Achromobacter xylosoxidans strain SKLAN202311ZW and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to an achromobacter xylosoxidans strain SKLAN202311ZW and application thereof. The preservation number of the Achromobacter xylosoxidans strain is CGMCC (China General Microbiological Culture Collection Center) No.28892, and the preservation number of the Achromobacter xylosoxidans strain is CGMCC No.28892. The strain can effectively reduce the content of solanine in the potato stems and leaves, significantly reduces the toxicity of the potato stems and leaves, and provides a basis for subsequent development and utilization. The method is suitable for the fields of large-scale agricultural production and feed processing, and improves the resource utilization rate and economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Achromobacter xylosoxidans SKLAN202311ZW and an application thereof. Background Art

[0002] The development and utilization of potato stems and leaves as animal feed can alleviate feed pressure. However, potato stems and leaves contain a certain amount of toxic alkaloid solanine. Generally speaking, solanine content in feed exceeding 6 mg / kg can cause poisoning. The main toxicity of solanine comes from α-solanine and α-chaconine. It is particularly important to find efficient, environmentally friendly and economical methods for degrading solanine. In recent years, microbial degradation as a green biotechnology has gradually attracted attention. By screening and utilizing bacteria with specific metabolic capabilities, effective degradation of solanine can be achieved, thereby improving the utilization rate of potato stems and leaves and reducing its toxicity risk. Summary of the Invention

[0003] The invention aims to provide a bacterium capable of degrading solanine in potato stems and leaves.

[0004] Another object of the present invention is to provide applications of the above strains.

[0005] Another object of the present invention is to provide a method for degrading solanine in potato stems and leaves.

[0006] The invention uses a specific Achromobacter Xylosoxidans strain screened out from potato fields. The strain can efficiently decompose solanine and significantly reduce its content.

[0007] The present invention provides a bacterial agent containing the Achromobacter xylosoxidans strain SKLAN202311ZW.

[0008] The microbial agent according to the present invention can be a liquid microbial agent or a solid microbial agent, and can be prepared by conventional technical means and by adding auxiliary materials permitted in the field of microbial preparations.

[0009] The present invention has the beneficial effect of identifying and isolating a strain of Achromobacter xylosoxidans, SKLAN202311ZW, from potato-growing soil. This strain can effectively reduce the solanine content in potato stems and leaves, significantly reducing their toxicity, providing a foundation for subsequent development and utilization. It is suitable for large-scale agricultural production and feed processing, improving resource utilization and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Show the morphology and colony morphology of the strains screened in this application;

[0011] Figure 2 Liquid chromatography-mass spectrometry detection showing that the strain of the present application degrades α-solanine, α-chaconine and solanidine;

[0012] Figure 3 Displays α-rhamnosidase, β-galactosidase, and β-glucosidase enzyme activity assays;

[0013] Figure 4 Showing the liquid chromatography-mass spectrometry detection of α-solanine and α-chaconine in potato stems and leaves.

[0014] The Achromobacter xylosoxidans strain SKLAN202311ZW was deposited on November 7, 2023 at the General Microbiology Center of the China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Achromobacter xylosoxidans, with the deposit number CGMCC No. 28892. DETAILED DESCRIPTION

[0015] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0016] Example 1 Isolation and Identification of Achromobacter xylosoxidans

[0017] In Jiehe Town, Tengzhou, Shandong Province, a potato field, freshly harvested and with the seedlings and leaves discarded, was randomly selected. Using the five-point method, 20g of soil samples were collected from the top, middle, and bottom layers of the soil at the four corners and the center. An equal volume of approximately 20mL of bacterial preservative solution was added to a 50mL centrifuge tube. After processing, the samples were placed on dry ice and stored in a -80°C freezer in the laboratory until use.

[0018] Preliminary screening of bacterial colonies: Add 25ug / mL of α-solanine and α-chaconine (purity>99.5%) to the LB broth, and add 100uL of bacterial solution of appropriate concentration. Incubate in an incubator at 37°C and 200rpm for 24 hours. Then spread the bacterial solution on the LB medium, use an inoculation loop to pick up the LB bacterial solution and streak it on the LB solid medium until a single colony is obtained. Select three single strains that can survive for enrichment and re-streak, namely Achromobacter xylosoxidans strain, strain 2 and strain 3. Observe the colony morphology under a microscope ( Figure 1 ).

[0019] Strain purification and enrichment: Pick a single colony from a streaked plate and streak it again, then culture it at 37°C overnight. Repeat this process three times to obtain a single, purified strain. Then, pick a single colony and culture it in 5 mL of LB broth on a shaker at 37°C for two days to fully enrich the strain. After that, perform 16S rRNA analysis. The remaining colony is mixed with saline containing 50% glycerol and stored at -80°C.

[0020] Screening of bacterial colonies with degradation effects: The three purified single colonies were enriched, and then 25ug / mL of α-solanine and α-chaconine were added to 10mL MSM culture medium as the sole carbon source, and a control group was set up. The control group only contained MSM culture medium, the empty group had no bacterial treatment, and only 25ug / mL of α-solanine or α-chaconine was added. The treatment groups were added with 1000uL of the corresponding bacterial solution (OD 600 The cells were incubated at 37°C and 200 rpm for 48 hours. The surviving bacteria were then placed in a 4°C incubator for further testing and verification.

[0021] Detection of solanine: Pipette 1 mL of bacterial solution into a centrifuge tube, add 5% acetic acid solution, shake thoroughly and mix well, then centrifuge at 5000g, 4°C for 15 minutes. The bacterial solution with degradation effect was subjected to HPLC-MS-MS to detect the content of α-solanine, α-chaconine and the final metabolite solanidine in the experimental group and the blank group respectively. The results showed that compared with strain 2 and strain 3, the Achromobacter Xylosoxidans strain significantly reduced the content of α-solanine (73%) and α-chaconine (99%), and the content of the final metabolite solanidine was significantly increased (p < 0.001). Figure 2 shown.

[0022] Activity detection of key degradation enzymes: centrifuge 1mL of bacterial solution, suspend with 0.08ml buffer, add 0.08mg lysozyme, ice bath for 30min, and then ultrasonicate (power 300W). Ultrasonication time is 5 seconds, interval is 5 seconds, ultrasonication is performed 5 times, and the interval time is slightly longer than the ultrasonication time, which is beneficial to protect the stability of the target indicator; after the ultrasonication is completed, centrifuge and take the supernatant, the centrifugal speed is 5000 rpm, and the centrifugal time is 15 minutes. Then, the Elisa kits of three key enzymes were used to determine the enzyme activity: α-rhamnosidase, β-galactosidase and β-glucosidase. The results showed that compared with the control group without the addition of bacterial solution, the enzyme activity of the three enzymes showed a significant upward trend, especially on the third day (p<0.05). The results are as follows Figure 3 shown.

[0023] Purification and Identification: Through multiple passages and morphological analysis, a highly efficient xylose-degrading strain, Achromobacter xylosoxidans, was obtained and named SKLAN202311ZW. It was identified by 16S rRNA sequencing and deposited with the China General Center for Microbiological Culture Collection (CGMCC No. 28892).

[0024] Example 2 Detection of solanine degradation characteristics of the isolated Achromobacter xylosoxidans strain in fresh stems and leaves.

[0025] Fresh potato stems and leaves were crushed and mixed, and 1 g was added into 100 μl of enriched Achromobacter Xylosoxidans strain (OD 600 The culture was carried out with a 50% flavonoid (value = 0.3) and residual α-solanine and α-chaconine were detected by liquid chromatography-mass spectrometry on days 1, 2, and 3. Compared with the blank group without bacterial solution, the content of α-solanine and α-chaconine decreased significantly, and the content was significantly lower after three days (p < 0.001). Figure 4 shown.

[0026] This invention provides an efficient, environmentally friendly, and economical method for degrading solanine in potato stems and leaves. By screening and applying a specific strain of Achromobacter xylosoxidans, the researchers successfully reduced the toxicity of solanine and improved resource utilization. This method has broad application prospects in agricultural production and feed processing, providing a new approach to addressing potato waste disposal.

Claims

1. Achromobacter xylosoxidans strain, characterized in that The preservation number of the Achromobacter xylosoxidans strain is CGMCC No. 28892.

2. Use of the Achromobacter xylosoxidans strain according to claim 1.

3. The use according to claim 2, characterized in that The Achromobacter xylosoxidans strain is used to degrade solanine.

4. The use according to claim 3, characterized in that The Achromobacter Xylosoxidans strain is used to degrade solanine in potato leaves.

5. A bacterial agent comprising the Achromobacter xylosoxidans strain according to claim 1.

6. A method for degrading solanine in potato leaves, characterized in that: The method comprises the step of co-culturing the bacterial solution of the Achromobacter xylosoxidans strain according to claim 1 with potato leaves.

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