Bacillus subtillis and application thereof

Abstract

The invention relates to the technical field of microorganisms and application thereof, in particular to bacillus subtillis and application thereof. The bacillus subtilis DL192 strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 11, 2024, and the preservation number is CGMCC No.32185; the nucleotide sequence table of the DL192 bacillus subtilis is as shown in SEQ ID No 1. According to the production method and application of the special superoxide dismutase microecological preparation for the rice, provided by the invention, after the special superoxide dismutase microecological preparation for the rice is used, the produced superoxide dismutase has relatively strong temperature resistance, heat resistance and stress resistance, has strong tolerance to ozone, high temperature and high acid, and is free of toxic and side effects. The superoxide dismutase rice keeps the excellent characters of original rice, the content of superoxide dismutase is obviously increased, the superoxide dismutase rice has the effects of enhancing the immunity of a human body and delaying senescence, and the superoxide dismutase rice can still keep high superoxide dismutase activity in the deep processing process.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and their application, in particular to Bacillus subtilis and its application. Background Art

[0002] Rice (Oryza sativa L.), also known as rice, paddy, etc., belongs to the Gramineae family, the genus Oryza, and is one of the three major food crops in the world. It is divided into indica rice and japonica rice. The total annual rice production in the world is as high as 986.53 million tons, which is the main food source for about 4 billion people.

[0003] Superoxide dismutase (SOD) is widely present in living organisms, plays an extremely important role in the self-protection system of living organisms, and also has important functions in the immune system. It has unique functions in radiation protection, anti-aging, and tumor prevention and treatment. In addition, as an important antioxidant enzyme, SOD also plays a key protective role in plants, which can remove superoxide free radicals and reduce the damage of oxidative stress to plants, thereby improving the stress resistance of plants.

[0004] To this end, we designed a Bacillus subtilis and its application to provide another technical solution to the above technical problems. Summary of the invention

[0005] Based on this, it is necessary to provide a method for producing a superoxide dismutase microecological preparation for rice in order to solve the technical problems raised in the above-mentioned background technology.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A DL192 Bacillus subtilis strain, wherein the Bacillus subtilis DL192 strain (bacillus subtilis) was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on October 11, 2024, with a deposit number of CGMCC No.32185;

[0008] The nucleotide sequence of the DL192 Bacillus subtilis is shown in SEQ ID No 1.

[0009] The use of Bacillus subtilis DL192 as claimed in claim 1 in a superoxide dismutase microecological preparation.

[0010] A superoxide dismutase microecological preparation specially used for rice, comprising the DL192 Bacillus subtilis as claimed in claim 1 and additives.

[0011] As a preferred embodiment of the superoxide dismutase microecological preparation for rice provided by the present invention, the DL192 Bacillus subtilis is 50 billion CFU / g.

[0012] As a preferred embodiment of the superoxide dismutase microecological preparation for rice provided by the present invention, the additive is light calcium carbonate or peat or humic acid or rare earth mineral powder;

[0013] The fineness of the light calcium carbonate or peat or humic acid or rare earth mineral powder is 0.09 mm.

[0014] The superoxide dismutase microecological preparation according to claim 3 is used in rice seedling cultivation and / or planting.

[0015] As a preferred embodiment of the application of the superoxide dismutase microecological preparation provided by the present invention in rice seedling cultivation, the content of the superoxide dismutase is 470 I U / g.

[0016] As a preferred embodiment of the application of the superoxide dismutase microecological preparation provided by the present invention in rice seedling cultivation, the application method is as follows:

[0017] The rice seeds are soaked in a superoxide dismutase preparation and the superoxide dismutase preparation is sprayed during the growth period of the rice.

[0018] As a preferred embodiment of the application of the superoxide dismutase microecological preparation provided by the present invention in rice seedling cultivation, rice seeds are soaked in a superoxide dismutase preparation at a dilution ratio of 1:300-500, and then dried and sown after the soaking is completed.

[0019] As a preferred embodiment of the application of the superoxide dismutase microecological preparation provided by the present invention in rice seedling cultivation, the superoxide dismutase preparation is sprayed during the growth of rice, and the steps are as follows:

[0020] At the rice seedling stage, spray the superoxide dismutase preparation at a 1:800-1200 times dilution on the plants, with an interval of 15-20 days each time, for a total of 2-3 times;

[0021] After transplanting, spray 800-1200 times diluted superoxide dismutase preparation for rice during the heading, flowering and filling stages.

[0022] It can be seen without a doubt that the above-mentioned technical solution of the present application can definitely solve the technical problem to be solved by the present application.

[0023] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0024] 1. The present invention provides a Bacillus subtilis and its application, which allows the special SOD preparation for rice to propagate and transfer in the rice plant through the plant's beneficial endophytic Bacillus subtilis, thereby improving the SOD activity and content in the rice. After using the special SOD preparation for rice, the SOD enzyme produced by the rice has strong heat resistance and stress resistance. When treated at 70°C for 1 hour, the enzyme activity is more than 80% of the original enzyme activity; when treated at 100°C for 20 minutes, the enzyme activity is more than 70% of the original enzyme activity; the rice has strong resistance to ozone, high temperature and high acid. The special SOD preparation for rice is prepared from the beneficial symbiotic Bacillus subtilis in the plant and has a high degree of safety. Therefore, the rice planted with the special SOD preparation for rice has a higher SOD enzyme activity and can still maintain a higher SOD enzyme activity during the deep processing process.

[0025] 2. The present invention applies a special biological agent for superoxide dismutase rice in the production process of rice, combined with rice cultivation and management technology, to obtain rice with high SOD enzyme content; SOD rice not only maintains the excellent traits of the original rice, but also significantly improves the SOD content, and has the effects of enhancing human immunity and delaying aging; therefore, SOD rice has a good market prospect and is in line with the development direction of green food and functional food.

[0026] 3. The special SOD preparation for rice of the present invention can be implemented in combination with other cultivation management measures without increasing working hours. It can also be mixed with other pesticides, fungicides (except bactericides), plant growth regulators, foliar fertilizers, chemical fertilizers and other agricultural chemicals without affecting the use effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 It is a schematic diagram of the phylogenetic tree of the strains of the present invention; DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions in the embodiments may be combined with each other.

[0031] The preparation of dedicated superoxide dismutase (SOD) biological preparation has not been achieved. We cultivated a Bacillus subtilis and named it DL192 Bacillus subtilis. The Bacillus subtilis DL192 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 11, 2024, with the deposit number CGMCC No.32185.

[0032] We have also sequenced the gene sequence of the DL192 Bacillus subtilis, and its nucleotide sequence is shown in SEQ NO 1 (see sequence listing).

[0033] Figure 1 Schematic diagram of the phylogenetic tree of the strains of the present invention, which was constructed using the neighbor-joining method.

[0034] Embodiment 1

[0035] Production of superoxide dismutase (SOD) biological preparation specifically for rice.

[0036] 1. Fermentation production of strains

[0037] 1. Preparation conditions

[0038] Sterilization conditions: temperature 121-125℃, pressure 0.11-0.14Mpa, time 30min;

[0039] Culture conditions: temperature 28-34°C, pressure 0.05Mpa, ventilation value 1:1, pH value 7-7.5;

[0040] Canning indicators: bacterial content ≥ 8 billion / mL, spore formation ≥ 90%, and miscellaneous bacteria rate ≤ 0.3%.

[0041] 2. Strains production

[0042] Pick a small amount from the slant of the original strain of Bacillus subtilis (strain name) and streak it in a spare slant test tube, place it in a constant temperature incubator at 28-30℃ and culture it for 24 hours, take it out and check to confirm that there is no contamination from foreign bacteria. When the bacteria grow neatly, continue to culture for 24-48 hours, then smear and stain it, and observe it under the oil lens of a microscope. When there is no contamination from foreign bacteria and the spores are neat, it can be used as the strain for fermentation production.

[0043] Scrape a small amount of bacterial moss from the plate of the cultured strain and put it into a shake flask containing 500 mL of liquid culture medium. Culture it in a full-temperature shaking incubator at 28°C and 180 rpm for 24 hours. Observe the growth status of the strain under a microscope. When it reaches the logarithmic growth phase, it can be cultured in a seed tank.

[0044] 3. Fermentation production

[0045] Fermentation production is divided into two parts: seed tank culture and fermentation tank culture. When the fermentation liquid reaches the specified index, the tank is released in time. Among them, seed tank and fermentation tank culture are existing mature technologies.

[0046] 2. Preparation of Biological Agents

[0047] 1. Add adsorbent

[0048] Adding light calcium carbonate or peat or humic acid or rare earth mineral powder with a fineness of 0.09 mm and a weight of 1 / 10 of the fermentation liquid into the obtained fermentation liquid, and then mechanically stirring for 3-6 hours to allow Bacillus subtilis to be fully adsorbed by the carrier;

[0049] Preferably, the amount of light calcium carbonate, peat or humic acid or rare earth mineral powder is 5-7 times that of Bacillus subtilis cells;

[0050] Preferably, the fineness of light calcium carbonate, peat or humic acid is 0.08-0.1 mm.

[0051] 2. Filter Press

[0052] Use open flow or undercurrent plate filter press to filter out excess water.

[0053] 3. Drying

[0054] Dry at low temperature (not exceeding 70°C) to make the moisture content ≤5%.

[0055] 4. Crushing

[0056] Use an ultra-fine grinder to grind, the fineness requirement is that 100% passes through a 0.09mm aperture sieve. The purpose of controlling the fineness is to prevent the nozzle from being blocked during use.

[0057] 5. Packaging

[0058] The obtained SOD preparation is white or black powder, the content of Bacillus subtilis is 50 billion CFU / g, the moisture content is ≤5%, and the fineness is 100% passing through a 0.09 mm sieve.

[0059] Embodiment 2

[0060] Cultivation of superoxide dismutase (SOD) rice.

[0061] Option 1: Soak the seeds in 300-500 times diluted rice-specific SOD preparation for 30 minutes, dry them and sow them, and plant and manage them using conventional methods. This can be combined with other cultivation and management techniques throughout the rice growth period without any conflicts.

[0062] Option 2: Soak the seeds in 300-500 times diluted rice-specific SOD preparation for 30 minutes, dry them and sow them, and plant them in the conventional way; spray with 800-1200 times diluted rice-specific SOD preparation 15 days after the rice seedlings emerge, spray once every 15-20 days, and spray 2-3 times in total; after transplanting, spray 800-1200 times diluted rice-specific SOD preparation once during the heading stage, flowering stage and filling stage of rice, and use 50g per mu each time; when spraying, the diluted solution should be evenly attached to the entire plant. The rice-specific SOD preparation can be combined with other cultivation management during the spraying process without any conflicts;

[0063] Preferably, the active ingredient of the sprayed rice-specific SOD preparation is 50 billion CFU / g of Bacillus subtilis.

[0064] Furthermore, the superoxide dismutase rice contained 470 IU / g of superoxide dismutase.

[0065] Embodiment 3

[0066] Superoxide dismutase (SOD) Determination of superoxide dismutase (SOD) activity in rice 1. Determination of SOD activity

[0067] The SOD activity was determined by NBT photochemical method.

[0068] The rice cultured under the same conditions as the cultivation and management measures for SOD rice seedlings in Example 2 was used as a control, with clean water replacing the rice-specific SOD preparation. The SOD enzyme activity of the SOD rice and the control rice was determined as follows:

[0069] 1. Equipment: 752 spectrophotometer; desktop centrifuge; light incubator; refrigerator; 10mL beaker, etc.

[0070] 2. Reagent: Nitro blue tetrazolium chloride (NBT) - produced by Shanghai Qianjin Pharmaceutical Factory

[0071] L-methionine (L-Met)—produced by Shanghai Kangda Amino Acid Factory;

[0072] Ethylenediaminetetraacetic acid (EDTA)—produced by Beijing Chemical Plant;

[0073] Riboflavin (VB2)—produced by Beijing Xizhong Chemical Plant.

[0074] 3. Preparation of reaction solution

[0075] Preparation of NBT reaction solution: prepare 1000 mL of phosphate buffer with a pH of 7.8 and 50 mmol / L, add 1.939 g of L-Met (L-methionine), and after it is completely dissolved, add 5.151 mg of nitro blue tetrazolium chloride (NBT), 0.49 mg of riboflavin (prepare a 0.49 mg / mL solution, pipette 1 mL and add) and 29.0 mg of ethylenediaminetetraacetic acid (EDTA). The final concentration of the solution is: Met is 13 mmol / L; NBT is 6.3×10 -3 mmo l / L; riboflavin (VB2) is 1.3×10 -3 0.1 mmol / L EDTA, placed in a brown reagent bottle, covered with a black cloth to block light, can be stored in a 4°C refrigerator for 1 month.

[0076] 4. Material pretreatment

[0077] The rice grains were ground in a mortar, and buffer was added in a ratio of pulp to 50 mM phosphate buffer = 1:2. The mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken for SOD enzyme activity determination.

[0078] 5. Determination method

[0079] 30 μL of sample was taken and placed in a 10 mL transparent glass beaker, 3 mL of NBT reaction solution was added, and mixed. In a 28°C SOD photochemical reaction chamber, 2×15W fluorescent light was used for 20 min (the light was 4000 lux), and the absorbance was measured at 560 nm. The NBT reaction solution was used as a blank control. Each group was repeated 3 times. The entire measurement process was carried out under light-proof or weak light conditions except for the photochemical reaction. At the same time, the standard enzymes of the American "Sigma" company and Shanghai Baoan Company and the SOD oral solution produced by Guizhou Laolaifu Pharmaceutical Company were used as test controls during the test.

[0080] 6. Calculation

[0081]

[0082] Note: A CK is the absorbance of the control tube, A E is the absorbance of the sample tube, V is the total volume of the sample solution (mL), V t is the amount of sample used in the determination (mL), and W is the fresh weight of the sample (g).

[0083] The results of the SOD enzyme activity determination in rice are shown in Table 1. The SOD enzyme activity in SOD rice is significantly higher than that in ordinary rice, and the difference is significant at the α=0.05 level. This technology can produce SOD health function rice with high SOD enzyme activity.

[0084] Table 1 Results of SOD enzyme activity determination in SOD rice

[0085]

[0086] Note: A and B are the methods of expressing differences in variance analysis, indicating that the SOD enzyme activity of SOD rice and the control ordinary rice is significantly different.

[0087] Example 4: Superoxide dismutase (SOD) Determination of the heat resistance of superoxide dismutase (SOD) in rice.

[0088] The rice cultured under the conditions that the SOD preparation was replaced with clean water and other cultivation and management measures were consistent with the cultivation and management measures of the SOD rice in Example 2 was used as the control; the SOD enzyme activity determination method of Example 3 was used. The SOD rice samples were kept at 70°C and 100°C for 1h and 20min respectively, and the SOD enzyme activity of the SOD rice and the control rice was determined. The results are shown in Table 2. After heat treatment, the SOD rice still maintained a high SOD enzyme activity, but the SOD enzyme activity contained in the control was extremely low. It shows that the SOD rice produced by this technology is suitable for storage and deep processing at room temperature, and can still maintain a high SOD activity.

[0089] Table 2 Determination of heat resistance of SOD in SOD rice

[0090]

[0091] Note: A and B are the methods of expressing differences in variance analysis, indicating that the SOD enzyme activity of SOD rice and the control ordinary rice is significantly different.

[0092] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A DL192 Bacillus subtilis, characterized in that The Bacillus subtilis DL192 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 11, 2024, with the deposit number CGMCC No.32185; The nucleotide sequence of the DL192 Bacillus subtilis is shown in SEQ ID No 1.

2. Application of DL192 Bacillus subtilis as claimed in claim 1 in superoxide dismutase microecological preparation.

3. A superoxide dismutase microecological preparation for rice, characterized in that: The superoxide dismutase preparation comprises the DL192 Bacillus subtilis as claimed in claim 1 and additives.

4. The superoxide dismutase microecological preparation for rice according to claim 3, characterized in that: The DL192 Bacillus subtilis is 50 billion CFU / g.

5. The superoxide dismutase microecological preparation for rice according to claim 3, characterized in that: The additive is light calcium carbonate or peat or humic acid or rare earth mineral powder; The fineness of the light calcium carbonate or peat or humic acid or rare earth mineral powder is 0.09 mm.

6. Use of the superoxide dismutase probiotic preparation according to claim 3 in rice seedling cultivation and / or planting.

7. The use of the superoxide dismutase probiotic preparation in rice seedling cultivation according to claim 6, characterized in that: The content of superoxide dismutase is 470 IU / g.

8. The use of the superoxide dismutase microecological preparation in rice seedling cultivation according to claim 6, characterized in that: The application method is as follows: The rice seeds are soaked in a superoxide dismutase preparation and the superoxide dismutase preparation is sprayed during the growth period of the rice.

9. The use of the superoxide dismutase probiotic preparation in rice seedling cultivation according to claim 8, characterized in that: The rice seeds are soaked in a superoxide dismutase preparation at a dilution ratio of 1:300-500, and then dried and sown.

10. The use of the superoxide dismutase microecological preparation in rice seedling cultivation according to claim 8, characterized in that: Spraying superoxide dismutase preparation during rice growth, the steps are as follows: At the rice seedling stage, spray the superoxide dismutase preparation at a 1:800-1200 times dilution on the plants, with an interval of 15-20 days each time, for a total of 2-3 times; After transplanting, spray 800-1200 times diluted superoxide dismutase preparation for rice during the heading, flowering and filling stages.

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