Title of Invention

A METHOD FOR PREPARING A NATURAL RUBBER LATEX FREE FROM PROTEINS

Abstract

A natural rubber latex containing substantially no proteins which is specified by 14, 31 and 4 5 kDa bands in SDS-PAGE measurement, and a process producing this latex by saponification of natural rubber latex with alkaline hydroxide in the presence of a surfactant. The natural rubber latex of the present invention can be suitably applied to the production of rubber products such as catheter, rubber glove, condom, and foam rubber, because it contains substantially no proteins which cause Type I allergy.

Full Text

DESCRIPTION NATURAL RUBBER LATEX FREE FROM PROTEINS, METHOD FOR PREPARING THE SAME AND USE THEREOF Field of the Invention The present invention relates to natural rubber latex free from proteins, production process thereof and use thereof. More specifically, it relates to natural rubber latex which substantially does not contain proteins with specific molecular weight which are characteristic of natural rubber latex, and a manufacturing and use thereof. Background of the Invention Heretofore, natural rubber has been applied to a wide variety of products ranging from industrial •products such as tires for automobiles and aircraft, and conveyer belts. Natural rubber is collected as latex containing water, proteins, inorganic salts and the like in addition to the rubber component and this latex is coagulated to give crude rubber (crepe rubber or smoked sheet rubber). A target rubber product is produced from this crude rubber through mastication, addition of compounding agents, molding-, and vulcanization. In addition, various kinds of industrial products are manufactured from natural rubber latex itself. For example, natural rubber latex is applied to the production of toy balloons, medical supplies such as medical gloves, catheter and condoms, foam rubber, elastic bands, rubber tubes, adhesives, and coating agents for paper processing. Fresh natural rubber latex contains non-rubber components such as proteins, lipids, carbohydrates and inorganic substances in addition to about 2 8 to 3 0% w/v (by weight/volume) of rubber components. Solid natural rubber (crude rubber) obtained by coagulating the fresh latex with formic acid contains about 6% w/v of the non-rubber components. These non-rubber components are known to be important for natural rubber to exhibit specific physical properties. However, it has become a social problem around 1990 that some of proteins contained in natural rubber latex products, gloves in particular, cause Type I acute allergy, and Food and Drug Administration (FDA) in U.S.A. has issued a medical alert toward manufacturers of rubber products to reduce soluble proteins contained in latex products. Following methods are known as a technique for reducing proteins in latex, (i) a method of successive centrifuging the latex, (ii) a method of treating the latex with a proteolytic enzyme, and (iii) a method of treating the latex with alkali. However, even after treatments by these (i), (ii), and (iii) methods, the resulting latex still contains a substantial amount of nitrogenous compounds and they are not able to be free from allergic reactions. As a result of extensive studies on the proteins contained in natural rubber latex, the present inventors have disclosed that it is difficult.to remove all the proteins by a normal proteolytic enzyme in the serum and on the surface of rubber particles in natural rubber latex, the latter of which are present as a bi-layer composed of proteins and lipids covering and stabilizing on the surface of rubber particles. Thus, by the method (i) of centrifuging the latex, the proteins on the surfaces of the rubber particles cannot be removed, although a part of the proteins in the serum can be removed. Meanwhile, by the method (ii) of treating the latex with a proteolytic enzyme, a part of the proteins on the surfaces of the rubber particles can be decomposed. However, the resulting deproteinized latex always contains small amounts of residual proteins. In addition, the proteolytic enzyme used cannot be completely removed from the latex, with result that the presence of residual proteins is inevitable, which may cause some allergic reaction. Moreover, by the method (iii) of treating the latex with alkali, most part of proteins on the surface of rubber particles can be decomposed. However, the reaction is always accompanied with the coagulation of rubber particles during the treatment, which makes it difficult to carry out the reaction keeping a stable latex state. Under the circumstances, one of the present inventors has made intensive studies. As a result, he has found a method of producing natural rubber containing a reduced amount of nitrogen such as 0.02% or less, the content of which is known to be an index of the protein content, and already filed a patent application for the invention (refer to JP-A 6-5 6 902 (1994)). The method comprises the steps of treating natural rubber latex with a surfactant and a proteolytic enzyme and then concentrating and washing the treated latex once or twice by centrifugation. The method provided the solid natural rubber obtained by coagulation of the resulting latex which was containing the residual nitrogen content of 0.02% or less. This indicates that the rubber included in the cream fraction of latex contains the nitrogen 0.02% or less in solid rubber. Since the latex obtained by this method is highly purified, gloves prepared by use of the low-protein natural rubber latex showed a lower possibility for allergic reactions. However, it has been confirmed by a clinical test with a scratch method, which is a more strict testing method, that about 8% of patients are still positive for the Type I allergy with respect to gloves made of the low-protein natural rubber latex obtained by this method (R. Hayakawa et. al,, Environ. Dermatol., 6, 10 (1999)). This indicates that the deproteinization by this method is not complete. Detailed Description of the Invention Therefore, an object of the present invention is to provide natural rubber latex free from substances determined to be the cause of Type I allergic reactions. Another object of the present invention is to provide a manufacturing method of the natural rubber latex mentioned above favorable for industrial production. Still another object of the present invention is to provide rubber products comprising natural rubber latex of the present invention such as catheter, rubber glove, condom, and foam rubber. Other objects and advantages of the present invention will be apparent from the following description. According to the present invention, firstly, the above objects and advantages of the present invention are achieved by natural rubber latex which contains substantially no proteins specified by the bands of 14, 31 and 45 kDa by an SDS-PAGE method. Further, according to the present invention, secondly, the above objects and advantages of the present invention are achieved by the manufacturing method of natural rubber latex of the present invention saponified with alkaline hydroxide in the presence of a surfactant or surfactants. Finally, according to the present invention, thirdly, the above objects and advantages of the present invention are achieved by rubber products produced by the use of natural rubber latex of the present invention such as catheter, x-ubber glove, condom, and foam rubber. Brief Description of the Drawings Figure 1 is a diagram showing the result of measurement of proteins in saponified natural rubber latex in Example 1 by SDS-PAGE measurement: Figure 2 is a diagram showing the result of measurement of proteins in fresh natural rubber latex by SDS-PAGE: Figure 3 is a diagram showing the result of measurement of proteins in cream fraction of the deproteinized natural rubber latex with the proteolytic enzyme by SDS-PAGE: Figure 4 is a diagram showing the result of measurement of proteins by SDS-PAGE for cream fraction of the natural rubber latex obtained Examples 2 to 4 by saponification under various conditions: Figure 5 is a photo showing the gloves; 1. A part of the glove prepared by using the saponified natural rubber latex. 2 . A part of the glove prepared by using the deproteinized natural rubber latex obtained by deproteinization. Best Mode for Carrying out the Invention Hereinafter, the present invention will be further described. Firstly, a description will be given to a method for producing the natural rubber latex of the present invention. The concentration of rubber in fresh natural rubber latex obtained is about 3 0% DRC (Dry rubber content). The fresh natural rubber latex is treated with an alkaline hydroxide under a certain condition in the presence of a surfactant or surfactants to have the protein therein subjected to hydrolysis. The resulting latex is concentrated to about 60% DRC by centrifugation, the process of which is expressed as concentration and washing. The concentrated saponified natural rubber latex is used as commercial latex or industrial raw material for the production of various industrial products. The production process of natural rubber latex containing a low nitrogen by a proteolytic enzyme, which was proposed by an inventor of the present invention, is required to be reacted latex after dilution to about 10% DRC with a proteolytic enzyme. At that point the enzymatic deproteinization method is different from the present invention. The present invention provides a profitable method from the viewpoint of mass production. Moreover, the present invention is favorable to reduce the content of proteins having special molecular weight. Hereinafter, the method for producing in the present invention will be further described. The method for producing the natural rubber latex of the present invention is carried out by saponifying natural rubber latex with an alkaline hydroxide in the presence of at least one kind of cationic surfactant, anionic surfactant or nonionic surfactant, followed by washing the decomposed proteins by centrifugation, for instance. When natural rubber latex is saponified with the alkaline hydroxide, coagulation of the latex can be prevented by use of the cationic surfactant, anionic surfactant or nonionic surfactant as described above. However, a nonionic surfactant and/or an anionic surfactant can be preferably used in this case. Illustrative examples of usable nonionic surfactants include polyoxyalkylene ethers, polyoxyalkylene esters, polyhydric alcohol fatty acid esters, sugar fatty acid esters and alkylpolyglycosides. Specific examples of the polyoxyalkylene ether nonionic surfactants include polyoxyalkylene alkyl ethers, polyoxyalkylene alkyl phenyl ethers, polyoxyalkylene polyol alkylene ethers, polyoxyalkylene styrenated phenol ethers, polyoxyalkylene distyrenated phenol ethers, and polyoxyalkylene tristyrenated phenol ethers. The polyoxyalkylene polyol of the above polyoxyalkylene polyol alkylene ethers can be a polyhydric alcohol having 2 to 12 carbon atoms. Illustrative examples thereof include propylene glycol, glycerin, sorbitol, sucrose, pentaerythritol, and sorbitan. Illustrative examples of the polyoxyalkylene ester nonionic surfactants include a polyoxyalkylene fatty acid ester. Illustrative examples of the polyhydric alcohol fatty acid ester nonionic surfactants include a fatty acid ester of a polyhydric alcohol having 2 to 12 carbon atoms and a fatty acid ester of a polyoxyalkylene polyhydric alcohol. Specific examples thereof include sorbitol fatty acid ester, sorbitan fatty acid ester, fatty acid monoglyceride, fatty acid diglyceride, and a polyglycerin fatty acid ester. Further, their polyalkylene oxide adducts such as a polyoxyalkylene sorbitan fatty acid ester and a polyoxyalkylene glycerin fatty acid ester can also be used. Illustrative examples of the sugar fatty acid ester nonionic surfactants include fatty acid esters of sucrose, glucose, maltose, fructose and polysaccharides. Their polyalkylene oxide adducts can also be used. Illustrative examples of the alkylpolyglycoside nonionic surfactants include an alkylglucoside, an alkylpolyglucoside, a polyoxyalkylene alkylglucoside and a polyoxyalkylene alkylpolyglucoside. Further, their polyalkylene oxide adducts can also be used. Preferred examples of the fatty acids of the above polyhydric alcohol fatty acid ester and sugar fatty acid ester surfactants are a linear or branched saturated or unsaturated fatty acid having 4 to 30 carbon atoms. The alkyl group in the surfactant is exemplified by an alkyl group having 4 to 3 0 carbon atoms. Further, the polyoxyalkylene group is exemplified by one having an alkylene group having 2 to 4 carbon atoms, more specifically, one with about 1 to 50 added moles of ethylene oxide. Illustrative examples of the anionic surfactant include carboxylic acid based anionic surfactants, sulfonic acid based anionic surfactants, sulfuric ester anionic surfactants and phosphoric ester anionic surfactants. Illustrative examples of the carboxylic acid based surfactants include a fatty acid salt, polyvalent carboxylate, rosinace and tall oil fatty acid salt which have 6 to 3 0 carbon atoms. A carboxylate having 10 to 2 0 carbon atoms is preferred. When the number of carbon atoms is 6 or less, dispersion and emulsification of proteins and impurities are insufficient, while when the number of carbon atoms is 30 or more, the surfactant becomes difficult LO be dispersed in water. Illustrative examples of the sulfonic acid based surfactants include an alkyl benzene sulfonate, an alkyl sulfonate, an alkyl naphthalene sulfonate, naphthalene sulfonate and diphenylether sulfonate. Illustrative examples of the sulfuric ester surfactants include an alkyl sulfate, a polyoxyalkylene alkyl sulfate, a polyoxyalkylene alkyl phenyl ether sulfate, tristyrenated phenol sulfate and a polyoxyalkylene distyrenated phenol sulfate. Illustrative examples of the salts of these compounds include metal salts such as Na, K, Ca, Mg and Zn, ammonium salts and amine salts such as a triethanolamine salt. Illustrative examples of the phosphoric ester surfactants include an alkyl phosphate and a polyoxyalkylene phosphate. Illustrative examples of the salts of these compounds include metal salts such as Na, K, Ca, Mg and Zn, ammonium salts and amine salts such as a triethanolamine salt. The above described surfactant is preferably added in an amount of 0.01 to 5.0% (w/v), more preferably 0.03 to 3.0%, particularly preferably 0.05 to 2.0%, based on the rubber latex. When the amount is smaller than the lower limit, the effect of the surfactant is not sufficient, while when the amount is larger than the upper limit, it is useless. A necessary amount of the surfactants mentioned above can be added after concentrating natural lubber latex in order to increase the mechanical stability of the natural rubber latex during storage. Further, as the alkaline hydroxide for saponifying the natural rubber latex, sodium hydroxide or potassium hydroxide is preferably used, for example. The amount of the alkaline hydroxide is preferably 0.1 to 10% (w/v) based on the rubber latex. When the amount is smaller than 0.1%, the reaction time becomes too long, while when the amount is larger than 10%, the coagulation reaction is liable to occur. A more preferred amount is 0.3 to 8%. Fresh latex or concentrated high ammonia latex can be used as natural rubber latex to be saponified with the alkali hydroxide and the surfactant. Although the reaction time is not particularly limited, the reaction is preferably carried out from several minutes to about one day. Further, during the reaction, the latex can be stirred or left to stand. However, the latex is preferably stirred so as to accelerate the reaction. In addition, the temperature can be adjusted as required, and a suitable temperature is 5 to 90°C, more preferably 20 to 70°C The resulting deproteinized natural rubber latex is concentrated to 50 to 70% DRC after saponification reaction. This process solubilizes the decomposed proteins into water, which can be transferred into serum and removed more effectively as increasing the degree of concentration. Although any method can be applied to the concentration of saponified latex, concentration by heating, centrifugation, dialysis or ultrafiltration can be used. If it is necessary, the concentrated natural rubber latex can be concentrated after dilution to over 10 % followed by recentrifugation to get purified natural rubber latex containing lesser amounts of decomposed proteins. However, it is not essential to remove residual decomposed proteins from saponified natural rubber latex in usual method of production. During these processes, it is necessary to satisfactorily hold the stability of natural rubber latex. At this point the kind and amount of surfactant used for saponification reaction are important factors. In general, the MST value (mechanical stability time, ASTM D1076-97) is used as an index of the stability of commercially available natural rubber latex. The MST value of the concentrated saponified natural rubber latex as goods should be equal to or higher than that of natural rubber latex. The selection of the kind and amount of surfactant is very important. The deproteinized natural rubber latex, which is produced by the above method of the present invention, is characterized by the fact that it contains substantially no proteins specified by the bands of 14, 31 and 4 5 kDa as analyzed by SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis). In this regard, the natural rubber latex is different from the conventionally known natural rubber latex with reduced nitrogen content. Here, “it contains substantially no specified proteins in the natural rubber latex” means as follows: The aqueous SDS (Sodium dodecyl sulfate) extracted from the natural rubber latex is dialyzed with a membrane having cut-off molecular weight of 3.5 kDa. The dialyzed solution is treated with acetone including 10% trichloroacetic acid to precipitate the proteins. The precipitate is collected by centrifugation. After washing with acetone, the precipitate is dissolved into aqueous urea solution to make the extract solution corresponding to 6 times concentration. The analysis for the extract solution by SDS-PAGE shows no protein band. That is, it has been confirmed by the analysis with SDS-PAGE that the natural rubber latex having reduced nitrogen content of 0.02% or less which has been produced by the conventional method, using a surfactant and a proteolytic enzyme, still shows these bands indicating the presence of residual specific proteins. More specifically, when the natural rubber latex of the present invention is compared with the natural rubber latex obtained by the above conventional method on the same level of nitrogen content, it has been disclosed that the bands of 14, 31 and 45 kDa substantially or completely disappeared in the natural rubber latex of the present invention, as analyzed by SDS-PAGE. On the other hand, the above bands, though in very small amounts, are still present in the natural rubber latex obtained by the above conventional method. Further, when the natural rubber latex treated with a proteolytic enzyme by the above conventional method is subjected to centrifugation, the bands of proteins peculiar to natural rubber latex are clearly found in its serum phase, ensuring that undecomposed proteins remain in the latex. Meanwhile, when the natural rubber latex treated by the method of the present invention is subjected to centrifugation, no such bands are found in its serum phase. Hence, it is easily confirmed that the coagulated natural rubber latex after the reactions is free from these proteins. Further, it is confirmed from studies by the present Inventors that the natural rubber latex which is substantially free from the bands of 14, 31 and 45 kDa, when analyzed by SDS-PAGE, can provide rubber products such as catheter, rubber glove and condom, which can be used safely on patients having Type I allergy even if the natural rubber contains a slightly large amount of nitrogen. It is disclosed that the reason why the natural rubber latex of the present invention as described above is different from the conventional natural rubber latex deproteinized by using a proteolytic enzyme is as follows. The conventional enzymatic treatment of natural rubber latex can reduce the nitrogen content by selective decomposition of some of the amide bonds in the proteins and rubbers by the proteolytic enzyme. On the other hand, the deproteinization by saponification using an alkaline hydroxide according to the present invention hydrolyzes the amide bonds in the proteins and rubbers decomposes unselectively and stoichiometrically, which results in the formation of low molecular weight peptides. Therefore, the natural rubber latex of the present invention is characterized in that it is not limited by the content of remaining nitrogen and is distinguished by substantially free from the proteins showing the bands of 14, 31 and 45 kDa when analyzed by SDS-PAGE in the natural rubber latex. Further, it was found that the natural rubber latex of the present invention was able to produce high quality rubber gloves of soft and comfortable. The properties of these gloves are comparable to those of gloves made of fresh natural rubber latex. It is remarkable that the rubber glove made of the conventional enzymatically deproteinized natural rubber latex shows a striped pattern as shown in Figure 5, which is undesirable from the viewpoint of quality control, whereas the saponified natural rubber latex of the present invention is excellent for making rubber products from this viewpoint. Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention shall not be limited by these Examples in any way. Examples Example 1 To 1,9 L of fresh latex (abbreviated as FL-latex) adjusted to a DRC of 30%, 100 mL of aqueous solution of NaOH containing 3 0g and 4 g of Emulgen-7 0 (Polyoxyethylene nonylphenyl ether) as a nonionic surfactant were added, and the saponification reaction was carried out at 70°C for 3 hours. The resulting latex was centrifuged at 13,000 rpm for 8 min to separate a cream fraction. The cream fraction was adjusted to 60% DRC by adding water and 0.5 g ammonium laurate was added. The saponified natural rubber latex obtained in this way was subjected to the analysis of proteins in the cream and serum fraction by using SDS-PAGE. The proteins in the cream fraction of the saponified natural rubber latex were analyzed as follows: The cream fraction 17 g was extracted by use of 2% (w/v) aqueous SDS (Sodium Dodecyl Sulfate) 17 mL at room temperature for 24 hr. The extract was dialyzed by use of a membrane having a cutoff molecular weight of 3.5 kDa for 24 hr at a room temperature under stirring in cold water. To 300 μL of this solution, 100 μL of acetone containing 10% trichloroacetic acid was added to precipitate the proteins. The precipitate was collected by centrifugation, washed with acetone, and then dissolved in 50 μL of 8 H urea aqueous solution so as to obtain an extract corresponding to six-fold concentration. This extract was subjected to SDS-PAGE measurement. The analysis of proteins in the serum fraction was carried out in a similar way. The result was given in Figure 1. In Figure 1, Lane 1 denotes standard molecular weight markers, Lane 2 corresponds to the cream fraction, and Lane 3 corresponds to the serum fraction. For comparison, the result of measurement by SDS-PAGE of the cream and serum fraction obtained from centrifugation of fresh natural rubber latex in the same manner as above was given in Figure 2, Further, comparative experiments were done for the deproteinized natural rubber latex. Fresh natural rubber latex with a DRC of 10% was deproteinized with Alcalase 2.0T (NOVO Nordisk Bioindustry Co.) as a proteolytic enzyme in the presence of 1.0% SDS at room temperature for 24 hr. The resulting deproteinized latex was concentrated to 60% DRC by centrifugation at 15,000 rpm. The concentrated deproteinized natural rubber latex was washed by repeating the centrifugation twice in a similar way. The cream fractions were separated form this deproteinized natural rubber latex and subjected to SDS-PAGE analysis. The result of SDS-PAGE measurement is shown in Figure 3. In Figure 1, Lane 2 (cream fraction) showed no bands characteristic of the proteins in the natural rubber latex (cf. Lane 3 in Figure 2). It was revealed that the cream fraction of this saponified natural rubber latex contained no proteins showing the bands of 14, 31 and 45 kDa by SDS-PAGE. Furthermore, Lane 3 in Figure 1 (the serum fraction) showed a broad band in the low molecular weight region in SDS-PAGE. This can be interpreted that the proteins in natural rubber latex are hydrolyzed by sodium hydroxide according to saponification to be decomposed to low molecular weight proteins and eluted in the serum fraction. In Figure 3, the cream fraction in the deproteinized natural rubber latex by proteolytic enzyme showed a slight band at 31 and 4 5 kDa. Examples 2 to 4 These Examples were given for changing the saponification conditions of natural rubber latex. The saponification conditions are shown in Table 1. Experiments were carried out in a similar way as Example, except for the saponification conditions. The cream fraction of these saponified natural rubber latices showed no band corresponding to 14, 31 and 45 kDa in SDS-PAGE measurement. In Table 1, the saponification conditions indicate the concentration of NaOH (w/v %), reaction temperature and reaction time. Figure 4 shows the result of SDS-PAGE analysis of the cream fraction of the resulting latices by using the conditions of the measurement mentioned in Example 1. No band was observed at 14, 31 and 4 5 kDa in these measurements of Examples 2, 3 and 4. This clearly indicated that these proteins were perfectly removed under these saponification conditions. In Figure 4, Lane 1 denotes SDS-PAGE of standard molecular weight markers and Lanes 2, 3, and 4 correspond to the cream fraction of the saponified natural rubber latex of Examples 2, 3 and 4, respectively. Examples 5 and 6 Saponification reactions were conducted in the same manner of Example 1, except for the use of surfactants instead of Emulgen-70 as given in Table 2. The result showed that all the saponified 1atices in these Examples contained no protein corresponding to the bands at 14, 31 and 45 kDa in SDS-PAGE. Table 2 Example 7 The deproteined natural rubber latex by saponificaion (SAP-NR) was subjected to the allergy tests to check whether it contained Type I acute allergy antigens. The experiments were conducted by the analysis of proteins of the saponified natural rubber latex with enzyme-linked immunosorbent assay (ELISA) using FIT Kit of FIT BIOTECH CO., LTD. The results are shown in Table 3. As a comparison, fresh natural rubber latex (FL-latex) was analyzed in a similar condition. Table 3 (Hev bl (MW 14.5 kDa), Hev b3 (MW 22.3 kDa), Hev b5 (MW 17.5kDa), and Hev b6.02 (MW 4.7 kDa) are proteins called Rubber elongation factor, Small rubber particle protein, Acidic latex protein, and Mature Hevein, respectively) No protein was detected in deproteined natural rubber latex by saponification. Thus, it was confirmed that the said latex was free from the possibility of causing allergy. Example 8 Natural rubber latex is traded on the market as latex. The stability of commercial latex during preservation is an important factor. The stability was checked for the saponified natural rubber latex of the present invention. Emal E 70-C or Emulgen-70 was used as a surfactant for the saponification of natural rubber latex. Saponification was carried out by adding 30 g of NaOH in 100 mL aqueous solution and 4 g of the surfactant mentioned above to 1.9L of fresh latex (FL-latex) with a DRC of 30% at 70°C for 3 hr. The resulting latex was centrifuged at 13,000 rpm for 8 min to separate cream fraction. The separated cream fraction was adjusted to 60% DRC by adding water and 0.5 g ammonium laurate and 12 g aqueous ammonia (2 8% w/v) were added to the latex. The Zeta potential (mV) of the resulting latex was -44 mV for both Emal E 70-C and Emulgen-70. The Zeta potential (mV) of natural rubber latex was -4 6 mV. This indicated that the saponified natural rubber latex of the present invention has almost i the same colloidal stability as natural rubber latex. The MST (Mechanical Stability Test: ASTM D1076-97) value was 1,230 sec and 657 sec for Emal E 70-C and Emulgen x-70, respectively, after preservation- of the latex at room temperature for 13 days. Under the same conditions, concentrated high ammonia natural rubber latex showed the MST value of 520 sec. This indicated that the saponified natural rubber latex of the present invention shows the colloidal stability higher than that of ordinary natural lubber latex. Example 9 The physical properties of vulcanized rubber film obtained from the saponified natural rubber latex are indicated. The physical properties of the film prepared from the saponified natural rubber latex (F-l) and the film prepared from those compounded according to the composition given in Table 4 (F-2) were analyzed. The saponified natural rubber latex was prepared in a similar way as Example 1. The film F-l was prepared by casting the saponified natural rubber latex on a glass plate. As for the film F-2 the compounded latex with the composition given in Table 4 was precured for 2 days at room temperature. The compounded latex was cast on a glass plate and kept for two days to make a thin film. The dried film was cured by heating at 120°C for 15 min. The physical properties of the films F-l and F-2 are tabulated in Table 5 and Table 6, respectively. Table 4 Antioxidant; Wingstay L Example 10 Rubber gloves were prepared by using the saponified natural rubber latex. The compounded latex (F-2) in Example 9 was precured for 2 days. A mold of glove, which was dried at 10 0°C after dipping in aqueous coagulant solution (calcium nitrate) for 20 sec, was dipped in the precured latex for 25 sec, taken out and post cured by heating at 120°C for 30 min. After washing with water for 3 0 to 60 sec, the glove was removed from the mold. Figure 5-1 shows the photograph of resulting rubber glove. For comparison, a rubber glove was prepared in a similar way by the use of the deproteinized natural rubber latex with a proteolytic enzyme and took a photograph as shown in Figure 5-2. It showed a clear stripe pattern. CLAIMS 1. Natural rubber latex which contains substantially no proteins specified by the bands of 14, 31 and 45 kDa by SDS-PAGE. 2. The natural rubber latex of claim 1, in which the natural rubber has a nitrogen content of 0.02 to 0.3% by weight based on said natural rubber. 3. A method for preparing the natural rubber latex of claim 1, which comprises saponifying natural rubber latex with alkaline hydroxide in the presence of a surfactant. 4. A rubber glove, catheter, condom or foam rubber made from the natural rubber latex of claim 1.

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3489-CHENP-2006 EXAMINATION REPORT REPLY RECEIVED 24-09-2012.pdf

3489-CHENP-2006 AMENDED PAGES OF SPECIFICATION 22-12-2011.pdf

3489-CHENP-2006 AMENDED CLAIMS 22-12-2011.pdf

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3489-CHENP-2006 EXAMINATION REPORT REPLY RECEIVED 10-10-2012.pdf

3489-CHENP-2006 EXAMINATION REPORT REPLY RECEIVED 20-09-2012.pdf

3489-CHENP-2006 FORM-1 20-11-2012.pdf

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3489-CHENP-2006 POWER OF ATTORNEY 22-12-2011.pdf

3489-CHENP-2006 AMENDED CLAIMS 20-11-2012.pdf

3489-CHENP-2006 AMENDED PAGES OF SPECIFICATION 20-11-2012.pdf

3489-CHENP-2006 CORRESPONDENCE OTHERS 28-10-2011.pdf

3489-CHENP-2006 EXAMINATION REPORT REPLY RECEIVED 22-12-2011.pdf

3489-CHENP-2006 FORM-1 22-12-2011.pdf

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