A polysaccharide-producing rhizobium mutant strain and its application

Abstract

The present invention discloses a polysaccharide-producing rhizobium mutant strain and its application, specifically belonging to the technical field of mutagenized rhizobia; the polysaccharide-producing rhizobium mutant strain is named rhizobium mutant strain Y1-12, which is obtained by optimizing and screening through ARTP plasma mutagenesis technology; compared with traditional rhizobia, the polysaccharide yield of rhizobium mutant strain Y1-12 is significantly improved; the feasibility of the polysaccharide-producing rhizobium mutant strain Y1-12 in industrial production is verified in the fermentation tank scale-up culture experiment, wherein the highest content of β-glucan can reach 14.441 g / L, which is 34.55% higher than that of the original strain, laying a certain foundation for the subsequent application of this mutant strain in polysaccharide synthesis and polysaccharide fermentation industrialization.

Description

Technical Field

[0001] The invention belongs to the technical field of mutated rhizobia, and in particular relates to a polysaccharide-producing rhizobium mutant strain and application thereof. Background Art

[0002] Rhizobia are extremely important symbiotic microorganisms in nature. They form nodules with a variety of plants, especially leguminous plants, and provide plants with essential nitrogen sources through nitrogen fixation, which is of immeasurable value to the sustainable development of agriculture. In recent years, in addition to its nitrogen fixation properties, the exopolysaccharides (EPS) produced by rhizobia have gradually become a research hotspot in scientific research and industry due to their unique physical and chemical properties and wide range of biological activities. EPS, as a new type of food additive, drug carrier and biomaterial, has gradually shown great market potential, which has prompted scientists to continuously seek effective ways to increase the production of rhizobium polysaccharides.

[0003] Although traditional rhizobia have inherent polysaccharide production capabilities, the yield and quality are often difficult to meet the growing market demand. Therefore, screening out rhizobia strains with high polysaccharide production through mutagenesis technology has become a key task. Mutagenesis technology introduces gene mutations through physical, chemical or biological methods to create a strain library with rich genetic diversity, thus having the opportunity to obtain mutagenized strains with significant improvements in polysaccharide synthesis. This targeted screening strategy can not only discover strains with higher yields, but also discover new types of polysaccharides and broaden their application areas.

[0004] In view of this, we explore efficient Rhizobium mutant strain screening and cultivation technology, find and cultivate Rhizobium mutant strains that can efficiently synthesize polysaccharides, aiming to efficiently produce more and higher-quality polysaccharides to meet the urgent needs of biotechnology, agriculture, medicine and other fields, and promote technological innovation and green development of related industries. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a rhizobium mutant strain capable of producing polysaccharides, and based on the rhizobium mutant strain, provide more efficient polysaccharide output in terms of polysaccharide fermentation and polysaccharide synthesis technology.

[0006] In order to achieve the above object, the present invention discloses the following technical solutions:

[0007] In the first aspect, the present invention provides a polysaccharide-producing Rhizobium mutant strain, the Rhizobium mutant strain is named Rhizobium mutant strain Y1-12, the preservation name is Rhizobium sp. Y1-12, the preservation number is CCTCC NO: M 20241902, the preservation date is September 2, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation unit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] In a second aspect, the present invention provides the use of the polysaccharide-producing rhizobium mutant strain described in the first aspect in the preparation of extracellular polysaccharide.

[0009] In a third aspect, the present invention provides the use of the polysaccharide-producing rhizobium mutant strain described in the first aspect in the preparation process of extracellular polysaccharide fermentation.

[0010] In a fourth aspect, the present invention provides a method for preparing a polysaccharide fermentation product based on the polysaccharide-producing rhizobium mutant strain described in the first aspect, comprising the following steps:

[0011] (1) Take monoclonal colonies of the well-growing rhizobium mutant strain Y1-12 and inoculate them into YMA liquid medium for cultivation until the bacteria reach the logarithmic growth phase to obtain the activated bacterial liquid;

[0012] (2) Inoculate the activated bacterial liquid into the seed medium for cultivation according to an inoculation amount of 0.5 v / v% to further amplify the number of bacteria and obtain the seed liquid;

[0013] (3) Select the well-growing seed liquid and inoculate it into the fermentation medium according to an inoculation amount of 6.7% and ferment for 30-36 h to obtain the fermentation broth;

[0014] (4) Extract and purify the polysaccharide from the fermentation broth to obtain the polysaccharide fermentation product.

[0015] Preferably, the components of the YMA liquid medium include solvent deionized water, 10 g / L of sucrose, 3 g / L of yeast powder, 0.25 g / L of KH2PO4, 0.25 g / L of K2HPO4, 0.2 g / L of MgSO4·7H2O, 0.1 g / L of NaCl, and pH = 6.8-7.0.

[0016] Preferably, the components of the seed medium include 10 g / L of sucrose, 3 g / L of yeast powder, 0.25 g / L of K2HPO4, 0.25 g / L of KH2PO4, 0.2 g / L of MgSO4·7H2O, 0.1 g / L of NaCl, solvent deionized water, and pH = 6.8-7.0.

[0017] Preferably, the components of the fermentation medium include 35-45 g / L of sucrose, 9 g / L of yeast powder, 0.25 g / L of K2HPO4, 0.25 g / L of KH2PO4, 0.2 g / L of MgSO4·7H2O, 0.1 g / L of NaCl, solvent deionized water, and pH = 6.8-7.0.

[0018] Preferably, the cultivation conditions in step (1) are cultivation at 30 °C and 200 r / min for 10-12 h to make the bacteria reach the logarithmic growth phase.

[0019] Preferably, the culture conditions in step (2) are to culture for 12 h at 30 °C and 200 r / min to further amplify the number of bacteria and obtain a seed solution.

[0020] Preferably, the fermentation conditions in step (3) are as follows: the rotation speed is ≥200 r / min, the fermentation temperature is 30 °C, the ventilation rate is ≥1.5 vvm, the dissolved oxygen content is 28 - 32%, and the pH value is 6.8 - 7.2.

[0021] Advantages of the present invention:

[0022] 1. Through the ARTP plasma mutagenesis technology, a rhizobium mutagenesis strain Y1-12 with high polysaccharide yield was screened out. Compared with traditional rhizobia, the polysaccharide yield was significantly improved. Among them, after 48-hour shake flask culture, the average polysaccharide concentration of the Y1-12 mutagenesis strain reached 2.19 g / L, an increase of 29.59% compared with 1.69 g / L of the original strain.

[0023] 2. By comparing the polysaccharide yields at different culture times, it was found that 48 hours was the optimal harvesting time for the Y1-12 mutagenesis strain in the shake flask culture stage. In the fermentation tank scale-up culture stage, the optimal harvesting time could be advanced to 30 - 36 hours. At this time, the polysaccharide yield reached the peak and then decreased, so the high-yield state could be locked in time, reducing energy consumption and costs, and improving the turnover rate of production facilities.

[0024] 3. Through the fermentation tank scale-up culture experiment, the feasibility of the high-polysaccharide-producing rhizobium mutagenesis strain Y1-12 in industrial production was verified. The highest β-glucan content could reach 14.441 g / L, an increase of 34.55% compared with the original strain, laying a foundation for subsequent industrial application.

[0025] 4. The high sucrose content in the fermentation medium can increase the polysaccharide yield of rhizobia, which has a promoting effect on both the original strain and the mutagenesis strain. The polysaccharide yield of the original strain also reached a relatively high level, further improving the polysaccharide yield of the mutagenesis strain. Description of the Drawings

[0026] In order to more clearly illustrate the technical solution of the present invention, the drawings required for description in the embodiments will be briefly introduced below.

[0027] Figure 1 It is the standard curve graph of the β-glucan standard product of the rhizobium mutagenesis strain Y1-12;

[0028] Figure 2 It is the high performance liquid chromatography graph of the β-glucan content in the fermentation broth of the rhizobium mutagenesis strain Y1-12.

[0029] Figure 3It is the standard curve graph of the β-glucan standard product of the original rhizobium strain;

[0030] Figure 4 It is the high performance liquid chromatography graph of the β-glucan content in the fermentation broth of the original rhizobium strain. Specific implementation manners

[0031] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the following further describes the present invention in detail with reference to specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention, not all of them. For clarity, not all features of the actual embodiments are described.

[0032] Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all fall within the protection scope of the present invention.

[0033] In the present invention:

[0034] The Latin scientific name of the original rhizobium strain Rhizobium pusense , was isolated from the soil in Changzhi area, Shanxi Province;

[0035] The genetically stable rhizobium mutant strain Y1-12 was deposited at the China Center for Type Culture Collection (abbreviation: CCTCC; address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, postal code: 430072) on September 2, 2024. The deposit name is Rhizobium sp. Y1-12, and the deposit number is CCTCC NO: M 20241902. The taxonomic naming of the rhizobium mutant strain Y1-12 is Rhizobium pusense, and the Latin scientific name is Rhizobium sp.

[0036] Preparation of the rhizobium mutant strain Y1-12

[0037] 1. ARTP mutagenesis treatment:

[0038] Use an ARTP plasma mutagenesis instrument for mutagenesis. Atmospheric and room temperature plasma (ARTP) is a plasma formed by the ionization of carrier gas at normal temperature and atmospheric pressure. The ARTP technology induces DNA damage to biomolecules such as DNA more evenly and efficiently through oxidative ions and free radicals, thereby inducing gene mutations in strains. Its principle is similar to that of traditional mutagenic agents such as ultraviolet rays and X-rays, but its radiation range is larger, and the mutagenesis efficiency can reach dozens of times that of traditional technologies.

[0039] (1) Preparation of the bacterial suspension: Centrifuge and collect the bacterial liquid cultured to the logarithmic phase, wash it twice with physiological saline, and then appropriately dilute it with physiological saline to prepare a bacterial suspension with an OD600 value between 0.6 and 0.8.

[0040] (2)ARTP mutagenesis combined mutagenesis: After adding the mutagen to the bacterial suspension, the plasma flow generated by the ARTP mutagenesis instrument was used to treat it for 20 s, 40 s, and 60 s for mutagenesis. After mutagenesis, the bacterial solution was diluted 10 5 times and then spread, and cultured at 30 °C until single colonies grew.

[0041] 2. Screening and culturing of mutant strains:

[0042] 2.1 Selecting single colonies for culture

[0043] Independent single colonies were selected one by one from the bacterial population that had been mutagenized and evenly spread on the YMA solid medium plate. These single colonies were then separately inoculated onto new YMA solid medium plates for culture to ensure their purity and growth characteristics. After this series of screening and culturing work, more than 1830 single colony cultures were finally selected.

[0044] YMA medium: Solvent deionized water, sucrose 10 g / L, yeast powder 3 g / L, KH2PO4 0.25 g / L, K2HPO4 0.25 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.1 g / L, pH = 6.8 - 7.0. On the basis of the liquid YMA medium, agar powder (usually about 15 g / L) was added, and it solidified after heat sterilization to form the YMA solid medium.

[0045] 2.2 Culturing mutant rhizobia in 96-well deep well plates

[0046] The previously mutagenized rhizobia strains were selected and cultured using 96-well deep well plates.

[0047] 1 mL of universal medium was added to the 96-well deep well plate, and single colonies were picked and inoculated into the medium. They were cultured at 30 °C and 200 rpm for three days (72 h). Then, the diameters of the polysaccharide circle outside the rhizobia and the colony diameter inside the circle in each deep well were measured respectively, and the ratio was calculated. Subsequently, the cultures in the deep wells were extracted separately, and the cultures were inactivated, that is, heated at 100 °C for 10 min to kill the cells. Next, the heat-treated bacterial solution was aspirated, and separated by centrifugation (centrifugation parameters 12000 rpm, 5 min), and only the supernatant part was retained and collected for subsequent experiments or analyses.

[0048] Universal medium: Solvent deionized water, sucrose 20 g / L, yeast powder 9 g / L, KH2PO4 0.25 g / L, K2HPO4 0.25 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.1 g / L, pH = 6.8 - 7.0.

[0049] 2.3 Screening of Mutant Strains with High Polysaccharide Yield

[0050] The color reaction characteristics between the rhizobium bacterial solution and the congo red solution were used to identify and screen mutant strains capable of efficiently producing polysaccharides.

[0051] The specific steps are as follows: First, 100 μL of the supernatant was aspirated from the centrifuged bacterial solution and mixed with an equal volume (i.e., 100 μL) of congo red solution with a concentration of 0.3 mg / mL in a 96-well plate, and left to react for 20 min to allow full reaction and color change. The absorbance value of the reaction system was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a specific wavelength (490 nm). 490 nm is the absorbance of the congo red dye solution, and the polysaccharide content was judged according to the absorbance value. 600 nm was used to measure the cell concentration of rhizobia.

[0052] 2.4 Flask Culture Experiment of Mutant Strains

[0053] Six strains with the highest absorbance and the original strain were cultured in a 250 mL YMA medium flask to obtain seed liquid. When the OD value reached between 0.6 and 0.8, 1 mL was taken and inoculated into a flask containing 500 mL of general medium for culture. The OD values of the bacterial solution were measured at 24 h, 48 h, and 72 h during the culture process. The bacterial solution was diluted to an appropriate multiple, and the absorbance at a wavelength of 600 nm was detected using a spectrophotometer. The absorbance was between 0.2 and 0.8, and then sent for liquid phase detection, alcohol precipitation, freeze-drying, and weighing. Alcohol precipitation and freeze-drying: 20 mL of the original strain and mutant strain bacterial solutions were taken respectively, centrifuged at 12000 rpm for 10 min, 10 mL of the supernatant was taken, and 4 times the volume of 95% ethanol was added for alcohol precipitation overnight. Centrifuge at 8000 rpm for 10 min, remove the supernatant, and freeze-dry.

[0054] Each of the above strains was cultured in 4 groups. The average OD(600) value and polysaccharide concentration after 48 hours of culture of each strain are shown in Table 1:

[0055] Table 1 Detection Values of Flask Culture for 48 h of Screened Strains and Original Strains

[0056]

[0057] glj① (2-E11) had the best performance, which was used as the target strain for preservation and named mutant strain Y1-12. Hereinafter, it is all referred to as Y1-12.

[0058] 2.5 Detection Results and Analysis

[0059] Table 2 is a detailed record of the growth status and polysaccharide production of the original strain and the Y1-12 mutagenized strain (4 groups each) at different time points (24h, 48h, 72h) in 2.4 above. The performance of the two was evaluated by comparing three indicators: the polysaccharide concentration obtained by alcohol precipitation and freeze-drying, the optical density (OD value) of the bacterial solution, and the peak area of the liquid chromatography detection of the polysaccharide in the bacterial solution.

[0060] Table 2 Detection values of the original strain (JJ-157) and the Y1-12 mutagenized strain

[0061]

[0062] Result analysis of Table 2:

[0063] (1) Polysaccharide obtained by alcohol precipitation and freeze-drying (g / L)

[0064] After culturing for 24h: The average polysaccharide concentration of the original strain was 1.48 g / L, while that of the Y1-12 mutagenized strain was 1.61 g / L, and the increase in the average polysaccharide concentration was 8.78%.

[0065] After culturing for 48h: The average polysaccharide concentration of the original strain increased to 1.69 g / L, while that of the Y1-12 mutagenized strain reached 2.19 g / L, and the increase in the average polysaccharide concentration was 29.59%.

[0066] After culturing for 72h: The average polysaccharide concentration of the original strain decreased slightly to 1.77 g / L, while that of the Y1-12 mutagenized strain was 1.88 g / L, and the increase in the average polysaccharide concentration slowed down to 6.21%.

[0067] At the same time, the reduction in polysaccharide production of Y1-12 from 48h to 72h was recorded. The polysaccharide production of Y1-12 at 72h decreased by an average of 0.31 g / L compared to 48h, and the average reduction rate was 14.16%.

[0068] Therefore, during the shake-flask culture stage, culturing for 48h is the optimal polysaccharide harvesting time for the Y1-12 strain, and the polysaccharide production reaches the highest value range.

[0069] (2) OD value (optical density)

[0070] From 24h to 72h: As the culture time increased, the OD values of both the original strain and the Y1-12 mutagenized strain showed an upward trend, indicating an increase in cell density. The growth rate of the average OD value of the Y1-12 mutagenized strain compared to the original strain at each time point was 2.52% (24h), 11.24% (48h), and 6.09% (72h), showing that its cell density increased more rapidly.

[0071] (3) Peak area of liquid-phase detection of polysaccharide in the bacterial solution

[0072] 24 h and 48 h: The Y1-12 mutagenized strain also showed outstanding performance in the liquid polysaccharide detection. The growth rate of the peak area far exceeded that of the original strain. The average growth rates of the mutagenized strain relative to the original strain were 76.94% (24 h) and 42.43% (48 h) respectively. The polysaccharide yield of the Y1-12 mutagenized strain was significantly higher than that of the original strain.

[0073] Preparation of Fermenter Culture Examples and Comparative Examples

[0074] I. Preparation of Example 1

[0075] 1. Fermenter culture of mutagenized strain

[0076] 1.1 Strain and medium preparation

[0077] (1) Strain preparation:

[0078] Use the rhizobium mutagenized strain Y1-12 obtained above.

[0079] (2) Activation medium preparation:

[0080] Use YMA medium as the activation medium.

[0081] (3) Seed medium preparation:

[0082] The seed medium includes sucrose (10 g / L), yeast powder (3 g / L), K2HPO4 (0.25 g / L), KH2PO4 (0.25 g / L), MgSO4·7H2O (0.2 g / L), NaCl (0.1 g / L). Add the weighed chemical components to deionized water, stir well to mix all components, adjust the pH value of the medium to 6.8 - 7, and sterilize the medium using a high-pressure steam sterilizer to ensure aseptic conditions. The sterilized medium should be stored under aseptic conditions until use.

[0083] (4) Fermentation medium preparation:

[0084] The fermentation medium includes sucrose (40 g / L), yeast powder (9 g / L), K2HPO4 (0.25 g / L), KH2PO4 (0.25 g / L), MgSO4·7H2O (0.2 g / L), NaCl (0.1 g / L). Add the weighed chemical components to deionized water, stir well to mix all components, adjust the pH value of the medium to 6.8 - 7, and sterilize the medium using a high-pressure steam sterilizer to ensure aseptic conditions. The sterilized medium should be stored under aseptic conditions until use.

[0085] 1.2 Activation and seed culture

[0086] (1) Seed activation:

[0087] Select monoclonal colonies of Rhizobium Y1-12 with good growth from the YMA solid plate, inoculate them into 4 mL of YMA liquid medium, and culture them overnight (10 - 12 h) in a constant temperature shaker at 30 °C and 200 r / min to make the bacteria reach the logarithmic growth phase.

[0088] (2)Seed culture:

[0089] Inoculate the activated bacterial liquid into a 500 mL shake flask containing 200 mL of seed medium at an inoculation amount of 0.5 v / v%, and continue to culture it at 30 °C and 200 r / min for about 12 hours to further amplify the number of bacteria and obtain the seed liquid.

[0090] 1.3 Fermentation tank culture process:

[0091] (1)Inoculation: Select the seed liquid with good growth and inoculate it into a 5 L fermentation tank containing fermentation medium that has been verified to be free of contaminants by microscopic examination at an inoculation amount of 6.7%. Among them, the inoculated bacterial liquid is 0.2 L, the fermentation medium is 2.8 L, and the total volume of the fermentation broth is 3 L.

[0092] (2)Initial culture condition setting: Set the initial rotation speed of the fermentation tank to 200 r / min, and gradually increase the rotation speed as the bacteria grow, and set the temperature to 30 °C. The aeration ratio is set to 3:2 (the volume ratio of air passing through the unit volume of culture medium per minute, V / V·min), and then gradually increase it to ensure sufficient oxygen supply.

[0093] (3)Monitoring and control of dissolved oxygen and pH: Use a dissolved oxygen probe to monitor the dissolved oxygen level in the tank to ensure that the dissolved oxygen content is controlled at about 30%. Use NaOH and KH2PO4 to adjust the pH value to keep the pH at about 7.0.

[0094] (4)Obtain the fermentation broth: The polysaccharide can reach the highest level after fermentation for about 30 - 36 h. At this time, the subsequent extraction and purification steps can be carried out on the fermentation broth.

[0095] 1.4 Detection of β-glucan content

[0096] Use high performance liquid chromatography to detect the β-glucan content in the supernatant of the fermentation broth, and use the purchased β-glucan standard as a control.

[0097] High performance liquid chromatography conditions:

[0098] Instrument: Waters 2690 / 5 high performance liquid chromatograph, equipped with a 2414 differential refractive index detector;

[0099] Chromatographic column: PL aquagel-OH 60 (8um, 300×7.5mm)

[0100] Column temperature: 40°C

[0101] Mobile phase: 0.1mol / L sodium nitrate solution

[0102] Flow rate: 1.0mL / min

[0103] Detection temperature: 35°C

[0104] External standard method standard curve:

[0105] Standard curve equation of the mutagenized strain: Y = 226525X - 1133R 2 = 0.999111; See the standard curve graph in Figure 1 .

[0106] Calculated by the external standard method, the highest yield of β-glucan can reach 14.441 g / L. See the chromatogram in Figure 2 .

[0107] II. Preparation of Comparative Example 1

[0108] Fermentation tank culture of the original strain

[0109] The original strain of rhizobia was cultured. The culture conditions and process were exactly the same as those in Example 1. Finally, calculated by the external standard method, the highest yield of β-glucan was 10.733 g / L. See the chromatogram in Figure 4 .

[0110] Standard curve equation of the original strain: Y = 216556X + 445R 2 = 0.999785; See the standard curve graph in Figure 3 .

[0111] III. Result analysis of Example 1 and Comparative Example 1

[0112] (1) Through the ARTP plasma mutagenesis technology, a high-polysaccharide-producing rhizobia mutagenized strain Y1-12 was screened out. Compared with the traditional rhizobia, the polysaccharide yield was significantly improved. Among them, after 48-hour shake flask culture, the average polysaccharide concentration of the Y1-12 mutagenized strain reached 2.19 g / L, an increase of 29.59% compared with 1.69 g / L of the original strain.

[0113] (2) By comparing the polysaccharide yields at different culture times, it was found that 48 hours was the optimal harvesting time for the Y1-12 mutant strain during the shake flask culture stage; in the fermenter scale-up culture stage, the optimal harvesting time could be advanced to 30-36 hours. At this time, the polysaccharide yield reached a peak, and the yield decreased thereafter, so the high-yield state could be locked in time, reducing energy consumption and costs, and improving the turnover rate of production facilities.

[0114] (3) Through the fermenter scale-up culture experiment, the feasibility of the high-polysaccharide-producing rhizobium mutant strain Y1-12 in industrial production was verified. The highest content of β-glucan could reach 14.441 g / L, which was 34.55% higher than that of the original strain, laying a foundation for subsequent industrial applications.

[0115] (4) The high sucrose content in the fermentation medium can increase the polysaccharide yield of rhizobia, which has a promoting effect on both the original strain and the mutant strain. The polysaccharide yield of the original strain also reached a relatively high level, further increasing the polysaccharide yield of the mutant strain.

[0116] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that this is only an example. The protection scope of the present invention is defined by the appended claims. Without departing from the principle and essence of the present invention, those skilled in the art can make various changes or modifications to these embodiments, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A polysaccharide-producing rhizobium mutant strain, characterized in that: The rhizobium mutant strain is named as rhizobium mutant strain Y1-12, the preservation name is Rhizobium sp. Y1-12, the preservation number is CCTCC NO: M 20241902, the preservation date is September 2, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation unit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Use of the polysaccharide-producing rhizobium mutant strain according to claim 1 in the preparation of extracellular polysaccharides.

3. Use of the polysaccharide-producing rhizobium mutant strain according to claim 1 in the fermentation process for preparing extracellular polysaccharides.

4. A method for preparing a polysaccharide fermentation product based on the polysaccharide-producing rhizobium mutant strain according to claim 1, characterized in that: The following steps are involved: (1) Take a monoclonal colony of the well-growing rhizobium mutant strain Y1-12 and inoculate it into YMA liquid medium to culture the bacteria until they reach the logarithmic growth phase and obtain an activated bacterial solution; (2) inoculating the activated bacterial solution into the seed culture medium at an inoculum volume of 0.5 v / v% to further amplify the bacterial count and obtain the seed solution; (3) Selecting seed liquid with good growth and inoculating it into the fermentation medium at an inoculation rate of 6.7%, fermenting for 30 to 36 hours to obtain fermentation liquid; (4) extracting and purifying polysaccharides from the fermentation broth to obtain the polysaccharide fermentation product; The components of the YMA liquid culture medium include solvent deionized water, 10 g / L sucrose, 3 g / L yeast powder, 0.25 g / L KH2PO4, 0.25 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.1 g / L NaCl, and pH=6.8-7.

0.

5. The preparation method according to claim 4, characterized in that: The components of the seed culture medium include 10 g / L sucrose, 3 g / L yeast powder, 0.25 g / L K2HPO4, 0.25 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 0.1 g / L NaCl, and solvent deionized water, with a pH value of 6.8-7.

0.

6. The preparation method according to claim 4, characterized in that: The components of the fermentation medium include 35-45 g / L sucrose, 9 g / L yeast powder, 0.25 g / L K2HPO4, 0.25 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 0.1 g / L NaCl, and solvent deionized water, with a pH of 6.8-7.

0.

7. The preparation method according to claim 4, characterized in that: The culture conditions in step (1) are culturing at 30° C. and 200 r / min for 10 to 12 hours to allow the bacteria to reach a logarithmic growth phase.

8. The preparation method according to claim 4, characterized in that: The culture conditions in step (2) are culturing at 30° C. and 200 r / min for 12 h to further amplify the number of bacteria and obtain a seed solution.

9. The preparation method according to claim 4, characterized in that: The fermentation conditions in step (3) are: rotation speed ≥ 200 r / min, fermentation temperature 30°C, ventilation volume ≥ 1.5 vvm, dissolved oxygen content 28-32%, and pH value 6.8-7.2.

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