Bradyrhizobium sp. QY124aN and microbial agent

Abstract

The present application provides a Bradyrhizobium sp. QY124aN and a bacterial agent, and belongs to the technical field of microorganisms. The present application provides a Bradyrhizobium sp. QY124aN and a bacterial agent, and solves the problems of complex nutritional substances of the existing Bradyrhizobium sp. culture medium and poor stability of the Bradyrhizobium sp. bacterial agent. The Bradyrhizobium sp. QY124aN provided by the present application is isolated from organic fertilizer and has the ability of self-nitrogen fixation, and the required nutritional substances are simple. The Bradyrhizobium sp. QY124aN can degrade corn stalk lignin, increase the water solubility of corn stalk lignin, enhance the antioxidant activity and ultraviolet resistance of metabolic products, protect the roots of plants, promote the growth of plants, and improve the stress resistance of plants in agricultural production. Moreover, the Bradyrhizobium sp. QY124aN can produce hyaluronic acid, increase the viscosity of the fermentation broth, and is beneficial to the stability of the Bradyrhizobium sp. QY124aN bacterial agent.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a Rhizobiumpus ense QY12 4aN. Background Technology

[0002] Rhizobium sp. is a group of Gram-negative bacteria widely distributed in soil. Under suitable conditions, it can convert atmospheric nitrogen into bioavailable nitrogen that can be absorbed and utilized by specific plants through a symbiotic relationship. In the laboratory, rhizobium can grow on yeast-mannitol agar. Furthermore, rhizobium can improve soil fertility, enhance soil health, inhibit the growth of plant pathogenic fungi, and reduce crop disease incidence. Rhizobium inoculants are commonly used agricultural microbial agents, such as soybean rhizobium inoculants, peanut rhizobium inoculants, and legume and forage rhizobium inoculants. In crop cultivation, rhizobium inoculants are widely used because they increase the yield of specific crops and are one of the alternatives to chemical fertilizers, contributing to the sustainable development of agriculture.

[0003] Rhizobium pusense, a microorganism belonging to the genus Rhizobium, was first isolated and purified from the root nodule soil of chickpea (Cicer arietinum L.) grown in Pusa, New Delhi, India. According to literature, Rhizobium pusense is a plant growth-promoting rhizobacteria (PGPR) with characteristics that promote plant growth, such as producing siderophores, indoleacetic acid (IAA), ammonia, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and effectively dissolving phosphates. 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, isolated from sandy ginger black soil in Anhui Province, which has the ability to produce IAA and CMC enzymes and is applied to straw decomposition and crop growth promotion. Application publication number CN 112358985A, entitled "Pusa rhizobium and its application in the preparation of water-soluble β-1,3-glucan," discloses a strain of Pusa rhizobium isolated 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. Invention patent application publication number CN 116286550A discloses a strain of Pusa rhizobium TYQ1 and its inoculum preparation. It was isolated from the rhizosphere soil of cucumbers infected with root-knot nematodes and cultured in LB medium. Its fermentation supernatant showed strong contact killing ability against root-knot nematodes and also exhibited salt tolerance, the ability to dissolve insoluble inorganic phosphorus, and the ability to secrete auxins. This strain not only significantly promoted the growth of cucumber seedlings but also slowed down the development of root-knot nematodes within the cucumber root system. Although there are reports of the application of *Pusa rhizobium* in the preparation of inoculants, the nutrients required for inoculant production are complex, the culture medium is expensive, and there are no reports on *Pusa rhizobium* producing hyaluronic acid and degrading lignin. Summary of the Invention

[0004] This invention provides a strain of Rhizobium pusa QY12 4aN and its inoculum, which solves the problems of complex nutrients in the culture medium required for existing Rhizobium pusa culture and poor stability of Rhizobium pusa inoculum.

[0005] A strain of Rhizobium pusa, namely Rhizobium pusa QY12 4aN, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29921 and deposit date of March 1, 2024.

[0006] A QY12 4aN bacterial agent, wherein the bacterial agent is prepared using the above-mentioned Rhizobium pusa QY12 4aN.

[0007] The microbial agent includes hyaluronic acid and lignin.

[0008] A method for preparing a QY12 4aN bacterial agent is as follows:

[0009] Step 1: Inoculate the above-mentioned Rhizobium pusa QY12 4aN onto Ashube agar plates for resuscitation and obtain single colonies;

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

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

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

[0013] The inoculum size for the culture described in step four is 5%.

[0014] The formula for the CSL liquid culture medium in step four is as follows: CSL 5g, K2HPO4 1g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.02g, ZnSO4·7H2O 0.04g, glucose 10.0g, and H2O 1000mL; the culture medium is sterilized at 121℃ for 15min before use.

[0015] The culture conditions described in step four are 39℃, 200r / min, and 72h.

[0016] Beneficial effects

[0017] 1. This invention provides a strain of Rhizobium pusa QY12 4aN, which is isolated from organic fertilizer and has the ability to fix nitrogen by itself, requiring simple nutrients.

[0018] 2. This invention provides a bacterial agent prepared by *Rhizobium pusa* QY12 4aN. The production process of *Rhizobium pusa* QY12 4aN bacterial agent is stable and easy to control.

[0019] 3. This invention provides a method by which *Pusa rhizobium* QY12 4aN degrades corn stalk lignin. Corn stalk lignin is an amorphous polyphenol polymer with a highly complex structure, composed of three phenylpropane units: p-hydroxyphenyl (H), eugenyl (S), and guaiacol (G), exhibiting antioxidant, antibacterial, and UV-resistant biological activities. *Pusa rhizobium* QY12 4aN degrades corn stalk lignin, producing low-molecular-weight lignin, increasing the water solubility of corn stalk lignin, enhancing the antioxidant and UV-resistant activities of its metabolites, protecting plant roots, promoting plant growth, and improving plant stress resistance in agricultural production.

[0020] 4. This invention provides a method for *Pulsatilla chinensis* QY12 4aN to bioconvert small molecule compounds such as glucose, mannitol, and nitrogen from 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), linked together by alternating β-1,3 and β-1,4 glycosidic bonds. Since *Pulsatilla chinensis* 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 *Pulsatilla chinensis* QY12 4aN inoculum. Attached Figure Description

[0021] Figure 1 Flowchart for the graded separation of lignin from corn stalks;

[0022] Figure 2 The colony morphology of Rhizobium pusa QY12 4aN on Assoube agar plates;

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

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

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

[0026] Figure 6 shows the infrared spectrum of the lyophilized hyaluronic acid powder sample. Figure 6A Infrared spectrum of hyaluronic acid standard. Figure 6B The image shows the infrared spectrum of the lyophilized powder sample.

[0027] Figure 7 Preparation process of Rhizobium pusa QY12 4aN inoculum;

[0028] Figure 8 The hyaluronic acid-lignin complex after alcohol precipitation in Example 4. Detailed Implementation

[0029] Example 1. Preparation of lignin from corn stalks.

[0030] 1. Preparation of lignin from corn stalks.

[0031] (1) Corn straw powder (<40 mesh sieve) and 0.2M NaOH were mixed evenly at a solid-liquid ratio of 1:2:4 (w / v), stirred at 80℃ for 2 hours, and then centrifuged at 10000 r / min for 10 min to obtain the supernatant and precipitate. The precipitate was washed twice with 0.2M NaOH solution at 80℃, centrifuged, and then the solution from washing the 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 mixture 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 deionized water at 70℃ until neutral, freeze dry to obtain dark brown powder, which is lignin-carbohydrate complex (CSALCC) extracted from corn straw.

[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 10000 r / min for 10 min to obtain the supernatant. Add 2 M H2SO4 solution to the supernatant to adjust the pH to 3, and let it stand at room temperature for 5 min to precipitate the lignin. Then centrifuge at 10000 r / min for 10 min, discard the supernatant, wash the precipitate with deionized water at 70℃ until neutral, freeze-dry to obtain the freeze-dried powder, named corn straw lignin (CSL). The CSL fractionation process flow chart is shown below. Figure 1 As shown.

[0034] 2. Major component analysis in CSALCC and CSL, the specific methods are as follows:

[0035] (1) Determination of lignin content.

[0036] Total lignin content was determined according to the NREL method of the National Energy Laboratory (NEL). Total lignin content is the sum of acid-soluble lignin and acid-insoluble lignin. 300 mg of CSALCC and 300 mg of CSL were placed separately in stoppered colorimetric tubes, and 3 mL of 72% sulfuric acid solution was added to each. After heating in a water bath at 30°C for 1 hour, 84 mL of deionized water was added to each, and the mixture was stirred. The mixture was then heated in a boiling water bath for 2 hours, filtered, and the residue was obtained. The residue was washed with hot water until neutral to obtain the washing liquid. The residue was dried, weighed, and the acid-insoluble lignin content was calculated using the following formula:

[0037]

[0038] In the formula: m is the mass of filter residue (mg), and 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, and measure the absorbance of the diluted solution at a wavelength of 320 nm, ensuring the absorbance is between 0.2 and 0.8. Calculate the acid-soluble lignin content using the following formula:

[0040]

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

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

[0043] Accurately weigh CSALCC and CSL, dissolve them in DMSO to prepare solutions of appropriate concentrations, and determine the total sugar content using the sulfuric acid-phenol method. Use glucose as a standard to plot 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 can be seen from Table 1, after fractionation separation with 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 screening corn straw lignin-degrading bacteria.

[0046] Table 1. Compositional analysis of lignin from corn stalks.

[0047]

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

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

[0050] First, nitrogen-fixing bacteria were isolated and purified using Ashube medium. The Ashube medium formula is as follows: KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaCO3 5.0g, mannitol 10.0g, CaSO4 0.1g, agar 15g, H2O 1000mL, sterilized at 121℃ for 15min to prepare Ashube plates. Organic fertilizer from Heilongjiang Ruiyuan Biotechnology Co., Ltd. was inoculated onto Ashube plates, and nitrogen-fixing bacteria were enriched at 39℃. After colonies grew, they were picked and repeatedly streaked on Ashube plates to obtain single colonies, which were then observed under a microscope to confirm the acquisition of pure cultures.

[0051] The pure culture was then inoculated onto CSL bilayer plates for incubation. The formulation of the CSL bilayer plate is as follows:

[0052] The formula for the lower layer culture medium is as follows: 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℃ for 15 min;

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

[0054] Colony morphology as Figure 2 and 3 As shown, on the Assumption plate ( Figure 2 ) and CSL culture medium double-layer plates ( Figure 3 On the surface, the colonies are transparent, colorless, and mucous-like, with smooth, moist surfaces and neat edges. Their morphology is as follows: Figure 4 As shown, the bacterial cells are rod-shaped, short, and without spores.

[0055] Single colonies picked from CSL double-layer plates were inoculated into yeast extract peptone glucose (YPD) medium. The YPD medium formulation was as follows: 5g tryptone, 2.5g yeast extract, 1g glucose, 1000mL H2O, and sterilized at 121℃ for 15min. The culture was then carried out at 39℃ and 180r / min. The bacterial cells were collected and their 16S rRNA gene sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results, confirmed by BLAST alignment, identified the strain as *Rhizobium pusense*, and named it *Rhizobium pusense* QY12 4aN. The strain was preserved using the glycerol preservation method at -80℃ and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29921 on March 1, 2024.

[0056] Assumption medium is a nitrogen-free, selective synthetic medium where mannitol is the sole carbon source, and it is commonly used to enrich and isolate free-living nitrogen-fixing bacteria. Therefore, *Pussa rhizobium* QY12 4aN can grow well on Assumption plates and be isolated and purified, such as... Figure 2 As shown, this indicates that *Pusarobacter pusa* QY12 4aN can use mannitol as a carbon source and utilize free nitrogen from the air to synthesize nitrogen-containing organic matter that it needs, i.e., it has a self-generating nitrogen fixation function.

[0057] According to the culture medium formulation, the upper layer of the CSL double-layer plate consists of CSL and inorganic salts, with no nitrogen source added. *Pulsatilla chinensis* QY12 4aN can grow well on the CSL double-layer plate, indicating that *Pulsatilla chinensis* QY12 4aN can utilize CSL as a carbon source for metabolism, obtaining the energy and carbon required for its growth. Simultaneously, it utilizes free nitrogen from the air to synthesize nitrogen-containing organic matter, further demonstrating its ability to fix nitrogen spontaneously.

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

[0059] Rhizobium pusa QY12 4aN expansion culture. Bacterial cells frozen at -80℃ were picked and inoculated onto Assoube plates for thawing. After single colonies grew, the cells were picked and inoculated onto YPD medium. After continuous expansion culture, the cells were centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed twice with sterile physiological saline to prepare a bacterial suspension.

[0060] Synthesis of hyaluronic acid. The prepared bacterial suspension was inoculated into a hyaluronic acid (HA) preparation medium with the following formula: 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, glucose 10.0 g, H₂O 1000 mL; sterilized at 121℃ for 15 min. The final concentration of *Rhizobium pusa* QY12 4aN cells was 1.0 OD / mL. The culture was carried out at 30℃ and 180 rpm with shaking for 2 days. After the culture was completed, the culture was centrifuged at 10000 rpm for 10 min, and the supernatant was collected. Hyaluronic acid in the supernatant was separated using D101 macroporous adsorption resin exchange column chromatography. The supernatant was mixed with deionized water at a 1:1 (v / v) ratio and loaded onto the column. The elution flow rate was 2 mL / min. During elution, the eluent was monitored online at 220 nm using a UV detector. The distribution of total sugars and glucuronic acid in the eluent was tracked using a microplate method. The glucuronic acid fraction was collected and concentrated using a rotary evaporator. The concentrated fraction was then subjected to alcohol precipitation. Three 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 8000 r / min for 5 min, discard the supernatant, and dissolve the precipitate in deionized water. Repeat the alcohol precipitation-deionized water dissolution step twice, and obtain lyophilized powder by vacuum freeze drying.

[0061] The hyaluronic acid content in the above-mentioned freeze-dried powder was determined according to the People's Republic of China Light Industry Standard QB / T4576-2023 Sodium Hyaluronate, using the spectrophotometric method-sulfuric acid-carbazole method, with slight modifications. A standard curve was plotted using glucuronic acid as the standard. The hyaluronic acid content was calculated based on the glucuronic acid standard curve using the following formula:

[0062]

[0063] In the formula:

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

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

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

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

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

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

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

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

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

[0073] w3: Moisture content of the sample, in grams per 100 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. Using borax as a catalyst, hyaluronic acid is acid-hydrolyzed with sulfuric acid to produce glucuronic acid and N-acetyl-D-glucosamine monomers. Glucuronic acid reacts with carbazole to form an organic complex, which exhibits a characteristic purple color. Its absorbance is directly proportional to the concentration of glucuronic acid. By measuring the absorbance, the content of hyaluronic acid can be determined.

[0076] The results determined by the above method show that the hyaluronic acid content in the freeze-dried powder is 57.68%.

[0077] Determination of infrared spectra of lyophilized powder samples. The functional groups of hyaluronic acid were determined using a Bruker EQUINOX 55 infrared spectrometer. An appropriate amount of lyophilized powder sample was mixed with KBr powder at a mass ratio of 1:100, ground evenly in a mortar, and then compressed into tablets. The spectra were measured at 4000-4000 cm⁻¹. -1 Spectral scanning was performed within the wavenumber range with a resolution of 4 cm⁻¹. -1 Scanned 32 times against an air background. Sodium hyaluronate standard (catalog number: 53747, Sigma-Aldrich) was used as a control.

[0078] The infrared spectrum of the lyophilized powder sample is shown in Figure 6. The infrared spectrum is at 3416 cm⁻¹. -1 A strong peak is observed at 2921 cm⁻¹, indicating the presence of OH tensile vibration. -1 The bands shown correspond to the presence of the extended symmetrical methyl group CH in glucuronic acid; at 1726 and 1637.00 cm⁻¹ -1 The peak value at 1566 cm⁻¹ indicates the presence of C=O carboxylamide I extension. -1 The peak values ​​at 1406 and 1375 cm⁻¹ indicate the presence of bending vibrations in NH₃; peak values ​​at 1406 and 1375 cm⁻¹ further indicate the presence of bending vibrations in NH₃. -1 The peaks at 1163, 1073, and 1045 cm⁻¹ indicate the presence of CO groups combined with C=O; -1 The peaks at 902 and 608 cm⁻¹ indicate the presence of COC, CO, and COH extension; -1 The presence of a peak at a certain point indicates the presence of COC extension. In summary, the infrared spectrum of the lyophilized powder exhibits the characteristic peaks of the infrared spectrum of hyaluronic acid and is similar to that of the hyaluronic acid standard, confirming that the main component of the lyophilized powder is hyaluronic acid.

[0079] Example 4. Preparation of Rhizobium pusa QY12 4aN inoculum.

[0080] Rhizobium pusa QY12 4aN cells frozen at -80℃ were inoculated onto Assoube agar plates for revival. After single colonies grew, single colonies were picked and inoculated into YPD for 24 h. Inoculation was performed twice at a 5% inoculum rate. The resulting culture was then inoculated into CSL liquid medium at a 5% (v / v) concentration. The CSL liquid medium formulation was as follows: CSL 5g, K2HPO4 1g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.02g, ZnSO4·7H2O 0.04g, glucose 10.0g, H2O 1000mL; sterilized at 121℃ for 15 min. The culture was carried out at 39℃ and 200 r / min for 72 hours. The fermentation broth was then aliquoted and packaged to obtain the *Rhizobium pusa* QY12 4aN inoculum. The pH, viscosity, polysaccharide, glucuronic acid, hyaluronic acid, and total polyphenol content of the fermentation broth were measured, along with its antioxidant activity and viable cell count. The flowchart is shown below. 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. Hyaluronic acid can adsorb lignin in the fermentation broth, turning it brown.

[0082] The specific analytical methods for the component analysis, antioxidant activity determination, and total colony count determination of the fermentation broth of *Rhizobium pusa* QY12 4aN inoculum are as follows:

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

[0084] (2) Determination of the viscosity of the fermentation broth. The viscosity was measured 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, add 3 times the volume of anhydrous ethanol, mix well, let stand at 4℃ for 2 h, then centrifuge at 8000 r / min for 5 min, discard the supernatant, and obtain the precipitate. Dissolve the precipitate in 4 mL of purified water, centrifuge, collect the supernatant, and dilute appropriately. The water-soluble polysaccharide content is determined using the sulfuric acid-phenol method. A standard curve is plotted using glucose as the standard. The water-soluble polysaccharide content in the fermentation broth is calculated based on the glucose standard curve.

[0086] (4) Determination of glucuronic acid content in fermentation broth. Take 1 mL of fermentation broth, add 3 times the volume of anhydrous ethanol, mix well, let stand at 4℃ for 2 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, dissolve the precipitate in 4 mL of purified water, centrifuge again, collect the supernatant, and dilute appropriately. Determine the glucuronic acid content using the sulfuric acid-carbazole method. Use glucuronic acid as a standard to plot a standard curve. Calculate the glucuronic acid content in the fermentation broth based on the glucuronic acid standard curve.

[0087] (5) Determination of hyaluronic acid content in 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 using the spectrophotometric method-sulfuric acid-carbazole method. A standard curve was plotted using glucuronic acid as the standard. The content of hyaluronic acid in the fermentation broth was calculated based on the glucuronic acid standard curve, using the following formula:

[0088] C HA = c × N × 1.9542 × 10 -3

[0089] In the formula:

[0090] C HA Hyaluronic acid content in the sample, expressed in grams per liter (g / L);

[0091] c: Based on 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 the sample solution;

[0093] 1.9542: The 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-Ciocalteu method was used. A standard curve was plotted using gallic acid as the standard. 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. An enzyme-linked immunosorbent assay (ELISA) reader was used for measurement. A suitable concentration of test solution was prepared from the lyophilized fermentation broth of Example 4 after 72 hours of fermentation. Before testing, the A concentration of the ABTS working solution was diluted with 50 mmol / L Tris-HCl (pH 7.4). 734nmDilute to 0.9 ± 0.1, mix 40 μL of sample solution with 160 μL of ABTS working solution, and react at room temperature in the dark for 5 min. Measure the absorbance of the reaction solution at 734 nm using a Safire 2 microplate reader. Use 50 mmol / L Tri-HCl (pH 7.4) as a blank control and Trolox as a positive control. The ABTS cationic radical scavenging activity is determined by the concentration of the sample solution that scavenges 50% of ABTS cationic radicals, i.e., the IC50 value. 50 It is expressed in (μg / mL).

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

[0098] (8) Total colony count determination. The determination was performed according to GB 20287-2006, the method for determining the number of viable bacteria in agricultural microbial agents. Plate counting agar medium and the pour method were used for the determination.

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

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

[0101]

[0102] Table 2 shows that after culturing *Rhizobium pusa* QY12 4aN in CSL liquid medium for 72 h, the contents of water-soluble polysaccharides increased from 0.00 g / L to 2.01 g / L, glucuronic acid from 0.00 g / L to 0.11 g / L, and hyaluronic acid from 0.00 g / L to 0.23 g / 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). The ability of *Rhizobium pusa* QY12 4aN to synthesize hyaluronic acid further indicates that its nitrogen originates from the nitrogen-fixing activity of *Rhizobium pusa* QY12 4aN. Simultaneously, the viscosity of the fermentation broth increased from 0 cP to 144 cP, indicating that the production of hyaluronic acid increased the viscosity of the fermentation broth. The total phenol content in 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 Folin-Ciocalteu method for determining the total phenol content in the fermentation broth can reflect the lignin content or the degree of lignin degradation. The decreasing trend in the total phenol content of the fermentation broth indicates that lignin has been degraded.

[0103] The above (7) results show that the ABTS cationic free radical scavenging activity of the freeze-dried ABTS cationic free radical scavenging powder of Rhizobium pusa QY12 4aN after 72 h of fermentation was [IC value missing].50 =527.67±24.01 μg / mL, IC50 value of ABTS cationic free radical scavenging activity of the initial fermentation broth lyophilized powder. 50 =1058.00±11.36 μg / mL. After fermentation with Rhizobium pusa QY12 4aN for 72 h, the ABTS cationic free radical scavenging activity of the fermentation broth was enhanced. This indicates that the fermentation broth has a certain antioxidant effect.

[0104] According to the above (8) test results, the effective viable count (cfu) in the Rhizobium pusa QY12 4aN inoculum is ≥2.0×10⁻⁶. 8 per mL.

Claims

1. A QY124aN bacterial agent, characterized in that, The bacterial agent was prepared using Rhizobium pusa QY124aN; The *Pusa rhizobium* QY124aN strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29921 and deposit date of March 1, 2024.

2. The microbial agent according to claim 1, characterized in that, The microbial agent includes hyaluronic acid and lignin.

3. A method for preparing the QY124aN bacterial agent according to any one of claims 1 to 2, characterized in that, The method is as follows: Step 1: Inoculate Rhizobium pusa QY124aN onto Ashube agar plates for resuscitation and obtain single colonies; Step 2: Inoculate a single colony into YPD medium and incubate for 24 hours to obtain bacterial culture; Step 3: Expand the bacterial culture twice to obtain the culture; Step 4: Inoculate the culture into CSL liquid medium and culture to obtain QY124aN bacterial agent.

4. The method according to claim 3, characterized in that, The inoculum size for the culture described in step four is 5%.

5. The method according to claim 3, characterized in that, The formulation of the CSL liquid culture medium in step four is as follows: CSL 5g, K2HPO4 1g, 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.

6. The method according to claim 3, characterized in that, The culture conditions described in step four are 39°C, 200 r / min, and culture for 72 h.

Images