Lactobacillus plantarum WCSF1-0012 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus plantarum WCSF1-0012 and application thereof.The lactobacillus plantarum WCSF1-0012 is preserved in the China General Microbiological Culture Collection Center on December 19, 2024, the preservation number is CGMCC No.33131, and the lactobacillus plantarum WCSF1-0012 is preserved in the China General Microbiological Culture Collection Center on December 19, 2024. The special gene sequence of the lactobacillus plantarum WCSF1-0012 is as shown in SEQ ID NO. 1; the lactobacillus plantarum WCSF1-0012 is used for preparing medicines, health-care products or food drinks for preventing, inhibiting or treating oral tumors. The lactobacillus plantarum WCSF1-0012 is a safe probiotic free of toxic and side effects, can effectively inhibit oral tumors, can play an anti-tumor role by activating macrophages, has long-term practical safety, is low in cost, high in safety and high in reliability, and can be used for preparing medicines or food drinks for preventing and even treating oral tumors.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Lactobacillus plantarum WCSF1-0012 and an application thereof. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck, accounting for more than 90% of oral tumors. According to statistics in 2020, there are approximately 380,000 new cases of malignant tumors in the oral cavity and tongue worldwide, and approximately 180,000 deaths. Enhancing the host's anti-tumor function by regulating the immune system is an emerging frontier in the field of tumor treatment.

[0003] In recent years, the immunomodulatory function of microorganisms has attracted widespread attention. Microorganisms, especially lactic acid bacteria, are characterized by low cost, high safety and high reliability. As one of the few microorganisms rated as recognized non-toxic by the U.S. Food and Drug Administration and the China Food and Drug Administration, Lactobacillus has good biosafety. Lactobacillus plantarum WCSF1 is a probiotic derived from the oral cavity. How to further prepare lactic acid bacteria that can activate the immune system efficiently, enhance the host's anti-tumor ability and do not introduce exogenous gene sequences through genetic engineering is a current technical difficulty. For this purpose, a strain of Lactobacillus plantarum WCSF1-0012 and its application are provided. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Lactobacillus plantarum WCSF1-0012 and its application in view of the defects of the prior art, so as to solve the problems raised by the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a Lactobacillus plantarum WCSF1-0012, wherein the Lactobacillus plantarum ( Lactobacillus plantarum )WCSF1-0012, was deposited in the General Microbiology Center of China Microorganism Culture Collection on December 19, 2024, with the deposit number CGMCC No.33131, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides a gene sequence of Lactobacillus plantarum WCSF1-0012 as shown in SEQ ID NO.1.

[0007] In addition, the present invention also provides an application of Lactobacillus plantarum WCSF1-0012, wherein the Lactobacillus plantarum WCSF1-0012 is used to prepare medicines or food and beverages for preventing, inhibiting or treating oral tumors.

[0008] Furthermore, the medicine includes a live strain of Lactobacillus plantarum WCSF1-0012.

[0009] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0010] Furthermore, the dosage form of the drug is tablets, capsules, oral liquid or lyophilized powder.

[0011] Furthermore, the food and beverage is a product prepared by adding an effective amount of Lactobacillus plantarum WCSF1-0012 to a lactic acid bacteria beverage, yogurt, fermented soy product, bacterial powder or milk powder.

[0012] Furthermore, the content of Lactobacillus plantarum WCSF1-0012 in the medicine or food and beverage is 10 8 CFU~10 11 CFU.

[0013] Furthermore, the medicine or food and drink is a medicine or food and drink that activates monocytes-macrophages and inhibits oral tumors.

[0014] Furthermore, the medicine or food and drink is a medicine or food and drink that efficiently activates monocyte-macrophage NF-κB and ISRE pathways and inhibits oral tumors.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention discloses Lactobacillus plantarum WCSF1-0012 which can effectively inhibit oral tumors. The Lactobacillus plantarum WCSF1-0012 is a safe lactic acid bacterium with no toxic side effects, which can effectively inhibit oral tumors and even treat oral tumors, that is, it can activate monocytes-macrophages and promote macrophages to kill tumor cells. In addition, it has long-term practical safety, low cost, high safety and high reliability, and can be used to prepare medicines or food and beverages for preventing or even treating oral tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the relative content of c-di-AMP in the bacteria of Lactobacillus plantarum WCSF1-0012 in Example 2 of the present invention; Figure 2 This is a diagram showing the activation of NF-κB and ISRE pathways in monocytes and macrophages by WCSF1-0012 in Example 3 of the present invention; Figure 3In Example 4 of the present invention, WCSF1-0012 promotes macrophages to clear oral tumor cells better than the wild type; wherein, Cal-27 only is the oral tumor cell group, Ctrl is the macrophage and oral tumor co-culture group, 10 ng / mL LPS is the lipopolysaccharide-treated macrophage and oral tumor co-culture group; 10 µg / mL c-di-AMP is the cyclic di-AMP-treated macrophage and oral tumor co-culture group; WT 50MOI is the Lactobacillus plantarum WCSF1-treated macrophage and oral tumor co-culture group; 0012 is the Lactobacillus plantarum WCSF1-0012-treated macrophage and oral tumor co-culture group; Figure 4 The resistance of WCSF1-0012 to antibiotics in Example 5 of the present invention did not show significant changes compared to the parent. The figure shows the OD values ​​of WCSF1 and WCSF1-0012 after being treated with different concentrations of antibiotics overnight. 600 nm Readings, antibiotics were erythromycin and chloramphenicol. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0018] In the following examples, the required strains are as follows: Lactobacillus plantarum ( Lactobacillus plantarum WCSF1-0012) strain, the plant lactobacillus WCSF1-0012 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 19, 2024, with the deposit number CGMCC No.33131, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; In the following examples, the required strains and cell lines are as follows: Lactobacillus plantarum WCSF1 ( L. plantarum WCSF1) Human monocyte-macrophage cell line THP-1 with NFκB and ISRE luciferase reporter systems; human OSCC cell line Cal-27 with luciferase-green fluorescent protein fusion protein; In the following examples, the required reagents are as follows: MRS medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; RPMI medium, DMEM medium, and fetal bovine serum were purchased from GIBCO (Thermo Fisher Scientific); High-purity D-luciferin potassium salt was purchased from Bio-Tech; Lipopolysaccharide (LPS) was purchased from Bio-Tech. c-di-AMP disodium was purchased from MCE; Mouse M-CSF recombinant protein was purchased from Proteintech; Chloramphenicol was purchased from Beijing Suolaibao Technology Co., Ltd.; Erythromycin was purchased from Maclean.

[0019] In the following examples, the nucleotide sequences are as follows: Lactobacillus plantarum ( Lactobacillus plantarum )The special gene sequence of WCSF1-0012 is shown in SEQ ID NO.1: ; Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0020] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0021] Example 1: Lactobacillus plantarum ( Lactobacillus plantarum WCSF1-0012) culture and preservation methods; (1) Culture method of bacterial strains; Step 1. Take 100 µl of Lactobacillus plantarum ( Lactobacillus plantarum WCSF1-0012) glycerol bacteria were spread on MRS solid medium plates containing 1.5% agar, cultured anaerobically at 37 degrees Celsius for 72 hours, and single colonies were picked. Lactobacillus plantarum WCSF1-0012) were inoculated into MRS liquid medium and cultured at 37°C for 16 h before use.

[0022] (2) Methods for preservation and activation of bacterial strains; The Lactobacillus plantarum ( Lactobacillus plantarum Add 50% glycerol to the bacterial solution of WCSF1-0012 to obtain a mixture with a final glycerol concentration of 25%. Mix well and place in a -80 ℃ refrigerator. It can be stored for 2-3 years. When activating, take out the glycerol cryovial, melt it in an ice bath, use a sterile inoculation loop to inoculate the bacterial solution into the pre-prepared MRS culture dish, place it in an anaerobic incubator to grow for 48 h-72 h, or until complete colonies grow in the culture dish, then it can be subcultured.

[0023] Example 2: Detection of the relative content of c-di-AMP in Lactobacillus plantarum WCSF1-0012; After activation and subculture of Lactobacillus plantarum WCSF1-0012 and its comparative strain Lactobacillus plantarum WCSF1 solid plates, single colonies were picked up with an inoculation needle and placed in 10 mL of MRS seed solution, and cultured anaerobically at 37 °C for 12-16 h. Then, 2% of the seed solution was added to a centrifuge tube containing 10 mL of MRS culture medium and cultured overnight for 12-16 h.

[0024] The culture solution was adjusted to OD 600 ≈0.5, take 30 mL of bacterial solution, 4 degrees Celsius, 4,000 x g , centrifuged for 5 min, resuspended in 250 µL extraction buffer, stored at -20°C for 30 min, and 15,000 x g , 4 degrees Celsius, centrifuged for 10 min, the supernatant was extracted, -20 degrees Celsius, as solution 1; the pellet was resuspended in 125 μL extraction buffer, stored at -20 degrees Celsius for 15 min, and 15,000 x g , 4 degrees Celsius, centrifuge for 5 min, and use it as solution 2. Thaw solution 1 at 4 degrees Celsius, mix with solution 2, and centrifuge at 15,000 x g , 4 degrees Celsius, centrifuged for 5 min, and 100 μL of the supernatant was taken as a sample for targeted detection of c-di-AMP. LC-MS was used for detection, and the test results were as follows Figure 1 shown.

[0025] Example 3: Lactobacillus plantarum WCSF1-0012 has monocyte-macrophage activation function; After activation and subculture of Lactobacillus plantarum WCSF1-0012 and its comparative strain Lactobacillus plantarum WCSF1 solid plates, single colonies were picked up with an inoculation needle and placed in 10 mL of MRS seed solution, and cultured anaerobically at 37 °C for 12-16 h. Then, 2% of the seed solution was added to a centrifuge tube containing 10 mL of MRS culture medium and cultured overnight for 12-16 h.

[0026] The absorbance (OD) at 600 nm was measured using a UV-visible spectrophotometer. 600 ) and cultured to the logarithmic growth phase (OD 600 ≈0.5~0.6) of bacterial suspension by centrifugation, discard the supernatant, add 10 mL PBS to mix and wash.

[0027] Prepare 10 8 CFU / mL, 5x10 7 CFU / mL and 2.5x10 7 CFU / mL RPMI bacterial solution (the concentration of bacterial solution in RPMI medium is 10 8 CFU / mL) for later use. 0.1 mL of RPMI bacterial solution, blank RPMI culture medium, and RPMI culture medium containing LPS and c-di-AMP were added to THP1 reporter cells and incubated at 37° 5% CO 2 The cells were treated for 2 h under the same conditions, centrifuged at 1500 rpm for 5 min, sterilized, resuspended in 0.2 mL PBS, centrifuged, and re-added with RPMI medium at 37° 5% CO 2 After treatment for 8 h, the culture medium was removed by centrifugation, the cells were resuspended in 0.2 mL PBS and centrifuged again. D-luciferin potassium solution was added again, the autofluorescence value was read, and the relative autofluorescence intensity unit RLU was recorded to obtain the comparative results of the activation levels of NF-κB and ISRE by stimulants of different strains and the control group.

[0028] like Figure 2 As shown, the activation of WCSF1-0012 at MOI 25, 50, and 100 was stronger than that of WCSF1 lactobacillus.

[0029] Example 4: Lactobacillus plantarum WCSF1-0012 promotes macrophages to kill oral tumor cells; Mouse primary bone marrow-derived M-CSF-induced macrophages were treated with blank medium, 10 ng / ml LPS, 10 μg / ml c-di-AMP, MOI 50 of Lactobacillus plantarum WCSF1, and MOI 50 of Lactobacillus plantarum WCSF1-0012 for 2 h, respectively. The treatment solution was removed and the cells were washed twice with PBS. 5 Cal-27 cell line expressing luciferase-green fluorescent protein fusion protein and 10 5 Macrophages and 2x10 5 Cal-27 cells expressing luciferase-green fluorescent protein fusion protein were co-cultured at 37°C with 5% CO 2 Remove the medium, resuspend in 0.2 mL PBS, centrifuge, add D-luciferin potassium solution, read the autofluorescence value, and record the relative autofluorescence intensity unit (RLU) for comparing the survival rate of Cal-27 cells.

[0030] like Figure 3 As shown, when WCSF1-0012-treated macrophages were co-cultured with Cal-27 cell lines, the viability of the Cal-27 cell line was reduced.

[0031] Example 5: The growth of Lactobacillus plantarum WCSF1-0012 can be inhibited by antibiotics; After activation and subculture of Lactobacillus plantarum WCSF1-0012 and its comparative bacterium Lactobacillus plantarum WCSF1 solid plates, single colonies were picked up with an inoculation needle and placed in 10 mL of MRS seed solution. The plates were anaerobically cultured at 37 °C for 12-16 h, and then 2% of the seed solution was added to MRS medium containing different concentrations of erythromycin and chloramphenicol. The plates were cultured overnight at 37 °C in a 96-well plate. The absorbance of the cultured bacterial solution at a wavelength of 600 nm was recorded to evaluate the inhibition of Lactobacillus plantarum WCSF1-0012 by antibiotics.

[0032] like Figure 4 As shown, there was no significant difference in the degree of inhibition of different concentrations of antibiotics on Lactobacillus plantarum WCSF1-0012 and its comparative strain WCSF1.

[0033] In the above examples, through in vitro experiments on monocyte-macrophage activation, oral tumor cell killing and antibiotic resistance detection, it was found that the Lactobacillus plantarum WCSF1-0012 of the present invention can activate immunity and inhibit oral tumors.

[0034] In addition, the plant lactobacillus WCSF1-0012 of the present invention is a plant lactobacillus that can effectively inhibit oral tumors. The lactobacillus is a safe lactic acid bacterium with no toxic side effects. It can effectively inhibit oral tumors and even treat oral tumors. In addition, it is safe, low-cost, high-safety, and high-reliability, and can be used to prepare medicines or food and beverages for preventing or even treating oral tumors.

[0035] The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum WCSF1-0012, characterized in that: The Lactobacillus plantarum ( Lactobacillus plantarum WCSF1-0012), was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 19, 2024, with the deposit number CGMCC No.33131.

2. A gene sequence of Lactobacillus plantarum WCSF1-0012 as claimed in claim 1, characterized in that: The gene sequence of the Lactobacillus plantarum WCSF1-0012 is shown in SEQ ID NO.

1.

3. Use of the Lactobacillus plantarum WCSF1-0012 as claimed in claim 1, characterized in that: The Lactobacillus plantarum WCSF1-0012 is used for preparing medicines or food and beverages for preventing, inhibiting or treating oral tumors.

4. According to the application of plant lactobacillus WCSF1-0012 described in claim 3, it is characterized in that, The medicine comprises a live strain of Lactobacillus plantarum WCSF1-0012.

5. According to the application of Lactobacillus plantarum WCSF1-0012 described in claim 3, it is characterized in that, The medicine includes pharmaceutically acceptable excipients.

6. The application of Lactobacillus plantarum WCSF1-0012 according to claim 3, characterized in that: The dosage form of the medicine is tablet, capsule, oral solution or lyophilized powder.

7. The application of Lactobacillus plantarum WCSF1-0012 according to claim 3, characterized in that: The food and beverage is a product prepared by adding an effective amount of Lactobacillus plantarum WCSF1-0012 to lactic acid bacteria beverage, yogurt, fermented bean products, bacterial powder or milk powder.

8. The application of Lactobacillus plantarum WCSF1-0012 according to claim 3, characterized in that: The content of Lactobacillus plantarum WCSF1-0012 in the medicine or food and beverage is 10 8 CFU~10 11 CFU.

9. The application of Lactobacillus plantarum WCSF1-0012 according to claim 3, characterized in that: The medicine or food and drink is a medicine or food and drink that can efficiently activate monocytes-macrophages and inhibit oral tumors.

10. The use of Lactobacillus plantarum WCSF1-0012 according to claim 3, characterized in that: The medicine or food and drink is a medicine or food and drink that activates monocyte-macrophage NF-κB and ISRE pathways and inhibits oral tumors.

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