A deproteinized natural rubber latex, its preparation method and application

Abstract

This invention provides a deproteinized natural rubber latex, its preparation method, and its application, belonging to the field of rubber latex technology. This invention introduces a multi-enzyme system combination immobilization technology to form a synergistic effect of multiple enzymes, allowing high-concentration enzymes to contact proteins in natural rubber latex, greatly improving enzyme catalytic efficiency. Addressing the complex and diverse nature of latex proteins, the catalytic characteristics of different enzymes are effectively combined to achieve a multi-enzyme cascade reaction, gradually and systematically hydrolyzing the proteins thoroughly. Simultaneously, an immobilized enzyme filter is introduced. Through a rising and falling circulating filter, the rubber particles collide with the high-density enzyme carrier while being uniformly stirred, reducing damage to the rubber particles during surface protein removal, thus maintaining the original physical properties of the latex. This invention also offers advantages such as high stability, reusability, easy separation, continuous operation, and environmental friendliness.

Description

Technical Field

[0001] This invention relates to the field of rubber latex technology, and in particular to a deproteinized natural rubber latex, its preparation method, and its application. Background Technology

[0002] Natural rubber latex, with its green biological origin, excellent film strength, high elasticity, and memory properties, is irreplaceable by other materials, making it the preferred material for expandable elastic products such as medical devices, catheters, tapes, tourniquets, gloves, and condoms. However, natural rubber latex contains latex proteins that can cause skin sensitization, and allergic reactions caused by these proteins are the biggest problem facing medical natural rubber latex products. Studies have found that the number of allergy sufferers among most people who frequently come into contact with natural rubber latex and its products has reached epidemic levels. These individuals experience allergic reactions such as rhinitis, conjunctivitis, contact urticaria, and bronchial asthma upon contact with these products, and some even experience sudden death. Currently, the available way to avoid protein allergies in natural rubber latex is to remove the proteins. However, the problem of performance degradation caused by protein removal in natural rubber latex remains unresolved, limiting the application of deproteinized rubber products. Research on protein removal from natural rubber latex has been ongoing for decades. Although various methods for removing proteins from natural rubber latex are known, including enzymatic protein removal patents such as CN 102702393 A, CN 86106292 A, CN 1246485 A, CN 112010996 A, CN102268106 A, CN 102002119 A, CN 105017446 A, and CN 1353116 A, these methods all use free proteases. These enzymes are difficult to separate from the product and can cause pollution from residual enzyme waste liquid. Therefore, the amount and type of enzyme used are limited, resulting in incomplete protein removal. At the same time, it will also damage the flowability and viscosity of the latex to varying degrees. The resulting natural rubber latex products are difficult to meet the requirements for use in products such as gloves and condoms, both in terms of protein content and physical properties.

[0003] In addition to rubber hydrocarbons and water, natural rubber latex contains approximately 5% non-rubber substances, of which proteins account for 1-2%. About 20% of these proteins are distributed on the surface of the rubber particles, serving as an important component of the protective layer; 65% are dissolved in the whey; and the remainder are attached to the bottom layer of the latex. Although proteins constitute a relatively small portion of the overall latex system, their diverse types and complex structures significantly influence the properties of natural rubber latex. Currently, the main methods for deproteinizing natural rubber latex include: 1) Surface treatment, also known as chlorination, which reduces the content of water-soluble proteins in rubber gloves by releasing chlorine gas; 2) Leaching, also known as rinsing, which removes water-soluble proteins from rubber products by washing with water, including dry membrane leaching and wet gel leaching; 3) Adsorption-displacement method, also known as substitution adsorption, which involves adding certain small molecules to denature the protein, then adding an adsorbent to adsorb it, and removing it from the latex by centrifugation; 4) Multiple centrifugation, which involves repeatedly diluting the latex with water and centrifuging to prepare latex with extremely low non-rubber content; 5) Chemical degradation, which uses strong acids or alkalis to degrade natural rubber latex. The process involves several steps: 6) Radiation method: irradiating natural rubber latex with gamma rays or electron beams causes oil-soluble bound proteins in the latex to become water-soluble proteins, which dissolve in the whey phase and can be removed by centrifugation; 7) Electrophoresis method: each protein has a unique isoelectric point and properties, which can be identified and separated by electrophoresis; 8) Emulsion graft copolymerization technology: preparing deproteinized natural rubber latex through emulsion copolymerization; 9) Enzymatic hydrolysis method: under alkaline conditions, proteases decompose proteins in natural rubber latex, transforming them into water-soluble polypeptides or amino acids. High-speed centrifugation separates the water-soluble polypeptides and amino acids from the rubber particles, removing proteins from the natural rubber latex.

[0004] Of the methods mentioned above, methods 1) and 2) are mainly suitable for removing proteins from natural rubber products, including gloves, but not for preparing deproteinized natural rubber latex. Methods 3) to 9) can obtain natural rubber latex with low protein content. However, due to their simplistic approach, it is difficult to completely remove the complex and diverse proteins in the latex. If these methods are overused (excessive centrifugation, radiation, chemical degradation, graft copolymerization, enzymatic hydrolysis, etc.), although they may help remove proteins, the rubber particles will be damaged to a greater extent, causing the latex to lose its original fluidity and viscosity. Even with the addition of emulsifiers, this is irreversible. The product will no longer have the film-forming properties, film strength, and high elasticity of natural rubber latex. During filtration, a large amount of flocculent material similar to tofu dregs will be produced. The resulting film is prone to cracking, flow marks, pinholes, and poor strength, making it unsuitable as a latex raw material for impregnation film formation in surgical gloves, condoms, and protective films. Summary of the Invention

[0005] The purpose of this invention is to provide a deproteinized natural rubber latex, its preparation method, and its application. The deproteinized natural rubber latex has a low protein content and excellent physical properties.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing deproteinized natural rubber latex, comprising the following steps:

[0008] By combining mesh gauze with an adsorption medium solution, adsorption is carried out to obtain enzyme carrier gauze.

[0009] The enzyme carrier gauze was immersed in different enzyme solutions for enzyme adsorption. The resulting gauze was then cross-linked and fixed in a cross-linking agent solution to obtain single-piece immobilized enzyme filter layers corresponding to different enzymes. The single-piece immobilized enzyme filter layers corresponding to different enzymes were stacked to obtain an immobilized enzyme filter screen. The enzymes used in the different enzyme solutions were protease, peptidase, phospholipase and lipase, respectively.

[0010] Natural rubber latex, water, and nonionic surfactant are mixed, and gluconolactone solution is added to the resulting mixture. The mixture is then filtered using the immobilized enzyme filter in a lifting and lowering cycle until the pH value is 3-6, thus obtaining the enzymatically hydrolyzed latex.

[0011] The enzymatically hydrolyzed latex, water-soluble polymer, and anionic surfactant are mixed, the pH is adjusted to 9-11, and after solubilization, the mixture is centrifuged to obtain deproteinized natural rubber latex.

[0012] Preferably, the mesh gauze is woven from any one or more of silk braided yarn, pure cotton braided yarn, and pineapple fiber yarn; the mesh size of the mesh gauze is 0.1–2.0 cm, the braided yarn strength is >25 MPa, and the elongation is <20%; the adsorption medium solution includes one or more of regenerated silk protein solution, polyethyleneimine solution, dopamine hydrochloride solution, sodium alginate solution, and chitosan solution; the adsorption temperature is 0–45°C, and the time is 10–300 min.

[0013] Preferably, the protease includes one or more of alkaline protease, neutral protease, bromelain, papain, trypsin, thermophilic protease, staphylococcal protease, and clostridium protease; the peptidase includes one or more of exopeptidase, aminopeptidase, and carboxypeptidase; and the phospholipase includes one or more of phospholipase A, phospholipase B, phospholipase C, and phospholipase D.

[0014] Preferably, the crosslinking agent in the crosslinking agent solution includes one or more of glutaraldehyde, glyoxal, terephthalaldehyde, and genipin; the concentration of the crosslinking agent solution is 0.05–3.5 wt%, the pH value is 7.0–9.5, and the dosage is 3–10 g / g mesh gauze; the temperature for enzyme adsorption and crosslinking fixation is independently 0–45°C, and the time for enzyme adsorption and crosslinking fixation is independently 10–300 min.

[0015] Preferably, the natural rubber latex contains 1.0-65% dry rubber by mass and has a pH value of 8.0-12.0.

[0016] Preferably, the nonionic surfactant is any one or more selected from Tween, Span, trehalose, stachyose, glucoside, nonylphenol polyoxyethylene ether, isomeric 13 alcohol polyoxyethylene ether, and alkyl polyglucoside, and the mass of the nonionic surfactant is 0.01-10% of the mass of the natural rubber latex; the concentration of the glucono-delta-lactone solution is 1-10 wt%, and the mass of glucono-delta-lactone in the solution is 0.01-10% of the mass of the natural rubber latex.

[0017] Preferably, during the cyclic filtration, the immobilized enzyme filter screen automatically rises and falls under the vertical axis, with a rising and falling speed of 1 to 20 cm / s, and the cyclic filtration time is 3 to 6 hours; after the cyclic filtration is completed, it is left to stand for ≥3 hours.

[0018] Preferably, the water-soluble polymer comprises one or more of polyethylene glycol, methoxy polyethylene glycol, polypropylene glycol, methyl polypropylene glycol, polyethylene oxide, polyethylene oxide ether, polyvinyl alcohol, polyvinyl alcohol copolymer, ethyl hydroxyethyl cellulose, methyl cellulose, polyacrylamide, polyacrylamide copolymer, polyacrylic acid, sodium polyacrylate, polypropylene oxide, and polyglycerol glycidyl ether polyvinyl alcohol, wherein the mass of the water-soluble polymer is 0.01-10% of the mass of natural rubber latex; the anionic surfactant comprises diphenyl ether sulfonate, alkylbenzene sulfonate, alkylnaphthalene sulfonate, and naphthalene sulfonate. The product comprises one or more of the following: alkyl sulfonates, dialkyl sulfosuccinates, α-alkene sulfonates, α-sulfonated fatty acid salts, alkyl sulfates, polyoxyalkylene stilbene phenol sulfates, polyoxyalkylene sulfates, tristilbene phenol sulfates, polyoxyalkylene phenyl ether sulfates, alkyl sulfates, polyoxyethylene alkyl sulfates, polyoxyethylene alkylphenyl ether sulfates, polyoxyethylene tristilbene phenol sulfates, and polyoxyethylene stilbene phenol sulfates; the mass of the anionic surfactant is 0.01–10% of the dry mass of the natural rubber latex; the solubilization time is 3–6 h.

[0019] The present invention provides a deproteinized natural rubber latex prepared by the preparation method described in the above technical solution.

[0020] This invention provides the application of the deproteinized natural rubber latex described in the above-mentioned technical solution in natural rubber latex products.

[0021] This invention provides a method for preparing deproteinized natural rubber latex. The method introduces a multi-enzyme system immobilization technique to achieve synergistic effects, allowing high-concentration enzymes to contact proteins in the natural rubber latex, significantly improving enzyme catalytic efficiency. Addressing the complex and diverse nature of latex proteins, the method effectively combines the catalytic characteristics of different enzymes to achieve a multi-enzyme cascade reaction, gradually and systematically hydrolyzing the proteins thoroughly. Simultaneously, an immobilized enzyme filter is introduced. Through a rising and falling circulating filter, the rubber particles collide with the high-density enzyme carrier, achieving uniform mixing and reducing damage to the rubber particles during surface protein removal, thus maintaining the original physical properties of the latex. This method offers advantages such as high stability, reusability, easy separation, continuous operation, and environmental friendliness.

[0022] The deproteinized natural rubber latex prepared by the method of the present invention can achieve a water-extractable protein content of ≤45μg / g and an antigen protein content of ≤8μg / g. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the multi-layer mesh filter device in Example 4;

[0024] Figure 2 This is a schematic diagram of the multi-layer filter assembly structure in Example 4;

[0025] Figure 3 This is a schematic diagram of the single-layer filter structure in Example 4. Detailed Implementation

[0026] This invention provides a method for preparing deproteinized natural rubber latex, comprising the following steps:

[0027] By combining mesh gauze with an adsorption medium solution, adsorption is carried out to obtain enzyme carrier gauze.

[0028] The enzyme carrier gauze was immersed in different enzyme solutions for enzyme adsorption. The resulting gauze was then cross-linked and fixed in a cross-linking agent solution to obtain single-piece immobilized enzyme filter layers corresponding to different enzymes. The single-piece immobilized enzyme filter layers corresponding to different enzymes were stacked to obtain an immobilized enzyme filter screen. The enzymes used in the different enzyme solutions were protease, peptidase, phospholipase and lipase, respectively.

[0029] Natural rubber latex, water, and nonionic surfactant are mixed, and gluconolactone solution is added to the resulting mixture. The mixture is then filtered using the immobilized enzyme filter in a lifting and lowering cycle until the pH value is 3-6, thus obtaining the enzymatically hydrolyzed latex.

[0030] The enzymatically hydrolyzed latex, water-soluble polymer, and anionic surfactant are mixed, the pH is adjusted to 9-11, and after solubilization, the mixture is centrifuged to obtain deproteinized natural rubber latex.

[0031] In this invention, unless otherwise specified, the raw materials or reagents required for preparation are all commercially available products well known to those skilled in the art.

[0032] This invention combines mesh gauze with an adsorption medium solution for adsorption, thereby obtaining enzyme carrier gauze.

[0033] In this invention, the mesh fabric is preferably woven from any one or more of silk yarn, pure cotton yarn, and pineapple fiber yarn. The mesh size of the mesh fabric is preferably 0.1–2.0 cm, the yarn strength is preferably >25 MPa, and the elongation is preferably <20%. This invention does not impose any special limitations on the weaving method; the mesh fabric meeting the above conditions can be obtained by following a well-known process.

[0034] Silk braided yarn, pure cotton braided yarn, and pineapple fiber yarn are natural fibers composed of proteins and polysaccharides. They have the structural advantage of rich natural fibrillary layers. In particular, they exhibit unique affinity and adsorption properties for proteases, silk fibroin, polyethyleneimine, chitosan, dopamine hydrochloride, sodium alginate, and chitosan. Even after enzyme fixation treatment, they still maintain softness and porosity, high mechanical strength, and excellent air permeability, water absorption and water resistance, permeability, and high capillary absorption.

[0035] In this invention, the adsorption medium solution preferably includes one or more of the following: regenerated silk protein solution, polyethyleneimine solution, dopamine hydrochloride solution, sodium alginate solution, and chitosan solution; when the adsorption medium solution is two or more of the above, this invention does not have a special limitation on the ratio of different types of adsorption medium solutions, and can be adjusted according to actual needs.

[0036] In this invention, the regenerated silk fibroin solution is preferably obtained by dissolving degummed silk in a ternary solvent of CaCl2 / CH3CH2OH / H2O and dialysis using a semi-permeable membrane. The concentration of the regenerated silk fibroin solution is preferably 0.5–5 wt%, more preferably 3.6 wt%, and the pH value is preferably 6–8, more preferably 7.0. This invention preferably uses a phosphate buffer solution to adjust the pH of the regenerated silk fibroin solution. This invention does not have specific limitations on the different solvent ratios in the CaCl2 / CH3CH2OH / H2O ternary solvent; adjustments can be made according to actual needs. In an embodiment of the present invention, a ternary solvent is obtained by mixing CaCl2 / CH3CH2OH / H2O at a mass ratio of 1:2:8. Degummed raw silk is placed in the ternary solvent at a mass ratio of 1:20 (raw silk: ternary solvent). The mixture is allowed to stand at 50°C for 6 hours to swell, and then stirred at 70°C until the degummed raw silk is completely dissolved. The resulting solution is then dialyzed using a semi-permeable membrane dialysis bag, i.e., dialyzed in pure water at room temperature, with the water changed every 4 hours. The conductivity of the dialysis water is measured until the conductivity is ≤1.0 μS / cm. The dialyzed silk fibroin solution is then concentrated with a 10 wt% polyethylene glycol solution to obtain a regenerated silk fibroin solution with a concentration of 3.6 wt% and a pH of 7.0.

[0037] In this invention, the concentration of the polyethyleneimine solution is preferably 1-10 wt%, the pH is preferably 8.0, and it is prepared using an acetate-sodium hydroxide buffer solution with a pH of 4-10; the concentration of the dopamine hydrochloride solution is preferably 1.0-4.0 mg / mL, and it is prepared using a 0.05-0.20 M (more preferably 0.1 M) tris(hydroxymethyl)aminomethane (Tris) buffer solution with a pH of 8.0-9.0; the concentration of the sodium alginate solution is preferably 0.1-3.0 wt%, more preferably 0.11 wt%, the pH is 7.0, and it is prepared using a sodium phosphate buffer solution with a pH of 4-6; the concentration of the chitosan solution is 0.1-3.0 wt%, more preferably 0.21 wt%, the pH is preferably 5.5, and it is prepared using a sodium acetate buffer solution with a pH of 4-6. This invention does not impose any special limitations on the specific preparation process of the adsorption medium solution; the above-mentioned adsorption medium solution can be obtained by following a process well known in the art.

[0038] The present invention does not have a special limitation on the ratio of the mesh gauze to the adsorption medium solution, as long as the mesh gauze is fully immersed in the adsorption medium solution.

[0039] Before combining the mesh gauze with the adsorption medium solution, the present invention preferably pre-treats the mesh gauze. The pre-treatment preferably includes immersing the mesh gauze in a 70wt% isopropanol solution, ultrasonically washing for 40 minutes, then ultrasonically cleaning with deionized water for 20 minutes to remove surface grease and dust, and then drying it in a 50℃ vacuum drying oven.

[0040] In this invention, the preferred method for combining the mesh gauze with the adsorption medium solution is immersion or spraying; the preferred adsorption temperature is 0–45°C, more preferably 25–30°C, and the preferred adsorption time is 10–300 min, more preferably 120 min. This invention achieves adsorption of the adsorption medium solution by the mesh gauze through adsorption.

[0041] After the adsorption is completed, the present invention preferably washes the obtained gauze with deionized water until the filtrate is colorless and transparent, and then dries it overnight in a vacuum oven at 50°C to obtain enzyme carrier gauze.

[0042] The adsorption medium solution used in this invention contains regenerated silk fibroin, polyethyleneimine, chitosan, dopamine hydrochloride, and sodium alginate chitosan, all of which possess excellent gelling properties and readily form membranes, porous microspheres, or gels. These materials can be processed into various forms and all serve as high-performance immobilized enzyme carriers. For example, regenerated silk fibroin transforms from a soluble structure to an insoluble p-sheet structure while simultaneously immobilizing the enzyme, significantly distinguishing it from other immobilized enzyme carriers. Chitosan and polyethyleneimine molecules have numerous amino groups. When pH < 10, these amino groups are mostly in a protonated state, making them positively charged polyelectrolytes. Most enzymes are negatively charged at pH < 10; therefore, under certain pH conditions, they exhibit electrostatic attraction to enzyme molecules, leading to ion adsorption and immobilization. Dopamine hydrochloride undergoes oxidative self-polymerization in aerobic, weakly alkaline solutions to generate polydopamine with strong adhesion properties. This polydopamine can adhere to any solid substrate surface, forming strong and durable bonds, thus giving the filler better adhesion, wettability, and surface roughness, which is more conducive to enzyme immobilization. The G unit of the sodium alginate molecular chain readily reacts with Ca... 2+ Its function is to form a hole between the G units of the two molecular chains, allowing Ca to bind. 2+ The "egg box" model forms an irreversible gel, thereby immobilizing the enzyme in the gel network to form an immobilized enzyme.

[0043] After obtaining the enzyme carrier gauze, the present invention immerses the enzyme carrier gauze in different enzyme solutions for enzyme adsorption, and then crosslinks and fixes the obtained gauze in a crosslinking agent solution to obtain single immobilized enzyme filter layers corresponding to different enzymes. The single immobilized enzyme filter layers corresponding to different enzymes are stacked and laid to obtain an immobilized enzyme filter screen.

[0044] In this invention, the enzymes used in the different enzyme solutions are proteases, peptidases, phospholipases, and lipases, respectively. The proteases preferably include one or more of alkaline proteases, neutral proteases, bromelain, papain, trypsin, thermophilic proteases, staphylococcal proteases, and clostridium proteases. The peptidases preferably include one or more of exopeptidases, aminopeptidases, and carboxypeptidases. The phospholipases preferably include one or more of phospholipase A, phospholipase B, phospholipase C, and phospholipase D. When the proteases or peptidases are two or more of the above, this invention does not have a special limitation on the ratio of different types of proteases or peptidases; adjustments can be made according to actual needs.

[0045] In this invention, when the enzyme is a protease, the concentration of the enzyme solution is preferably 10-60 mg / mL, more preferably 15.5-16.5 mg / mL, the amount added is 500-1500 mg / g mesh gauze, more preferably 1000 mg / g mesh gauze, and the pH value is preferably 7.0-10.0, more preferably 9.0; when the enzyme is a peptidase, the concentration of the enzyme solution is preferably 10-60 mg / mL, more preferably 12.9 mg / mL, the amount added is preferably 500-1500 mg / g mesh gauze, more preferably 1000 mg / g mesh gauze, and the pH value is preferably 7.0-9.5, more preferably 10.0 ... The pH value is preferably 7.5; when the enzyme is phospholipase, the concentration of the enzyme solution is preferably 5-30 mg / mL, more preferably 8.5 mg / mL, the addition amount is preferably 200-1200 mg / g mesh gauze, more preferably 1000 mg / g mesh gauze, and the pH value is preferably 7.0-9.5, more preferably 9.0; when the enzyme is lipase, the concentration of the enzyme solution is preferably 5-30 mg / mL, more preferably 8.9 mg / mL, the addition amount is preferably 200-1200 mg / g mesh gauze, more preferably 1000 mg / g mesh gauze, and the pH value is preferably 5.0-9.0, more preferably 9.0.

[0046] In this invention, the solvent used for the enzyme solution is preferably water; other solutions used in this invention, unless otherwise specified, are all aqueous solutions.

[0047] Natural rubber latex contains various proteins, such as α-globulin adsorbed on the surface of rubber particles, as well as rubber proteins, basic proteins, and fibrous proteins. At least 15 types of proteins are found in whey alone. Rubber particles are spherical particles with a double-membrane-core structure. The core is composed of rubber hydrocarbons, with phospholipids coating the core surface to form a phospholipid layer, and proteins coating the phospholipid layer to form a protein layer. Phospholipids and proteins together constitute the double-membrane structure of the rubber particle. This invention employs a complex enzyme to hydrolyze proteins, specifically using a complex enzyme (protease, peptidase, phospholipase, and lipase) to enzymatically hydrolyze the phospholipids and proteins in the double-membrane structure at multiple levels, causing them to peel off into water-soluble substances which are then removed by centrifugation.

[0048] Different proteases act on different sites on the peptide chain, resulting in varying cleavage frequencies. Alkaline proteases, neutral proteases, bromelain, papain, trypsin, thermophilic proteases, staphylococcal proteases, and clostridium proteases each have their own specific peptide bond cleavage sites, exhibiting high hydrolytic activity. This invention allows for the combined use of different proteases to effectively hydrolyze various proteins in latex. However, these proteases are endopeptidases, meaning they only cleave bonds within the peptide chain and not at the terminal bonds. Consequently, the proteins hydrolyzed by this enzyme still retain a high molecular weight (number average molecular weight). Proteins with a pH of 4500 or higher and their breakdown products are prone to causing allergies and have limited water solubility. The peptidase used in this invention is an exopeptidase, capable of cleaving amino acids one by one from the N-terminus or C-terminus of the peptide chain, releasing free amino acids. Its function is to completely hydrolyze the protein peptide chain into small amino acids. This invention introduces lipase and phospholipase to decompose and detach fats and phospholipids adhering to the protein, minimizing interference from oily substances in protein hydrolysis.

[0049] In this invention, the crosslinking agent in the crosslinking agent solution preferably includes one or more of glutaraldehyde, glyoxal, terephthalaldehyde, and genipin; the concentration of the crosslinking agent solution is preferably 0.05–3.5 wt%, more preferably 0.5–3 wt%, the pH value is preferably 7.0–9.5, more preferably 7.5–9.0, and the dosage is preferably 3–10 g / g mesh gauze, more preferably 5 g / g; the solvent used in the crosslinking agent solution is preferably water.

[0050] In this invention, when the enzyme is a protease, the preferred method for preparing the monolithic immobilized enzyme filter layer includes: immersing an enzyme carrier gauze in a protease solution, shaking it in a constant-temperature shaker to adsorb the enzyme, allowing the protease to adsorb onto the enzyme carrier gauze, removing the supernatant, then immersing it in a cross-linking agent solution to cross-link and fix the adsorption medium for the adsorbed protease, washing it with phosphate buffer until no more protease is eluted, and then freezing or drying it at room temperature to obtain the monolithic immobilized protease filter layer. This invention does not have a specific limitation on the freezing or drying process; any process well-known in the art can be followed. This invention also does not have a specific limitation on the phosphate buffer; any commercially available product well-known in the art can be used.

[0051] In this invention, when the enzymes are peptidase, phospholipase, and lipase in sequence, the protease solution is replaced by peptidase solution, phospholipase solution, and lipase solution in sequence, and the above method is repeated to obtain a single immobilized peptidase filter layer, a single immobilized phospholipase filter layer, and a single immobilized lipase filter layer, respectively.

[0052] In this invention, the temperature for enzyme adsorption and cross-linking fixation is preferably 0–45°C, more preferably 25–30°C (i.e., room temperature), and the time for enzyme adsorption and cross-linking fixation is preferably 10–300 min, more preferably 120 min.

[0053] In this invention, the preferred method for stacking the single-layer immobilized enzyme filter layers corresponding to the different enzymes is to select one or more combinations of single-layer immobilized protease filter layers, single-layer immobilized peptidase filter layers, single-layer immobilized phospholipase filter layers, and single-layer immobilized lipase filter layers to form a multi-layer immobilized enzyme filter mesh with interlayer spacing. The interlayer spacing is preferably 2 to 50 cm, more preferably 5 cm, and each type of single-layer immobilized protease filter layer (single-layer immobilized protease filter layer, single-layer immobilized peptidase filter layer, single-layer immobilized phospholipase filter layer, and single-layer immobilized lipase filter layer) is at least one layer.

[0054] In this invention, the immobilized enzyme loading in the immobilized enzyme filter is preferably 93-375 mg / g filter.

[0055] After obtaining the immobilized enzyme filter, the present invention mixes natural rubber latex, water and nonionic surfactant, adds gluconolactone solution to the resulting mixture, and uses the immobilized enzyme filter to perform up-and-down circulation filtration until the pH value is 3-6 (more preferably 5.0) to obtain enzymatically hydrolyzed latex.

[0056] In this invention, the natural rubber latex is preferably fresh latex of the Brazilian rubber tree or its concentrated latex or its clear latex; the mass content of dry rubber in the natural rubber latex is preferably 1.0-65%, more preferably 25-60%, and the pH value is preferably 8.0-12.0.

[0057] The present invention preferably uses steel bars to form steel rings as a frame to fix the filter screen, and then uses three equally divided vertical axes (the angle between the connecting lines is 120°) to fix each filter screen respectively, forming a multi-layer filter screen with different layer spacing. The filter screen is placed in a cylindrical tube and automatically lifted and lowered by a mechanical device.

[0058] In this invention, the preferred method for mixing natural rubber latex, water, and nonionic surfactant, and then adding glucono-delta-lactone solution to the resulting mixture is to place the natural rubber latex in a cylindrical container (height / inner diameter = 100cm / 40cm), add water to make the latex concentration ≤30wt%, then add the nonionic surfactant, start the automatic lifting and lowering of the filter screen at a speed of 10cm / s, maintain for 4 hours, let it stand for 8 hours, start the automatic lifting and lowering of the filter screen again at a speed of 10cm / s, maintain the lifting and lowering state, and start slowly adding glucono-delta-lactone solution after 1 hour.

[0059] In this invention, the nonionic surfactant is preferably any one or more of Tween, Span, trehalose, stachyose, glucoside, nonylphenol polyoxyethylene ether (OP-10), isomeric 13 alcohol polyoxyethylene ether, and alkyl polyglucoside. The mass of the nonionic surfactant is preferably 0.01-10% of the mass of natural rubber latex, more preferably 5%. When the nonionic surfactant is two or more of the above, this invention does not have a special limitation on the ratio of different types of nonionic surfactants, and can be adjusted according to actual needs.

[0060] In this invention, the concentration of the glucono-delta-lactone solution is preferably 1-10 wt%, more preferably 6 wt%; the solvent used in the glucono-delta-lactone solution is preferably water; the mass of glucono-delta-lactone in the glucono-delta-lactone solution is preferably 0.01-10% of the mass of the natural rubber latex, more preferably 5%. The glucono-delta-lactone solution is preferably added dropwise; the dropwise addition rate is preferably 133 g / min. During the dropwise addition of glucono-delta-lactone, a lifting filter is activated to achieve stirring, allowing the glucono-delta-lactone to disperse rapidly and avoiding flocculation caused by high concentration in the microenvironment.

[0061] In this invention, the shape and size of the immobilized enzyme filter are preferably matched to the inner cavity of the container holding natural rubber latex, so that it can automatically rise and fall while fixed on the vertical axis.

[0062] After adding the glucono-delta-lactone solution, the present invention preferably initiates a circulating filtration and stirring process using an immobilized enzyme filter, maintaining this for 3–6 hours. This allows the pH value of the latex system to slowly decrease, gradually changing from 10 to 3–6, and stabilizing at 3–6, more preferably 5. The present invention stabilizes the pH by controlling the amount of glucono-delta-lactone solution added, as the hydrolysis of glucono-delta-lactone causes a decrease in the solution pH, the degree of which depends on the amount of glucono-delta-lactone used. During the circulating filtration, the immobilized enzyme filter automatically rises and falls while fixed on a vertical axis, with a preferred rising and falling speed of 1–20 cm / s, more preferably 10 cm / s. The preferred circulating filtration time is 3–6 hours.

[0063] During the cyclic filtration process, the natural rubber latex is allowed to fully interact with the immobilized enzyme through cyclic filtration, decomposing the proteins and phospholipids in the latex. The natural rubber latex is protected with nonionic surfactants, and the natural rubber latex is slowly acidified by gluconolactone hydrolysis, which changes the hydrophilicity and hydrophobicity of the enzymatically hydrolyzed proteins and phospholipids, causing them to lose their function as a protective layer for the rubber particles and detach from the surface of the rubber particles. The nonionic surfactants are then adsorbed onto the surface of the rubber particles in their place.

[0064] The immobilized enzyme filter of this invention has multiple functions, including stirring, enhancing the contact between rubber particles and enzymes, preventing the residue of free enzymes in latex, and allowing for the reuse of immobilized enzymes. The more filter layers the immobilized enzyme filter contains, the more complete the protein decomposition; or the longer the circulation filtration time, the more complete the protein decomposition.

[0065] After completing the cyclic filtration, the present invention preferably allows the obtained latex to stand, removes the immobilized enzyme filter, washes it with water, freezes or dries it at room temperature, and reuses it; the standing time is preferably ≥3h.

[0066] After obtaining the enzymatically hydrolyzed latex, the present invention mixes the enzymatically hydrolyzed latex, water-soluble polymer and anionic surfactant, adjusts the pH value to 9-11, solubilizes, and then separates by centrifugation to obtain deproteinized natural rubber latex.

[0067] In this invention, the water-soluble polymer preferably includes one or more of the following: polyethylene glycol, methoxy polyethylene glycol, polypropylene glycol, methyl polypropylene glycol, polyethylene oxide, polyethylene oxide ether, polyvinyl alcohol, polyvinyl alcohol copolymer, ethyl hydroxyethyl cellulose, methyl cellulose, polyacrylamide, polyacrylamide copolymer, polyacrylic acid, sodium polyacrylate, polypropylene oxide, and polyglycerol glycidyl ether polyvinyl alcohol. When the water-soluble polymer is two or more of the above types, this invention does not have a special limitation on the ratio of different types of water-soluble polymers, and can be adjusted according to actual needs.

[0068] In this invention, the mass of the water-soluble polymer is preferably 0.01 to 10% of the mass of the natural rubber latex, and more preferably 3%.

[0069] In this invention, the anionic surfactant preferably includes one or more of the following: diphenyl ether sulfonate, alkylbenzene sulfonate, alkylnaphthalene sulfonate, naphthalene sulfonate, alkyl sulfonate, dialkyl sulfosuccinate, α-alkene sulfonate, α-sulfonated fatty acid salt, alkyl sulfate, polyoxyalkylene stilbene phenol sulfate, polyoxyalkylene sulfate, tristyrene phenol sulfate, polyoxyalkylene phenyl ether sulfate, alkyl sulfate salt, polyoxyethylene alkyl sulfate salt, polyoxyethylene alkylphenyl ether sulfate salt, polyoxyethylene tristyrene phenol sulfate salt, and polyoxyethylene stilbene phenol sulfate salt. When the anionic surfactant is two or more of the above types, this invention does not have a special limitation on the ratio of different types of anionic surfactants, and can be adjusted according to actual needs.

[0070] In this invention, the mass of the anionic surfactant is preferably 0.01 to 10% of the dry mass of the natural rubber latex, more preferably 3%.

[0071] This invention utilizes anionic surfactants and water-soluble polymers to restore or even enhance the water solubility of protein and phospholipid decomposition products, allowing them to enter the aqueous phase. At the same time, it increases the specific gravity difference between the clear rubber and the latex, facilitating subsequent centrifugation to remove most of the non-rubber components, including proteins and phospholipids, to obtain deproteinized natural rubber latex.

[0072] The present invention does not have any special limitations on the mixing of the enzymatically hydrolyzed latex, water-soluble polymer and anionic surfactant. The materials can be mixed evenly according to a process known in the art.

[0073] In this invention, the pH value is preferably adjusted to 9-11 (preferably 10) using KOH solution. After solubilization under stirring, the mixture is allowed to stand for at least 5 hours, and then centrifuged to obtain deproteinized natural rubber latex. The solubilization time is preferably 3-6 hours. This invention does not impose any special limitations on the stirring and centrifugation speeds; procedures well-known in the art can be followed. The concentration of the KOH solution is not particularly limited; it can be adjusted according to actual needs.

[0074] This invention, after obtaining deproteinized natural rubber latex (purity ≥99%, dry rubber content / total solids content) by centrifugation, preferably further includes diluting the prepared deproteinized natural rubber latex with deionized water to below 30%, followed by centrifugation for concentration. This process of dilution and centrifugation is repeated sequentially. Through the action of different immobilized enzyme filters, the water-extractable protein content in the natural rubber latex can be ≤45μg / g (test standard ASTM D 5712), and the antigen protein content can be ≤8μg / g (test standard ASTM D 6499).

[0075] This invention immobilizes proteases, peptidases, phospholipases, and lipases on a mesh gauze with filtration function to create an immobilized enzyme filter. Through circulating filtration, the immobilized enzymes fully interact with the natural rubber latex, decomposing the proteins and phospholipids in the latex. A nonionic surfactant is used for protection, and the natural rubber latex is slowly acidified by gluconolactone hydrolysis, altering the hydrophilicity and hydrophobicity of the enzymatically hydrolyzed proteins and phospholipids, causing them to lose their protective layer function and detach from the rubber particle surface. The nonionic surfactant is then adsorbed onto the rubber particle surface. The resulting latex is adjusted to alkalinity, and anionic surfactants and water-soluble polymers are added to restore or even enhance the water solubility of the protein and phospholipid decomposition products. Finally, centrifugation is used to remove the proteins, yielding deproteinized natural rubber latex.

[0076] The protein content in fresh natural rubber latex accounts for 1-2% of its mass. Approximately 20% of the protein is distributed on the surface of the rubber particles, 65% is dissolved in the whey, and the remaining protein is attached to the bottom layer of the natural rubber latex. The structure of a rubber particle typically consists of three layers: the innermost layer is the sol layer, composed of rubber hydrocarbon molecules with a low degree of polymerization; the middle layer is the gel layer, composed of rubber hydrocarbon molecules with a higher degree of polymerization, which may have branched or cross-linked structures. Proteins and phospholipids bond to the ends of the rubber hydrocarbon molecules, further bonding or associating with more advanced branched structures. The outermost layer is a protective layer formed by proteins and lipids, which helps maintain the dispersion and stability of the rubber particles. The protein and lipid molecules in the protective layer contain many polar groups and highly electronegative elements. Since water molecules in the whey are also polar, permanent dipole attraction and hydrogen bonding can cause hydration on the surface of the rubber particles, forming a hydration film. This hydration film is sensitive to pH, stable under alkaline conditions, but easily destroyed under acidic conditions. This invention utilizes the synergistic effect of proteases, peptidases, phospholipases, and lipases to effectively decompose proteins and lipids in different structural layers of natural rubber latex. While reducing the branching degree of rubber molecules, proteins and lipids more easily detach from the surface of the rubber particles, releasing water-soluble protein and phospholipid decomposition products. In particular, immobilizing multiple enzymes on a mesh gauze with filtration function provides superior stability, high activity, and operability compared to free enzymes, and allows for reusability. This also improves enzyme utilization efficiency and allows for significant dosage increases, ensuring that the immobilized enzymes do not remain in the latex. The circulating filtration of the mesh gauze greatly increases the probability of contact between rubber particles and enzymes, improving reaction efficiency from both chemical and physical perspectives.

[0077] Even after immobilized enzyme decomposition, a significant portion of the protein and phospholipid products remain adsorbed on the surface of the rubber particles, carrying a negative charge and being sensitive to pH. This invention incorporates gluconolactone and a nonionic surfactant into natural latex. The gluconolactone undergoes gentle hydrolysis, releasing hydrogen ions very uniformly and slowly, gradually lowering the pH. This allows the nonionic surfactant to adsorb onto the rubber particle surface at a rate greater than the rate at which the protein and phospholipid decomposition products are acidified. This enables the nonionic surfactant to replace proteins and lipids as a protective layer, maintaining the dispersion stability of the rubber particles. This solves the common problem encountered in latex alkali-acid reversal, where an acid solution is typically added dropwise to an alkaline latex. The dropwise addition is too rapid to diffuse quickly enough, creating a microenvironment with excessively high acid concentration. In this environment, rubber particles are easily demulsified, causing localized particle coagulation. This damage is irreversible, ultimately affecting the film strength of the latex and even leading to film defects.

[0078] Existing enzymatic hydrolysis methods all use free proteases, resulting in some enzyme residue in the product and some being released into the environment, polluting the environment. This limits the amount and type of enzyme used, leading to low hydrolysis efficiency, incomplete protein removal, complex operation, difficulty in control, and challenges for industrial production. This invention uses immobilized enzymes, which are enzymes that can be fixed on a carrier and catalyze reactions within a certain spatial range. Most enzyme-catalyzed reactions occur in aqueous solutions, while immobilized enzymes are treated with physical or chemical methods to make them insoluble in water, yet still retain enzyme activity. Compared to free enzymes, immobilized enzymes are more stable, less affected by external factors such as temperature, organic solvents, and pH, and are particularly easy to separate from the reaction system, allowing for continuous operation, easier control of the catalytic reaction, simplified purification process, reduced environmental pollution, reusability, lower costs, and facilitating industrial production. This invention immobilizes multiple enzymes separately on different gauze carriers and multiple enzymes separately on the same carrier, combining them into an ordered multi-enzyme complex. This effectively combines the catalytic properties of different enzymes, achieving a multi-enzyme cascade reaction, thereby greatly improving the catalytic efficiency of the enzymes.

[0079] Existing methods employ mechanical stirring. If the stirring is gentle, homogenization is not achieved, and the density difference between the dispersed phase and the dispersion medium leads to uneven protein removal from the latex. If the stirring is intensified, the high-speed shearing and mechanical impact forces cause varying degrees of damage to the morphology and structure of the rubber particles. This invention, however, aims to achieve isotropic and uniform mixing to the greatest extent possible through a fixed enzyme filter while minimizing damage to the rubber particles. This reduces concentration differences between different microenvironments within the container. The fixed enzyme filter circulates at a uniform speed within the latex. The collision between the rubber particles and the filter coincides with the contact between the protein on the particle surface and the fixed enzyme. Furthermore, the filter area covers the entire cross-section of the container. During its circulation, each collision between the rubber particles and the filter represents contact between the protein and the high-density enzyme, allowing enzymatic hydrolysis and homogenization to occur simultaneously.

[0080] The present invention provides a deproteinized natural rubber latex prepared by the preparation method described in the above technical solution.

[0081] This invention provides the application of the deproteinized natural rubber latex described above in natural rubber latex products. This invention does not specifically limit the method of application; any method well-known in the art can be used.

[0082] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0083] Example 1

[0084] Preparation of adsorption medium solution

[0085] (1) Preparation of regenerated silk fibroin solution: CaCl2 / CH3CH2OH / H2O were mixed at a mass ratio of 1:2:8 to obtain a ternary solvent. Degummed raw silk was placed in the ternary solvent at a mass ratio of 1:20 (raw silk: ternary solvent). The solution was allowed to stand at 50°C for 6 hours to swell. Then, it was stirred at 70°C until the degummed raw silk was completely dissolved. The resulting solution was dialyzed through a semi-permeable membrane dialysis bag, i.e., dialyzed in pure water at room temperature. The water was changed every 4 hours, and the conductivity of the dialysis water was measured until the conductivity was ≤1.0μS / cm. The dialyzed silk fibroin solution was concentrated with 10wt% polyethylene glycol solution to obtain a regenerated silk fibroin solution with a concentration of 3.6wt% and a pH of 7.0.

[0086] (2) Preparation of dopamine hydrochloride solution: 1.817 kg of tris(hydroxymethyl)aminomethane (Tris) was dissolved in deionized water to obtain a 0.1 M Tris buffer solution. Hydrochloric acid was added to adjust the pH to 8.0. 1.0 kg of dopamine was dissolved in the Tris buffer solution to obtain a dopamine hydrochloride solution with a concentration of 1.9 mg / mL.

[0087] (3) Preparation of polyethyleneimine solution: Polyethyleneimine was dissolved in deionized water and acetic acid-sodium hydroxide with pH 5 was used as buffer solution to prepare a 1.42 wt% polyethyleneimine solution with pH 8.0.

[0088] (4) Preparation of sodium alginate solution: Sodium alginate was dissolved in deionized water and sodium phosphate buffer with pH 5 was used as buffer to prepare a 0.11 wt% sodium alginate solution with pH 7.0.

[0089] (5) Chitosan solution preparation: Chitosan was dissolved in 1% acetic acid aqueous solution, and sodium acetate with pH 5 was used as buffer solution to prepare a 0.21 wt% chitosan solution with pH 5.5.

[0090] Example 2

[0091] Preparation of enzyme carrier gauze

[0092] Table 1 Woven Mesh Fabric

[0093]

[0094]

[0095] (1) Place the silk thread woven mesh in a 70wt% isopropanol solution and ultrasonically wash for 40 min, then ultrasonically clean it with deionized water for 20 min to remove surface grease and dust. Take it out and place it in a 50℃ vacuum drying oven to dry. Immerse the cleaned and dried silk thread woven mesh in the above-mentioned regenerated silk protein solution and maintain it at room temperature for 120 min. Take out the silk thread woven mesh, wash it with deionized water until the filtrate is colorless and transparent, and place it in a 50℃ vacuum oven to dry overnight to obtain a silk thread woven mesh with adsorbed silk protein. Replace the silk protein solution with the above-mentioned hydrochloric acid dopamine solution to obtain a silk thread woven mesh with adsorbed hydrochloric acid dopamine.

[0096] (2) The pure cotton woven mesh was placed in a 70wt% isopropanol solution and ultrasonically washed for 40 min, then ultrasonically cleaned with deionized water for 20 min to remove surface grease and dust. It was then placed in a 50℃ vacuum drying oven to dry. The cleaned and dried pure cotton woven mesh was immersed in the above-mentioned dopamine hydrochloride solution and maintained at room temperature for 120 min. The pure cotton woven mesh was then removed, washed with deionized water until the filtrate was colorless and transparent, and placed in a 50℃ vacuum oven to dry overnight, obtaining a pure cotton woven mesh adsorbed with dopamine hydrochloride. The dopamine hydrochloride solution was replaced with the above-mentioned regenerated silk protein solution, polyethyleneimine solution, sodium alginate solution, and chitosan solution to obtain corresponding pure cotton woven meshes adsorbed with regenerated silk protein, polyethyleneimine, sodium alginate, and chitosan.

[0097] (3) Place the pineapple fiber woven mesh in a 70wt% isopropanol solution and ultrasonically wash for 40 min, then ultrasonically clean it with deionized water for 20 min to remove the grease and dust on the surface. Take it out and put it into a 50℃ vacuum drying oven to dry. Immerse the cleaned and dried pineapple fiber woven mesh in the above chitosan solution and maintain it at room temperature for 120 min. Take out the pineapple fiber woven mesh, wash it with deionized water until the filtrate is colorless and transparent, and put it in a 50℃ vacuum oven to dry overnight to obtain the pineapple fiber woven mesh with adsorbed chitosan.

[0098] Example 3

[0099] Preparation of immobilized enzymes

[0100] (1) Preparation of papain solution: Dissolve papain in sodium phosphate buffer solution with pH 5.5 to prepare papain solution with a concentration of 15.5 mg / mL and pH value of 9.0;

[0101] (2) Preparation of alkaline protease solution: Dissolve alkaline protease in sodium phosphate buffer solution with pH 9.5 to prepare alkaline protease solution with a concentration of 15.5 mg / mL and a pH of 9.0;

[0102] (3) Preparation of bromelain solution: Dissolve bromelain in sodium phosphate buffer solution with pH 7.5 to prepare an alkaline protease solution with a concentration of 15.0 mg / mL and a pH of 9.0;

[0103] (4) Preparation of trypsin solution: Dissolve trypsin in sodium phosphate buffer solution with pH 8.0 to prepare a trypsin solution with a concentration of 16.5 mg / mL and a pH of 9.0;

[0104] (5) Preparation of exopeptidase solution: 12 kg of fresh sheep kidneys were added to 100 L of water and crushed and autolyzed at room temperature for 6 h. The autolyzed kidney juice was centrifuged at 5000 r / min for 20 min, and the supernatant was collected and filtered. Ammonium sulfate was added to the filtrate to 50% saturation, the precipitate was collected, the precipitate was dissolved in water, and after dialysis and desalting, the concentration of the compound exopeptidase solution was maintained at 12.9 mg / mL and the pH was 7.5.

[0105] (6) Preparation of compound phospholipase solution and lipase solution: Weigh 8 kg of phospholipase A, 8 kg of phospholipase B and 2 kg of lipase respectively, extract them with 100 L of glycine-sodium hydroxide buffer solution with pH 9.5 for 1 h, and then centrifuge at 2000 r / min for 30 min to remove impurities. Take the supernatant as compound phospholipase solution (concentration 8.5 mg / mL, pH 9.0) and lipase solution (8.9 mg / mL, pH 9.0) respectively, and keep them in a refrigerator at 4℃ for later use.

[0106] Immobilized enzymes:

[0107] The silk woven web with adsorbed regenerated silk fibroin was immersed in the above-mentioned papain solution and shaken at 300 rpm at room temperature until the solution became clear. The supernatant was decanted, and the silk woven web with adsorbed silk fibroin and papain was crosslinked with 3.0 wt% glutaraldehyde solution (pH 9.0) for 120 min. After crosslinking, the woven web was removed, washed twice with glycine buffer (pH 6.0), and then washed with water until the pH stabilized. It was dried at room temperature to obtain silk fibroin woven web with immobilized papain. The supernatant, crosslinked solution, and eluent were collected and mixed, and the protein content was determined to calculate the immobilized papain loading (based on SN / T). Standard 2497.20-2010, "Safety Test Methods for Imported and Exported Hazardous Chemicals, Part 20: Bradford Method for the Determination of Protein Content," describes a method where Coomassie Brilliant Blue G250 combines with basic amino acids (arginine) and aromatic amino acids in protein molecules in an acidic solution to form a blue complex. Within a certain range, the intensity of the color is directly proportional to the protein concentration. A protein reference solution is used as a standard curve, and the protein content in the test sample is determined by colorimetry.

[0108] Immobilized enzyme load: The amount of protease immobilized per unit mass of woven mesh, in mg / g;

[0109] Immobilized enzyme activity: Under experimental conditions, the amount of enzyme consumed to produce 1 μmol of product per unit time is defined as 1 U. For example, a protease hydrolyzes a casein substrate under certain temperature and pH conditions. Then, reagents such as trichloroacetic acid are added to terminate the enzyme reaction, and the unhydrolyzed casein is precipitated. The filtrate absorbs ultraviolet light, and the activity can be determined by ultraviolet spectrophotometry. The enzyme activity can be calculated based on the absorbance.

[0110] The immobilized enzyme woven mesh and its corresponding parameters are shown in Table 2.

[0111] Table 2 shows the immobilized enzyme-woven webs obtained using different enzyme solutions and cross-linking agents (conditions not listed in Table 2 are the same as above):

[0112]

[0113]

[0114]

[0115] Examples 4-16 (including Examples 4-1 and 4-2)

[0116] Table 3. Filter mesh layer combination sequence in Example 4

[0117] Ⅰ Silk fibroin web with immobilized papain Ⅱ Silk fibroin woven web with immobilized alkaline protease Ⅲ Dopamine hydrochloride immobilized silk thread braided web Ⅳ Dopamine hydrochloride silk yarn woven mesh with immobilized exopeptidase enzyme Ⅴ Silk protein pure cotton yarn woven mesh with immobilized phospholipase and lipase Ⅵ Dopamine hydrochloride immobilized exopeptidase pure cotton yarn braided mesh Ⅹ Pineapple fiber woven web with chitosan immobilized bromelain

[0118] (1) As Figures 1-3 As shown, steel rings with a diameter of 30cm are made using steel bars with a diameter of 0.5cm. These rings serve as the frame for fixing the filter screens listed in Table 2. Each filter screen is then fixed using three equally spaced vertical axes (with a 120° angle between the connecting lines), forming a multi-layered filter screen with a mesh spacing of 5cm. This is placed inside a cylindrical tube with a diameter of 40cm and a height of 100cm, and automatically raised and lowered using a mechanical device. 40kg of commercially available high-ammonia natural rubber latex (obtained by centrifugation and concentration of fresh latex from Brazilian rubber trees, with a dry rubber content of 60wt%, a total solids content of 61.9wt%, and a pH of 12) is placed in the cylindrical tube. Deionized water is added to adjust the latex concentration to 25wt% (for Examples 10-12, 83kg of fresh latex from Brazilian rubber trees, with a dry rubber content of 29wt%, a total solids content of 33wt%, is added; deionized water is added to adjust the latex concentration to 25wt%, and ammonia is used to adjust the pH to 11). Then, 5wt% of a nonionic surfactant (specific type) is added based on the mass of the natural rubber latex. (See Table 4, all proportions in Table 4 are mass ratios) 19.2 kg, start the automatic lifting and lowering of the filter screen at a speed of 10 cm / s, maintain for 4 hours, then let it stand for 8 hours; start the automatic lifting and lowering of the filter screen again at a speed of 10 cm / s, maintain the lifting and lowering state for 1 hour, then start adding 2.4 kg of glucono-delta-lactone solution (the concentration of glucono-delta-lactone solution is 6 wt%, and the mass of glucono-delta-lactone accounts for 5% of the mass of natural rubber latex) at a rate of 133 g / min, continue to maintain the circulation filtration for 6 hours, then let it stand for 3 hours, and measure the pH value to be 5.0; remove the filter screen, wash it with deionized water, and dry it at room temperature for reuse.

[0119] (2) Add 17 kg of anionic surfactant potassium laurate (3 wt% of the natural rubber latex mass) and 18.4 kg of water-soluble polymer (polyethylene glycol: methoxy polyethylene glycol mass ratio = 1:1) (3 wt% of the natural rubber latex mass) to the latex, adjust the pH value to 10 with 1 wt% KOH solution, mechanically stir for 3 h, let stand for 6 h, and centrifuge at high speed to obtain deproteinized natural rubber latex with a purity ≥ 99% (dry rubber content / total solids content).

[0120] Performance testing

[0121] 1) The mechanical stability of the deproteinized natural rubber latexes obtained in Examples 4 to 16 was determined according to ISO 35 Method for Determination of Mechanical Stability of Concentrated Natural Rubber Latex. The results are shown in Table 4.

[0122] 2) Determination of protein content extracted by water extraction

[0123] The prepared deproteinized natural rubber latex was sampled according to ISO 123. A casting process was used, employing commercially available polypropylene petri dishes with an inner diameter of 90 mm. The petri dishes were placed horizontally on a support, and a certain amount of natural rubber latex was poured into the dishes, spreading it evenly across the bottom. The latex was then allowed to air dry at room temperature until transparent. The finished film was then peeled off the bottom of the petri dish; the thickness of the dried film was 0.3 ± 0.05 mm. The protein content extracted by water from the air-dried film was determined according to the method described in ASTM D5712, and the results are shown in Table 4.

[0124] Table 4. Protein content of aqueous extracts from Examples 4-16 and Comparative Examples 1-7 and the resulting latexes.

[0125]

[0126]

[0127]

[0128]

[0129] Generally, the more centrifugations, the greater the damage to the latex. Table 4 shows that, with only one centrifugation, multilayer networks consisting of protease fixation networks, peptidase fixation networks, and phospholipase and lipase fixation networks simultaneously exhibit the best protein removal effect, as seen in Examples 4, 6, and 9. Furthermore, these networks retain their activity and original protein removal capacity even after repeated use, as in Examples 4-1 and 4-2. In contrast, multilayer networks lacking phospholipase and lipase networks or lacking peptidase networks show slightly weaker protein removal capabilities, as seen in Examples 5, 7, and 8. Multilayer networks containing only one protease exhibit even weaker protein removal capabilities, as seen in Examples 11, 12, and 1. 3. Example 16: Multilayer networks containing only peptidase or only phospholipase and lipase have the weakest protein removal ability, as in Examples 14 and 15. In comparative examples, for the same enzyme, the protein removal ability of using free enzymes is much weaker than that of multilayer networks composed of protease fixation networks, peptidase fixation networks, and phospholipase and lipase fixation networks, and even weaker than that of multilayer networks containing only one protease. Adding different free enzymes in steps and centrifuging multiple times can improve the protein removal ability, but the effect is not as good as that of multilayer networks composed of protease fixation networks, peptidase fixation networks, and phospholipase and lipase fixation networks. More importantly, the latex particles are damaged to a greater extent.

[0130] Application examples

[0131] Pre-vulcanization of deproteinized natural rubber latex and preparation of gloves thereof:

[0132] Weigh 15 parts of a 10% potassium hydroxide aqueous solution; weigh 40.5 parts of ZDC, 13.5 parts of PX, 90 parts of sulfur, 67.5 parts of zinc oxide, 90 parts of antioxidant Wingstay L, 11.5 parts of cetyltrimethylammonium bromide, 2 parts of nano calcium carbonate, 0.1 parts of Tween 40, 0.1 parts of Span-80 and deionized water, mix and grind for 6 hours to prepare a 50% mixed vulcanizing agent suspension.

[0133] Weigh 15,000 parts of the deproteinized natural rubber latex obtained from Examples 4-16 and Comparative Examples 1-7, and add it to a jacketed vulcanizing reactor. Under stirring conditions, add 5,000 parts of deionized water, 15 parts of 10% potassium hydroxide aqueous solution, and 626 parts of 50% mixed vulcanizing agent suspension. After stirring evenly, heat the deproteinized natural rubber latex to 50°C in a water bath for 60 minutes, then cool it to 45°C for 4 hours. Let it stand at 30°C for 2-5 days, and monitor the chloroform value. The degree of vulcanization of the deproteinized natural rubber latex, expressed as chloroform value, is considered complete when it reaches the third degree of vulcanization. + Pre-vulcanized deproteinized natural rubber latex was obtained. The pre-vulcanized deproteinized natural rubber latex was diluted with deionized water to a total solids mass fraction of 40%. The chloroform value was monitored for three consecutive days and remained within the range of III. + —Between the four initial stages.

[0134] 1) Following the conventional glove molding and dipping process, the hand mold is heated to 70°C and then immersed in a coagulation solution (42g calcium nitrate, 4.5g calcium carbonate, 0.8g wetting agent, 4.5g release agent, and 500g water, stirred and heated to 95°C, then naturally cooled to room temperature). After 20 seconds, the hand mold is removed and dried at 110°C for 5 minutes. The dried hand mold is then cooled to 60°C and immersed in deproteinized natural rubber latex. After 20 seconds, the hand mold is removed and vulcanized and dried at 100°C for 10 minutes. After cooling to 50°C, the hand mold is immersed in polyurethane emulsion. After 12 seconds, the hand mold is removed and heated at 80°C for 18 minutes. After cleaning, the hand mold is demolded to obtain deproteinized natural rubber latex gloves. Its physical properties were measured according to GB 7543-2020 / ISO 10282:2014 and GB / T 21869-2008 / ISO 21171:2006 standards, and the results are shown in Table 5.

[0135] Table 5. Physical properties of 13 deproteinized natural rubber latex gloves

[0136]

[0137]

[0138] Note: The gloves listed in Table 5 are made of the materials used in the examples, and 13 gloves of each type were taken. Their dimensions are 7.0 (width 89±5mm; length ≥270mm; single layer thickness ≥0.11mm), and the protein content extracted from water according to the standard test is less than 11μg / g.

[0139] Table 5 shows that the changes in physical properties are consistent with the results in Table 3. For example, the multi-layer network composed of protease immobilization network, peptidase immobilization network, and phospholipase and lipase immobilization network has the best protein removal effect. This results in the lowest 300% constant elongation load of the deproteinized latex glove, indicating that it is soft, skin-friendly, and relaxed without tightness. At the same time, the highest elongation at break indicates that it is softer and more elastic, and there are no defects such as pinholes or cracks. In contrast, the protein removal ability of other multi-layer network combinations is weakened. That is, the water-extracted protein content in the latex increases, corresponding to an increase in the 300% constant elongation load of the glove and a decrease in the elongation at break. In the comparative example, using free enzymes and multiple centrifugations can remove most of the water-extracted protein, but the latex particles are damaged. Although the 300% constant elongation load of the glove is small, the elongation at break is reduced, and water leakage occurs, indicating that the glove has defects such as pinholes or cracks, that is, poor latex film-forming properties.

[0140] 2) Assessment of the film-forming properties of deproteinized natural rubber latex

[0141] The pre-cured latex obtained in the above different embodiments was immersed in a 5cm diameter glass test tube preheated to 50°C for 5 seconds. The mold was then lifted out at a speed of 750mm / min, and dried on the surface of the mold to form a rubber film. The mold was then preheated to 50°C again and immersed in the aforementioned compound latex for 5 seconds, before being pulled out at a speed of 1600mm / min. After pulling out, the rubber film on the surface of the mold was vulcanized at 100°C for 30 minutes to obtain a rubber film with a thickness of 0.1mm. The evaluation was conducted by measuring the thickness (mm) of the rubber film formed on the surface of the mold and visually observing for any liquid unevenness and film uniformity.

[0142] The evaluation criteria for whether or not the liquid is evenly distributed are as follows:

[0143] A: No uneven liquid distribution was observed at all;

[0144] B: Very little uneven liquid distribution can be observed;

[0145] C: Obvious uneven liquid distribution occurs.

[0146] The evaluation criteria for the uniformity of film thickness are as follows:

[0147] A: The thickness of the membrane is roughly uniform;

[0148] B: The thickness of the film is somewhat uneven, but not to the extent that it affects actual use.

[0149] C: The unevenness in membrane thickness reaches a level that is clearly and easily observed.

[0150] The results are shown in Table 6.

[0151] Table 6. Evaluation of the film-forming properties of deproteinized natural rubber latex

[0152]

[0153]

[0154] As shown in Table 6, latex with a lower protein content after one centrifugation and water extraction has a more uniform liquid distribution and better film uniformity. In contrast, latex with a slightly higher protein content after one centrifugation and water extraction has some instability in liquid distribution and film uniformity. For latex that has been centrifuged multiple times, the liquid distribution and film uniformity are very poor, making it difficult to use for impregnating film-forming products such as gloves and condoms.

[0155] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing deproteinized natural rubber latex, characterized in that, Includes the following steps: By combining mesh gauze with an adsorption medium solution, adsorption is carried out to obtain enzyme carrier gauze. The enzyme carrier gauze was immersed in different enzyme solutions for enzyme adsorption. The resulting gauze was then cross-linked and fixed in a cross-linking agent solution to obtain single-piece immobilized enzyme filter layers corresponding to different enzymes. The single-piece immobilized enzyme filter layers corresponding to different enzymes were stacked to obtain an immobilized enzyme filter screen. The enzymes used in the different enzyme solutions were protease, peptidase, phospholipase and lipase, respectively. Natural rubber latex, water, and a nonionic surfactant were mixed, and a glucono-delta-lactone solution was added to the resulting mixture. The immobilized enzyme filter is used for vertical and circular filtration until the pH value is 3-6, to obtain the enzymatically hydrolyzed latex. The enzymatically hydrolyzed latex, water-soluble polymer, and anionic surfactant are mixed, the pH is adjusted to 9-11, and after solubilization, the mixture is centrifuged to obtain deproteinized natural rubber latex.

2. The preparation method according to claim 1, characterized in that, The mesh gauze is woven from any one or more of silk yarn, pure cotton yarn, and pineapple fiber yarn. The mesh size of the mesh gauze is 0.1~2.0cm, the yarn strength is >25MPa, and the elongation is <20%. The adsorption medium solution includes one or more of regenerated silk protein solution, polyethyleneimine solution, dopamine hydrochloride solution, sodium alginate solution, and chitosan solution. The adsorption temperature is 0~45℃, and the time is 10~300min.

3. The preparation method according to claim 1, characterized in that, The protease includes one or more of alkaline protease, neutral protease, bromelain, papain, trypsin, thermophilic protease, staphylococcal protease, and clostridium protease; the peptidase includes one or more of exopeptidase, aminopeptidase, and carboxypeptidase; and the phospholipase includes one or more of phospholipase A, phospholipase B, phospholipase C, and phospholipase D.

4. The preparation method according to claim 1, characterized in that, The crosslinking agent solution contains one or more of glutaraldehyde, glyoxal, terephthalaldehyde, and genipin; the concentration of the crosslinking agent solution is 0.05~3.5wt%, the pH value is 7.0~9.5, and the dosage is 3~10g / g mesh gauze; the temperature for enzyme adsorption and crosslinking fixation is independently 0~45℃, and the time for enzyme adsorption and crosslinking fixation is independently 10~300min.

5. The preparation method according to claim 1, characterized in that, The natural rubber latex contains 1.0-65% dry rubber by mass and has a pH value of 8.0-12.

0.

6. The preparation method according to claim 1, characterized in that, The nonionic surfactant is any one or more selected from Tween, Span, trehalose, stachyose, glucoside, nonylphenol polyoxyethylene ether, isomeric 13 alcohol polyoxyethylene ether, and alkyl polyglucoside, and the mass of the nonionic surfactant is 0.01~10% of the mass of the natural rubber latex; the concentration of the glucono-delta-lactone solution is 1~10wt%, and the mass of glucono-delta-lactone in the glucono-delta-lactone solution is 0.01~10% of the mass of the natural rubber latex.

7. The preparation method according to claim 1, characterized in that, During the cyclic filtration, the immobilized enzyme filter screen automatically rises and falls under the vertical axis, with a rising and falling speed of 1~20cm / s, and the cyclic filtration time is 3~6h; after the cyclic filtration is completed, it is left to stand for ≥3h.

8. The preparation method according to claim 1, characterized in that, The water-soluble polymer includes one or more of polyethylene glycol, methoxy polyethylene glycol, polypropylene glycol, methyl polypropylene glycol, polyethylene oxide, polyethylene oxide ether, polyvinyl alcohol, polyvinyl alcohol copolymer, ethyl hydroxyethyl cellulose, methyl cellulose, polyacrylamide, polyacrylamide copolymer, polyacrylic acid, sodium polyacrylate, polypropylene oxide, and polyglycerol glycidyl ether polyvinyl alcohol, wherein the mass of the water-soluble polymer is 0.01-10% of the mass of natural rubber latex; the anionic surfactant includes diphenyl ether sulfonate, alkylbenzene sulfonate, alkylnaphthalene sulfonate, naphthalene sulfonate, alkyl... The product comprises one or more of the following: alkyl sulfonate, dialkyl sulfosuccinate, α-alkene sulfonate, α-sulfonated fatty acid salt, alkyl sulfate, polyoxyalkylene stilbene phenol sulfate, polyoxyalkylene sulfate, tristilbene phenol sulfate, polyoxyalkylene phenyl ether sulfate, alkyl sulfate ester, polyoxyethylene alkyl sulfate ester, polyoxyethylene alkylphenyl ether sulfate ester, polyoxyethylene tristilbene phenol sulfate ester, and polyoxyethylene stilbene phenol sulfate ester; the mass of the anionic surfactant is 0.01~10% of the dry mass of the natural rubber latex; the solubilization time is 3~6h.

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