Method for increasing content of metal ions in probiotic spores and application

Abstract

The invention provides a method for increasing the content of metal ions in probiotic spores and application, and belongs to the field of microorganisms, the method comprises the following steps: inoculating spore-producing probiotics into a spore-producing culture medium for spore-producing culture to obtain a mixed bacteria solution; killing spore-producing probiotics in the mixed bacterial liquid, centrifuging, and collecting spore precipitate; the spore precipitate is washed, and probiotic spores are obtained; the spore production culture medium contains metal ions, and the metal ions comprise but are not limited to at least one of K < + >, Ca < 2 + >, Mg < 2 + >, Cu < 2 + >, Zn < 2 + >, Se < 4 + > and Mn < 2 + >; the concentration of the metal ions is 0.01 to 1 mg / mL; the spore-producing probiotics comprise but are not limited to any one of clostridium butyricum, bacillus coagulans, bacillus licheniformis and bacillus subtilis. The probiotic spores prepared by the method have high metal ion content, can be used for improving or treating colitis or colorectal cancer, and provide a new choice for treatment of colitis and colorectal cancer.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a method for increasing the content of metal ions in probiotic spores and an application thereof. Background Art

[0002] Colitis refers to inflammatory lesions of the colon caused by various reasons. It can be caused by bacteria, fungi, viruses, parasites, protozoa and other organisms, as well as by allergic reactions and physical and chemical factors. According to the different causes, it can be divided into specific inflammatory lesions and nonspecific inflammatory lesions. The former refers to infectious colitis, ischemic colitis and pseudomembranous colitis, etc., and the latter includes ulcerative colitis and colon Crohn's disease. The main clinical manifestations are diarrhea, abdominal pain, mucus stool and bloody stool, tenesmus, even constipation, and inability to defecate for several days; often accompanied by symptoms such as weight loss and fatigue, and often recurrent attacks. At present, the incidence of colitis in my country is gradually increasing, the course of the disease is long, and there is a risk of inducing colorectal cancer, so it has received more and more attention. Summary of the invention

[0003] In order to obtain a new treatment approach for colitis, the present invention provides a method for increasing the metal ion content in probiotic spores. The probiotic spores prepared by this method have a higher metal ion content and can be used to improve or treat colitis or colorectal cancer, providing a new option for the treatment of the disease.

[0004] The invention also provides probiotic spores with high metal ion content and application thereof.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for increasing the content of metal ions in probiotic spores, the method comprising:

[0007] Inoculating spore-forming probiotics into a spore-forming medium for spore-forming culture to obtain a mixed bacterial solution;

[0008] Killing the spore-forming probiotics in the mixed bacterial solution, then centrifuging to collect the spore precipitate;

[0009] washing the spore precipitate to obtain probiotic spores;

[0010] The spore-forming medium contains metal ions, including but not limited to K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ 、Se 4+ and Mn 2+ At least one of;

[0011] The metal ion concentration is 0.01 to 1 mg / mL;

[0012] The spore-forming probiotics include, but are not limited to, any one of Clostridium butyricum (CB), Bacillus coagulans (BC), Bacillus licheniformis (BL) and Bacillus subtilis (BS).

[0013] Furthermore, the spore-forming probiotics are inoculated into a spore-forming medium for spore-forming culture to obtain a mixed bacterial solution, which specifically includes:

[0014] The spore-forming probiotic culture solution is inoculated into the spore-forming medium for spore-forming culture, and the spore-forming culture time is 48±2h to obtain a mixed bacterial solution;

[0015] The concentration of the spore-forming probiotic culture solution is 7.5×10 7 CFU / mL~1.5×10 8 CFU / mL, the volume ratio of the spore-forming probiotic culture solution to the spore-forming medium is 4:100;

[0016] The killing of the spore-forming probiotics in the mixed bacterial solution, followed by centrifugation, and collecting the spore precipitate specifically includes:

[0017] The mixed bacterial solution was heated to 70-80° C. and kept warm for 20-30 min to remove spore-forming probiotics. After cooling to room temperature, the mixed bacterial solution was centrifuged at 12000±200 rpm for 10±2 min to collect spore precipitates.

[0018] Furthermore, the spore precipitate is washed to obtain probiotic spores, which specifically includes:

[0019] The spore precipitate is washed with sodium chloride solution and ultrapure water in sequence to obtain probiotic spores;

[0020] Alternatively, the spore precipitate is washed with a sodium chloride solution and ultrapure water in sequence, and then freeze-dried to obtain a probiotic spore freeze-dried powder.

[0021] Furthermore, the spore-forming medium also includes but is not limited to at least one of peptone, beef extract, sodium chloride and brain heart extract.

[0022] Preferably, the spore-forming culture medium used by different spore-forming probiotics is as follows:

[0023] The sporulation medium used for Clostridium butyricum was: water, 38.5 mg / mL brain heart infusion powder, 0.5 mg / mL MnSO4;

[0024] The spore-forming medium used for Bacillus coagulans was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 0.5 mg / mL mg MnSO4;

[0025] The spore-forming medium used for Bacillus licheniformis was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 0.5 mg / mL MnSO4;

[0026] The spore-forming medium used for Bacillus subtilis: water, 5 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, and 0.15 mg / mL MnSO4.

[0027] Preferably, the spore-forming culture medium used by different spore-forming probiotics is as follows:

[0028] The sporulation medium used for Clostridium butyricum was: water, 38.5 mg / mL brain heart infusion powder, 1 mg / mL CaCl2;

[0029] The spore-forming medium used for Bacillus coagulans was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 1 mg / mL CaCl2;

[0030] The sporulation medium used for Bacillus licheniformis was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 1 mg / mL CaCl2;

[0031] The spore-forming medium used for Bacillus subtilis: water, 5 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, and 1 mg / mL CaCl2.

[0032] Based on the same inventive concept, the present invention provides probiotic spores with high metal ion content, and the probiotic spores are prepared by the above-mentioned method for increasing the metal ion content in probiotic spores.

[0033] Alternatively, based on the same inventive concept, the present invention provides probiotic spores with a high metal ion content, wherein the probiotic spores include but are not limited to at least one of Clostridium butyricum spores, Bacillus coagulans spores, Bacillus licheniformis spores and Bacillus subtilis spores, and the content of metal ions in the probiotic spores is 14800-51200 μg / g;

[0034] The metal ions include but are not limited to K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ 、Se 4+ and Mn 2+At least one of .

[0035] In the present invention, the content of metal ions in probiotic spores is 14800-51200 μg / g. As an essential component of various enzymes and metabolic reactions, metal ions can enhance cell activity and antioxidant capacity, improve the ecology of intestinal flora, support the growth and metabolism of probiotics, and help treat inflammatory diseases and tumors. Too low ion content can affect the metabolic activity of probiotics, reduce its therapeutic potential, and may weaken the inhibitory effect of intestinal flora on pathogens. When the content is too high and exceeds the tolerance threshold of cells, it can cause cytotoxicity or increased oxidative stress.

[0036] Based on the same inventive concept, the present invention provides the use of probiotic spores with high metal ion content in the preparation of drugs for preventing or treating colitis.

[0037] Based on the same inventive concept, the present invention also provides the use of probiotic spores with high metal ion content in the preparation of drugs for preventing or treating colorectal cancer.

[0038] Based on the same inventive concept, the present invention also provides the use of probiotic spores with high metal ion content in the preparation of any one of feed additives, veterinary drugs, fertilizer additives and pesticides.

[0039] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0040] 1. A method for increasing the content of metal ions in probiotic spores of the present invention, wherein a certain concentration of metal ions is added to the spore-forming medium of spore-forming probiotics, wherein the metal ions include but are not limited to K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2 + 、Se 4+ and Mn 2+ At least one of the above can obtain probiotic spores with high metal ion content under the premise of increasing spore yield and shortening fermentation time. The high level of calcium dipicolinate (DPA) in the spore core can react with metal ions such as Ca in the spore production medium. 2+ Complexation to form Ca 2+ -DPA complex is stored in the spores. When the probiotic spores absorb enough nutrients and water from the intestine, the germination process is triggered. During this process, the spore nucleus swells and absorbs water, the hydrophobic protein shell breaks down and falls off, and the nutrient cells are regenerated. At the same time, the metal cation-DPA is gradually released into the intestinal environment. Under the synergistic effect of metal cations and probiotics, colitis and colorectal cancer can be improved or treated, providing a new option for the treatment of colitis and colorectal cancer.

[0041] 2. The probiotic spores with high metal ion content of the present invention contain metal ions, including but not limited to K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ 、Se 4+ and Mn 2+ At least one of the above, and the metal ion content reaches 51200μg / g, which can be used to treat colitis or colorectal cancer. After oral administration into the intestine, the probiotic spores germinate, and during the germination process, the spore nucleus expands and absorbs water, the hydrophobic protein shell cleaves and falls off, and the nutrient cells are regenerated. At the same time, the metal cations are gradually released into the intestinal environment. In the presence of metal cations such as Ca 2+ , Mn 2+ Under the synergistic effect with probiotics, it can improve or treat colitis and colorectal cancer, and can be used to prepare drugs for treating colitis or colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0043] Figure 1 The content of manganese ions in Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis extracted from spore-forming culture media with different Mn ion concentrations in the present invention;

[0044] Figure 2 The calcium ion content in Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis extracted from spore-forming culture media with different Ca ion concentrations in the present invention;

[0045] Figure 3 This is a curve diagram of weight changes in mice with orthotopic colorectal cancer that were gavaged with high-Mn-content probiotic spores in Examples 1-3 of the present invention;

[0046] Figure 4 The CD8 in situ colon tumor cells were obtained after intragastric administration of high Mn content probiotic spores in Example 1-3 of the present invention. + T cell infiltration;

[0047] Figure 5 The results of H&E staining of in situ colon tumor sections after intragastric administration of high-Mn-content probiotic spores in Example 1-3 of the present invention;

[0048] Figure 6This is a graph showing the weight change of colitis mice that were gavaged with high-Ca-content probiotic spores in Examples 1-3 of the present invention;

[0049] Figure 7 This is a curve diagram showing changes in DAI scores of colitis mice gavaged with high-Ca-content probiotic spores in Examples 1-3 of the present invention;

[0050] Figure 8 These are the results of H&E staining of colon sections of colitis mice that were gavaged with high-Ca content spores in Examples 1-3 of the present invention. DETAILED DESCRIPTION

[0051] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0052] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0053] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] The technical principles of the present invention are as follows:

[0056] Metal ions are an important component of all life forms. Most enzymes require metal ions for their function and participate in almost all critical basic biological processes. More and more evidence shows that metal ions play a key role in regulating the host immune system against pathogen invasion. Therefore, "metal immunology" has emerged based on the study of the key role of metal ions and their compounds in immune cell differentiation, development and immune response.

[0057] Currently, the role of metal ion signals in the immune system has been widely studied. From the initial recognition of pathogen / damage-associated pattern molecules (PAMPs / DAMPs) to the final generation of protective immunity and immune memory, successful immune responses against infection or tumors are invariably operated by a series of transcellular and intracellular signaling cascades, in which different metal cations include but are not limited to K + , Ca 2+ Mg 2+ , Fe2+ 、Zn 2+ 、Se 4+ and Mn 2+ etc., and play an important role in transmitting various signals. As a metal "recycling station", microorganisms can effectively reduce the concentration and toxicity of heavy metal elements in the environment by chelating metal ions, or make them completely harmless. Compared with the commonly used physical and chemical remediation methods, the biggest advantage is that it is economical and efficient, and will not produce secondary pollution. It is a natural, environmentally friendly and efficient remediation method. With the intensification of environmental pollution, my country has achieved certain results in using microorganisms to treat heavy metal pollution. Bacillus is a type of microorganism widely found in ecological environments such as cultivated land, woodland, and grassland, and is an important agricultural growth-promoting bacteria. At present, Bacillus has "showed its prowess" in the treatment of heavy metal pollution. It can produce a large amount of extracellular secretions such as organic acids, proteins, polypeptides and other anionic groups and heavy metals through complexation, chelation, adsorption and other methods to form precipitates, thereby effectively reducing harmful heavy metal pollution in the environment. In addition, its dormant body, one of the most persistent static structures in biology, spores, has excellent ability to tolerate extremely harsh environments, so it has a wide range of applications in agriculture, environmental protection, food, medicine and other fields.

[0058] When Bacillus is under environmental stress such as nutrient deficiency, it will form spores with strong stress resistance after being highly concentrated and dehydrated. Studies have shown that the spore structure includes the outer wall, outer membrane, cortex, inner membrane and core, each of which plays a different role. The inventors found that the high level of calcium dipicolinate (DPA) in the spore core can react with Ca in the adversity. 2+ Complexation and formation of Ca 2+ -DPA complex and stored in the core, the complex can not only maintain the normal dormancy of spores, but also play a key role in the spores' tolerance to strong acid and alkali environments, toxic chemicals, ionizing radiation, proteases, lysozymes and surfactants. It was also found that DPA can be combined with Ca 2+ It can form 1:1 complexes with various divalent cations and even monovalent cations.

[0059] In order to explore the regulatory effect of different metal ions on the immune system in the environment of intestinal inflammation or tumors, in order to obtain a new treatment approach for intestinal inflammation or cancer, a certain concentration of metal cations is added during the probiotic fermentation spore production process, and a method for obtaining probiotic spores with high metal ion content is obtained. Experiments have found that by adding metal ions to the spore production medium, probiotic spores with high metal ion content can be obtained under the premise of increasing spore yield and shortening fermentation time, and when the spores absorb enough nutrients and water, the germination process will be triggered, during which the spore nucleus expands and absorbs water, the hydrophobic protein shell is cracked and falls off, and the nutrient cells are regenerated. At the same time, the metal cation-DPA is gradually released into the environment to regulate the intestinal immune system, and under the synergistic effect with the probiotics, the improvement and treatment of colon inflammation and cancer are achieved.

[0060] The following is a detailed description of a method for increasing the metal ion content in probiotic spores and its application in combination with examples and experimental data.

[0061] Example 1

[0062] This embodiment provides a method for increasing the content of Mn and Ca ions in probiotic spores.

[0063] 1. The method for increasing the content of Mn ions in probiotic spores is as follows:

[0064] 1) Probiotics in the logarithmic growth phase were inoculated into a spore-forming medium with MnSO4 concentrations of 0, 0.01, and 0.15 mg / mL at an inoculation ratio of 4%. After 48 hours of cultivation, the bacterial liquid was collected and heated at 80°C for 30 minutes to remove the remaining bacteria.

[0065] 2) After cooling to room temperature, centrifuge at 12000 rpm for 10 min to collect the spore precipitate, wash the obtained spore precipitate twice with sodium chloride solution to remove impurities, and then wash it twice with ultrapure water. After further removing impurities, disperse it in an appropriate amount of ultrapure water and freeze-dry it to obtain spore powder.

[0066] Among them, the probiotics in this embodiment are Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis. The spore-forming culture medium corresponding to different probiotics after removing MnSO4 is as follows:

[0067] The spore-forming medium used for Bacillus coagulans: 100 mL of distilled water, 1.0 g of peptone, 0.3 g of beef extract, and 0.5 g of sodium chloride;

[0068] The spore-forming medium used for Bacillus licheniformis: 100 mL of distilled water, 1.0 g of peptone, 0.3 g of beef extract, and 0.5 g of sodium chloride;

[0069] The spore-forming medium used for Bacillus subtilis: 100 mL of distilled water, 0.5 g of peptone, 0.3 g of beef extract, and 0.5 g of sodium chloride.

[0070] The overall ion content of the obtained spores was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES). Figure 1 . As the concentration of Mn ions increased, the Mn ion content of the obtained spores also increased. Compared with the control group spores, the Mn ion content in the coagulation and subtilis spores increased significantly, by about 796 times; the increase in Bacillus licheniformis was particularly significant, by about 2347 times. The Mn ion content of the spores reached 14800μg / g~34500μg / g

[0071] 2. The method for increasing the content of Ca ions in probiotic spores is as follows:

[0072] 1) Probiotics in the logarithmic growth phase were inoculated into a spore-forming medium with a CaCl2 concentration of 0 and 1 mg / mL at an inoculation ratio of 4%. After 48 hours of cultivation, the bacterial liquid was collected and heated at 80°C for 30 minutes to kill the remaining bacteria.

[0073] 2) After cooling to room temperature, centrifuge at 12000 rpm for 10 min to collect the spore precipitate, wash the obtained spore precipitate twice with sodium chloride solution to remove impurities, and then wash it twice with ultrapure water. After further removing impurities, disperse it in an appropriate amount of ultrapure water and freeze-dry it to obtain spore powder.

[0074] Among them, the probiotics are Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis. The spore-forming culture medium corresponding to different probiotics after removing CaCl2 is as follows:

[0075] The spore-forming medium used for Bacillus coagulans: 100 mL of distilled water, 1.0 g of peptone, 0.3 g of beef extract, and 0.5 g of sodium chloride;

[0076] The spore-forming medium used for Bacillus licheniformis: 100 mL of distilled water, 1.0 g of peptone, 0.3 g of beef extract, and 0.5 g of sodium chloride;

[0077] The spore-forming medium used for Bacillus subtilis: 100 mL of distilled water, 0.5 g of peptone, 0.3 g of beef extract, and 0.5 g of sodium chloride.

[0078] ICP-OES was used to measure the ion content of the obtained spores. The results were as follows Figure 2 As shown. With the increase of Ca ion concentration, the Ca ion content in the obtained spores also increased. Compared with the spores in the control group, the Ca ion content in the spores increased significantly, by about 7.5 times. The Ca ion content of the spores reached 16699μg / g~51230μg / g.

[0079] Example 2

[0080] This embodiment provides a use of probiotic spores with a high Mn content in the treatment of colorectal cancer, comprising the following steps:

[0081] In this example, 6-8 week old female Balb / c mice (Beijing Sibeifu Biological Co., Ltd.) were used to cut the subcutaneous tumor tissue into 2 mm 3 The tumor tissue blocks on the left and right sides were glued to the cecum of mice using biological tissue glue to construct an orthotopic colon cancer homologous transplantation model. The mice were randomly divided into 8 groups, namely, the healthy control group (Normal), colon cancer group (CRC), Bacillus coagulans spore group (BCs), Bacillus licheniformis spore group (BLs), Bacillus subtilis spore group (BSs), high manganese Bacillus coagulans spore group (BCs-Mn), high manganese Bacillus licheniformis spore group (BLs-Mn), and high manganese Bacillus subtilis spore group (BSs-Mn). Oral gavage of spores 1×10 9 CFU / day, and the therapeutic effect of the high Mn probiotic spores prepared in Example 1 was detected after three weeks of treatment. The weight of the mice was recorded every day, and the tumor tissues were collected after the treatment, and pathological analysis was performed by H&E staining. The effect of Mn on the infiltration of immune cells in tumor tissues was further analyzed by immunofluorescence staining.

[0082] The experimental results show that there is almost no difference in the weight of mice in each group. Probiotic spore treatment with high Mn content promotes the maturation of DCs and further promotes the infiltration of effector T cells in tumors. + T cell infiltration increased significantly ( Figure 4 ), H&E results also showed that the tumor tissues of mice gavaged with probiotic spores with high Mn content showed obvious necrosis ( Figure 5 ). This indicates that the effect of probiotic-based immunotherapy on in situ colorectal cancer can be enhanced under the action of Mn ions.

[0083] Example 3

[0084] This embodiment provides a use of probiotic spores with high calcium content in the treatment of colitis, comprising the following steps:

[0085] This experiment used 6-8 week old C57 male mice (Beijing Sibeifu Biological Co., Ltd.), and dextran sulfate sodium salt (DSS) was used to induce colitis model. The colitis model mice were randomly divided into 8 groups, namely healthy control group (Normal), colon cancer group (CRC), Bacillus coagulans spore group (BCs), Bacillus licheniformis spore group (BLs), Bacillus subtilis spore group (BSs), high calcium Bacillus coagulans spore group (BCs-Ca), high calcium Bacillus licheniformis spore group (BLs-Ca), high calcium Bacillus subtilis spore group (BSs-Ca). Oral gavage of spores 1×10 9 CFU / day, and the therapeutic effect of the high-Ca probiotic spores (1 mg / mL) prepared in Example 1 was tested after three weeks of treatment. The weight of the mice and the disease activity index (DAI) score were recorded every day. After the treatment, the colon tissues were collected and sliced ​​for H&E staining for further analysis.

[0086] According to the experimental results ( Figure 6 ) It can be seen that the weight of mice in the healthy control group increased slightly, and the DAI remained at a low level, indicating that the mice were in good health. The weight loss of mice treated with DSS reached more than 15%, and the DAI value was the highest. Treatment with high-Ca content spores was able to curb the weight loss of colitis mice, and even showed an upward trend on the 9th day of treatment, reducing the DAI value. It is worth mentioning that spores without added ions also have a certain therapeutic effect, but not as good as probiotic spores with high Ca content ( Figure 7 ). In addition, pathological analysis of colon tissue sections revealed Figure 8 As shown in the figure, the colon tissue of the healthy control group showed no signs of destruction or inflammation in terms of cell morphology and arrangement. After DSS induction, the colon tissue showed severe mucosal ulcers, crypt deformation and disappearance, goblet cell loss, and inflammatory cell infiltration, indicating epithelial damage and colonic inflammation. High Ca treatment significantly alleviated the inflammatory condition and almost preserved the complete colon microstructure morphology, indicating that the release of Ca ions, in conjunction with probiotic spores, can significantly improve the therapeutic effect of colitis.

[0087] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0088] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for increasing the metal ion content in probiotic spores, characterized in that: The method comprises: Inoculating spore-forming probiotics into a spore-forming medium for spore-forming culture to obtain a mixed bacterial solution; Killing the spore-forming probiotics in the mixed bacterial solution, then centrifuging to collect the spore precipitate; washing the spore precipitate to obtain probiotic spores; The spore-forming medium contains metal ions, including but not limited to K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ 、Se 4+ and Mn 2+ At least one of; The metal ion concentration is 0.01 to 1 mg / mL; The spore-forming probiotics include, but are not limited to, any one of Clostridium butyricum, Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis.

2. A method for increasing the metal ion content in probiotic spores according to claim 1, characterized in that: The step of inoculating the spore-forming probiotics into a spore-forming culture medium for spore-forming culture to obtain a mixed bacterial solution specifically comprises: The spore-forming probiotic culture solution is inoculated into the spore-forming medium for spore-forming culture, and the spore-forming culture time is 48±2h to obtain a mixed bacterial solution; The concentration of the spore-forming probiotic culture solution is 7.5×10 7 CFU / mL~1.5×10 8 CFU / mL, the volume ratio of the spore-forming probiotic culture solution to the spore-forming medium is 4:100; The killing of the spore-forming probiotics in the mixed bacterial solution, followed by centrifugation, and collecting the spore precipitate specifically includes: The mixed bacterial solution was heated to 70-80° C. and kept warm for 20-30 min to remove spore-forming probiotics. After cooling to room temperature, the mixed bacterial solution was centrifuged at 12000±200 rpm for 10±2 min to collect spore precipitates.

3. A method for increasing the metal ion content in probiotic spores according to claim 1, characterized in that: The step of washing the spore precipitate to obtain probiotic spores specifically comprises: The spore precipitate is washed with sodium chloride solution and ultrapure water in sequence to obtain probiotic spores; Alternatively, the spore precipitate is washed with a sodium chloride solution and ultrapure water in sequence, and then freeze-dried to obtain a probiotic spore freeze-dried powder.

4. A method for increasing the metal ion content in probiotic spores according to claim 1, characterized in that: The spore-forming medium further comprises but is not limited to at least one of peptone, beef extract, sodium chloride and brain heart infusion.

5. A method for increasing the metal ion content in probiotic spores according to claim 1, characterized in that: The spore-forming culture media used for different spore-forming probiotics are as follows: The sporulation medium used for Clostridium butyricum was: water, 38.5 mg / mL brain heart infusion powder, 0.5 mg / mL MnSO4; The spore-forming medium used for Bacillus coagulans was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 0.5 mg / mL MnSO4; The spore-forming medium used for Bacillus licheniformis was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 0.5 mg / mL MnSO4; The spore-forming medium used for Bacillus subtilis: water, 5 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, and 0.15 mg / mL MnSO4.

6. A method for increasing the metal ion content in probiotic spores according to claim 1, characterized in that: The spore-forming culture media used for different spore-forming probiotics are as follows: The sporulation medium used for Clostridium butyricum was: water, 38.5 mg / mL brain heart infusion powder, 1 mg / mL CaCl2; The spore-forming medium used for Bacillus coagulans was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 1 mg / mL CaCl2; The sporulation medium used for Bacillus licheniformis was: water, 10 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, 1 mg / mL CaCl2; The spore-forming medium used for Bacillus subtilis: water, 5 mg / mL peptone, 3 mg / mL beef extract, 5 mg / mL sodium chloride, and 1 mg / mL CaCl2.

7. Probiotic spores with high metal ion content, characterized in that: The probiotic spores are prepared by a method for increasing the content of metal ions in probiotic spores according to any one of claims 1 to 6.

8. Probiotic spores with high metal ion content, characterized in that: The probiotic spores include but are not limited to at least one of Clostridium butyricum spores, Bacillus coagulans spores, Bacillus licheniformis spores and Bacillus subtilis spores, and the content of metal ions in the probiotic spores is 14800-51200 μg / g; The metal ions include but are not limited to K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ 、Se 4+ and Mn 2+ At least one of .

9. Use of the probiotic spores with high metal ion content as claimed in claim 7 or 8 in the preparation of a drug for preventing or treating colitis.

10. Use of the probiotic spores with high metal ion content as claimed in claim 7 or 8 in the preparation of a drug for preventing or treating colorectal cancer.

11. Use of the probiotic spores with high metal ion content as claimed in claim 7 or 8 in the preparation of any one of feed additives, veterinary drugs, fertilizer additives and pesticides.

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