A probiotic compound preparation, a preparation method and application thereof

Abstract

The present application provides a probiotic compound preparation, which is prepared by directly mixing a probiotic compound colony dry powder with inorganic material, wherein the inorganic material is inorganic mineral material subjected to dispersion treatment; and provides a preparation method and application thereof.The probiotic compound preparation provided by the present application can play a role through simple methods such as oral administration and intragastric administration.The inorganic mineral material subjected to dispersion treatment can effectively improve its carrying capacity and colonization capacity, and can also change the microenvironment in the host body through the inorganic mineral material itself, so as to achieve the purpose of changing the composition and quantity of intestinal microflora, and then through the change of the composition and quantity of intestinal microflora, the purpose of preventing, relieving and treating various diseases caused by intestinal flora imbalance can be achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a probiotic compound preparation, its preparation method, and its application. Background Technology

[0002] The gut is the largest component of the immune system. Over 99% of the microorganisms in the gut are bacteria, numbering approximately 100 trillion, comprising 500-1000 different species, including trillions of symbiotic bacteria. These vast numbers of bacteria can be broadly categorized into three groups: harmful bacteria, neutral bacteria, and beneficial bacteria (probiotics).

[0003] Harmful bacteria, if they grow out of control and in large numbers, can cause a variety of diseases, produce carcinogens and other harmful substances, or affect the function of the immune system.

[0004] Neutral bacteria, also known as bacteria with dual functions, such as Escherichia coli and Enterococcus, are beneficial to health under normal circumstances. However, if they proliferate uncontrollably or migrate from the intestines to other parts of the body, they may cause many problems.

[0005] Beneficial bacteria, also known as probiotics, mainly consist of various Bifidobacteria and Lactobacilli. They are essential for human health, synthesizing various vitamins, participating in food digestion, promoting intestinal peristalsis, inhibiting the growth of pathogenic bacteria, and decomposing harmful and toxic substances. Probiotics can colonize the host's body, altering the composition of the host's gut microbiota in a specific area. They can also promote nutrient absorption and maintain intestinal health by regulating the host's mucosal and systemic immune functions or by adjusting the balance of gut microbiota.

[0006] Human health is closely related to the structure of beneficial bacteria in the gut. Through long-term evolution, individual adaptation and natural selection have maintained a dynamic balance among different species within the gut microbiota, between the microbiota and the host, and between the microbiota, the host, and the environment. This forms a mutually dependent and mutually restrictive system. Therefore, under normal circumstances, the gut microbiota structure is relatively stable and does not cause disease in the host. However, when the internal and external environment changes, this balance is disrupted, which can accelerate the development of diseases such as cancer, enteritis, and obesity.

[0007] Bifidobacterium is a Gram-positive, rod-shaped, sometimes forked anaerobic probiotic widely found in the digestive tract, vagina, and oral cavity of humans and animals. Bifidobacterium can interact with human immune cells, regulating specific signaling pathways related to innate and adaptive immunity, and promoting Th1 immune responses. Bifidobacterium enhances the function of dendritic cells in the tumor microenvironment and recruits CD8+ T cells. On the other hand, because patients with malignant tumors undergo various treatments, these treatments also have a significant impact on the composition of the gut microbiota. Therefore, helping patients with malignant tumors rebuild and optimize their gut microbiota structure is of positive significance.

[0008] Lactobacillus acidophilus belongs to the genus Lactobacillus, is a Gram-positive bacillus with rounded ends, and is mainly found in the small intestine. Lactobacillus acidophilus can secrete avidin-like substances (acidolin, acidophilin, and lactobacillus), which antagonize pathogenic microorganisms in the intestine.

[0009] Montmorillonite is a layered mineral composed of hydrated aluminosilicate particles, with the molecular formula (Al,Mg)₂[Si₄O₂]. 10 Montmorillonite (OH)₂·nH₂O has a three-layered, plate-like structure consisting of a central aluminum-oxygen octahedron and upper and lower silicon-oxygen tetrahedra. It contains exchangeable cations between the crystal layers, exhibiting high ion exchange capacity and strong water absorption and swelling ability. Montmorillonite particles are small and colloidally dispersed, with plate-like crystals visible under an electron microscope. Montmorillonite can coat the digestive tract mucosa, fixing and inhibiting viruses, bacteria, and their toxins within the digestive tract. It also binds to mucus glycoproteins, repairing and enhancing the protective function of the mucosal barrier.

[0010] Attapulgite (Mg, Al)2Si4O 10 (OH)·4(H2O) is a hydrous magnesium aluminum silicate clay mineral with a chain-like layered structure. In each 2:1 unit structural layer, the vertices of tetrahedral crystals are inverted at certain intervals, forming a chain-like structure. Channels parallel to the chains are formed between the tetrahedral strips, and these channels are filled with water of crystallization.

[0011] Currently, the main methods for regulating gut microbiota are oral probiotics and fecal microbiota transplantation (FMT), but both have certain drawbacks: 1) A major characteristic of probiotics is that they cannot tolerate gastric acid and bile salts. Therefore, oral probiotics cannot cross the gastric acid barrier and are difficult to colonize the gut, making it extremely difficult to change the composition of gut microbiota; 2) As an emerging technology, fecal microbiota transplantation (FMT) can better change the composition of gut microbiota, but this method has the potential risk of pathogen infection, and its safety still needs to be verified. Summary of the Invention

[0012] The purpose of this invention is to provide a probiotic compound preparation that can be used in a manner similar to simple oral administration, thereby promoting the stable colonization of probiotics in the host's intestine.

[0013] The above-mentioned objectives of the present invention can be achieved by the following technical solutions:

[0014] A probiotic compound preparation, wherein the probiotic compound preparation is prepared by directly mixing probiotic compound colony dry powder with inorganic materials, wherein the inorganic materials are inorganic mineral materials that have undergone dispersion treatment.

[0015] Probiotics, as a type of microorganism, have long been proven to have beneficial effects on humans. However, previous studies have found that oral probiotics have significant drawbacks. The main reason is that oral administration requires digestion by gastric acid and bile, but most probiotics cannot tolerate gastric acid and bile salts. Therefore, the number of probiotics after digestion by gastric acid and bile is greatly reduced, and their activity is correspondingly lowered. Although gastric acid is neutralized by secretions produced by the duodenum in the later stages, in some cases, hosts with excessive gastric acid secretion may even cause a small amount of gastric acid to flow into the intestines. This not only seriously affects the colonization of probiotics that enter the intestines with digestive juices, but in severe cases, it can even affect the number and distribution of existing probiotic communities in the intestines. At the same time, gastric acid can erode the intestines, forming ulcers, causing an imbalance in the intestinal flora environment. Harmful bacteria multiply in large numbers and enter the bloodstream through the ulcers, thus triggering a series of subsequent serious diseases. The probiotic compound preparation provided by this invention can effectively solve the above-mentioned problems.

[0016] Furthermore, in the aforementioned probiotic compound preparation, the probiotic compound colonies include, but are not limited to, colonies obtained from Bacillus strains and / or actinomycete colonies obtained from Actinomycete strains.

[0017] Furthermore, in the aforementioned probiotic compound preparation, the Bacillus strain includes, but is not limited to, one or more of Lactobacillus, Bacillus, and cocci.

[0018] Furthermore, in the aforementioned probiotic compound preparation, the lactobacillus includes, but is not limited to, one or more of Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii.

[0019] Furthermore, in the aforementioned probiotic compound preparation, the Bacillus species include, but are not limited to, one or more of Bacillus licheniformis, Bacillus cereus, Clostridium butyricum, Bacillus coagulans, and Bacillus subtilis.

[0020] Furthermore, in the aforementioned probiotic compound preparation, the cocci include, but are not limited to, one or more of Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium.

[0021] Furthermore, in the aforementioned probiotic compound preparation, the actinomycete strain is Bifidobacterium.

[0022] Furthermore, in the aforementioned probiotic compound preparation, the Bifidobacterium includes, but is not limited to, one or more of Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium infantis.

[0023] Furthermore, in the aforementioned probiotic compound preparation, the Bifidobacterium is preferably Bifidobacterium longum.

[0024] In the above-mentioned probiotic compound preparations, the probiotics can be a single strain or a mixture of strains.

[0025] The preferred probiotics are Bifidobacterium and / or Lactobacillus acidophilus.

[0026] In the compound probiotic freeze-dried powder, the mass ratio of Lactobacillus acidophilus freeze-dried powder and Bifidobacterium freeze-dried powder is (0-2):(2-0); preferably 1:1.

[0027] Furthermore, in the above-mentioned probiotic compound preparation, the inorganic mineral material is dispersed by adding a dispersant to the inorganic mineral material.

[0028] The dispersant is selected from formulations that simultaneously possess both lipophilic and hydrophilic properties within the molecule. It can promote the uniform dispersion of particles / fiber bundles aggregated in inorganic mineral materials in the medium, forming a stable suspension morphology, thereby achieving affinity, encapsulation and protection of probiotic dry powder.

[0029] Furthermore, in the aforementioned probiotic compound preparation, the inorganic mineral material is a silicate mineral material.

[0030] Furthermore, in the aforementioned probiotic compound preparation, the inorganic silicate mineral material includes, but is not limited to, one or more of the following: cation exchange resin with microporous structure, fluorite, dioctahedral smectite, trioctahedral smectite, diatomite, kaolinite, attapulgite, illite, chlorite, sepiolite, zeolite, and talc.

[0031] The preferred inorganic materials are attapulgite and / or montmorillonite.

[0032] In the inorganic material mixture, the mass ratio of attapulgite and montmorillonite is (0-2):(2-0); preferably 1:1.

[0033] The inorganic materials selected in this invention are those with cation exchange capacity and high surface area. These materials should have a certain particle size, which is on the same order of magnitude as the size of the probiotic cells in the mixed culture, so that they can effectively carry and protect the probiotics.

[0034] Furthermore, in the above-mentioned probiotic compound preparation, the dispersant is an inorganic dispersant or an organic dispersant.

[0035] Furthermore, in the above-mentioned probiotic compound preparation, the inorganic dispersant is a silicate dispersant or an alkali metal phosphate dispersant.

[0036] Furthermore, in the aforementioned probiotic compound preparation, the silicate dispersant is water glass; and the alkali metal phosphate dispersant is sodium tripolyphosphate, sodium hexametaphosphate, or sodium pyrophosphate.

[0037] Furthermore, in the aforementioned probiotic compound preparation, the organic dispersant is a low-molecular-weight polymer dispersant.

[0038] Furthermore, in the aforementioned probiotic compound preparation, the low-molecular-weight polymer dispersant is polyethylene glycol.

[0039] The dispersant is selected from sodium hexametaphosphate, sodium pyrophosphate and / or polyethylene glycol, preferably sodium pyrophosphate and polyethylene glycol; in the dispersant, the mass ratio of sodium pyrophosphate and polyethylene glycol is (0-2):(2-0); preferably 1:1.

[0040] In the dispersed inorganic material, the inorganic material and the dispersant are in a mass ratio of (5-15):(0.5-1.5), preferably 9:1.

[0041] Probiotic compound preparations can be in all suitable dosage forms, such as powders, capsules, and tablets.

[0042] Inorganic materials can be natural mineral materials, processed natural materials (such as modified materials), or artificially prepared materials.

[0043] The second inventive point of this invention is that it provides a method for preparing the above-mentioned probiotic compound preparation, which involves culturing the probiotic compound colonies, freeze-drying them, and then mixing them with sterilized and dispersed inorganic materials.

[0044] The preparation method provided by this invention is simple to operate. After the probiotic complex colonies are freeze-dried, they can be powdered and directly mixed with the dispersed inorganic materials. While ensuring the effect of the finished product, the cost and demand of industrial manufacturing are greatly reduced.

[0045] Furthermore, in the above-mentioned method for preparing a probiotic compound preparation, the culture time of each strain in the probiotic compound colony is 2-8 hours.

[0046] Furthermore, in the preparation method of the above-mentioned probiotic compound preparation, the dispersion treatment method of the inorganic material is as follows: the inorganic material is mixed with water, magnetically stirred and ultrasonically treated, then a dispersant is added and heated in a water bath, and finally washed, dried, pulverized and sieved.

[0047] Furthermore, in the above-mentioned method for preparing a probiotic compound preparation, the method for mixing the probiotic compound colony freeze-dried agent with the dispersed inorganic material is as follows: the probiotic compound colony freeze-dried agent is mixed at 10... 7 -10 14 The ratio of CFU / g of the dispersed inorganic material is added to the dispersed inorganic material for mixing; preferably 10. 10 cfu / g.

[0048] The present invention discloses a method for preparing a probiotic compound preparation as follows:

[0049] Step 1: Culture multiple probiotic strains under appropriate culture conditions for 2-8 hours, wash, examine smears under a microscope, extract 16S rDNA and sequence to determine the purity of the strains. After confirming that there is no contamination by other bacteria, add a freeze-drying protectant, freeze-dry and mix to obtain a probiotic compound colony freeze-dried powder.

[0050] Step 2: Weigh an appropriate amount of inorganic material and soak it in disinfectant (ethanol) for disinfection; then sterilize it by ultraviolet irradiation, radiation, etc., and finally add a dispersant to disperse it to obtain dispersed inorganic material.

[0051] The dispersion process is as follows: Weigh a certain amount of inorganic material, dissolve it in 200ml of deionized water, stir magnetically for 5 minutes, sonicate for 15 minutes, then add 10% mass fraction of dispersant, heat and stir in a 60℃ water bath for 2 hours, wash with deionized water 3 times, and finally dry, pulverize, pass through a 200-mesh sieve, and store for later use.

[0052] Step 3: The lyophilized powder prepared from one or more probiotic strains is then processed at 10... 7 -10 14 After dispersing the CFU / g inorganic materials, the proportions and dosages are mixed to obtain the probiotic compound preparation. During this process, buffers, excipients, solubilizers, flavoring agents, and other excipients are added according to the required dosage form.

[0053] The third inventive point of this invention is that it provides the application of the above-mentioned probiotic compound preparation in the preparation of a drug for promoting probiotic intestinal colonization.

[0054] The fourth inventive point of this invention is to provide the application of the above-mentioned probiotic compound preparation in the preparation of disease treatment drugs.

[0055] The fifth inventive point of this invention is to provide the application of the above-mentioned probiotic compound preparation in the preparation of drugs for the treatment of malignant tumors.

[0056] Furthermore, in the above-mentioned applications, the malignant tumor treatment drugs also include therapeutic drugs that can be used in combination.

[0057] Furthermore, in the above applications, the therapeutic agents that can be used in combination are doxorubicin or PD-1.

[0058] Furthermore, in the above applications, the malignant tumor is malignant melanoma, breast cancer, colorectal cancer, sarcoma, gastric cancer, liver cancer, lung cancer, cervical cancer, pancreatic cancer, thyroid cancer, bladder cancer, skin cancer, esophageal cancer, prostate cancer, nasopharyngeal cancer, or oral cancer.

[0059] The sixth inventive point of this invention is that it provides the application of the above-described preparation method in the preparation of drugs that inhibit the growth of malignant tumors.

[0060] Furthermore, in the above applications, the malignant tumor is malignant melanoma, breast cancer, colorectal cancer, sarcoma, gastric cancer, liver cancer, lung cancer, cervical cancer, pancreatic cancer, thyroid cancer, bladder cancer, skin cancer, esophageal cancer, prostate cancer, nasopharyngeal cancer, or oral cancer.

[0061] The seventh inventive point of this invention is that it provides the use of the above-mentioned probiotic compound preparation in the preparation of a medicament for treating any one of the following conditions: diarrhea, constipation, indigestion, hypertension, lactase deficiency, lactose intolerance, vaginal infection, cirrhosis, peritoneal inflammation, enterogenic endotoxemia, atopic dermatitis, allergy, irritable bowel syndrome, periodontitis, mental illness, ulcerative colitis, and polycystic ovary syndrome.

[0062] The eighth inventive point of this invention is that it provides a medicament for treating malignant tumors, the medicament containing the aforementioned probiotic compound preparation.

[0063] Furthermore, the aforementioned drugs also include chemotherapy drugs and / or immunotherapy drugs used to treat malignant tumors.

[0064] Experiments have demonstrated that the probiotic compound preparation disclosed in this invention can effectively promote the colonization of probiotics in the host's intestines and regulate the composition of the host's intestinal flora. Based on this technical principle, it can be deduced that as long as regulating the composition of the intestinal microbiota in the host can treat or alleviate these diseases, then the preparation method disclosed in this invention can certainly be used to treat these diseases. These diseases include at least: diarrhea, constipation, indigestion, hypertension, lactase deficiency, lactose intolerance, vaginal infection, cirrhosis, abdominal inflammation, enterogenic endotoxemia, atopic dermatitis, allergies, irritable bowel syndrome, periodontitis, mental illnesses, ulcerative colitis, and polycystic ovary syndrome.

[0065] The features and advantages of this invention are as follows: The probiotic compound preparation provided by this invention is prepared by directly mixing probiotic compound colony dry powder with inorganic materials. This preparation method eliminates the probiotic cultivation process; it only requires directly mixing the active probiotic dry powder with the dispersed inorganic mineral materials that act as a carrier. The operation is simpler and more suitable for large-scale industrial production. The probiotic compound preparation prepared by this method can exert its effects through simple methods such as oral administration or gavage. After dispersion treatment, the inorganic mineral materials can effectively improve their carrying capacity (adsorption capacity) and colonization capacity. At the same time, the inorganic mineral materials themselves can change the microenvironment (such as acidity and alkalinity) in the host. Based on this adjustment, the active ingredients of the dry powder compounded with multiple probiotic colonies are then colonized in the host's intestines, thereby changing the composition and quantity of the intestinal microbiota. In this way, the changes in the composition and quantity of the intestinal microbiota can prevent, alleviate, and treat various diseases caused by intestinal flora imbalance.

[0066] This invention can also directly mix freeze-dried powders of probiotics such as Lactobacillus acidophilus, Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Streptococcus thermophilus, Enterococcus faecalis, Bacillus licheniformis, Bacillus cereus, Clostridium butyricum, Bacillus coagulans, Bacillus subtilis, and Enterococcus faecalis with dispersed inorganic silicate materials such as montmorillonite and attapulgite to prepare probiotic compound preparations. Utilizing inorganic silicate materials as a medium, a more favorable ecological niche is provided for the colonization of these probiotics in the intestine, thereby playing a role in the treatment of diseases related to intestinal flora imbalance and reduced probiotic counts. Specifically promoting the growth and reproduction of these probiotics with the activity of activating host immune cells can fundamentally solve the problems of difficult probiotic colonization in the intestine and the potential infection risks associated with exogenous fecal microbiota transplantation, ultimately achieving the effect of specifically altering the composition of the intestinal flora according to the patient's condition. Attached Figure Description

[0067] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0068] Figure 1 The image shows a microscopic view of the combination of Lactobacillus acidophilus and dispersed montmorillonite.

[0069] Figure 2 The image shows a microscopic view of the combination of Bifidobacterium and dispersed montmorillonite.

[0070] Figure 3 The image shows a microscopic view of the combination of Lactobacillus acidophilus, Bifidobacterium, and dispersed montmorillonite.

[0071] Figure 4 The image shows a microscopic view of the combination of Lactobacillus acidophilus and dispersed attapulgite.

[0072] Figure 5 The image shows a microscopic view of the combination of Bifidobacterium and dispersed attapulgite.

[0073] Figure 6 The image shows a microscopic view of the combination of Lactobacillus acidophilus, Bifidobacterium, and dispersed attapulgite.

[0074] Figure 7 The image shows a microscopic representation of the combination of Lactobacillus acidophilus, Bifidobacterium, dispersed montmorillonite, and dispersed attapulgite. Detailed Implementation

[0075] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0076] Example 1:

[0077] A probiotic compound preparation is prepared by directly mixing probiotic compound colony dry powder with inorganic materials. The probiotic compound colony includes, but is not limited to, colonies obtained by culturing Bacillus strains and / or actinomycete colonies obtained by culturing Actinomycete strains. The inorganic materials are inorganic mineral materials that have undergone dispersion treatment.

[0078] The composite formulation provided by this invention contains inorganic mineral materials that have undergone dispersion treatment to form a suspension, improving their affinity and encapsulation properties. This effectively promotes the colonization of probiotics. Furthermore, since the inorganic mineral materials are themselves particulate alkaline substances, they can adhere to the surface of the probiotic powder, forming an alkaline protective layer. This significantly reduces the harmful effects of gastric acid or bile salts on probiotics and effectively neutralizes excess gastric acid. By altering the microenvironment of the gastrointestinal tract, this formulation promotes probiotic colonization, enhances probiotic activity, and effectively enriches the quantity and types of probiotics in the gastrointestinal tract. Consequently, it effectively regulates the balance of the gastrointestinal flora, not only treating some diseases caused by gastrointestinal flora imbalance but also effectively preventing various diseases caused by harmful bacteria in the gastrointestinal tract.

[0079] Example 2:

[0080] A probiotic compound preparation is prepared by directly mixing probiotic compound colony dry powder with inorganic materials. The probiotic compound colony includes, but is not limited to, colonies obtained by culturing Bacillus strains and / or actinomycete colonies obtained by culturing Actinomycete strains; the inorganic materials are inorganic mineral materials that have undergone dispersion treatment.

[0081] The Bacillus strains included include, but are not limited to, one or more of Lactobacillus, Bacillus, and cocci; Lactobacillus may be selected from one or more of Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii; Bacillus may be selected from one or more of Bacillus licheniformis, Bacillus cereus, Clostridium butyricum, Bacillus coagulans, and Bacillus subtilis; and cocci may be selected from one or more of Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium.

[0082] The actinomycete strain is Bifidobacterium; the Bifidobacterium can be selected from one or more of the following: Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium infantis.

[0083] Probiotics can be a single strain or a mixture of strains.

[0084] The preferred probiotics are Bifidobacterium and / or Lactobacillus acidophilus.

[0085] In the compound probiotic freeze-dried powder, the mass ratio of Lactobacillus acidophilus freeze-dried powder and Bifidobacterium freeze-dried powder is (0-2):(2-0); preferably 1:1.

[0086] The method for dispersing inorganic mineral materials is to add a dispersant to the inorganic mineral materials.

[0087] The dispersant can be either an inorganic or an organic dispersant;

[0088] The inorganic dispersant is a silicate dispersant or an alkali metal phosphate dispersant; the silicate dispersant is water glass; the alkali metal phosphate dispersant is sodium tripolyphosphate, sodium hexametaphosphate, or sodium pyrophosphate.

[0089] The organic dispersant is a low-molecular-weight polymer dispersant; polyethylene glycol can be selected as an example.

[0090] The dispersant is selected from sodium hexametaphosphate, sodium pyrophosphate and / or polyethylene glycol, preferably sodium pyrophosphate and polyethylene glycol; in the dispersant, the mass ratio of sodium pyrophosphate and polyethylene glycol is (0-2):(2-0); preferably 1:1.

[0091] The inorganic mineral material in the probiotic compound preparation is a silicate mineral material; it can be selected from one or more of the following: cation exchange resin with microporous structure, fluorite, dioctahedral smectite subgroup, trioctahedral smectite subgroup, diatomite, kaolinite, attapulgite, illite, chlorite, sepiolite, zeolite and talc.

[0092] The preferred inorganic materials are attapulgite and / or montmorillonite.

[0093] In the inorganic material mixture, the mass ratio of attapulgite and montmorillonite is (0-2):(2-0); preferably 1:1.

[0094] In the dispersed inorganic material, the inorganic material and the dispersant are in a mass ratio of (5-15):(0.5-1.5), preferably 9:1.

[0095] Probiotic compound preparations can be in all suitable dosage forms, such as powders, capsules, and tablets.

[0096] The present invention also provides a method for preparing a probiotic compound preparation, which involves culturing the probiotic compound colonies, freeze-drying them, and then mixing them with sterilized and dispersed inorganic materials.

[0097] The culture time for each strain in the probiotic compound colony is 2-8 hours.

[0098] The method for dispersing inorganic materials is as follows: mix the inorganic materials with water, stir magnetically, then ultrasonically treat, add a dispersant, heat in a water bath, and finally wash, dry, pulverize, and sieve.

[0099] The mixing method of probiotic compound colony lyophilized agent and dispersed inorganic material is as follows: the probiotic compound colony lyophilized agent is mixed at 10... 7 -10 14 The ratio of CFU / g of the dispersed inorganic material is added to the dispersed inorganic material for mixing; preferably 10.10 cfu / g.

[0100] The specific preparation method of the probiotic compound preparation disclosed in this invention is as follows:

[0101] Step 1: Culture multiple probiotic strains under appropriate culture conditions for 2-8 hours, wash, examine smears under a microscope, extract 16S rDNA and sequence to determine the purity of the strains. After confirming that there is no contamination by other bacteria, add a freeze-drying protectant, freeze-dry and mix to obtain a probiotic compound colony freeze-dried powder.

[0102] Step 2: Weigh an appropriate amount of inorganic material and soak it in disinfectant (ethanol) for disinfection; then sterilize it by ultraviolet irradiation, radiation, etc., and finally add a dispersant to disperse it to obtain dispersed inorganic material.

[0103] The dispersion process is as follows: Weigh 10g of inorganic material, dissolve it in 200ml of deionized water, stir magnetically for 5min, sonicate for 15min, then add 10% mass fraction of dispersant, heat and stir in a 60℃ water bath for 2h, wash with deionized water 3 times, and finally dry, pulverize, pass through a 200-mesh sieve, and store for later use.

[0104] Step 3: The lyophilized powder prepared from one or more probiotic strains is then processed at 10... 7 -10 14 After dispersing the CFU / g inorganic materials, the proportions and dosages are mixed to obtain the probiotic compound preparation. During this process, buffers, excipients, solubilizers, flavoring agents, and other excipients are added according to the required dosage form.

[0105] This invention also provides the application of probiotic compound preparations in the preparation of drugs for promoting probiotic intestinal colonization, in the preparation of drugs for treating diseases, and in the preparation of drugs for treating malignant tumors.

[0106] This invention also provides the application of the above preparation method in the preparation of drugs that inhibit the growth of malignant tumors.

[0107] Malignant tumors include malignant melanoma, breast cancer, colorectal cancer, sarcoma, stomach cancer, liver cancer, lung cancer, cervical cancer, pancreatic cancer, thyroid cancer, bladder cancer, skin cancer, esophageal cancer, prostate cancer, nasopharyngeal cancer, or oral cancer.

[0108] This invention also provides the use of probiotic compound preparations in the preparation of drugs for treating any one of the following conditions: diarrhea, constipation, indigestion, hypertension, lactase deficiency, lactose intolerance, vaginal infection, cirrhosis, abdominal inflammation, enterogenic endotoxemia, atopic dermatitis, allergy, irritable bowel syndrome, periodontitis, mental illness, ulcerative colitis, and polycystic ovary syndrome.

[0109] The present invention also provides a medicament for treating malignant tumors, the medicament comprising the aforementioned probiotic compound preparation.

[0110] The drug also includes chemotherapy drugs and / or immunotherapy drugs used to treat malignant tumors.

[0111] Example 3:

[0112] Preparation for efficacy experiments of probiotic freeze-dried powder + dispersed inorganic material system:

[0113] 1) Microbial culture and preparation:

[0114] To provide effective comparison, the following microorganisms were selected: *Escherichia coli* (negative control) 1', *Bifidobacterium* 2', *Lactobacillus acidophilus* 3', *Lactobacillus bulgaricus* 4', *Lactobacillus delbrueckii* 5', *Bacillus licheniformis* 6', *Bacillus cereus* 7', *Clostridium butyricum* 8', *Bacillus coagulans* 9', *Bacillus subtilis* 10', *Streptococcus thermophilus* 11', *Enterococcus faecalis* 12', *Enterococcus faecium* 13', *Bifidobacterium adolescentis* 14', *Bifidobacterium longum* 15', and *Bifidobacterium infantis* 16'. All these microorganisms were obtained from the China General Microbiological Culture Collection Center (CGMCC). Table 1 shows the selection of culture media and culture conditions for anaerobic culture of the microorganisms. After culture, they were used in subsequent experiments, as detailed in the table below.

[0115]

[0116]

[0117] The freeze-drying process of the above-mentioned probiotics is as follows: the cultured bacterial colonies are pre-frozen at -40℃ for 3 hours, and then quickly transferred to -60℃ for freeze-drying for 28 hours to obtain freeze-dried probiotic powder with a moisture content of ≤2%.

[0118] In the compound probiotic freeze-dried powder, the mixture of Lactobacillus acidophilus freeze-dried powder and Bifidobacterium freeze-dried powder is in a mass ratio of (0-2):(2-0), preferably 1:1.

[0119] 2) Preparation of dispersed inorganic materials:

[0120] Preparation of several inorganic materials: To make effective comparisons, the following inorganic materials were prepared for the experiment: montmorillonite (1″), diatomaceous earth (2″), kaolinite (3″), attapulgite (4″), mesoporous silica (5″), metal-framework silica MOF-5 (6″), a mixture of montmorillonite and diatomaceous earth (7″), a mixture of montmorillonite and attapulgite (8″), and a mixture of montmorillonite and metal-framework silica (9″). The preparation processes for these materials are as follows:

[0121] The inorganic material is first dispersed. The specific process is as follows: weigh 10g of inorganic material, dissolve it in 200ml of deionized water, stir magnetically for 5min, sonicate for 15min, then add 10% mass fraction of dispersant, heat and stir in a 60℃ water bath for 2h, wash with deionized water 3 times, and finally dry, pulverize, pass through a 200-mesh sieve, and store for later use.

[0122] Among them, montmorillonite (1″) was purchased from Sinopharm, diatomite (2″), kaolinite (3″), attapulgite (4″) was purchased from Alibaba, and mesoporous silica (5″) and metal skeleton silica MOF-5 (6″) were purchased from Sigma-Aldrich, Inc.

[0123] In the inorganic material mixture, the montmorillonite and diatomaceous earth mixture (7″) is in a mass ratio of (0-2):(2-0); preferably 1:1; the montmorillonite and attapulgite mixture (8″) is in a mass ratio of (0-2):(2-0); preferably 1:1; the montmorillonite and metallic silica mixture (9″) is in a mass ratio of (0-2):(2-0); preferably 1:1.

[0124] The dispersant is selected from sodium hexametaphosphate, sodium pyrophosphate and / or polyethylene glycol, namely sodium hexametaphosphate (a), sodium pyrophosphate (b), polyethylene glycol (c), a mixture of sodium hexametaphosphate and sodium pyrophosphate (d), a mixture of sodium hexametaphosphate and polyethylene glycol (e), and a mixture of sodium pyrophosphate and polyethylene glycol (f), preferably a mixture of sodium pyrophosphate and polyethylene glycol (f); in the dispersant, the mixture of sodium hexametaphosphate and sodium pyrophosphate (d), the mixture of sodium hexametaphosphate and polyethylene glycol (e), and the mixture of sodium pyrophosphate and polyethylene glycol (f) are all in a mass ratio of (0-2):(2-0); preferably 1:1.

[0125] Sodium hexametaphosphate, sodium pyrophosphate, and polyethylene glycol were all purchased from Alibaba and are food grade.

[0126] In the dispersed inorganic material, the inorganic material and the dispersant are in a mass ratio of (5-15):(0.5-1.5), which can be selected as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, with 9:1 being the preferred ratio.

[0127] Weigh an appropriate amount of the dispersed inorganic material and soak it in a disinfectant (ethanol); then sterilize it by means of ultraviolet irradiation, radiation, etc.

[0128] Sedimentation rate refers to the ratio of the weight of particles on the settling plate to the total weight of particles in the detection area of ​​the dispersed formulation per unit time, expressed as a percentage. A lower percentage indicates fewer particles on the settling plate, meaning better dispersion. Table 2 shows the selection of dispersants and inorganic materials based on sedimentation rate, as detailed below:

[0129]

[0130] In Table 2 above, the dispersants are (a)-(f) in sequence, and the inorganic materials are (1″)-(9″) in sequence. Among the inorganic materials, the mixing ratio of (7″)-(9″) is 1:1; among the dispersants, the mixing ratio of (d)-(f) is 1:1; the ratio of inorganic materials to dispersants by mass is 9:1.

[0131] As can be seen from Table 2 above, the sedimentation rate is the lowest, about 0.1%, when the inorganic material and dispersant are selected as 8″+f. Therefore, the mixture of montmorillonite and attapulgite (8″) is selected as the inorganic material and the mixture of sodium pyrophosphate and polyethylene glycol (f) is selected as the dispersant, and the dispersion treatment is carried out at a weight ratio of 9:1.

[0132] 3) Probiotic freeze-dried powder + dispersed inorganic materials:

[0133] Probiotic freeze-dried powder at 10 7 -10 14 After CFU / g dispersion, the inorganic materials are mixed in the required proportions, and during this process, buffers, excipients, solubilizers, flavoring agents and other excipients are added according to the required dosage form.

[0134] Table 3 shows the proportions of various material systems, as detailed in the table below:

[0135]

[0136] 4) Mouse experiment:

[0137] To verify the effectiveness of this invention, various disease model mice were prepared, and the subjects used in the experiment included:

[0138] Pure phosphate-buffered saline (PBS), Bifidobacterium lyophilized powder (represented by A in the table below), Lactobacillus acidophilus lyophilized powder (represented by B in the table below), and a 1:1 lyophilized powder mixture of Bifidobacterium and Lactobacillus acidophilus (represented by C in the table below).

[0139] Dispersed silicate materials: montmorillonite (represented by 1 in the table below), attapulgite (represented by 2 in the table below), montmorillonite + attapulgite 1:1 (represented by 3 in the table below), and the preparations obtained by mixing microbial freeze-dried powder with dispersed inorganic materials are represented by A+(1-3), B+(1-3), and C+(1-3) in the table below.

[0140] Figures 1-7 The images show the microstructure of the combination of probiotic freeze-dried powder and dispersed inorganic materials, where the probiotic freeze-dried powder consists of Lactobacillus acidophilus and / or Bifidobacterium freeze-dried powder, and the dispersed inorganic materials consist of dispersed montmorillonite and / or dispersed attapulgite.

[0141] To demonstrate that the technical solution of probiotic freeze-dried powder + dispersed inorganic materials used in this invention is superior to the suspensions described in the prior art (inventor's prior patents CN112972503 and CN112972504), this invention also sets up suspension comparative examples (referred to as Comparison 1 and Comparison 2, respectively) based on the technical solutions disclosed in CN112972503 and CN112972504. The ratios of bacterial suspension and inorganic materials are selected from the optimal embodiment data in the above-mentioned publications, and are respectively referred to as D and E. At the same time, in order to highlight the technical effects of "freeze-drying" and "dispersion", comparative examples of "bacterial suspension + dispersed inorganic materials" and "freeze-dried bacteria + undispersed inorganic materials" are set up, respectively referred to as F and G.

[0142] In comparisons 1 and 2 of CN112972503, the biological culture medium contained Bifidobacterium (A), Lactobacillus (B), and montmorillonite + attapulgite (8). The mixed solutions SA+8 and SB+8 were obtained after the microbial culture medium was mixed with the inorganic material suspension (S). Therefore...

[0143] In Example 4 (melanoma), D should be selected from the better of SA+8 (62.1) and SB+8 (60.4), that is, D = SA+8 (62.1);

[0144] In Example 6 (breast cancer), D should be selected from the better of SA+8 (58.3) and SB+8 (55.1), that is, D = SA+8 (58.3);

[0145] In Example 7 (colon cancer), D should be selected from the better of SA+8 (49.7) and SB+8 (57.4), that is, D = SB+8 (57.4);

[0146] In Example 8 (osteosarcoma), D should be selected from the better of SA+8 (51.6) and SB+8 (59.3), that is, D = SB+8 (59.3);

[0147] In Example 9 (liver cancer), D should be selected from the better of SA+8 (57.3) and SB+8 (56.1), that is, D = SA+8 (57.3);

[0148] In Example 10 (lung cancer), D should be the better of SA+8 (57.8) and SB+8 (56.2), i.e., D = SA+8 (57.8);

[0149] In Example 11 (cervical cancer), D should be selected from the better of SA+8 (54.8) and SB+8 (53.5), that is, D = SA+8 (54.8).

[0150] Furthermore, since the effects of probiotic mixtures are produced through the digestive system of the organism, cell experiments cannot be used to verify their efficacy; in vivo experiments are necessary. Therefore, this invention employs common mouse experiments to verify the effects. Specifically, the change in tumor weight between the control and treatment groups is used to assess the tumor inhibition rate; tumor inhibition rate = (tumor weight of control group - tumor weight of treatment group) ÷ tumor weight of control group × 100%. In some cases, the subjects tested in the treatment group may be ineffective, resulting in a negative tumor inhibition rate. For ease of data processing, negative numbers and tumor inhibition rates below 3% are uniformly represented as 0. These cases indicate that the subjects tested in the treatment group have no effect.

[0151] In addition, 10 replicates were set up for each experimental subject (i.e., 10 mice were used in each system), and the average tumor weight was used as the basis for calculation.

[0152] In addition, in order to verify the combined effects of various subjects with commonly used chemotherapy drugs and immunotherapy agents, the inventors also set up two sets of experiments: one was to use A+(1-3), B+(1-3), C+(1-3) in combination with doxorubicin (ADM), and the other was to use A+(1-3), B+(1-3), C+(1-3) in combination with PD1.

[0153] The conditions for using doxorubicin are as follows: 18-20g mice are injected intraperitoneally with doxorubicin at a dose of 3mg / kg, starting on the fourth day after tumor transplantation, and injected once every two days.

[0154] The conditions for using PD1 are as follows: 18-20g mice are injected with PD1 antibody via the tail vein at a dose of 10mg / kg, starting on the fourth day after tumor transplantation, and injected once every two days.

[0155] The probiotic compound preparation disclosed in this invention and the method for regulating intestinal flora using this preparation mainly involve administering a probiotic freeze-dried powder-inorganic mineral material compound preparation to mice by gavage, thereby directly altering the composition of the intestinal flora in mice.

[0156] Example 4:

[0157] Melanoma mouse experiment:

[0158] B16-F10 cells were obtained from the Cell Resource Center of the 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.

[0159] The above-mentioned experimental subjects were administered the drug via gavage to the stomachs of C57BL / 6J mice. Each mouse received 0.01g of mineral material, administered every two days for two weeks. On day fifteen, B16-F10 tumor cells were counted for viable cells using a erythrocyte counting chamber, and the cell count was then adjusted to 5 × 10⁻⁶ cells / mL. 6 A tumor model was established by subcutaneous inoculation of the right forelimb axilla of mice with a dose of 0.1 ml / mouse. Twenty days after tumor implantation, mice were euthanized by cervical dislocation 24 hours after the last administration. The tumors were dissected and weighed. The tumor inhibition rate of the mineral material and the probiotic-mineral material composite preparation was calculated based on the change in tumor weight to evaluate the therapeutic effect. The results are shown in Table 4 below.

[0160]

[0161]

[0162] As can be seen from Table 4, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SA+8 was selected as D and E, which is better; while the inhibition rate of C+3 group of the present invention, 63.5%, is significantly better than that of D and E groups, 62.1%. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0163] Example 5:

[0164] Ulcerative colitis mouse model:

[0165] SPF-grade C57BL / 6J mice weighing 18-20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0166] After C57BL / 6J mice were given a 3% DSS aqueous solution (sodium dextran sulfate, which can induce enteritis) for 7 days, significant weight loss, diarrhea, and bloody stools were observed. The prepared oral solution was administered to the stomachs of C57BL / 6J mice via gavage, with each mouse receiving 0.05g of mineral material once daily for 7 days. Treatment efficacy was assessed after 7 days; mice were considered cured if they no longer experienced diarrhea or bloody stools and their weight recovered on the last day. The results are shown in Table 5 below.

[0167]

[0168] As can be seen from Table 5, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SB+8 is selected as D and E, which is better; while the cure rate of the C+3 group of the present invention is equivalent to 100% of the D and E groups, which means that it has a very good therapeutic effect. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0169] Example 6:

[0170] Breast cancer mouse experiment:

[0171] 4T1 cells were obtained from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; SPF-grade BALB / c mice weighing 18-20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0172] The above-mentioned experimental subjects were administered the drug via gavage to BALB / c mice. Each mouse received 0.01g of mineral material, administered every two days for two weeks. On day fifteen, 4T1 tumor cells were counted for viable cells using a erythrocyte counting chamber, and the cell count was then adjusted to 5 × 10⁻⁶ cells / mL. 6 A tumor model was established by subcutaneous inoculation of the right forelimb axilla of mice with a dose of 0.1 ml / mouse. Twenty-five days after tumor implantation, mice were euthanized by cervical dislocation 24 hours after the last administration. The tumors were dissected and weighed. The tumor inhibition rate of the mineral material and the probiotic-mineral material composite preparation was calculated based on the change in tumor weight to evaluate the therapeutic effect. The results are shown in Table 6 below.

[0173]

[0174]

[0175] As can be seen from Table 6, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SA+8 was selected as D and E, which is better; while the inhibition rate of C+3 group of the present invention, 60.1%, is significantly better than that of D and E groups, 58.3%. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0176] Example 7:

[0177] Colon cancer mouse experiment:

[0178] MC38 cells were obtained from the Cell Resource Center of the 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.

[0179] The above-mentioned experimental subjects were administered the drug via gavage to the stomachs of C57BL / 6J mice. Each mouse received 0.01g of mineral material, administered every two days for two weeks. On day fifteen, MC38 tumor cells were counted for viable cells using a erythrocyte counting chamber, and the cell count was then adjusted to 5 × 10⁻⁶ cells / mL. 6 A tumor model was established by subcutaneous inoculation of the right forelimb axilla of mice with a dose of 0.1 ml / mouse. Twenty-five days after tumor implantation, mice were euthanized by cervical dislocation 24 hours after the last administration. The tumors were dissected and weighed. The tumor inhibition rate of the mineral material and the probiotic-mineral material composite preparation was calculated based on the change in tumor weight to evaluate the therapeutic effect. The results are shown in Table 7 below.

[0180]

[0181]

[0182] As can be seen from Table 7, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SB+8 is selected as D and E, which is better; while the inhibition rate of group C+3 of the present invention, 58.6%, is significantly better than that of groups D and E, 57.4%. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0183] Example 8:

[0184] Osteosarcoma mouse experiment:

[0185] S180 cells were obtained from the Cell Resource Center of the 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.

[0186] The above-mentioned experimental subjects were administered the drug via gavage to the stomachs of C57BL / 6J mice. Each mouse received 0.01g of mineral material, administered every two days for two weeks. On day fifteen, S180 tumor cells were counted for viable cells using a erythrocyte counting chamber, and the cell count was then adjusted to 5 × 10⁻⁶ cells / mL. 6 A tumor model was established by subcutaneous inoculation of the right forelimb axilla of mice with a dose of 0.1 ml / mouse. Thirty days after tumor implantation, mice were euthanized by cervical dislocation 24 hours after the last administration. The tumors were dissected and weighed. The tumor inhibition rate of the mineral material and the probiotic-mineral material composite preparation was calculated based on the change in tumor weight to evaluate the therapeutic effect. The results are shown in Table 8 below.

[0187]

[0188]

[0189] As can be seen from Table 8, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SB+8 is selected as D and E, which is better; while the inhibition rate of C+3 group of the present invention, 61.2%, is significantly better than that of D and E groups, 59.3%. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0190] Example 9:

[0191] Liver cancer mouse experiment:

[0192] Heps cells were obtained 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.

[0193] The above-mentioned experimental subjects were administered the drug via gavage to the stomachs of C57BL / 6J mice. Each mouse received 0.01g of mineral material, administered every two days for two weeks. On day fifteen, viable cells of the Heps tumor cells were counted using a erythrocyte counting chamber, and the cell count was then adjusted to 5 × 10⁻⁶ cells / mL. 6 A tumor model was established by subcutaneous inoculation of the right forelimb axilla of mice with a dose of 0.1 ml / mouse. Twenty-five days after tumor implantation, mice were euthanized by cervical dislocation 24 hours after the last administration. The tumors were dissected and weighed. The tumor inhibition rate of the mineral material and the probiotic-mineral material composite preparation was calculated based on the change in tumor weight to evaluate the therapeutic effect. The results are shown in Table 9 below.

[0194]

[0195]

[0196] As can be seen from Table 9, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SA+8 was selected as D and E, which is better; while the inhibition rate of group C+3 of the present invention, 59.6%, is significantly better than that of groups D and E, 57.3%. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0197] Example 10:

[0198] Lung cancer mouse experiment:

[0199] LLC cells were obtained from the Cell Resource Center of the 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.

[0200] The above-mentioned experimental subjects were administered the drug via gavage to the stomachs of C57BL / 6J mice. Each mouse received 0.01g of mineral material, administered every two days for two weeks. On day fifteen, LLC tumor cells were counted using a erythrocyte counting chamber, and the cell count was then adjusted to 5 × 10⁻⁶ cells / mL. 6 A tumor model was established by subcutaneous inoculation of the right forelimb axilla of mice with a dose of 0.1 ml / mouse. Twenty-five days after tumor implantation, mice were euthanized by cervical dislocation 24 hours after the last administration. The tumors were dissected and weighed. The tumor inhibition rate of the mineral material and the probiotic-mineral material composite preparation was calculated based on the change in tumor weight to evaluate the therapeutic effect. The results are shown in Table 10 below.

[0201]

[0202]

[0203] As can be seen from Table 10, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SA+8 was selected as D and E, which is better; while the inhibition rate of C+3 group of the present invention, 61.4%, is significantly better than that of D and E groups, 57.8%. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0204] Example 11:

[0205] Cervical cancer mouse experiment:

[0206] HeLa cells were obtained from the Cell Resource Center of the 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.

[0207] The above-mentioned experimental subjects were administered the drug via gavage to BALB / c Nude mice. Each mouse received 0.01g of mineral material, administered every two days for two weeks. On day fifteen, HeLa tumor cells were counted using a erythrocyte counting chamber, and the cell count was then adjusted to 5 × 10⁻⁶ cells / mL. 6 A tumor model was established by subcutaneous inoculation of the right forelimb axilla of mice with a dose of 0.1 ml / mouse. Thirty days after tumor implantation, mice were euthanized by cervical dislocation 24 hours after the last administration. The tumors were dissected and weighed. The tumor inhibition rate of the mineral material and the probiotic-mineral material composite preparation was calculated based on the change in tumor weight to evaluate the therapeutic effect. The results are shown in Table 11 below.

[0208]

[0209] As can be seen from Table 11, among Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SA+8 was selected as D and E, which is better; while the inhibition rate of group C+3 of the present invention, 56.3%, is significantly better than that of groups D and E, 54.8%. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0210] Example 12:

[0211] Diarrhea mouse experiment:

[0212] SPF-grade C57BL / 6J mice weighing 18-20 grams were purchased from the Animal Experiment Center of Yangzhou University.

[0213] Mice were fasted for 4 hours, then administered 0.5 ml of senna leaf extract every 12 hours for four consecutive days. Administration was stopped after observing obvious diarrhea symptoms in the mice. The prepared experimental solution was administered via gavage to C57BL / 6J mice, with each mouse receiving 0.05 g of mineral material, twice daily for two days. Treatment efficacy was assessed after three days; mice were considered cured if they no longer experienced diarrhea on the last day. Results are as follows.

[0214] As shown in Table 12:

[0215]

[0216] As can be seen from Table 12, in Comparison 1 (CN112972503) and Comparison 2 (CN112972504), SA+8 / SB+8 was selected as D and E; while the cure rate of the C+3 group of the present invention is equivalent to 100% of the D and E groups. C+3 is [(C) freeze-dried powder mixture of Bifidobacterium + Lactobacillus acidophilus 1:1] + [(3) montmorillonite + attapulgite 1:1].

[0217] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probiotic compound preparation, characterized in that, The probiotic compound preparation is prepared by directly mixing probiotic compound colony dry powder with inorganic materials, wherein the inorganic materials are inorganic mineral materials that have undergone dispersion treatment; The dispersion treatment method for the inorganic mineral material is as follows: a dispersant is added to the inorganic mineral material; The dispersant is a mixture of sodium pyrophosphate and polyethylene glycol; The inorganic mineral material is montmorillonite or a mixture of montmorillonite and attapulgite. The probiotics are Bifidobacterium and Lactobacillus acidophilus.

2. A method for preparing a probiotic compound preparation, characterized in that, The probiotic compound preparation is the probiotic compound preparation according to claim 1. Its preparation method is to culture the probiotic compound colonies, freeze-dry them, and then mix them with inorganic materials that have been sterilized and dispersed.

3. The method for preparing a probiotic compound preparation according to claim 1, characterized in that, The culture time for each strain in the probiotic complex colony is 2-8 hours.

4. The method for preparing a probiotic compound preparation according to claim 1, characterized in that, The method for dispersing the inorganic material is as follows: the inorganic material is mixed with water, magnetically stirred and then ultrasonically treated, then a dispersant is added and heated in a water bath, and finally washed, dried, pulverized and sieved.

5. The method for preparing a probiotic compound preparation according to claim 3 or 4, characterized in that, The method for mixing the probiotic compound colony freeze-dried agent with the dispersed inorganic material is as follows: the probiotic compound colony freeze-dried agent is mixed at 10... 7 -10 14 The proportion of CFU / g dispersed inorganic material is added to the dispersed inorganic material for mixing.

6. The application of the probiotic compound preparation according to claim 1 in the preparation of drugs for treating malignant tumors, characterized in that, The malignant tumors mentioned are malignant melanoma, breast cancer, colorectal cancer, osteosarcoma, liver cancer, lung cancer, and cervical cancer.

7. The application according to claim 6, characterized in that, The drugs for treating malignant tumors also include therapeutic agents that can be used in combination.

8. The application according to claim 7, characterized in that, The therapeutic agents that can be used in combination are doxorubicin or PD-1.

9. The preparation method according to any one of claims 2-5 is used in the preparation of drugs for inhibiting the growth of malignant tumors, characterized in that, The malignant tumors mentioned are malignant melanoma, breast cancer, colorectal cancer, osteosarcoma, liver cancer, lung cancer, and cervical cancer.

10. A medicament for treating malignant tumors, said medicament comprising the probiotic compound preparation of claim 1, characterized in that, The malignant tumors mentioned are malignant melanoma, breast cancer, colorectal cancer, osteosarcoma, liver cancer, lung cancer, and cervical cancer.

11. The medicament according to claim 10, characterized in that, The drug also includes chemotherapy drugs and / or immunotherapy drugs used to treat malignant tumors.

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