Application of selenium-enriched lactobacillus rhamnosus microbial agent in preparation of antitumor drugs

Abstract

The invention discloses an application of a selenium-rich lactobacillus rhamnosus microbial inoculum in preparation of antitumor drugs, and relates to the field of biotechnology and tumor treatment, the selenium-rich lactobacillus rhamnosus is prepared through a selenium-rich fermentation process, inorganic selenium difficult to absorb can be converted into an organic form suitable for body absorption, and bioavailability is improved. The selenium-enriched lactobacillus rhamnosus disclosed by the invention can inhibit tumor growth in an oral administration manner, and a new carrier is provided for probiotic anti-tumor research.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and tumor treatment, and particularly relates to the application of selenium-enriched Lactobacillus rhamnosus in the preparation of anti-tumor drugs. Technical Background

[0002] As a common physiological probiotic in the human body, Lactobacillus rhamnosus is widely used in the fields of food, medicine, and scientific research. Lactobacillus rhamnosus exists in the intestines of humans and many other mammals, and has multiple functions such as maintaining the balance of the intestinal microecology, inhibiting the invasion and colonization of pathogenic bacteria, regulating the body's immune ability, and removing toxins from the body. In recent years, the research on Lactobacillus rhamnosus in the anti-tumor field has gradually increased. It can inhibit tumor growth by activating immune cells such as macrophages, NK cells, and T cells in the body, and promoting the release of inflammatory cytokines. As a probiotic, Lactobacillus rhamnosus can regulate the intestinal flora, inhibit the growth of tumor-promoting flora, and reduce the risk of tumor occurrence. In addition, the bacteria itself and its metabolites also have the ability to inhibit tumor proliferation. However, although Lactobacillus rhamnosus shows potential in anti-tumor, there are still many problems such as large differences in the effects of different strains and insufficient research on the long-term application safety. Further research support is still needed for its further application. In order to further improve the anti-tumor efficacy of probiotics, strategies such as combining with chemotherapy, immunotherapy drugs, or engineering probiotics are currently often used to enhance the ability of probiotics to treat tumors.

[0003] Selenium is an essential trace element for the human body. Appropriate intake of selenium can improve human immunity, scavenge free radicals, remove toxins from the body, and prevent diabetes, cancer, and cardiovascular and cerebrovascular diseases. In anti-tumor research, selenium can inhibit tumor growth and metastasis through various pathways such as antioxidant, inducing apoptosis, inhibiting cell proliferation, inhibiting angiogenesis, and enhancing immune function. Selenium in food mainly exists in two forms: inorganic and organic. Inorganic selenium is difficult to be absorbed by the body and has a low bioavailability.

[0004] Nano-selenium has a small particle size and is easy to penetrate the cell membrane, thus improving the absorption efficiency. The present invention uses selenium-enriched fermentation and the probiotic Lactobacillus rhamnosus to carry nano-selenium. Nano-selenium is easy to penetrate the cell membrane of probiotics, thereby improving its absorption and distribution in probiotic cells, and at the same time synthesizing corresponding selenium proteins. Selenium proteins induce tumor cell cycle arrest and apoptosis by balancing the intracellular redox state and maintaining the stability of DNA. The present invention not only improves the bioavailability of selenium, but also synergizes with the probiotic effect of Lactobacillus rhamnosus, enhances the body's immunity, and jointly exerts anti-tumor ability. This combination provides a new idea for the development of new functional foods and drugs. Summary of the Invention

[0005] To solve the problems of the prior art, the object of the present invention is to provide an application of a selenium-enriched Lactobacillus rhamnosus bacterium agent in the preparation of an anti-tumor drug.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, the present application provides an application of a selenium-enriched Lactobacillus rhamnosus bacterium agent in the preparation of an anti-tumor drug.

[0007] In the second aspect, the present application provides a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus bacterium agent.

[0008] The first aspect of the present application provides an application of a selenium-enriched Lactobacillus rhamnosus bacterium agent in the preparation of an anti-tumor drug.

[0009] Furthermore, the selenium-enriched Lactobacillus rhamnosus bacterium agent is prepared by selenium-enriched fermentation of Lactobacillus rhamnosus, which converts nano-selenium in the culture medium into organic selenium, increasing the absorption efficiency of the body, and the selenium content is 5-100 μg / g. After Lactobacillus rhamnosus is revived in a solid medium, single colonies are picked and transferred to 200 mL of a liquid medium, and cultured at a constant temperature of 40 °C for 24 h. The activated Lactobacillus rhamnosus seed liquid is inoculated into 1 L of the medium at a concentration of 1:50, and a nano-selenium solution that has been sterilized by filtration is added to a final concentration of 2-20 mg / L, and statically cultured at 40 °C for 24 h, and the fermented bacteria are collected by centrifugation at 8000 g for 10 min.

[0010] Furthermore, the tumor is a solid tumor.

[0011] Even further, the solid tumor includes any one of melanoma, breast cancer, colorectal cancer, lymphoma, liver cancer, bladder cancer or lung cancer.

[0012] Furthermore, the fermentation medium of the selenium-enriched Lactobacillus rhamnosus contains the following components: vitamin C, 4 g / L; NaCl, 2.4 g / L; Na 2 HPO 4 5.8 g / L; KH 2 PO 4 0.528571 g / L; glucose, 5 g / L; yeast extract, 3 g / L; beef extract powder, 1.7 g / L; soy peptone, 2 g / L; beef peptone, 20 g / L; nano-selenium solution, 2-20 mg / L.

[0013] Furthermore, the bacterial content of Lactobacillus rhamnosus is not less than 1×10 9 CFU / g.

[0014] Even further, the administration method and time interval of the selenium-enriched Lactobacillus rhamnosus are once a day by gavage.

[0015] Furthermore, the administration dose of the selenium-enriched Lactobacillus rhamnosus is 3×108 CFU / kg, 3×10 9 CFU / kg or 3×10 10 CFU / kg, and the gavage volume each time is 100 μL.

[0016] Furthermore, after treatment with selenium-enriched Lactobacillus rhamnosus, the growth of malignant tumors in mice was significantly inhibited, with slow growth and no obvious adverse reactions after oral administration.

[0017] The second aspect of the present application provides a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent, and the dosage form of the probiotic preparation includes any one of freeze-dried powder, tablets, capsules, granules, dairy products or yogurt.

[0018] Furthermore, the freeze-dried powder is diluted with maltodextrin or skim milk powder or other fillers, and the active colony count of the Lactobacillus rhamnosus is 1×10 9 to 5×10 10 CFU / g, and the selenium content is 5 - 100 μg / g.

[0019] The application of the selenium-enriched Lactobacillus rhamnosus of the present invention in the prevention and / or treatment of tumors, and the selenium-enriched Lactobacillus rhamnosus has a significantly better therapeutic effect on tumors than the non-treatment group mice and the transgenic Bifidobacterium longum expressing the classical anti-tumor gene endostatin.

[0020] The administration dose of selenium-enriched Lactobacillus rhamnosus is 3×10 8 CFU / kg, 3×10 9 CFU / kg or 3×10 10 CFU / kg, preferably 3×10 9 CFU / kg, and the gavage volume each time is 100 μL. The administration frequency and interval of selenium-enriched Lactobacillus rhamnosus are: start administration after the tumor volume reaches 20 mm 3 and administer once a day for 7 consecutive times. The selenium-enriched Lactobacillus rhamnosus is used for treatment by intravenous injection, intraperitoneal injection, subcutaneous injection, oral administration, intratumoral administration, and oral administration is preferred.

[0021] The application of the selenium-enriched Lactobacillus rhamnosus of the present invention in the combined prevention and / or treatment of tumors with conventional antibodies, small molecule drugs, immune checkpoint inhibitors, chemotherapy drugs, and biological agents.

[0022] In this invention, C57BL / 6 mice were used as experimental animals, and the test operations were carried out in accordance with the internationally unified standard operation of tumor animal models and the principles of animal welfare. B16F10 cells, 4T1 cells, CT26 and MC38 cells, A20 cells, MB49 cells, LLC cells, and H22 cells were successively implanted subcutaneously to establish mouse B16F10 melanoma model, 4T1 breast cancer model, CT26 and MC38 colorectal cancer model, A20 lymphoma model, MB49 bladder cancer model, LLC lung cancer model, and H22 liver cancer model respectively, which was recorded as day 0, and the change in tumor volume was monitored. When the average tumor volume reached 20 mm 3 on the 8th day, the mice were evenly grouped according to the tumor size, and were respectively intragastrically administered with sterilized pure water and selenium-enriched Lactobacillus rhamnosus bacterial solution every day. The body weight and tumor size of the mice were recorded at the same time every day. When the tumor volume of some mice reached 1500 mm 3 on the 14th day, the treatment was terminated, and the tumors, liver and spleen were weighed and photographed. The results showed that oral administration of selenium-enriched Lactobacillus rhamnosus had a significant anti-tumor effect. After 7 consecutive days of oral administration, compared with the control group, the tumor volume was 18.0% of that of the control group, and the tumor weight was 18.0% of that of the control group, showing an excellent anti-tumor effect. During the treatment period, the body weight of the mice taking probiotics did not show obvious fluctuations, and there was no obvious enlargement of the liver and spleen in the mice after the treatment, showing good oral safety.

[0023] Beneficial effects: The selenium-enriched Lactobacillus rhamnosus of the present invention can improve the absorption efficiency of selenium by the body and enhance the immune system function by orally delivering selenoprotein, cooperate with the anti-tumor ability of the probiotic itself, significantly inhibit the growth of melanoma, and has high safety.

[0024] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses the probiotic Lactobacillus rhamnosus as a delivery carrier for selenium, can efficiently deliver selenoprotein by oral administration, improve the bioavailability of selenium, reduce adverse reactions, and has high patient compliance and low production cost, which is suitable for industrial production.

[0025] (2) The selenium-enriched Lactobacillus rhamnosus of the present invention can effectively inhibit the growth of solid tumors (melanoma, breast cancer, colorectal cancer, lymphoma, liver cancer, bladder cancer, lung cancer) when used alone. Compared with the non-treatment group, the average tumor volume decreased by 82.0%; its curative effect is significantly better than that of transgenic Bifidobacterium longum expressing the classical anti-tumor gene endostatin; a significantly unexpected curative effect has been produced. It is expected to be used in combination with other drugs as a carrier for anti-tumor drugs. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the test examples of this application, the following will briefly introduce the attached drawings required for the description of the test examples or the prior art. Obviously, the attached drawings in the following description are only some test examples of this application. For those of ordinary skill in the art, without creative efforts, other attached drawings can also be obtained based on these attached drawings.

[0027] Abbreviation Explanation of the Attached Drawings

[0028] 1: Tumor non-treatment group; 2: Bifidobacterium longum expressing endostatin; 3: Lactobacillus rhamnosus enriched with selenium;

[0029] Figure 1 It is the experimental flow chart of the treatment of mouse melanoma by the strain of the present invention.

[0030] Figure 2 shows the treatment effect of the strain of the present invention on mouse melanoma.

[0031] Figure 2A It is the representative image of mouse melanoma tumor after treatment with the strain of the present invention.

[0032] Figure 2B It is the statistical result of the weight of mouse melanoma tumor after treatment with the strain of the present invention.

[0033] Figure 3 It is the tumor growth curve of mice during the treatment with the strain of the present invention

[0034] Figure 4 It is the statistical result of the weight change of mice during the treatment with the strain of the present invention.

[0035] Figure 5 shows the changes of mouse organs after treatment with the strain of the present invention.

[0036] Figure 5A It is the representative image of mouse liver and spleen after treatment with the strain of the present invention.

[0037] Figure 5B It is the statistical result of the weight of mouse liver and spleen after treatment with the strain of the present invention. Detailed Embodiments

[0038] The present invention will be described in detail below in conjunction with the attached drawings and specific embodiments. The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. The experimental methods in the following examples of the present invention that do not specify specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. All kinds of commonly used chemical reagents used in the examples are commercially available products.

[0039] Unless otherwise defined, all technical and scientific terms used in this invention have the same meanings as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the specification of this invention are only for the purpose of describing specific embodiments and are not intended to limit this invention.

[0040] The terms "comprise" and "have" and any variations thereof in this invention are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products or equipment.

[0041] As used in this invention, "a plurality of" means two or more. "And / or" describes the relationship between associated objects and indicates that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0042] The first aspect of the embodiments of this application provides an application of a selenium-enriched Lactobacillus rhamnosus bacterium agent in the preparation of an anti-tumor drug.

[0043] In some embodiments, the selenium-enriched Lactobacillus rhamnosus bacterium agent is prepared by selenium-enriched fermentation of Lactobacillus rhamnosus, which converts nano-selenium in the culture medium into organic selenium and increases the absorption efficiency of cells for selenium. The selenium content is 5-100 μg / g. After Lactobacillus rhamnosus is revived on a solid medium, single colonies are picked and transferred to 200 mL of liquid medium, and cultured at a constant temperature of 40 °C for 24 h. The activated Lactobacillus rhamnosus seed liquid is inoculated into 1 L of medium at a concentration of 1:50, and a nano-selenium solution that has been sterilized by filtration is added to a final concentration of 2-20 mg / L, and statically cultured at 40 °C for 24 h. The fermented bacteria are collected by centrifugation at 8000 g for 10 min.

[0044] In some embodiments, the tumor is a solid tumor.

[0045] In some embodiments, the solid tumor includes any one of melanoma, breast cancer, colorectal cancer, lymphoma, liver cancer, bladder cancer or lung cancer.

[0046] In some embodiments, the fermentation medium of the selenium-enriched Lactobacillus rhamnosus contains the following components: vitamin C, 4 g / L; NaCl, 2.4 g / L; Na 2 HPO 4 ,5.8 g / L; KH 2 PO 4, 0.528571 g / L; glucose, 5 g / L; yeast extract, 3 g / L; beef extract powder, 1.7 g / L; soy peptone, 2 g / L; beef peptone, 20 g / L; nano-selenium solution, 2 - 20 mg / L.

[0047] In some embodiments, the content of Lactobacillus rhamnosus bacteria is not less than 1×10 9 CFU / g.

[0048] In some embodiments, the administration method and time interval of selenium-enriched Lactobacillus rhamnosus are once a day by gavage.

[0049] In some embodiments, the administration dose of selenium-enriched Lactobacillus rhamnosus is 3×10 8 CFU / kg, 3×10 9 CFU / kg or 3×10 10 CFU / kg, and the volume of each gavage is 100 μL.

[0050] In some embodiments, after the treatment with selenium-enriched Lactobacillus rhamnosus, the growth of malignant tumors in mice is significantly inhibited, with slow growth, and no obvious adverse reactions occur after oral administration.

[0051] The second aspect of the embodiments of the present application provides a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent, and the dosage form of the probiotic preparation includes any one of freeze-dried powder, tablets, capsules, granules, dairy products or yogurt.

[0052] In some embodiments, the freeze-dried powder is diluted with maltodextrin or skim milk powder or other fillers, and the active colony number of the Lactobacillus rhamnosus is 1×10 9 to 5×10 10 CFU / g, and the selenium content is 5 - 100 μg / g.

[0053] The application of the selenium-enriched Lactobacillus rhamnosus of the present invention in the prevention and / or treatment of tumors, and the treatment effect of the selenium-enriched Lactobacillus rhamnosus on solid tumors is significantly better than that of the non-treatment group of mice. The administration dose of the selenium-enriched Lactobacillus rhamnosus is 3×10 8 CFU / kg, 3×10 9 CFU / kg or 3×10 10 CFU / kg, preferably 3×10 9 CFU / kg, and the volume of each gavage is 100 μL. The administration frequency and interval of the selenium-enriched Lactobacillus rhamnosus are: start administration after the tumor volume reaches 20 mm 3 , once a day for 7 consecutive times. The selenium-enriched Lactobacillus rhamnosus is used for treatment by intravenous injection, intraperitoneal injection, subcutaneous injection, oral administration, intratumoral administration, preferably oral administration.

[0054] Use of the selenium-enriched Lactobacillus rhamnosus of the present invention in combination with conventional antibodies, small molecule drugs, immune checkpoint inhibitors, chemotherapeutic drugs, and biological agents for the prevention and / or treatment of tumors.

[0055] Example 1

[0056] Use of a selenium-enriched Lactobacillus rhamnosus agent of the present invention in the preparation of an anti-tumor drug. The tumor is a solid tumor (such as melanoma).

[0057] The selenium-enriched Lactobacillus rhamnosus agent is prepared by selenium-enriched fermentation of Lactobacillus rhamnosus, which converts nano-selenium in the culture medium into organic selenium and increases the absorption efficiency of cells for selenium. The selenium content is 5 - 100 μg / g. After Lactobacillus rhamnosus is revived on a solid medium, a single colony is picked and transferred to 200 mL of liquid medium, and cultured at a constant temperature of 40 °C for 24 h. The activated Lactobacillus rhamnosus seed solution is inoculated into 1 L of medium at a concentration of 1:50, and a nano-selenium solution that has been sterilized by filtration is added to a final concentration of 2 - 20 mg / L, and statically cultured at 40 °C for 24 h. The fermented bacteria are collected by centrifugation at 8000 g for 10 min.

[0058] The fermentation medium of the selenium-enriched Lactobacillus rhamnosus contains the following components: vitamin C, 4 g / L; NaCl, 2.4 g / L; Na 2 HPO 4 5.8 g / L; KH 2 PO 4 0.528571 g / L; glucose, 5 g / L; yeast extract, 3 g / L; beef extract powder, 1.7 g / L; soy peptone, 2 g / L; beef peptone, 20 g / L; nano-selenium solution, 2 - 20 mg / L.

[0059] The bacterial content of Lactobacillus rhamnosus is not less than 1×10 9 CFU / g.

[0060] The administration method and time interval of the selenium-enriched Lactobacillus rhamnosus are once a day by gavage.

[0061] The administration dose of the selenium-enriched Lactobacillus rhamnosus is 3×10 8 CFU / kg, 3×10 9 CFU / kg or 3×10 10 CFU / kg, and the volume of each gavage is 100 μL.

[0062] After treatment with the selenium-enriched Lactobacillus rhamnosus, the growth of malignant tumors in mice was significantly inhibited, and the growth was slow. No obvious adverse reactions occurred after oral administration.

[0063] Preparation method of the selenium-enriched Lactobacillus rhamnosus of the present invention

[0064] (1) Prepare 3 L of Lactobacillus rhamnosus medium, adjust the pH value to 7.4 with NaOH, and transfer it into a 5-L fermenter. The medium contains the following components:

[0065]

[0066]

[0067] (2) Sterilize at 121 °C for 20 minutes and cool to room temperature.

[0068] (3) Inoculate 1 mL of thawed Lactobacillus rhamnosus seed liquid into 200 mL of the medium and conduct activation culture at 40 °C for 24 hours.

[0069] (4) Transfer the activated Lactobacillus rhamnosus seed liquid to 1 L of the medium at an inoculation amount of 3%, and add the nano-selenium solution filtered through a 0.22-μm filter membrane to a final concentration of 2 - 20 mg / L (the preparation method refers to the applied patents: A Preparation Method of Nano-Selenium Solution, Application No.: 2024110944130; A Selenium-Enriched Lactobacillus rhamnosus Bacterial Agent and Its Preparation Method, Application No.: 2024110944520). Conduct static culture at 40 °C for 24 hours. The Lactobacillus rhamnosus of the present invention is from the China Center for Industrial Culture Collection, and the strain number is: CICC 6137.

[0070] (5) Centrifuge at 4 °C and 8000 g for 10 minutes, collect the fermented bacteria, and obtain the selenium-enriched Lactobacillus rhamnosus bacterial agent.

[0071] Example 2

[0072] The difference between Example 2 and Example 1 is that the solid tumor is breast cancer.

[0073] Example 3

[0074] The difference between Example 3 and Example 1 is that the solid tumor is lymphoma.

[0075] Example 4

[0076] The difference between Example 4 and Example 1 is that the solid tumor is colorectal cancer.

[0077] Example 5

[0078] The difference between Example 5 and Example 1 is that the solid tumor is liver cancer.

[0079] Example 6

[0080] The difference between Example 6 and Example 1 is that the solid tumor is bladder cancer.

[0081] Example 7

[0082] Example 7 is different from Example 1 in that the solid tumor is lung cancer.

[0083] Example 8

[0084] A probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent of the present invention, and the dosage form of the probiotic preparation is a freeze-dried powder. The freeze-dried powder is diluted with maltodextrin or skim milk powder or other fillers, and the viable colony count of Lactobacillus rhamnosus is 1×10 9 to 5×10 10 CFU / g, and the selenium content is 5 - 100 μg / g.

[0085] Example 9

[0086] Example 9 is different from Example 8 in that a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent of the present invention, and the dosage form of the probiotic preparation is a tablet.

[0087] Example 10

[0088] Example 10 is different from Example 8 in that a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent of the present invention, and the dosage form of the probiotic preparation is a capsule.

[0089] Example 11

[0090] Example 11 is different from Example 8 in that a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent of the present invention, and the dosage form of the probiotic preparation is a granule.

[0091] Example 12

[0092] Example 12 is different from Example 8 in that a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent of the present invention, and the dosage form of the probiotic preparation is a dairy product.

[0093] Example 13

[0094] Example 13 is different from Example 8 in that a probiotic preparation containing a selenium-enriched Lactobacillus rhamnosus agent of the present invention, and the dosage form of the probiotic preparation is yogurt.

[0095] Example 14

[0096] Application of the selenium-enriched Lactobacillus rhamnosus of the present invention in the prevention and / or treatment of tumors. The therapeutic effect of the selenium-enriched Lactobacillus rhamnosus on melanoma is significantly better than that of the non-treatment group of mice. The dosage of the selenium-enriched Lactobacillus rhamnosus is 3×10 8 CFU / kg, 3×10 9 CFU / kg or 3×10 10 CFU / kg, preferably 3×10 9CFU / kg, and the gavage volume each time is 100 μL. The administration frequency and interval of Lactobacillus rhamnosus - selenium are as follows: when the tumor volume reaches 20 mm 3 treatment starts, once a day for 7 consecutive days. Lactobacillus rhamnosus - selenium is used for treatment by intravenous injection, intraperitoneal injection, subcutaneous injection, oral administration, and intratumoral administration, and preferably by oral administration.

[0097] Application of the Lactobacillus rhamnosus - selenium of the present invention in the combined prevention and / or treatment of tumors with conventional antibodies, small molecule drugs, immune checkpoint inhibitors, chemotherapeutic drugs, and biological agents.

[0098] Experimental Example 1

[0099] Inhibitory effect of Lactobacillus rhamnosus - selenium on solid tumors

[0100] The Lactobacillus rhamnosus - selenium in the present invention is obtained by selenium - rich fermentation of Lactobacillus rhamnosus and its derivatives or genetically modified strains, and its intestinal colonization ability remains unchanged. Hereinafter, only Lactobacillus rhamnosus is taken as an example.

[0101] Establishment of solid tumor models: In the present invention, B16F10 melanoma models, 4T1 breast cancer models, CT26 and MC38 colorectal cancer models, A20 lymphoma models, MB49 bladder cancer models, LLC lung cancer models, and H22 liver cancer models were successively established for efficacy verification. The culture methods of the above - mentioned tumor cell lines are the same, the production process of tumor model inoculation also follows the internationally unified standard operation, and the growth and efficacy judgment of tumor animals also follow the internationally prevailing animal welfare principles. Therefore, here only the B16F10 melanoma model is taken as a representative, and no redundant description will be repeated one by one.

[0102] Establishment of melanoma model: 15 SPF - grade female C57BL / 6 mice at 6 - 8 weeks old (19 ± 1 g) were purchased from Changzhou Cavens Experimental Animal Co., Ltd. They were raised in an environment with a temperature of 25 ± 2°C and a humidity of 50 ± 5%, with 12 - hour light - dark alternation every day, and free access to sterilized water. They were adaptively fed for 7 days.

[0103] After B16F10 mouse melanoma cells were cultured to the logarithmic growth phase in 1640 medium, the medium was discarded, washed twice with sterile PBS, 0.5% trypsin was added, and digested at 37°C for 1 min. 1640 medium was added to terminate digestion, and the cell suspension was obtained by pipetting. After centrifugation at 800 rpm for 5 min, the supernatant medium was removed, washed twice with sterile PBS, and then cell counting was performed. The cells were resuspended with sterile PBS, and the cell concentration was adjusted to 2×10 6 cells / mL, and 100 μL was subcutaneously injected into each mouse at the axillary fat pad. After inoculation, the mice were raised in a clean - grade animal room. When the tumor volume of the mice grew to 20 mm 3 subsequently, follow - up experiments were carried out.

[0104] Experimental grouping: According to the tumor size, the mice were evenly divided into 3 groups, with 5 mice in each group; the 3 groups were as follows: 1: Tumor non-treatment group; 2: Bifidobacterium longum expressing endostatin (positive control group, a strain that has been proven to have tumor inhibitory effects in previous studies in our laboratory, expressing recombinant human endostatin protein through plasmid pBV222; Frontiers in Microbiology, 2022, 13:927277.); 3: Lactobacillus rhamnosus enriched with selenium.

[0105] Treatment plan: During the treatment period, the control mice (group 1) were gavaged with 200 μL of sterile PBS buffer daily, and the mice in the probiotic treatment groups (groups 2 and 3) were gavaged with 200 μL of probiotic solution daily (Bifidobacterium longum expressing endostatin or Lactobacillus rhamnosus enriched with selenium resuspended in sterile PBS buffer, 3×10 9 CFU / kg). The body weights of the mice were weighed daily, and the tumor size was observed and recorded. The tumor volume calculation formula was: volume = length × width 2 ×0.52. When the tumors of the control mice grew to 1500 mm 3 , the experiment was terminated. After anesthetizing and sacrificing the mice, the complete tumors were dissected, representative images were taken, and the weights were measured ( Figure 1 ).

[0106] During the experiment, the tumors of the control mice (group 1) grew rapidly. On the 10th day after tumor inoculation, they entered the logarithmic growth phase. On the 14th day, the tumor volumes of some mice had reached 1500 mm 3 , so the experiment was terminated and visceral samples of each group were taken. At the end of the experiment, the average tumor volume of the control mice was 1018 mm 3 , and the average tumor weight reached 0.74 g (Figure 2, Figure 3 ). The tumors in the treatment group of Bifidobacterium longum expressing endostatin (group 2) decreased. At the end of the experiment, the average tumor volume of the mice was 397 mm 3 , and the average tumor weight was 0.37 g; compared with the untreated control group, the tumor volume decreased by 61% and the tumor weight decreased by 50%. The treatment group of Lactobacillus rhamnosus enriched with selenium (group 3) had obvious curative effects, and the tumor growth rate was significantly inhibited. At the end of the experiment, the average tumor volume of the mice in the Lactobacillus rhamnosus enriched with selenium treatment group was 207 mm 3, the average tumor weight was 0.16 g; compared with the untreated control group, the tumor volume decreased by about 80%, and the weight was only 22% of the control group, a reduction of 78%; compared with the treatment group of Bifidobacterium longum expressing endostatin (Group 2), the tumor volume decreased by about 48%, and the weight decreased by about 57%. The results indicate that Lactobacillus rhamnosus enriched with selenium can serve as a carrier of selenium, increase the bioavailability of selenium, and at the same time enhance the anti-tumor effect of probiotics, inhibiting the growth of melanoma after oral administration. The anti-tumor efficacy of Lactobacillus rhamnosus enriched with selenium is even significantly better than that of Bifidobacterium longum expressing endostatin.

[0107] In different tumor models (B16F10 melanoma model, 4T1 breast cancer model, CT26 and MC38 colorectal cancer models, A20 lymphoma model, MB49 bladder cancer model, LLC lung cancer model, H22 liver cancer model), the treatment group in Group 2 showed similar treatment effects as in the B16F10 melanoma model, that is, oral administration of Lactobacillus rhamnosus enriched with selenium has significantly better anti-tumor ability than Bifidobacterium longum expressing endostatin.

[0108] Oral administration of probiotics does not bring obvious adverse reactions to the host. During the treatment period, the body weights of the mice in the control group and the probiotic treatment group fluctuated within 5%. After the treatment ended, the average body weight of the mice in the Lactobacillus rhamnosus enriched with selenium treatment group was 102% of the initial body weight, and it did not cause obvious changes in the body weight of the mice. During the process of treating tumors with bacteria, their metabolites and the bacteria themselves often cause an excessive immune response in the host, leading to autoimmunity and inflammation, thus causing hepatosplenomegaly and impairing the functions of the liver and spleen. As bacteria widely present in the intestines of healthy humans or other mammals, probiotics do not cause obvious adverse reactions after oral administration. The experimental results show that after continuous oral administration of Lactobacillus rhamnosus enriched with selenium for seven days, compared with the non-treatment group, the changes in the weights of the livers and spleens of the mice were not significant, indicating that oral treatment with Lactobacillus rhamnosus enriched with selenium does not cause hepatosplenomegaly, showing good safety.

[0109] The above results indicate that oral administration of Lactobacillus rhamnosus enriched with selenium can enhance the body's resistance to solid tumors, and its anti-tumor effect is better than that of Bifidobacterium longum expressing endostatin. While inhibiting tumors, it does not cause serious adverse reactions and has high safety, and is expected to be developed as an ideal carrier for anti-tumor drug delivery or combination, with broad application prospects.

[0110] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above test examples. What is described in the above test examples and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. Application of a selenium-enriched Lactobacillus rhamnosus agent in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The selenium-enriched Lactobacillus rhamnosus agent is prepared by fermenting Lactobacillus rhamnosus through selenium enrichment, and the selenium content is 5-100 μg / g.

3. The use according to claim 1, characterized in that: The tumor is a solid tumor.

4. The use according to claim 3, characterized in that: The solid tumor includes any one of melanoma, breast cancer, colorectal cancer, lymphoma, liver cancer, bladder cancer or lung cancer.

5. The use according to claim 2, characterized in that: The fermentation medium of the selenium-enriched Lactobacillus rhamnosus contains the following components: vitamin C, 4 g / L; NaCl, 2.4 g / L; Na2HPO4, 5.8 g / L; KH2PO4, 0.528571 g / L; glucose, 5 g / L; yeast extract, 3 g / L; beef extract powder, 1.7 g / L; soy peptone, 2 g / L; beef peptone, 20 g / L; and nano-selenium solution, 2-20 mg / L.

6. The use according to claim 1, characterized in that: The content of Lactobacillus rhamnosus is not less than 1×10 9 CFU / g.

7. The use according to claim 1, characterized in that: The administration method and time interval of the selenium-enriched Lactobacillus rhamnosus are once a day, intragastric administration; the dosage of the selenium-enriched Lactobacillus rhamnosus is 3×10 8 CFU / kg, 3×10 9 CFU / kg or 3×10 10 CFU / kg, and the volume of each gavage was 100 μL.

8. The use according to claim 7, characterized in that: After treatment with selenium-enriched Lactobacillus rhamnosus, the growth of malignant tumors in mice was significantly inhibited, the growth was slow, and no obvious adverse reactions occurred during oral administration.

9. A probiotic preparation containing the selenium-enriched Lactobacillus rhamnosus agent according to claim 1, characterized in that: The dosage form of the probiotic preparation includes any one of freeze-dried powder, tablets, capsules, granules, dairy products or yogurt.

10. The probiotic preparation according to claim 9, characterized in that: The freeze-dried powder is diluted with maltodextrin or skimmed milk powder or other fillers, and the active colony count of Lactobacillus rhamnosus is 1×10 9 Up to 5×10 10 CFU / g, and the selenium content is 5-100μg / g.

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