Streptococcus lactis strain SKLAN202403XW for degrading solanine and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to a streptococcus lactis strain SKLAN202403XW for degrading solanine and application of the streptococcus lactis strain SKLAN202403XW. The preservation number of the strain is CGMCC No.30021. The strain and a preparation thereof can effectively reduce the content of solanine in potato stems and leaves, significantly reduce the toxicity of the potato stems and leaves, and provide a basis for subsequent development and utilization.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Streptococcus lactis strain SKLAN202403XW capable of degrading solanine and an application thereof. Background Art

[0002] Utilizing potato stems and leaves as animal feed can alleviate the pressure on imports and consumption of traditional feed. However, potato stems and leaves contain a certain amount of the toxic alkaloid solanine, which is toxic to humans and animals and can cause symptoms such as nerve paralysis and hemolysis. Traditional processing methods include high-temperature treatment or chemical decomposition, but these methods are not only costly but also place a heavy burden on the environment.

[0003] Therefore, finding an efficient, environmentally friendly, and economical method for solanine degradation is particularly important. In recent years, microbial degradation has gradually attracted attention as a green biotechnology. By screening and utilizing bacteria with specific metabolic capabilities, effective degradation of solanine can be achieved, thereby increasing the utilization rate of potato stems and leaves and reducing their toxicity risk. Streptococcus lactis is a probiotic used in food and industrial applications, but there are currently no reports on its solanine degradation. Summary of the Invention

[0004] The invention aims to provide a bacterium capable of degrading solanine in potato stems and leaves.

[0005] Another object of the present invention is to provide applications of the above strains.

[0006] Another object of the present invention is to provide a method for degrading solanine in potato leaves.

[0007] The invention uses a specific lactic acid streptococcus (Enterococcus lactis) screened out from potato fields, the preservation number of which is CGMCC No. 30021. The strain can efficiently decompose solanine and significantly reduce its content.

[0008] The present invention provides a bacterial agent containing the above-mentioned Enterococcus lactis strain SKLAN202403XW.

[0009] The microbial agent according to the present invention can be a liquid preparation or a solid preparation, and can be prepared by conventional technical means and by adding auxiliary materials permitted in the field of microbial preparations.

[0010] The beneficial effects of the present invention are as follows: the present invention obtains a Streptococcus lactis (Enterococcus lactis) strain SKLAN202403XW by identifying and isolating it from potato-growing soil; the strain and its preparation can effectively reduce the solanine content in potato stems and leaves, significantly reducing the toxicity of potato stems and leaves, and providing a basis for subsequent development and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Show the bacterial morphology and colony morphology of the strains isolated in this application;

[0012] Figure 2 Liquid chromatography mass spectrometry test results showing that the strain of the present application degrades α-solanine and α-chaconine;

[0013] Figure 3 Displays α-rhamnosidase, β-galactosidase, and β-glucosidase enzyme activity assays;

[0014] Figure 4 Liquid chromatography-mass spectrometry detection results show that the strain of the present application degrades α-solanine and α-chaconine in potato stems and leaves.

[0015] The Enterococcus lactis strain SKLAN202403XW was deposited on March 13, 2024 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, China). The classification name is Enterococcus lactis, and the deposit number is CGMCC No. 30021. DETAILED DESCRIPTION

[0016] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0017] Example 1 Isolation and Identification of Enterococcus lactis Strain

[0018] A potato field, freshly harvested and with the seedlings and leaves discarded, was randomly selected in Jiehe Town, Tengzhou, Shandong Province. 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 incubation solution was added to a 50mL centrifuge tube. After processing, the samples were placed on dry ice and stored at -80°C in the laboratory until use.

[0019] Preliminary screening of bacterial colonies: Add 25ug / mL of α-solanine and α-chaconine (purity > 99.5%) to 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 LB medium. Use an inoculation loop to pick up the LB bacterial solution and streak it on LB solid medium until a single colony is obtained. Select three single surviving strains for enrichment and re-streaking, namely, Enterococcus lactis strain 2 and strain 3. Observe the colony morphology under a microscope ( Figure 1 ).

[0020] 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.

[0021] Example 2 Detection of solanine degradation characteristics of isolated Enterococcus lactis strains

[0022] 1. Screening of bacterial colonies with degradation effects: The three single colonies purified above were enriched, and then 25ug / mL of α-solanine and α-chaconine were added to 10mL MSM medium as the sole carbon source, and a control group was set up. The control group only contained MSM medium, and the empty group had no bacterial treatment and only added 25ug / mL of α-solanine or α-chaconine. 1000uL of the enriched bacterial solution (OD 600 Value = 0.3), and cultured in an incubator at 37°C and 200 rpm for 48 hours. The bacteria were then placed in a 4°C incubator for further testing and verification;

[0023] 2. Detection of solanine: Pipette 1 mL of bacterial culture 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 culture with degradation effect was subjected to HPLC-MS-MS to detect the content of α-solanine and α-chaconine in the experimental group and the blank group respectively. The results showed that compared with strain 2 and strain 3, the Enterococcus lactis strain significantly reduced the content of α-solanine (72%) and α-chaconine (100%), and the content of the final metabolite solanidine was significantly increased (p < 0.001). Figure 2shown.

[0024] 3. 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 centrifugation 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 (p<0.01), such as Figure 3 shown.

[0025] Purification and Identification: Through multiple passages and morphological analysis, a highly efficient degrading bacterium, Enterococcus lactis SKLAN202403XW, was obtained. It was identified by 16S rRNA sequencing and deposited with the China General Center for Microbiological Culture Collection (CGMCC No. 30021).

[0026] Example 3 Detection of solanine degradation characteristics of isolated Enterococcus lactis strains in fresh stems and leaves

[0027] Fresh potato stems and leaves were crushed and mixed, and 1 g was added into 100 μl of the enriched Enterococcus lactis strain (OD 600 The culture was performed with a 0.3 value and the 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.

[0028] Example 4 Whole genome and transcriptome screening and identification of Enterococcus lactis genes.

[0029] DNA was extracted from a logarithmically growing strain of Streptococcus lactis using a high-purity bacterial genomic DNA extraction kit. Whole-genome sequencing was performed using a PacBio Sequel IIe and an Illumina sequencer. Genes potentially involved in solanine degradation, including carbohydrases, were annotated in the CAZyme library.

[0030] The strain was inoculated into LB medium containing solanine (1 mg / mL), while the control group was a solanine-free medium. Total RNA was extracted from the tissue using the CTAB method, and genomic DNA was removed. High-quality RNA samples were used for subsequent library construction. Through the whole genome and transcriptome analysis, the key coding genes gene0604 and gene2370 capable of degrading solanine were identified. The obtained genes were inserted into a vector suitable for expression in the target host Escherichia coli TOP10.

[0031] The vector containing the target gene is introduced into the host Escherichia coli BL21(DE3) strain. Antibiotic selection is performed to ensure that only successfully transformed bacteria are retained. The recombinant plasmid is introduced into the host strain using competent cells or chemical methods. Expression of the target gene is induced. The activity of the recombinant protein is measured using techniques such as ELISA, and culture conditions are adjusted based on the results to improve expression efficiency.

[0032] Purify the recombinant protein using affinity chromatography or other purification techniques to ensure its high purity and activity. Analyze the purified protein by SDS-PAGE or mass spectrometry to confirm its molecular weight and integrity.

[0033] The recombinant protein is mixed with potato stem and leaf samples containing solanine and incubated under suitable conditions. The solanine content in the treated samples is measured using high performance liquid chromatography (HPLC) or other detection methods to evaluate the degradation effect.

Claims

1. An Enterococcus lactis strain, characterized in that The deposit number of the Streptococcus lactis strain is CGMCC No.30021.

2. Use of the Enterococcus lactis strain according to claim 1.

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

4. The use according to claim 3, characterized in that The Enterococcus lactis strain was used to degrade solanine in potato leaves.

5. A bacterial agent comprising the Enterococcus lactis 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 Enterococcus lactis strain according to claim 1 with potato leaves.

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