Exiguobacterium aurantiacum and application of microbial inoculum thereof in production of high-performance tire rubber

Abstract

The invention discloses an application of Exiguobacterium aurantiacum and a microbial inoculum thereof in production of high-performance tire rubber, and belongs to the field of primary processing of natural rubber. The active components of the Exiguobacterium aurantiacum microbial inoculum are Exiguobacterium aurantiacum active thalli and protease secreted in the culture process, the Exiguobacterium aurantiacum microbial inoculum can degrade rubber protein, the microbial inoculum is not limited by the ammonia content in latex when being applied to solidification of natural rubber latex, the Exiguobacterium aurantiacum continues to grow and secrete protease, and the rubber protein can be degraded. The production process is simplified, the production cost is reduced, and the production efficiency and the flexibility are remarkably improved; the tensile strength of a natural rubber product produced by using the microbial agent as a coagulant is greater than or equal to 22.0 MPa, the nitrogen content is 0.15-0.25% (w / w), and the method improves the performance of rubber, reduces the introduction of impurities, and has a wide application prospect in the production of natural rubber raw rubber.

Description

Technical Field

[0001] The invention specifically relates to application of Exiguobacterium aureum and its bacterial agent in the production of high-performance tire rubber, and belongs to the field of primary processing of natural rubber. Background Art

[0002] As an important industrial raw material, natural rubber occupies an irreplaceable position in tire manufacturing and other rubber products industries. Its excellent elasticity, wear resistance and anti-aging properties make it a key factor in improving product performance. Fresh natural latex contains about 30% rubber hydrocarbons, about 60% water and about 6% non-rubber components. The protein content in the non-rubber components is the highest, accounting for 1-2% of the entire latex system. The main chemical structure of rubber hydrocarbons in fresh latex is cis-polyisoprene. Natural rubber is collected from rubber trees. Fresh latex is coagulated by acid or naturally coagulated, and then the coagulation is dehydrated and dried to make raw rubber products. The processing technology will have an important impact on the comprehensive performance of natural rubber. In the process of collecting fresh latex, in order to maintain its freshness and prevent microbial contamination, farmers usually add ammonia water as a preservative and antibacterial agent to obtain fresh latex raw materials with varying ammonia content. Although this practice effectively extends the shelf life of latex, it also introduces the problem of high ammonia content, which puts higher requirements on the subsequent rubber production process.

[0003] The coagulation methods of natural rubber latex can be divided into three categories:

[0004] (1) Natural coagulation of latex: After the latex flows out of the rubber tree, it is placed in the complex environment of the rubber plantation. The bacteria in the environment grow in the latex and continuously produce organic acids, which reduces the pH value of the latex. Some bacteria secrete proteases, which act on the protein layer of the rubber particles in the latex, destroying the adsorption layer on the surface of the rubber particles, causing the rubber particles to lose stability and stick to each other. The above two factors work together to cause the latex to coagulate naturally. The main disadvantages of preparing natural rubber products by the natural coagulation method are long coagulation cycle, severe odor of the product, and serious wastewater pollution.

[0005] (2) Chemical coagulation method: adding chemical coagulants such as acid and salt. Formic acid coagulation method is the main coagulation method currently used for standard rubber in China. However, this method has the disadvantages of formic acid and acetic acid being highly irritating to operators and serious corrosion of equipment. At present, the standard natural rubber products produced by the formic acid coagulation method have a tensile strength of 14.0-20.0MPa in pure rubber formula, a Mooney viscosity of 60-70ML (1+4) 100°C, a plastic initial value P0 between 30 and 40, and a plastic retention index PRI between 75 and 85%. The above standard rubber products are difficult to be used in the production of high-performance aviation tires and high-quality all-steel radial tire products.

[0006] (3) Biocoagulation method: The rubber latex is coagulated by the action of the original or added microorganisms or enzymes. There are three main methods for producing natural rubber products using the biocoagulation method:

[0007] 1) Adding carbohydrates to latex, the naturally existing microorganisms in the latex decompose and utilize the carbohydrates to produce acid, which lowers the pH value of the latex and reaches the isoelectric point of the rubber protein, causing the latex to coagulate.

[0008] CN100347198C discloses a process of mixing and coagulating natural rubber latex with brown sugar fermentation solution and burnt glutinous rice flour; Patent CN1223611C discloses a method of coagulating raw latex with biological assistance, using white sugar as an additive to coagulate latex through biological fermentation; CN1223611C discloses a method of coagulating natural rubber latex with biological assistance. It adds a coagulant coagulant of 0.1% to 0.3% sodium alkylbenzene sulfonate and 0.6% to 0.8% white sugar to natural rubber latex, claiming that it can produce radial tire rubber and aviation tire rubber. Sodium alkylbenzene sulfonate is an anionic surfactant that can improve the stability of natural rubber latex; white sugar is a carbon source that can promote the growth of microorganisms, but in this patent, the so-called biological assistance is that the added white sugar promotes the growth of microorganisms naturally present in natural rubber latex to achieve the purpose of latex coagulation. It can indeed promote the coagulation of natural rubber latex, but it is impossible to produce products that meet the technical requirements of radial tire rubber and aircraft tire rubber;

[0009] 2) Add proteases from microorganisms (alkaline proteases, lysozymes, etc.) or plant sources (papain, bromelain, etc.) to the latex. The proteases act on the protein layer of the rubber particles in the latex, destroying the adsorption layer on the surface of the rubber particles, causing the rubber particles to lose stability and stick to each other and coagulate.

[0010] Patents that use this principle to coagulate natural rubber include: CN1077895C uses protease and starch as coagulants and hydroxylamine sulfate as a viscosity regulator to coagulate latex to produce radial tire rubber. In this production process, the additionally added chemical hydroxylamine sulfate can improve the constant viscosity of natural rubber in some cases, but it has potential health and environmental risks due to its strong acidity and strong corrosiveness; CN102002119B uses the characteristic of protease decomposing protein to destroy the stability of latex to coagulate fresh natural rubber latex. The initial value of the plasticity of the natural rubber obtained is low, and the tensile strength of the product is not listed. degree; CN111718922A discloses a rubber-specific enzyme composed of enzyme preparation 1 (composed of 50% protease and 50% papain) and enzyme preparation 2 (composed of 50% acid protease and 50% neutral protease) to coagulate natural rubber latex, but the performance of the product produced by this method is unknown; CN1222528A discloses a process for producing radial tire rubber using plant protease or microbial protease as a biological coagulant, and the tensile strength of the produced natural rubber raw rubber is between 19.4 and 21.0 MPa, which still does not meet the technical requirements of aviation tire tread rubber.

[0011] 3) Artificially cultured microorganisms are inoculated into latex to decompose carbohydrates in the latex to produce acid, thereby lowering the pH value of the latex and reaching the isoelectric point of the rubber protein, causing the latex to coagulate.

[0012] CN104497174B discloses a method for coagulating natural rubber latex using a combination of Lactobacillus bulgaricus and Bacillus natto, but the combination of microorganisms needs to coagulate the latex under low ammonia conditions with an ammonia content of less than 0.1%; CN100482690C and CN114230690A use a culture solution of fungus (Trichoriella ornithopoda Oorschotet de hoog) mixed with calcium chloride to coagulate fresh natural rubber latex to produce high-performance natural rubber, but the coagulation process in the patent requires the assistance of calcium chloride. Calcium chloride is a salt commonly used in divalent metal salts for coagulating natural rubber latex, and has the effect of rapidly coagulating latex. However, this coagulant will increase the ash content of rubber, limiting the application of natural rubber. CN105936882A and CN105968234A disclose a method for producing natural rubber raw rubber by coagulating natural latex with a microbial flora composed of a symbiotic flora and coagulant bacteria. The symbiotic microbial flora mainly includes photosynthetic bacteria, acetic acid bacteria, lactic acid bacteria, yeast and actinomycetes, and the coagulant bacteria is a fungus (Trichoriella ornithopoda Oorschot et de hoog), but does not list the technical indicators of the natural rubber produced by the method; CN103709271A A method for producing high-performance natural rubber, which is to take fresh natural latex under the action of natural microorganisms or add protease or acid to obtain a high-performance natural rubber product, wherein the raw material of this prior art requires fresh latex with less than 0.05% ammonia, and the fresh latex needs to be additionally pretreated, the production process is cumbersome, and is not conducive to the industrial production of rubber;

[0013] Some documents or patents have also adopted a biological coagulation process that simultaneously adds carbohydrates, microorganisms and / or proteases. For example, Deng Dayu et al. studied the effects of using enzyme-assisted laboratory-made microbial coagulation solution and alkaline protease to coagulate fresh latex on the physical and chemical indicators of natural rubber. Their biological coagulation process requires the joint participation of proteases and microorganisms, but does not specify which microorganisms are used in the experiment. Moreover, the performance of the prepared product mainly depends on the amount of enzyme added (Effect of Enzyme-Assisted Microbial Coagulation on the Performance of Natural Rubber Elastomers, 2023, 33(1): 1-4.); CN102093492B uses a microbial culture solution with acetic acid bacteria, yeast and lactic acid bacteria as the main flora and additional ethanol, acetic acid and Sugar source is added to rubber latex to promote the coagulation of latex particles, and the pH value of microbial culture solution is required to be less than 4, and the technical indicators of biological coagulation of natural rubber are not listed; CN117384314A uses a coagulant obtained by mixing protease, active bacteria (Bifidobacterium or Lactobacillus) and sugar to prepare a high-performance domestic aviation tire tread rubber with high strength, wear resistance and heat resistance. The production process requires the addition of multiple substances and additional sugar, and the process is cumbersome; at the same time, the prior art also mentions that the process has certain requirements for the raw materials used, preferably natural latex without gel and with an ammonia content of less than 0.05wt%; and the increase of lactic acid bacteria in the latex before coagulation will reduce the PRI (Microbial CN102504050A discloses a method for producing high-elasticity and shock-absorbing natural rubber, which comprises sequentially adding ammonium laurate, alkali, protease, microbial agent and sugar to fresh natural latex for biological coagulation to produce natural rubber with high elasticity and mechanical strength, wherein the microbial agent is a culture fluid obtained by artificially culturing a variety of naturally existing microorganisms in whey wastewater after coagulation of natural latex, and the ammonia content in the raw material fresh latex used in this prior art is limited to 0-0.09%, and the ammonia content in the fresh latex needs to be additionally pretreated.

[0014] At present, high-end products such as aviation and radial automobile tires have put forward new requirements for the comprehensive performance of natural rubber, but the technical indicators of domestic standard natural rubber are poor, and high-performance natural rubber is almost completely dependent on imports. High-performance natural rubber products are in urgent need of localization, which poses new challenges to the domestic natural rubber processing technology. When the rubber production system in the prior art processes the raw material fresh latex with high ammonia content, it is often necessary to pre-treat the raw material to adjust its temperature, pH value and ammonia content to a suitable production range. These pre-treatment steps not only increase production costs, but also limit production efficiency and flexibility, which is not conducive to the industrial production and wide application of natural rubber. Summary of the invention

[0015] In view of the current situation mentioned in the background technology, in order to solve the above technical problems, the present invention provides a process for producing high-performance natural rubber using Exiguobacterium aurantium or its bacterial agent.

[0016] Exiguobacterium aurantiacum is a type of Gram-positive, non-spore-forming, facultative anaerobic bacteria. Exiguobacterium aurantiacum CGMCC 1.6137 can grow and secrete enzymes such as proteases under a wide range of temperature and pH conditions. When used in the coagulation of fresh latex, it is not limited by the ammonia content in the latex. It can be directly produced in the outdoor natural environment using fresh latex raw materials with high ammonia content that have not been specially treated, simplifying the production process, reducing production costs, and significantly improving production efficiency and flexibility. The Exiguobacterium aurantiacum inoculant contains Exiguobacterium aurantiacum and the protease it produces. The Exiguobacterium aurantiacum contained in the inoculant will continue to grow using the maltodextrin and nitrogen source in the culture medium, and continuously secrete proteases. During the coagulation process of natural raw rubber, it improves the mechanical properties of raw rubber by degrading some proteins, providing a solution to solve the poor mechanical properties of natural rubber in my country and realize the localization of special rubber for high-end products.

[0017] The first object of the present invention is to provide a method for producing high-performance natural rubber, wherein the method uses Exiguobacterium aurantium CGMCC 1.6137 or a bacterial agent containing Exiguobacterium aurantium CGMCC 1.6137 and its metabolites as a biological coagulant to produce natural rubber.

[0018] In one embodiment of the present invention, the high performance refers to at least one of the following:

[0019] Nitrogen content ≤0.6%; initial plasticity value ≥36; plasticity retention rate ≥70%; 73≤Mooney viscosity ≤93; tensile strength ≥22.0Mpa; elongation at break ≥800%.

[0020] In one embodiment of the present invention, the method is specifically: mixing the filtered fresh latex and the bacterial agent of Exiguobacterium aureum CGMCC 1.6137 in parallel into a coagulation tank, standing for coagulation, and producing high-performance natural rubber according to the natural rubber production process after the curd is matured.

[0021] In one embodiment of the present invention, the filtered fresh latex is obtained by filtering through a 35-45 mesh sieve to remove impurities.

[0022] In one embodiment of the present invention, the dry rubber content in the fresh latex is 20-25% (w / w), and the pH value is 6.5-7.5.

[0023] In one embodiment of the present invention, the added amount of the bacterial agent is 0.05% to 0.10% (w / w) of the dry rubber content.

[0024] In one embodiment of the present invention, the bacterial agent needs to be diluted or dissolved with 8 to 10 times the weight of water.

[0025] In one embodiment of the present invention, the standing time is 2 to 8 hours.

[0026] In one embodiment of the present invention, the aging time is 14 to 24 hours.

[0027] In one embodiment of the present invention, the natural rubber production process comprises the steps of thinning and dehydrating, washing, tableting, granulating and drying the matured coagulated mass.

[0028] In one embodiment of the present invention, the drying condition is 110-115° C. for 8-12 hours.

[0029] In one embodiment of the present invention, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0030] In one embodiment of the present invention, the viable count of Exiguobacterium aureum CGMCC 1.6137 in the bacterial agent is 10 7 ~10 9 CFU / mL.

[0031] In one embodiment of the present invention, the bacterial concentration of Exiguobacterium aurantium CGMCC 1.6137 in the bacterial agent is in the range of 10 7 ~10 9 CFU / mL.

[0032] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Exiguobacterium aurantiacum CGMCC 1.6137 and then expanding the culture.

[0033] In one embodiment of the present invention, the bacterial agent contains active bacteria of Exiguobacterium aurantiacum CGMCC 1.6137 and protease secreted by Exiguobacterium aurantiacum CGMCC 1.6137.

[0034] In one embodiment of the present invention, the bacterial agent is a powder obtained by drying a bacterial liquid containing Exiguobacterium aurantiacum CGMCC 1.6137.

[0035] In one embodiment of the present invention, the drying refers to spray drying, freeze drying, vacuum drying or other bacterial liquid drying processes.

[0036] In one embodiment of the present invention, the liquid bacterial agent is obtained by the following method:

[0037] The seed liquid of Exiguobacterium aurantium CGMCC 1.6137 was inoculated into a liquid fermentation medium to obtain a fermentation liquid, and the fermentation liquid was concentrated to obtain a liquid bacterial agent.

[0038] In one embodiment of the present invention, the seed solution inoculation amount is 5-10% (v / v); preferably, the inoculation amount is 8%.

[0039] In one embodiment of the present invention, the culture conditions are: temperature 25-28°C, stirring speed 180-200r / min, fermentation time 24-36h, fermentation tank pressure 0.05-0.08Mpa, fermentation liquid ventilation ratio (v / v.min) is 1:(0.20-0.30); preferably, the culture conditions are: temperature 28°C, stirring speed 200r / min, fermentation time 36h, fermentation tank pressure 0.06Mpa, fermentation liquid ventilation ratio (v / v.min) is 1:0.25.

[0040] In one embodiment of the present invention, maltodextrin accounts for 1.5-3.5% (w / v) in the liquid fermentation medium, and the remainder is whey; preferably, the maltodextrin accounts for 2.0-3.0% (w / v); more preferably, the maltodextrin accounts for 2% (w / v).

[0041] In one embodiment of the present invention, the whey is wastewater after natural rubber latex is coagulated.

[0042] In one embodiment of the present invention, the specific steps of the concentration are: ultrafiltration at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 2500 to 3500 at 0.1 to 0.3 MPa to obtain a concentrated fermentation broth that is concentrated 8 to 10 times;

[0043] Preferably, ultrafiltration is performed at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 3000 at 0.3 MPa to obtain a concentrated fermentation broth that is concentrated 8 times.

[0044] In one embodiment of the present invention, the solid bacterial agent is obtained by the following method:

[0045] The liquid bacterial agent is added with maltodextrin and then dried to obtain a solid bacterial agent containing Exiguobacterium aurantium CGMCC 1.6137 and the protease secreted by it.

[0046] In one embodiment of the present invention, the added amount of maltodextrin is 220-240 g / L.

[0047] In one embodiment of the present invention, the drying method is spray drying; the conditions of the spray drying are: the air outlet temperature is 55±2°C, and the feed rate is 10-60 mL / min.

[0048] In one embodiment of the present invention, the protease activity in the solid bacterial agent is 500-1000 U / g, and the viable bacteria count of Exiguobacterium aurantium is (0.5-1.0)×10 8 CFU / g.

[0049] The second object of the present invention is to provide a method for producing natural rubber with high tensile strength, wherein the method uses Exiguobacterium aurantium CGMCC 1.6137 or a bacterial agent containing Exiguobacterium aurantium CGMCC 1.6137 as a biological coagulant to produce natural rubber.

[0050] In one embodiment of the present invention, the high tensile strength refers to a tensile strength ≥ 22.0 MPa.

[0051] In one embodiment of the present invention, the method is specifically: mixing the filtered fresh latex and the bacterial agent of Exiguobacterium aureum CGMCC 1.6137 in parallel into a coagulation tank, standing for coagulation, and producing high-performance natural rubber according to the natural rubber production process after the curd is matured.

[0052] In one embodiment of the present invention, the filtered fresh latex is obtained by filtering through a 35-45 mesh sieve to remove impurities.

[0053] In one embodiment of the present invention, the dry rubber content in the fresh latex is 20-25% (w / w), and the pH value is 6.5-7.5.

[0054] In one embodiment of the present invention, the added amount of the bacterial agent is 0.05% to 0.10% (w / w) of the dry rubber content.

[0055] In one embodiment of the present invention, the bacterial agent needs to be diluted or dissolved with 8 to 10 times the weight of water.

[0056] In one embodiment of the present invention, the standing time is 2 to 8 hours.

[0057] In one embodiment of the present invention, the aging time is 14 to 24 hours.

[0058] In one embodiment of the present invention, the natural rubber production process comprises the steps of thinning and dehydrating, washing, tableting, granulating and drying the matured coagulated mass.

[0059] In one embodiment of the present invention, the drying condition is 110-115° C. for 8-12 hours.

[0060] In one embodiment of the present invention, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0061] In one embodiment of the present invention, the viable count of Exiguobacterium aureum CGMCC 1.6137 in the bacterial agent is 10 7 ~10 9 CFU / mL.

[0062] In one embodiment of the present invention, the bacterial concentration of Exiguobacterium aurantium CGMCC 1.6137 in the bacterial agent is in the range of 10 7 ~10 9 CFU / mL.

[0063] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Exiguobacterium aurantiacum CGMCC 1.6137 and then expanding the culture.

[0064] In one embodiment of the present invention, the bacterial agent contains active bacteria of Exiguobacterium aurantiacum CGMCC 1.6137 and protease secreted by Exiguobacterium aurantiacum CGMCC 1.6137.

[0065] In one embodiment of the present invention, the bacterial agent is a powder obtained by drying a bacterial liquid containing Exiguobacterium aurantiacum CGMCC 1.6137.

[0066] In one embodiment of the present invention, the drying refers to spray drying, freeze drying, vacuum drying or other bacterial liquid drying processes.

[0067] In one embodiment of the present invention, the liquid bacterial agent is obtained by the following method:

[0068] The seed liquid of Exiguobacterium aurantium CGMCC 1.6137 was inoculated into a liquid fermentation medium to obtain a fermentation liquid, and the fermentation liquid was concentrated to obtain a liquid bacterial agent.

[0069] In one embodiment of the present invention, the seed solution inoculation amount is 5-10% (v / v); preferably, the inoculation amount is 8%.

[0070] In one embodiment of the present invention, the culture conditions are: temperature 25-28°C, stirring speed 180-200r / min, fermentation time 24-36h, fermentation tank pressure 0.05-0.08Mpa, fermentation liquid ventilation ratio (v / v.min) is 1:(0.20-0.30); preferably, the culture conditions are: temperature 28°C, stirring speed 200r / min, fermentation time 36h, fermentation tank pressure 0.06Mpa, fermentation liquid ventilation ratio (v / v.min) is 1:0.25;

[0071] In one embodiment of the present invention, maltodextrin accounts for 1.5-3.5% (w / v) in the liquid fermentation medium, and the remainder is whey; preferably, the maltodextrin accounts for 2.0-3.0% (w / v); more preferably, the maltodextrin accounts for 2% (w / v).

[0072] In one embodiment of the present invention, the whey is wastewater after natural rubber latex is coagulated.

[0073] In one embodiment of the present invention, the specific steps of the concentration are: ultrafiltration at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 2500 to 3500 at 0.1 to 0.3 MPa to obtain a concentrated fermentation broth that is concentrated 8 to 10 times;

[0074] Preferably, ultrafiltration is performed at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 3000 at 0.3 MPa to obtain a 10-fold concentrated fermentation broth.

[0075] In one embodiment of the present invention, the solid bacterial agent is obtained by the following method:

[0076] The liquid bacterial agent is added with maltodextrin and then dried to obtain a solid bacterial agent containing Exiguobacterium aurantium CGMCC 1.6137 and the protease secreted by it.

[0077] In one embodiment of the present invention, the added amount of maltodextrin is 220-240 g / L.

[0078] In one embodiment of the present invention, the drying method is spray drying; the conditions of the spray drying are: the air outlet temperature is 55±2°C, and the feed rate is 10-60 mL / min.

[0079] In one embodiment of the present invention, the protease activity in the solid bacterial agent is 500-1000 U / g, and the viable bacteria count of Exiguobacterium aurantium is (0.5-1.0)×10 8CFU / g.

[0080] The third object of the present invention is to provide a high-performance tire rubber, which is prepared by the above method.

[0081] Beneficial effects:

[0082] The invention provides an application of a strain of Exiguobacterium aureum CGMCC 1.6137 and a bacterial agent prepared therefrom in the production of high-performance tire rubber. The active components of the bacterial agent are active bacterial bodies of Exiguobacterium aureum and proteases secreted during the cultivation process, which can degrade rubber protein and have excellent application effects in high-performance natural rubber.

[0083] (1) After the bacterial agent is added to the natural rubber latex as a biological coagulant instead of chemical coagulants such as formic acid, the protease in the bacterial agent quickly degrades the protein in the latex, destroying the stability of the latex; at the same time, the golden orange yellow Exiguobacterium contained in the bacterial agent continues to grow using the maltodextrin and nitrogen source in the culture medium, and continuously secretes protease; the natural rubber latex coagulates into blocks within 2 to 8 hours, and after 12 to 18 hours of maturation, the milk is turbid and not white, and the rubber hydrocarbon has been completely coagulated;

[0084] (2) Exiguobacterium aurantium CGMCC 1.6137 can grow and reproduce in a wide pH range, and is not limited by the ammonia content in the latex when used for coagulation of natural latex, and absorbs ammonia as an inorganic nitrogen source during the growth process. That is, the coagulation of latex in the present invention can be carried out under relatively high alkalinity (ammonia content 0-0.15%), without the need to add acid to neutralize ammonia before coagulation; the fresh latex raw material with high ammonia content can be directly used without pretreatment, which simplifies the production process, reduces production costs, and significantly improves production efficiency and flexibility;

[0085] (3) The Exiguobacterium aurantium CGMCC 1.6137 provided by the present invention can stably grow and reproduce in a wide temperature range, so that the production of natural rubber is no longer restricted by region and season and can be carried out under a wider range of environmental conditions;

[0086] (4) The growth of Exiguobacterium aurantium CGMCC 1.6137 has a low demand for nutrients. The nitrogen source it needs comes entirely from the wastewater after the coagulation of natural rubber latex. There is no need to add organic nitrogen sources such as yeast powder and bean cake powder, thus avoiding the problem of these organic nitrogen sources not being fully utilized and increasing the burden on sewage treatment.

[0087] (5) The present invention has experimentally confirmed that the protease secreted by the above-mentioned strain has an excellent degradation effect on natural rubber protein and has great application potential in the production of high-performance natural rubber products;

[0088] (6) The method of the present invention does not require special pretreatment of the raw materials, and does not require the addition of other nutrients or chemical treatment agents, thereby reducing operational risks, reducing the generation and emission of harmful substances, and improving the working environment;

[0089] (7) The product prepared by the method of producing high-performance natural rubber using the bacterial agent of the present invention as a coagulant has a tensile strength of ≥22.0 MPa, a nitrogen content of 0.15-0.25%, and other indicators meet the technical requirements of "GJB 7269-2011 General Specification for Natural Rubber for Aviation Tires" and "NY / T 459-2022 Natural Raw Rubber Radial Tire Rubber", which improves the performance of rubber and reduces the introduction of impurities, and has broad application prospects in the production of natural rubber raw rubber. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 This is a graph showing the effects of different carbon sources on the protease production of Exobacillum aurantium.

[0091] Figure 2 This is the result graph showing the effect of maltodextrin addition on the protease production during the fermentation of Exobacillum aurantium.

[0092] Figure 3 This is the result diagram of the optimal catalytic temperature of protease.

[0093] Figure 4 This is the result diagram of the optimal catalytic pH of the protease. DETAILED DESCRIPTION

[0094] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0095] The main detection methods used in the present invention are:

[0096] (1) Determination of protease activity:

[0097] Tyrosine standard curve: Prepare a tyrosine standard solution with a final concentration of 1 mg / mL using 50 mmol / L Tris-HCl (pH 8.0) as solvent, and dilute to 0, 20, 40, 60, 80, 100 μg / mL tyrosine solutions; take 1 mL of each concentration of tyrosine solution and add 5 mL of 0.4 mol / L Na 2 CO 3The solution was mixed with 1 mL of Folin-phenol reagent, and the color was developed in a 40°C water bath for 20 min. The absorbance was measured at 660 nm, and a standard curve was prepared with the tyrosine solution concentration as the abscissa and the absorbance value as the ordinate.

[0098] Fermentation broth protease activity assay: The fermentation broth was centrifuged at 10000r / min for 10min, 1mL of supernatant was added with 1mL of 2% (w / w) casein solution, and color was developed in a 40℃ water bath for 20min; 2mL of 0.4mol / L trichloroacetic acid was added to terminate the reaction, and the mixture was kept at 40℃ for 10min. After the end of the incubation, the mixture was centrifuged at 10000r / min for 10min. 1mL of supernatant was taken and 5mL of 0.4mol / L Na 2 CO 3 and 1mL Folin-phenol reagent, color development at 40℃ for 20min; using inactivated fermentation broth as control, measure absorbance at 660nm. The enzyme activity unit U is defined as: the amount of enzyme required to release 1μg of tyrosine per minute when hydrolyzing casein per milliliter of enzyme solution under the above conditions.

[0099] (2) The nitrogen content was determined in accordance with the method of GB / T 8088 Determination of nitrogen content of natural rubber and natural rubber latex.

[0100] (3) The initial value of plasticity shall be determined in accordance with the method of GB / T3510 Rapid plasticity test method for determination of plasticity of unvulcanized rubber.

[0101] (4) The plasticity retention rate is determined according to the method of "GB / T3517 Determination of Plasticity Retention Rate (PRI) of Natural Rubber"

[0102] (5) The Mooney viscosity (ML(1+4)100°C) was determined in accordance with the method of GB / T1232.1 Determination of unvulcanized rubber by disc shear viscometer Part 1: Determination of Mooney viscosity.

[0103] (6) The tensile strength and elongation at break shall be tested in accordance with the method of GB / T528 Determination of tensile stress-strain properties of vulcanized rubber or thermoplastic rubber.

[0104] The Exiguobacterium aurantium involved in the present invention is purchased from China General Microbiological Culture Collection Center, with the collection number being CGMCC 1.6137.

[0105] The fresh latex involved in the following embodiments is collected from rubber trees. During the collection process of the fresh latex, the collectors may add ammonia water to obtain a fresh latex raw material with an ammonia content of about 0.05%.

[0106] Example 1: Preparation method of Exiguobacterium aurantium seed solution

[0107] (1) Bacterial Activation

[0108] Taking Exiguobacterium aureum as the starting strain, Exiguobacterium aureum was transferred to a slant to recover the strain; the slant culture medium components were: beef powder 10g / L, peptone 10g / L, sodium chloride 5g / L, yeast extract 3.0g / L, glucose 5g / L, agar 15g / L, pH 7.0.

[0109] (2) Seed culture

[0110] The activated Exobacterium aureum was inoculated into the seed culture medium and cultured at 30°C and 200 r / min for 24 h to serve as the seed solution.

[0111] The components of the liquid seed culture medium are: beef powder 10g / L, peptone 10g / L, sodium chloride 5g / L, yeast extract 3g / L, glucose 5g / L, and the pH is adjusted to 7.0 with 2mol / L sodium hydroxide solution;

[0112] The seed liquid was inoculated into the liquid fermentation medium at an inoculation rate of 8% (v / v), in which maltodextrin accounted for 2.5% (w / v) and the rest was whey, which was the wastewater after the coagulation of natural latex; the fermentation conditions were: temperature 28°C, stirring speed 200r / min, fermentation time 36h; fermentation tank pressure 0.06Mpa, fermentation liquid ventilation ratio (v / v.min) 1:0.25;

[0113] After the fermentation is completed, the obtained product is a fermentation liquid containing live Exiguobacterium aurantium and protease.

[0114] Example 2: Effects of different carbon sources on protease production by Exiguobacterium aurantiacum

[0115] An experimental analysis was conducted on the types of carbon sources in the fermentation medium. The carbon sources included corn starch, glucose, waste molasses, white sugar and maltodextrin, all added in an amount of 2% (w / v). The remaining component of the medium was whey, which was the wastewater after the coagulation of natural latex.

[0116] The specific implementation method is the same as that of Example 1, except that the maltodextrin in the culture medium is replaced with corn starch, glucose, molasses or white sugar.

[0117] After fermentation, the activity of proteases in the fermentation broth with different carbon sources was tested. Figure 1 shown.

[0118] Under the same dosage, the protease activity of the fermentation broth using maltodextrin as the carbon source was the highest, which was 52.5U / L (Table 1).

[0119] Table 1 Effects of different carbon sources on protease production by Exococcobacillus aurantium

[0120] Carbon source Corn starch glucose Molasses white sugar Maltodextrin Protease activity (U / L) 32.1 9.6 24.5 10.1 52.5

[0121] Example 3: Effect of maltodextrin addition on protease production during fermentation of Exiguobacterium aurantium

[0122] After determining that the optimal carbon source is maltodextrin, the inventors further analyzed the effects of different dosages of maltodextrin on the enzyme activity of the fermentation broth. The dosages of maltodextrin were 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% (w / v), respectively.

[0123] The specific implementation is the same as Example 1, except that the added amount of maltodextrin is replaced with 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or 4% (w / v).

[0124] After fermentation, the protease activity in the fermentation broth was tested. Figure 2 shown.

[0125] Different dosages of maltodextrin have different effects on the enzyme activity of the fermentation broth. The appropriate dosage of maltodextrin is 1.5-3.5%, and the optimal dosage range is 2-3%. The protease activity is 51.4-52.3U / L (Table 2).

[0126] Table 2 Effect of maltodextrin addition on protease production during fermentation of Exiguobacterium aurantium

[0127] Maltodextrin dosage 0.5％ 1％ 1.5％ 2％ 2.5％ 3％ 3.5％ 4％ Protease activity (U / L) 31.8 40.6 49.7 52.3 51.4 51.5 45.2 42.1

[0128] Example 4: Preparation of Exiguobacterium aurantium

[0129] The fermentation broth obtained in Example 3 and cultured with 2% maltodextrin was ultrafiltered at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 3000 and a concentration of 10 times was obtained, and the enzyme activity was 515 U / mL, which was a liquid bacterial agent.

[0130] Add maltodextrin as an auxiliary material to the liquid bacterial agent at an addition amount of 230 g / L. The mixed concentrated enzyme solution was spray dried, the dryer outlet air temperature was 55 ± 2 °C, the feed rate was 40 mL / min, and the protease activity was 500 U / g and the number of live bacteria of Exiguobacterium aurantium was 0.8 × 10 8 CFU / g solid bacterial agent, the product is uniform powder and has good dispersion and solubility.

[0131] Example 5: Optimal Action Temperature and Optimal Action pH of Protease in Bacteria Agents

[0132] 1. Screening of the optimal action temperature:

[0133] Using 2% casein as substrate, the concentrated enzyme solution prepared in Example 4 was diluted to an appropriate concentration with pH 8.0 Tris-HCl buffer, and then mixed with the substrate at a ratio of 1:1, and the enzyme activity was determined at pH 8.0. The reaction temperature range was 30-60°C, and one point was measured every 5°C. The highest enzyme activity was taken as 100%, and the relative enzyme activity at other time points was calculated.

[0134] Figure 3 The results showed that the activity of the protease in catalyzing the substrate was the highest at 40°C, which was 52.5 U / L, and the enzyme activity at 30-50°C was 75% or more of the highest enzyme activity (Table 3).

[0135] Table 3 Optimal catalytic temperature of protease

[0136] temperature 30℃ 35℃ 40℃ 45℃ 50℃ 55℃ 60℃ Protease activity (U / L) 39.2 48.1 52.5 47.6 39.2 35.0 11.0 Relative enzyme activity (%) 75 92 100 91 75 67 21

[0137] 2. Screening of the optimal pH:

[0138] Prepare buffer systems with different pH values, Tris-HCl (pH 6.0-9.0), Gly-NaOH (pH 9.0-10.0). Prepare 2% casein with buffer systems with pH 6.0-10.0, and dilute the concentrated enzyme solution mixed in Example 4 to appropriate multiples with different buffer systems, and then measure the activity of the protease at 40°C to determine its optimal pH. Take the highest enzyme activity as 100% to calculate the relative enzyme activity at other pH values.

[0139] Figure 4 The results showed that the activity of the protease in catalyzing the substrate was the highest at pH 8.0, which was 52.5 U / L. The activity of the protease at pH 6.0-9.5 was above 65% of the highest activity (Table 4).

[0140] Table 4 Optimal catalytic pH of proteases

[0141] pH 6 6.5 7 7.5 8 8.5 9 9.5 10 Protease activity (U / L) 35.0 38.2 42.9 47.1 52.5 47.6 40.8 36.6 32.4 Relative enzyme activity (%) 67 73 82 90 100 91 78 70 62

[0142] Example 6: Method for preparing high-performance natural rubber using solid bacterial agent

[0143] 2000 kg of fresh latex filtered through a 40-mesh sieve to remove impurities was taken, and its dry rubber content was determined to be 34.5% (w / w), and the pH value was 6.8. Water was added to adjust the dry rubber concentration to 23% (w / w) to obtain diluted latex; 0.08% (w / w) of the dry rubber weight of the solid bacterial agent obtained in Example 4 was taken, and the bacterial agent was dissolved in 10 times the weight of water in advance;

[0144] The dissolved inoculant and the diluted latex were mixed in parallel and put into the coagulation tank. The latex and inoculant were mixed at an ambient temperature of 32°C for 180 minutes to form flocculation, and the curd without white water was formed after aging for 17 hours. The curd was thinned and dehydrated, washed, sheeted, granulated, and dried at 110°C for 11 hours to obtain a high-performance natural rubber product. The quality indicators and properties of the natural rubber were determined (Table 5).

[0145] Example 7: Method for preparing high-performance natural rubber using liquid bacterial agent

[0146] 5000 kg of fresh latex filtered through a 40-mesh sieve to remove impurities was taken, and its dry rubber content was determined to be 31.8% (w / w), and the pH value was 6.5. Water was added to adjust the dry rubber concentration to 20% (w / w) to obtain diluted latex; 0.10% (w / w) of the dry rubber weight of the liquid bacterial agent obtained in Example 4 was taken, and the bacterial agent was diluted with 10 times the weight of water in advance;

[0147] The diluted inoculant and the diluted latex were mixed and put into the coagulation tank. The latex and the inoculant were mixed at an ambient temperature of 22°C for 205 minutes to form flocculation, and the curd without white water was formed after aging for 16 hours. The curd was thinned and dehydrated, washed, sheeted, granulated, and dried at 115°C for 8 hours to obtain a high-performance natural rubber product. The quality indicators and properties of the natural rubber were determined (Table 5).

[0148] Example 8: Method for preparing high-performance natural rubber using liquid bacterial agent

[0149] The specific implementation method is the same as Example 6, except that the pH value of the latex is 7.5, and the reaction temperature after the bacterial agent and the latex are mixed is 25°C.

[0150] Example 9: Method for preparing high-performance natural rubber using solid bacterial agent

[0151] The specific implementation method is the same as Example 7, except that the pH value of the latex is 8.0, and the reaction temperature after the bacterial agent and the latex are mixed is 30°C.

[0152] Comparative Example 1:

[0153] The dissolved bacterial agent in Example 6 was replaced with 3% formic acid solution, and the other process parameters and steps were the same as those in Example 6 to prepare natural rubber, and the quality indicators and properties of the rubber were measured (Table 5).

[0154] Comparative Example 2:

[0155] (1) The concentrated fermentation broth in Example 4 was centrifuged at 10,000 rpm for 20 min, and the precipitate was collected and spray-dried with maltodextrin as a coagulant (the main component was active bacteria of Exiguobacterium aurantium) according to the method in Example 4, wherein the number of live bacteria of Exiguobacterium aurantium was 0.8×10 8CFU / g;

[0156] (2) The solid bacterial agent in Example 6 was replaced by the coagulant obtained in step (1), and the other process parameters and steps were the same as those in Example 6 to prepare high-performance rubber, and the quality indicators and properties of the rubber were measured (Table 5).

[0157] Comparative Example 3:

[0158] (1) The concentrated fermentation broth in Example 4 was centrifuged at 10,000 rpm for 20 min, and the supernatant (the main component of which was the protease secreted by Exiguobacterium aurantiacum) was collected. The enzyme activity of the supernatant was 515 U / mL;

[0159] (2) The liquid bacterial agent in Example 7 was replaced by the supernatant obtained in step (1), and the other process parameters and steps were the same as in Example 7 to prepare high-performance rubber, and the quality indicators and properties of the rubber were measured (Table 5).

[0160] Comparative Example 4:

[0161] The solid bacterial agent in Example 7 was replaced by commercially available alkaline protease (derived from Bacillus licheniformis, produced by Nanning Pangbo Bioengineering Co., Ltd., 100,000 U / g). The other process parameters and steps were the same as those in Example 7. A natural rubber product was prepared, and the quality indicators and properties of the rubber were determined (Table 5).

[0162] The technical indicators of the natural rubber products in the embodiments and comparative examples are shown in Table 5.

[0163] Table 5 Technical indicators of natural rubber products

[0164]

[0165]

[0166] Tensile strength is one of the important indicators for measuring tire rubber performance. When the tire is impacted by external force or the internal pressure increases, natural rubber with high tensile strength can prevent the tire from rupturing or tearing, thereby avoiding safety accidents caused by tire failure; natural rubber with high tensile strength can better maintain the shape and pattern of the tire tread, thereby increasing the friction between the tire and the ground and enhancing the grip; and natural rubber with high tensile strength has better wear resistance, which can reduce the degree of wear of the tire during long-term use. As shown in Table 5, the tensile strength of the natural rubber (Examples 6 to 9) obtained by using the solid bacterial agent and liquid bacterial agent prepared by the present invention is greater than 22.0Mpa, which is significantly better than the natural rubber obtained in Comparative Examples 1 to 4.

[0167] The nitrogen content in the natural rubber product obtained by using the solid bacterial agent and the liquid bacterial agent prepared by using the present invention is significantly lower than that in Comparative Examples 1 to 3. The aurantium absorbs ammonia in the latex as an inorganic nitrogen source and secretes protease into the fermentation liquid to decompose the protein in the latex, which is more effective than using formic acid as a coagulant or only using protease as a coagulant.

[0168] In addition, the initial plasticity value P0, tensile stress, Mooney viscosity and tensile elongation of the natural rubber obtained in Examples 6 to 9 are significantly better than those of the natural rubber prepared using formic acid in Comparative Example 1; the plasticity retention rate PRI and tensile stress of the natural rubber obtained in Examples 6 to 9 are significantly better than those of the natural rubber prepared using only Exiguobacterium aurantiacum bacteria or only using the protease secreted by Exiguobacterium aurantiacum in Comparative Example 2 and Comparative Example 3, and the natural rubber obtained using commercially available alkaline protease in Example 4.

[0169] The above shows that the bacterial agent prepared by using the Exiguobacterium aurantium of the present invention can effectively improve the rubber performance, and the prepared natural rubber meets the technical standards of NY / T 459-2022 natural raw rubber radial tire rubber and GJB 7269-2011 general specification of natural rubber for aviation tires.

[0170] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for producing high-performance natural rubber, characterized in that: The method uses Exiguobacterium aurantium CGMCC1.6137 or a bacterial agent containing Exiguobacterium aurantium CGMCC 1.6137 and its metabolites as a biological coagulant to produce natural rubber.

2. The method according to claim 1, characterized in that The method specifically comprises the following steps: mixing the filtered fresh latex and the bacterial agent of Exiguobacterium aureum CGMCC 1.6137 in parallel into a coagulation tank, allowing the mixture to stand for coagulation, and producing high-performance natural rubber according to the natural rubber production process after the coagulation is matured.

3. The method according to claim 1 or 2, characterized in that: The added amount of the bacterial agent is 0.05% to 0.10% w / w of the dry glue content.

4. The method according to claim 1 or 2, characterized in that: The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

5. The method according to claim 1 or 2, characterized in that: The bacterial agent contains active bacteria of Exiguobacterium aurantiacum CGMCC1.6137 and protease secreted by Exiguobacterium aurantiacum CGMCC 1.6137.

6. The method according to claim 1 or 2, characterized in that: The bacterial agent is prepared by inoculating the seed liquid of Exiguobacterium aurantium CGMCC1.6137 into a liquid fermentation medium to obtain a fermentation liquid, and concentrating the fermentation liquid to obtain the bacterial agent.

7. The method according to claim 6, characterized in that The liquid fermentation medium contains 1.5-3.5% (w / v) maltodextrin, and the remainder is whey; preferably, the maltodextrin accounts for 2.0-3.0% (w / v); more preferably, the maltodextrin accounts for 2% (w / v); the whey is the wastewater after the coagulation of natural latex.

8. The method according to claim 1 or 2, characterized in that: The natural rubber production process comprises the following steps: thinning and dehydrating the matured coagulated mass, washing, tableting, granulating and drying the mass.

9. A method for producing natural rubber with high tensile strength, characterized in that: The method uses Exiguobacterium aurantium CGMCC 1.6137 or a bacterial agent containing Exiguobacterium aurantium CGMCC 1.6137 and its metabolites as a biological coagulant to produce natural rubber; the high tensile strength refers to a tensile strength of ≥22.0MPa.

10. A high performance tire rubber, characterized in that: The rubber is prepared by the method according to claims 1 to 9.

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