Pantoea agglomerans and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to pantoea agglomerans and application thereof, and provides a pantoea agglomerans DSX-01, a pantoea strain with good cellulose decomposition capacity is obtained through methods such as purification and separation, the pantoea strain is identified as the pantoea agglomerans DSX-01, and through an experiment for detecting the activity of cellulose decomposition enzyme of the pantoea agglomerans DSX-01, the content of the cellulose decomposition enzyme of the pantoea agglomerans DSX-01 is determined to be higher than the content of the cellulose decomposition enzyme of the pantoea agglomerans DSX-01. The pantoea agglomerans DSX-01 has good cellulose decomposition capability and is preserved in the China Center for Type Culture Collection on March 6, 2025, and the preservation address is Luojia mountain on eight road of Wuchang District, Wuhan City, Hubei Province; the preservation number of the strain is CCTCC M 2025390; the Pantoea agglomerans DSX-01 is inoculated into a liquid culture medium to obtain a Pantoea agglomerans DSX-01 liquid strain, then the Pantoea agglomerans or Pantoea agglomerans fermentation liquor is inoculated into wood chips, the wood chips are fermented and proportioned into a fermentation material, the phallus impudicus strain is inoculated into the fermentation material to be compared with a blank material, and the promoting effect of the phallus impudicus strain on growth of phallus impudicus mycelia is remarkably higher than that of the blank material.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Pantoea agglomerans and an application thereof. Background Art

[0002] Winter mulberry is an edible fungus that has high requirements for the growth environment. After fermentation, the fermentation material can provide the fungus with richer nutrients and a suitable growth environment. During the fermentation process, the organic matter in the culture medium is decomposed, releasing nutrients such as carbon source, nitrogen source and minerals required for the growth of mycelium. After pretreatment, the physical properties of the fermentation material are improved, such as enhanced air permeability and water retention, which helps the rapid growth and expansion of mycelium. The use of fermentation material can also reduce the contamination of bacteria in the mushroom sticks and improve the purity and vitality of the strains.

[0003] Fermentation bacteria can decompose complex organic substances (such as lignin, cellulose, etc.) in the fermentation material and convert them into simple carbon and nitrogen sources that can be used by winter iris hyphae, thereby providing rich nutrition for the growth of winter iris. Fermentation bacteria in fermentation materials can interact with other microorganisms to form a complex microbial community. These microorganisms continuously evolve during the fermentation process, decompose organic matter, release nutrients, and inhibit the growth of harmful microorganisms, thereby maintaining the microbial ecological balance in the fermentation material and reducing the risk of bacterial stick contamination. The metabolic activity of fermentation bacteria can accelerate the fermentation process, shorten the fermentation time, and improve the quality and stability of the fermentation material. In addition, fermentation bacteria can also produce some beneficial metabolites, such as enzymes, organic acids, etc., which can further promote the growth of winter iris hyphae. In summary, fermentation bacteria in winter iris fermentation material not only provide nutrition by decomposing organic matter, but also improve the physical and chemical properties of the fermentation material and the structure of the microbial community, thereby creating a more suitable environment for the growth of winter iris hyphae.

[0004] The invention adopts purification and separation methods to obtain a fermentation bacteria strain suitable for large-scale factory production of winter iris fermentation material from winter iris fermentation material. Summary of the invention

[0005] The purpose of the present invention is to provide a clumping Pantoea agglomerata DSX-01 suitable for winter strychnos radix fermented materials and its application, so as to realize the large-scale factory production of winter strychnos radix fermented materials.

[0006] One of the objects of the present invention is to provide a Pantoea agglomerans DSX-01, which was deposited in the China Center for Type Culture Collection on March 6, 2025, with the deposit address at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province; deposit number: CCTCC M2025390.

[0007] Bacteria species name: Pantoea agglomerans;

[0008] Latin name: Pantoea agglomerans;

[0009] Strain number: DSX-01.

[0010] The second object of the present invention is to provide a liquid strain of Pantoea agglomerans DSX-01, which is obtained by inoculating the above-mentioned Pantoea agglomerans DSX-01 into a liquid culture medium;

[0011] Preferably, the culture medium is a liquid culture medium of Pantoea agglomerata DSX-01, and the liquid culture medium of Pantoea agglomerata DSX-01 comprises a carbon source, a nitrogen source, and an inorganic salt;

[0012] Preferably, the carbon source is a mixture of 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 a mixture of 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 a mixture of one or more of potassium nitrate, corn syrup 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 object of the present invention is to provide an application of Pantoea agglomerans or a fermentation liquid of Pantoea agglomerans in promoting the growth of mycelium of Ilex sphaerocephala, specifically, the Pantoea agglomerans or the fermentation liquid of Pantoea agglomerans is inoculated into sawdust, and the sawdust is fermented to form a fermentation material;

[0019] Preferably, the sawdust is miscellaneous sawdust;

[0020] Preferably, the fermented sawdust used in the fermentation material ratio is fermented by inoculating the agglomerating Pantoea;

[0021] Preferably, the fermentation material formula contains sawdust, wheat bran (or sawdust), cornmeal, sugar, light calcium carbonate (or lime), and gypsum;

[0022] Preferably, the inoculated winter clover variety is Qianmi No. 1 or Qianzhuang No. 1, and the winter clover strains tested are the winter clover strains Qianzhuang No. 1 and Qianmi No. 1 provided by the Guizhou Institute of Crop Variety Resources.

[0023] The invention has the beneficial effects of separating and purifying the fermentation bacteria strains in the existing winter iris fermentation material, obtaining a strain of Pantoea with good cellulose decomposition ability, and identifying the strain as Pantoea agglomerans DSX-01. The experiment of detecting the activity of its cellulose decomposition enzyme shows that Pantoea agglomerans DSX-01 has good cellulose decomposition ability. At the same time, the strain is inoculated into sawdust for fermentation, and the winter iris strain is inoculated into the fermentation material for comparison with the blank material, and the effect of promoting the growth of winter iris mycelium is significantly higher than that of the blank. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To determine the cellulose degradation enzyme capacity of Pantoea agglomerans DSX-01;

[0025] Figure 2 This is the colony picture of Pantoea agglomerans DSX-01;

[0026] Figure 3 The promotion effect of Pantoea agglomerans DSX-01 on the mycelial growth of Qianzhuang No. 1 (CK on the right);

[0027] Figure 4 This is the promoting effect of Pantoea agglomerata DSX-01 on the mycelial growth of Qianmi No. 1 (CK on the left). DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0029] Case study 1: Screening and identification of Pantoea agglomerans

[0030] 1 Test strain

[0031] 1.1 Test strains

[0032] The test strain Pantoea agglomerans DSX-01 was isolated from the fermentation material prepared by Jinchan Dashan Technology Co., Ltd. in Nayong County, Bijie City. After successful isolation, it was stored at 4°C and deposited in the China Center for Type Culture Collection, which is abbreviated as CCTCC, with the collection number: CCTCC M 2025390.

[0033] 1.2 Test culture medium

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

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

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

[0037] 2. Strain Screening

[0038] 2.1 Sample processing

[0039] The collected fermentation material is sampled and rinsed with deionized water to remove surface impurities and residues; the cleaned samples are placed in an oven for drying to remove moisture; the dried samples are ground into powder; the ground samples are appropriately diluted with sterile water to increase the number of microorganisms in the samples; the diluted samples are evenly spread on a solid culture medium to form a single colony.

[0040] 2.2 Separation and purification

[0041] From the initially screened colonies, single colonies are further isolated by streaking or other methods. These single colonies are 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 Cellulase-producing strain screening medium

[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, pH7.

[0045] 2.3.2 Isolation and screening of cellulose-degrading bacteria

[0046] Each strain was replicated 3 times and cultured at 30℃ for 3 days. 4mL of 1mg / mL Congo red solution was evenly covered on the plate, and the Congo red solution was discarded after staining for 30min. Then, it was rinsed with 1mol / L NaCl solution for 30min. The colony diameter d (mm) and transparent circle D (mm) were measured by the cross method. The strains with large ratios were selected according to the H value (H=D / d) for enzyme activity determination. The better cellulase-producing strain DSX-01 was selected from the strains isolated from the fermentation material sample. Figure 1 shown.

[0047] 3. Strain identification

[0048] 3.1 Strain morphology

[0049] Description of colony morphology: Pantoea agglomerans are Gram-negative rods with rod-shaped cells. On LB medium, colonies are round with a diameter of 1-5 mm. Colonies are mucous or smooth; Figure 2 shown.

[0050] 3.2 16S rRNA gene sequence analysis

[0051] The 16S rRNA gene sequence of strain DSX-01 was sequenced using the sequencer ABI3730-XL, and the data in the NCBI BLAST database were compared with the partial sequence of Pantoea agglomerans strain RSG116S ribosomal RNA gene in the gene library, with the highest homology of 98.27%, so strain DSX-01 was identified as Pantoea agglomerans.

[0052] Case study 2: Fermentation culture of Pantoea agglomerans

[0053] 1. Bacteria activation

[0054] Inoculate the glycerol tube or freeze-dried Pantoea DSX-01 into the prepared LB medium plate and culture it in a 32°C constant temperature incubator for 12-16 hours;

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

[0056] 2. Shake flask seed preparation

[0057] Pick 1-2 loops of the activated plate bacteria and inoculate them into the shake flask culture medium, and culture them in a constant temperature shaking shaker at 30°C for 12-14 hours to obtain the liquid bacteria of Pantoea agglomerata.

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

[0059] 3. Fermentation broth preparation

[0060] The shake flask seed liquid was inoculated into the fermentation medium at an inoculation rate of 5‰, the temperature was 30°C-35°C, the rotation speed was 80-100rpm, the air volume was 0.5vvm / min, and the culture was carried out 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. Fermentation material preparation

[0063] 4.1 Raw material processing

[0064] Layered stacking: Generally, it is divided into three layers, with the first and second layers 60 cm thick each, and the third layer 40 cm thick. Sprinkle the fermentation agent (agglomerated pantothecin fermentation liquid, urea) evenly between the layers and on the third layer, with the ratio of 4:4:2 from bottom to top.

[0065] Raw material ratio: 1 ton of sawdust is matched with 1L of agglomerated pantoea fermentation liquid and 5 kg of urea to meet the nitrogen requirements of microbial fermentation and reasonably adjust the carbon-nitrogen ratio.

[0066] 4.2 Adjust moisture

[0067] Soak the sawdust thoroughly before stacking, with the ratio of dry sawdust to water being 1:1.8, so that the moisture content reaches 60%-70%; it is best if liquid drips out when holding the material in your hand.

[0068] 4.3 Fermentation material composting

[0069] The width of the pile is generally 1.6-2 meters, the 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 dark brown in color, with a soft texture and no obvious plant remains. When you squeeze out the juice from the compost with your hands, it is colorless and has a foul odor.

[0072] Case study 3: Growth-promoting effect of Pantoea agglomerans on winter safflower

[0073] 1 Test materials

[0074] The tested winter clover strains were the winter clover strains Qianzhuang No. 1 and Qianmi No. 1 provided by the Guizhou Institute of Crop Varieties Resources, the fermentation feed with Pantoea agglomerans DSX-01 and the fermentation feed without adding Pantoea agglomerans DSX-01 (CK).

[0075] 2 Test methods

[0076] Preparation of inoculated test tubes: The fermented material with Pantoea agglomerans DSX-01 and the fermented material without Pantoea agglomerans DSX-01 (CK) were filled into test tubes with a length of 20 cm and sterilized at 120°C and 0.125 MPa for 120 minutes.

[0077] The tested winter strychnine strains were inoculated into test tubes, placed in a constant temperature incubator at 25°C, and cultured in the dark for 30 days. The growth length of the winter strychnine hyphae was observed every week.

[0078] 3 Test results

[0079] Compared with the control group, the experimental group showed Figure 3 , Figure 4 From the test data, it can be clearly seen that the fermentation material of Pantoea agglomerata DSX-01 has a significant effect on promoting the growth of mycelium of Winter mulberry. Figure 4 It can be clearly seen that the growth rate of Oxytropis fasciata mycelium in the fermentation material using Pantoea agglomerata DSX-01 is significantly higher than that of the control, further verifying the effect of this patented strain on the growth of Oxytropis fasciata mycelium.

[0080] Table 1 Growth-promoting effect of Pantoea agglomerata DSX-01 mycelium

[0081]

[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Pantoea agglomerata, characterized in that: The Pantoea agglomerans was named as DSX-01 and deposited in China Center for Type Culture Collection with the deposit number: CCTCC M 2025390.

2. The agglomerated Pantoea according to claim 1, characterized in that: The colony morphology of Pantoea agglomerans is described as follows: Pantoea agglomerans is a Gram-negative bacillus, the cells are rod-shaped, and on LB culture medium, the colonies are round with a diameter of 1-5 mm, and the colonies are mucous or smooth.

3. A method for preparing a liquid strain of Pantoea agglomerans, characterized in that: include: The above-mentioned Pantoea agglomerans DSX-01 is inoculated into a liquid culture medium to obtain a liquid strain of Pantoea agglomerans DSX-01. The culture medium is a liquid culture medium of Pantoea agglomerans DSX-01, and the liquid culture medium of Pantoea agglomerans DSX-01 includes a carbon source, a nitrogen source, and an inorganic salt.

4. The method for preparing a liquid strain of Pantoea agglomerans according to claim 3, wherein the carbon source is a mixture of one or more of xylose, maltose, sodium carboxymethyl cellulose, glucose, mannose, lactose and brown sugar.

5. The method for preparing a liquid strain of Pantoea agglomerans according to claim 4, wherein the carbon source is a mixture of glucose, sodium carboxymethyl cellulose and xylose.

6. The method for preparing a liquid strain of Pantoea agglomerans according to claim 3, wherein the nitrogen source is a mixture of one or more of yeast extract, peptone, ammonium oxalate, and soybean meal. 7 . The method for preparing a liquid strain of Pantoea agglomerans according to claim 6 , wherein the nitrogen source is a mixture of peptone and yeast extract.

8. The method for preparing a liquid strain of Pantoea agglomerans according to claim 3, wherein the inorganic salt is a mixture of one or more of potassium nitrate, corn syrup powder, potassium dihydrogen phosphate, magnesium sulfate, and dipotassium hydrogen phosphate. 9 . The method for preparing a liquid strain of Pantoea agglomerans according to claim 8 , wherein the inorganic salt is a mixture of potassium dihydrogen phosphate and magnesium sulfate.

10. An application of Pantoea agglomerans, characterized in that: The agglomerating Pantoea DSX-01 is suitable for fermentation material of Winter Globe. Specifically, the above-mentioned Pantoea agglomerata or Pantoea agglomerata fermentation liquid is inoculated into sawdust, and the sawdust is fermented into fermentation material to promote the growth of Winter Globe mycelium.

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