Rhizobium pustatae QY124aN and fungicide

Abstract

The invention provides a strain of rhizobium pustatae QY124aN and a microbial inoculum, and belongs to the technical field of microorganisms. By providing the rhizobium pustatae QY124aN and the fungicide, the problems that a culture medium required by existing rhizobium pustatae is complex in nutrient substance, and the stability of the rhizobium pustatae fungicide is poor are solved. The rhizobium massa QY124aN provided by the invention is separated from an organic fertilizer, has a self-living nitrogen fixation capability, needs simple nutrient substances, can degrade corn straw lignin, increase the water solubility of the corn straw lignin, enhance the antioxidant activity and uvioresistant activity of metabolites, has a protection effect on plant roots, promotes plant growth, and has a good application prospect. The stress resistance of plants in agricultural production is improved. And hyaluronic acid can be produced, the viscosity of fermentation liquor can be increased, and the stability of the rhizobium pustatae QY124aN microbial inoculum is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Rhizobiumpusense QY12 4aN (Rhizobiumpusense QY12 4aN). Background Art

[0002] Rhizobium sp. is a type of Gram-negative bacteria widely distributed in the soil. Under suitable conditions, it can convert free nitrogen in the air into combined nitrogen that can be absorbed and utilized by plants in a symbiotic relationship with specific plants. In the laboratory, rhizobia can grow on yeast mannitol agar medium. In addition, rhizobia can also improve soil fertility, improve soil quality and health, inhibit the growth of plant pathogenic fungi, and reduce the incidence of crops. Rhizobium agents are a type of commonly used agricultural microbial agents, such as soybean rhizobia agents, peanut rhizobia agents, and legume and forage rhizobia agents. In crop cultivation, rhizobia agents are widely used because they increase the yield of specific crops. They are one of the substitutes for chemical fertilizers and are conducive to the sustainable development of agriculture.

[0003] Rhizobium pusense is a microorganism belonging to the genus Rhizobium. It was first isolated and purified from the nodule soil of chickpea (Cicer arietinum L.) grown in Pusa, New Delhi, India. According to literature reports, Rhizobium pusense is a plant growth-promoting rhizobacteria (PGPR) with plant growth-promoting properties, such as producing siderophores, indoleacetic acid (IAA), ammonia and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and can effectively dissolve phosphate. The invention patent "A strain of Rhizobium pusense X2 producing IAA and CMC enzymes and its application" (CN113088471A) discloses a strain of Rhizobium pusense X2 producing IAA and CMC enzymes, which was isolated from the black soil of Shajiang in Anhui Province, has the ability to produce IAA and CMC enzymes, and is used to promote straw rot and crop growth. The application publication number CN 112358985A "Pusa rhizobium and its application in the preparation of water-soluble β-1,3 glucan" discloses a strain of Pusha rhizobium obtained by sampling and isolating from Hengshui Lake wetland, which has the ability to produce water-soluble β-1,3 glucan and its application in the preparation of β-1,3 glucan. The invention patent with the application publication number CN 116286550 A discloses a strain of Pusha rhizobium TYQ1 and its bacterial agent preparation, which was isolated from the rhizosphere soil of cucumbers infected with root-knot nematodes and cultured in LB medium. Its fermentation supernatant has a strong contact killing ability against root-knot nematodes and has the functions of salt tolerance, dissolving insoluble inorganic phosphorus and secreting auxin. This strain can not only significantly promote the growth of cucumber seedlings, but also retard the development process of root-knot nematodes in the cucumber root system. Although there have been reports on the use of Rhizobium pusa in the preparation of microbial agents, the nutrients required for the production of microbial agents are complex, the cost of the culture medium is high, and there are no reports on the ability of Rhizobium pusa to produce hyaluronic acid and degrade lignin. Summary of the invention

[0004] The invention provides a Pusha rhizobium QY12 4aN and a bacterial agent thereof, which solve the problems that the nutrients of the culture medium required by the existing Pusha rhizobium are complex and the stability of the Pusha rhizobium bacterial agent is poor.

[0005] A strain of Rhizobium pusa, wherein the Rhizobium pusa QY12 4aN is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.29921 and a deposit date of March 1, 2024.

[0006] A QY12 4aN bacterial agent is prepared by using the above-mentioned Rhizobium spp. QY12 4aN.

[0007] The bacterial agent comprises hyaluronic acid and lignin.

[0008] A preparation method of QY12 4aN bacterial agent is as follows:

[0009] Step 1: inoculate the above-mentioned Rhizobium pusa QY12 4aN onto the Axubei plate for recovery to obtain a single colony;

[0010] Step 2: Inoculate a single colony into YPD medium and culture for 24 hours to obtain bacterial solution;

[0011] Step 3: Expand the bacterial solution twice to obtain a culture;

[0012] Step 4: inoculate the culture into CSL liquid culture medium to obtain QY12 4aN bacterial agent;

[0013] The inoculation size of the culture in step 4 is 5%.

[0014] Step 4: The formula of the CSL liquid culture medium is: CSL 5 g, K2HPO4 1 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.02 g, ZnSO4·7H2O 0.04 g, glucose 10.0 g, H2O 1000 mL; the culture medium is sterilized at 121°C for 15 min before use.

[0015] The culture conditions in step 4 are 39° C. and 200 r / min for 72 h.

[0016] Beneficial Effects

[0017] 1. The present invention provides a Rhizobium pusa QY12 4aN strain, which is separated from organic fertilizer and has the ability of self-generated nitrogen fixation and requires simple nutrients.

[0018] 2. The present invention provides a bacterial agent prepared by using Rhizobium spp. QY12 4aN. The production process of the Rhizobium spp. QY12 4aN bacterial agent is stable and easy to control.

[0019] 3. The present invention provides a Pusha rhizobium QY12 4aN that can degrade corn straw lignin. Corn straw lignin is an amorphous polyphenol polymer with a highly complex structure, composed of three phenylpropane units of p-hydroxyphenyl (H), syringyl (S) and guaiacyl (G), and has biological activities such as antioxidant, antibacterial and anti-ultraviolet. Pusha rhizobium QY12 4aN degrades corn straw lignin to produce low molecular weight lignin, increase the water solubility of corn straw lignin, enhance the antioxidant activity and anti-ultraviolet activity of metabolites, have a protective effect on plant roots, promote plant growth, and improve the stress resistance of plants in agricultural production.

[0020] 4. The present invention provides a Pusha rhizobium QY12 4aN that can bioconvert small molecule compounds such as glucose, mannitol and nitrogen in the air into hyaluronic acid during its growth. Hyaluronic acid, as a natural linear glycosaminoglycan, is mainly composed of repeating units of β-(1,4)-glucuronic acid (GA) and β-(1,3)-acetylglucosamine (NAG), which are connected together by alternating β-1,3 and β-1,4 glycosidic bonds. Since Pusha rhizobium QY12 4aN can produce hyaluronic acid, the hyaluronic acid in its fermentation broth can increase the viscosity of the fermentation broth, which is beneficial to the stability of the Pusha rhizobium QY12 4aN bacterial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of corn stover lignin fractionation;

[0022] Figure 2 This is the colony morphology of Rhizobium pusa QY12 4aN on the Axubei plate;

[0023] Figure 3 This is the colony morphology of Rhizobium pusa QY12 4aN on CSL double-layer plates;

[0024] Figure 4 The bacterial morphology of Rhizobium pusa QY12 4aN (10×100);

[0025] Figure 5 The hyaluronic acid after alcohol precipitation in Example 3;

[0026] FIG6 is an infrared spectrum of a hyaluronic acid freeze-dried powder sample, Fig. 6A This is the infrared spectrum of hyaluronic acid standard. Figure 6B is the infrared spectrum of the freeze-dried powder sample;

[0027] Figure 7 The preparation process of the Pusha rhizobium QY12 4aN inoculant;

[0028] Figure 8 This is the hyaluronic acid-lignin complex after alcohol precipitation in Example 4. DETAILED DESCRIPTION

[0029] Example 1. Preparation of corn straw lignin.

[0030] 1. Preparation of corn straw lignin.

[0031] (1) Corn stalk powder (<40 mesh sieve) and 0.2 M NaOH were mixed evenly at a solid-liquid ratio of 1:2 4 (w / v), stirred at 80°C for 2 h, and then centrifuged at 10000 r / min for 10 min to obtain a supernatant and a precipitate. The precipitate was washed twice with 80°C 0.2 M NaOH solution, centrifuged, and then the solution of the washed precipitate was combined with the supernatant to obtain a mixed solution, which was then cooled to room temperature.

[0032] (2) Add 2M H2SO4 solution to the mixed solution while stirring, adjust the pH to 3, and let it stand at room temperature to precipitate lignin. Then centrifuge at 10000r / min for 10min, discard the supernatant, wash the precipitate with 70℃ deionized water until it is neutral, and freeze-dry to obtain a dark brown powder, which is the corn straw alkali-extracted lignin-carbohydrate complex (CSALCC).

[0033] (3) Weigh 1.0 g of CSALCC and add it to 150 mL of 5% sodium bicarbonate solution. Stir and dissolve at room temperature for 30 min, then centrifuge at 10,000 r / min for 10 min to obtain a supernatant. Add 2 M H2SO4 solution to the supernatant, adjust the pH to 3, and let it stand at room temperature for 5 min to precipitate lignin. Then centrifuge at 10,000 r / min for 10 min, discard the supernatant, wash the precipitate with 70°C deionized water until it is neutral, freeze-dry, and obtain freeze-dried powder, which is named corn straw lignin (CSL). The CSL fractionation flow chart is as follows: Figure 1 shown.

[0034] 2. Analysis of the main components in CSALCC and CSL. The specific methods are as follows:

[0035] (1) Determination of lignin content.

[0036] The total lignin content was determined according to the NREL method of the National Energy Laboratory of the United States. The total lignin content is the sum of acid-soluble lignin and acid-insoluble lignin. Take 300 mg of CSALCC and 300 mg of CSL and put them into stoppered colorimetric tubes, add 3 mL of 72% sulfuric acid solution respectively, heat them in a water bath at 30°C for 1 hour, add 84 mL of deionized water respectively, mix them, heat them in a boiling water bath for 2 hours, filter them, and obtain the filter residue. Wash the filter residue with hot water until it is neutral to obtain washing liquid. Dry the filter residue, weigh it, and calculate the acid-insoluble lignin content. The calculation formula is as follows:

[0037]

[0038] Where: m is the mass of filter residue (mg), M is the mass of CSALCC or CSL weighed (mg).

[0039] Combine the filtrate and washing liquid, dilute with deionized water to an appropriate concentration, measure the absorbance of the diluted solution at a wavelength of 320nm, make the absorbance between 0.2-0.8, and calculate the acid-soluble lignin content. The calculation formula is as follows:

[0040]

[0041] Where: A 320 is the absorbance of the diluted solution at 320 nm, V is the volume of the diluted solution, B is the dilution factor, ε is the photometric coefficient 30 (L / g·cm), and M is the mass of CSALCC or CSL weighed (mg).

[0042] (2) Determination of total sugar content.

[0043] Weigh CSALCC and CSL accurately, dissolve them in DMSO to prepare solutions of appropriate concentrations, and determine them using the sulfuric acid-phenol method. Use glucose as the standard to draw a standard curve. Calculate the total sugar content in CSALCC and CSL based on the glucose standard curve.

[0044] (3) Experimental results

[0045] The results are shown in Table 1. As shown in Table 1, after fractionation by 0.2M NaOH solution and 5% sodium bicarbonate solution, the total lignin content in corn straw lignin (CSL) was 86.40±1.38% (w / w), and the total sugar content was 11.59±0.76 (w / w), which can be used for the screening of the following corn straw lignin-degrading bacteria.

[0046] Table 1 Composition analysis of corn straw lignin

[0047]

[0048] Example 2. Isolation and identification of Rhizobium pusa QY12 4aN.

[0049] 1. Isolation and identification of Rhizobium spp. QY12 4aN.

[0050] First, the nitrogen-fixing bacteria were isolated and purified using Axubei medium. The formula of Axubei medium is as follows: KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaCO3 5.0g, mannitol 10.0g, CaSO4 0.1g, agar 15g, H2O1000mL, sterilized at 121℃ for 15min, and Axubei plates were prepared. The organic fertilizer of Heilongjiang Ruiyuan Biotechnology Co., Ltd. was inoculated on the Axubei plate, and the nitrogen-fixing bacteria were enriched at 39℃. After the colonies grew, the colonies were picked and repeatedly streaked on the Axubei plate to obtain single colonies, which were observed under a microscope to confirm that pure culture was obtained.

[0051] Then the pure culture was inoculated into a CSL medium double-layer plate for cultivation. The formula of the CSL medium double-layer plate is as follows:

[0052] The formula of the lower culture medium is: K2HPO4 1.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.02 g, ZnSO4·7H2O 0.04 g, agar 15 g, H2O 1000 mL, sterilized at 121 °C for 15 min;

[0053] The formula of the upper culture medium is: CSL 5 g, K2HPO4 1 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.02 g, ZnSO4·7H2O 0.04 g, agar 15 g, H2O 1000 mL, sterilized at 121°C for 15 min.

[0054] Colony morphology Figure 2 and 3 As shown, on the Ashubei plate ( Figure 2 ) and CSL medium double-layer plates ( Figure 3 ) on the surface, the colonies are transparent, colorless, and mucous. The surface of the colonies is smooth and moist, and the edges of the colonies are neat. Figure 4 As shown, the bacteria are rod-shaped, short and have no spores.

[0055] The single colony picked from the CSL medium double-layer plate was inoculated into yeast extract peptone glucose (YPD) medium. The YPD medium formula is as follows: 5g tryptone, 2.5g yeast extract, 1g glucose, 1000mL H2O, sterilized at 121℃ for 15min. The culture was carried out at 39℃ and 180r / min, and the bacteria were collected and commissioned to Sangon Biotechnology (Shanghai) Co., Ltd. for 16s rRNA gene sequencing. The sequencing results were compared by Blast, and the strain was assigned to Rhizobium pusense and named Rhizobium pusense QY12 4aN. The glycerol preservation method was used and stored in a -80℃ refrigerator. It was also preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration, with the preservation number CGMCC NO.29921 and the preservation date of March 1, 2024.

[0056] Axubei medium is a nitrogen-free, selective synthetic medium with mannitol as the only carbon source. It is usually used to enrich and isolate self-generated nitrogen-fixing bacteria. Therefore, Rhizobium pusaensis QY12 4aN can grow well on Axubei plates and be isolated and purified. Figure 2 As shown, it shows that Rhizobium spp. QY12 4aN can use mannitol as a carbon source and utilize free nitrogen in the air to synthesize nitrogen-containing organic matter it needs, that is, it has autogenous nitrogen fixation.

[0057] According to the formula of the culture medium, the upper layer of the CSL double-layer plate is composed of CSL and inorganic salts, and no nitrogen source is added. Rhizobium pusa QY12 4aN can grow well on the CSL double-layer plate, indicating that Rhizobium pusa QY12 4aN can use CSL as a carbon source for metabolism to obtain the energy and carbon required for its own growth, and at the same time use the free nitrogen in the air to synthesize the nitrogen-containing organic matter it needs, further proving its ability to autogenously fix nitrogen.

[0058] Example 3. Verification of the synthesis of hyaluronic acid by Rhizobium spp. QY12 4aN.

[0059] Rhizobium Pusha QY12 4aN expansion. Pick the bacteria frozen at -80℃ and inoculate them on the Axubei plate for recovery. After a single colony grows, pick the bacteria and inoculate them into the YPD medium. After continuous expansion, centrifuge at 6000r / min for 5min, discard the supernatant, wash the precipitate twice with sterile saline, and prepare the bacterial suspension.

[0060] Synthesis of hyaluronic acid. The prepared bacterial suspension was inoculated into the hyaluronic acid (HA) preparation medium, the formula of which is as follows: K2HPO4 1.0g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.02g, ZnSO4·7H2O 0.04g, glucose 10.0g, H2O 1000mL; 121℃, sterilized for 15min. The final concentration of Rhizobium pusa QY12 4aN was 1.0OD / mL, and the culture was shaken at 30℃ and 180r / min for 2 days. After the culture was completed, centrifuged at 10000r / min for 10min and the supernatant was taken. The hyaluronic acid in the supernatant was separated by D101 macroporous adsorption resin exchange column chromatography. The supernatant was mixed with deionized water at a ratio of 1:1 (v / v) and then loaded. The elution flow rate was 2 mL / min. During the elution process, an ultraviolet detector was used to detect the eluate online at 220 nm. The distribution of total sugar and glucuronic acid in the eluate was tracked and detected by the microplate method. The glucuronic acid fraction was collected and concentrated by a rotary evaporator to obtain the concentrated fraction. The concentrated fraction was precipitated with alcohol, and 3 times the volume of anhydrous ethanol was added to the concentrated fraction, mixed, and allowed to stand at 4°C for 2 hours to obtain the hyaluronic acid after alcohol precipitation. Figure 5As shown, centrifuge at 8000r / min for 5min, discard the supernatant, and dissolve the precipitate in deionized water. Repeat the alcohol precipitation-deionized water dissolution steps twice, and obtain freeze-dried powder by vacuum freeze drying.

[0061] The determination of the hyaluronic acid content in the above freeze-dried powder was carried out with reference to the light industry standard QB / T4576-2023 of the People's Republic of China for sodium hyaluronate, using the spectrophotometer method-sulfuric acid-carbazole method with slight modifications. Glucuronic acid was used as the standard to draw a standard curve. The content of hyaluronic acid was calculated based on the glucuronic acid standard curve, and the calculation formula is as follows:

[0062]

[0063] Where:

[0064] X1: The content of hyaluronic acid in the sample (on a dry basis), in grams per hundred grams (g / 100g);

[0065] ρ1: According to the absorbance of the sample, find the corresponding mass concentration of glucuronic acid from the standard curve, in micrograms per milliliter (μg / mL);

[0066] w1: mass of sample solution, in grams (g);

[0067] 10 -6 : Conversion factor between micrograms and grams;

[0068] V: volume of the sample, in milliliters (mL);

[0069] 100: Conversion factor between grams and hundred grams;

[0070] 379.3: relative molecular weight of hyaluronic acid disaccharide structural unit;

[0071] w2: the mass of the sample solution pipetted, in grams (g);

[0072] m: sample mass, in grams (g);

[0073] w3: moisture content of the sample, in grams per hundred grams (g / 100g);

[0074] 194.1: Relative molecular mass of D-glucuronic acid.

[0075] According to QB / T 4576-2023, the principle of the spectrophotometer-sulfuric acid-carbazole method for determining hyaluronic acid is as follows: hyaluronic acid contains N-acetyl-D-glucosamine and glucuronic acid in equal molar ratios. Borax is used as a catalyst to hydrolyze hyaluronic acid with sulfuric acid to produce glucuronic acid and N-acetyl-D-glucosamine monomers. Glucuronic acid reacts with carbazole to form an organic complex, which shows a unique purple color, and its absorbance is proportional to the concentration of glucuronic acid. By measuring the absorbance, the content of hyaluronic acid can be determined.

[0076] The result of determination according to the above method shows that the content of hyaluronic acid in the above freeze-dried powder is 57.68%.

[0077] Determination of infrared spectrum of freeze-dried powder sample. The functional groups of hyaluronic acid were determined by Bruker EQUINOX 55 infrared spectrometer. An appropriate amount of freeze-dried powder sample was mixed with KBr powder at a mass ratio of 1:100, ground evenly in a mortar, and pressed into tablets. -1 Spectral scanning is performed within the wavenumber range with a resolution of 4 cm -1 , scanned 32 times, with air as the background, and sodium hyaluronate standard (Cat. No. 53747, Sigma-Aldrich) as the control.

[0078] The infrared spectrum of the freeze-dried powder sample is shown in Figure 6. The infrared spectrum is at 3416 cm -1 A strong peak is shown at 2921 cm -1 The bands at 1726 and 1637.00 cm -1 The peak at 1566 cm -1 The peaks at 1406 and 1375 cm -1 The peaks at 1163, 1073 and 1045 cm -1 The peaks at 902 and 608 cm -1 A peak appears at , indicating the presence of COC stretching. In summary, the infrared spectrum of the freeze-dried powder shows characteristic peaks of the infrared spectrum of hyaluronic acid, and is similar to the infrared spectrum of the hyaluronic acid standard. It can be determined that the main component of the freeze-dried powder is hyaluronic acid.

[0079] Example 4. Preparation of Rhizobium Pusha QY12 4aN bacterial agent.

[0080] Pick the -80℃ frozen Pusha rhizobium QY12 4aN bacteria and inoculate them on the Axubei plate for recovery. After a single colony grows, pick a single colony and inoculate it into YPD for 24h. Inoculate at 5% inoculation volume and expand the culture twice continuously. Inoculate the culture obtained by expansion into CSL liquid culture medium at 5% (v / v). The formula of CSL liquid culture medium is as follows: CSL 5g, K2HPO41g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.02g, ZnSO4·7H2O 0.04g, glucose 10.0g, H2O 1000mL; sterilize at 121℃ for 15min. The culture was carried out at 39°C and 200 r / min. The culture was terminated after 72 hours of culture. The fermentation broth was divided and packaged to obtain the Pusa rhizobium QY12 4aN bacterial agent. The pH, viscosity, content of polysaccharides, glucuronic acid, hyaluronic acid and total polyphenols of the fermentation broth, as well as the antioxidant activity and effective viable count of the fermentation broth were tested. The flow chart is shown in FIG. Figure 7 .

[0081] According to Example 3, the fermentation broth was subjected to alcohol precipitation to obtain a hyaluronic acid-lignin complex, such as Figure 8 As shown in Figure 2, hyaluronic acid can absorb lignin in the fermentation liquid and appear brown.

[0082] The composition analysis, antioxidant activity determination and total colony count determination of the fermentation broth of Rhizobium pusa QY12 4aN inoculum were carried out as follows:

[0083] (1) Determination of pH of fermentation broth: The pH was determined using a SevenExcellence multi-parameter tester (Mettler-Toledo, Switzerland).

[0084] (2) Determination of the viscosity of the fermentation broth: The viscosity was determined using a DV-II+pro viscometer (Brookfield, USA).

[0085] (3) Determination of water-soluble polysaccharide content in fermentation broth. Take 1 mL of fermentation broth and add 3 times the volume of anhydrous ethanol, mix well, let stand at 4°C for 2 hours, centrifuge at 8000r / min for 5 minutes, discard the supernatant, and obtain a precipitate. Dissolve the precipitate in 4 mL of pure water, centrifuge and take the supernatant, then dilute it appropriately. The water-soluble polysaccharide content is determined by the sulfuric acid-phenol method. Use glucose as the standard and draw a standard curve. Calculate the water-soluble polysaccharide content in the fermentation broth based on the glucose standard curve.

[0086] (4) Determination of glucuronic acid content in fermentation broth. Take 1 mL of fermentation broth and add 3 times the volume of anhydrous ethanol, mix well, let stand at 4°C for 2 hours, centrifuge at 8000 r / min for 5 minutes, discard the supernatant, dissolve the precipitate with 4 mL of pure water, centrifuge and take the supernatant, dilute it appropriately, and determine it by sulfuric acid-carbazole method. Use glucuronic acid as the standard and draw a standard curve. Calculate the glucuronic acid content in the fermentation broth based on the glucuronic acid standard curve.

[0087] (5) Determination of the content of hyaluronic acid in the fermentation broth. Referring to the China Food Industry Association group standard T / CNFIA155-2022 Sodium hyaluronate beverage, the content of sodium hyaluronate in the fermentation broth was determined by the spectrophotometer method-sulfuric acid-carbazole method. Glucuronic acid was used as the standard to draw a standard curve. The content of hyaluronic acid in the fermentation broth was calculated according to the glucuronic acid standard curve, and the calculation formula is as follows:

[0088] C HA =c×N×1.9542×10 -3

[0089] Where:

[0090] C HA : The content of hyaluronic acid in the sample, in grams per liter (g / L);

[0091] c: According to the absorbance of the sample, find the corresponding glucuronic acid concentration from the standard curve, in micrograms per milliliter (μg / mL);

[0092] N: dilution factor of sample solution;

[0093] 1.9542: Ratio of the relative molecular mass of the repeating disaccharide unit of hyaluronic acid (379.3) to the relative molecular mass of glucuronic acid (194.1).

[0094] 10 -3 : Conversion factor between micrograms per milliliter (μg / mL) and grams per liter (g / L).

[0095] (6) Determination of total polyphenol content in fermentation broth. The Folin-phenol method was used for determination. Gallic acid was used as the standard substance to draw a standard curve. The total polyphenol content in the supernatant was calculated based on the gallic acid standard curve.

[0096] (7) Determination of antioxidant activity of fermentation broth. The ABTS method was used. The assay was performed using an ELISA instrument. The lyophilized powder of the fermentation broth fermented for 72 h in Example 4 was used to prepare a test solution of appropriate concentration. Before the test, the A of the ABTS working solution was diluted with 50 mmol / L Tris-HCl (pH 7.4) solution. 734nmDilute to 0.9±0.1, mix 40μL sample solution with 160μL ABTS working solution, react for 5min at room temperature in the dark, and use Safire2 microplate reader to measure the absorbance of the reaction solution at 734nm. Use 50mmol / L Tric-HCl (pH7.4) as blank control and Trolox as positive control. The ABTS cation radical scavenging activity is the concentration of the sample solution when it scavenges 50% of the ABTS cation radicals, i.e., IC 50 (μg / mL).

[0097] ABTS cation radical scavenging rate (%) = (blank absorbance value - sample absorbance value) / blank absorbance value × 100%

[0098] (8) Determination of total colony count. Refer to GB 20287-2006 Determination of effective viable count in agricultural microbial agents. In the determination, plate count agar medium and pouring method are used.

[0099] The results of the above (1)-(6) measurements are shown in Table 2

[0100] Table 2 Component analysis of fermentation broth of Rhizobium pusa QY12 4aN

[0101]

[0102] As shown in Table 2, after culturing Rhizobium pusa QY12 4aN in CSL liquid medium for 72h, the water-soluble polysaccharide content increased from 0.00g / L to 2.01g / L, the glucuronic acid content increased from 0.00g / L to 0.11g / L, and the hyaluronic acid content increased from 0.00g / L to 0.23g / L. Hyaluronic acid is a linear glycosaminoglycan, mainly composed of repeating units of β-(1,4)-glucuronic acid (GA) and β-(1,3)-acetylglucosamine (NAG). Rhizobium pusa QY12 4aN can synthesize hyaluronic acid, which further indicates that its nitrogen comes from the nitrogen fixation of Rhizobium pusa QY12 4aN. At the same time, the viscosity of the fermentation broth increased from 0cP to 144cP, indicating that the production of hyaluronic acid increases the viscosity of the fermentation broth. The total phenol content of the fermentation broth decreased from 0.36 mg / mL to 0.19 mg / mL, indicating that CSL in the fermentation broth was degraded. This is because lignin is the most abundant renewable natural aromatic polymer in nature and is rich in phenolic hydroxyl groups. The total phenol content of the fermentation broth was determined by the Folin-phenol method, which can reflect the content of lignin in the fermentation broth or the degradation of lignin. The total phenol content in the fermentation broth showed a downward trend, indicating that lignin was degraded.

[0103] The above (7) test results show that the scavenging activity of ABTS cation radicals of the lyophilized powder of the fermentation broth of Rhizobium pusa QY12 4aN after 72h of fermentation is IC50 =527.67±24.01μg / mL, scavenging activity of ABTS cation radicals of initial fermentation broth lyophilized powder, IC 50 =1058.00±11.36μg / mL. After fermentation with Rhizobium pusa QY12 4aN for 72h, the scavenging activity of ABTS cation free radicals in the fermentation broth was enhanced, indicating that the fermentation broth had a certain antioxidant effect.

[0104] The above (8) test results show that the effective viable count (cfu) of Rhizobium pusa QY12 4aN inoculum is ≥ 2.0×10 8 Pieces / mL.

Claims

1. A strain of Rhizobium pusa, characterized in that: The Pusha rhizobium QY124aN is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.29921 and the deposit date March 1, 2024.

2. A QY124aN bacterial agent, characterized in that: The bacterial agent is prepared by using the Rhizobium spp. QY124aN described in claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent comprises hyaluronic acid and lignin.

4. A method for preparing a QY124aN bacterial agent, characterized in that: The method is: Step 1: inoculating the Pusha rhizobium QY124aN described in claim 1 onto an Axubei plate for recovery to obtain a single colony; Step 2: Inoculate a single colony into YPD medium and culture for 24 hours to obtain bacterial solution; Step 3: Expand the bacterial solution twice to obtain a culture; Step 4: Inoculate the culture into CSL liquid culture medium to obtain QY124aN bacterial agent.

5. The method according to claim 4, characterized in that The inoculum size of the culture in step 4 is 5%.

6. The method according to claim 4, characterized in that Step 4: The formula of the CSL liquid culture medium is: CSL 5g, K2HPO4 1 g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.02g, ZnSO4·7H2O 0.04g, glucose 10.0g, H2O 1000mL; the culture medium is sterilized at 121°C for 15min before use.

7. The method according to claim 4, characterized in that The culture conditions in step 4 are 39° C. and 200 r / min for 72 h.

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