A method for extracting saxifrage active components by enzymatic coupling and membrane separation for shoe and clothing antibacterial agents

Abstract

The application discloses a kind of Saxifrage active components of shoe and clothes antibacterial agent enzymatic coupling membrane separation extraction method, it includes after Saxifrage crushing treatment, water is stirred, then add composite plant hydrolytic enzyme and carry out enzymolysis pretreatment, after enzymolysis is completed, enzyme solution is killed enzyme treatment, then it is carried out high-speed centrifugation and removes coarse residue, and obtains crude extract liquid;Crude extract liquid is input to membrane separation system;Then collect the retention liquid of ultrafiltration membrane and / or nanofiltration membrane, carry out drying treatment, obtain high-activity Saxifrage enrichment component, the present scheme is combined with cellulase and pectinase and is carried out enzymolysis auxiliary extraction, can specifically destroy Saxifrage cell wall cellulose, pectin structure, break the bondage of active ingredient by cell wall, significantly improve the dissolution rate of flavonoids, phenolic acid and other active substances in cell;And enzymolysis reaction condition is mild (40-55 ℃, pH 4.5-6.0), can effectively avoid the destruction of heat-sensitive active ingredient by strong heat, strong acid and alkali and other conditions, maximum limit retains the original activity of active component.

Description

Technical Field

[0001] This invention relates to the field of plant active ingredient extraction and separation technology, and in particular to an enzymatic hydrolysis coupled membrane separation and extraction method for active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel. Background Technology

[0002] Saxifraga stolonifera, a perennial herb belonging to the genus Saxifraga in the family Saxifragaceae, is widely distributed in East, South, and Southwest my country. It is a plant resource with both medicinal and ornamental value. The entire plant can be used medicinally. Traditional Chinese medicine believes it has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and dispelling wind and dampness. It is often used to treat sore throat, eczema, otitis media, and carbuncles. Modern pharmacological studies have shown that Saxifraga stolonifera contains abundant flavonoids (such as quercetin, quercetin-3-rhamnoside, and bergenin), phenolic acids (such as chlorogenic acid and caffeic acid), triterpenoids, volatile oils, and antimicrobial peptides, exhibiting broad-spectrum antibacterial, anti-inflammatory, antioxidant, antiviral, and anti-allergic pharmacological activities. Based on its excellent biological activity, Saxifraga stolonifera has shown broad application prospects in cosmetics (such as anti-allergy, acne-removing, and antioxidant products), oral care products (such as mouthwash and toothpaste), and pharmaceuticals (such as antibacterial and anti-inflammatory preparations).

[0003] However, the extraction and separation technology of active components from Saxifraga stolonifera still has many shortcomings. Currently, the main methods for extracting and separating active components from Saxifraga stolonifera include traditional organic solvent extraction, reflux extraction, and column chromatography. Among these, organic solvent extraction (such as extraction with ethanol, methanol, and ethyl acetate) is the most commonly used method, but it has the following prominent problems: First, the use of large amounts of organic solvents not only causes serious environmental pollution but also increases the cost of solvent recovery and treatment; second, organic solvents may remain in the product, affecting the safety and compliance of the final product, especially in cosmetics and oral care products, where solvent residues are strictly limited; third, organic solvents may cause the degradation or inactivation of some heat-sensitive active ingredients (such as phenolic acids and antimicrobial peptides) during high-temperature concentration, leading to the loss of active components and reducing the efficacy of the product.

[0004] While column chromatography (such as macroporous adsorption resins and silica gel column chromatography) can achieve a certain degree of component separation and purification, it suffers from problems such as low separation efficiency, cumbersome operation, long cycle time, high solvent consumption, and high equipment investment, making it difficult to achieve large-scale, continuous production. Furthermore, column chromatography lacks selectivity in enriching active components, often targeting only a specific type of component (such as total flavonoids), failing to retain the multi-component synergistic activity characteristics inherent in Saxifraga stolonifera. It is noteworthy that Saxifraga stolonifera has a dense cell wall structure, primarily composed of cellulose, hemicellulose, and pectin, which constitute the main barrier to the dissolution of intracellular active components. Traditional extraction methods (such as water extraction and alcohol extraction) lack specific means to disrupt the cell wall structure, resulting in low dissolution rates of intracellular active components such as flavonoids and phenolic acids. A large amount of effective components are bound within the cell wall, leading to raw material waste and low extraction efficiency. Although some studies in recent years have attempted to use physical enhancement methods such as ultrasound-assisted extraction and microwave-assisted extraction, these methods have problems such as high equipment costs, difficulty in scale-up, and local overheating that damages active ingredients, making it difficult to meet the needs of clean industrial production.

[0005] Furthermore, existing separation methods generally lack an "activity-oriented" design concept, meaning they do not monitor changes in the bioactivity of each component during separation, making it impossible to precisely retain highly active components. This results in significant fluctuations in the activity of the final product and poor quality stability. This poses a major technical bottleneck for cosmetic and pharmaceutical intermediates that require clearly defined efficacy and controllable quality. Therefore, developing a green, efficient, low-cost, activity-oriented method for enriching the active components of Saxifraga stolonifera, achieving effective cell wall disruption, efficient dissolution of active ingredients, selective retention of target components, and ensuring high product activity and safety, has become an urgent technical problem to be solved in the field of Saxifraga stolonifera deep processing. Summary of the Invention

[0006] In view of this, this invention addresses the shortcomings of existing technologies in the extraction and separation of active components from Saxifraga stolonifera, such as residual organic solvents, loss of active ingredients, low separation efficiency, and lack of activity targeting. It proposes an enzymatic hydrolysis-coupled membrane separation and extraction method for active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel. This method is environmentally friendly, has high separation efficiency, and low energy consumption. It can effectively improve the dissolution rate and enrichment effect of active components while ensuring the high activity and safety of the product. This invention also provides the Saxifraga stolonifera active concentrate prepared by this method and its applications, expanding the application scenarios of Saxifraga stolonifera.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows: A method for enzymatic hydrolysis coupled with membrane separation and extraction of active components of Saxifraga stolonifera for use in antibacterial agents of footwear and apparel includes: sequentially enzymatically hydrolyzing the Saxifraga stolonifera raw material, centrifuging, membrane system separation, and drying to finally obtain the active enriched components of Saxifraga stolonifera; specifically, it includes: pulverizing Saxifraga stolonifera, adding water to form a slurry, adding a compound plant hydrolytic enzyme for enzymatic hydrolysis pretreatment, after enzymatic hydrolysis, inactivating the enzyme in the hydrolysate, and then centrifuging it at high speed to remove coarse residue to obtain a crude extract; inputting the crude extract into a membrane separation system equipped with a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane; then collecting the retentate from the ultrafiltration membrane and / or nanofiltration membrane, and drying it to obtain highly active enriched components of Saxifraga stolonifera.

[0008] As a preferred implementation method, this solution preferably includes the following steps: (1) Enzymatic hydrolysis pretreatment: The raw material of Saxifraga stolonifera is crushed and mixed with water. The pH of the mixture is adjusted to 4.5-6.0. Cellulase and pectinase are then added. The mixture is enzymatically hydrolyzed at 40-55℃ for 60-120 minutes. Then, the mixture is heated to inactivate the enzymes and obtain the enzymatic hydrolysate. The raw material of Saxifraga stolonifera is fresh Saxifraga stolonifera or whole Saxifraga stolonifera dried in the shade. The amount of cellulase added is 0.1%-0.5% of the mass of the raw material of Saxifraga stolonifera and the amount of pectinase added is 0.1%-0.3% of the mass of the raw material of Saxifraga stolonifera. (2) Centrifugation: The enzymatic hydrolysate is centrifuged and the supernatant is collected to obtain crude extract of Saxifraga stolonifera; (3) Membrane system separation: The crude extract is pumped into a membrane separation system with microfiltration membrane, ultrafiltration membrane and nanofiltration membrane; in the membrane separation system, the crude extract first passes through a microfiltration membrane with a pore size of 0.1-1.0 μm, and then the microfiltration permeate is collected; the microfiltration permeate is then passed through an ultrafiltration membrane with a molecular weight cutoff of 8000-15000 Da, and the ultrafiltration retentate is collected; the ultrafiltration permeate is further passed through a nanofiltration membrane with a molecular weight cutoff of 150-300 Da for concentration and desalination, and the nanofiltration retentate is collected; wherein, the microfiltration membrane is a ceramic membrane or a polyvinylidene fluoride membrane; the ultrafiltration membrane is a polyethersulfone membrane or a regenerated cellulose membrane; (4) Drying: Detect the membrane separation components obtained in step (3), which include the ultrafiltration retentate and the nanofiltration retentate. Based on the detection results, perform vacuum freeze-drying or spray drying on the ultrafiltration retentate and / or the nanofiltration retentate to obtain the active enrichment of Saxifraga stolonifera.

[0009] As a preferred implementation method, preferably, in step (3) of this scheme, the DPPH free radical scavenging rate of each component is detected in real time during the membrane separation process to determine the optimal active component retention range; Accordingly, in step (4), the membrane separation component with the highest free radical scavenging activity is subjected to vacuum freeze drying or spray drying to obtain the active enrichment of Saxifraga stolonifera.

[0010] Based on the above, this solution also proposes an active ingredient of Saxifraga stolonifera for footwear and apparel antibacterial agents, which is obtained by the extraction method described above. Its total flavonoid content is ≥25%, its total phenolic acid content is ≥10%, and it is also rich in active ingredients such as quercetin and bergenin, thus retaining the original broad-spectrum antibacterial and anti-inflammatory activities of Saxifraga stolonifera.

[0011] Based on the above, this solution also proposes a footwear and apparel antibacterial finishing agent containing active enrichment of Saxifraga stolonifera, which includes the Saxifraga stolonifera active component for footwear and apparel antibacterial agents mentioned above; preferably, the mass percentage of the Saxifraga stolonifera active component in the footwear and apparel antibacterial agent is 15%; the footwear and apparel antibacterial agent also includes a crosslinking agent and a dispersant.

[0012] Based on the above, this solution also proposes the application of the antibacterial finishing agent for footwear and apparel containing active extracts of Saxifraga stolonifera, wherein the antibacterial agent is applied to the footwear and apparel fabric or the lining of the footwear material by padding, impregnation or spraying.

[0013] The aforementioned antibacterial agent for footwear and apparel is prepared using a mixture of saxifrage active concentrate aqueous solution as the main ingredient, a mixture of water-soluble PU high-efficiency crosslinking agent and sodium lignosulfonate as auxiliary ingredients, and diethylenetriamine latent liquid microcapsule curing agent as a liquid additive to enhance adhesion through a stepwise variable-speed stirring method. This finishing agent can be firmly adhered to footwear and apparel fabrics through padding, impregnation, or spraying processes. According to the antibacterial test standard GB / T 20944.3-2008, the treated fabric achieved inhibition rates of 93.6% against Staphylococcus aureus and 92.0% against Escherichia coli, both exceeding the national standard (≥90%) requirements for antibacterial textiles. This demonstrates excellent antibacterial application value in footwear and apparel, expands the new applications of saxifrage in the field of antibacterial footwear and apparel, and has promising market prospects.

[0014] The active components of Saxifraga stolonifera obtained by this method can also be used as intermediates in the preparation of cosmetics, oral care products and pharmaceuticals, and are especially suitable for the preparation of products with antibacterial, anti-inflammatory and antioxidant effects.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: (1) Upgraded cell wall breaking technology to improve the dissolution rate of active ingredients: This invention combines cellulase and pectinase for enzymatic hydrolysis-assisted extraction, which can specifically destroy the cellulose and pectin structure of Saxifraga stolonifera cell wall, break the cell wall binding of active ingredients, and significantly improve the dissolution rate of active substances such as flavonoids and phenolic acids in the cells; and the enzymatic hydrolysis reaction conditions are mild (40-55℃, pH4.5-6.0), which can effectively avoid the destruction of heat-sensitive active ingredients by strong heat, strong acid and alkali conditions, and retain the original activity of active components to the maximum extent.

[0016] (2) Green and environmentally friendly, achieving clean production: This solution replaces the traditional organic solvent extraction and column chromatography with a three-stage membrane separation technology of "microfiltration-ultrafiltration-nanofiltration". No organic solvents are used throughout the process, there is no solvent residue, and environmental pollution is avoided. At the same time, the membrane separation process has low energy consumption and simple operation, which can realize large-scale clean production and reduce production costs.

[0017] (3) Precise enrichment to ensure high product activity: The membrane separation system used in this solution can be cut step by step according to the molecular weight of the active component, which can efficiently enrich the active components of specific molecular weight ranges (such as antimicrobial peptides or flavonoids of 500-1500 Da), while removing impurities such as large molecular proteins, polysaccharides and small molecular inorganic salts; combined with offline DPPH antioxidant activity tracking and HPLC fingerprinting, the membrane component with the best molecular weight cutoff can be accurately determined to ensure that the final enriched product has high activity and quality stability.

[0018] (4) The product has wide application and strong practicality: The total flavonoids and total phenolic acids in the active extract of Saxifraga stolonifera prepared by this invention are high, and it retains broad-spectrum antibacterial, anti-inflammatory and antioxidant activities. It can be used as an intermediate in the field of antibacterial finishing agents for footwear and clothing. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The Fourier transform infrared (FT-IR) spectra of the products obtained in Examples 1-5 are superimposed images, obtained using a Thermo Scientific Nicolet iS50 infrared spectrometer (KBr pellet, 4000-400 cm⁻¹). - ¹); Figure 2 The image shows the superimposed UV-Vis absorption spectra of the products obtained in Examples 1-5, obtained using a Shimadzu UV-2600 spectrophotometer (200-600 nm scan). Figure 3 The above is a comparison chart of the DPPH free radical scavenging rates of each membrane separation component in Examples 1-5 (517 nm colorimetric method). Figure 4 This is a comparison chart of the inhibition rates of Staphylococcus aureus and Escherichia coli in different treatment groups in Example 7 of the present invention. Detailed Implementation The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 This embodiment proposes an enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel, which includes: First, take 1 kg of dried whole Saxifraga stolonifera, crush it through a 40-mesh sieve, add 10 L of purified water, and stir well. Adjust the pH to 5.0 with citric acid, and add 3 g of cellulase (10000 U / g) and 2 g of pectinase (30000 U / g). Incubate the solution in a 50°C water bath for 90 minutes, stirring constantly. After incubation, rapidly raise the temperature to 95°C and maintain it for 10 minutes to inactivate the enzyme. Then, centrifuge the hydrolysate at 15000 rpm using a tubular centrifuge and collect the centrifuged liquid.

[0022] The centrifuged liquid is first passed through a 0.2 μm ceramic microfiltration membrane, and the permeate is collected to remove bacteria and fine particles.

[0023] The microfiltration permeate was pumped into a spiral-wound ultrafiltration membrane module with a molecular weight cutoff of 10,000 Da at an operating pressure of 0.4 MPa. The ultrafiltration retentate (rich in large protein and polysaccharide molecules) and the ultrafiltration permeate were collected. The ultrafiltration permeate was then concentrated using a nanofiltration membrane with a molecular weight cutoff of 200 Da at an operating pressure of 1.5 MPa to obtain the nanofiltration retentate.

[0024] The ultrafiltration retentate was freeze-dried to obtain 120g of solid powder. HPLC analysis showed that it contained 8.2% quercetin-3-rhamnoside, 3.5% bergenin, and 28% total flavonoids.

[0025] Example 2 This embodiment proposes an enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel, which includes: First, take 2 kg of fresh whole Saxifraga stolonifera, wash and cut it into sections, crush it through a 20-mesh sieve, add 20 L of purified water, and stir well. Adjust the pH to 5.5 with hydrochloric acid, and add 6 g of cellulase (10000 U / g) and 4 g of pectinase (30000 U / g). Incubate the mixture in a 45℃ water bath for 120 minutes, stirring every 30 minutes. After incubation, raise the temperature to 90℃ and maintain it for 15 minutes to inactivate the enzyme. Then, centrifuge the hydrolysate at 12000 rpm using a disc centrifuge, collecting the supernatant to obtain the crude Saxifraga stolonifera extract.

[0026] The crude extract was then passed sequentially through a polyvinylidene fluoride microfiltration membrane with a pore size of 0.45 μm, and the microfiltration permeate was collected. The microfiltration permeate was then pumped into a polyethersulfone ultrafiltration membrane module with a molecular weight cutoff of 8000 Da at an operating pressure of 0.35 MPa, and the ultrafiltration retentate was collected.

[0027] Finally, the ultrafiltration retentate was freeze-dried under vacuum to obtain 165g of Saxifraga stolonifera active concentrate. Analysis showed that the total flavonoid content was 26.8% and the total phenolic acid content was 11.2%.

[0028] Example 3 This embodiment proposes an enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel, which includes: Take 500g of dried whole Saxifraga stolonifera, crush it through a 60-mesh sieve, add 5L of deionized water, and stir well. Adjust the pH to 4.8 with phosphoric acid, and add 2g of cellulase (10000U / g) and 1.5g of pectinase (30000U / g). Incubate the solution in a 55℃ water bath for 60 minutes, and after the incubation, raise the temperature to 95℃ to inactivate the enzyme for 10 minutes. Centrifuge the hydrolysate at 10000r / min for 20 minutes and collect the supernatant.

[0029] The supernatant was passed through a 0.1 μm ceramic microfiltration membrane, and the microfiltration permeate was collected. The microfiltration permeate was then passed through a regenerated cellulose ultrafiltration membrane with a molecular weight cutoff of 15000 Da, and the ultrafiltration permeate was collected. Finally, the ultrafiltration permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 200 Da (operating pressure 1.2 MPa), and the nanofiltration retentate was collected, simultaneously removing inorganic salts and small molecule impurities.

[0030] Finally, the nanofiltration retentate was spray-dried (inlet air temperature 160℃, outlet air temperature 80℃) to obtain 52g of Saxifraga stolonifera active concentrate. The DPPH free radical scavenging rate IC50 was 18.5μg / mL, and the total phenolic acid content was 13.6%.

[0031] Example 4 This embodiment proposes an enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel, which includes: Take 3 kg of fresh whole Saxifraga stolonifera, crush it through a 30-mesh sieve, and add 30 L of purified water. Adjust the pH to 5.2 with acetic acid, add cellulase (0.3%, w / w) and pectinase (0.2%, w / w), and enzymatically hydrolyze at 48℃ for 100 minutes. The enzyme inactivation conditions are 90℃ for 15 minutes; then use a continuous centrifuge at 14000 r / min to collect the supernatant.

[0032] The supernatant was passed through a 0.5 μm polyvinylidene fluoride microfiltration membrane. The microfiltration permeate was then passed sequentially through a three-stage ultrafiltration membrane module with molecular weight cutoffs of 10000 Da, 5000 Da, and 1000 Da.

[0033] The retentate and permeate from each ultrafiltration stage were collected, and the DPPH free radical scavenging rate was measured. The results showed that the 10000 Da ultrafiltration retentate had the highest scavenging rate (92.3%), followed by the 5000 Da retentate (78.6%), while the 1000 Da permeate had the lowest scavenging rate (35.2%). Therefore, the 10000 Da ultrafiltration retentate was selected as the target component. Finally, the retentate was freeze-dried under vacuum to obtain 210 g of Saxifraga stolonifera active concentrate. Analysis revealed a total flavonoid content of 25.3% and a total phenolic acid content of 10.8%.

[0034] Example 5 This embodiment proposes an enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel, which includes: First, take 800g of dried whole Saxifraga stolonifera, crush it through a 50-mesh sieve, and add 8L of purified water. Adjust the pH to 4.5 with lactic acid, add cellulase (0.4%, w / w) and pectinase (0.25%, w / w), and enzymatically hydrolyze at 42℃ for 110 minutes. The enzyme inactivation conditions are 95℃ for 10 minutes. Centrifuge at 12000r / min for 15 minutes and collect the supernatant. Pass the supernatant through a 0.8μm ceramic microfiltration membrane. Pass the microfiltration permeate through an ultrafiltration membrane with a molecular weight cutoff of 8000Da, and collect the ultrafiltration permeate (rich in small molecule active peptides and flavonoid aglycones). Then, concentrate the ultrafiltration permeate through a nanofiltration membrane with a molecular weight cutoff of 150Da to obtain the nanofiltration retentate, removing water and some inorganic salts.

[0035] The nanofiltration retentate was freeze-dried under vacuum to obtain 68 g of Saxifraga stolonifera active concentrate. HPLC-MS analysis showed that the concentrate contained abundant low molecular weight antimicrobial peptides (molecular weight 800-1500 Da) and quercetin aglycone, and exhibited strong antimicrobial activity against Staphylococcus aureus with a MIC of 62.5 μg / mL.

[0036] Example 6 To facilitate comparison of the products obtained in each embodiment, this scheme provides a unified characterization of the products obtained in Examples 1-5, using the following method: (1) FT-IR analysis: A Thermo Scientific Nicolet iS50 infrared spectrometer was used, KBr pellet method, scanning range 4000-400 cm⁻¹ - ¹, resolution 4 cm - ¹, Scanned 32 times, with background subtraction using a KBr blank as the background, the results are as follows Figure 1As shown.

[0037] (2) UV-Vis analysis: Each product was prepared into a 0.5 mg / mL aqueous solution, and a Shimadzu UV-2600 UV-Vis spectrophotometer was used to scan in the range of 200-600 nm. Deionized water was used as a blank control. The results are as follows: Figure 2 , Figure 3 As shown.

[0038] (3) DPPH scavenging rate determination: Each membrane separation component (crude extract, microfiltration permeate, ultrafiltration retentate, ultrafiltration permeate, nanofiltration retentate) was diluted with deionized water and mixed with an equal volume of 0.1 mmol / L DPPH ethanol solution. The mixture was reacted at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm. The scavenging rate was calculated according to the formula. The results are as follows: Figure 4 As shown.

[0039] Example 7 7.1 Composition and Proportion of Antibacterial Agents for Footwear and Apparel This embodiment provides an antibacterial finishing agent for footwear and apparel containing active extracts of Saxifraga stolonifera. The main ingredient is an aqueous solution of active extracts of Saxifraga stolonifera, and the auxiliary ingredients are a crosslinking agent and a dispersant. A latent microcapsule curing agent is used as a liquid additive to enhance adhesion over time.

[0040] Specifically, the allocation of each group by volume is as follows: Main ingredient mixture: The active concentrate of Saxifraga stolonifera prepared in Example 1 (total flavonoid content 28%, quercetin-3-rhamnoside content 8.2%) was prepared into a 5% aqueous solution with deionized water and used as the main ingredient mixture; The auxiliary mixture is composed of a water-soluble PU high-efficiency crosslinking agent and a dispersant, wherein the dispersant is sodium lignosulfonate, and the volume ratio of the water-soluble PU high-efficiency crosslinking agent to sodium lignosulfonate is 5:1. Liquid additive: a diethylenetriamine latent liquid microcapsule curing agent with a latency of more than 1 month and a purity of ≥90%; The volume ratio of the main ingredient mixture, the auxiliary ingredient mixture, and the liquid additive is 100:2:4.

[0041] 7.2 Preparation method of antibacterial finishing agent for footwear and apparel Based on the above-mentioned formulation of the antibacterial finishing agent for footwear and apparel, its preparation method includes the following steps: (1) Pour the aqueous solution of active extract of Saxifraga stolonifera (main material mixture) into the reaction vessel and stir at a stirring speed of 600-900 r / min for 3-5 min to make it evenly distributed and form fine particles. (2) Increase the stirring speed of the reactor to 1000-1300 r / min, and slowly add the auxiliary mixture obtained by mixing water-soluble PU high-efficiency crosslinking agent and sodium lignosulfonate salt at a volume ratio of 5:1 into the reactor within 2-3 minutes, while maintaining the stirring speed, so that the auxiliary mixture is evenly distributed in the main mixture. (3) Adjust the stirring speed of the reactor to 300-500 r / min, and slowly add diethylenetriamine latent liquid microcapsule curing agent within 3-5 min. During this period, maintain the stirring speed so that the liquid additive is evenly and without damage distributed into the prepared mixture of main and auxiliary materials, thus obtaining the saxifrage-based antibacterial finishing agent for footwear and clothing.

[0042] 7.3 Application methods of antibacterial finishing agents on footwear and apparel The above-mentioned antibacterial finishing agent is applied to the footwear and apparel fabric using a padding method. The specific steps are as follows: (1) Select commercially available pure cotton knitted fabric (180g / m²) as the base material and cut it into 20cm×20cm test pieces; (2) Immerse the test piece in the antibacterial finishing agent working solution (diluted to 1.5 times the original concentration), bath ratio 1:20, and soak at room temperature for 30 minutes; (3) Use a small rolling mill for two dips and two rolls, and control the roll residue rate at 75±5%; (4) Place the treated fabric in a 120℃ oven for pre-drying for 5 minutes, and then cure it at 150℃ for 2 minutes; (5) After curing, the fabric is gently washed once with deionized water and dried at room temperature to become antibacterial shoe and clothing fabric.

[0043] As an alternative application method, the antibacterial finishing agent of the present invention can also be uniformly sprayed onto the surface of the lining or insole of the finished footwear and apparel by spraying, or treated with the lining material of the footwear by impregnation.

[0044] 7.4 Antibacterial efficacy testing and parallel control verification Referring to the national standard GB / T 20944.3-2008 "Evaluation of antimicrobial properties of textiles - Part 3: Shaking method", typical Gram-positive bacteria (Staphylococcus aureus ATCC 6538) and Gram-negative bacteria (Escherichia coli ATCC 8739) were selected for testing.

[0045] Set up the following parallel controls: Blank control group: Untreated pure cotton fabric (after sterilization); Sex control group: Fabrics treated with only blank finishing agent (deionized water + water-soluble PU crosslinking agent + sodium lignosulfonate + microcapsule curing agent) without saxifrage active enrichment; Experimental group: Antibacterial footwear and apparel fabrics prepared according to the methods described in 6.2 and 6.3 above.

[0046] The test results are shown in the table below:

[0047] Figure 4 This is a comparison chart of the inhibition rates of Staphylococcus aureus and Escherichia coli in different treatment groups. Figure 4 af corresponds to the number af in the table above.

[0048] The above results indicate that the colony counts in both the blank control group and the negative control group were significantly increased compared to the initial inoculation number, suggesting that neither the fabric itself nor the finishing agent without active components possesses antibacterial ability. However, the experimental group achieved inhibition rates of 93.6% against Staphylococcus aureus and 92.0% against Escherichia coli, both exceeding the national standard (GB / T 20944.3-2008 requiring an inhibition rate ≥90%) for antibacterial textiles. This demonstrates that the active extract of Saxifraga stolonifera extracted in this invention has significant and reliable antibacterial efficacy in footwear and apparel antibacterial agents.

[0049] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for enzymatic hydrolysis and membrane separation extraction of active components of Saxifraga stolonifera for use in antibacterial agents for footwear and apparel, characterized in that, It includes: The raw material of Saxifraga stolonifera was subjected to enzymatic pretreatment, centrifugation, membrane system separation, and drying in sequence to obtain the active enriched components of Saxifraga stolonifera.

2. The enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel as described in claim 1, characterized in that, It includes: After crushing and processing Saxifraga stolonifera, water is added to form a slurry, and then a compound plant hydrolytic enzyme is added for enzymatic pretreatment. After enzymatic hydrolysis, the enzyme inactivation treatment is performed on the hydrolysate, and then the hydrolysate is centrifuged at high speed to remove coarse residue, resulting in a crude extract. The crude extract is then fed into a membrane separation system equipped with microfiltration membrane, ultrafiltration membrane, and nanofiltration membrane. The retentate from the ultrafiltration membrane and / or nanofiltration membrane is then collected and dried to obtain highly active Saxifraga stolonifera enriched components.

3. The enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel as described in claim 1 or 2, characterized in that, It includes the following steps: (1) Enzymatic hydrolysis pretreatment: After crushing the raw material of Saxifraga stolonifera, mix it with water, adjust the pH of the mixture to 4.5-6.0, add cellulase and pectinase, and enzymatically hydrolyze at 40-55℃ for 60-120 minutes, then heat up to inactivate and obtain the enzymatic hydrolysate; (2) Centrifugation: The enzymatic hydrolysate is centrifuged and the supernatant is collected to obtain crude extract of Saxifraga stolonifera; (3) Membrane system separation: The crude extract is pumped into a membrane separation system with microfiltration membrane, ultrafiltration membrane and nanofiltration membrane; in the membrane separation system, the crude extract first passes through a microfiltration membrane with a pore size of 0.1-1.0 μm, and then the microfiltration permeate is collected; the microfiltration permeate is then passed through an ultrafiltration membrane with a molecular weight cutoff of 8000-15000 Da, and the ultrafiltration retentate is collected; the ultrafiltration permeate is further passed through a nanofiltration membrane with a molecular weight cutoff of 150-300 Da for concentration and desalination, and the nanofiltration retentate is collected; (4) Drying: Detect the membrane separation components obtained in step (3), which include the ultrafiltration retentate and the nanofiltration retentate. Based on the detection results, perform vacuum freeze-drying or spray drying on the ultrafiltration retentate and / or the nanofiltration retentate to obtain the active enrichment of Saxifraga stolonifera.

4. The enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel as described in claim 3, characterized in that, The raw material of Saxifraga stolonifera is fresh Saxifraga stolonifera or whole Saxifraga stolonifera dried in the shade; the amount of cellulase added is 0.1%-0.5% of the mass of the raw material of Saxifraga stolonifera, and the amount of pectinase added is 0.1%-0.3% of the mass of the raw material of Saxifraga stolonifera.

5. The enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel as described in claim 3, characterized in that, The microfiltration membrane is a ceramic membrane or a polyvinylidene fluoride membrane; the ultrafiltration membrane is a polyethersulfone membrane or a regenerated cellulose membrane.

6. The enzymatic hydrolysis-coupled membrane separation and extraction method for the active components of Saxifraga stolonifera used in antibacterial agents for footwear and apparel as described in claim 3, characterized in that, In step (3), during the membrane separation process, the DPPH free radical scavenging rate of each component is detected in real time to determine the optimal retention range of the active component; In step (4), the membrane separation component with the highest free radical scavenging activity is subjected to vacuum freeze drying or spray drying to obtain the active enrichment of Saxifraga stolonifera.

7. A saxifrage active ingredient for footwear and apparel antibacterial agents, characterized in that, It is obtained by the extraction method described in any one of claims 1 to 6, and its total flavonoid content is ≥25% and its total phenolic acid content is ≥10%.

8. A footwear and apparel antibacterial finishing agent containing active extracts of Saxifraga stolonifera, characterized in that, It includes the active ingredient of Saxifraga stolonifera for footwear and apparel antibacterial agents as described in claim 7.

9. The antibacterial finishing agent for footwear and apparel containing active extracts of Saxifraga stolonifera according to claim 8, characterized in that, The active ingredient of Saxifraga stolonifera accounts for 15% of the mass of the antibacterial agent in footwear and apparel; the antibacterial agent in footwear and apparel also includes a crosslinking agent and a dispersant.

10. The application of the antibacterial finishing agent for footwear and apparel containing active extracts of Saxifraga stolonifera according to claim 9, characterized in that: The antibacterial agent for footwear and apparel is applied to the footwear and apparel fabric or the lining of the footwear material by padding, impregnation or spraying.

Images