Culture medium for increasing yield of azotobacter caulo-fixing rhizobium ORS571 exopolysaccharide as well as preparation method and application of culture medium

Abstract

The invention belongs to the technical field of microbial culture, and particularly relates to a culture medium for increasing the yield of extracellular polysaccharide of azotobacter stem nodule ORS571 as well as a preparation method and application of the culture medium. The calcium ions are used as a second messenger to regulate and control various biological processes of the azotobacter stem nodule ORS 571, so that the production of the extracellular polysaccharide of the azotobacter stem nodule ORS 571 is promoted. Results of the embodiment show that when the culture medium containing the calcium ions is used for fermenting the azotobacter stem nodule ORS571, the yield of the extracellular polysaccharide of the azotobacter stem nodule ORS571 reaches 0.424-0.567 mg / mL, the content of the extracellular polysaccharide of the azotobacter stem nodule ORS571 is remarkably increased compared with that of a treatment group without the calcium ions, and the content of the extracellular polysaccharide of the azotobacter stem nodule ORS571 is remarkably increased in the purified crude polysaccharide obtained after purification, so that the content of the extracellular polysaccharide of the azotobacter stem nodule ORS571 is greatly increased. And the purity of the extracellular polysaccharide of the azotobacter japonicus ORS571 can reach 30 to 40 percent.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture, and particularly relates to a culture medium for improving the extracellular polysaccharide yield of stem nodule nitrogen-fixing rhizobium ORS571, and a preparation method and application thereof. Background Art

[0002] Exopolysaccharides (EPS) are a class of complex biopolymer compounds. They are water-soluble polysaccharides that are secreted outside the cell wall by some special microorganisms during their growth and metabolism, easily separated from the bacteria, and secreted into the environment. They are secondary metabolites of microorganisms and are of great significance to the growth of microorganisms. Different microbial exopolysaccharides have different characteristics and uses. More than 100 microorganisms have been found in nature to produce exopolysaccharides, mainly bacteria, but also a few fungi.

[0003] Azorhizobium caulinodans ORS571 belongs to α-proteobacteria and was originally isolated by Dreyfus et al. from the stem nodules of Sesbania rostrata, a tropical leguminous plant. Its host plants are very limited. Although it can form nodules symbiotically with a variety of Sesbania plants, it only forms effective nodules on S. rostrata. Like other rhizobia, ORS571 provides nitrogen nutrients to the host by fixing free nitrogen in the atmosphere, thereby reducing the host's dependence on nitrogen fertilizers in the soil. Unlike other rhizobia, ORS571 can form nodules on the roots and stems of plants at the same time. It can not only symbiotically fix nitrogen with its natural host, but also autogenously or as an endophyte to fix nitrogen in other plants. Because ORS571 has both the common characteristics of rhizobia and the characteristics of self-fixing nitrogen, it is more valuable for research than other rhizobia.

[0004] However, the low exopolysaccharide production of stem-nodular nitrogen-fixing rhizobium ORS571 is the main factor restricting its large-scale production of exopolysaccharides. The production of exopolysaccharides is easily affected by different factors, which is related to the genetic characteristics and culture conditions of stem-nodular nitrogen-fixing rhizobium ORS571, and is regulated by related genes. Therefore, improving the exopolysaccharide production of stem-nodular nitrogen-fixing rhizobium ORS571 is particularly important for the mass production of bacterial exopolysaccharides. Summary of the invention

[0005] The purpose of the present invention is to make up for the deficiencies of the prior art, increase the extracellular polysaccharide yield of stem nodule nitrogen-fixing rhizobium ORS571, and prepare the extracellular polysaccharide yield of stem nodule nitrogen-fixing rhizobium ORS571 with higher purity.

[0006] In order to achieve the above object, the present invention provides the use of calcium ions in improving the extracellular polysaccharide production of stem nodule nitrogen-fixing rhizobium ORS571.

[0007] The present invention also provides the use of calcium ions in preparing a culture medium for increasing the extracellular polysaccharide production of stem nodule nitrogen-fixing rhizobium ORS571;

[0008] The calcium ion concentration in the culture medium is 0.272-4.45 mmol / L.

[0009] Preferably, the calcium ion concentration in the culture medium is 2.72 mmol / L.

[0010] The present invention also provides a culture medium for improving the extracellular polysaccharide yield of stem nodule nitrogen-fixing rhizobium ORS571, the culture medium comprising 0.5% to 1% DL sodium lactate, 5 to 10 mmol / L K-Pi buffer, 0.1% v / v 1000×Mg-Na-Mo mixed solution, 5 to 10 mmol / L NH4Cl, 0.272 to 4.45 mmol / L CaCl2·2H2O, 5.4 mg / LFeCl3·6H2O, 0.1% v / v 1000× vitamin mixed solution and the balance of water;

[0011] The K-Pi buffer includes 0.615 mol / L K2HPO4 and 0.3851 mol / L KH2PO4;

[0012] The 1000×Mg-Na-Mo mixed solution includes: 100 g / L MgSO4·7H2O, 50 g / L NaCl and 5 g / L Na2MoO4·2H2O;

[0013] The 1000× vitamin mixture includes 2 g / L biotin, 4 g / L niacin and 4 g / L pantothenic acid.

[0014] The present invention also provides a method for preparing the culture medium described in the above technical solution, comprising the following steps:

[0015] DL-lactate sodium, K-Pi buffer, 1000×Mg-Na-Mo mixed solution, NH4Cl and water are mixed and sterilized to obtain a first premix;

[0016] The first premix, CaCl2·2H2O, FeCl3·6H2O and 1000× vitamin mixture are mixed to obtain the culture medium.

[0017] The present invention also provides the use of the culture medium described in the above technical solution in preparing extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571.

[0018] The present invention also provides a method for preparing extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571, comprising the following steps:

[0019] The nodule nitrogen-fixing rhizobium ORS571 is inoculated into the culture medium described in the above technical solution, cultured at 30-37° C. with shaking, and the culture solution is collected; the culture solution contains the extracellular polysaccharide of the nodule nitrogen-fixing rhizobium ORS571.

[0020] Preferably, the shaking culture is carried out at a rotation speed of 150 to 180 rpm and for a time of 12 to 48 hours.

[0021] Preferably, after obtaining the culture solution, the method further comprises: concentrating the culture solution to obtain a concentrated solution;

[0022] performing alcohol precipitation extraction on the concentrated solution to obtain an extract;

[0023] The extract is purified to obtain purified extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571.

[0024] Preferably, the purification step comprises: enzymatically hydrolyzing the extract using papain to obtain an enzymatic hydrolysis product;

[0025] The enzymatic hydrolysis temperature is 55-65°C and the time is 12-48h;

[0026] performing alcohol precipitation on the enzymatic hydrolysis product and collecting a first precipitate;

[0027] Redissolving the first precipitate with water, adjusting the pH to 2.5, and collecting the second precipitate;

[0028] The second precipitate and the potassium acetate solution are mixed, and the mixture is allowed to stand at 4° C., and the third precipitate is collected; the solvent of the potassium acetate solution is a 75% v / v ethanol solution;

[0029] The third precipitate is concentrated and dried to obtain purified extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571.

[0030] Beneficial effects:

[0031] The present invention provides an application of calcium ions in increasing the production of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571. Calcium ions, as second messengers, regulate various biological processes of stem nodule nitrogen-fixing rhizobium ORS571, thereby promoting the production of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571 and increasing the production of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571. The results of the embodiment show that the present invention uses a culture medium containing calcium ions to ferment stem nodule nitrogen-fixing rhizobium ORS571, which can increase the content of extracellular polysaccharides produced by stem nodule nitrogen-fixing rhizobium ORS571. The results of the embodiment show that the production of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571 reaches 0.424-0.567 mg / mL, and compared with the treatment group without adding calcium ions, the content of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571 is significantly increased, and after purification, the purity of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571 reaches 30-40%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0033] Figure 1 is the concentration of extracellular polysaccharide produced by different treatment groups; ** indicates extremely significant difference in variance analysis;

[0034] Figure 2 is the total sugar of extracellular polysaccharide after purification in different treatment groups; among them, * indicates significant difference in variance analysis, and ** indicates extremely significant difference in variance analysis. DETAILED DESCRIPTION

[0035] The invention provides application of calcium ions in improving the extracellular polysaccharide yield of stem nodule nitrogen-fixing rhizobium ORS571.

[0036] In the present invention, as an embodiment, the calcium ion is used as a component of the culture medium to increase the extracellular polysaccharide production of stem nodule nitrogen-fixing rhizobium ORS571. As an embodiment, the calcium ion concentration in the culture medium is 0.272-4.45 mmol / L; as another embodiment, the calcium ion concentration in the culture medium is 2.72 mmol / L

[0037] The invention provides a culture medium for improving the extracellular polysaccharide yield of stem nodule nitrogen-fixing rhizobium ORS571, wherein the culture medium comprises 0.5% to 1% DL sodium lactate, 5 to 10 mmol / L K-Pi buffer, 0.1% v / v 1000×Mg-Na-Mo mixed solution, 5 to 10 mmol / L NH4Cl, 0.272 to 4.45 mmol / L CaCl2·2H2O, 5.4 mg / L FeCl3·6H2O, 0.1% v / v 1000× vitamin mixed solution and the balance of water;

[0038] The K-Pi buffer includes 0.615 mol / L K2HPO4 and 0.3851 mol / L KH2PO4;

[0039] The 1000×Mg-Na-Mo mixed solution includes: 100 g / L MgSO4·7H2O, 50 g / L NaCl and 5 g / L Na2MoO4·2H2O;

[0040] The 1000× vitamin mixture includes 2 g / L biotin, 4 g / L niacin and 4 g / L pantothenic acid.

[0041] The present invention also provides a method for preparing the culture medium described in the above technical solution, comprising the following steps:

[0042] DL-lactate sodium, K-Pi buffer, 1000×Mg-Na-Mo mixed solution, NH4Cl and water are mixed and sterilized to obtain a first premix;

[0043] The first premix, CaCl2·2H2O, FeCl3·6H2O and 1000× vitamin mixture are mixed to obtain the culture medium.

[0044] As an embodiment, the culture medium includes 1% DL sodium lactate, 10mmol / L K-Pi buffer, 0.1% v / v 1000×Mg-Na-Mo mixed solution, 10mmol / L NH4Cl, 0.272~4.45mmol / L CaCl2·2H2O, 5.4mg / LFeCl3·6H2O, 0.1% v / v 1000× vitamin mixed solution and the remainder water.

[0045] As an embodiment, the sterilization temperature is 111-134°C; as another embodiment, the sterilization temperature is 121°C. As another embodiment, the sterilization time is 20-50 minutes; as another embodiment, the sterilization time is 30 minutes. In a specific embodiment of the present invention, the sterilization conditions can be 121°C, 20-30 minutes, 134°C, 20 minutes, or 111°C, 50 minutes.

[0046] As an embodiment, the sterilization pressure is 0.1-0.22MPa; as another embodiment, the sterilization pressure is 0.2MPa. The present invention first mixes DL-lactate sodium, K-Pi buffer, 1000×Mg-Na-Mo mixed solution, NH4Cl and water, and then mixes with CaCl2·2H2O, FeCl3·6H2O and 1000× vitamin mixed solution after sterilization, which can provide trace elements for microbial growth; the CaCl2·2H2O, FeCl3·6H2O and 1000× vitamin mixed solution is the CaCl2·2H2O, FeCl3·6H2O and 1000× vitamin mixed solution filtered through a 0.22μm filter membrane.

[0047] The present invention also provides the use of the culture medium described in the above technical solution in preparing extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571. The present invention has no strict requirements for the stem nodule nitrogen-fixing rhizobium ORS571, and it can be obtained conventionally. The prior art disclosing the stem nodule nitrogen-fixing rhizobium ORS571 includes prior art 1 (Dreyfus BL, Elmerich C, Dommergues Y. 1983. Free-living Rhizobium strain able to grow on N2 as the sole nitrogen source. Appl. Environ. Microbiol. 45: 711-713.), prior art 2 (Dreyfus B, Garcia JL, Gillis M. 1988. Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int. J. Syst. Bacteriol. 38: 89-98.) and prior art 3 (Dreyfus B, Dommergues Y. 1981. Nitrogen-fixing nodules induced by Rhizobium on the stem of the tropical legume Sesbania rostrata. FEMS Microbiol. Lett. 10:313-317.) etc.

[0048] The present invention also provides a method for preparing extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571, comprising the following steps:

[0049] The nodule nitrogen-fixing rhizobium ORS571 is inoculated into the culture medium described in the above technical solution, cultured at 30-37° C. with shaking, and the culture solution is collected; the culture solution contains the extracellular polysaccharide of the nodule nitrogen-fixing rhizobium ORS571.

[0050] In the present invention, as an embodiment, the rotation speed of the shaking culture is 150-180rpm; as another embodiment, the rotation speed of the shaking culture is 130rpm. As an embodiment, the shaking culture time is 12-48h; as another embodiment, the shaking culture time is 20-40h; as another embodiment, the shaking culture time is 25-35h; as another embodiment, the shaking culture time is 30h.

[0051] As an embodiment, after obtaining the culture solution, the present invention concentrates the culture solution to obtain a concentrated solution and then extracts the concentrated solution by alcohol precipitation to obtain an extract. In the present invention, as an embodiment, the concentration method is rotary evaporation. As an embodiment, the alcohol precipitation extraction uses 95% v / v ethanol.

[0052] As an embodiment, after obtaining the extract, the present invention purifies the extract to obtain purified extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571. In the present invention, as an embodiment, the purification step includes: enzymolyzing the extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571 with papain to obtain an enzymolysis product; precipitating the enzymolysis product with alcohol to collect a first precipitate; re-dissolving the first precipitate with water and adjusting the pH to 2.5 to collect a second precipitate; mixing the second precipitate with a potassium acetate solution, standing at 4°C, and collecting a third precipitate; concentrating the third precipitate and drying it to obtain purified extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571. As an embodiment, the temperature of the enzymolysis is 55-65°C; as another embodiment, the temperature of the enzymolysis is 60°C. As an embodiment, the enzymatic hydrolysis time is 12 to 48 hours; as another embodiment, the enzymatic hydrolysis time is 20 to 40 hours; as another embodiment, the enzymatic hydrolysis time is 25 to 35 hours; as another embodiment, the enzymatic hydrolysis time is 30 hours. As an embodiment, the solvent of the potassium acetate solution is a 75% v / v ethanol solution; the concentration of potassium acetate in the 75% v / v ethanol solution containing potassium acetate is 2 mol / L.

[0053] The present invention utilizes a culture medium containing calcium ions to ferment stem nodule nitrogen-fixing rhizobium ORS571, and can increase the content of extracellular polysaccharides produced by stem nodule nitrogen-fixing rhizobium ORS571. The results of the embodiment show that the extracellular polysaccharide yield of stem nodule nitrogen-fixing rhizobium ORS571 reaches 0.424-0.567 mg / mL, and compared with a treatment group without adding calcium ions, the extracellular polysaccharide content of stem nodule nitrogen-fixing rhizobium ORS571 is significantly improved, and the purity of the extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571 can reach 30-40% in the crude polysaccharide obtained after enzymatic hydrolysis and purification by papain.

[0054] To further illustrate the present invention, a culture medium for improving the extracellular polysaccharide production of stem nodule nitrogen-fixing rhizobium ORS571 provided by the present invention, as well as its preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] 1. Reagents

[0057] (1) 50% DL sodium lactate: 70% DL sodium lactate (71 mL) + distilled water (29 mL), prepare 100 mL and store at 4°C.

[0058] (2) 1M K-Pi buffer (pH 7.0): 1M K2HPO4 (61.5 mL) + 1M KH2PO4 (38.5 mL), prepare 100 mL and store at 4°C.

[0059] (3) 1000×Mg-Na-Mo mixed solution: MgSO4·7H2O 10 g + NaCl 5 g + Na2MoO4·2H2O (sodium molybdate) 0.5 g, prepare 100 mL and store at 4°C.

[0060] (4) 1M NH4Cl: prepare 100 mL and store at 4°C.

[0061] (5) 40 g / L CaCl2·2H2O (1000×CaCl2): Prepare 50-100 mL, sterilize by filtration (0.22 μm), and store at 4°C.

[0062] (6) 5.4 g / LFeCl3·6H2O (1000×FeCl3): Prepare 50-100 mL, filter sterilize (0.22 μm), and store at 4°C.

[0063] (7) 1000× vitamin mixture: Weigh 0.2 g of biotin, 0.4 g of niacin, and 0.4 g of pantothenic acid, make up to 100 mL, filter sterilize (0.22 μm), divide into 1 mL portions in 1.5 mL test tubes, and store at -20°C.

[0064] 2. Prepare L3 culture medium

[0065] (1) Based on the total volume of L3 medium of 100 mL, add 2 mL of 50% DL-Lactate, 1 mL of 1M K-Pi buffer (pH 7.0), 100 μL of 1000×Mg-Na-Mo mixed solution and 1 mL of 1M NH4Cl into a beaker, make up the rest with distilled water, seal the bottle, and place it in an autoclave for high pressure sterilization;

[0066] (2) After cooling, add 100 μL of 1000×CaCl2, 100 μL of 1000×FeCl3 and 100 μL of 1000×vitamin mixture to obtain L3 medium, wherein Ca 2+ The concentration is 0.272mmol / L.

[0067] Example 2

[0068] Same as Example 1, except that when preparing L3 medium, 1000 μL 1000×CaCl2 was added to make the Ca content in L3 medium 2+ The concentration is 2.72mmol / L.

[0069] Comparative Example 1

[0070] Same as Example 1, except that when preparing L3 medium, 1000×CaCl2 was replaced with an equal amount of distilled water to make the Ca in L3 medium 2+ The concentration is 0.

[0071] Test Example 1

[0072] This test case conducts three processing experiments, as follows:

[0073] Treatment 1 (calcium-free group): 500 mL of L3 medium prepared in Comparative Example 1 was cultured and placed in a 1 L conical flask, and 5 mL of OD 600 Azorhizobium caulinodans ORS571 bacterial solution with a value of 1.0;

[0074] Treatment 2 (normal calcium group): 500 mL of L3 medium prepared in Example 1 was cultured and placed in a 1 L conical flask, and 5 mL of OD 600 Azorhizobium caulinodans ORS571 bacterial solution with a value of 1.0;

[0075] Treatment 3 (excess calcium group): 500 mL of L3 medium prepared in Example 2 was cultured and placed in a 1 L conical flask, and 5 mL of OD600 Azorhizobium caulinodans ORS571 bacterial solution with a value of 1.0;

[0076] After inoculation of each treatment group, the cells were cultured in an air bath thermostat shaker at 37°C and 150 rpm for 24 h.

[0077] 1. Determination of total sugar content of extracellular polysaccharides

[0078] The phenol-sulfuric acid method is used to measure the total sugar content of extracellular polysaccharides. The principle is that sugar will be hydrolyzed into monosaccharides under the action of concentrated sulfuric acid. Monosaccharides react with phenol at the high temperature generated by concentrated sulfuric acid dissolved in water to form orange-yellow compounds. This compound has a maximum absorption peak at 490nm, and its absorbance is proportional to the sugar content. By comparing with a sugar standard of known concentration, the sugar content in the sample to be tested can be calculated. This method is suitable for the determination of various carbohydrates, including monosaccharides, oligosaccharides and polysaccharides, and has the advantages of simple operation, high sensitivity and good repeatability. The specific steps are as follows:

[0079] (1) Take glucose as the standard, weigh 32 mg of glucose into a test tube, add 10 mL of deionized water to dissolve, mix thoroughly, and prepare a glucose solution with a concentration of 3200 μg / mL. Take out 5 mL of the 3200 μg / mL glucose solution into another test tube, add deionized water to dilute to 10 mL, mix thoroughly, and prepare a 1600 μg / mL glucose solution. Take another 5 mL of the 1600 μg / mL glucose solution and repeat the above steps to obtain 800, 400, 200, and 100 μg / mL glucose solutions.

[0080] (2) Take 20 μL of glucose solution of different concentrations and place it in a 96-well plate. Then add 20 μL of 5% phenol solution and 100 μL of concentrated sulfuric acid. Use a pipette to quickly pipette and mix well. Repeat six times for each concentration group.

[0081] (3) Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 490 nm, establish a regression equation, and draw a standard curve.

[0082] (4) Pipette 20 μL of each fermentation broth into a 96-well plate, then add 20 μL of 5% phenol solution to ensure thorough mixing. Subsequently, quickly add 100 μL of concentrated sulfuric acid and immediately use a pipette to mix thoroughly. Repeat three times to ensure that the sugar and reagent react fully and maximize the reaction efficiency.

[0083] (5) The reaction system was placed at room temperature for 30 minutes to allow the color to fully develop. Finally, the absorbance value of each reaction system was measured at a wavelength of 490 nm using an enzyme marker. According to the pre-established glucose standard curve, the measured absorbance value was substituted into the regression equation to calculate the total sugar content in each group of fermentation broth. The specific results are shown in Table 1 and Figure 1 shown.

[0084] Table 1 Determination results of total sugar content in fermentation broth

[0085]

[0086] According to Table 1 and Figure 1 It can be seen that as the calcium ion concentration in the culture medium increases, the content of exopolysaccharide secreted by stem nodule nitrogen-fixing rhizobium ORS571 also increases.

[0087] 2. Isolation and Extraction of Extracellular Polysaccharides

[0088] After 1 day of cultivation in each treatment group, the fermentation liquid was centrifuged at 5000rpm for 5min using a high-speed centrifuge, and the supernatant was taken. The supernatant was transferred to a rotary evaporator, and rotary evaporation was performed at 60°C to concentrate it to 200-400mL. The extracellular polysaccharide solution was then crudely purified by water extraction and alcohol precipitation. The principle is to use the difference in solubility of proteins in different solvents, change the solvent environment, reduce the solubility of proteins, and precipitate them from the solution, thereby achieving the purpose of separation and purification. The specific operation steps are: pour the concentrated solution after rotary evaporation into a 2L beaker, add 2 times the volume of 95v / v% ethanol, seal with plastic wrap, precipitate overnight at 4°C, centrifuge, take the precipitate, and dissolve the precipitate with 400-600mL deionized water. In this way, the sample can be crudely purified to obtain a crude polysaccharide solution, which is then concentrated by rotary evaporation until viscous, and then vacuum freeze-dried to obtain a crude polysaccharide extract, which is marked and stored for later use.

[0089] 3. Purification of Extracellular Polysaccharides

[0090] The Sevage method will cause a large amount of sample loss, which is not conducive to subsequent experiments, while the papain enzymatic hydrolysis method has milder conditions, can reduce product loss during the purification process, and improve the yield. Therefore, the papain enzymatic hydrolysis method is used instead of the Sevage method for polysaccharide purification. The specific operation steps are: dissolve the obtained polysaccharide crude extract with a small amount of deionized water, add 10% papain, stir and enzymolyze at 55-65°C for 12-48h, then boil at 100°C for 10min to inactivate papain, centrifuge at 5000rpm for 15min, and take the supernatant. Add 2 volumes of 95v / v% ethanol to the supernatant, seal with plastic wrap, keep overnight at 4°C, centrifuge again, take the precipitate, add deionized water to redissolve, adjust pH to 2.5 with 1M HCl to produce acidic protein precipitate, centrifuge to take the supernatant, add 3 volumes of 75v / v% ethanol containing 2M KAc, keep overnight at 4°C, centrifuge to take the precipitate, dissolve with deionized water, centrifuge, concentrate by rotary evaporation until viscous, transfer to EP tube for vacuum freeze drying, and detect the concentration of extracellular polysaccharide by referring to the above-mentioned phenol-sulfuric acid method. The results are shown in Table 2 and Figure 2 shown.

[0091] Table 2 Total sugar content of water-soluble extracellular polysaccharides after purification (mg / mL)

[0092]

[0093] According to Table 2 and Figure 2 It can be seen that by purifying the extracellular polysaccharides secreted by rhizobia through the above process, a water-soluble polysaccharide component with higher purity can be obtained.

[0094] According to the above content, it can be seen that calcium ions can promote the production of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571 and increase the yield of extracellular polysaccharides of stem nodule nitrogen-fixing rhizobium ORS571.

[0095] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of calcium ions in increasing the production of extracellular polysaccharides from Azorhizobium caulinodans ORS571.

2. Application of calcium ions in the preparation of a culture medium for increasing the production of extracellular polysaccharides from stem nodule nitrogen-fixing rhizobium ORS571; The calcium ion concentration in the culture medium is 0.272-4.45 mmol / L.

3. The use according to claim 2, characterized in that: The calcium ion concentration in the culture medium is 2.72 mmol / L.

4. A culture medium for increasing the production of extracellular polysaccharides from stem nodule nitrogen-fixing rhizobium ORS571, characterized in that: The culture medium comprises 0.5% to 1% DL sodium lactate, 5 to 10 mmol / L K-Pi buffer, 0.1% v / v 1000×Mg-Na-Mo mixed solution, 5 to 10 mmol / L NH4Cl, 0.272 to 4.45 mmol / L CaCl2·2H2O, 5.4 mg / L FeCl3·6H2O, 0.1% v / v 1000× vitamin mixed solution and the balance of water; The K-Pi buffer includes 0.615 mol / L K2HPO4 and 0.3851 mol / L KH2PO4; The 1000×Mg-Na-Mo mixed solution includes: 100 g / L MgSO4·7H2O, 50 g / L NaCl and 5 g / L Na2MoO4·2H2O; The 1000× vitamin mixture includes 2 g / L biotin, 4 g / L niacin and 4 g / L pantothenic acid.

5. The method for preparing the culture medium according to claim 4, characterized in that: The steps include: Mix DL sodium lactate, K-Pi buffer, 1000×Mg-Na-Mo mixed solution, NH4Cl and water, sterilize and obtain a first premix; The first premix, CaCl2·2H2O, FeCl3·6H2O and 1000× vitamin mixture are mixed to obtain the culture medium.

6. Use of the culture medium according to claim 4 or the culture medium obtained by the preparation method according to claim 5 in preparing extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571.

7. A method for preparing extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571, characterized in that: The steps include: The nodule nitrogen-fixing rhizobium ORS571 is inoculated into the culture medium according to claim 4 or the culture medium obtained by the preparation method according to claim 5, cultured at 30-37° C. with shaking, and the culture solution is collected; the culture solution contains the extracellular polysaccharide of the nodule nitrogen-fixing rhizobium ORS571.

8. The preparation method according to claim 7, characterized in that: The shaking culture has a rotation speed of 150 to 180 rpm and a time of 12 to 48 hours.

9. The preparation method according to claim 7 or 8, characterized in that: After obtaining the culture solution, the method further includes: concentrating the culture solution to obtain a concentrated solution; performing alcohol precipitation extraction on the concentrated solution to obtain an extract; The extract is purified to obtain purified extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571.

10. The preparation method according to claim 9, characterized in that: The purification step comprises: enzymolyzing the extract with papain to obtain an enzymolysis product; The enzymatic hydrolysis temperature is 55-65°C and the time is 12-48h; performing alcohol precipitation on the enzymatic hydrolysis product and collecting a first precipitate; Redissolving the first precipitate with water, adjusting the pH to 2.5, and collecting the second precipitate; The second precipitate and the potassium acetate solution are mixed, and the mixture is allowed to stand at 4° C., and the third precipitate is collected; the solvent of the potassium acetate solution is a 75% v / v ethanol solution; The third precipitate is concentrated and dried to obtain purified extracellular polysaccharide of stem nodule nitrogen-fixing rhizobium ORS571.

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