Probiotics systems and their applications

Abstract

This provides a system in which exogenous probiotics can form a stable microenvironment within the host, can be administered by a simple method such as oral administration, and can exert the effect of changing the composition of intestinal microbial communities, ultimately playing an active role in the process of malignant tumor treatment.

Description

Technical Field

[0001] The present invention relates to probiotics, particularly a probiotic system that can be used for medical purposes, and more particularly a probiotic system that can be used for the treatment of malignant tumors.

Background Art

[0002] Probiotics are beneficial active microorganisms that colonize the human body and change the composition of the flora in a certain part of the host. Mainly there are lactic acid bacteria, bifidobacteria, actinomycetes, yeasts, etc. These have the effect of promoting nutrient absorption and maintaining intestinal health by regulating the host mucosa and systemic immune function, or by regulating the balance of the intestinal flora. Probiotics are generally considered to have many advantages, such as enhancing the body's immunity, maintaining the structural balance of the intestinal flora, increasing the body's antioxidant level, suppressing intestinal inflammation, and protecting the intestinal mucosal barrier.

[0003] According to some reports, intestinal probiotics also have the effect of suppressing tumor growth and have a significant effect on the treatment of malignant tumors. For example, according to the study by Gopalakrishnan et al. in 2018, the anti-tumor effect of probiotics is related to immune activation. The intestinal flora of tumor patients is different from that of healthy people. In healthy people, bifidobacteria are the dominant flora, but in some cancer patients, the number of bifidobacteria has significantly decreased.

[0004] Bifidobacterium is a gram-positive, rod-shaped, anaerobic probiotic that may have branched ends and is widely present in the digestive tract, vagina, and oral cavity of humans and animals. Bifidobacterium interacts with the body's immune cells, regulates specific signaling pathways related to innate and adaptive immunity, and can promote Th1-type immune responses. Bifidobacterium has the functions of enhancing dendritic cells in the tumor microenvironment and recruiting CD8+ T cells. On the other hand, patients with malignant tumors receive various treatments, and these treatment methods also have a significant impact on the composition of the microbiota. Therefore, it is of positive significance to help reconstruct and optimize the structure of the intestinal flora in patients with malignant tumors.

[0005] However, in order to reconstruct the gut microbiota and enable probiotics to exert positive effects in the human body, it is necessary to change the composition of the gut microbiota. The currently reported methods for changing the composition of the gut microbiota mainly include fecal microbial transplantation and taking probiotics. Fecal microbial transplantation is not a preferred treatment option because it is costly, complex to operate, and has a high risk of pathogen infection. At the same time, from current various practices and attempts, the effect of directly orally administering probiotics is very slight, and taking probiotics only temporarily changes the composition of the gut microbiota, and this time is shown to be so short that these probiotics cannot exert their effects. The reason for this phenomenon is that foreign bacteria have a low ability to inhabit the host intestine and cannot form a stable aggregation and self-protection system, so they are easily directly removed from the gastrointestinal environment.

[0006] Therefore, the present invention hopes to invent a system that has the ability to stably grow probiotics, can help form a stable microenvironment for foreign probiotics in the host, can be administered by a simple method such as oral administration, can exert the effect of changing the composition of the gut microbiota, and can ultimately play a positive role in the process of malignant tumor treatment.

Summary of the Invention

Problems to be Solved by the Invention

[0007] To solve the problems described in the background art, the present inventors creatively proposed the concept of a system that uses an inorganic material system to form a long-term stable flora in vivo for exogenous probiotics, and formed and optimized this system through a series of studies. This system has great potential and value in medical applications.

Means for Solving the Problems

[0008] The present invention discloses a medically significant probiotic system containing probiotics and inorganic materials. Among them, the probiotics may be a single species or a mixed species; the inorganic material may be a natural mineral material, a processed natural material, or an artificially manufactured material.

[0009] Preferably, the probiotics constituting the probiotic system are probiotics that produce an acidic environment during the growth process, and the probiotics that produce an acidic environment here mean that the probiotics form an acidic environment around the colony during the metabolism process.

[0010] More preferably, the probiotics constituting the probiotic system are lactic acid bacteria.

[0011] Most preferably, the probiotics constituting the probiotic system are one or more of Lactobacillus, Bifidobacterium, Clostridium butyricum, or Streptococcus faecalis.

[0012] The inorganic materials constituting the probiotics system are preferably materials having cation exchange capacity and high surface area. These materials can be selected from any of cation exchange resins having a microporous structure, fluorite, and dioctahedral smectite subgroups. For example, they can be beidellite, montmorillonite, nontronite, any of the trioctahedral smectites subgroup such as saponite, sauconite, hectorite, stevensite, diatomaceous earth, kaolin, attapulgite, illite, chlorite, sepiolite, zeolite, talc.

[0013] More preferably, the inorganic materials constituting the probiotics system are materials having a positively charged surface in an acidic environment.

[0014] Most preferably, the inorganic materials constituting the probiotics system are materials having no positive charge on the surface in a neutral or alkaline environment and having a positive charge on the surface in an acidic environment. The artificial material can be mesoporous silica with a pore diameter of 2 - 50 nm; or a metal-organic framework compound selected from any of ZIF-67, UiO-66(Zr), MOF-74-Mg, Co-MOF-74, MIL-53(Fe), MIL-101, MOF-74-Ni, Cu-BTC, MOF-74-Fe, ZIF-8(Basolite Z1200), RMOF-1, MIL-100(Cr), MOF-5, Ce-BTC, MgDOBDC, MIL-53(Al).

[0015] In some examples, as the inorganic materials constituting the probiotics system, natural or certain artificially treated mineral materials derived from the smectite family are selected. More specifically, natural or certain artificially treated mineral materials derived from the montmorillonite subgroup are selected.

[0016] In some other embodiments, the inorganic material constituting the probiotics system further selects any one of diatomaceous earth, kaolin, or attapulgite.

[0017] In some other embodiments, the inorganic material constituting the probiotics system further selects a material that is artificially manufactured and has a positive charge on its surface in an acidic environment.

[0018] In order for the probiotics and the inorganic material to form a system that can be stably used, the inventors mixed the probiotics culture solution and the inorganic material suspension, then continued the culture for a certain period of time, and finally obtained a mixed system that can be stably used.

[0019] In order to form a probiotics-inorganic material system that can be stably used, it can be manufactured by the following procedure.

[0020] S1: Activation and culture amplification of probiotics: Anaerobic culture is carried out for 10 min - 4 h under the conditions of 25 - 40 °C and 0 - 200 rpm. S2: Activation of inorganic materials: Stir the inorganic materials in an isotonic buffer solution for 10 min - 1 h; S3: Adsorption of probiotics onto the surface of inorganic materials, formation and stabilization of probiotics biofilms: Mix the inorganic materials and probiotics at a ratio of 10:1 - 1:10, and carry out anaerobic culture for 4 h - 48 h under the conditions of 25 - 40 °C and 0 - 200 rpm; S4: After separating and purifying the finished product, freeze-dry it and store it in an environment of 4 °C.

[0021] For the convenience of later administration, many options are provided for the administration method. However, the form of the probiotics system described in the present invention is any one of an extractable solid form, a chewable solid form, an injectable injection solution, an oral liquid that can be orally administered, and an inhalable gaseous form. To obtain the above forms, the stable probiotics system formed by the above method can be processed by a known ordinary processing process.

[0022] The probiotic system obtained by the method disclosed in the present invention has been experimentally proven to stably, rapidly, and permanently colonize the host, and to reconstruct and optimize the intestinal microbiota composition within the host. Naturally, if it is possible to reconstruct and optimize the intestinal microbiota composition within the host to treat or alleviate these diseases, the probiotic system of the present invention is inevitably expected to be used for the treatment of these diseases. These diseases include at least diarrhea, constipation, dyspepsia, hypertension, lactase deficiency, lactose intolerance, vaginal infections, cirrhosis, peritoneal inflammation, endotoxemia, atopic dermatitis, allergies, irritable bowel syndrome, periodontitis, mental disorders, ulcerative colitis, polycystic ovary syndrome.

[0023] Also, a series of experiments have proven that the probiotic system according to the present invention has a broad therapeutic effect and tumor growth inhibitory effect on malignant tumors. These malignant tumors include at least melanoma, breast cancer, rectal cancer, sarcoma, gastric cancer, liver cancer, lung cancer, cervical cancer, pancreatic cancer.

[0024] The probiotic system according to the present invention can also be used as a drug for treating various malignant tumors, and can also play an auxiliary role in the current existing treatment system. On the other hand, the probiotic system according to the present invention can be used as a health food to assist in the recovery and reconstruction of the intestinal microbiota of patients during the process of chemotherapy and immunotherapy. On the other hand, it can also be used as an adjuvant therapeutic drug for chemotherapy and immunotherapy. It has been experimentally proven that the probiotic system described in the present invention can significantly enhance the effects of currently known chemotherapy and immunotherapy.

[0025] The present invention can form composite microspheres such as "bacteria-inorganic material-biofilm" in which probiotics in vivo are combined with an inorganic material system in vitro and co-cultured, and the inhabiting ability of foreign bacteria in the host intestine is low and they are easily removed directly from the gastrointestinal environment. These microspheres can be well adsorbed on the intestinal surface of the host, provide a safe and reliable environment for the growth of probiotics, and will not be swept away by the host's own digestive system. Therefore, if it is a disease that has a certain therapeutic effect by reconstructing or optimizing human intestinal probiotics, the technology described in the present invention is expected to have a certain effect.

[0026] Not only creatively proposing such a design concept, the inventors of the present invention have conducted a lot of research, discovered the mechanism by which probiotics and the inorganic material system can stably combine and finally form "bacteria-inorganic material-biofilm" composite microspheres, and summarized the characteristics of inorganic materials suitable for providing a stable growth environment for probiotics. Such inorganic materials should have cation exchange ability and a high surface area. More importantly, this material itself should have a positive charge, and a more preferable choice is to have no positive charge in a neutral or alkaline environment and have a positive charge in an acidic environment.

[0027] In addition, the inventors of the present invention have also discovered that the most preferable inorganic materials can selectively promote the growth and proliferation of specific probiotics. For example, Lactobacillus and Bifidobacterium are probiotics that are selectively promoted.

[0028] Lactobacillus and Bifidobacterium are Gram-positive bacteria, and the teichoic acid in their cell walls can be recognized by Toll-like receptor 2 (TLR2). Lactobacillus and Bifidobacterium formed in these intestines interact with dendritic cells via TLR2, activate the dendritic cells, and cause them to enter the tumor. At the same time, the activated dendritic cells play a central role in the process of initiating T cells as important antigen-presenting cells (APCs). Further research by the inventors of the present invention has shown that the number of cytotoxic T cells (CD8+) has increased in both the blood and the tumor. In addition, when the probiotic system was administered, the expression of several cytokines (IFN-γ, IL-12) that promote the anti-cancer immune response increased. Therefore, it can be seen that the most preferred inorganic material of the present invention and its specific characteristics can maximize the action of the probiotic system in the tumor treatment process, selectively stimulate specific probiotics, and maintain the appearance of other undesirable fungi at a low level, ensuring safety.

Brief Description of the Drawings

[0029]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0030] Combined with the following examples, the present invention can be better understood.

[0031] Preparation of the effect experiment of the probiotic + inorganic material system: (1) Cultivation and preparation of microorganisms. To conduct an effective comparison, Bifidobacterium (A), Lactobacillus (B), and Escherichia coli (negative control) (C) are selected. All three types of these microorganisms are derived from the China General Microbiological Culture Collection Center (CGMCC), and anaerobic culture is performed as shown in the following table and then used in subsequent experiments.

[0032]

Table 1

[0033] (2) Preparation of inorganic materials. Preparation of several inorganic materials. To conduct an effective comparison, inorganic materials including smectite (1), diatomaceous earth (2), kaolin (3), attapulgite (4), mesoporous silica (5), metal-organic framework silica MOF-5 (6), a mixture of smectite and diatomaceous earth (7), a mixture of smectite and attapulgite (8), and a mixture of smectite and metal-organic framework (9) are prepared for experiments, and the preparation processes of these materials are as follows respectively.

[0034] The aseptically treated inorganic material particles were mixed with phosphate buffer solution (PBS) and stirred to obtain the desired mineral material suspension. The aseptic treatment of the inorganic materials adopted the ultraviolet irradiation method.

[0035] Among them, smectite (1) was purchased from Xianfa Pharmaceutical, diatomaceous earth (2), kaolin (3), and attapulgite (4) were purchased from Alibaba, mesoporous silica (5) and metal-organic framework silica MOF-5 (6) were purchased from Sigma-Aldrich, USA.

[0036] The particle size of the inorganic material is 300 - 600 mesh, about 20 - 50 microns, and the concentration and ratio of each material system are as shown in the following table.

[0037]

Table 2

[0038] (3) Preparation of the mixed system of microorganisms and inorganic materials The microorganism may be cultured for a certain period of time by a method during the preparation of the microorganism, and then an inorganic material suspension is added to the culture solution at a certain ratio, and mixed and cultured at 37°C for a certain period of time. The specific ratio and mixed culture time are shown in the following table.

[0039]

Table 3

[0040] (4) Separate and purify the finished product The combination of the microorganism and the inorganic material and the liquid were separated by a centrifugation method, and the centrifugation conditions were 1000 rpm and 5 min. The precipitate obtained by centrifugation with the isotonic buffer solution used in step (2) was washed away to obtain a purified combination of the microorganism and the inorganic material. The combination of the microorganism and the inorganic material was resuspended in the buffer solution to obtain a suspension with a concentration of 0.01 g / ml.

[0041] Figure 1 and Figure 2 are electron micrographs of a biofilm formed on the surface of Bifidobacterium smectite and an electron micrograph of a biofilm formed on the surface of Lactobacillus smectite. It has been explained that the combination of the microorganism and the inorganic material predicted as above was obtained.

[0042] (5) Mouse experiment To verify the effects of the present invention, a plurality of pathological model mice were prepared. The experimental subjects were pure PBS, pure microbial culture solution of Bifidobacterium (denoted as A in the following table), Lactobacillus (denoted as B in the following table), Escherichia coli (denoted as C in the following table), pure inorganic material suspension: smectite (denoted as 1 in the following table), diatomaceous earth (denoted as 2 in the following table), kaolin (denoted as 3 in the following table), attapulgite (denoted as 4 in the following table), artificially manufactured pores and metal-organic framework silica MOF-5 (denoted as 5 and 6 in the following table), smectite + diatomaceous earth (denoted as 7 in the following table), smectite + attapulgite (denoted as 8 in the following table), smectite + metal-organic framework silica mixture (denoted as 9 in the following table). The mixed solutions immediately after mixing the microbial culture solution and the inorganic material suspension are denoted as FA+(1)-(9), FB+(1)-(9), FC+(1)-(9) in the following table, and the mixed solutions SA+(1)-(9), SB+(1)-(9), SC+(1)-(9) obtained after mixed culture of the microbial culture solution and the inorganic material suspension.

[0043] In addition, since the action of the probiotic mixed system acts through the living digestive system, it is impossible to verify the effect by cell experiments, and it is necessary to adopt in vivo experiments. Therefore, the present invention verified the effect using a commonly seen mouse experiment. Specifically, the tumor inhibition rate = (tumor weight of the control group tumor - tumor weight of the treatment group tumor) ÷ tumor weight of the control group tumor × 100% was used to evaluate the change in tumor weight between the control group and the treatment group. In individual cases, the test object of the treatment group may be ineffective, and a negative tumor inhibition rate may appear. To facilitate data processing, it was agreed that when a negative value appears and the tumor inhibition rate is less than 3%, it is uniformly represented by 0. In these cases, it indicates that the test object of this treatment group has no effect.

[0044] In addition, 10 repetitions were set for each subject (that is, 10 mice were used for each system), and the average value of the tumor weight was used as the basis for calculation.

[0045] In addition, to verify the combined effects of each subject with current common chemotherapy drugs and immunotherapy agents, the invention team set up two groups of experiments: the combination of SA+(1)-(9), SB+(1)-(9), SC+(1)-(9) with adriamycin (ADM), and the combination of SA+(1)-(9), SB+(1)-(9), SC+(1)-(9) with PD1. Among them, the usage conditions of adriamycin are as follows. For mice weighing 18 - 20 g, adriamycin is injected intraperitoneally, the dosage is 3 mg / kg, and it is injected once every two days starting from the 4th day after tumor transplantation.

[0046] The usage conditions of PD1 are as follows: for mice weighing 18 - 20 g, the PD1 antibody is injected via the tail vein, the dosage is 10 mg / kg, and it is injected once every two days starting from the 4th day after tumor transplantation.

[0047] Example 1: Experiment on melanoma mice B16-F10 cells are derived from the Cell Resource Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences; SPF-grade C57BL / 6J mice weighing 18 - 20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0048] The subjects of the above experiments were injected into the stomachs of C57BL / 6J mice by intragastric administration. The dosage was calculated based on the amount of 0.01 g of mineral material per mouse, and the intragastric administration was carried out once every two days for a total of two weeks. On the 15th day, B16-F10 tumor cells were counted for viable cells through a hemocytometer, and then the cells were adjusted to 5×106 / ml and inoculated subcutaneously into the right anterior limb flank of the mice (0.1 ml / mouse) to create a tumor model. Twenty days after tumor transplantation and 24 hours after the final administration, the cervical vertebrae were removed to cause death, the tumors were dissected and weighed, and the tumor inhibition rates of the mineral material and the probiotic mineral material composite preparation were calculated from the change in tumor weight to evaluate the therapeutic effect of the tumor. The results are as shown in the following table.

[0049]

Table 4

[0050] Example 2: Experiment on breast cancer mice The 4T1 cells were derived from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; SPF-grade BALB / c mice weighing 18 - 20 g were purchased from the Animal Experiment Center of Yangzhou University.

[0051] The subjects of the above experiment were injected into the stomachs of BALB / c mice by intragastric administration. The dosage was calculated based on the amount of 0.01 g of mineral material per mouse, and intragastric administration was performed once every two days for a total of two weeks. On the 15th day, the 4T1 tumor cells were used to count the living cells through a hemocytometer. Then, the cells were adjusted to 5×106 / ml and inoculated subcutaneously into the right anterior limb flank of the mice (0.1 ml / mouse) to establish a tumor model. 25 days after tumor transplantation and 24 hours after the final administration, the mice were sacrificed by dislocation of the cervical vertebrae. The tumors were dissected and weighed, and the tumor inhibition rates of the mineral material and the probiotic mineral material composite preparation were calculated from the changes in tumor weight to evaluate the therapeutic effect of the tumor. The results are as follows in the table.

[0052]

Table 5

[0053] Example 3: Experiment on Colorectal Cancer Mice The MC38 cells were derived from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; SPF-grade C57BL / 6J mice weighing 18 - 20 g were purchased from the Animal Experiment Center of Yangzhou University.

[0054] The subjects of the above experiment were injected into the stomachs of C57BL / 6J mice by intragastric administration. The dosage was calculated based on the amount of 0.01 g of mineral material per mouse, and intragastric administration was performed once every two days for a total of two weeks. On the 15th day, the MC38 tumor cells were used to count the living cells through a hemocytometer. Then, the cells were adjusted to 5×106 / ml and inoculated subcutaneously into the right anterior limb flank of the mice (0.1 ml / mouse) to establish a tumor model. 25 days after tumor transplantation and 24 hours after the final administration, the mice were sacrificed by dislocation of the cervical vertebrae. The tumors were dissected and weighed, and the tumor inhibition rates of the mineral material and the probiotic mineral material composite preparation were calculated from the changes in tumor weight to evaluate the therapeutic effect of the tumor. The results are as follows in the table.

[0055]

Table 6

[0056] Example 4: Experiment on Osteosarcoma Mice S180 cells are derived from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; SPF-grade C57BL / 6J mice weighing 18 - 20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0057] The subjects of the above experiment were injected into the stomachs of C57BL / 6J mice by intragastric administration. The dosage was calculated based on the amount of 0.01 g of mineral material per mouse, and intragastric administration was performed once every two days for a total of two weeks. On the 15th day, S180 tumor cells were counted for viable cells through a hemocytometer, and then the cells were adjusted to 5×106 / ml and inoculated subcutaneously into the right anterior limb flank of the mice (0.1 ml / mouse) to establish a tumor model. 30 days after tumor transplantation, after finally administering for 24 hours, the cervical vertebrae were removed to cause death, the tumors were dissected and weighed, and the tumor inhibition rates of the mineral material and the probiotics mineral material composite preparation were calculated from the changes in tumor weight to evaluate the therapeutic effect of the tumor. The results are as shown in the following table.

[0058]

Table 7

[0059] Example 5: Experiment on Liver Cancer Mice Heps cells are derived from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; SPF-grade C57BL / 6J mice weighing 18 - 20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0060] The subjects of the above experiments were injected into the stomachs of C57BL / 6J mice by intragastric administration. The dosage was calculated based on the amount of 0.01 g of mineral material per mouse and administered intragastrically once every two days for a total of two weeks. On the 15th day, live cells of heps tumor cells were counted through a hemocytometer. Subsequently, the cells were adjusted to 5×106 / ml and inoculated subcutaneously into the right anterior limb flank of the mice (0.1 ml / mouse) to establish a tumor model. 25 days after tumor transplantation and 24 hours after the final administration, the cervical vertebrae were removed to cause death, the tumors were dissected and weighed, and the tumor inhibition rates of the mineral material and the probiotics-mineral material composite preparation were calculated from the changes in tumor weight to evaluate the therapeutic effect of the tumors. The results are as shown in the following table.

[0061]

Table 8

[0062] Example 6: Experiment on Lung Cancer Mice LLC cells were derived from the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences; SPF-grade C57BL / 6J mice weighing 18 - 20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0063] The subjects of the above experiments were injected into the stomachs of C57BL / 6J mice by intragastric administration. The dosage was calculated based on the amount of 0.01 g of mineral material per mouse and administered intragastrically once every two days for a total of two weeks. On the 15th day, live cells of LLC tumor cells were counted through a hemocytometer. Subsequently, the cells were adjusted to 5×106 / ml and inoculated subcutaneously into the right anterior limb flank of the mice (0.1 ml / mouse) to establish a tumor model. 25 days after tumor transplantation and 24 hours after the final administration, the cervical vertebrae were removed to cause death, the tumors were dissected and weighed, and the tumor inhibition rates of the mineral material and the probiotics-mineral material composite preparation were calculated from the changes in tumor weight to evaluate the therapeutic effect of the tumors. The results are as shown in the following table.

[0064]

Table 9

[0065] Example 7: Experiment on Mice with Cervical Cancer Hela cells are derived from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; SPF-grade BALB / c Nude mice weighing 18 - 20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0066] The subjects of the above experiment were injected into the stomachs of BALB / c Nude mice by intragastric administration. The dosage was calculated based on the amount of 0.01 g of mineral material per mouse and administered intragastrically once every two days for a total of two weeks. On the 15th day, Hela tumor cells were counted for viable cells through a hemocytometer, and then the cells were adjusted to 5×106 / ml and inoculated subcutaneously into the right anterior limb flank of the mice (0.1 ml / mouse) to establish a tumor model. 30 days after tumor transplantation and 24 hours after the final administration, the cervical vertebrae were removed to cause death, the tumors were dissected and weighed, and the tumor inhibition rates of the mineral material and the probiotics mineral material composite preparation were calculated from the changes in tumor weight to evaluate the therapeutic effect of the tumor. The results are as shown in the following table.

[0067]

Table 10

[0068] Example 8: Experiment on Diarrheal Mice SPF-grade C57BL / 6J mice weighing 18 - 20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0069] After 4 hours of fasting, the mice were administered 0.5 ml (senna leaf extract) every 12 hours for 4 consecutive days. After obvious diarrhea symptoms were observed, the administration was stopped. The orally administered solution, which was the subject of the prepared experiment, was injected into the stomachs of C57BL / 6J mice by intragastric administration. The dosage was calculated based on the amount of 0.05 g of mineral material per mouse and administered intragastrically twice a day for a total of two days. The therapeutic effect was tabulated 3 days later, and if the mice stopped having diarrhea on the last day, it was considered cured.

[0070]

Table 11

[0071] Example 9: Ulcerative Colitis Mouse Model SPF-grade C57BL / 6J mice weighing 18 - 20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0072] After C57BL / 6J mice drank 3% DSS aqueous solution for 7 days, obvious symptoms such as weight loss, diarrhea, and bloody stools were observed. The orally administered solution, which was the subject of the prepared experiment, was injected into the stomach of C57BL / 6J mice by intragastric administration. The dosage was calculated based on the amount of 0.05 g of mineral material per mouse and administered intragastrically once a day for a total of 7 days. After 7 days, the treatment effects were aggregated. If the mice did not have diarrhea or bloody stools and their weight recovered on the last day, it was considered cured.

[0073]

Table 12

[0074] Example 10: Experiment on forming a film on the surface of smectite after treatment under different conditions by microorganisms

[0075] To modify smectite with 3-triethoxysilylpropyl succinic anhydride (TESSA), 1 g of smectite was taken, dispersed in 100 mL of dd H2O, gently shaken for 30 minutes, and then 1 mL of TESSA was added. The mixture was placed in a reflux apparatus and heated with stirring at 60 °C for 24 hours. Then, the product was washed with ethanol and dd H2O to obtain TESSA-MMT. TESSA, chemical formula: C13H24O6Si, purchased from Macklin, reagent number: T849358-5g. The procedure for modifying smectite with 3-aminopropyltriethoxysilane (APTES) was as follows: 1 g of smectite was taken, dispersed in 100 mL of dd H2O, gently shaken for 30 minutes, and then 1.6 mL of APTES was added. The mixture was placed in a reflux apparatus and heated with stirring at 60 °C for 24 hours. Then, the product was washed with ethanol and dd H2O to obtain APTES-MMT. APTES, chemical formula: H2nNCH2CH2CH2Si(OC2H5)3, purchased from 3Achem, reagent number: A11384. In this way, three types of smectites were obtained: the untreated smectite surface has a positive charge in an acidic environment, the APTES-treated smectite surface always has a positive charge, and the TESSA-treated smectite surface never has a positive charge.

[0076] When the pH of the medium was adjusted to around 7 and acidophilic Lactobacillus was cultured together with untreated smectite, acidophilic Lactobacillus could not form a biofilm on the smectite particles. When the pH of the medium for Escherichia coli and Bacillus subtilis was less than 5.5, Escherichia coli and Bacillus subtilis could form a biofilm on the untreated smectite particles.

[0077] The TESSA-treated smectite can maintain a negative charge at pH 5.5. When acidophilic Lactobacillus was cultured together with TESSA-MMT, acidophilic Lactobacillus did not form a biofilm on the particle surface.

[0078] The surface of the APTES-treated smectite always has a positive charge, and acidophilic Lactobacillus, Escherichia coli, Bacillus subtilis, etc. can all form a biofilm on the APTES-MMT surface. Figure 3 is an electron micrograph of Escherichia coli forming a biofilm on the surface of the smectite after APTES treatment, but Escherichia coli and Bacillus subtilis cannot form a biofilm on the unmodified smectite surface.

[0079] From the above experimental results, it was found that the positive charge on the particle surface plays an important role in the biofilm formation process. The potential mechanism by which smectite selectively supports acid-producing microorganisms to form a biofilm on its surface may be due to the acidic environment produced by specific acid-producing microorganisms, which accordingly changes the surface positive charge.

[0080] As described above, the concept, principle, and idea of the present invention have been described in detail with reference to examples and specific embodiments including the examples. Those skilled in the art will understand that the embodiments of the present invention are not limited to the several forms as described above, and after reading the present application documents, it is possible to make improvements, substitutions, and equivalent forms to the steps, methods, devices, and components in the above-described embodiments as much as possible, and it will be understood that these improvements, substitutions, and equivalent forms are considered to be included within the scope of the present invention. The protection scope of the present invention is only subject to the scope of the claims.

Claims

1. A probiotic system characterized by comprising probiotics and an inorganic material, wherein the probiotics are probiotics capable of creating an acidic environment during the growth process, the probiotics are lactic acid bacteria, the lactic acid bacteria are one or more selected from Lactobacillus, Bifidobacterium, Fusobacterium, and Enterococcus faecalis, the inorganic material is at least one of a cation exchange resin having a pore structure, fluorite, dioctahedral smectites subgroup, trioctahedral smectites subgroup, diatomaceous earth, kaolin, attapulgite, illite, chlorite, sepiolite, zeolite, and talc, and the inorganic material is an artificially manufactured material having a positive charge on the surface and not having a positive charge on the surface in a neutral or alkaline environment. A probiotic system characterized by this.

2. The probiotic system according to Claim 1, wherein the inorganic material is artificially manufactured and is a material having a positive charge on the surface at all times.

3. The probiotic system according to Claim 1 or 2, which is a conjugate of the probiotics and the inorganic material.

4. The form of the probiotic system is any one of an extractable solid form, a chewable solid form, an injectable injection solution, an orally administrable oral solution, and an inhalable gaseous form. The probiotic system according to Claim 1 or 2, characterized by this.

5. Use of the probiotic system according to Claim 1 or 2 in the manufacture of a therapeutic agent for diseases.

6. Use of the probiotic system according to Claim 1 or 2 in the manufacture of a therapeutic agent for malignant tumors.

7. The malignant tumor is malignant melanoma, breast cancer, rectal cancer, sarcoma, gastric cancer, liver cancer, lung cancer, cervical cancer, pancreatic cancer, thyroid cancer, cervical cancer, bladder cancer, skin cancer, esophageal cancer, prostate cancer, nasopharyngeal cancer, or oral cancer. The use according to Claim 6, characterized by this.

8. Use of the probiotic system according to Claim 1 or 2 in the manufacture of a drug for suppressing the growth of malignant tumors.

9. The use according to claim 8, wherein the malignant tumor is melanoma, breast cancer, rectal cancer, sarcoma, gastric cancer, liver cancer, lung cancer, cervical cancer, pancreatic cancer, thyroid cancer, cervical cancer, bladder cancer, skin cancer, esophageal cancer, prostate cancer, nasopharyngeal cancer, or oral cancer.

10. Use of the probiotic system according to claim 1 or 2 in the manufacture of a therapeutic agent for treating malignant tumors, in combination with chemotherapy and immunotherapy.

11. The use according to claim 10, wherein the malignant tumor is melanoma, breast cancer, rectal cancer, sarcoma, gastric cancer, liver cancer, lung cancer, cervical cancer, pancreatic cancer, thyroid cancer, cervical cancer, bladder cancer, skin cancer, esophageal cancer, prostate cancer, nasopharyngeal cancer, or oral cancer.

12. Use of the probiotic system according to claim 1 or 2 in the manufacture of a therapeutic agent for treating any one of the diseases of diarrhea, constipation, dyspepsia, hypertension, lactase deficiency, lactose intolerance, vaginal infection, cirrhosis, peritoneal inflammation, endotoxemia, atopic dermatitis, allergy, irritable bowel syndrome, periodontitis, mental illness, ulcerative colitis, polycystic ovary syndrome.

13. A therapeutic agent for treating malignant tumors, comprising the probiotic system according to claim 1 or 2.

14. The therapeutic agent according to claim 13, further comprising a chemotherapeutic agent and / or an immunotherapeutic agent for treating malignant tumors.

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