Application of basophilic bacillus and microbial inoculum thereof in production of standard natural rubber No. 10 rubber by taking skim gum as raw material

Abstract

The invention discloses application of basophilic bacillus and a microbial agent thereof in production of standard natural rubber 10 # rubber by taking skim gum as a raw material, and belongs to the field of primary processing of natural rubber. According to the invention, the bacillus alcalophilus CICC 23037 and the microbial inoculum thereof are used for producing standard natural rubber No. 10 rubber by taking skim gum as a raw material; the bacillus alcaliphilus CICC 23037 grows well in the skim skim with high ammonia content, continuously secretes protease to remove rubber protein in the skim skim, simultaneously secretes organic acid, neutralizes ammonia in the skim skim, reduces the pH value of the skim skim and destroys the stability of latex, so that the skim skim is solidified in a range that the pH value is less than or equal to 9.0; the technical indexes of the skim rubber obtained by the method provided by the invention meet the performance requirements of No. 10 rubber in GB / T 8081-2018 natural raw rubber technology graded rubber (TSR) specification guide rule.

Description

Technical Field

[0001] The invention relates to application of alkaliphilic bacillus and bacterial agent thereof in production of standard natural rubber No. 10 with skim latex as raw material, belonging to the technical field of natural rubber processing. Background Art

[0002] Natural rubber is the core industrial raw material in tire manufacturing and other rubber products industries. Natural rubber comes from the fresh latex of rubber trees. The fresh latex undergoes a coagulation process, and then the coagulated block is dehydrated and dried, and finally converted into a raw rubber product. In the collection of fresh latex, in order to ensure its freshness and prevent the invasion of microorganisms, farmers routinely add ammonia water as a preservative and antibacterial agent, and the ammonia content in the fresh latex raw materials obtained is uneven. Skim latex is a by-product of the production of concentrated latex by centrifugation of natural rubber fresh latex. After centrifugation, the larger rubber particles are distributed in the concentrated latex. The particles in the skim latex are small and the content is low. The rubber hydrocarbon content in the skim latex is about 3-7%. After centrifugation, a large amount of non-rubber components enter the skim latex, making the content of non-rubber components (such as protein (content 8-15%), phospholipids and metal ions, etc.) in the skim latex higher than that of fresh latex. These substances enter into skim rubber along with the flocculation of skim rubber; the polyisoprene content in skim rubber is less than 80%, and due to the high protein content, it is easy to mildew during storage, vulcanizes quickly, and is easy to scorch.

[0003] Since the particle size of skim rubber particles is small and the specific surface area of ​​the particles is small, the overall content of non-rubber components adsorbed on the surface is high, and the density of non-rubber components is greater than that of rubber hydrocarbons. Therefore, compared with latex, the particle size of skim rubber particles is small, the Brownian motion is stronger, the probability of rubber particle collision is large, and the stability is poor, but the concentration of its particles is low and flocculation is difficult. In order to speed up the flocculation speed and reduce the cost, sulfuric acid flocculation is commonly used. The skim rubber produced by sulfuric acid flocculation has the disadvantages of high nitrogen content and low PRI. The amount of wastewater during production is large, and because it contains sulfate ions, it is difficult to handle in the later stage, and the environmental protection pressure is high. At the same time, sulfuric acid is highly corrosive, unsafe to operate, and seriously corrodes equipment.

[0004] Ammonia is a preservative for fresh latex. It is easily soluble in water. After centrifugation, it enters the skim latex with the whey phase in large quantities, making the ammonia content in the skim latex higher, above 0.25% (pH>10). When the skim latex is coagulated, acid is usually added to neutralize the ammonia and reduce the pH value to make the rubber particles unstable and demulsify and flocculate. The concentration of rubber particles in the skim latex is low, and it is difficult for a chain reaction to occur. Therefore, the amount of acid required to flocculate the skim latex is more than that of flocculating fresh latex. In addition, the performance of the skim latex rubber itself is poor, and adding excessive acid will also have an adverse effect on its performance and increase the cost.

[0005] At present, there are mainly the following ways of flocculation of skim clear:

[0006] (1) Treat skim latex with protease to improve the performance of skim latex rubber.

[0007] Microbial flocculation uses proteases to decompose proteins on the surface of colloid particles, destroying the protective layer of the colloid particles and causing the colloid particles to become unstable and break the emulsion and flocculate.

[0008] (2) Preparation of skim rubber by hollow fiber column concentration method: removing metal ions (such as copper, manganese, etc.) and improving the mechanical properties and anti-aging properties of skim rubber.

[0009] (3) Nano-composite materials are prepared by adding nano-fillers, such as clay, carbon black, and white carbon black, to the rubber to flocculate the rubber.

[0010] Chinese patent CN103232559B discloses a method for improving the technical indicators of skim rubber to meet the requirements of standard rubber No. 10: first, the skim is treated with protease, a surfactant is added for surface replacement, sodium metabisulfite is added for antioxidant, and then coagulation; the wet gel is soaked in sodium hydroxide, oxalic acid and calcium carbonate solutions respectively, and finally dried to obtain skim rubber with various technical indicators meeting the requirements of standard rubber No. 10. The process technology is very complicated.

[0011] Chinese patent CN103772532A provides a method for processing skim rubber, which uses organic acid to neutralize ammonia in skim rubber, adds protease and flows down a tank with the organic acid solution, and performs coagulation and maturation in a coagulation tank. However, the nitrogen content of the skim rubber product is >0.6%, and PRI is <50, which does not meet the requirements of the SCR10 technical indicators in the national standard GB / T8081-2018.

[0012] In Chinese patent CN104693321A and the dissertation "Study on Quality Improvement and Performance of Skim Rubber" (Zhang Zhe, Master's thesis of Hainan University, 2013), hollow fiber columns are used to filter out small molecular weight non-rubber components, and the skim emulsion with a dry rubber content of 10-50% is concentrated. After papain is added to the purified skim, solid skim rubber is obtained by acid coagulation, which meets the performance index requirements of domestic No. 5 standard rubber. However, this technology requires major modifications to existing process equipment and the process flow is complicated, which is not conducive to industrial production.

[0013] Chinese patent CN112048029A adopts the method of adding four kinds of proteases, namely alkaline protease, papain, acid protease and neutral protease, to the skim latex to enzymatically hydrolyze the skim latex respectively, and then adding acid to coagulate the enzymatically hydrolyzed skim latex. The obtained wet rubber particles need to be soaked in a 10% sodium mercaptobenzothiazole salt aqueous solution for 20 to 30 minutes and then dried to obtain standard rubber. The production process is complicated and not conducive to industrial application.

[0014] The skim rubber production system in the prior art often needs to add a large amount of acid or protease to adjust its pH value or ammonia content to a suitable production range, and use other complex means to obtain natural rubber No. 10 (SCR10) that meets national standards when processing skim. The production process is complicated, which not only increases the production cost, but also limits the production efficiency and flexibility, which is not conducive to the industrial production and wide application of natural rubber. In order to solve the problems of large amount of skim, difficulty in flocculation, cumbersome processing process, poor performance of skim rubber obtained, and difficulty in wastewater treatment, it is an important task to find a reasonable way to process skim to prepare high-performance skim 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 strain of Bacillus alcalophilus, which is purchased from the China Industrial Microbiological Culture Collection Center and has a collection number of CICC 23037. The present invention adopts a biological flocculation method to make the performance of skim rubber equivalent to that of ordinary natural rubber No. 10 (SCR10), increase the yield of natural rubber, simplify the process, and reduce the difficulty of wastewater post-treatment, so that the production process can achieve environmental protection.

[0016] The first object of the present invention is to provide an application of alkaliphilic Bacillus CICC 23037 or a bacterial agent containing alkaliphilic Bacillus CICC23037 and its metabolites in the production of standard natural rubber No. 10 (SCR10) using skim latex as a raw material.

[0017] In one embodiment of the present invention, the natural rubber No. 10 rubber meets the technical requirements of No. 10 rubber in "GB / T 8081-2018 Specification Guidelines for Technical Graded Rubber (TSR) of Natural Raw Rubber" (P0≥30, PRI≥50, nitrogen content≤0.75%).

[0018] In one embodiment of the present invention, the application is specifically: adjusting the pH of skim latex, mixing skim latex and alkaliphilic Bacillus CICC 23037 bacterial agent in parallel into a coagulation tank, standing for coagulation, and after the rubber block is matured, producing skim latex rubber according to the natural rubber production process.

[0019] In one embodiment of the present invention, the skim latex is obtained by filtering through a 35-45 mesh sieve to remove impurities.

[0020] In one embodiment of the present invention, the dry rubber content in the skim latex is 4% to 6% (w / w).

[0021] In one embodiment of the present invention, the step of adjusting the pH of the skim latex is to adjust the pH of the skim latex to ≤9 using 10% formic acid.

[0022] 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 in the skim latex.

[0023] In one embodiment of the present invention, the bacterial agent needs to be diluted or dissolved with 8 to 10 times the mass of water.

[0024] In one embodiment of the present invention, the aging time is 14 to 24 hours.

[0025] In one embodiment of the present invention, the natural rubber production process comprises the steps of thinning and dehydrating, washing, sheeting, granulating and drying the matured rubber blocks.

[0026] In one embodiment of the present invention, the drying condition is 110-115° C. for 7-12 hours.

[0027] In one embodiment of the present invention, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0028] In one embodiment of the present invention, the viable count of alkaliphilic Bacillus CICC 23037 in the bacterial agent is 10 7 ~10 9 CFU / mL.

[0029] In one embodiment of the present invention, the bacterial concentration of alkaliphilic Bacillus CICC 23037 in the bacterial agent is in the range of 10 7 ~10 9 CFU / mL.

[0030] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of alkaliphilic Bacillus CICC 23037 and then expanding the culture.

[0031] In one embodiment of the present invention, the active components of the bacterial agent are active bacteria of alkaliphilic Bacillus CICC 23037 and proteases secreted during the culture process.

[0032] In one embodiment of the present invention, the bacterial agent is a powder obtained by drying a bacterial liquid containing alkalophilic Bacillus CICC 23037.

[0033] In one embodiment of the present invention, the drying refers to spray drying, freeze drying, vacuum drying or other bacterial liquid drying processes.

[0034] In one embodiment of the present invention, the liquid bacterial agent is obtained by the following method:

[0035] The alkaliphilic Bacillus seed liquid is inoculated into a liquid fermentation medium to obtain a fermentation liquid, and the fermentation liquid is concentrated to obtain a liquid bacterial agent.

[0036] In one embodiment of the present invention, the seed solution inoculation amount is 5-10% (v / v); preferably, the inoculation amount is 8%.

[0037] In one embodiment of the present invention, the liquid fermentation medium components are: soluble starch 40-60 g / L, beef powder 2-4 g / L, peptone 4-6 g / L, sodium chloride 4-6 g / L, glucose 0.5-1.0 g / L, and ammonia water is used to adjust the pH to 7.5-8.5;

[0038] Preferably, the liquid fermentation medium components are: 40 g / L soluble starch, 3 g / L beef powder, 5 g / L peptone, 5 g / L sodium chloride, 0.5 g / L glucose, and the pH is adjusted to 8.0 with ammonia water.

[0039] In one embodiment of the present invention, the culture conditions are: temperature 35-37°C, stirring speed 180-200 r / min, fermentation time 24-36 h; fermentation tank pressure 0.05-0.08 MPa, fermentation liquid ventilation ratio (v / v.min) 1: (0.20-0.30);

[0040] Preferably, the culture conditions are: temperature 37° C., stirring speed 200 r / min, fermentation time 36 h, fermentation tank pressure 0.07 MPa, and fermentation liquid ventilation ratio (v / v.min) of 1:0.25.

[0041] In one embodiment of the present invention, the specific step of concentration is: ultrafiltration is performed at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 2500 to 3500 and a pressure of 0.1 to 0.3 MPa to obtain a concentrated fermentation broth that is concentrated 8 to 10 times.

[0042] In one embodiment of the present invention, the solid bacterial agent is obtained by the following method:

[0043] Maltodextrin is added to the above-mentioned liquid bacterial agent and then dried to obtain a solid bacterial agent containing alkaliphilic Bacillus and the protease secreted by the bacterium.

[0044] In one embodiment of the present invention, the added amount of maltodextrin is 220-240 g / L.

[0045] 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.

[0046] In one embodiment of the present invention, the protease activity in the solid bacterial agent is 500-1000 U / g, and the number of viable alkaliphilic Bacillus is (0.5-1.0)×10 8 CFU / g.

[0047] The second object of the present invention is to provide a method for preparing natural rubber with a nitrogen content of ≤0.6%, an initial plasticity value P0≥30, a plasticity retention rate PRI≥50%, a tensile stress of ≥2.0Mpa and / or a tensile strength of ≥25Mpa, wherein the natural rubber is prepared by using alkaliphilic Bacillus CICC 23037 or a bacterial agent containing alkaliphilic Bacillus CICC 23037 and its metabolites.

[0048] In one embodiment of the present invention, the method specifically comprises: adjusting the pH of skim latex, mixing skim latex and alkaliphilic Bacillus CICC 23037 inoculant in parallel into a coagulation tank, standing for coagulation, and after the rubber block is matured, producing skim latex rubber according to the natural rubber production process.

[0049] In one embodiment of the present invention, the skim latex is obtained by filtering through a 35-45 mesh sieve to remove impurities.

[0050] In one embodiment of the present invention, the dry rubber content in the skim latex is 4% to 6% (w / w).

[0051] In one embodiment of the present invention, the step of adjusting the pH of the skim latex is to adjust the pH of the skim latex to ≤9 using 10% formic acid.

[0052] 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 in the skim latex.

[0053] In one embodiment of the present invention, the bacterial agent needs to be diluted or dissolved with 8 to 10 times the mass of water.

[0054] In one embodiment of the present invention, the aging time is 14 to 24 hours.

[0055] 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.

[0056] In one embodiment of the present invention, the drying condition is 110-115° C. for 7-12 hours.

[0057] In one embodiment of the present invention, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0058] In one embodiment of the present invention, the viable count of alkaliphilic Bacillus CICC 23037 in the bacterial agent is 10 7~10 9 CFU / mL.

[0059] In one embodiment of the present invention, the bacterial concentration of alkaliphilic Bacillus CICC 23037 in the bacterial agent is in the range of 10 7 ~10 9 CFU / mL.

[0060] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of alkaliphilic Bacillus CICC 23037 and then expanding the culture.

[0061] In one embodiment of the present invention, the active components of the bacterial agent are active bacteria of alkaliphilic Bacillus CICC 23037 and proteases secreted during the culture process.

[0062] In one embodiment of the present invention, the bacterial agent is a powder obtained by drying a bacterial liquid containing alkalophilic Bacillus CICC 23037.

[0063] In one embodiment of the present invention, the drying refers to spray drying, freeze drying, vacuum drying or other bacterial liquid drying processes.

[0064] In one embodiment of the present invention, the liquid bacterial agent is obtained by the following method:

[0065] The alkaliphilic Bacillus seed liquid is inoculated into a liquid fermentation medium to obtain a fermentation liquid, and the fermentation liquid is concentrated to obtain a liquid bacterial agent.

[0066] In one embodiment of the present invention, the seed solution inoculation amount is 5-10% (v / v); preferably, the inoculation amount is 8%.

[0067] In one embodiment of the present invention, the liquid fermentation medium components are: soluble starch 40-60 g / L, beef powder 2-4 g / L, peptone 4-6 g / L, sodium chloride 4-6 g / L, glucose 0.5-1.0 g / L, and ammonia water is used to adjust the pH to 7.5-8.5;

[0068] Preferably, the liquid fermentation medium components are: 40 g / L soluble starch, 3 g / L beef powder, 5 g / L peptone, 5 g / L sodium chloride, 0.5 g / L glucose, and the pH is adjusted to 8.0 with ammonia water.

[0069] In one embodiment of the present invention, the culture conditions are: temperature 35-37°C, stirring speed 180-200 r / min, fermentation time 24-36 h; fermentation tank pressure 0.05-0.08 MPa, fermentation liquid ventilation ratio (v / v.min) 1: (0.20-0.30);

[0070] Preferably, the culture conditions are: temperature 37° C., stirring speed 200 r / min, fermentation time 36 h, fermentation tank pressure 0.07 MPa, and fermentation liquid ventilation ratio (v / v.min) of 1:0.25.

[0071] In one embodiment of the present invention, the specific step of concentration is: ultrafiltration is performed at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 2500 to 3500 and a pressure of 0.1 to 0.3 MPa to obtain a concentrated fermentation broth that is concentrated 8 to 10 times.

[0072] In one embodiment of the present invention, the solid bacterial agent is obtained by the following method:

[0073] The liquid bacterial agent is added with maltodextrin and then dried to obtain a solid bacterial agent containing alkaliphilic Bacillus and protease.

[0074] In one embodiment of the present invention, the added amount of maltodextrin is 220-240 g / L.

[0075] 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.

[0076] In one embodiment of the present invention, the protease activity in the solid bacterial agent is 500-1000 U / g, and the number of viable alkaliphilic Bacillus is (0.5-1.0)×10 8 CFU / g.

[0077] The third object of the present invention is to provide a standard natural rubber No. 10 (SCR10) produced with skim latex as raw material, wherein the standard natural rubber No. 10 (SCR10) is prepared by the above application or method.

[0078] Beneficial effects:

[0079] Adding nutrients or microorganisms to skim latex to secrete proteases and produce organic acids can accelerate the flocculation of skim latex rubber. However, it is difficult to select the type of bacteria, and bacteria have strict requirements on the living environment. In skim latex with high ammonia content, it is difficult for general microorganisms to survive, and it has not been realized at present. The present invention provides a strain of alkaliphilic Bacillus and a bacterial agent prepared therefrom, which can degrade rubber protein and has excellent application effect in the production of No. 10 standard natural rubber using skim latex as raw material. There is no report on the application of Bacillus bacterial agents in the production of natural rubber using skim latex as raw material.

[0080] (1) The alkaliphilic Bacillus CICC 23037 used in the present invention can grow in an alkaline pH range, and the coagulation of skim latex can be carried out under relatively high alkaline conditions (ammonia content>0.05%), and can tolerate an ammonia content of 0-0.15%. Therefore, when the alkaliphilic Bacillus is used in the coagulation of skim latex, it is not limited by the excessively high ammonia content in the skim latex. It only needs to adjust the pH of the skim latex to ≤9.0, and the amount of added acid required to neutralize ammonia before coagulation is greatly reduced, thereby simplifying the production process, reducing the production cost, and significantly improving the production efficiency and flexibility;

[0081] (2) During the growth of alkaliphilic Bacillus CICC 23037, ammonia in the skim latex is absorbed as an inorganic nitrogen source, and protease is secreted into the continuous fermentation liquid. The protease decomposes the protein in the skim latex and secretes organic acid to reduce the pH of the skim latex, thereby accelerating the coagulation of the skim latex.

[0082] (3) The alkaliphilic Bacillus CICC 23037 provided by the present invention can stably grow and reproduce in a wide temperature range, which makes the production of natural rubber no longer restricted by region and season and can be carried out under a wider range of environmental conditions;

[0083] (4) The protease secreted by alkaliphilic Bacillus CICC 23037 has excellent degradation effect on natural rubber protein and has great application potential in the production of skim natural rubber products;

[0084] (5) Compared with the traditional sulfuric acid flocculation method, the wastewater of the method of the present invention does not contain sulfate ions, which reduces the difficulty of post-treatment of wastewater and the production process is environmentally friendly;

[0085] (6) When the bacterial agent of the present invention is used as a coagulant in the process of producing standard natural rubber No. 10 (SCR10) using skim as raw material, the production process is simple and the production cost is reduced. The indicators of the obtained skim rubber all meet the technical requirements of No. 10 rubber in the "GB / T8081-2018 Specification Guidelines for Technical Graded Rubber (TSR) of Natural Raw Rubber" (P0≥30, PRI≥50, nitrogen content≤0.75%). BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 :Effects of different carbon sources on protease production by alkaliphilic Bacillus.

[0087] Figure 2 :Effect of soluble starch addition on protease production by alkalophilic Bacillus.

[0088] Figure 3 : The optimal catalytic temperature of protease.

[0089] Figure 4 : The optimal catalytic pH of proteases. DETAILED DESCRIPTION

[0090] 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.

[0091] The main detection methods used in the present invention are:

[0092] (1) Determination of protease activity:

[0093] 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 it into 0, 20, 40, 60, 80, and 100 μg / mL tyrosine solutions; take 1 mL of tyrosine solution of each concentration, add 5 mL of 0.4 mol / L Na2CO3 solution and 1 mL of Folin-phenol reagent, respectively, develop color in a 40°C water bath for 20 min, measure the absorbance at 660 nm, and prepare the standard curve with the tyrosine solution concentration as the horizontal axis and the absorbance value as the vertical axis.

[0094] Fermentation broth protease activity determination: centrifuge the fermentation broth at 10000r / min for 10min, take 1mL of supernatant and add 1mL of 2% (w / w) casein solution, and color it in a 40℃ water bath for 20min; add 2mL of 0.4mol / L trichloroacetic acid to terminate the reaction, keep it at 40℃ for 10min, and centrifuge it at 10000r / min for 10min after the end of the insulation. Take 1mL of supernatant, add 5mL of 0.4mol / L Na2CO3 and 1mL of Folin-phenol reagent, and color it at 40℃ for 20min; use the inactivated fermentation broth as a control and measure the 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.

[0095] (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.

[0096] (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.

[0097] (4) The plasticity retention rate is determined in accordance with the method of GB / T 3517 Determination of the plasticity retention rate (PRI) of natural raw rubber.

[0098] (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.

[0099] (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.

[0100] The alkaliphilic bacillus (Bacillus alcalophilus) involved in the present invention is purchased from the China Industrial Microbiological Culture Collection Center, and the collection number is CICC 23037.

[0101] The skim latex used in the following examples is a product separated when natural rubber latex is concentrated by centrifugation, and the ammonia content of the skim latex is 0.12% to 0.15%.

[0102] Example 1: Preparation method of alkaliphilic Bacillus seed solution

[0103] (1) Bacterial Activation

[0104] Using alkaliphilic Bacillus CICC 23037 as the starting strain, the alkaliphilic Bacillus was transferred to a slant to recover the strain; the slant culture medium components were: beef powder 3g / L, peptone 5g / L, sodium chloride 5g / L, glucose 5g / L, agar 15g / L, pH 7.0.

[0105] (2) Seed culture

[0106] The activated alkaliphilic Bacillus was inoculated into the seed culture medium and cultured at 37°C and 200 r / min for 24 h to serve as the seed solution;

[0107] The components of the liquid seed culture medium are: 3 g / L beef powder, 5 g / L peptone, 5 g / L sodium chloride, 5 g / L glucose, and the pH is adjusted to 7.0 with 2 mol / L sodium hydroxide solution.

[0108] The seed liquid was inoculated into the liquid fermentation medium at an inoculation rate of 8% (v / v).

[0109] The fermentation medium components are: 40 g / L soluble starch, 3 g / L beef powder, 5 g / L peptone, 5 g / L sodium chloride, 0.5 g / L glucose, and the pH is adjusted to 8.0 with ammonia water.

[0110] The fermentation conditions were as follows: temperature 37°C, stirring speed 200 r / min, fermentation time 36 h; fermentation tank pressure 0.07 MPa, fermentation liquid ventilation ratio (v / v.min) 1:0.25;

[0111] After the fermentation is completed, the obtained product is a fermentation liquid containing live alkaliphilic Bacillus and protease.

[0112] Example 2: Effect of different carbon sources on protease production by alkaliphilic Bacillus

[0113] The types of carbon sources in the fermentation medium were experimentally analyzed. The carbon sources included soluble starch, glucose, molasses, white sugar or maltodextrin, and the added amount was 4% (w / v).

[0114] The specific implementation method is the same as that of Example 1, except that the soluble starch is replaced by glucose, molasses, white sugar or maltodextrin.

[0115] After fermentation, the activity of proteases in the fermentation broth with different carbon sources was tested. Figure 1 shown.

[0116] Under the same dosage conditions, the protease activity of the fermentation broth using soluble starch as the carbon source was the highest, which was 125.3 U / L (Table 1).

[0117] Table 1 Effects of different carbon sources on protease production by alkaliphilic Bacillus

[0118] Carbon source Soluble starch glucose Maltodextrin Waste Honey white sugar Protease activity (U / L) 125.3 21.4 36.1 63.5 20.1

[0119] Example 3: Effect of Soluble Starch Addition on Protease Production during Alkaliphilic Bacillus Fermentation

[0120] After determining that soluble starch is the most suitable carbon source in the fermentation medium, the inventors further analyzed the effects of different amounts of soluble starch on the enzyme activity of the fermentation broth. The amounts of soluble starch were 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% (w / v), respectively.

[0121] The specific implementation is the same as Example 1, except that the added amount of soluble starch is replaced by 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8% (w / v).

[0122] After fermentation, the protease activity in the fermentation broth was tested. Figure 2 shown.

[0123] right Figure 2 The analysis showed that different dosages of soluble starch had different effects on the enzyme activity of the fermentation broth. The optimal dosage range of soluble starch was 4-5%, and its protease activity was 122.1-125.3 U / L (Table 2).

[0124] Table 2 Effect of soluble starch addition on protease production during alkaliphilic Bacillus fermentation

[0125] Carbon source dosage 1％ 2％ 3％ 4％ 5％ 6％ 7％ 8％ Protease activity (U / L) 86.5 90.2 99.3 125.3 122.1 96.2 92.5 91.0

[0126] Example 4: Preparation of alkaliphilic Bacillus CICC 23037 bacterial agent

[0127] The fermentation broth obtained in Example 1 was ultrafiltered at low temperature using an ultrafiltration membrane with a molecular weight cutoff of 3000 and a concentration of 0.3 MPa to obtain a fermentation broth concentrated 10 times with an enzyme activity of 1250 U / mL, which was a liquid bacterial agent.

[0128] Add maltodextrin as an auxiliary material to the liquid bacterial agent at an addition amount of 220 g / L. The mixed concentrated enzyme solution was spray dried at a dryer outlet temperature of 55 °C and a feed rate of 30 mL / min. The protease activity was 1250 U / g and the number of viable alkaliphilic Bacillus was 0.8 × 10 8 CFU / g solid bacterial agent, the product is uniform powder and has good dispersion and solubility.

[0129] Example 5: Optimal catalytic temperature and optimal catalytic pH of protease in bacterial agent

[0130] 1. Optimum catalytic temperature:

[0131] Using 2% casein as substrate, the mixed concentrated enzyme solution 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 measured at pH 8.0. The catalytic 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.

[0132] Figure 3 The results showed that the activity of the protease in catalyzing the substrate was the highest at 45°C, which was 139.2 U / L, and the enzyme activity at 35-50°C was more than 80% of the highest enzyme activity (Table 3).

[0133] Table 3 Optimal catalytic temperature of protease in bacterial agent

[0134] temperature 30℃ 35℃ 40℃ 45℃ 50℃ 55℃ 60℃ Protease activity (U / L) 90.5 112.8 125.3 139.2 118.3 97.4 29.2 Relative enzyme activity (%) 65 81 95 100 85 70 21

[0135] 2. Optimal catalytic pH:

[0136] 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 mixed concentrated enzyme solution in Example 4 to appropriate multiples with different buffer systems, then measure the activity of the protease to determine its optimal pH. Take the highest enzyme activity as 100% to calculate the relative enzyme activity at other pH values.

[0137] Figure 4The results showed that the activity of the protease in catalyzing the substrate was the highest at pH 8.0, which was 125.3 U / L, and the enzyme activity was relatively high at pH 7.0-9.0 (Table 4).

[0138] Table 4 Optimal catalytic pH of protease in bacterial agent

[0139] pH 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 Protease activity (U / L) 33.8 70.2 106.5 115.3 125.3 116.5 102.7 67.7 28.8 Relative enzyme activity 27 56 85 92 100 93 82 54 23

[0140] Example 6: Method for preparing No. 10 standard natural rubber using solid bacterial agent as coagulant and skim latex as raw material

[0141] 2000 kg of the latex filtered through a 40-mesh sieve to remove impurities was taken, and the dry rubber content was determined to be 4.5% (w / w). The pH value was adjusted to 8.5 with 10% formic acid, and 0.08% (w / w) of the solid bacterial agent obtained in Example 4 by weight of dry rubber was taken. The bacterial agent was dissolved in 10 times the weight of water in advance;

[0142] The dissolved bacterial agent and skim latex were mixed and put into the coagulation tank. The skim latex and bacterial agent were mixed at an ambient temperature of 28°C for 22 hours to form flocculation, and the curd without white water was formed after aging for 24 hours. The curd was thinned and dehydrated, washed, sheeted, granulated, and dried at 110°C for 7.5 hours to obtain skim latex rubber, and its quality indicators and properties were measured (Table 5).

[0143] Example 7: Method for preparing No. 10 standard natural rubber using liquid bacterial agent as coagulant and skim latex as raw material

[0144] 5000 kg of the latex filtered through a 40-mesh sieve to remove impurities was taken, and the dry rubber content was determined to be 5.2% (w / w). The pH value was adjusted to 7.5 with 10% formic acid, and 0.10% (w / w) of the liquid bacterial agent obtained in Example 4 by weight of the dry rubber was taken. The bacterial agent was diluted with 10 times the weight of water in advance;

[0145] The diluted bacterial agent and skim latex were mixed and put into the coagulation tank. The skim latex and bacterial agent were mixed at an ambient temperature of 22°C for 16 hours to form flocculation, and the curd without white water was formed after aging for 24 hours. The curd was thinned and dehydrated, washed, sheeted, granulated, and dried at 115°C for 8 hours to obtain skim latex rubber, and its quality indicators and properties were measured (Table 5).

[0146] Example 8: Method for preparing No. 10 standard natural rubber using solid bacterial agent as coagulant and skim latex as raw material

[0147] The specific implementation method is the same as Example 6, except that the pH value of the skim latex is adjusted to 7.0 with 10% formic acid, and the reaction temperature after the bacterial agent and the skim latex are mixed is 25°C.

[0148] Example 9: Method for preparing No. 10 standard natural rubber using liquid bacterial agent as coagulant and skim latex as raw material

[0149] The specific implementation method is the same as Example 7, except that the pH value of the skim latex is adjusted to 6.5 with 10% formic acid, and the reaction temperature after the bacterial agent and the skim latex are mixed is 22°C.

[0150] Comparative Example 1:

[0151] The diluted bacterial agent in Example 6 was replaced with 15% sulfuric acid solution, and the 15% sulfuric acid solution and skim latex were mixed and fed into a coagulation tank. The other process parameters and steps were the same as those in Example 6 to prepare skim latex rubber, and the quality indicators and properties of the rubber were measured (Table 5).

[0152] Comparative Example 2:

[0153] The concentrated fermentation broth in Example 4 was centrifuged at 10000 rpm for 20 min, and the supernatant (the main component of which was the protease secreted by the alkaliphilic Bacillus) was collected as a coagulant, and the enzyme activity of the supernatant was 1250 U / mL;

[0154] The liquid bacterial agent in Example 7 was replaced by the supernatant, and the other process parameters and steps were the same as those in Example 7 to prepare skim rubber, and the quality indicators and properties of the rubber were measured (Table 5).

[0155] Comparative Example 3:

[0156] The concentrated fermentation broth in Example 4 was centrifuged at 10000 rpm for 20 min, and the precipitate was collected. Maltodextrin was added as an auxiliary material according to the method in Example 4 and spray-dried as a coagulant (the main component was active cells of alkaliphilic Bacillus), wherein the number of active cells of alkaliphilic Bacillus was 0.8×10 8 CFU / g;

[0157] The solid bacterial agent in Example 6 was replaced by the above coagulant, and the other process parameters and steps were the same as those in Example 6 to prepare skim rubber, and the quality indicators and properties of the rubber were measured (Table 5).

[0158] Comparative Example 4:

[0159] The solid bacterial agent in Example 6 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 6 to prepare skim rubber, and the quality indicators and properties of the rubber were measured (Table 5).

[0160] The performance indicators of the skim rubber in the embodiments and comparative examples are shown in Table 5.

[0161] Table 5 Performance indexes of skim rubber products

[0162]

[0163] When the solid bacterial agent and the liquid bacterial agent prepared by the alkaliphilic Bacillus of the present invention are used to coagulate the skim latex (Examples 6 to 9), the coagulation time is significantly lower than that of the comparative examples 2 to 4. From the performance indicators of the skim rubber in Table 5, the skim rubber (Examples 6 to 9) prepared by the solid bacterial agent and the liquid bacterial agent prepared by the alkaliphilic Bacillus of the present invention are better than the skim rubber obtained in comparative examples 1 to 4 in various indicators; nitrogen content ≤ 0.6%, initial plasticity P0 ≥ 30, plasticity retention rate PRI ≥ 50%, tensile stress > 2.0 MPa, Mooney viscosity > 80 ML (1 + 4) 100 ° C, tensile strength > 25 MPa, all indicators meet the technical requirements of No. 10 rubber (SCR10) in GB / T 8081-2018 Specification Guidelines for Technical Graded Rubber (TSR) of Natural Raw Rubber.

[0164] 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. Application of alkaliphilic Bacillus CICC 23037 or a bacterial agent containing alkaliphilic Bacillus CICC 23037 and its metabolites in the production of standard natural rubber No. 10 using skim latex as raw material.

2. The use according to claim 1, characterized in that: The application is specifically as follows: adjusting the pH of skim latex, mixing skim latex and alkaliphilic Bacillus CICC 23037 bacterial agent in parallel into a coagulation tank, standing for coagulation, and after the rubber block is matured, producing skim latex rubber according to the natural rubber production process.

3. The use 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 in the skim latex.

4. The use according to claim 1 or 2, characterized in that: The step of adjusting the skim latex pH is to use formic acid to adjust the skim latex pH to ≤ 9.

5. The use according to claim 1 or 2, characterized in that: The active components of the bacterial agent are active bacterial bodies of alkaliphilic Bacillus CICC 23037 and proteases secreted during the culture process.

6. The use according to claim 1 or 2, characterized in that: The protease activity in the bacterial agent is 500-1000 U / g, and the number of live alkaliphilic Bacillus is (0.5-1.0)×10 8 CFU / g.

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

8. The use according to claim 1 or 2, characterized in that: The preparation method of the bacterial agent is as follows: Inoculating the alkaliphilic Bacillus seed liquid into a liquid fermentation medium to obtain a fermentation liquid, and concentrating the fermentation liquid to obtain a bacterial agent; Preferably, the seed solution inoculation amount is 5-10% (v / v); Preferably, the liquid fermentation medium component contains soluble starch; preferably, the soluble starch is added in an amount of 4% to 5%.

9. A method for preparing natural rubber with a nitrogen content of ≤0.6%, an initial plasticity value P0 ≥30, a plasticity retention rate PRI ≥50%, a tensile stress ≥2.0 MPa and / or a tensile strength ≥25 MPa, characterized in that: The method is prepared by utilizing alkaliphilic Bacillus CICC 23037 or a bacterial agent containing alkaliphilic Bacillus CICC 23037 and its metabolites.

10. A standard natural rubber No. 10 produced from skim latex as raw material, characterized in that: The standard natural rubber No. 10 is prepared according to the application described in claims 1 to 8 or by the method described in claim 9.

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