Soybean rhizobium freeze-dried powder as well as preparation method and application thereof

Abstract

The invention relates to freeze-dried soybean rhizobium powder as well as a preparation method and application thereof. The freeze-dried soybean rhizobium powder contains thalli and an auxiliary agent, the thalli contain rhizobium japonicum; the auxiliary agent comprises at least one of skim milk powder, maltodextrin, trehalose, ascorbic acid, sodium glutamate, glycerol, Ectoin and polyhydroxyalkanoate. The freeze-dried powder is used for soybean seed dressing, can keep the high activity of rhizobium strains, and has important significance for improving the yield and quality of soybeans.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular to a soybean rhizobium freeze-dried powder and a preparation method and application thereof. Background Art

[0002] Leguminous plants can convert molecular nitrogen in the air into ammonia nitrogen through the nitrogen fixation of symbiotic rhizobia, providing nutrients for crops. Inoculation of leguminous crops with rhizobia can increase crop yields, especially soybeans, peanuts, etc., and rhizobia inoculation technology has become a conventional agricultural technology. The use of rhizobia agents is conducive to improving the number, quality and nitrogen fixation of leguminous plant nodules, which can effectively reduce the use of chemical fertilizers, improve the quality of soil and ecological environment, increase the yield and quality of crops, and facilitate the adjustment of agricultural industrial structure. Therefore, the research and application of rhizobia will play an important role in increasing crop yields, reducing fertilizer usage and production costs, reducing water and soil pollution and soil-borne diseases, and establishing ecological balance. Rhizobia are usually stored and transported in liquid dosage forms, but the existing rhizobia have poor heat resistance. After being stored at room temperature, the number of live bacteria is often significantly reduced, affecting the performance of their biological efficacy.

[0003] Rhizobia are also made into freeze-dried powder for seed dressing to increase the yield of crops such as soybeans. This freeze-dried powder has the advantages of easy storage and transportation, so it has broad development prospects. However, the existing rhizobia have poor tolerance in low temperature environments. After freeze-drying, the number of effective live bacteria will drop significantly, which limits its use.

[0004] Therefore, there is an urgent need to provide a new freeze-dried powder that is more resistant to low temperatures and a freeze-drying process that can improve low-temperature resistance, so as to increase the survival rate of rhizobia, better utilize the biological fertilizer effect of rhizobia, and increase crop yields. Summary of the invention

[0005] The purpose of the present invention is to provide a new freeze-dried powder that is more resistant to low temperatures and a freeze-drying process that can improve low temperature resistance, which are applied to the crop seed dressing process to increase the survival rate of soybean rhizobia, thereby increasing crop yields.

[0006] In order to achieve the above-mentioned object, the present disclosure provides a soybean rhizobium freeze-dried powder in a first aspect, wherein the soybean rhizobium freeze-dried powder contains bacteria and an adjuvant; The bacterial body contains soybean rhizobium; the auxiliary agent includes at least one of skimmed milk powder, maltodextrin, trehalose, ascorbic acid, sodium glutamate, glycerol, ectoine and polyhydroxyalkanoate (PHA).

[0007] Optionally, calculated on a dry matter basis, the weight ratio of the bacterial cells to the auxiliary agent is 1:(1-2); Wherein, the auxiliary agent, relative to 10-20 parts by weight of skimmed milk powder, further contains 10-50 parts by weight of maltodextrin, 10-60 parts by weight of trehalose, 0.1-1.5 parts by weight of ascorbic acid, 5-15 parts by weight of sodium glutamate, 5-10 parts by weight of glycerol, 0.01-0.1 parts by weight of icodone and 0.01-0.1 parts by weight of polyhydroxyalkanoate.

[0008] In another aspect, the present disclosure provides a method for preparing soybean rhizobium freeze-dried powder, the preparation method comprising the following steps: S1. Inoculate soybean rhizobia on a plate culture medium and culture in a constant temperature incubator at 26-30°C for 12-36 hours; S2, picking a single colony from the strain cultured in step S1, inoculating it into a seed culture medium, and culturing it at 26-30° C. and 150-220 rpm for 12-24 h to obtain a seed solution; S3, inoculating the seed liquid into the fermentation medium at an inoculum amount of 1-5%, culturing for 12-36 hours at 26-30° C., 150-220 rpm, 0.04-0.06 MPa, and a pH value of 6.9-7.1 to obtain a fermentation liquid; if the pH value rises during the fermentation process, adding glucose to the fermentation medium so that the glucose content in the fermentation medium is 0.1-0.5 g / L; S4, cooling the fermentation broth to 10-15°C, centrifuging and collecting the cells; adding 1-5 times of 4-10°C purified water to the collected cells for redissolution, and centrifuging and collecting the cells again; repeating the above operation until the supernatant is colorless, and collecting the muddy cells; S5, uniformly mixing the mud-like bacterial cells with the auxiliary agent, and subjecting them to vacuum freeze-drying treatment; The auxiliary agent includes at least one of skimmed milk powder, maltodextrin, trehalose, ascorbic acid, sodium glutamate, glycerol, ectoine and polyhydroxyalkanoate.

[0009] The preparation method provided by the present invention greatly improves the number, activity and stability of the soybean rhizobium freeze-dried powder by controlling the temperature and time of the freeze-drying process. The freeze-dried bacterial powder is more than 80 billion / g, and the activity is maintained at more than 80% and 60% respectively when stored at 4°C and 25°C for 360 days, and the bacterial quantity can be rapidly expanded from 100 million to more than 1 billion within 20 hours; after fumigation at 50°C for 20 minutes, the survival rate is still 87.67%, and it can still be fermented to 1.74 billion CFU / mL when inoculated into the fermentation medium at a 2% inoculation rate.

[0010] Optionally, calculated on a dry matter basis, the weight ratio of the mud-like bacterial bodies to the auxiliary agent is 1:(1-2); Preferably, the auxiliary agent contains 10-20 g / L skim milk powder, 10-50 g / L maltodextrin, 10-60 g / L trehalose, 0.1-1.5 g / L ascorbic acid, 5-15 g / L sodium glutamate, 5-10 g / L glycerol, 0.01-0.1 g / L icodole and 0.01-0.1 g / L polyhydroxyalkanoate.

[0011] Optionally, the plate culture medium contains: sorbitol 9-11 g / L, yeast extract powder 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, sodium chloride 0.08-0.12 g / L, calcium chloride 0.04-0.06 g / L, boric acid 0.01-0.03 g / L, sodium molybdate monohydrate 0.01-0.03 g / L, agar 17-19 g / L, pH 6.9-7.1, and the culture medium is sterilized at 115-118°C for 20-30 min.

[0012] Optionally, the seed culture medium contains: sorbitol 9-11 g / L, yeast extract powder 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, sodium chloride 0.08-0.12 g / L, calcium chloride 0.04-0.06 g / L, boric acid 0.01-0.03 g / L, sodium molybdate monohydrate 0.01-0.03 g / L, pH 6.9-7.1, and the culture medium is sterilized at 115-118°C for 20-30 min.

[0013] Optionally, the fermentation medium contains: sorbitol 9-11 g / L, yeast extract 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, sodium chloride 0.08-0.12 g / L, calcium chloride 0.04-0.06 g / L, boric acid 0.01-0.03 g / L, sodium molybdate monohydrate 0.01-0.03 g / L, pH 6.9-7.1, and the culture medium is sterilized at 115-118°C for 20-30 min.

[0014] Optionally, the centrifugation is performed by a high-speed centrifuge or a centrifuge.

[0015] On the other hand, the present disclosure provides the use of the above-mentioned soybean rhizobium freeze-dried powder or the soybean rhizobium freeze-dried powder prepared by the above-mentioned method in crop seed dressing, preferably the crop is a leguminous crop, and more preferably the leguminous crop is soybean.

[0016] On the other hand, the present disclosure provides a seed coating agent, which contains the above-mentioned soybean rhizobium freeze-dried powder or the soybean rhizobium freeze-dried powder prepared by the above-mentioned method.

[0017] On the other hand, the present disclosure provides a coated seed, wherein the coated seed contains the above-mentioned soybean rhizobium freeze-dried powder or the soybean rhizobium freeze-dried powder prepared by the above-mentioned method.

[0018] Through the above technical scheme, the present disclosure provides a soybean rhizobium freeze-dried powder and a preparation method of the freeze-dried powder. The freeze-dried powder contains soybean rhizobium and an auxiliary agent with specific ingredients. It is applied to soybean seed dressing and can form nitrogen-fixing nodules with higher activity at the roots of soybeans. The expansion effect is stable, which is of great significance for improving the yield and quality of soybeans.

[0019] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 It is the Gram staining picture of the fermentation test of soybean rhizobium freeze-dried powder of Example 2.

[0021] Figure 2 It is a dilution coating count chart of the soybean rhizobium freeze-dried powder fermentation test of Example 2. DETAILED DESCRIPTION

[0022] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0023] In one aspect, the present disclosure provides a soybean rhizobium freeze-dried powder, wherein the soybean rhizobium freeze-dried powder contains bacteria and an adjuvant; The bacterial body contains soybean rhizobium; the auxiliary agent includes at least one of skimmed milk powder, maltodextrin, trehalose, ascorbic acid, sodium glutamate, glycerol, ectoine and polyhydroxyalkanoate.

[0024] The present disclosure provides a specific combination of adjuvants, in which the components cooperate with each other and work together to provide certain nutrients for the bacteria, and significantly improve the stability and biological activity of soybean rhizobium freeze-dried powder. When applied to crop seed dressing, it can play a nitrogen-fixing role and promote crop growth.

[0025] Optionally, calculated on a dry matter basis, the weight ratio of the bacterial cells to the auxiliary agent is 1:(1-2); Wherein, the auxiliary agent, relative to 10-20 parts by weight of skimmed milk powder, further contains 10-50 parts by weight of maltodextrin, 10-60 parts by weight of trehalose, 0.1-1.5 parts by weight of ascorbic acid, 5-15 parts by weight of sodium glutamate, 5-10 parts by weight of glycerol, 0.01-0.1 parts by weight of icodone and 0.01-0.1 parts by weight of polyhydroxyalkanoate.

[0026] The inventors of the present disclosure prepared auxiliary agents according to the above specific ratios, which further improved the stability and biological activity of the lyophilized powder.

[0027] In another aspect, the present disclosure provides a method for preparing soybean rhizobium freeze-dried powder, the preparation method comprising the following steps: S1. Inoculate soybean rhizobia on a plate culture medium and culture in a constant temperature incubator at 26-30°C for 12-36 hours; S2, picking a single colony from the strain cultured in step S1, inoculating it into a seed culture medium, and culturing it at 26-30° C. and 150-220 rpm for 12-24 h to obtain a seed solution; S3, inoculating the seed liquid into the fermentation medium at an inoculum amount of 1-5%, culturing for 12-36 hours at 26-30° C., 150-220 rpm, 0.04-0.06 MPa, and a pH value of no more than 6.9-7.1 to obtain a fermentation liquid; If the pH value rises during the fermentation process, glucose is added to the fermentation medium so that the glucose content in the fermentation medium is 0.1-0.5 g / L; S4, cooling the fermentation broth to 10-15°C, centrifuging and collecting the cells; adding 1-5 times of 4-10°C purified water to the collected cells for redissolution, and centrifuging and collecting the cells again; repeating the above operation until the supernatant is colorless, and collecting the muddy cells; S5, uniformly mixing the mud-like bacterial cells with the auxiliary agent, and subjecting them to vacuum freeze-drying treatment; The auxiliary agent includes at least one of skimmed milk powder, maltodextrin, trehalose, ascorbic acid, sodium glutamate, glycerol, ectoine and polyhydroxyalkanoate.

[0028] The present disclosure regulates the freeze-drying parameters such as temperature, rotation speed, and culture time in the above freeze-drying process to further ensure the quality of the freeze-dried powder and the freeze-drying effect, so that the amount of soybean rhizobia in the freeze-dried powder is more than 80 billion / g, and the number of live bacteria is kept at more than 80% and 60% respectively at 4°C and 25°C for 360 days, and the bacteria can be quickly expanded from 100 million to more than 1 billion within 20 hours. And the prepared freeze-dried powder can still reach a survival rate of more than 85% after fumigation at 50°C for 20 minutes.

[0029] Optionally, calculated on a dry matter basis, the weight ratio of the sludge cells to the auxiliary agent is 1:(1-2); the auxiliary agent contains 10-20 parts by weight of skimmed milk powder, 10-50 parts by weight of maltodextrin, 10-60 parts by weight of trehalose, 0.1-1.5 parts by weight of ascorbic acid, 5-15 parts by weight of sodium glutamate, 5-10 parts by weight of glycerol, 0.01-0.1 parts by weight of icodone and 0.01-0.1 parts by weight of polyhydroxyalkanoate.

[0030] Optionally, the plate culture medium contains: sorbitol 9-11 g / L, yeast extract powder 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, sodium chloride 0.08-0.12 g / L, calcium chloride 0.04-0.06 g / L, boric acid 0.01-0.03 g / L, sodium molybdate monohydrate 0.01-0.03 g / L, agar 17-19 g / L, pH 6.9-7.1, and the culture medium is sterilized at 115-118°C for 20-30 min.

[0031] Optionally, the seed culture medium contains: sorbitol 9-11 g / L, yeast extract powder 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, sodium chloride 0.08-0.12 g / L, calcium chloride 0.04-0.06 g / L, boric acid 0.01-0.03 g / L, sodium molybdate monohydrate 0.01-0.03 g / L, pH 6.9-7.1, and the culture medium is sterilized at 115-118°C for 20-30 min.

[0032] Optionally, the fermentation medium contains: sorbitol 9-11 g / L, yeast extract 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, sodium chloride 0.08-0.12 g / L, calcium chloride 0.04-0.06 g / L, boric acid 0.01-0.03 g / L, sodium molybdate monohydrate 0.01-0.03 g / L, pH 6.9-7.1, and the culture medium is sterilized at 115-118°C for 20-30 min.

[0033] Optionally, the centrifugation is performed by a high-speed centrifuge or a centrifugal tube centrifuge at a centrifugation speed of 8000-15000 rpm; through high-speed centrifugation, effective separation of the bacteria can be achieved in a shorter time.

[0034] On the other hand, the present disclosure provides the use of the above-mentioned soybean rhizobium freeze-dried powder or the soybean rhizobium freeze-dried powder prepared by the above-mentioned method in crop seed dressing, preferably the crop is a leguminous crop, and more preferably the leguminous crop is soybean.

[0035] On the other hand, the present disclosure provides a seed coating agent, which contains the above-mentioned soybean rhizobium freeze-dried powder or the soybean rhizobium freeze-dried powder prepared by the above-mentioned method.

[0036] On the other hand, the present disclosure provides a coated seed, wherein the coated seed contains the above-mentioned soybean rhizobium freeze-dried powder or the soybean rhizobium freeze-dried powder prepared by the above-mentioned method.

[0037] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0038] The plate culture medium used in the present disclosure includes: 10 g / L sorbitol, 1 g / L yeast extract, 0.2 g / L magnesium sulfate, 0.5 g / L potassium hydrogen phosphate, 0.1 g / L sodium chloride, 0.05 g / L calcium chloride, 0.02 g / L boric acid, 0.02 g / L sodium molybdate monohydrate, 18 g / L agar, pH 7.0±0.1, and the culture medium is sterilized at 115-118°C for 20-30 min.

[0039] The seed culture medium includes: sorbitol 10g / L, yeast extract powder 1g / L, magnesium sulfate 0.2g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.1g / L, calcium chloride 0.05g / L, boric acid 0.02g / L, sodium molybdate monohydrate 0.02g / L, pH is 7.0±0.1, and the culture medium is sterilized at 115-118°C for 20-30min.

[0040] The fermentation medium includes the following components: sorbitol 10g / L, yeast extract powder 1g / L, magnesium sulfate 0.2g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.1g / L, calcium chloride 0.05g / L, boric acid 0.02g / L, sodium molybdate monohydrate 0.02g / L, pH 7.0±0.1, and the culture medium is sterilized at 115-118°C for 20-30min.

[0041] Example 1 Preparation of soybean rhizobium freeze-dried powder: (1) Preparation of strains: Soybean rhizobium (the soybean rhizobium used in this example was purchased from China Agricultural University with a product number of CCBAU 45436, and its detailed information is recorded in the literature Li YZ, Wang D, Feng XY, Jiao J, Chen WX, Tian CF. Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants. Appl Environ Microbiol. 2015 Dec18;82(4):1305-15) was inoculated on a plate culture medium and cultured in a constant temperature incubator at 28°C for 20 h; (2) Seed culture: a single colony is selected from the strain cultured in step (1), inoculated into a seed culture medium, and cultured at 28° C. and 180 rpm for 18 h to obtain a seed solution; (3) Fermentation culture: According to the inoculation amount of 2%, the cultured seed liquid was transferred to the sterilized fermentation medium under aseptic operation. The culture conditions were: tank pressure 0.05MPa, 28℃, stirring speed 180rpm; during the fermentation process, when the pH value rose, glucose was added immediately to control the glucose content of the fermentation liquid to <0.5g / L, and fermentation was continued until the number of soybean rhizobia nodules no longer increased; after the fermentation was completed, the temperature of the fermentation liquid was immediately reduced to 10±1℃; (4) Centrifugation: The fermentation broth was centrifuged at 8000 rpm for 5 min in a tabletop centrifuge to collect the bacterial cells; 2 times 10°C purified water was added to the bacterial cells, and the mixture was thoroughly stirred and then centrifuged at 8000 rpm for 5 min in a tabletop centrifuge. This was repeated twice to collect the sludge bacterial cells; (5) Compounding: Weigh an appropriate amount of muddy bacterial cells, add 1.5 times (by weight) of auxiliary materials, mix thoroughly and evenly to obtain an emulsion; The auxiliary material formula is: skimmed milk powder 15g / L, trehalose 10g / L, maltodextrin 7g / L, ascorbic acid 0.8g / L, sodium glutamate 10g / L, glycerol 8g / L, icodopine 0.1g / L, PHA 0.1g / L; (6) Drying: The material obtained in step (5) is evenly added into a 20 mL vial and subjected to vacuum freeze drying to obtain a lyophilized powder; (7) Packaging: The vial is stoppered in a freeze dryer under vacuum. After the vial is taken out, it is capped with an aluminum cap to prepare soybean rhizobium freeze-dried powder.

[0042] Example 2 This example is used to measure the fermentation effect of the soybean rhizobium freeze-dried powder prepared in Example 1.

[0043] In a vial containing 1g of soybean rhizobium freeze-dried powder, inject 9mL of sterile water and fully dissolve it. Inoculate the dissolved soybean rhizobium into the fermentation medium at a 2% inoculation rate and culture at 28°C and 180rpm for 24 hours. After the fermentation is completed, the fermentation status is detected by Gram staining and dilution coating counting. For detailed results, see Figure 1-2 .

[0044] from Figure 1-2 It can be seen that the soybean rhizobium freeze-dried powder can grow when inoculated into the fermentation medium, and the number of live bacteria can reach 10 after 24 hours of cultivation. 9 CFU / mL.

[0045] Example 3 This example tests the storage temperature of soybean rhizobium freeze-dried powder.

[0046] The soybean rhizobium freeze-dried powder was stored at 4°C and 25°C, and the number of live bacteria was tested every 80 days until 360 days.

[0047] Method for detecting viable bacterial count: Take a vial containing 1g of soybean rhizobium freeze-dried powder and inject 9mL of sterile water to fully dissolve it. Take 1mL of the above liquid and add it to 9mL of sterile water to obtain 10 -2 Repeat the process until you get 10 -8 Take 100 μL of each gradient dilution and spread it evenly on the plate, and culture it at 28°C for 36 hours.

[0048] After the soybean rhizobium freeze-dried powder was stored at 4°C and 25°C for 360 days, the survival rate was still 68%-83%, as shown in Table 1.

[0049] Table 1 Changes in the number of live bacteria of soybean rhizobia at 4℃ and 25℃

[0050] When the soybean rhizobium freeze-dried powder was stored in an environment of 4°C for 360 days, the number of live bacteria gradually decreased, and the final survival rate reached 83.41%; when it was stored in an environment of 25°C for 360 days, the number of live bacteria gradually decreased, and the final survival rate reached 68.75%.

[0051] Example 4 This example is used to determine the heat resistance of soybean rhizobium freeze-dried powder.

[0052] The soybean rhizobium freeze-dried powder was fumigated at 50, 60, 70 and 80°C for 20 min, and then the number of live bacteria and the fermentation level were detected.

[0053] Method for detecting viable bacterial count: Take a vial containing 1g of soybean rhizobium freeze-dried powder and inject 9mL of sterile water to fully dissolve it; take 1mL of the above liquid and add it to 9mL of sterile water to obtain 10 -2 Repeat the process until you get 10 -8 The dilutions were taken, and 100 μL of each gradient dilution was evenly spread on the plate and cultured at 28° C. for 36 hours; the fermentation level detection method was carried out according to Example 2, and the results are shown in Table 2.

[0054] Table 2 Changes in the number of live bacteria of soybean rhizobium 45436 under high temperature treatment of 50-80℃

[0055] The number of live bacteria of soybean rhizobium freeze-dried powder decreased after fumigation at 50-80℃ water vapor for 20 minutes. After fumigation at 50℃, the survival rate was still 87.67%. When inoculated into the fermentation medium at a 2% inoculation rate, it could still ferment to 1.74×10 9 CFU / mL.

[0056] The above results indicate that the freeze-dried powder of soybean rhizobium provided in the present disclosure has good survival rate and stability, and can be used for soybean seed dressing to maintain high activity of rhizobium strains.

[0057] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0058] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0059] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A soybean rhizobium freeze-dried powder, characterized in that: The soybean rhizobium freeze-dried powder contains bacteria and adjuvants; The bacterial body contains soybean rhizobium; the auxiliary agent includes at least one of skimmed milk powder, maltodextrin, trehalose, ascorbic acid, sodium glutamate, glycerol, ectoine and polyhydroxyalkanoate.

2. The soybean rhizobium freeze-dried powder according to claim 1, wherein Calculated on a dry matter basis, the weight ratio of the bacterial cells to the auxiliary agent is 1:(1-2); Wherein, in the auxiliary agent, relative to 10-20 weight parts of skimmed milk powder, the auxiliary agent also contains 10-50 weight parts of maltodextrin, 10-60 weight parts of trehalose, 0.1-1.5 weight parts of ascorbic acid, 5-15 weight parts of sodium glutamate, 5-10 weight parts of glycerol, 0.01-0.1 weight parts of icodone and 0.01-0.1 weight parts of polyhydroxyalkanoate.

3. A method for preparing soybean rhizobium freeze-dried powder, characterized in that: The preparation method comprises the following steps: S1. Inoculate soybean rhizobia on a plate medium and culture at 26-30°C for 12-36h; S2, picking a single colony from the strain cultured in step S1, inoculating it into a seed culture medium, and culturing it at 26-30° C. and 150-220 rpm for 12-24 h to obtain a seed solution; S3, inoculating the seed liquid into the fermentation medium at an inoculum amount of 1-5%, culturing for 12-36 hours at 26-30°C, 150-220rpm, 0.04-0.06MPa, and pH 6.9-7.1 to obtain a fermentation liquid; if the pH value rises during the fermentation process, adding glucose to the fermentation medium so that the glucose content in the fermentation medium is 0.1-0.5g / L; S4, cooling the fermentation broth to 10-15°C, centrifuging and collecting the cells; adding 1-5 times of 4-10°C purified water to the collected cells for redissolution, and centrifuging and collecting the cells again; repeating the above operation until the supernatant is colorless, and collecting the muddy cells; S5, uniformly mixing the mud-like bacterial cells with the auxiliary agent, and subjecting them to vacuum freeze-drying treatment; The auxiliary agent includes at least one of skimmed milk powder, maltodextrin, trehalose, ascorbic acid, sodium glutamate, glycerol, ectoine and polyhydroxyalkanoate.

4. The method according to claim 3, wherein: Calculated on a dry matter basis, the weight ratio of the mud-like bacterial body to the auxiliary agent is 1:(1-2); The auxiliary agent contains 10-20 parts by weight of skimmed milk powder, 10-50 parts by weight of maltodextrin, 10-60 parts by weight of trehalose, 0.1-1.5 parts by weight of ascorbic acid, 5-15 parts by weight of sodium glutamate, 5-10 parts by weight of glycerol, 0.01-0.1 parts by weight of icodone and 0.01-0.1 parts by weight of polyhydroxyalkanoate.

5. The method according to claim 3, wherein: The plate culture medium contains: 9-11 g / L of sorbitol, 0.8-1.2 g / L of yeast extract powder, 0.1-0.3 g / L of magnesium sulfate, 0.4-0.6 g / L of dipotassium hydrogen phosphate, 0.08-0.12 g / L of sodium chloride, 0.04-0.06 g / L of calcium chloride, 0.01-0.03 g / L of boric acid, 0.01-0.03 g / L of sodium molybdate monohydrate, 17-19 g / L of agar, and has a pH of 6.9-7.

1.

6. The method according to claim 3, wherein: The seed culture medium contains: 9-11 g / L sorbitol, 0.8-1.2 g / L yeast extract powder, 0.1-0.3 g / L magnesium sulfate, 0.4-0.6 g / L dipotassium hydrogen phosphate, 0.08-0.12 g / L sodium chloride, 0.04-0.06 g / L calcium chloride, 0.01-0.03 g / L boric acid, 0.01-0.03 g / L sodium molybdate monohydrate, and has a pH of 6.9-7.

1.

7. The method according to claim 3, wherein: The fermentation medium contains: 9-11 g / L sorbitol, 0.8-1.2 g / L yeast extract powder, 0.1-0.3 g / L magnesium sulfate, 0.4-0.6 g / L dipotassium hydrogen phosphate, 0.08-0.12 g / L sodium chloride, 0.04-0.06 g / L calcium chloride, 0.01-0.03 g / L boric acid, 0.01-0.03 g / L sodium molybdate monohydrate, and has a pH of 6.9-7.

1.

8. Use of the soybean rhizobium freeze-dried powder according to any one of claims 1 to 2 or the soybean rhizobium freeze-dried powder prepared by the method according to any one of claims 3 to 7 in crop seed dressing, wherein the crop is preferably a leguminous crop, and more preferably the leguminous crop is soybean.

9. A seed coating agent, wherein: The seed coating agent contains the soybean rhizobium freeze-dried powder according to any one of claims 1-2 or the soybean rhizobium freeze-dried powder prepared by the method according to any one of claims 3-7.

10. A coated seed, wherein: The coated seeds contain the soybean rhizobium freeze-dried powder according to any one of claims 1-2 or the soybean rhizobium freeze-dried powder prepared by the method according to any one of claims 3-7.

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