, with a stirring speed of 450 rpm.

[0124] C2. Condensation reaction The three batches of systems obtained in step C1 were stirred and reacted at 25°C for 4 h, maintaining pH 6.2, and the reaction was carried out in the dark. The reaction endpoint was determined by the stabilization of the free amino content measured by the TNBS method and the stabilization of the aldehyde content measured by the DNPH method.

[0125] C3. Reduction Stabilization The three batches of the system obtained in step C2 were all cooled to 5°C. Based on 100 parts by weight of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate, 2 parts by weight of sodium borohydride were added in four batches, with a 6-minute interval between each batch. During the addition, the mixture was stirred at 5°C and 400 rpm. The reduction stabilization time was 1.5 hours. After completion, borates and small molecule salts were removed by ultrafiltration. The process was terminated when the conductivity of the permeate was below 80 μS / cm.

[0126] C4. High-pressure homogenization The three batches of composite nanocomplex dispersions obtained in step C3 were combined and homogenized four times under high pressure at 80 MPa, with the feed temperature maintained at 12 °C. Cooling was performed 6 minutes after each homogenization. Dynamic light scattering was used for analysis, and the Z-average particle size was 250 nm, with a PDI of 0.22.

[0127] S4. Preparation of stock solution Based on 100 parts by weight of the solid composite nanocomplex obtained in step C4, add 0.5 parts by weight of N-acetylglucosamine, 0.1 parts by weight of yeast β-glucan, and 0.9 parts by weight of sodium chloride. First, dissolve N-acetylglucosamine in a portion of purified water, then add sodium chloride aqueous solution, followed by yeast β-glucan dispersion, and finally add the composite nanocomplex dispersion system. Stir at 450 rpm for 50 min at 25°C.

[0128] S5. pH Adjustment The pH of the system obtained in step S4 was adjusted to 6.00 using an aqueous solution of citric acid and trisodium citrate dihydrate, while maintaining stirring at 25°C and 450 rpm during the adjustment process. A pH fluctuation of no more than 0.05 within 10 minutes was considered the completion criterion.

[0129] S6. Filtration and Filling The system obtained in step S5 was first pre-filtered through a 1000-mesh filter medium, and then terminally filtered through a 0.45μm filter medium, maintaining a filtration pressure of 0.10MPa. After filtration, purified water was added to bring the total volume to 1200g, resulting in a solids content of 12.00wt% for the stock solution. The stock solution was then filled into single-use bags (8g per bag) under low-bacterial conditions. After filling, the bags were sealed, and the packaging specifications were recorded.

[0130] Quality testing methods and results Three samples of the stock solution were taken for testing in this embodiment. The pH was measured using a calibrated pH meter, and the result was 6.00±0.04; the solid content was determined by drying at 105℃ to constant weight, and the result was 12.00±0.12wt%; the average particle size of the composite nanocomplex was determined by dynamic light scattering, and the Z-average average particle size was 250±8nm, and the PDI was 0.22±0.03; the aldehyde content of the A1-derived plant oligosaccharide intermediate was 1.00±0.04mmol / g; the binding rate of the B1-derived collagen-hyaluronic acid intermediate was 5.0±0.5wt%; the difference in solid content before and after filtration was 0.06wt%, and the change in average particle size after filtration was 5nm.

[0131] Features and application scenarios of this embodiment This embodiment adopts a multi-batch composite nano-complex dispersion system merging method, so that the ratio of different A1-derived plant oligosaccharide intermediates and B1-derived collagen-hyaluronic acid intermediates can be coordinated in the same finished product. It is suitable for demonstrating feasibility under a wide process window, and is also suitable for composite care stock solutions with high A1 content, moderate solids content and bag packaging.

[0132] Comparative Example 1: It is basically the same as Example 1, except that 3.0g of sodium periodate is not added in step A3, the dropping solution is replaced with an equal mass of purified water, and it is treated with the same dropping time, temperature, light protection conditions and stirring conditions as in Example 1, while other conditions remain unchanged.

[0133] Comparative Example 2: It is basically the same as Example 1, except that the amount of sodium periodate weighed in steps A1 and A3 is adjusted from 3.0g to 1.0g. It is still prepared as an aqueous solution and added dropwise to the oligosaccharide enrichment part within 20min. Other conditions remain unchanged.

[0134] Comparative Example 3: It is basically the same as Example 1, except that in step A2, after the filtrate passes through a membrane with a molecular weight cutoff of 50kDa and the permeate is collected, it is no longer subjected to secondary fractionation through a membrane with a molecular weight cutoff of 1kDa. Instead, the permeate from the 50kDa membrane is directly used as the oligosaccharide enrichment part and enters step A3. Other conditions remain unchanged.

[0135] Comparative Example 4: It is basically the same as Example 1, except that in step B2, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride added is adjusted from 2.0g to 0.5g, and N-hydroxysuccinimide is still 1.0g, while other conditions remain unchanged.

[0136] Comparative Example 5: It is basically the same as Example 1, except that after adding 20.0g of hydrolyzed collagen in step B3, the coupling reaction time was adjusted from 2h to 0.5h, while the reaction temperature, stirring speed and pH maintenance method remained unchanged, and other conditions remained unchanged.

[0137] Comparative Example 6: It is basically the same as Example 1, except that in step C3, the amount of sodium borohydride added is adjusted from 0.1 parts by mass to 0.05 parts by mass based on 100 parts by mass of the total solids of A1-derived plant oligosaccharide intermediate and B1-derived collagen-hyaluronic acid intermediate, and is still added in two batches, with other conditions remaining unchanged.

[0138] Comparative Example 7: It is basically the same as Example 1, except that the high-pressure homogenization pressure in step C4 is adjusted from 40MPa to 20MPa, the number of homogenizations is still 2, the feed temperature and cooling interval remain unchanged, and other conditions remain unchanged.

[0139] Comparative Example 8: It is basically the same as Example 1, except that in step S5, an aqueous solution of citric acid and trisodium citrate dihydrate is used to adjust the pH of the system to 3.80. The pH stability criterion is still that the fluctuation does not exceed 0.05 within 10 minutes, and other conditions remain unchanged.

[0140] Comparative Example 9: Essentially the same as Example 1, except that in step C1, the A1-derived plant oligosaccharide intermediate was removed, and only the B1-derived collagen-hyaluronic acid intermediate dispersion was added to a pH 4.8 buffer system at a concentration of 2.00 g solids. Subsequent steps C2 to C4 and S4 to S6 were performed under the conditions corresponding to Example 1, with other conditions remaining unchanged. This comparative example was used to verify the synergistic effect between the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate.

[0141] Comparative Example 10: Essentially the same as Example 1, except that in step C1, the B1-derived collagen-hyaluronic acid intermediate was removed, and only the A1-derived plant oligosaccharide intermediate dispersion was added to a pH 4.8 buffer system at a concentration of 2.00 g solids. Subsequent steps C2 to C4 and S4 to S6 were performed under the conditions corresponding to Example 1, with other conditions remaining unchanged. This comparative example was used to verify the synergistic effect between the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate.

[0142] Comparative Example 11: Essentially the same as Example 1, except that step C1 still involves adding 1.50 g of solid A1-derived plant oligosaccharide intermediate and 0.50 g of solid B1-derived collagen-hyaluronic acid intermediate. However, the aldehyde-amino condensation reaction in step C2 and the sodium borohydride reduction stabilization in step C3 are not performed. After mixing in step C1, the mixture directly proceeds to step C4 for high-pressure homogenization, with other conditions remaining unchanged. This comparative example is used to verify the synergistic effect of the condensation-reduction interface construction mechanism between the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate.

[0143] Characterization and performance testing: The particle size and PDI (particulate density index) of the composite nanocomplex were tested to evaluate the dispersion scale and distribution uniformity of the plant bioreaction composite stock solution. The test principle is based on the inversion of the scattering intensity fluctuations of Brownian motion particles by dynamic light scattering. The sample was diluted with the same pH buffer to a transparent state, equilibrated at 25°C for 2 min, and the Z-average particle size, PDI, and three replicate results were recorded. The results were expressed as mean ± standard deviation, referring to ISO 22412:2025 or the equivalent DLS method. The results were used to determine the target particle size of 80-300 nm and the changes before and after storage.

[0144] Apparent viscosity and filtration compatibility tests were used to evaluate the processing flowability of the plant bioreaction compound concentrate during filling, filtration, and use. A rotational rheometer or rotational viscometer was used at 25°C and a shear rate of 10 s⁻¹. - ¹、50s - ¹ and 100s - ¹ Record the viscosity curve; take another 100g sample and pre-filter it through a 200-mesh filter and then filter it through a 0.45μm filter. Record the filtration time, pressure, difference in solids before and after filtration, and particle size change. Refer to ASTM D2196 or the equivalent rotational viscosity method. The data are expressed as mean ± standard deviation.

[0145] Low irritation and in vitro cell compatibility tests were used to evaluate the mildness and biocompatibility of the plant bioreaction compound stock solution in relevant application scenarios. Cell viability was determined using the HaCaT cell or fibroblast extract contact method, and irritation was evaluated using an in vitro reconstructed tissue model. Contact time, elution, incubation, and MTT assay were performed according to ISO 10993-5 and equivalent in vitro compatibility or irritation evaluation methods. Results were recorded as cell viability, tissue viability, and concordance with positive and negative controls.

[0146] The moisture retention rate test was used to evaluate the contribution of the composite nano-complex, N-acetylglucosamine, and yeast β-glucan in the plant bioreaction complex stock solution to moisture retention. Equal amounts of sample were applied to a standard moisture retention test membrane or an ex vivo biological substrate and placed at 25°C and 50% relative humidity for 0 h, 2 h, 4 h, and 8 h. The water loss and residual moisture rate were measured and calculated. Each group had n=3 samples. The moisture retention rate results are expressed as mean ± standard deviation and corrected against a blank substrate.

[0147] The aldehyde and free amino group change assay was used to quantify the condensation and reduction stabilization of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate. Samples were taken after mixing in C1, after C2, and after C3. The aldehyde content was determined by hydroxylamine hydrochloride titration or the DNPH method, and the free amino group content was determined by the TNBS method. The data were recorded in mmol / g or μmol / g, and the consumption rate was calculated based on the difference before and after the reaction. The results are expressed as mean ± standard deviation.

[0148] The binding rate of B1-derived collagen-hyaluronic acid intermediates and the residual small molecule assay were used to evaluate the degree of coupling and the adequacy of purification. The purified B1 intermediates were used to separate free hydrolyzed collagen or free gelatin, and the binding rate was calculated based on hyaluronic acid. For the same sample, HPLC was used to record residual 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. External standard curves, recoveries, and repeatability were established. Results are expressed as wt%, mg / kg, and mean ± standard deviation.

[0149] Accelerated storage stability testing is used to evaluate the dispersion stability, pH stability, and microbial control status of plant bioreactive compound concentrates under packaging and transportation conditions. Samples are filled into single-use bottles, containers, or bags and stored at 4°C, 25°C, and 45°C for 30 days. pH, solids content, Z-average particle size, PDI, appearance, and microbial limits are measured at 0, 7, 14, and 30 days. Data are expressed as variation and mean ± standard deviation to determine the presence of significant aggregation or precipitation.

[0150] Figure 1 The images show the DLS particle size distribution of Examples 1, 7, and 11 of this invention. Figure 2 This is a cumulative particle size distribution diagram of DLS particles in Embodiment 1, Comparative Example 7, and Comparative Example 11 of the present invention. Figure 1 and Figure 2 It can be seen that Example 1 exhibits a narrow single-peak particle size distribution around 80 nm, with a PDI of approximately 0.19, and the cumulative distribution curve is mainly concentrated within 100 nm, indicating that the obtained composite nanocomplex has a small particle size, concentrated distribution, and a low proportion of large particles. In contrast, the particle size peaks of Comparative Examples 7 and 11 shift to around 220 nm and 450 nm, respectively, accompanied by broadening of the distribution and an overall shift of the cumulative curve towards larger particle sizes. This indicates that when homogenization conditions are insufficient or there is a lack of effective aldehyde-amino condensation reduction interface construction, the system is more prone to forming larger particles and heterogeneous aggregates. The above results demonstrate that appropriate high-pressure homogenization treatment and interface stabilization reaction can synergistically promote the refinement and uniform dispersion of composite nanocomplexes, providing a structural basis for subsequent storage stability, filtration permeability, and performance.

[0151] Figure 3 This is a graph showing the Z-average average particle size storage variation for Examples 1, 7, and 11 of the present invention. Figure 4 This diagram illustrates the changes in PDI storage in Embodiment 1, Comparative Example 7, and Comparative Example 11 of the present invention. Figure 3 and Figure 4 It can be seen that in Example 1, the Z-average particle size increased only from about 80 nm to about 86 nm during the 0–30 day storage period, and the PDI remained at about 0.19–0.21, indicating that no significant particle growth, flocculation, or uncontrolled particle size distribution occurred in this system during storage. Comparative Examples 7 and 11, under the same storage conditions, saw their average particle sizes increase to about 262 nm and about 522 nm, respectively, and their PDI increased to about 0.39 and about 0.50, respectively, showing more pronounced particle aggregation and wider particle size distribution. These results further demonstrate that by controlling the homogenization pressure and introducing aldehyde-amino condensation and reduction stabilization processes, a more stable composite structure can be formed at the particle interface, thereby effectively suppressing collisional aggregation and particle size drift of nanoparticles during storage.

[0152] Figure 5 This is a graph showing the stage-wise changes in aldehyde content in Example 1, Comparative Example 1, and Comparative Example 11 of the present invention. Figure 6 This is a graph showing the stage-wise changes in free amino group content in Example 1, Comparative Example 1, and Comparative Example 11 of the present invention. Figure 7 This is a graph showing the change in condensation-reduction consumption rate for Embodiment 1, Comparative Example 1, and Comparative Example 11 of the present invention. Figures 5 to 7 It can be seen that in Example 1, the aldehyde content decreased from approximately 0.20 mmol / g in the C1 stage to approximately 0.05 mmol / g in the C3 stage, and the free amino content decreased from approximately 100 μmol / g to approximately 50 μmol / g. Furthermore, the overall consumption rate in the C3 stage reached over 60%, indicating that the active aldehyde groups in the A1-derived plant oligosaccharide intermediate and the free amino groups in the B1-derived collagen-hyaluronic acid intermediate reacted effectively, forming a relatively stable composite linkage structure in the subsequent reduction and stabilization steps. In Comparative Example 1, due to the change in the aldehyde source, the initial aldehyde content was low, leading to insufficient consumption in the subsequent reaction. Comparative Example 11 showed a smaller increase in the consumption of aldehyde and amino groups, indicating that the interfacial binding efficiency significantly decreased when the necessary condensation-reduction stabilization process was lacking. These stage-wise changes demonstrate, from the reaction process perspective, that the aldehyde source, the degree of amino participation, and the reduction-stabilization treatment jointly affect the construction efficiency of the composite nanocomplex, and correspond to the aforementioned results of particle size refinement and storage stability.

[0153] Figure 8 The graphs show the apparent viscosity-shear rate changes of Embodiment 1, Comparative Example 3, and Comparative Example 7 of the present invention. Figure 9 This is a graph showing the difference in filtered solids content for Embodiment 1, Comparative Example 3, and Comparative Example 7 of the present invention. Figure 8 It can be seen that in Example 1, the time range is 1–100 s. - ¹The apparent viscosity decreased from approximately 12.0 mPa·s to 6.4 mPa·s within the shear rate range, exhibiting lower viscosity and slight shear thinning characteristics, indicating good flowability and processing compatibility in aqueous systems. Combined with Figure 9 It can be seen that the difference in solids before and after filtration in Example 1 is approximately 0.03 wt%, significantly lower than that in Comparative Example 3 (approximately 0.35 wt%) and Comparative Example 7 (approximately 0.18 wt%), indicating that the system exhibits lower retention of active ingredients and particle loss during final filtration. Comparative Example 3, due to its higher viscosity, is more prone to increased filtration resistance and solids retention; Comparative Example 7, with its wider particle distribution and higher proportion of large particles, also suffers from reduced filtration permeability. This demonstrates that this solution, while maintaining the fluidity of the low-solids aqueous system, can reduce filtration loss through particle size control and interface stabilization, making it suitable for forming a stable, homogeneous, and easily fillable stock solution system.

[0154] Figure 10This is a dose-response graph of HaCaT cell viability in Example 1, Comparative Example 6, and Comparative Example 8 of the present invention. Figure 11 This is a viability-dose response diagram of in vitro tissue models from Examples 1, 6, and 8 of the present invention. Figure 10 It can be seen that within the extraction concentration range of 25%–100%, the viability of HaCaT cells treated in Example 1 remained at approximately 96%–99%, indicating that it did not exhibit significant cytotoxicity to the test cells. Figure 11 It was found that, under 100% extraction conditions, the RhE tissue viability after treatment in Example 1 was still approximately 93%, higher than approximately 86% in Comparative Example 6 and approximately 80% in Comparative Example 8. This result indicates that appropriate reduction stabilization and weakly acidic buffer pH control can reduce the risk of irritation from unreacted active groups or pH shifts in the system, enabling the composite nanocomplex to exhibit good mildness and compatibility at both the cellular and in vitro tissue model levels.

[0155] Figure 12 This is a graph showing the relationship between moisture residue and apparent viscosity for Examples 1, 9, and 10 of the present invention. Figure 13 This is a graph showing the relationship between moisture residue and apparent viscosity for Examples 1, 9, and 10 of the present invention. Figure 12 It can be seen that the moisture retention rate of Example 1 at 8 h was approximately 66%, which is higher than that of Comparative Example 9 (approximately 55%) and Comparative Example 10 (approximately 48%). This indicates that the composite nano-complex has a certain synergistic water-retention effect with N-acetylglucosamine and yeast β-glucan, and can delay moisture loss. Further combined with... Figure 13 It can be seen that Example 1, while maintaining a moisture retention rate of approximately 66% after 8 hours, has an apparent viscosity of approximately 8.5 mPa·s, exhibiting both good moisture retention and low viscosity flowability. Comparative Example 9, although having higher viscosity, did not provide sufficient improvement in moisturizing effect, while Comparative Example 10 showed a decrease in moisture retention rate due to its lower system viscosity and insufficient synergistic components. These results indicate that this solution does not simply rely on increasing viscosity to achieve a moisturizing effect, but rather achieves a comprehensive balance of moisturizing performance, flowability, and processing compatibility through the synergistic configuration of nano-complex structures and functional moisturizing components.

[0156] Figure 14 This is a macroscopic optical photograph of the plant bioreaction composite stock solution from Example 1 of the present invention. Figure 14As can be seen, the sample of Example 1 was a light yellow, transparent to microemulsified aqueous stock solution, filled into single-use bottles, each containing 5 g. No visible precipitation, stratification, or coarse flocculent matter was observed. The stock solution had a solids content of 1.00 ± 0.04 wt%, a pH of 4.20 ± 0.03, an average particle size of 80 ± 3 nm for the composite nanocomplex DLS Z-average, and a PDI of 0.19 ± 0.02. This macroscopic appearance is consistent with the aforementioned DLS particle size distribution, low PDI, and difference in filtration solids, indicating that the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate can form a stable nano-dispersion system in a weakly acidic, low-solids aqueous phase, meeting the appearance requirements of a transparent to microemulsified stock solution and the final filtration process requirements.

[0157] Figure 15 This is a scanning electron microscope (SEM) image of the composite nanocomplex stock solution from Example 1 of the present invention after dilution, drying, and conductive treatment. Figure 15 Low-magnification SEM images show that the dry solids form a continuous or semi-continuous coating layer on the substrate surface, with no obvious micron-sized coarse aggregates observed. This indicates that the 40 MPa high-pressure homogenization treatment and 0.45 μm terminal filtration effectively control large particle residue. Figure 15 b and Figure 15 High-magnification SEM images (c) reveal that the composite nanocomplex is mainly composed of near-spherical or slightly collapsed nanobuilding units. The particles exhibit random packing and localized contact. The dry particle size ranges from tens to hundreds of nanometers, showing a good correlation with the hydrated particle size of 80 ± 3 nm. Figure 15 The cross-sectional or magnified SEM images show that the contact between the dry deposition layer and the substrate is relatively continuous, and no through cracks are observed. This indicates that the composite nanocomplex can still maintain good film-forming properties and stacking integrity after drying, which further supports its stable dispersion and interfacial bonding characteristics from a morphological perspective.

[0158] Figure 16 This is a transmission electron microscope (TEM) image of the composite nanocomplex from Example 1 of the present invention. Figure 16 Bright-field TEM images show that the composite nanocomplexes exhibit a near-spherical or ellipsoidal soft nanoparticle morphology with relatively gentle particle boundaries, consistent with the structural characteristics of nanocomplexes formed by organic components such as plant oligosaccharides, collagen, and hyaluronic acid. Figure 16 The magnified TEM image (b) shows that the internal contrast of the particles is relatively uniform, and no obvious hollow cavities or inorganic grain stacking structures were observed, indicating that it is closer to a solid or gel-like organic composite nanostructure. Figure 16 High-resolution TEM images show that no clear periodic lattice fringes were observed in the sample; Figure 16The d-area electron diffraction pattern shows diffuse halos or weak diffuse scattering characteristics, indicating that the main body of the composite nanocomplex is an amorphous organic structure and lacks long-range crystalline order. These TEM results are consistent with the preparation route of low-temperature aqueous condensation, reduction stabilization, and high-pressure homogenization, and further demonstrate at the microstructural level that this method can obtain a uniform, stable composite complex system with soft organic nanoscale characteristics.

[0159] Table 1 Performance of Examples and Comparative Examples

[0160] As can be seen from the performance of the examples and comparative examples in Table 1, Example 1 maintains a low PDI, small filtration loss, and high cell viability under low solids and small particle size conditions, demonstrating the fluidity and mildness of the low-load system. In Examples 2 to 4, with increasing amounts of solids, A1-derived plant oligosaccharide intermediates, and B1-derived collagen-hyaluronic acid intermediates, the residual moisture content increased, but the viscosity also increased simultaneously, while maintaining acceptable particle size distribution and stability. In Comparative Examples 1 to 8, changes in oxidative oligomerization, membrane fractionation, coupling, reduction stabilization, high-pressure homogenization, or pH conditions resulted in adverse changes in particle size distribution, storage particle size variation, filtration difference, or irritation indicators. In Comparative Examples 9 to 11, after disassembling the A1 / B1 synergistic components or disrupting the condensation-reduction interface construction method, the balance between dispersion stability, moisture retention, and mildness became difficult to maintain, indicating that the proportions of core technical units, interface construction, and post-processing sequence need to be jointly controlled.

[0161] 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 plant bioreaction complex stock solution, characterized in that, The plant bioreaction complex stock solution comprises the following components: excluding purified water, the remaining components are calculated based on the total solids of the plant bioreaction complex stock solution. The content of A1-derived plant oligopolysaccharide intermediates is 15-45 wt%, and the A1-derived plant oligopolysaccharide intermediates are aldehyde-based oligosaccharides of Saussurea involucrata obtained by oxidative oligomerization of Saussurea involucrata culture powder. The content of B1-derived collagen-hyaluronic acid intermediate is 5-25 wt%, and the B1-derived collagen-hyaluronic acid intermediate is a conjugated intermediate obtained by coupling hydrolyzed collagen or gelatin with sodium hyaluronate. The content of N-acetylglucosamine is 0.1-10 wt%; The content of yeast β-glucan is 0.1-10 wt%; The sodium chloride content is 0.1-1.5 wt%; The total content of one or both of citric acid and trisodium citrate dihydrate is 0.05-1.0 wt%; based on the total mass of the plant bioreaction compound stock solution, The remainder is purified water.

2. The plant bioreaction composite stock solution of claim 1, wherein, The A1-derived plant oligosaccharide intermediate was prepared through the following steps: A1. Raw material preparation: By weight, the raw materials include 100 parts by weight of snow lotus culture powder, 3-30 parts by weight of sodium periodate, 1-10 parts by weight of glycerol, and 1000-3000 parts by weight of purified water; A2. Extraction and grading: Disperse snow lotus culture powder in purified water and extract for 1-4 hours at a temperature of 45-65℃. After filtration, pass the filtrate through a membrane with a molecular weight cutoff of 50kDa and collect the permeate. Then pass the permeate through a membrane with a molecular weight cutoff of 1kDa and collect the retentate as the oligosaccharide enrichment portion. A3. Oxidative oligomerization: Adjust the pH of the oligosaccharide enrichment obtained in step A2 to 4.0-7.0, and react it with sodium periodate at 10-30℃ under light-protected conditions for 0.5-4 hours; A4. Termination and Post-processing: Glycerol was added to terminate the reaction, followed by dialysis or ultrafiltration to remove salt and concentration, to obtain the A1-derived plant oligosaccharide intermediate with an aldehyde content of 0.20-1.50 mmol / g and a weight-average molecular weight of 1-20 kDa based on dry basis.

3. The plant bioreaction composite stock solution of claim 1, wherein, The B1-derived collagen-hyaluronic acid intermediate was prepared through the following steps: B1. Raw material preparation: By weight, the raw materials include 10-40 parts sodium hyaluronate, 20-80 parts hydrolyzed collagen or gelatin, 2-20 parts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-15 parts N-hydroxysuccinimide, and 500-3000 parts buffer solution composed of citric acid and trisodium citrate dihydrate. B2. Dissolution and activation: Sodium hyaluronate is dissolved in the buffer solution with a pH of 4.5-6.0, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially at 10-25°C, and the mixture is activated for 0.5-2 hours. B3. Coupling reaction: Add hydrolyzed collagen or gelatin and react at 15-35℃ for 2-12 hours; B4. Purification: Remove unreacted small molecule reagents by ultrafiltration or dialysis, then concentrate or freeze-dry to obtain the B1-derived collagen-hyaluronic acid intermediate with a binding rate of 5-30 wt% based on hyaluronic acid and a residual amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride not exceeding 100 mg / kg.

4. The plant bioreaction composite stock solution of claim 1, wherein, The composite nanocomplex was prepared by the following steps: C1. The A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate are mixed in a solid mass ratio of 4:1 to 1:4 in a buffer system with a pH of 4.8-6.2 composed of citric acid and trisodium citrate dihydrate; C2. The condensation reaction of aldehyde and amino groups is carried out at 15-30℃ for 1-6 hours; C3. Based on 100 parts by weight of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate, add 0.1-5 parts by weight of sodium borohydride, reduce and stabilize at 0-10°C for 0.2-2 hours, and remove borates and small molecule salts by dialysis or ultrafiltration. C4. The composite nanocomplex with an average particle size of 80-300 nm is obtained by high-pressure homogenization treatment at 40-90 MPa 2-5 times.

5. The plant bioreaction composite stock solution of claim 1, wherein, Based on the total solids of the plant bioreaction complex stock solution, the content of the A1-derived plant oligosaccharide intermediate is 20-40 wt%, the content of the B1-derived collagen-hyaluronic acid intermediate is 8-20 wt%, the content of N-acetylglucosamine is 0.5-5 wt%, and the content of yeast β-glucan is 0.1-5 wt%. The A1-derived plant oligosaccharide intermediates have an aldehyde content of 0.20-1.00 mmol / g on a dry basis and a weight-average molecular weight of 2-10 kDa. The average molecular weight of the sodium hyaluronate is 5-300 kDa; When the protein component in the B1-derived collagen-hyaluronic acid intermediate is hydrolyzed collagen, its average molecular weight is 0.5-10 kDa; When the protein component in the B1-derived collagen-hyaluronic acid intermediate is gelatin, its Bloom strength is 100-300g; the snow lotus culture powder is derived from the callus culture of Tian Shan snow lotus, and its total flavonoid content in the dried product is not less than 7wt%, and its protein content is not less than 20wt%.

6. A method for preparing a plant bioreaction composite stock solution according to claim 1, characterized by, Includes the following steps: S1. Provide the prepared A1-derived plant oligosaccharide intermediate; S2. Provide the prepared B1-derived collagen-hyaluronic acid intermediate; S3. The A1-derived plant oligosaccharide intermediate provided in step S1 and the B1-derived collagen-hyaluronic acid intermediate provided in step S2 are mixed in a buffer system with a pH of 4.8-6.2 composed of citric acid and trisodium citrate dihydrate at a solid mass ratio of 4:1-1:

4. The aldehyde and amino groups are condensed, and the mixture is then reduced and stabilized with sodium borohydride and homogenized under high pressure to obtain a composite nanocomplex with an average particle size of 80-300 nm. S4. Add the composite nano-complex obtained in step S3, N-acetylglucosamine, yeast β-glucan, and sodium chloride to purified water. Based on the total solids of the plant bioreaction composite stock solution obtained in step S6, the amount of N-acetylglucosamine added is 0.1-10 wt%, the amount of yeast β-glucan added is 0.1-10 wt%, and the amount of sodium chloride added is 0.1-1.5 wt%. S5. Adjust the pH of the system obtained in step S4 to 4.2-6.2 using one or both of citric acid and trisodium citrate dihydrate; S6. The system obtained in step S5 is filtered using a combination of 200-1000 mesh filtration and 0.45μm terminal filtration, or it is filled in a low-bacterial environment where the microbial limit test results meet the requirements for release of low-bacterial stock solution, and purified water is added to bring the solid content of the obtained stock solution to 1-15wt% to obtain the plant bioreaction composite stock solution.

7. The production method according to claim 6, characterized by, In step S3, the solid mass ratio of the A1-derived plant oligosaccharide intermediate to the B1-derived collagen-hyaluronic acid intermediate is 2:1-1:2; the amount of sodium borohydride added is 0.2-2 wt% of the total solids of the A1-derived plant oligosaccharide intermediate and the B1-derived collagen-hyaluronic acid intermediate. The reduction stabilization temperature is 0-5℃; the high-pressure homogenization pressure is 50-80MPa, and the homogenization is performed 3-4 times. The average particle size of the resulting composite nanocomplex is 100-250nm.

8. The preparation method according to claim 6, characterized in that, In step S4, based on the total solids of the plant bioreaction complex stock solution obtained in step S6, the amount of N-acetylglucosamine added is 0.5-5 wt%, the amount of yeast β-glucan added is 0.1-5 wt%, and the amount of sodium chloride added is 0.3-0.9 wt%.

9. The preparation method according to claim 6, characterized in that, In step S6, the filtration process involves first performing a 200-1000 mesh pre-filtration, followed by a 0.45μm terminal filtration.

10. The preparation method according to claim 6, characterized in that, In step S6, the obtained plant bioreaction compound stock solution is filled into single-use bottles, cans or bags.

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