Pantoea agglomerans and uses thereof

Abstract

This invention relates to the field of microbial technology, specifically disclosing a *Pantotheca acuminata* strain and its applications. A *Pantotheca acuminata* strain DSX-01 is provided. Through purification and isolation methods, a strain with good cellulose-decomposing ability was obtained, identified as *Pantotheca acuminata* DSX-01. Experiments detecting its cellulose-decomposing enzyme activity showed that *Pantotheca acuminata* DSX-01 has good cellulose-decomposing ability. It was deposited on March 6, 2025, at the China Center for Type Culture Collection (CCTCC), address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province; accession number: CCTCC M 2025390. The *Pantotheca acuminata* DSX-01 strain was inoculated into a liquid culture medium to obtain a liquid strain. The *Pantotheca acuminata* strain or its fermentation broth was then inoculated into sawdust, and the sawdust was fermented to prepare a fermentation substrate. A comparison was made between the fermentation substrate and the blank substrate, showing that the *Pantotheca acuminata* strain inoculated into the substrate significantly promoted the growth of *Pantotheca acuminata* mycelium.

Description

A type of pantothenic bacteria and its application Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of pantothenic bacteria and its applications. Background Technology

[0002] Winter fungus is an edible fungus with relatively high requirements for its growth environment. Fermentation provides the fungus with richer nutrients and a more suitable growth environment. During fermentation, the organic matter in the substrate is decomposed, releasing nutrients such as carbon, nitrogen, and minerals necessary for mycelial growth. Pre-treatment of the fermentation substrate improves its physical properties, such as increased aeration and water retention, which promotes rapid mycelial growth and expansion. Using fermentation substrate also reduces contamination by other microorganisms in the substrate, improving the purity and vitality of the spawn.

[0003] Fermentation bacteria can decompose complex organic matter (such as lignin and cellulose) in the fermentation substrate, converting it into simple carbon and nitrogen sources usable by *Fructus medica* mycelium, thus providing abundant nutrients for its growth. Fermentation bacteria in the substrate can interact with other microorganisms, forming a complex microbial community. These microorganisms continuously evolve during fermentation, decomposing organic matter, releasing nutrients, and inhibiting the growth of harmful microorganisms, thereby maintaining the microbial ecological balance in the substrate and reducing the risk of contamination. The metabolic activities of fermentation bacteria can accelerate the fermentation process, shorten fermentation time, and improve the quality and stability of the substrate. Furthermore, fermentation bacteria can produce beneficial metabolites, such as enzymes and organic acids, which can further promote the growth of *Fructus medica* mycelium. In summary, fermentation bacteria in *Fructus medica* fermentation substrate not only provide nutrients by decomposing organic matter but also improve the physicochemical properties and microbial community structure of the substrate, thus creating a more suitable environment for the growth of *Fructus medica* mycelium.

[0004] This invention employs purification and separation methods to obtain a fermentation strain suitable for large-scale industrial production of *Fructus Epimedium* fermentation material. Summary of the Invention

[0005] The purpose of this invention is to provide a batching pantothecin DSX-01 suitable for winter fungus fermentation materials and its application, so as to realize the large-scale industrial production of winter fungus fermentation materials.

[0006] One of the objectives of this invention is to provide a cluster of pantothenic bacteria DSX-01, which was deposited on March 6, 2025, at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province; accession number: CCTCC M2025390.

[0007] Strain name: Pantotheca acuminata;

[0008] Latin name: Pantoea agglomerans;

[0009] Strain number: DSX-01.

[0010] The second objective of this invention is to provide a liquid culture of Pantotheca acuminata DSX-01, which is obtained by inoculating the above-mentioned Pantotheca acuminata DSX-01 into a liquid culture medium;

[0011] Preferably, the culture medium is a liquid culture medium of Pantotheca acuminata DSX-01, and the liquid culture medium of Pantotheca acuminata DSX-01 includes a carbon source, a nitrogen source, and inorganic salts;

[0012] Preferably, the carbon source is one or more of xylose, maltose, sodium carboxymethyl cellulose, glucose, mannose, lactose, and brown sugar;

[0013] Preferably, the optimal carbon source is a mixture of glucose, sodium carboxymethyl cellulose, and xylose;

[0014] Preferably, the nitrogen source is one or more of yeast extract, peptone, ammonium oxalate, and soybean meal;

[0015] Preferably, the optimal nitrogen source is a mixture of peptone and yeast extract;

[0016] Preferably, the inorganic salt is one or a mixture of potassium nitrate, corn steep liquor powder, potassium dihydrogen phosphate, magnesium sulfate, and dipotassium hydrogen phosphate;

[0017] Preferably, the optimal inorganic salt is a mixture of potassium dihydrogen phosphate and magnesium sulfate.

[0018] The third objective of this invention is to provide an application of Pantotheca acuminata or Pantotheca acuminata fermentation broth in promoting the growth of Dictamnus dasycarpus mycelium. Specifically, the above-mentioned Pantotheca acuminata or Pantotheca acuminata fermentation broth is inoculated into sawdust, and the fermented sawdust is proportioned to form a fermentation material.

[0019] Preferably, the wood chips are made from mixed wood chips;

[0020] Preferably, the fermented sawdust used in the fermentation material formulation is fermented by inoculating the clump of Pantothenic agaricus;

[0021] Preferably, the fermentation feed formulation contains sawdust, wheat bran (or sawdust), cornmeal, white sugar, light calcium carbonate (or lime), and gypsum.

[0022] Preferably, the inoculated *Fungiformis edulis* variety is Qianmi No. 1 or Qianzhuang No. 1, and the tested *Fungiformis edulis* strains are Qianzhuang No. 1 and Qianmi No. 1, provided by the Guizhou Provincial Institute of Crop Germplasm Resources.

[0023] The beneficial effects of this invention are as follows: By isolating and purifying the fermentation bacteria strains in existing *Fructus Hordei* fermentation materials, a *Pantotheca acuminata* strain with good cellulose-decomposing ability was obtained. The strain was identified as *Pantotheca acuminata* DSX-01. Experiments detecting its cellulose-decomposing enzyme activity showed that *Pantotheca acuminata* DSX-01 had good cellulose-decomposing ability. Simultaneously, the strain was inoculated into sawdust for fermentation. A comparison was made between the *Fructus Hordei* strain inoculated into the fermentation material and the control material; the promoting effect on *Fructus Hordei* mycelial growth was significantly higher in the former than in the latter. Attached Figure Description

[0024] Figure 1 shows the determination of the cellulase-degrading enzyme capacity of the pantothenic acid DSX-01;

[0025] Figure 2 shows the colony diagram of the clustered pantothecin DSX-01 bacteria;

[0026] Figure 3 shows the promoting effect of Pantotheca acuminata DSX-01 on the mycelial growth of Qianzhuang No. 1 (CK on the right);

[0027] Figure 4 shows the promoting effect of the clustered Pantotheca acuminata DSX-01 on the mycelial growth of Qianmi No. 1 (CK is on the left). Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0029] Implementation Case 1: Screening and Identification of Pantotheca agglomerans

[0030] 1 test strain

[0031] 1.1 Test strains

[0032] The tested strain, Pantotheca acuminata DSX-01, was isolated from fermentation material prepared by Jinchan Dashan Technology Co., Ltd. in Nayong County, Bijie City. After successful isolation, it was stored at 4℃ and deposited in the China Center for Type Culture Collection (CCTCC), with accession number CCTCC M 2025390.

[0033] 1.2 Test Culture Medium

[0034] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 1000 ml distilled water, pH 7.0.

[0035] PDA solid culture medium: 200 g / L potato, 20 g / L glucose, 15 g / L agar powder, 1000 ml distilled water, pH 7.0.

[0036] Liquid culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L glucose, 10 g / L sodium carboxymethyl cellulose, 5 g / L xylose, 3 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 1000 ml distilled water, pH 7.0.

[0037] 2. Strain screening

[0038] 2.1 Sample processing

[0039] The collected fermentation material was sampled, and the sample was rinsed with deionized water to remove surface impurities and residues. The cleaned sample was placed in an oven to dry to remove moisture. The dried sample was ground into powder. The ground sample was appropriately diluted with sterile water to increase the number of microorganisms in the sample. The diluted sample was evenly spread on a solid culture medium to form single colonies.

[0040] 2.2 Separation and Purification

[0041] From the initially screened colonies, single colonies are further isolated using streak plating or other methods. These single colonies are then cultured and isolated again, and this step is repeated 2-3 times to confirm the purity of the isolated strains.

[0042] 2.3 Isolation and Screening of Cellulose-Degrading Bacteria

[0043] 2.3.1 Screening medium for cellulase-producing strains

[0044] Sodium carboxymethyl cellulose (CMC-Na) 10g, yeast extract 5g, peptone 10g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, sodium chloride 5g, agar 10g, distilled water 1000mL, pH 7.

[0045] 2.3.2 Isolation and Screening of Cellulose-Degrading Bacteria

[0046] Each strain was replicated in triplicate and cultured at 30°C for 3 days. 4 mL of 1 mg / mL Congo red solution was evenly spread on the plate, stained for 30 min, and then the Congo red solution was discarded. The plate was then rinsed with 1 mol / L NaCl solution for 30 min. The colony diameter d (mm) and clear zone D (mm) were measured using the cross-hatching method. Strains with a high H value (H = D / d) were selected for enzyme activity assay. The cellulase-producing strain DSX-01 was selected from the strains isolated from the fermentation sample, as shown in Figure 1.

[0047] 3 strains identified

[0048] 3.1 Strain morphology

[0049] Colony morphology description: Pantotheca acuminata is a Gram-negative bacillus with rod-shaped cells. On LB medium, colonies are round, with a diameter between 1 and 5 mm, and are either slimy or smooth; as shown in Figure 2.

[0050] 3.2 16S rRNA gene sequence analysis

[0051] Sequencing was performed using an ABI 3730-XL sequencer. The 16S rRNA gene sequence of strain DSX-01 was compared with data in the NCBI BLAST database. The highest homology (98.27%) was found with the partial sequence of Pantoea agglomerans strain RSG116SribosomalRNAgene in the gene bank. Therefore, strain DSX-01 was identified as Pantoea agglomerans.

[0052] Implementation Case 2: Fermentation Culture of Pantothenia glutinosa

[0053] 1. Activation of bacterial strains

[0054] Inoculate the glycerol tubes or freeze-dried Pantotheca DSX-01 clumps onto prepared LB medium plates and incubate at 32°C for 12-16 hours.

[0055] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 1000 ml distilled water, pH 7.0.

[0056] 2. Preparation of shake flask seed

[0057] Take 1-2 loops of activated plate culture and inoculate it into a shake flask culture medium. Incubate at 30°C in a shaking incubator for 12-14 hours to obtain a liquid culture of Pantothenia gravis.

[0058] Liquid culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L glucose, 10 g / L sodium carboxymethyl cellulose, 5 g / L xylose, 3 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 1000 ml distilled water, pH 7.0.

[0059] 3. Preparation of fermentation broth

[0060] Inoculate the seed culture from the shake flask into the fermentation medium at an inoculation rate of 5‰, and incubate at a temperature of 30℃-35℃, a rotation speed of 80-100 rpm, an air flow rate of 0.5 vvm / min, for 48 hours.

[0061] Fermentation medium composition: peptone 10g / L, yeast extract 5g / L, glucose 10g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 5g / L.

[0062] 4. Preparation of fermentation material

[0063] 4.1 Raw material processing

[0064] Layered stacking: Generally, it is stacked in three layers. The first and second layers are each 60 cm thick, and the third layer is 40 cm thick. The fermentation agent (clustering pantothenic acid fermentation liquid and urea) is evenly sprinkled between the layers and on the third layer, with the amount from bottom to top in a ratio of 4:4:2.

[0065] Raw material ratio: 1 ton of sawdust is mixed with 1 L of clump-forming Pantothenic Acid Fermentation Broth and 5 kg of urea to meet the nitrogen requirements of microbial fermentation, and the carbon-nitrogen ratio is adjusted appropriately.

[0066] 4.2 Adjusting moisture

[0067] Before stacking, soak the sawdust thoroughly. The ratio of dry sawdust to water should be 1:1.8, so that the moisture content reaches 60%-70%; it is advisable that liquid drips out when the material is squeezed in the hand.

[0068] 4.3 Fermentation material preparation

[0069] The stack width is generally 1.6-2 meters, the stack height is 1.0-1.6 meters, and the length depends on the amount of material and the size of the site.

[0070] 4.4 Judgment of Fermentation Effect

[0071] Fully decomposed compost is dark brown to blackish-brown in color, soft in texture, with inconspicuous plant remains. When you squeeze the compost by hand, the juice that comes out after filtering is colorless and has a foul odor.

[0072] Case Study 3: Growth-promoting effect of *Panthera philoxeroides* on *Fungiella vesicatoria*

[0073] 1. Experimental Materials

[0074] The tested *Fungiella asiatica* strains were Qianzhuang No. 1 and Qianmi No. 1, provided by the Guizhou Provincial Institute of Crop Germplasm Resources. The fermentation materials containing *Pantotheca acuminata* DSX-01 and those without *Pantotheca acuminata* DSX-01 were used (CK).

[0075] 2. Experimental Methods

[0076] Preparation of inoculation test tubes: Fill the fermentation material containing clumps of pantothenic acid DSX-01 and the fermentation material without clumps of pantothenic acid DSX-01 (CK) into test tubes with a length of 20 cm, and sterilize at 120℃ and 0.125 MPa for 120 minutes.

[0077] The tested *Fungiformis sylvestris* strains were inoculated into test tubes and placed in a constant temperature incubator at 25°C for 30 days in the dark. The mycelial growth length of *Fungiformis sylvestris* was observed weekly.

[0078] 3. Experimental Results

[0079] Compared with the control group, as shown in Table 1, Figure 3, and Figure 4, the experimental data clearly show that the fermentation substrate of *Pantotheca acuminata* DSX-01 significantly promotes the growth of *Dictyophora indica* mycelium. Figures 3 and 4 clearly show that the growth rate of *Dictyophora indica* mycelium in the fermentation substrate using *Pantotheca acuminata* DSX-01 was significantly higher than that in the control group, further verifying the effect of this patented strain on the growth of *Dictyophora indica* mycelium.

[0080] Table 1. Effect of Pantotheca acuminata DSX-01 on the growth of D. coli mycelium.

[0081]

[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An application of a type of pantothenic aerosol, characterized in that, The *Pantoea agglomerans* species, designated DSX-01, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 2025390. The colony morphology of *Pantoea agglomerans* is described as follows: *Pantoea agglomerans* is a Gram-negative bacillus with rod-shaped cells. On LB medium, the colonies are round, with a diameter between 1-5 mm, and are either mucous or smooth. *Pantoea agglomerans* DSX-01 is suitable for fermenting *Fructus *Dictyophora indica*. Specifically, the *Pantoea agglomerans* or its fermentation broth is inoculated into sawdust and mixed with the fermented sawdust to form a fermentation medium to promote the growth of *Fructus *Dictyophora indica* mycelia.

2. The application of a clustered pantothecin according to claim 1, characterized in that, The preparation method of the Pantotheca agglomerata fermentation broth includes: inoculating the above-mentioned Pantotheca agglomerata DSX-01 into a liquid culture medium to obtain Pantotheca agglomerata DSX-01 fermentation broth, wherein the culture medium is a liquid culture medium of Pantotheca agglomerata DSX-01, and the liquid culture medium of Pantotheca agglomerata DSX-01 includes a carbon source, a nitrogen source and inorganic salts.

3. The application of a clustered pantothecin according to claim 2, characterized in that, The carbon source is one or more of xylose, maltose, sodium carboxymethyl cellulose, glucose, mannose, lactose, and brown sugar.

4. The application of a clustered pantothenic acid bacteria according to claim 3, wherein the carbon source is a mixture of glucose, sodium carboxymethyl cellulose and xylose.

5. The application of a clustered pantothenic acid bacteria according to claim 2, wherein the nitrogen source is one or more of yeast extract, peptone, ammonium oxalate, and soybean meal.

6. The application of a clustered pantothenic acid bacteria according to claim 5, wherein the nitrogen source is a mixture of peptone and yeast extract.

7. The application of Pantotheca agglomerata according to claim 2, wherein the inorganic salt is one or more of potassium nitrate, potassium dihydrogen phosphate, magnesium sulfate, and dipotassium hydrogen phosphate.

8. The application of Pantotheca agglomerata according to claim 7, wherein the inorganic salt is a mixture of potassium dihydrogen phosphate and magnesium sulfate.

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