Method for reducing anaphylaxis of natural latex by multi-enzyme cascade catalysis of rubber protein

Abstract

The invention relates to a method for reducing the anaphylaxis of natural latex by multi-enzyme cascade catalysis of rubber protein. The invention relates to the technical field of catalysis of rubber protein hydrolysis, in particular to a method for reducing the anaphylaxis of natural latex by thoroughly hydrolyzing rubber protein through multi-enzyme cascade catalysis. The method for reducing the anaphylaxis of the natural latex by catalyzing the rubber protein through the synergistic effect of multiple enzymes from the perspective of industrial production comprises the following steps: adding an enzyme A and an enzyme B into the natural latex for reaction so as to reduce the anaphylaxis of the natural latex, the enzyme A is alkaline protease; and the B enzyme is at least one of aminopeptidase, carboxypeptidase B, carboxypeptidase A and carboxypeptidase Y. According to the method disclosed by the invention, the enzyme A-endopeptidase and the enzyme B-exopeptidase are subjected to multi-enzyme-stage combined treatment and concerted catalysis, so that the enzyme A-endopeptidase and the enzyme B-exopeptidase are completely hydrolyzed or are completely hydrolyzed into more amino acid substances and short-chain peptides (oligopeptides) as far as possible, and the amino acid substances and the short-chain peptides (oligopeptides) are retained in latex. Protein is highly hydrolyzed into amino acid with higher safety, so that the possibility of I-type anaphylactic reaction caused by protein allergens is greatly reduced or even eliminated.

Description

Technical Field

[0001] The invention relates to the technical field of catalytic hydrolysis of rubber protein, in particular to a method for reducing the allergy of natural rubber latex by catalyzing rubber protein through multi-enzyme cascade. Background Art

[0002] Natural rubber latex is a biocompatible natural polymer extracted from the Hevea brasiliensis tree. It possesses excellent film-forming properties, durability, tear resistance, and elasticity, making it widely used in healthcare, daily life, and other fields. However, the water-soluble proteins present in natural rubber latex are known allergens. Existing reports indicate that direct contact with human skin and mucous membranes, or the use of medical products made of natural rubber latex, such as gloves, condoms, or catheters, can induce allergic dermatitis (such as measles). In severe cases, respiratory distress and even shock may occur. This is likely the result of residual water-soluble proteins on the skin surface forming histamine, which triggers a type I hypersensitivity reaction. Allergic reactions to natural rubber latex products in specific risk groups have become one of the most serious occupational hazards for medical professionals, latex product manufacturers, and other sensitive individuals. This has placed natural rubber latex and its product manufacturers and distributors in a predicament and, to a certain extent, has impacted the application of natural rubber.

[0003] Reducing water-soluble protein content and allergic reactions to latex present significant challenges, making deproteinization of natural rubber latex a key technology in the field of natural rubber science and technology. Research institutions and manufacturers worldwide are actively developing methods for producing low-protein natural rubber latex and its products. Possible deproteinization methods include chemical methods, low-temperature crystallization, high-speed multiple centrifugation, adsorption displacement, and radiation. These methods are often difficult to commercialize due to their complexity and high cost, lack of effective protein removal, or adverse effects on product quality. Currently, the most common method for producing low-protein concentrated natural rubber latex is protease hydrolysis. This involves adding proteases to fresh latex to degrade most of the protein in the natural rubber particles. Centrifugation is then performed to produce low-protein concentrated latex, which can reduce nitrogen content from approximately 0.50% to 0.02%-0.10% or even lower. However, even in deproteinized natural rubber, residual proteins and peptides still exist. Allergic patients will still have allergic reactions when they come into contact with the product, making the currently produced low-protein natural rubber latex still face the embarrassment of latex allergy (He Gong, et al. Industrial Crops and Products, 2024 (1), 118633). The original intention of protease hydrolysis is to cut the peptide bonds between proteins. Peptides including allergens will be cut into peptides or amino acids to reduce sensitization, but this will change the structure of the antigen epitope, which may lead to the exposure of more allergic antigens, and in turn increase the allergenicity of latex.

[0004] Obviously, the latex industry needs a new method that can completely hydrolyze rubber protein into safer amino acids to reduce the allergenicity of natural latex. Summary of the Invention

[0005] Based on the above, the present invention provides a method for reducing the allergenicity of natural rubber latex by using a multi-enzyme cascade to hydrolyze rubber protein. Using natural rubber latex as raw material, the method utilizes proteases and peptidases to hydrolyze rubber protein via a multi-enzyme cascade to generate more amino acids and oligopeptides. This method significantly reduces the allergenicity of natural rubber latex and various products produced using it, thereby expanding the application of natural rubber latex.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for reducing the allergenicity of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein, comprising the following steps:

[0008] Enzyme A and enzyme B are added to natural rubber latex to react, so that the protein allergens contained in the natural rubber latex are degraded into short peptide fragments and more amino acids that are non-allergenic to humans, thereby reducing the allergenicity of the natural rubber latex;

[0009] The enzyme A is an endopeptidase that acts on the inside of the polypeptide chain in an alkaline environment, i.e., an alkaline protease;

[0010] The B enzyme is an exopeptidase that acts on the end group of the polypeptide chain under a neutral or alkaline environment, and is at least one of aminopeptidase, carboxypeptidase B, carboxypeptidase Y, and carboxypeptidase A. In industry, aminopeptidase and carboxypeptidase Y are preferred because of their low price.

[0011] In a preferred embodiment of the present invention, the enzyme A and the enzyme B can be added simultaneously, or the enzyme A can be added first and then the enzyme B can be added.

[0012] In a preferred embodiment of the present invention, the method further comprises the steps of adjusting the pH of the natural latex and adding a surfactant to the natural latex before adding enzyme A to the natural latex for reaction.

[0013] In a preferred embodiment of the present invention, the added amount of the surfactant is 0.5% to 3.0% of the mass of the natural rubber latex.

[0014] In the present invention, the purpose of adding a surfactant is to stabilize the natural rubber latex.

[0015] The surfactant is an anionic surfactant or a nonionic surfactant. The anionic surfactant is one of sodium lauryl sulfate, sodium dodecylbenzene sulfonate, sodium lignin sulfonate, and sodium butylnaphthalene sulfonate; the nonionic surfactant is one of polyoxyethylene fatty alcohol ether, polyethylene glycol octylphenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, and alkylphenol polyoxyethylene ether.

[0016] In a preferred embodiment of the present invention, the enzyme activity of enzyme A in each kilogram of natural rubber latex is 30,000 U to 300,000 U.

[0017] In a preferred embodiment of the present invention, the enzyme activity of enzyme B in each kilogram of natural rubber latex is 6,000 U to 300,000 U.

[0018] In the present invention, enzyme A can be added by dissolving it in water and adding it to the natural rubber latex; enzyme B can be added by dissolving it in water or making it into a suspension and adding it to the natural rubber latex mixture.

[0019] In a preferred embodiment of the present invention, the temperature for adding enzyme A to carry out the reaction is 20-45° C. and the reaction time is 1 to 5 days.

[0020] In a preferred embodiment of the present invention, the temperature for adding enzyme B to carry out the reaction is 20-45° C. and the reaction time is 1 to 10 days.

[0021] After the reaction in step 1 is completed, a hydrolysis degree of about 20% to about 30% of soluble and insoluble proteins can be obtained; after the reaction in step 2 is completed, a hydrolysis degree of about 50% to about 60% or higher of soluble and insoluble proteins can be obtained.

[0022] After the reaction in step 2 is completed, the pH can be raised to 10-11 by adding concentrated ammonia water to inhibit the enzyme activity so that no further hydrolysis occurs. The latex can then be stored, compounded and used according to conventional methods.

[0023] The present invention also provides a low-allergen natural rubber latex prepared according to the method of reducing the allergenicity of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein.

[0024] The present invention also provides the application of the low-allergen natural latex in preparing rubber products that come into contact with the human body.

[0025] In the present invention, the types of rubber products can be various medical gloves, industrial / household protective gloves, finger cots, condoms, foam sponges, balloons, latex tubes, latex sheets / bags.

[0026] The low-allergen natural rubber latex produced by the method of the present invention can be used for all natural rubber products that require latex and coagulated rubber particles made from the natural latex.

[0027] The present invention discloses the following technical effects:

[0028] 1) The present method uses a multi-enzyme cascade catalytic treatment involving enzymes A (protease) and B (peptidase) to completely or as completely as possible hydrolyze the protein into more amino acids and short-chain peptides (oligopeptides). This converts the protein into safer amino acids, significantly reducing or even eliminating the risk of Type I allergic reactions caused by proteins in natural rubber latex.

[0029] 2) The present method eliminates the need for high-speed centrifugation to remove protein hydrolysis products from natural rubber latex during enzymatic hydrolysis, reducing the operational complexity and rubber loss associated with the centrifugation process. Studies have shown that each dilution and centrifugation step results in a 10% loss of rubber particles. Therefore, the production cost of the present method is significantly lower than that of other solutions to address protein allergens (such as denaturation, multiple centrifugation steps, enzymatic hydrolysis, and substitution) that involve decomposing or removing proteins, demonstrating significant economic benefits and potential for large-scale production.

[0030] 3) The present invention utilizes multi-stage enzymatic hydrolysis of rubber protein to generate a large amount of amino acids and oligopeptides, which are then stored in natural rubber latex. Amino acids are natural antioxidants that significantly improve the aging resistance of natural rubber. Furthermore, amino acids can form complexes with free metal ions such as copper, magnesium, calcium, and iron, further stabilizing natural rubber latex and extending the product's shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Schematic diagram of the method for determining the amino acid content in natural rubber latex of the present invention.

[0033] Figure 2 Schematic diagram of different enzymes acting on different peptide bonds in proteins to catalyze hydrolysis to produce polypeptides, oligopeptides and amino acids. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Unless otherwise specified, the "%" in the present invention refers to mass percentage.

[0040] The "room temperature" mentioned in the present invention, unless otherwise specified, refers to 20-30°C.

[0041] The present invention sequentially treats natural rubber latex with at least one enzyme with protease activity (i.e., enzyme A, which hydrolyzes rubber protein into polypeptides) and at least one enzyme with peptidase activity (i.e., enzyme B, which further hydrolyzes polypeptides into amino acids and oligopeptides). Through this multi-stage enzymatic treatment of natural rubber latex, the protein allergens contained therein are at least largely, or completely, degraded into oligopeptide fragments and amino acids with low molecular weight that are incapable of causing allergic reactions in humans, thereby fundamentally reducing the risk of rubber protein causing allergic reactions in humans.

[0042] Generally, peptides with less than 50 amino acid residues are called polypeptides, while peptides with more than 50 (some say more than 100) amino acid residues are called proteins. Amino acids and polypeptides generally do not produce allergic reactions. There is no definitive evidence that when the number of amino acids in a polypeptide or small molecule oligopeptide chain is below a certain value, the polypeptide or oligopeptide chain will not cause allergies to the human body. However, when the number of amino acid residues in a small molecule short peptide chain is smaller (10 or less in length, i.e., oligopeptides), the species specificity and antigenicity of the protein can be effectively eliminated, the possibility of causing allergic or toxic reactions in humans is lower, and the safety of contact is higher. The present invention is based on the fact that rubber proteins (including allergens) in natural rubber latex can be deeply hydrolyzed into more short-chain oligopeptides and amino acids by enzyme A (protease) and enzyme B (peptidase) through multiple stages, thereby reducing the risk of rubber proteins forming type I allergens in the human body.

[0043] In the method of the present invention, the steps of protease hydrolysis and peptidase hydrolysis of natural rubber latex can be carried out simultaneously, but preferably, the protease hydrolysis is carried out first, using the protease to destroy the spatial structure and a small amount of peptide bonds of the rubber protein, and the protease hydrolysis converts the protein into polypeptides and some amino acids; then, the peptidase acts on the peptide bonds, hydrolyzing the peptide chains one by one from the free carboxyl end or free amino end of the polypeptide to generate amino acids or oligopeptides.

[0044] In some embodiments of the present invention, prior to protease hydrolysis, the pH of the natural rubber latex is first adjusted to approximately 8.5-9.5 to ensure optimal protease activity. The protease is then added to the natural latex in a sealed container, and the mixture of natural rubber latex and protease is periodically stirred at room temperature, or more preferably, at 28-45°C, to allow for enzymatic protein hydrolysis. After the protease hydrolysis step is completed, a peptidase is added to the mixture and the natural rubber latex is treated in a sealed container. The natural rubber latex-peptidase mixture is again periodically stirred at room temperature or at 30-45°C to further hydrolyze the polypeptides into amino acids or oligopeptides.

[0045] As we know, enzymes with proteolytic functions can be divided into two major types: endopeptidases (EC 3.4.21–3.4.99) and exopeptidases (EC 3.4.11–3.4.19). Endopeptidases (also known as proteases) primarily act on peptide bonds within protein polypeptide chains, breaking down long protein chains into short peptide fragments. When proteases hydrolyze proteins, the site of action varies depending on the type of peptide bond. Using their substrate specificity, proteases can cleave peptide chains to yield polypeptides and some amino acids.

[0046] Exopeptidases (the aforementioned peptidases) are a general term for enzymes that further break down proteins and polypeptides into amino acids, including aminopeptidases, carboxypeptidases, and dipeptidases. Aminopeptidases (EC 3.4.11), which act on amino-terminal peptide bonds, are a class of exoproteases that selectively cleave amino acid residues from the N-termini of proteins and polypeptides to produce free amino acids. Carboxypeptidases (EC 3.4.12–3.4.19), which act on carboxy-terminal peptide bonds, catalyze the hydrolysis of the carboxyl-terminal amino acid in polypeptide chains. They are a class of exopeptidases that specifically degrade the C-terminus of the peptide chain, releasing free amino acids one by one. It should be noted that the hydrolysis or metabolism of peptides by peptidases is nonspecific and multifunctional. A single peptidase can metabolize many different substrates, or a single substrate can be metabolized by multiple peptidases in a sequential, multi-step degradation process, demonstrating relative specificity.

[0047] Schematic diagram of different enzymes acting on different peptide bonds of proteins to catalyze hydrolysis to produce polypeptides, oligopeptides and amino acids. Figure 2 shown.

[0048] The following describes in more detail the steps in some embodiments of the present invention:

[0049] 1. Selection of natural latex:

[0050] In theory, the natural latex to which the method of the present invention is applicable includes fresh latex (field latex), natural latex that has been centrifuged and not yet preserved by ammonia, and concentrated natural latex preserved by ammonia or other preservation systems.

[0051] As is well known, the aging (ripening) process causes a series of complex physical and chemical changes in the composition and structure of rubber particles, which is conducive to improving colloidal stability. As storage time increases, the adhesion of proteins to the outer layer of latex particles also increases. Therefore, it is most effective to immediately deproteinize fresh (field) natural rubber latex separated from rubber trees using an enzymatic method. Fresh natural rubber latex contains approximately 1% to 2% rubber protein, of which approximately 60% of the protein content is dissolved in the aqueous phase, approximately 20% is adsorbed on the surface of the rubber particles, and the remaining 20% ​​is bound to larger particles such as lutoids and Frey-Wyssling particles. Therefore, fresh (field) latex that has not been centrifuged contains a large amount of water and other non-rubber components, including ash impurities. The amount of enzyme required for enzyme treatment is large, and the numerous factors affecting the enzymatic hydrolysis process are difficult to effectively control. Further concentration and removal of impurities may also be required by centrifugation at a later stage. Therefore, concentrated natural rubber latex that has been centrifuged and stored by ammonia, in which most of the rubber protein has been removed, is preferred, while concentrated natural rubber latex that has been centrifuged but not yet stored by ammonia is less preferred.

[0052] 2. Adjust the pH value of latex

[0053] To maximize the hydrolysis reactions of proteases and peptidases, the pH of the natural rubber latex typically needs to be adjusted to match the optimal pH of the protease and peptidase solutions. The pH of concentrated natural rubber latex preserved with ammonia is typically between 9 and 11, which is too alkaline for optimal protease and peptidase activity. Therefore, the pH of the natural rubber latex needs to be appropriately lowered to match the optimal pH for protease and peptidase hydrolysis. The optimal pH for proteases is between 6 and 9, the optimal pH for aminopeptidases is near neutral, and carboxypeptidases have a wider optimal pH range (4 to 8.5). Therefore, the pH of the latex is preferably adjusted to approximately 6.0-9.0, with an optimal pH of 7.0-8.5. Of course, this step can be omitted by using certain proteases that exhibit high activity within the pH range of ammoniated natural rubber latex. The method of the present invention can also function without pH adjustment. However, full hydrolysis under suboptimal hydrolysis conditions requires a long time and is not economical.

[0054] The pH of latex can be lowered by a number of known methods. For example, the pH of ammoniated latex can be lowered by a very practical method, preferably by: (1) evaporation of ammonia, by heating, stirring, blowing air through the latex, etc.; (2) bubbling carbon dioxide or other weakly acidic substances through the latex; or (3) a combination of these two methods.

[0055] 3. Protease hydrolysis treatment

[0056] In the enzyme treatment step A, an aqueous solution of protease with an optimal pH close to that of natural latex is added to the natural latex, stirred evenly, and placed in a sealed container to allow the latex mixture to digest for a sufficient time to hydrolyze the protein contained therein, usually 1 to 5 days, with the mixture gently stirred every 2 to 12 hours.

[0057] Preferably, the aqueous solution of protease added to the latex produces a protease activity in the mixture equal to or greater than 1,000 U / 100 g of natural rubber latex, or an equivalent ratio thereof. The preferred enzyme activity of concentrated natural rubber latex with a dry rubber content of 60% is preferably equal to or greater than 100,000 U / kg, and more preferably 200,000 to 300,000 U / kg. Although small doses of protease will also produce a slow enzymatic hydrolysis reaction, the time required to achieve sufficient hydrolysis may be very long, resulting in low production efficiency and failing to meet the needs of modern production. The general procedure for protease treatment is as follows:

[0058] First, a sufficient amount of alkaline protease is dissolved in water to prepare a 10% aqueous solution, and a sufficient amount of alkaline protease is weighed out at a dosage of 10,000 to 300,000 U of enzyme activity per 1 kg of concentrated natural rubber latex with a dry rubber content of 60%. Then, the pH value of the alkaline protease solution is adjusted to 8.0 to 8.5 with 10% ammonia water or 5 mol / L NaOH solution to achieve optimal enzyme activity.

[0059] Add 0.5% to 3% of a surfactant to the concentrated natural rubber latex and stir evenly; reduce the pH of the latex to about 8.5 to 9.5 by stirring to evaporate ammonia, introducing carbon dioxide gas or adding a weak acid;

[0060] Add the protease solution to the natural rubber latex, mix thoroughly, and then perform enzymatic digestion. It is recommended to process the latex in a sealed container at room temperature to prevent the volatilization of water from the latex, which may cause coagulation and skinning on the latex surface. It is also easy to take measures to appropriately increase the temperature to 28-45°C, which can greatly accelerate the hydrolysis of the rubber.

[0061] The protease hydrolysis process requires stirring the latex mixture 2-5 times daily until the desired degree of hydrolysis (DH) is reached (the ratio of the number of peptide bonds cleaved during the hydrolysis process to the total number of peptide bonds in a given protein), which is approximately 20% to 30%. This typically takes 2 to 3 days for most conventional latexes. However, this required time is not fixed and depends on the differences in the chemical composition of the natural rubber latex being processed (due to agricultural factors such as the strain, origin, and tree age, processing methods, and storage system), the appropriate enzymatic hydrolysis conditions (dosage and pH), the amount of agitation, and the ambient temperature.

[0062] Common proteases include trypsin, chymotrypsin (chymotrypsin), thermolysin, pepsin, papain, bromelain, staphylococcal protease, subtilisin, and clostripain. Deproteinized natural rubber latex is usually prepared using alkaline proteases such as trypsin, bromelain, papain, and proteinase K. The protein content is significantly reduced by using alkaline proteases or in combination with other protein denaturants, surfactants, and removal processes.

[0063] 4. Further enzymatic hydrolysis by peptidase

[0064] The natural rubber latex mixture treated with the above-mentioned protease is further treated with peptidase to further hydrolyze the polypeptides produced by the protease to produce more amino acids and short peptide chains. The process is roughly the same as the above-mentioned protease treatment process. The general procedure for peptidase treatment of natural rubber latex is as follows:

[0065] First, one or more peptidases are dissolved in water or a buffer solution to form a solution of a certain concentration. The pH value is adjusted to be closest to the pH value of the latex treated with the protease (the pH value of the latex has usually dropped to around 8.0 at this point). Then, the peptidase aqueous solution or suspension is added to the natural rubber latex treated with the protease, stirred evenly, and placed in a sealed container at room temperature or preferably 28-45°C to digest the mixture for a sufficient time to hydrolyze the protein contained therein as much as possible. This is usually 1 to 10 days or even longer. The mixture is gently stirred every 2 to 12 hours, and measures can also be taken to appropriately increase the reaction temperature to promote complete hydrolysis.

[0066] Dosage of peptidase: It is recommended to add at least one or more peptidases to each 1 kg of concentrated natural rubber latex with a dry rubber content of 60%, so that its enzyme activity is 0.6 million U to 30 million U. Optionally, about 100,000 U or more per kilogram of concentrated natural rubber latex with a dry rubber content of 60% is added to the natural rubber latex after protease treatment to promote the hydrolysis of more polypeptides into amino acids. The reaction mixture is stirred 2 to 3 times a day, usually for 5-10 days, to ensure that the preferred protein hydrolysis degree of 50% to 60% or even higher is achieved. Similarly, the required enzymatic hydrolysis time will vary according to the parameters of any given treatment (such as the type and amount of peptidase added, ambient temperature, stirring times, etc.).

[0067] The present invention can also carry out the protease treatment and the peptidase treatment simultaneously, that is, the protease and peptidase solutions are added to the natural latex at the same time, as long as the amount of enzyme used is sufficient to complete the hydrolysis and digestion of the natural latex protein as much as possible.

[0068] After the multi-enzyme cascade catalytic treatment, the pH of the natural rubber latex can be raised to 10-11 by adding concentrated ammonia to inhibit enzyme activity, preventing further hydrolysis. The latex can then be stored, compounded, and used according to conventional methods. It can be formulated in the same manner as conventional natural rubber latex to produce any type of natural rubber latex product. When the multi-enzyme cascade catalytic treatment with proteases and peptidases produces a sufficient degree of protein hydrolysis, the allergenicity of the latex and products made from it is significantly reduced, making it more suitable for use in the manufacture of various medical products that come into contact with the human body, such as various medical gloves, industrial and household protective gloves, finger cots, condoms, foam sponges, balloons, latex tubes, latex sheets / bags, and the like.

[0069] The present invention utilizes different enzymes to catalyze and hydrolyze different portions of the various protein chains in natural rubber latex, thereby increasing the degree of protein hydrolysis and the specificity of the products. Using natural rubber latex as raw material, the rubber proteins therein are enzymatically hydrolyzed by proteases to yield polypeptides and a small amount of amino acids. Peptidases are then used to further hydrolyze the polypeptide chains into oligopeptides and amino acids that are non-allergenic to humans, thereby significantly reducing the risk of rubber proteins causing allergic reactions in humans. The present method does not alter existing natural rubber latex production procedures and does not utilize the conventional high-speed centrifugation required in deproteinization processes to remove proteins and their hydrolyzates. This method offers low cost, low energy consumption, high removal rates, and high quality. It avoids the environmental pollution and labor-intensive process associated with high-content rubber wastewater caused by multiple centrifugations, resulting in low manufacturing costs, a simple production process, and no secondary pollution. Furthermore, the natural rubber latex retains more amino acids, a natural antioxidant generated in situ, which significantly improves the anti-aging properties of natural rubber. It also forms complexes with free metal ions in whey, reducing factors that destabilize the latex and extending its shelf life.

[0070] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0071] The commercially available concentrated natural rubber latex used in the examples of the present invention was purchased from Maoming Branch of Guangken Rubber Group Co., Ltd. and met the technical requirements of GB / T 8289-2023 when it was shipped from the factory.

[0072] The alkaline protease used in the examples of the present invention is food-grade licheniformis protease 2709, purchased from Nanning Pangbo Bioengineering Co., Ltd., with an enzyme activity of 100,000 U / g, an optimum pH of 8.0, and an optimum temperature of 50°C; aminopeptidase is purchased from Ningxia Xiasheng Industrial Co., Ltd., with an enzyme activity of 5,000 U / g and an action pH range of 6.0-10.0; carboxypeptidase Y is yeast carboxypeptidase, purchased from Shanghai Shifeng Biology, with an enzyme activity of 50,000 U / g.

[0073] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0074] Example 1

[0075] 1.0 kg of commercially available concentrated natural rubber latex (0.7% ammonia content, 62.3% total solids content, pH 10.45) was added to 100 g of a 10% aqueous solution of sodium lauryl sulfate and stirred at 80 rpm. The mixture was then left open and stirred until the pH of the latex sample dropped to 9.5, yielding a stabilized latex. One gram of alkaline protease (100,000 U / g enzyme activity) was dissolved in water to form a 10% aqueous solution. The pH was raised to 9.00 by adding 25% aqueous ammonia to yield a protease solution. The protease solution was then added to the stabilized latex. After thorough stirring, the hydrolysis mixture was placed in a sealed container. The mixture was reacted at 25-30° C., with stirring at 50 rpm once or twice daily (the purpose of stirring was to allow the latex particles to fully contact the enzyme, encouraging both to participate in the enzymatic hydrolysis reaction and improving enzymatic efficiency). The reaction was continued for two days to yield a protease-treated latex mixture.

[0076] 3.2g of aminopeptidase (5,000 U / g, food grade) and 2g of yeast carboxypeptidase Y (50,000 U / g) were dissolved in 50ml of distilled water, and 25mg of carboxypeptidase A (bovine pancreas, 20 U / mg, sourced from Shanghai Yuanye) was added and stirred to form a suspension. This suspension was then added to the protease-treated latex mixture. The mixture was placed in a sealed container and reacted at 28-32°C, stirring once or twice daily at 50 rpm for 20-30 minutes each time for 7 consecutive days. The hydrolysis reaction was then terminated by adding 25% ammonia to raise the pH of the latex mixture to 11.

[0077] The following method was used to determine the composition and content of free amino acids in the whey of the natural rubber latex treated with the multi-enzyme cascade catalysis in Example 1, and an untreated original latex sample was used as a "control sample" for comparison. The test results are shown in Table 1.

[0078] Determination of amino acid content in natural rubber latex: Figure 1 Following the sample preparation steps, the amino acid composition and content were determined using a SYKAMS-433D amino acid analyzer. Chromatographic column: SYKAM strong acid cation exchange resin LCAK06 / Na. Mobile phase A: 0.12N sodium citrate, pH 3.45; mobile phase B: 0.2N sodium citrate, pH 10.85. Flow rate: ninhydrin solution at 0.25 mL / min, eluent at 0.45 mL / min. Detector 1: 570 nm, bipolar, 12123 mV, 6.25 samples per second; Detector 2: 440 nm, bipolar, 12123 mV, 6.25 samples per second; Reaction temperature: 138°C.

[0079] Table 1 Composition and content of free amino acids in natural rubber latex whey in Example 1

[0080]

[0081] It can be seen that the free amino acids in fresh latex are mainly aspartic acid, glutamic acid, alanine, glycine and serine. After multi-enzyme cascade catalytic hydrolysis, the proportion of glutamic acid and alanine is significantly reduced, and the proportion of serine, aspartic acid, glycine, leucine, etc. decreases to a certain extent. However, the total amount of amino acids in whey is much higher than that in fresh latex (about 4 times that of the control sample), indicating that the protein in natural latex can be highly catalytically hydrolyzed by multi-enzyme cascade catalysis, and more protein or polypeptide substances are converted into free amino acids or oligopeptides.

[0082] Comparison of the hydrolysis degree of natural rubber latex protein by different treatment methods in Example 2-6

[0083] Using commercially available concentrated natural rubber latex (0.7% ammonia, 62.3% total solids, pH 10.45) as the latex sample, carbon dioxide was first passed through the concentrated natural rubber latex to lower the pH to 9.5, thereby obtaining a pretreated latex. The pretreated latex was then divided into five portions and treated as follows:

[0084] Example 2

[0085] 10 g of sodium dodecylbenzenesulfonate was added to 1 kg of pretreated latex and stirred at 80 rpm. Then, 3 g of 2709 alkaline protease (enzyme activity 100,000 U / g) was added. The mixture was stored at 28° C. for three days, stirred twice a day at 50 rpm for 20-30 minutes each time. Then, 30 g of aminopeptidase (5,000 U / g) was added. The mixture was stirred twice a day at 50 rpm for 20-30 minutes each time. The reaction was continued for 6 days. The pH of the latex was then restored to 10.5 by adding 25% aqueous ammonia to obtain Sample A.

[0086] Example 3

[0087] To 1 kg of pretreated latex, 10 g of sodium dodecylbenzenesulfonate was added and stirred at 80 rpm. Then, 3 g of alkaline protease (enzyme activity 100,000 U / g) was added. The mixture was stored at 28°C for 9 days, stirring twice daily at 50 rpm for 20-30 minutes each time. The pH was then raised to 10.5 with aqueous ammonia to obtain Sample B.

[0088] Example 4

[0089] To 1 kg of pretreated latex, 100 g of a 10% aqueous sodium lauryl sulfate solution was added and stirred at 80 rpm. Then, 20 g of aminopeptidase (5,000 U / g, food grade) was added. The mixture was stored at 28°C for 9 days, stirring twice daily at 50 rpm for 20-30 minutes. The pH was then raised to 10.5 with aqueous ammonia to obtain Sample C.

[0090] Example 5

[0091] To 0.5 kg of pretreated latex, 0.5 kg of distilled water was first added and mixed uniformly. 10 g of sodium dodecylbenzenesulfonate was added and stirred uniformly at 80 rpm. Then, 3 g of 2709 alkaline protease (enzyme activity 100,000 U / g) was added. The mixture was stored at 28° C. for three days, stirred twice daily at 50 rpm for 20-30 minutes each time. Then, 30 g of aminopeptidase (5,000 U / g, food grade) was added. The mixture was stirred twice daily at 50 rpm for 20-30 minutes each time. The reaction was continued for 8 days. The pH of the latex was then restored to 10.5 by adding 25% aqueous ammonia to obtain Sample D.

[0092] Example 6. Pretreatment: The latex was maintained at 28° C. and stirred twice daily at a speed of 50 rpm for 20-30 minutes for 9 consecutive days. The pH of the latex was then restored to 10.5 with 25% ammonia water to obtain a control sample.

[0093] After the above-mentioned treatment steps, the free amino acid components in the whey samples of Examples 2-6 were extracted using the experimental method of Example 1, and the amino acid content and protein hydrolysis degree were tested.

[0094] The degree of hydrolysis (DH) refers to the degree to which a protein is broken down under specific hydrolysis conditions. There are many methods for determining the degree of protein hydrolysis, and the free amino acid method is used here. The definition of protein hydrolysis is the ratio of the number of peptide bonds cleaved during protein hydrolysis to the total number of peptide bonds in a given protein, that is:

[0095] DH=h / h tot ×100% (1)

[0096] Where h is the number of peptide bonds cleaved per gram of protein after hydrolysis, in millimoles (mmol / g); h tot It is the number of millimoles of peptide bonds per gram of original protein (mmol / g). When protein is specified, h tot is a constant. According to the literature, h of casein is totThe h value of natural rubber protein from plant sources is 8.2mmol / g, soy protein is 7.8mmol / g, wheat protein is 8.8mmol / g, gluten is 8.3mmol / g, cottonseed protein is 7.54mmol / g, peanut kernel is 7.54mmol / g, and gelatin is 11.1mmol / g. Therefore, a relatively moderate value of 8.0mmol / g is taken as the h value of natural rubber protein from plant sources. tot Generally, since each peptide bond cleavage can simultaneously generate a -NH2 and a -COOH, as long as the amount of -NH2 (amino acid amount) is quantitatively measured, the h value can be known, and the corresponding DH value can be roughly calculated, which can qualitatively measure the degree of hydrolysis of rubber protein under the action of enzyme reagents.

[0097] The resulting amino acid product is not a single substance, but a complex mixture. It is unrealistic to know exactly how many molecules there are in the mixed amino acid mixture and thus accurately calculate the total number of peptide bonds. Moreover, the number of peptide bonds varies with the composition of the amino acid product. The average molecular weight of the 20 common natural amino acids is 128 g / mol, so an estimate is used to calculate the number of millimoles of peptide bonds h:

[0098] h = amino acid mass / relative average molecular weight of amino acid = amino acid mass / 128 (2)

[0099] The natural rubber protein h tot Substituting the estimated value of into formula (1), we get:

[0100] DH=h / 8.0×100% (3)

[0101] Therefore, the results of the degree of hydrolysis of natural rubber latex protein in Examples 2-6 are shown in Table 2.

[0102] Table 2 Degree of protein hydrolysis of samples in Examples 1-6

[0103]

[0104] The results of the multi-enzyme cascade catalysis using a combination of proteases and peptidases on the degree of hydrolysis of rubber proteins are shown in Table 2. When used alone, these proteases or peptidases alone cannot effectively increase the degree of protein hydrolysis, and therefore cannot effectively reduce the content of protein allergens that cause type I allergies and thus reduce the risk of latex allergy.

[0105] The concentration of natural rubber latex affects the concentration of its protein as a substrate for enzymatic hydrolysis. Appropriately reducing the concentration can increase the degree of protein hydrolysis, but this should be determined based on the actual latex concentration required in production. If the concentration of natural rubber latex is reduced without a subsequent concentration process, it will inevitably affect its storage, transportation, convenience of use, and production costs. These results confirm that it is necessary and feasible to use a combination of protease and peptidase to hydrolyze natural rubber protein gradually and thoroughly over a long period of time to the greatest extent possible, which can significantly reduce the allergens in natural rubber latex. At the same time, instead of using the conventional high-speed centrifugation process to separate the enzymatic hydrolysis products, the final beneficial enzymatic hydrolysis products - amino acids and oligopeptides - are retained in the latex, reducing the production costs caused by the cumbersome high-speed centrifugation process and also reducing rubber loss. This has never been used in many previous natural rubber latex deproteinization processes.

[0106] The present invention is not limited to the specific reagents, such as proteases, peptidases, surfactants, and treatment times and temperatures illustrated and described above, but includes all modifications and equivalents thereof within the scope of the claims.

[0107] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for reducing the allergenicity of natural rubber latex by catalyzing rubber protein through multi-enzyme cascade, characterized in that: The following steps are involved: Adding enzyme A and enzyme B to natural rubber latex to react so as to reduce the allergenicity of natural rubber latex; The enzyme A is an endopeptidase that acts on the inside of the polypeptide chain in an alkaline environment, i.e., an alkaline protease; The B enzyme is an exopeptidase that acts on the end group of a polypeptide chain under a neutral or alkaline environment, and is at least one of aminopeptidase, carboxypeptidase B, carboxypeptidase Y, and carboxypeptidase A.

2. The method for reducing the allergic reaction of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein according to claim 1, characterized in that: The enzyme A and the enzyme B can be added at the same time, or the enzyme A can be added first and then the enzyme B can be added.

3. The method for reducing the allergic reaction of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein according to claim 2, characterized in that: Before adding enzyme A to the natural rubber latex for reaction, the method further includes adjusting the pH of the natural rubber latex and adding a surfactant to the natural rubber latex.

4. The method for reducing the allergic reaction of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein according to claim 3, characterized in that: The added amount of the surfactant is 0.5% to 3.0% of the mass of the natural rubber latex.

5. The method for reducing the allergic reaction of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein according to claim 2, characterized in that: The enzyme activity of enzyme A in each kilogram of natural rubber latex is 30,000 U to 300,000 U.

6. The method for reducing the allergic reaction of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein according to claim 2, characterized in that: The enzyme activity of B enzyme in each kilogram of natural rubber latex is 0.6 million U to 300,000 U.

7. The method for reducing the allergic reaction of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein according to claim 2, characterized in that: The temperature for adding enzyme A to react is 20-45° C., and the reaction time is 1 to 5 days.

8. The method for reducing the allergic reaction of natural rubber latex by using multi-enzyme cascade catalysis of rubber protein according to claim 2, characterized in that: The temperature for adding enzyme B to react is 20-45°C and the reaction time is 1 to 10 days.

9. Hypoallergenic natural rubber latex prepared by the method for reducing the allergenicity of natural rubber latex by using rubber protein through multi-enzyme cascade catalysis according to any one of claims 1 to 8.

10. The low-allergen natural rubber latex according to claim 9 is used for preparing rubber products that come into contact with the human body.

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