Lysobacter enzymogenes SYSU923 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lysobacter enzymogenes SYSU923 and application thereof. According to the invention, a dominant strain lysobacter enzymogenes SYSU923 with broad-spectrum bacteriostatic activity is obtained by culturing, separating and purifying a soil sample through a flat plate bacteriostatic zone method, and is identified as lysobacter enzymogenes through gram staining, microscopic examination, single colony morphology observation and molecular biology experiments, and the lysobacter enzymogenes SYSU923 can be used for preparing the lysobacter enzymogenes SYSU923 with broad-spectrum bacteriostatic activity. The lysobacter enzymogenes SYSU923 or the culture of the lysobacter enzymogenes SYSU923 is combined with the immune checkpoint inhibitor to carry out anti-tumor treatment on esophageal cancer implanted tumor mice, the lysobacter enzymogenes SYSU923 is found to have a remarkable effect in the aspect of enhancing the treatment effect of the immune checkpoint inhibitor, and through combined treatment, the in-situ growth of tumors can be effectively inhibited; the metastasis of tumor cells is also remarkably reduced, and a more effective treatment strategy is expected to be provided for esophageal cancer patients.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to an enzyme-producing Lysozyme Bacillus SYSU923 and an application thereof. Background Art

[0002] Esophageal cancer is a common malignant tumor worldwide. According to the 2020 global cancer statistics, the number of new cases of esophageal cancer reached 604,000 and the number of deaths reached 544,000. China is a high-incidence area of ​​esophageal cancer. Although the incidence and mortality rates are declining, it is still one of the major malignant tumors threatening the health of Chinese residents.

[0003] The treatment challenges facing esophageal cancer include how to improve the early diagnosis rate, how to optimize the treatment plan to improve the efficacy and quality of life of patients, and how to deal with the problem of drug resistance to immunotherapy. At present, the treatment model of esophageal cancer has gradually evolved from a single surgery or postoperative adjuvant treatment model to a multidisciplinary comprehensive treatment model including surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. This paradigm shift not only marks a comprehensive upgrade of the treatment strategy, but also reflects a deep concern for the quality of life of patients.

[0004] Immunotherapy drugs are currently the most popular means of treating tumors. Tumor immunotherapy mainly includes immune vaccines, immune checkpoint inhibitor therapy, adoptive immune cell therapy, cytokine therapy, etc. Among them, immune checkpoint inhibitor therapy has attracted much attention for its significant clinical efficacy.

[0005] Immune checkpoints are protective molecules in the human immune system, acting like brakes to prevent inflammatory damage caused by over-activation of T cells. Tumor cells take advantage of this characteristic of the human immune system and over-express immune checkpoint molecules to inhibit the response of the human immune system, escape human immune surveillance and killing, and thus promote the growth of tumor cells. Inhibiting the expression of immune checkpoint molecules and their ligands can enhance the killing effect of T cells on tumors and achieve the purpose of anti-tumor. The published immune checkpoints include CTLA-4, PD-1 / PD-L1, LAG-3, TIM-3, VISTA, A2aR, etc.

[0006] Programmed cell death protein 1 (PD-1) is expressed on a variety of lymphocytes, especially highly expressed on tumor-specific T cells. In the tumor microenvironment, it causes the expansion of malignant tumor cells by interfering with protective immune responses. It has two ligands, namely programmed cell death ligands 1 and 2 (PD-L1, PD-L2), of which PD-L1 is expressed by tumor cells to escape the anti-tumor response of the immune system against it. Blocking the interaction between PD-1 and PD-L1 can maintain the response of T cells after T cells enter the tumor microenvironment and ensure the anti-tumor effect of T cells. The application of antibodies against PD-1 / PD-L1 in the treatment of esophageal cancer has made significant progress. Drugs such as Nivolumab, Pembrolizumab, Atezolizumab, Avelumab and Cemiplimab have all been approved for the treatment of esophageal cancer. These drugs play an important role in the treatment of esophageal cancer by blocking the PD-1 / PD-L1 signaling pathway, activating the patient's own immune system, and enhancing the attack on tumor cells. In the first-line treatment of esophageal cancer, immunotherapy combined with chemotherapy has become one of the standard treatment options. For example, pembrolizumab combined with chemotherapy has shown good efficacy and safety in the first-line treatment of advanced esophageal cancer. In addition, atezolizumab has also shown positive therapeutic effects in the treatment of esophageal cancer. In terms of perioperative treatment, immunotherapy has also shown potential.

[0007] However, although PD-1 / PD-L1 antibody therapy has shown significant efficacy in the treatment of esophageal cancer, not all patients can benefit from it. According to clinical studies, only about 20-45% of patients respond to PD-1 / PD-L1 antibody therapy. In general, PD-1 / PD-L1 antibody therapy provides a new treatment option for patients with esophageal cancer, but further research is still needed to optimize the treatment strategy and improve the treatment effect. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an enzyme-producing lytic Bacillus SYSU923 and its application. The enzyme-producing lytic Bacillus SYSU923 can improve the state of immune cells and enhance the body's anti-tumor immune response by regulating immune factors, and can further enhance the therapeutic effect of esophageal cancer tumors when used in combination with immune checkpoint inhibitors.

[0009] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0010] In the first aspect, the present invention provides an enzyme-producing lysozyme Bacillus, which is the enzyme-producing lysozyme Bacillus SYSU923 strain, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No. 32493 and a deposit date of November 4, 2024.

[0011] Preferably, the 16S rRNA sequence of the Bacillus enzymolyticus SYSU923 strain is as shown in SEQ ID NO.1.

[0012] The invention collects soil samples, and cultures, separates and purifies the dominant strain Lysobacter enzymogenes SYSU923 in the soil with broad-spectrum antibacterial activity through a plate inhibition zone method. The dominant strain Lysobacter enzymogenes SYSU923 is identified as Lysobacter enzymogenes through Gram staining, microscopic examination, single colony morphology observation and molecular biology experiments.

[0013] In a second aspect, the present invention provides an enzyme-producing lysozyme bacillus culture, wherein the enzyme-producing lysozyme bacillus culture is prepared from the enzyme-producing lysozyme bacillus. The enzyme-producing lysozyme bacillus comprises live enzyme-producing lysozyme bacillus, inactivated bacteria with complete morphological structure, inactivated bacteria with incomplete morphological structure, biological materials containing live enzyme-producing lysozyme bacillus, and at least one of metabolites secreted during the growth process of live enzyme-producing lysozyme bacillus.

[0014] Preferably, the enzymolytic Lysozyme Bacillus culture is prepared by at least one of the following methods:

[0015] (1) scraping the activated enzyme-producing lysozyme bacillus into a sterile solvent to obtain an enzyme-producing lysozyme bacillus bacterial suspension, wherein the bacterial suspension is an enzyme-producing lysozyme bacillus culture;

[0016] (2) scraping the activated enzyme-producing Lysozyme Bacillus and inoculating it into a liquid culture medium for cultivation, centrifuging to obtain a supernatant, and then filtering the supernatant to obtain a fermentation filtrate, wherein the fermentation filtrate is the enzyme-producing Lysozyme Bacillus culture;

[0017] (3) Scrape the activated enzyme-producing lysozyme Bacillus into a sterile solvent to obtain an enzyme-producing lysozyme Bacillus bacterial suspension, crush the bacterial suspension, centrifuge to obtain the supernatant, and then filter the supernatant to obtain a lysate, which is the enzyme-producing lysozyme Bacillus culture.

[0018] In a third aspect, the present invention provides the use of the enzyme-producing Lysozyme Bacillus or the enzyme-producing Lysozyme Bacillus culture in the preparation of a product for preventing and / or treating esophageal cancer.

[0019] The product includes food or medicine; the food includes at least one of milk powder, cheese, curd, yogurt, ice cream or fermented cereal food; the dosage form of the medicine includes pills, tablets, granules, capsules, oral liquid or tube feeding preparations, and the medicine includes human medicine or animal medicine.

[0020] In a fourth aspect, the present invention provides the use of the enzyme-producing Bacillus, or the enzyme-producing Bacillus culture combined with an immune checkpoint inhibitor in the preparation of a drug for treating esophageal cancer.

[0021] The present invention uses the SYSU923 strain or its culture combined with immune checkpoint inhibitors to treat esophageal cancer tumor-bearing mice. It is found that the SYSU923 strain has a significant effect in enhancing the therapeutic effect of immune checkpoint inhibitors. Through combined treatment, it can not only effectively inhibit the growth of tumors in situ, but also significantly reduce the metastasis of tumor cells. This discovery provides a new idea for the immunotherapy of esophageal cancer, has great clinical application potential, and is expected to provide a more effective treatment strategy for esophageal cancer patients.

[0022] Preferably, the immune checkpoint inhibitor includes at least one of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, VISTA and A2aR antibodies.

[0023] Preferably, the immune checkpoint inhibitor is a PD-1 antibody and / or a PD-L1 antibody.

[0024] The PD-1 antibodies include Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab, Cindilimab, Camrelizumab and other substances that can bind to PD-1, block the PD-1 / PD-L1 signaling pathway, upregulate T cell activation, and activate endogenous anti-tumor immune responses. The PD-L1 antibodies include Atezolizumab, Avelumab, Durvalumab and other substances that can bind to PD-L1, block the PD-1 / PD-L1 signaling pathway, upregulate T cell activation, and activate endogenous anti-tumor immune responses.

[0025] In a fifth aspect, the present invention provides a product for preventing or treating esophageal cancer, wherein the product comprises the enzyme-producing Lysozyme Bacillus or the culture of the enzyme-producing Lysozyme Bacillus.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention collects soil samples, and isolates and purifies the dominant strain Lysobacter enzymogenes SYSU923 in the soil with broad-spectrum antibacterial activity by plate inhibition zone method, and identifies it as Lysobacter enzymogenes by Gram staining, microscopic examination, single colony morphology observation and molecular biology experiments;

[0028] 2. The present invention uses the single bacterium SYSU923 or its combination with immune checkpoint inhibitors to perform anti-tumor treatment on mice implanted with esophageal cancer. It is found that the SYSU923 screened by the present invention can significantly enhance the therapeutic effect of immune checkpoint inhibitors on esophageal cancer, and can not only inhibit the growth of tumors in situ but also inhibit the metastasis of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the colony morphology of Bacillus enzymolyticus SYSU923 on R2A agar plate;

[0030] Figure 2 This is the morphology of Bacillus enzymolyticus SYSU923 under the microscope after Gram staining;

[0031] Figure 3 This is the phylogenetic tree of Lysozyme Bacillus SYSU923;

[0032] Figure 4 This is a graph showing the effect of the enzyme-producing Bacillus SYSU923 strain in treating esophageal cancer in Effect Example 2. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Unless otherwise specified, the reagents and strains used in the examples are conventional reagents and strains in the art, and can be purchased through commercial channels. The experimental operations not specifically described in the examples are conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common knowledge they master.

[0035] The configuration method of the culture medium described in the embodiment is as follows:

[0036] LB solid culture medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, agar 20 g / L, sterilized at 121°C for 15 min.

[0037] LB liquid culture medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, sterilized at 121°C for 15 min.

[0038] R2A agar medium: R2A agar medium dry powder 18.1g / L, sterilized at 121℃ for 15min.

[0039] R2A liquid culture medium: R2A liquid culture medium dry powder 18.1g / L, sterilized at 121℃ for 15min.

[0040] BHI agar medium: BHI agar medium dry powder 18.1g / L, sterilized at 121℃ for 15min.

[0041] The indicator bacteria selected in the present invention are as follows:

[0042] Candida lusitaniae ATCC 34449, Candida guilliermondii ATCC 6260, Candida albicans ATCC 60193, Candida kruseic ATCC 6258, Candida glabrata ATCC 2001, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, ATCC 25923, Methicillin-resistant Staphylococcus aureus ATCC 43300, Staphylococcus epidermidis epidermidis ATCC 12228), Propionibacterium acne ATCC 6919, the above strains were from the laboratory strain bank; Candida auris, Trichophyton rubrum and Trichophyton mentagrophytes were all clinical strains from the dermatology laboratory of Guangzhou First People's Hospital.

[0043] Example 1

[0044] This embodiment provides a method for screening and identifying the enzyme-producing Lysozyme Bacillus SYSU923 strain, comprising the following steps:

[0045] (1) Screening of strains

[0046] 1. Sampling and culture of strains: Take 1.5g of soil with a depth of about 5cm or rhizosphere with a length of about 2cm (shake off the surface soil of the rhizosphere) to a 15mL centrifuge tube, add PBS to the 15mL mark; place the centrifuge tube horizontally for 30min, and then let it stand in the refrigerator overnight; take the supernatant, dilute it with PBS to 10 times, 20 times, and 100 times, take 20μL and apply it to R2A culture medium, place it in a 30℃ incubator, and culture it for 24h;

[0047] 2. Screening of dominant strains by plate inhibition zone method: obtain bacterial culture solutions of Escherichia coli (G- bacteria), Staphylococcus aureus (G+ bacteria), and Candida albicans (fungus) from the strain library, aspirate 20 μL and inoculate on a plate, and purify the bacteria by plate streak method; pick a single bacterium on the plate and dissolve it in 1 mL PBS to obtain the indicator bacterial solution; take 20 μL of the indicator bacterial solution and spread it evenly on the plate with a disposable coating stick, then inoculate the colonies on the above R2A plate (if the color, size, and shape are similar, do not pick them repeatedly) on the plates coated with the three indicator bacteria, respectively, place them in a 30°C incubator, and culture for 24 hours; pick colonies with obvious inhibition zones, purify the strains and repeat the antibacterial experiment to ensure that the purified strains have antibacterial effects.

[0048] Through the above plate inhibition zone method, 5-6 strains with broad-spectrum antibacterial activity were screened out from 76 samples (ie, the Lysozyme Bacillus SYSU923 strain of the present invention).

[0049] (2) Identification of strains

[0050] 1. Single colony observation and Gram staining: Streak the activated SYSU923 strain on an R2A agar plate and place it in a 30°C incubator for 48 hours. Observe the morphology of the colonies and take photos. Figure 1 It can be seen that after 24 hours of culture, the colonies are yellow mucus, indicating that the growth of the SYSU923 strain will produce pigments. After 48 hours of culture, the colonies are slightly yellow spherical. The microscopic examination of the strain after Gram staining is shown in the figure below. Figure 2 As shown, the SYSU923 strain is a Gram-negative bacillus that does not produce spores.

[0051] 2. 16S rDNA sequencing: PCR amplification was performed using 27F and 1492R as primers. The subsequent 16S rDNA sequence (the 16S rDNA sequence is shown in SEQ ID NO.1) was determined by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results of the SYSU923 strain were uploaded to NCBI for BLAST analysis to clarify the type of the SYSU923 strain. Multiple sequence homology comparison analysis was performed using MEGA11.0 software, and the phylogenetic tree was constructed using the neighbor-joining method.

[0052] After sequencing, the 16SrDNA sequence of strain SYSU923 was 1414bp long. The 16SrDNA sequence (shown in SEQ ID NO.1) was uploaded to NCBI for comparison. According to the degree of sequence homology, the top fifteen strains were selected to draw Table 1. The phylogenetic tree was constructed using MEGA11.0. Figure 3 It can be seen that the SYSU923 strain has the highest homology with L. enzymogenes strain SR01. Combined with the data in Table 1, the homology between the SYSU923 strain and L. enzymogenes strain SR01 is 99.93%, which further confirms that the SYSU923 strain is an enzymolytic bacillus.

[0053] The L. enzymogenes SYSU923 strain identified as L. enzymogenes is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.32493, the deposit date is November 4, 2024, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0054] Table 1 Comparison of strain homology

[0055]

[0056] Example 2

[0057] This example provides a method for preparing a culture of Bacillus lysozyme-producing SYSU923, including the following methods:

[0058] (1) Preparation of bacterial suspension: scrape the activated zymolytic Bacillus SYSU923 into sterile PBS to obtain a zymolytic Bacillus bacterial suspension, which is the zymolytic Bacillus culture;

[0059] (2) Preparation of fermentation filtrate: The activated enzyme-producing Lysobacterium SYSU923 was scraped and inoculated into a 75 cm flask containing 50 mL of LB liquid medium. 2The cell culture flask was continuously shaken and cultured at 30°C and 120 rpm; when the OD value of the bacterial solution minus the LB control was greater than 1.0, the culture was stopped; the bacterial solution was transferred to a 50 mL centrifuge tube, centrifuged at room temperature and 10,000 rpm for 15 min, and the fermentation filtrate was filtered using a 0.22 μm filter to obtain a fermentation filtrate, which was the culture of Bacillus lysozyme-producing SYSU923;

[0060] (3) Preparation of lysate: scrape the activated enzyme-producing Bacillus SYSU923, resuspend it in PBS at a weight ratio of 1:10, place it in a cell disruptor for 30 minutes, and then place the bacterial solution in a centrifuge at 4°C, 12000 rpm for 15 minutes. Take the supernatant and filter it with a 0.22 μm filter to obtain a lysate, which is the enzyme-producing Bacillus SYSU923 culture.

[0061] Effect Example 1

[0062] This effect example tests the antibacterial spectrum of the enzyme-producing Lysozyme Bacillus SYSU923 screened in Example 1, and the specific method is as follows:

[0063] The existing Gram-positive bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus, Enterococcus faecalis, Staphylococcus epidermidis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), fungi (Candida albicans, Candida guilliermondii, Candida krusei, Candida glabrata, Candida albicans) and three clinical fungi (Candida auris, Trichophyton rubrum, Trichophyton mentagrophytes) in the strain library were used as indicator bacteria, and the plate inhibition zone method was used to determine the inhibition spectrum. The specific results are shown in Table 2.

[0064] The results in Table 2 show that the SYSU923 strain has an inhibitory effect on most fungi and Gram-positive bacteria. Among them, Candida glabrata (ATCC 2001) cannot grow on the R2A plate; the SYSU923 strain can antagonize Staphylococcus aureus (ATCC 25923) and methicillin-resistant Staphylococcus aureus (ATCC 43300) on the R2A plate, but no inhibition zone appears on the LB plate, indicating that the antibacterial substances secreted by the strain are related to the nutrition of the culture medium. Under the condition of nutritional limitation, the SYSU923 strain can secrete antibacterial substances against methicillin-resistant Staphylococcus aureus.

[0065] Table 2 Determination of antibacterial spectrum

[0066]

[0067]

[0068] Effect Example 2

[0069] In order to verify the effect of the enzyme-producing Lysozyme Bacillus SYSU923 strain in treating tumors, this effect example uses esophageal cancer as a representative tumor to conduct animal experiments. The specific method is as follows:

[0070] Male C57BL / 6 mice aged 4-6 weeks were used as experimental subjects. Esophageal cancer cells AKR (1*10 6 The esophageal cancer model mice were obtained by subcutaneous tumor implantation of 1×10 / mouse. The model mice were divided into six groups, with 5 mice in each group. The control group was treated with PBS by intragastric administration every day after tumor implantation; the α-PD1 group was intraperitoneally injected with αPD-1 antibody (60 μg / mouse) every 2 days after tumor implantation for one week; the live lytic Bacillus group was intragastricly administered with live lytic Bacillus SYSU923 suspension (1×10 9 CFU / 100 μL / mouse) to the end point of the experiment; live lytic Bacillus + α-PD1 group: mice were gavaged with live lytic Bacillus SYSU923 suspension every day after tumor implantation, and αPD-1 antibody was intraperitoneally injected every 2 days after tumor implantation for one week (60 μg / mouse); dead lytic Bacillus group: mice were gavaged with lytic Bacillus SYSU923 suspension every day after tumor implantation (10 9 CFU / mouse) were crushed by cell disruptor and then centrifuged to obtain the supernatant (i.e., lysate); dead lysing Bacillus + α-PD1 group: mice were intragastrically administrated with lysing Bacillus SYSU923 suspension (10 9 CFU / mouse) was crushed by a cell disruptor and then centrifuged to obtain the supernatant. After one week of tumor implantation, αPD-1 antibody was intraperitoneally injected every 2 days (60 μg / mouse). After 30 days of tumor implantation, the mice were euthanized and the tumor tissues were removed. The tumor tissues were weighed and the volume was calculated. For specific operations, see Figure 4 A.

[0071] Figure 4 (BD) are the actual pictures of mouse tumor samples at the end of the experiment, and the statistical charts of tumor weight and volume. The results show that compared with the control group, the tumor growth of the live lysate and dead lysate groups was slower (P < 0.001), and the anti-tumor effect was significantly enhanced after combination with αPD-1 antibody (P < 0.001), and the tumor growth inhibition effect was obvious. The tumor weight of the live lysate combined with αPD-1 antibody was the smallest, indicating that the live lysate has a certain enhancement effect on the anti-tumor effect of αPD-1 antibody under oral administration.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A Lysozyme-producing Bacillus, characterized in that The enzyme-producing lysozyme bacillus is the enzyme-producing lysozyme bacillus SYSU923 strain, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.32493 and a deposit date of November 4, 2024.

2. The Lysozyme-producing Bacillus according to claim 1, characterized in that The 16SrRNA sequence of the Bacillus enzymolyticus SYSU923 strain is shown in SEQ ID NO.

1.

3. A culture of an enzyme-producing lysozyme bacillus, characterized in that The enzyme-producing Lysozyme Bacillus culture is prepared from the enzyme-producing Lysozyme Bacillus described in claim 1 or 2.

4. The culture of Bacillus lysozyme according to claim 3, characterized in that The enzymolytic Lysozyme Bacillus culture is prepared by at least one of the following methods (1)-(3): (1) scraping the activated enzyme-producing lysozyme bacillus into a sterile solvent to obtain an enzyme-producing lysozyme bacillus bacterial suspension, wherein the bacterial suspension is an enzyme-producing lysozyme bacillus culture; (2) scraping the activated enzyme-producing Lysozyme Bacillus and inoculating it into a liquid culture medium for cultivation, centrifuging to obtain a supernatant, and then filtering the supernatant to obtain a fermentation filtrate, wherein the fermentation filtrate is the enzyme-producing Lysozyme Bacillus culture; (3) Scrape the activated enzyme-producing lysozyme Bacillus into a sterile solvent to obtain an enzyme-producing lysozyme Bacillus bacterial suspension, crush the bacterial suspension, centrifuge to obtain the supernatant, and then filter the supernatant to obtain a lysate, which is the enzyme-producing lysozyme Bacillus culture.

5. Use of the enzyme-producing lytic Bacillus according to claim 1 or 2, or the enzyme-producing lytic Bacillus culture according to claim 3 or 4, in the preparation of a product for preventing and / or treating esophageal cancer.

6. Use of the Bacillus zymolyticus according to claim 1 or 2, or the Bacillus zymolyticus culture according to claim 3 or 4 in combination with an immune checkpoint inhibitor in the preparation of a drug for treating esophageal cancer.

7. The use according to claim 6, characterized in that The immune checkpoint inhibitor includes at least one of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, VISTA and A2aR antibodies.

8. The use according to claim 7, characterized in that The immune checkpoint inhibitor is a PD-1 antibody and / or a PD-L1 antibody.

9. A product for preventing or treating esophageal cancer, characterized in that: The product comprises the enzymolytic Bacillus according to claim 1 or 2, or the enzymolytic Bacillus culture according to claim 3 or 4.

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