JP2002145903A - Hypoallergenic natural rubber - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a natural rubber which has a very low risk of inducing allergy. SOLUTION: The hypoallergenic natural rubber of the present invention is a natural rubber which has been subjected to a protein decomposition treatment, and is characterized in that a protein having a number average molecular weight of 4500 or more and a protein degradation product are not detected. Such a natural rubber can be obtained by adding a protease having an exopeptidase activity to a natural rubber latex and subjecting it to a protein decomposition treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a natural rubber which has almost no risk of inducing allergy.

[0002]

2. Description of the Related Art Natural rubber has high elongation, high elasticity,
Since it has characteristics such as good film strength, it is widely used in household products such as gloves, surgical gloves, medical devices such as various catheters, nursing devices, contraceptive devices and the like. On the other hand, using medical devices such as surgical gloves and catheters made of natural rubber may cause immediate (type I) allergy such as dyspnea and anaphylaxis-like symptoms (angioedema, urticaria, cyanosis, etc.). It has been speculated that such immediate allergy is induced by a protein contained in natural rubber as an antigen.

[0003] In recent years, attempts have been made to remove proteins from natural rubber to a high degree.
No. 5 (JP-A-6-56902), a natural rubber latex is subjected to a proteolytic treatment by adding a proteolytic enzyme such as an alkaline protease and a surfactant, and then the latex is sufficiently centrifuged. A washing method has been proposed. According to the method described in the above-mentioned patent publication, proteins in natural rubber can be decomposed and removed at a high level. Specifically, the amount of protein contained in natural rubber can be determined by the Kjeldahl's method. When represented by the content (N%), it can be an extremely low value of 0.02% or less. In general, natural rubber is
The number average molecular weight <Mn> is a mixture of a high molecular weight component of 1,000,000 to 2.5 million and a low molecular weight component of 100,000 to 200,000. It is presumed that they are mutually linked via the contained peptide molecule and the like. Here, the molecular weight of the low molecular weight component considered to be produced in the original biosynthesis is 100,000, and one molecule of the peptide molecule intervening in the intermolecular bond, ie, a nitrogen atom ( Assuming that the atomic weight 14) is bonded by one atom, the nitrogen content of the natural rubber is 0.014%. Therefore, even if advanced deproteinization treatment is performed, 0.02
% Nitrogen is considered to remain.

[0004] When a deproteinization treatment is performed by the method described in the above-mentioned publication, a rubber film formed using natural rubber latex after the treatment gives a characteristic 3 polypeptide specific to the polypeptide.
No infrared absorption spectrum of 280 ^-1 cm is observed. Therefore, according to the method described in the above publication, protein degradation /
It can be seen that the removal is highly achieved.

[0005]

However, even when the protein is decomposed and removed through various conventionally known deproteinization treatments including the method described in the above-mentioned patent publication, the protein still shows allergy. Potential threats have been revealed by recent studies. Therefore, the present inventors (i) determined the time-of-flight (TOF) to confirm how much protein and its decomposed products remained in the natural rubber latex deproteinized by the conventional method.
Analysis by a protein analysis system that combines a mass spectrometer with a two-dimensional surface-modified chip, and (ii)
In order to confirm the allergenicity of the natural rubber latex deproteinized by the conventional method, an antigen-antibody using the serum of a human patient was used instead of the conventional in vivo measurement by a patch test or the like. In vitro measurements based on the reaction were performed.

As a result, even when the protein is deproteinized by the conventional method, (I) a protein or a protein degradation product having a number average molecular weight <Mn> of about 4500 to 4700 remains, Was not sufficiently degraded, and (II) it was revealed that even after the deproteinization treatment, it contained an allergenic protein in an amount sufficient to induce immediate allergy.

The term "allergenic protein" is defined in the present invention as follows. All proteins and their degradation products (hereinafter referred to as “total proteins”) present in the natural rubber latex sample include a group of “antigen proteins” capable of producing antibodies in human serum. In addition, antibodies produced in human serum can cause allergic reactions.
gE class antibody and Ig which does not induce allergic reaction
It is classified into E class antibodies. Here, "antigen protein"
Among them, an antigen protein that produces an IgE class antibody that may cause an allergic reaction is referred to as an "allergenic protein" to distinguish it from other antigen proteins.

Accordingly, an object of the present invention is to provide a natural rubber which has a high degree of protein degradation and has a very low possibility of inducing allergy.

[0009]

Means for Solving the Problems and Effects of the Invention The hypoallergenic natural rubber of the present invention is a natural rubber which has been subjected to a protein decomposition treatment, and has a number average molecular weight of 4500 or more and a protein decomposed product. Is not detected. The hypoallergenic natural rubber of the present invention has a low average allergic molecular weight <Mn> of 4500 or more, as is clear from the fact that protein and protein degradation products are not detected. The protein is more highly degraded. Therefore, in proteins,
Most of the sites that can serve as antigens for producing IgE class antibodies to human serum are considered to have disappeared or denatured by the degradation treatment, and the risk of causing allergy is extremely low.

In the present invention, the detection of proteins and protein degradation products is usually carried out by mass spectrometry, preferably a protein analysis system [for example, a combination of a time-of-flight (TOF) mass spectrometer and a two-dimensional surface-modified chip] Using a protein structure analyzer “Protein Chip ^™ System” manufactured by Cyphergen Biosystems Inc.]. According to the “protein chip ^™ system”, it is possible to detect even a minute amount of protein present in only about 0.1 μg per 1 mL of natural rubber latex.

[0011] The hypoallergenic natural rubber of the present invention comprises:
The protein is very highly degraded and therefore the risk of inducing allergies is significantly reduced. Therefore, it is suitable as a raw material for household products such as gloves, medical devices such as catheters, nursing devices, contraceptive devices and the like.

[0012]

Next, the hypoallergenic natural rubber according to the present invention will be described in detail. The hypoallergenic natural rubber of the present invention is a natural rubber that has been subjected to a protein decomposition treatment as described above, and has a number average molecular weight <Mn> of 45.
00 or more, preferably 2000 or more, more preferably 1
It is characterized in that a protein and a protein degradation product of 500 or more are not observed.

The hypoallergenic natural rubber of the present invention preferably has an allergenic protein content index for producing IgE-class antibodies in human serum of 10 μg / g or less, preferably 5 μg / g or less. More preferably, there is. As described above, when a protein having a number average molecular weight <Mn> of 4500 or more and protein degradation treatment has been performed to such an extent that protein degradation products are not detected, IgE is compared to human serum in the protein as described above. It is presumed that the site that can serve as an antigen for producing a class of antibodies has been sufficiently degraded or denatured. In general, when the content index of the allergenic protein is 10 μg / g or less, it is considered that the possibility of causing allergy due to the protein is extremely low.

Here, "content index of allergenic protein"
And, among the proteins present in natural rubber latex,
This is an index indicating the degree of protein content that can produce an IgE class antibody (can be an antigen) with respect to human serum based on general high ammonia latex (HA latex). This is the value shown in the table. As for the protein present in the natural rubber latex, the total amount and the total amount of the eluted protein can be determined by analysis, but the amounts of the allergenic protein and the non-allergenic protein cannot be individually quantified.
Therefore, the allergenicity of the natural rubber latex is evaluated as a relative value of the degree of allergy based on a normal natural rubber latex, in this case, HA latex.

The above "allergenic protein content index"
Is a competitive last immunoinhibition method using the Pharmacia Cap system [Competitive RAST-immunoinhibition method, (X.B.
aur et Al., Allergy, 52, 661-664 (1997))], and is specifically calculated as follows. First, HA as a reference sample
A latex extract (non-ammonia natural rubber latex in the above literature) was used, and this was added to IgE in human serum.
By mixing and aging the antibodies, the antigen-antibody reaction between the allergenic protein in the HA latex and the IgE antibody is advanced. As a source of the IgE antibody, serum of a person having latex allergy is used.
Next, the IgE antibody remaining without causing an antigen-antibody reaction was reacted with the solid-phase Immuno-Cap latex antigen, and the immobilized IgE antibody was labeled with an enzyme (β-D-galactosidase). After binding to the IgE antibody, the amount of the remaining IgE antibody is measured by measuring the fluorescence intensity. From this measured value, the degree of allergicity of the protein eluted from the HA latex is determined. The degree of allergy is measured for several samples having different dilutions of the HA latex, and a calibration curve is prepared. On the other hand, the degree of allergy is determined for the natural rubber latex as a measurement sample in the same manner as described above. As a result, for example, if the degree of allergy eluted by HA latex is equivalent to the degree of allergy when the amount is 10 μg per 1 g of rubber, the content index of the allergenic protein for the measurement sample is 10 μg / g.

In the present invention, the "allergenic protein content index" is used as an index indicating the degree of allergy of latex. As described above, the hypoallergenic natural rubber of the present invention, which has a very low allergenic protein content index, can be said to be substantially free of allergens caused by proteins. The hypoallergenic natural rubber of the present invention is obtained by subjecting a protein having an exopeptidase activity to a protein degradation treatment in place of a conventionally used protease (proteolytic enzyme).

[0017] Proteases (proteolytic enzymes) used in the conventional deproteinization treatment are mainly proteases produced by bacteria (bacteria), and these proteases have an optimum pH in an alkaline region. In addition, they had endopeptidase activity but did not show exopeptidase activity. In conventional deproteinization,
Primarily the above alkaline protease was used because (a) natural rubber field latex, once concentrated, is supplied as so-called ammonia latex to which ammonia is added for the purpose of preventing coagulation and decay of the latex. (B) Therefore, in order to avoid coagulation of rubber molecules, it is necessary to use a so-called alkaline protease having an optimum pH in an alkaline region in order to avoid coagulation of rubber molecules. And so on. Table 1 shows the characteristics of the commercially available alkaline protease used in the conventional deproteinization treatment.

[0018]

[Table 1]

On the other hand, the hypoallergenic natural rubber of the present invention is characterized in that the protein is subjected to a decomposition treatment using a protease having an exopeptidase activity. By performing treatment using such a protease, it is possible to surprisingly degrade proteins to such an extent that proteins having a number average molecular weight <Mn> of 4500 or more and protein degradation products are not substantially present. JP-A-9-71604 discloses that a surfactant is added to natural rubber latex, a neutralizing agent is further added to adjust the pH, and then a protease is added to decompose the protein in the latex. There is a statement to do. However, the above-mentioned publication only discloses a general description of a protein degradation treatment using an enzyme, and does not mention at all how much the molecular weight of the protein is reduced by such treatment. In addition, the invention described in the above-mentioned publication discloses that after a protein is decomposed by an enzyme, a vulcanizing agent is added to the latex to form and vulcanize, and then the polymer is immersed in dilute alkaline water to extract the decomposed protein. The feature is in the extraction processing.

[Production Method of Hypoallergenic Natural Rubber] The hypoallergenic natural rubber of the present invention is prepared by adding a protease having exopeptidase activity alone or in a mixture of two or more thereof to a natural rubber latex, and then reacting for several hours. It is obtained by aging for about one week and decomposing proteins in latex. More specifically, a protease having an exopeptidase activity is added at a ratio of about 0.001 to 10% by weight based on the rubber solid content of the natural rubber latex,
The aging may be performed at a temperature of 5 to 90 ° C, preferably 20 to 60 ° C, for several hours to one week. The protein decomposition treatment may be performed twice or more as necessary.

When adding a protease having exopeptidase activity, as described later, it is necessary to consider that most of the proteases have an optimum pH in a neutral region (specifically, pH 6 to 9). Then, the pH of the latex in advance to the neutral region, specifically, pH 6-9,
It is more preferable to adjust the pH to a range of 6.5 to 8.5. In order to adjust the pH of the latex to the neutral region, for example, sodium dihydrogen phosphate, formalin, dilute hydrochloric acid, etc. may be added to the latex.

The protein-decomposed latex may be further purified (removed) by centrifugation, coagulation of rubber, ultrafiltration, or the like, if necessary. When performing a purification treatment (removal treatment) by centrifugation, a surfactant may be added in a range of 0.01 to 10% by weight, and then centrifugation may be performed according to a conventional method. Although the number of times of centrifugation is sufficient once, the centrifugation may be repeated two or more times in order to enhance the effect of removing proteins and their degradation products.

[Raw material latex] The natural rubber latex used as a starting material for obtaining the hypoallergenic natural rubber of the present invention means a latex obtained from a natural rubber tree. Alternatively, any of commercially available ammonia-treated latex and the like can be used. [Protease having Exopeptidase Activity] Examples of the protein-degrading enzyme used for producing the hypoallergenic natural rubber of the present invention include proteases having exopeptidase activity. By performing degradation by endopeptidase together with exopeptidase, the effect of reducing the molecular weight of the protein and the efficiency of degradation can be further improved. Therefore, in order to produce the hypoallergenic natural rubber of the present invention, it is preferable to use, for example, a protease having both exopeptidase activity and endopeptidase activity.

If the protease having the above exopeptidase activity is limited by its origin, as described above, Aspergillus, a kind of filamentous fungus, is used.
s; a protease produced by a microorganism belonging to the genus Aspergillus or the genus Rhizopus; Examples of the microorganism belonging to the genus Aspergillus include Aspergillus oryzae (Aspergillus oryzae).
lus oryzae) and Aspergillus mels (Asp. mellus)
), Aspergillus niger, Aspergillus awamori, Aspergillus glaucus, Aspergillus flavus, Aspergillus sojae (A)
sp. sojae) and the like.

The microorganisms belonging to the genus Rhizopus include, for example, Rhizopus oryzae,
Rhizopus javanicus (Rhi. Javanicus), Rhizops delemar (Rhi. Delemar), Rhizopus nigricans (Rhi. Nigricans) and the like. The protease having an exopeptidase activity used in the present invention is, among the above, particularly Aspergillus oryzae (Asp. Oryza).
e), preferably those produced by microorganisms belonging to Aspergillus mels (Asp. mellus) or Rhizopus oryzae.

The protease having the above exopeptidase activity exhibits the characteristics of a commercially available protease.

[0027]

[Table 2]

In the proteolytic treatment for obtaining the hypoallergenic natural rubber of the present invention, a proteolytic treatment using a protease having the above exopeptidase activity may be used in combination with a proteolytic treatment using another protease.
The other proteases are not particularly limited, and include those derived from bacteria shown in Table 1 above, those derived from filamentous fungi (excluding proteases having exopeptidase activity), those derived from yeast, and the like. There are various known proteases.

[Surfactant] When the protein is decomposed to produce the hypoallergenic natural rubber of the present invention, the protein is stabilized before or during the treatment with a protease having exopeptidase activity. It is preferable to add a surfactant as an agent to the latex. In particular, when the pH of the ammonia-treated latex is adjusted to a neutral range or when protein is decomposed, it is desirable to add a surfactant to prevent coagulation of the rubber component.

Examples of the surfactant include various conventionally known anionic surfactants, nonionic surfactants, and cationic surfactants. Although not particularly limited, most of the proteases having exopeptidase activity are exemplified. Has an optimum pH in the neutral region, specifically, pH 6 to 9, more preferably pH 6.5 to 8.5. More preferably, those exhibiting stable surface activity in the pH range of 6.5 to 8.5 are preferably used.

Hereinafter, surfactants that can be used in the present invention will be described. The surfactants exemplified below can be used alone or in combination of two or more. (Anionic surfactant) Examples of the anionic surfactant include carboxylic acids, sulfonic acids, sulfates, and phosphates. Examples of the carboxylic acid-based anionic surfactant include a fatty acid salt having 6 to 30 carbon atoms, a polyvalent carboxylate, a rosinate, a dimer acid salt, a polymer acid salt, and a tall oil fatty acid salt. Among them, carboxylate having 10 to 20 carbon atoms is preferable. If the number of carbon atoms is less than 6, the dispersing and emulsifying effects of proteins and impurities may be insufficient.
If it exceeds 300, it may be difficult to disperse it in water.

Examples of the sulfonic acid-based anionic surfactant include alkyl benzene sulfonate, alkyl sulfonate, alkyl naphthalene sulfonate, naphthalene sulfonate, diphenyl ether sulfonate and the like. Examples of the sulfate-based surfactant include alkyl sulfates, polyoxyalkylene alkyl sulfates, polyoxyalkylene alkyl phenyl ether sulfates, tristyrenated phenol sulfates, and polyoxyalkylenedistyrenated phenol sulfates. And the like.

Examples of the phosphoric acid ester-based anionic surfactant include alkyl phosphoric acid ester salts and polyoxyalkylene phosphoric acid ester salts. Salts of these compounds include metal salts (Na, K, Ca, Mg, Zn
Etc.), ammonia salts, amine salts (such as triethanolamine salts) and the like. (Nonionic surfactant) Examples of the nonionic surfactant include polyoxyalkylene ethers, polyoxyalkylene esters, polyhydric alcohol fatty acid esters, sugar fatty acid esters, and alkyl polyglycoside.

Examples of the polyoxyalkylene ether nonionic surfactant include polyoxyalkylene alkyl ether, polyoxyalkylene alkyl phenyl ether, polyoxyalkylene polyol alkyl ether, polyoxyalkylene styrenated phenol ether, and polyoxyalkylene distyrene. Phenol ether, polyoxyalkylene tristyrene phenol ether, and the like. Examples of the polyol include polyhydric alcohols having 2 to 12 carbon atoms, such as propylene glycol, glycerin, sorbitol, sucrose, pentaerythritol, and sorbitan.

Examples of the polyoxyalkylene ester nonionic surfactant include polyoxyalkylene fatty acid esters. As the polyhydric alcohol fatty acid ester-based nonionic surfactant, the carbon number is 2 to 2.
And a fatty acid ester of a polyhydric alcohol or a fatty acid ester of a polyoxyalkylene polyhydric alcohol. More specifically, examples thereof include sorbitol fatty acid ester, sorbitan fatty acid ester, fatty acid monoglyceride, fatty acid diglyceride, and polyglycerin fatty acid ester. Further, these polyalkylene oxide adducts (for example, polyoxyalkylene sorbitan fatty acid ester, polyoxyalkylene glycerin fatty acid ester, etc.) can also be used. Examples of the sugar fatty acid ester-based nonionic surfactant include sucrose, glucose, maltose, fructose, and fatty acid esters of polysaccharides. Polyalkylene oxide adducts thereof can also be used.

Examples of the alkyl polyglycoside nonionic surfactant include alkyl glucoside, alkyl polyglucoside, polyoxyalkylene alkyl glucoside, polyoxyalkylene alkyl polyglucoside, and fatty acid esters thereof.
Further, these polyalkylene oxide adducts can also be used. Examples of the alkyl group in these nonionic surfactants include an alkyl group having 4 to 30 carbon atoms. Further, as the polyoxyalkylene group,
Those having an alkylene group having 2 to 4 carbon atoms,
For example, those having an addition mole number of ethylene oxide of about 1 to 50 mol can be mentioned. Examples of the fatty acid include linear or branched saturated or unsaturated fatty acids having 4 to 30 carbon atoms.

(Cationic Surfactant) Examples of the cationic surfactant include an alkylamine salt type, an alkylamine derivative type and a quaternary product thereof, and an imidazolinium salt type. Examples of the alkylamine salt type cationic surfactant include salts of primary amine, secondary amine and tertiary amine. The alkylamine derivative-type cationic surfactant has at least one of an ester group, an ether group, and an amide group in a molecule and includes, for example, polyoxyalkylene (AO)
Alkylamines and their salts, alkylesteramines (including AO adducts) and their salts, alkyletheramines (including AO adducts) and their salts, alkylamidoamines (including AO adducts) and their salts, alkylesteramidoamines (Including AO adducts) and salts thereof, and alkyl ether amidoamines (including AO adducts) and salts thereof.

Examples of the type of the salt include hydrochloride, phosphate, acetate, alkyl sulfate, alkylbenzene sulfonic acid, alkyl naphthalene sulfonic acid, fatty acid, organic acid, alkyl phosphate, alkyl ether carboxylic acid, and alkyl Examples include amide ether carboxylic acids, anionic oligomers, and anionic polymers. Among the alkylamine derivative-type cationic surfactants, specific examples of acetate include, for example, coconutamine acetate, stearylamine acetate and the like. The alkyl group in the alkylamine salt-type and alkylamine derivative-type cationic surfactants is not particularly limited, and examples thereof include a linear, branched or Guerbet-shaped one having usually 8 to 22 carbon atoms. .

As the quaternized alkylamine salt-type and alkylamine derivative-type cationic surfactants, the above-mentioned alkylamine salts and alkylamine derivatives can be converted to, for example, methyl chloride, methyl bromide, dimethyl sulfate, diethyl sulfate and the like. A quaternized one may be mentioned. Specifically, alkyltrimethylammonium halides such as lauryltrimethylammonium halide, cetyltrimethylammonium halide, stearyltrimethylammonium halide; dialkyldimethylammonium halides such as distearyldimethylammonium halide; trialkylmethylammonium halide; dialkylbenzylmethylammonium halide;
Alkylbenzyldimethylammonium halide and the like.

Examples of the imidazolinium salt type cationic surfactant include 2-heptadecenyl-hydroxylethylimidazoline and the like. Among the surfactants exemplified above, those which exhibit stable surface activity particularly when the pH is in the range of 6.5 to 8.5 include, for example, polyoxyethylene nonylphenyl ether which is a nonionic surfactant, anionic surfactant Examples of the activator include sodium polyoxyethylene alkylphenyl ether sulfate.

[Other Additives] In the production of the hypoallergenic natural rubber of the present invention, in addition to the above-mentioned components, other additives can be blended as required.
Such other additives include, for example, phosphates such as potassium phosphate monobasic, potassium phosphate dibasic, sodium phosphate, etc .; acetates such as potassium acetate, sodium acetate; sulfuric acid, acetic acid; Acids such as hydrochloric acid, nitric acid, citric acid and succinic acid and salts thereof; ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate and the like. Examples of the enzyme include lipase, esterase, amylase, laccase, and cellulase. Further, as a dispersant, styrene sulfonic acid copolymer, naphthalene sulfonic acid formalin condensate, lignin sulfonic acid, polycyclic aromatic sulfonic acid copolymer, homopolymer / copolymer of acrylic acid and maleic anhydride, Isobutylene-acrylic acid, isobutylene-maleic anhydride copolymer, and the like.

[Residual Protein Content and Allergenicity of Natural Rubber] The hypoallergenic natural rubber of the present invention has the same level of nitrogen content (N%) as that of the control natural rubber when compared by the Kjeldahl method. Protein remains. However, the hypoallergenic natural rubber of the present invention has extremely low contents of a protein having a high molecular weight and an allergenic protein as compared with deproteinized natural rubber which has been subjected to deproteinization by a conventional method. Specifically, from the hypoallergenic natural rubber obtained by the method of the present invention, the number average molecular weight <
Proteins with Mn> 4500 or more and protein degradation products are not detected.

In the case of the deproteinized natural rubber that has been subjected to the deproteinization treatment (one enzymatic treatment and one centrifugal separation treatment) according to the conventional method, the number average molecular weight <Mn> is apparent from the comparative examples described later. And a protein degraded product of which is not less than 4500 is detected. On the other hand, in the natural rubber that has been subjected to the protein decomposition treatment (one enzymatic treatment, no centrifugation treatment) by the method of the present invention, the number average molecular weight <Mn> is 4500 or more, as is clear from the examples described later. Certain proteins and protein degradation products are not detected.

As described above, the number average molecular weight <Mn> is 45.
A level at which no protein or protein degradation product of 00 or more is detected indicates that the protein has been degraded to such an extent that the allergy problem caused by the protein does not substantially occur. That is, although the hypoallergenic natural rubber of the present invention has a large amount of total protein remaining in the natural rubber, the possibility of causing immediate allergy caused by proteins is extremely low.

[0045]

Next, the hypoallergenic natural rubber of the present invention will be described with reference to Examples and Comparative Examples. In the following Examples and Comparative Examples, the natural rubber latex is a field latex produced in Malaysia or a high ammonia latex manufactured by Soctech (Malaysia) [rubber content: 60.2% by weight, ammonia content: 0.7%]
It was used.

As the surfactant, "Triton X-" manufactured by Toho Chemical Industry Co., Ltd. is used as a nonionic surfactant.
100 "was used for each. [Production of Hypoallergenic Natural Rubber] Example 1 (Protein degradation treatment only) 1.0 part by weight of nonionic surfactant and 0.12% sodium naphthenate were added to 100 parts by weight of rubber of field latex. After the latex was stabilized, the pH of the latex was adjusted to 7.0 with a 5% aqueous solution of sodium dihydrogen phosphate. Subsequently, 0.1 part by weight of a protease having an exopeptidase activity was added to 100 parts by weight of the rubber component of the latex, and the mixture was allowed to stand at 30 ° C. for 24 hours to be matured.
A hypoallergenic natural rubber latex treated with a protein was obtained.

The protease has an exopeptidase activity and an endopeptidase activity and has an optimum pH in a neutral region.
A protease produced by a microorganism belonging to Oryzae (Aspergillus oryzae) [trade name "Umamizyme" manufactured by Amano Pharmaceutical Co., Ltd.] was used (see Table 2). This protease has a peptidase activity of 70 u / g or more (pH 7.0,
LGG (L-Leucyl-Glycyl-Glycine) method).

Further, the rubber component of the thus obtained hypoallergenic natural rubber latex was coagulated and dried to obtain a hypoallergenic natural rubber subjected to a protein decomposition treatment. (Protein Decomposition Treatment and Removal Treatment) The hypoallergenic natural rubber latex subjected to the protein degradation treatment described above was diluted with a 1% aqueous solution of a nonionic surfactant to make the concentration of the rubber component of the latex 10%. After, 13000 rp
m for 30 minutes. The upper layer cream thus separated was taken out and redispersed in the same amount of water to obtain a hypoallergenic natural rubber latex subjected to a protein decomposition and removal treatment.

Further, the cream was dried to obtain a hypoallergenic natural rubber which had been subjected to a protein decomposition and removal treatment. Example 2 (Protein degradation treatment only) High ammonia latex (hereinafter referred to as “HA latex”) having a rubber content of 3
It is diluted to 0% by weight and stabilized by adding 1.0 part by weight of a nonionic surfactant and 0.12% of sodium naphthenate (stabilizer) to 100 parts by weight of the rubber component of the latex. After that, the pH of the latex was adjusted to 7.0 with a 5% aqueous sodium dihydrogen phosphate solution. Next, 0.1 part by weight of a protease ("Uramizyme" described above) was added to 100 parts by weight of the rubber component of this latex, and 3 parts by weight were added.
The mixture was allowed to stand at 0 ° C. for 24 hours to be aged, thereby obtaining a hypoallergenic natural rubber latex subjected to a protein decomposition treatment.

Further, the rubber component of the thus obtained hypoallergenic natural rubber latex was coagulated and dried to obtain a hypoallergenic natural rubber subjected to a protein decomposition treatment. (Protein Decomposition Treatment and Removal Treatment) The hypoallergenic natural rubber latex subjected to the protein degradation treatment described above was diluted with a 1% aqueous solution of a nonionic surfactant to make the concentration of the rubber component of the latex 10%. After, 13000 rp
m for 30 minutes. The upper layer cream thus separated was taken out and redispersed in the same amount of water to obtain a hypoallergenic natural rubber latex subjected to a protein decomposition and removal treatment.

On the other hand, the cream was dried to obtain a hypoallergenic natural rubber which had been subjected to a protein decomposition and removal treatment. Example 3 (Proteolytic treatment only) As a protease having exopeptidase activity, a protease produced by a microorganism belonging to Aspergillus mellus in place of the above-mentioned "Umamizyme" [trade name of Amano Pharmaceutical Co., Ltd.] “P“ Amano ”3G”] and the addition amount was 0.1 parts by weight with respect to 100 parts by weight of the rubber content of the latex, except that the field latex was used as a starting material. The protein (as a raw material) was decomposed to obtain a hypoallergenic natural rubber latex.

The protease "P" Amano "3G"
Has a protein digestibility of 10,000 u / g or more (pH 7.0,
As shown in Table 2, the protease has an exopeptidase activity and an endopeptidase activity and has an optimum pH in a neutral region. Further, the rubber component of the hypoallergenic natural rubber latex thus obtained was coagulated and dried to obtain a hypoallergenic natural rubber subjected to a protein decomposition treatment.

(Protein Decomposition Treatment and Removal Treatment) The hypoallergenic natural rubber latex treated by the above-mentioned protease “P“ Amano ”3G” was subjected to dilution treatment and centrifugation in the same manner as in Example 1. By performing the separation treatment and the redispersion treatment, a hypoallergenic natural rubber latex subjected to a protein decomposition and removal treatment was obtained. Further, the cream obtained by the centrifugation was dried to obtain a hypoallergenic natural rubber which had been subjected to a protein decomposition and removal treatment.

Example 4 (Proteolytic treatment only) As a protease having exopeptidase activity, the above-mentioned "P" Amano "3G" was used in place of the above-mentioned "Umamizyme", and the amount of addition was changed to latex rubber. The same procedure as in Example 2 was conducted except that 0.1 part by weight was used for 100 parts by weight (namely, HA
The protein was degraded (using latex as a starting material) to obtain a hypoallergenic natural rubber latex.

Further, the rubber component of the thus obtained hypoallergenic natural rubber latex was coagulated and dried to obtain a hypoallergenic natural rubber subjected to a protein decomposition treatment. (Protein degradation treatment and removal treatment) The hypoallergenic natural rubber latex subjected to the protein degradation treatment by the above-mentioned protease “P“ Amano ”3G” was subjected to a dilution treatment, a centrifugation treatment, and the like in the same manner as in Example 2. And a redispersion treatment to obtain a hypoallergenic natural rubber latex that has been subjected to a protein decomposition and removal treatment.

On the other hand, the cream fraction obtained by the above-mentioned centrifugation was dried to obtain a hypoallergenic natural rubber which had been subjected to a protein decomposition and removal treatment. Example 5 (Proteolytic treatment only) As a protease having exopeptidase activity, a protease produced by a microorganism belonging to Rhizopus oryzae in place of the above-mentioned "Umamizyme" [trade name of Amano Pharmaceutical Co., Ltd.] "Peptidase R"]], and the addition amount was 0.1 parts by weight with respect to 100 parts by weight of the rubber content of the latex (except that a field latex was used as a starting material). Perform protein degradation treatment,
A hypoallergenic natural rubber latex was obtained.

The protease “peptidase R” is
Peptidase activity of 420 u / g or more (pH 7.0, L
GG method), as shown in Table 2, a protease having an exopeptidase activity and an endopeptidase activity and having an optimum pH in a neutral region. Further, the rubber component of the hypoallergenic natural rubber latex thus obtained was coagulated and dried to obtain a hypoallergenic natural rubber subjected to a protein decomposition treatment.

(Protein Degradation Treatment and Removal Treatment) The hypoallergenic natural rubber latex treated with the above protease “peptidase R” for protein degradation was subjected to dilution treatment, centrifugation treatment, and the like in the same manner as in Example 1. And a redispersion treatment to obtain a hypoallergenic natural rubber latex that has been subjected to a protein decomposition and removal treatment. On the other hand, the cream component obtained by the centrifugation was dried to obtain a hypoallergenic natural rubber which had been subjected to a protein decomposition and removal treatment.

Example 6 (Proteolytic treatment only) As a protease having exopeptidase activity, the above-mentioned "peptidase R" was used instead of the above-mentioned "equinamizyme", and the amount added was 100% of the latex rubber content. Except that the amount was 0.1 part by weight with respect to part by weight, the same as in Example 2 (that is, HA
The protein was degraded (using latex as a starting material) to obtain a hypoallergenic natural rubber latex.

Further, the rubber component of the thus obtained hypoallergenic natural rubber latex was coagulated and dried to obtain a hypoallergenic natural rubber subjected to a protein decomposition treatment. (Protein Degradation Treatment and Removal Treatment) Example 2 was applied to a hypoallergenic natural rubber latex that had been subjected to protein degradation treatment with the above-mentioned protease "peptidase R".
By performing a dilution treatment, a centrifugal separation treatment, and a redispersion treatment in the same manner as described above, a hypoallergenic natural rubber latex subjected to a protein decomposition and removal treatment was obtained.

Further, the cream obtained by the centrifugation was dried to obtain a hypoallergenic natural rubber which had been subjected to a protein decomposition and removal treatment. Comparative Example 1 (Proteolytic treatment only) 0.1 part by weight of alkaline protease, 1.0 part by weight of nonionic surfactant, and 0.12%
Of sodium naphthenate was added for stabilization.

The alkaline protease is a protease derived from bacteria and produced by a microorganism belonging to Bacillus licheniformis which is a kind of Bacillus bacterium [trade name “Alcalase 2” manufactured by Novo Nordisk Bioindustry Co., Ltd.] .0
M "] was used. This alkaline protease has a titer of 2.0 AU / g (pH 8.3) and, as shown in Table 1, has an optimal pH in the alkaline region and has exopeptidase activity although it has endopeptidase activity. It has no activity.

Next, the pH was adjusted to 9.2 with sodium dihydrogen phosphate, and the mixture was allowed to stand at 30 ° C. for 24 hours to ripen, thereby obtaining a natural rubber latex to which a protein decomposition treatment was applied. . (Protein Decomposition Treatment and Removal Treatment) The natural rubber latex subjected to the protein decomposition treatment described above was diluted with a 1% aqueous solution of a nonionic surfactant to make the rubber content of the latex 10%, and then 13,000 rpm. For 30 minutes. The separated upper layer cream was redispersed in water to obtain a natural rubber latex that had been subjected to protein degradation and removal treatment.

Further, the cream was coagulated and dried to obtain a deproteinized natural rubber. [Evaluation of Proteolysis Treatment] (Measurement of Nitrogen Content) In the above Examples and Comparative Examples, a latex obtained by redispersing cream produced after centrifugation in water was used, and this latex was flowed on a glass plate. This was cast to produce a cast film.

These cast films were used as samples for measuring the nitrogen content. Example 1 and Comparative Example 1
As for (1), a cast film similar to the above was prepared using latex subjected to only the protein decomposition treatment, and this film was also used as a sample for measuring the nitrogen content. Furthermore, a cast film was prepared directly from the field latex and the HA latex that had not been subjected to the protein decomposition treatment or the washing treatment, and this was used as a control sample.

The nitrogen content (N%) of each of the samples of Examples, Comparative Examples and Controls was determined by the RRIM test method (Ru
bber Research Institute of Malaysia (1973). 'SMR Bu
lletin No. 7 '). Nitrogen content (N%)
Table 3 shows the measurement results. (Measurement of Content Index of Allergenic Protein) Based on the last inhibition method (RAST-inhibition method), the content index of the allergenic protein contained in the natural rubber latex obtained in the above Examples and Comparative Examples was measured. . The “Allergenic Protein Content Index” was measured using the Pharmacia Cap syst
BGFA by competitive immunoinhibition using em
(Occupational Health Research Institute).

In Example 1 and Comparative Example 1, the content index of the allergenic protein was similarly calculated for the latex subjected to only the protein decomposition treatment. Table 3 shows the measurement results of the content index of the allergenic protein.

[0068]

[Table 3]

As is evident from Table 3, the hypoallergenic natural rubber latex of Example 1 had a nitrogen content (N%) which was lower than that of the deproteinized natural rubber latex treated with the conventional enzyme in Comparative Example 1. ), But the content index of the allergenic protein was significantly lower. Further, in Example 1, even when only the enzyme treatment was performed, the content of the allergenic protein was significantly lower than that of the normal HA latex shown in Control 2, and the conventional enzyme treatment was performed. (No protein removal treatment of Comparative Example 1).

[0070] Therefore, it was found that the possibility of inducing allergy caused by proteins was significantly reduced. (Protein molecular weight analysis) The natural rubber latexes obtained in Example 1 and Comparative Example 1 were analyzed for flight time (TO
F) The number average molecular weight <Mn> is measured by a protein analysis system combining a two-dimensional surface-modified chip with a mass spectrometer (Protein Chip ^™ System, a protein structure analyzer manufactured by Ciphergen Biosystems). Was analyzed for the presence or absence of impurities mainly in the range of 4500 to 4700.

The protein molecular weight analysis using the “protein chip ^™ system” was performed as follows. (1)
First, a serum sample of natural rubber latex is adsorbed on the chip, and then (2) washed with a washing buffer so that only the adsorbed protein remains,
(3) An energy absorbing substance was applied on the adsorbed protein, and (4) the protein on the chip was ionized by irradiating a laser. Then, (5) the molecular weight was measured using a TOF mass spectrometer.

The analysis results of the number-average molecular weight <Mn> of the remaining protein and the degraded protein were measured with respect to the sample obtained from the hypoallergenic natural rubber latex subjected to only the proteolytic treatment in Example 1. FIG. 1 shows the results. FIG. 2 shows the measurement results of a sample obtained from the hypoallergenic natural rubber latex subjected to the proteolytic treatment and the removal treatment of Example 1, and FIG. The measurement results of the samples obtained from the deproteinized natural rubber latex by the treatment used are shown in FIG. 3, respectively.

As a control, a molecular weight analysis was performed on a sample obtained from the HA latex that had not been subjected to the proteolytic treatment and the washing treatment. FIG. 4 shows the measurement results. As is clear from the results of the molecular weight analysis, in Comparative Example 1 (FIG. 3), the number average molecular weight <Mn> was about 4700.
(A position indicating the presence of the protein). On the other hand, the number average molecular weight <Mn> of the case where only the proteolytic treatment of Example 1 was performed (FIG. 1) and the case where the protein and its decomposed product were further removed (FIG. 2) were both obtained. No peak was observed in the region of 4500 or more. In particular, when the protein and its degradation product were removed in addition to the protein degradation treatment (FIG. 2), no remarkable peak was observed even in the region where the number average molecular weight <Mn> was 1500 to 4500.

[Brief description of the drawings]

FIG. 1 shows the number-average molecular weight of remaining protein and degraded protein of the hypoallergenic natural rubber latex obtained in Example 1 and subjected to only proteolytic treatment <Mn.
> Is a chart showing the measurement results.

FIG. 2 is a chart showing the measurement results of the number-average molecular weight <Mn> of the remaining protein and protein degradation products in the hypoallergenic natural rubber latex obtained in Example 1 and subjected to the protein degradation treatment and the removal treatment. It is.

FIG. 3 shows the number-average molecular weight of remaining protein and degraded protein of the deproteinized natural rubber latex obtained in Comparative Example 1 which has been subjected to protein degradation and removal, <Mn.
> Is a chart showing the measurement results.

FIG. 4 is a chart showing the measurement results of the number average molecular weight <Mn> of the protein present in the high ammonia-treated latex.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshiaki Miyamoto 3-6-9, Wakihama-cho, Chuo-ku, Kobe-shi, Hyogo F-term in Sumitomo Rubber Industries, Ltd. 4C081 AC08 BA15 CB051 CD31 DA03

Claims (4)

[Claims] 1. A hypoallergenic natural rubber which is a natural rubber which has been subjected to a protein decomposition treatment, wherein a protein having a number average molecular weight of 4500 or more and a protein degradation product are not detected. 2. The hypoallergenic natural rubber according to claim 1, wherein a protein having a number average molecular weight of 1500 or more and a protein degradation product are not detected. 3. The hypoallergenic natural rubber according to claim 1, wherein the content index of the allergenic protein that produces IgE class antibodies in human serum is 10 μg / g or less. 4. The method according to claim 1, wherein the content index of the allergenic protein is 5 μm.
The hypoallergenic natural rubber according to claim 3, which is not more than g / g.