Application of probiotic bacillus cereus living bacterium agent, extracellular metabolite and protein complexing agent thereof in prevention and treatment of malignant tumors

Abstract

The invention discloses a group of probiotic bacillus cereus living bacteria agents. The number of living bacteria in the bacteria agents is not less than 2 * 10 < 9 > cfu / mL. The probiotic bacillus cereus comprises one or more strains with the number of CGMCC (China General Microbiological Culture Collection Center) No.04060.4, CMCC (China General Microbiological Culture Collection Center) 63305 and DM423. The invention further provides a preparation method of the extracellular metabolite of the probiotic bacillus cereus and the extracellular protein complexing agent of the probiotic bacillus cereus. The anti-tumor activity of the compound is further explored. An intragastric tumor-bearing mouse experiment verifies that the probiotic bacillus cereus living bacterium agent, the extracellular metabolite, the extracellular protein complexing agent and other products can inhibit proliferation of subcutaneous tumor cells of mice and prolong the service life of the tumor-bearing mice, have good anti-tumor activity and can be used for preventing or treating malignant tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a group of probiotic Bacillus cereus ( Bacillus cereus ) Application of live bacterial agents, extracellular metabolites and their protein complexes in the prevention and treatment of malignant tumors. Background Art

[0002] Malignant tumors pose a great threat to human health. They are a disease of abnormal cell proliferation that can invade surrounding tissues and organs, causing damage to body functions and even death. With the advancement of medical technology, the treatment status of malignant tumors has improved, but it still faces many challenges. At present, the treatment methods of malignant tumors mainly include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy, which have improved the overall survival rate and quality of life of patients to a certain extent. However, there are still some problems in the treatment process, such as high treatment costs, uneven distribution of medical resources, limited capacity of primary medical institutions, insufficient patient awareness, and side effects and complications of treatment. In addition, the recurrence and metastasis of malignant tumors are also difficult problems in treatment, especially for advanced malignant tumors, whose cure rate is still low.

[0003] Given the serious threat of malignant tumors to human health and the limitations of existing treatments, it is particularly important to develop new anti-tumor drugs that are safe and have no toxic side effects. Such drugs can not only more effectively control the growth and spread of tumors, but also relieve patients' pain and improve their quality of life. At the same time, the development of new anti-tumor drugs can also help solve the problem of drug resistance and provide new hope for patients who are ineffective with traditional treatments.

[0004] Bacillus cereus is a Gram-positive bacillus widely distributed in the air, soil, water and plants. Bacillus cereus has stress-resistant endogenous spores and is highly resistant to heat, ultraviolet rays, ionizing radiation and antibiotics. Most Bacillus cereus are considered food-borne spoilage bacteria. However, Bacillus cereus CGMCC No.04060.4, CMCC63305, DM423, etc. are used as probiotic Bacillus cereus because of their adaptability to the gastrointestinal environment and their inhibitory effects on pathogens. Probiotic Bacillus cereus can inhibit the growth of harmful bacteria in the intestine and reduce the risk of intestinal infection. It can also promote the repair of intestinal mucosa and relieve intestinal inflammation, so it has a good effect on the treatment of acute and chronic enteritis, diarrhea and other diseases. Probiotic Bacillus cereus can also promote the absorption and utilization of nutrients, increase the number of beneficial bacteria in the intestine, improve the structure of intestinal flora, and improve the overall health of the human body. In addition, the probiotic Bacillus cereus can also activate the immune system of animals (including humans), improve the body's immunity, and thus indirectly enhance the body's disease resistance.

[0005] At present, there are many probiotic products containing Bacillus cereus on the market, such as Siliankang Bifidobacterium tetravalent live bacteria tablets, Zhengchangsheng Licheniformis live bacteria capsules, Yunnan Baiyao probiotic powder, etc. These products are mainly concentrated in the treatment of diarrhea, constipation, functional dyspepsia and other aspects related to intestinal flora imbalance. In recent years, more and more studies have begun to focus on the application of microorganisms in cancer treatment. However, whether probiotic Bacillus cereus has anti-tumor effects, whether probiotic Bacillus cereus and its extracellular products can directly kill tumor cells, and then be used in the prevention and treatment of malignant tumors, there is no relevant report, and there is no related product or preparation. Based on this, this application has been developed. Summary of the invention

[0006] The present invention aims to provide a group of probiotic Bacillus cereus ( Bacillus cereus ) Application of live bacterial agents, extracellular metabolites and their protein complexes in the prevention and treatment of malignant tumors.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme: A probiotic Bacillus cereus live bacterial agent, wherein the total number of live bacteria in the agent is not less than 2×10 9 cfu / mL. The probiotic Bacillus cereus has tumor growth inhibition activity and can induce tumor cell apoptosis. The live bacterial agent can inhibit the proliferation of tumor cells and prolong the life of tumor-bearing mice.

[0008] The above-mentioned probiotic Bacillus cereus live bacteria agent, wherein the probiotic Bacillus cereus comprises one or more of Bacillus cereus with the preservation numbers of CGMCC No.04060.4, CMCC 63305, DM423, etc.

[0009] Furthermore, the present invention provides the above-mentioned probiotic Bacillus cereus live bacteria agent, which is prepared by the following specific steps: (1) Streak the frozen strain on TSB solid medium, pick a single colony and transfer it to TSB liquid medium for further activation culture; (2) Inoculate the rejuvenated cells into TSB liquid culture medium at a rate of 5-10%, culture in a shake flask at 37±2℃ for 20-24h until the stable phase, centrifuge and collect fresh cells; (3) Wash the fresh bacteria with sterile phosphate buffered saline (PBS, pH 7.0-7.2), and then fully suspend the bacteria with an appropriate amount of PBS to obtain a bacterial suspension. The density of the bacteria in the bacterial suspension should be no less than 2×10 9 cfu / mL, that’s it.

[0010] Specifically, in step 1), the streak culture temperature can be 37±2°C and the time can be 12-24 h; the activation culture is carried out until OD 600 The value is 0.6-0.8.

[0011] Furthermore, the present invention also provides a probiotic Bacillus cereus extracellular metabolite, which is prepared by the following steps: (1) Streak the frozen strain on TSB solid medium at 37±2℃ for 12-24h; pick a single colony and transfer it to TSB liquid medium to continue activation culture until OD 600 The value is 0.6-0.8; (2) Inoculate the rejuvenated cells into TSB liquid medium at a rate of 5-10%, culture in a shake flask at 37±2℃ for 16-18h until the end of the logarithmic phase, centrifuge, collect the fresh cells, wash them three times with sterile PBS, and then transfer the cells to a volume of TSB liquid medium equal to that before centrifugation for shaking culture; (3) Take the culture medium obtained above, centrifuge it, collect the supernatant, and filter it with a 0.22 μm sterile filter membrane to obtain the product.

[0012] Specifically, in step 2), the shaking culture temperature may be 37±2°C and the time may be 4-6 h.

[0013] Furthermore, the present invention also provides a probiotic Bacillus cereus extracellular protein complex, which is prepared by the following steps: (1) Streak the frozen strain on TSB solid medium, pick a single colony and transfer it to TSB liquid medium for further activation culture; inoculate the rejuvenated bacteria into TSB liquid medium at an inoculum rate of 3-5%, and culture in a shake flask at 37±2℃ for 16-18h until the mid-logarithmic phase to obtain the fermentation seed liquid; (2) Add the sterilized TSB liquid medium to the fermentation seed liquid at an inoculum rate of 5-10%, culture at 37±2℃ with shaking for 4-6 h, centrifuge, and collect the supernatant; (3) adding an equal volume of a low-concentration ammonium sulfate solution to grade the protein, allowing the mixture to stand, centrifuging and collecting the supernatant, further adding an equal volume of a high-concentration ammonium sulfate solution to grade the protein, allowing the mixture to stand, centrifuging and collecting the precipitate, purifying the collected precipitated protein by using a dextran gel column filtration chromatography, screening a protein component with anti-tumor activity from the multiple separated protein components by using a CCK8 experiment, collecting the active protein component, and further purifying and concentrating it through an ultrafiltration tube to obtain a protein concentrate; (4) Preparation of protein complex solution: Take the protein concentrate prepared above, add soy peptone and sodium chloride, mix well, and freeze-dry to obtain the product.

[0014] Specifically, in the preparation process of the above-mentioned probiotic Bacillus cereus extracellular protein complex, in step 3), the concentration of the low-concentration ammonium sulfate solution can be 38-42%, and the concentration of the high-concentration ammonium sulfate solution can be 75-85%; the molecular weight cutoff of the ultrafiltration tube can be 50-100 KD; in step 4), soy peptone and sodium chloride are added to final concentrations of 0.04-0.06% and 0.04-0.06%, respectively.

[0015] The present invention also provides the use of the above-mentioned probiotic Bacillus cereus live bacteria agent, extracellular metabolites and protein complex agent in the prevention and treatment of tumors. The tumors can be various malignant tumors.

[0016] Furthermore, in the above application, the malignant tumor includes but is not limited to one or more of esophageal cancer, intestinal cancer, nasopharyngeal cancer, gastric cancer, liver cancer, colorectal cancer, non-small cell lung cancer, breast cancer, melanoma, head and neck squamous cell carcinoma, lymphoma, etc.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses the anti-tumor activity of a probiotic Bacillus cereus live bacteria agent and its extracellular metabolites, extracellular protein complexes, bacterial cultures, inactivated bacterial products, lysates, or mixtures thereof, and also provides a preparation method of the probiotic Bacillus cereus extracellular metabolites and its extracellular protein complexes. Experiments on intragastric administration of tumor-bearing mice have verified that the probiotic Bacillus cereus live bacteria agent and its extracellular metabolites, extracellular protein complexes and other products of the present invention can inhibit the proliferation of subcutaneous tumor cells in mice, prolong the lifespan of tumor-bearing mice, and have good anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The inhibitory effects of probiotic Bacillus cereus CGMCC No.04060.4, CMCC 63305, and DM423 on esophageal cancer cells ECA109 (human) and AKR (mouse), brain glioma cells U87, and gastric cancer cells HGC-27; *, P<0.1, **, P<0.01, ***, P<0.001; Figure 2 To detect the effect of extracellular metabolites of probiotic Bacillus cereus CGMCC04060.4 on inducing apoptosis of esophageal cancer cells ECA109 and gastric cancer cells HGC-27 by flow cytometry; Figure 3 To detect the effect of extracellular metabolites of probiotic Bacillus cereus CMCC63305 on inducing apoptosis of esophageal cancer cell ECA109 and gastric cancer cell HGC-27 by flow cytometry; Figure 4To detect the effect of extracellular metabolites of probiotic Bacillus cereus DM423 on apoptosis of esophageal cancer cells ECA109 and gastric cancer cells HGC-27 by flow cytometry; Figure 5 (a) The size of subcutaneous tumor in mice injected with intratumoral injection (n=4); (b) The growth curve of subcutaneous tumor in mice (n=4); (c) The weight of subcutaneous tumor in mice; **, P<0.01, ***, P<0.001; Figure 6 (a) The size of subcutaneous tumor in mice in the oral gavage group (n=4); (b) The growth curve of subcutaneous tumor in mice (n=4); (c) The weight of subcutaneous tumor in mice; ns, P>0.05, **, P<0.01; Figure 7 Immunohistochemical staining was used to detect Ki-67 expression, (a) intratumoral injection group; (b) oral gavage group Scalebar: 50 μm. ns, P>0.05, ***, P<0.001; Figure 8 The effect of the probiotic Bacillus cereus CGMCC 04060.4 extracellular protein complex on the viability of esophageal cancer cell ECA109 and gastric cancer cell HGC-27 was detected by CCK8; Figure 9 The EdU experiment was used to detect the effect of the extracellular protein complex of probiotic Bacillus cereus CGMCC 04060.4 on the proliferation ability of esophageal cancer cell ECA109 and gastric cancer cell HGC-27; Scale bar: 100 μm, **, P<0.01, ***, P<0.001; Figure 10 Effects of intratumoral injection of probiotic Bacillus cereus CGMCC 04060.4 extracellular protein complex (BcE-pro) and probiotic Bacillus cereus extracellular metabolites (BcE-mix) on the growth of subcutaneous tumors of esophageal cancer; (a) subcutaneous tumor size of mice (n=4); (b) subcutaneous tumor growth curve of mice; (c) subcutaneous tumor weight of mice (n=4) ***, P<0.001; Figure 11 After intratumoral injection of probiotic Bacillus cereus CGMCC 04060.4 extracellular protein complex (BcE-pro) and extracellular metabolites (BcE-mix), immunohistochemical staining was used to detect the expression of proliferation-related molecule Ki-67; Scalebar: 50 μm. **, P<0.01; ***, P<0.001; Figure 12Biosafety assessment of mice after intratumoral injection of BcE-pro; (a) Weight changes of mice after intratumoral injection of BcE-pro (n=4); (b) HE images of liver, spleen and kidney; ns, P>0.05, Scale bar: 100 μm.

[0019] Specific implementation form The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0020] Strain Description Bacteria name: Bacillus cereus; Latin name: Bacillus cereus ; Strain numbers: CGMCC No.04060.4, CMCC 63305 and DM423.

[0021] Mechanism of action studies Using molecular biology, cell biology and other technical means, the anti-tumor mechanism of Bacillus cereus strains was studied. Through experiments, it was found that the strain can secrete a substance with anti-tumor activity, which can inhibit the proliferation and migration of tumor cells and induce tumor cell apoptosis.

[0022] In the following tumor experiments of the present invention, operations or steps not described in detail may be carried out using conventional techniques in the art, and are not the innovation of the present application and will not be described in detail.

[0023] Application in the prevention and treatment of tumors The Bacillus cereus strain was made into a pharmaceutical preparation and given to animals by oral administration or injection. The anti-tumor effect of the strain was observed through animal experiments and clinical trials. The experimental results showed that the strain could significantly reduce the incidence of tumors and prolong the survival of mice.

[0024] 1. Experimental Animals of the Present Invention Experimental animals: 5-week-old male BALB / c-nu mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010. The breeding environment is located in the animal breeding room of the School of Pharmacy of Henan University, with good ventilation. Male mice were raised in cages with 6 per cage, free access to food and water, and the temperature was maintained at 20-24°C and the relative humidity was 40%-70%.

[0025] 2. Experimental Materials 2.1 Experimental Reagents 2.1.1 Reagent preparation TSB liquid culture medium (taking the preparation of 1L as an example): 17g of tryptic peptone, 3g of soy peptone, 5g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose, make up to 1L with distilled water, adjust the pH value to about 7.3, then sterilize with high-pressure steam (121℃, 20 min), and use it for subsequent microbial culture after cooling.

[0026] TSB solid culture medium (taking the preparation of 1L as an example): 17g of tryptic peptone, 3g of soy peptone, 5g of sodium chloride, 2.5g of potassium dihydrogen phosphate, 2.5g of glucose, and 2% agar powder. Make up to 1L with distilled water, adjust the pH value to about 7.3, and then sterilize with high-pressure steam (121℃, 20 min). Cool to 50-60℃, pour into a culture dish in a sterile environment, cool and solidify, and prepare TSB solid plate, which can be used for subsequent streak culture of strains.

[0027] Preparation of tumor cell culture medium: commercially available DMEM medium, add 10% volume of fetal bovine serum.

[0028] 2.1.2 Other reagents CCK8 detection solution (Beijing Solebow Technology Co., Ltd.), EdU detection kit (Shanghai Biotech Co., Ltd.), immunohistochemistry kit (Shanghai Bioengineering Co., Ltd.), apoptosis kit (Shanghai Biotech Co., Ltd.), Ki-67 antibody (Proteintech (18704-1-AP 1:200), PBS, TBST.

[0029] 2.2. Instruments and consumables Flow cytometer (CytoFLEX LX), centrifuge (MiniSpeed ​​3300), microplate reader (Bio-Rad), fluorescence inverted microscope (Nikon), freezer (CDLJ-10W), shaker incubator (BSD-WX3350), clean bench workroom, pipette gun, 96-well culture plate, and 6-well culture plate.

[0030] 2.3 Cell lines Human esophageal cancer cells (ECA109), mouse esophageal cancer cells (AKR), liver cancer cells (97H), gastric cancer cells (HGC-27), colon cancer cells (SW480), lung cancer cells (A549), and brain glioma cells (U87).

[0031] 3. Statistical analysis All experimental results were repeated more than three times. The statistical graphs in this paper were completed using GraphPad Prism 8 software, and correlation analysis was performed using SPSS software for data statistical analysis. P<0.05 was statistically significant; ns, P >0.05; *, P <0.05; **, P <0.01; ***, P <0.001.

[0032] Example 1. Extracellular metabolites of probiotic Bacillus cereus have the activity of inhibiting tumor cell proliferation 1.1 The preparation method of the extracellular metabolites of probiotic Bacillus cereus is as follows: Probiotic Bacillus cereus CGMCC No.04060.4, CMCC 63305 and DM423 were streaked onto TSB solid plates for culture (37°C, 18 h), and single colonies were picked and transferred to TSB liquid medium for continued activation culture until OD 600 The value is about 0.8; the rejuvenated bacteria are inoculated into 100 mL TSB liquid culture medium according to an inoculum size of 10%, and the culture is shaken at 37°C for 16-18 hours to the end of the logarithmic phase, and the fresh bacteria are collected by centrifugation at 4°C and 12000rpm. The bacteria are washed 3 times with sterile PBS, and then the bacteria are transferred to 100 mL TSB liquid culture medium, and placed at 37°C and 200 rpm for shaking culture for 4 hours; the culture solution of the above shaking culture for 4 hours is taken, centrifuged at 4°C and 12000rpm, and the supernatant is collected. After filtering with a 0.22 μm sterile filter membrane, the supernatant is obtained, which is the extracellular metabolite of the probiotic Bacillus cereus, and stored at -20°C for standby use.

[0033] The experimental method is as follows: After digestion of tumor cells in good condition, they were plated in 96-well plates at a density of 9,000 cells per well. After being cultured in a CO2 incubator at 37°C for 24 h, they were divided into two groups. The experimental group was replaced with a cell culture medium containing 10% volume of probiotic Bacillus cereus extracellular metabolites, and the normal group continued to culture with normal cell culture medium. At the same time, a group of wells without cells and only with cell culture medium were set up as a blank control group. Then they were cultured in the above incubator for 24 h, 10 μL of CCK8 detection solution was added to each well, and then incubated in the incubator for 2 h, and placed in an ELISA reader to measure the OD 450 nm The values ​​were taken and the cell growth viability was calculated (cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of normal group - OD value of blank group) × 100%).

[0034] The results of CCK8 experiment showed that probiotics B. cereus CGMCC 04060.4, B. cereus CMCC63305 and B.cereus The extracellular metabolites of DM423 can inhibit the proliferation of esophageal cancer, gastric cancer and brain glioma cells. Figure 1 Compared with the blank control group NC, B. cereus The inhibition rates of CGMCC 04060.4 on the activity of esophageal cancer cells ECA109 (human) and AKR (mouse), brain glioma cells U87, and gastric cancer cells HGC-27 were 23.25%, 35%, 70%, and 48.1%, respectively; B. cereus The inhibition rates of CMCC63305 on the cell activity of esophageal cancer cells ECA109 (human) and AKR (mouse), brain glioma cells U87, and gastric cancer cells HGC-27 were 31.2%, 41.3%, 45.6%, and 55.8%, respectively; B. cereus The inhibition rates of DM423 on the activity of esophageal cancer cells ECA109 (human) and AKR (mouse), brain glioma cells U87, and gastric cancer cells HGC-27 were 42.5%, 52.1%, 75.6%, and 56.8%, respectively.

[0035] Example 2. Extracellular metabolites of probiotic Bacillus cereus induce apoptosis in esophageal and gastric cancer cells 2.1 The experimental method for detecting cell apoptosis level by flow cytometry is as follows: Tumor cells with good growth status were taken, digested, centrifuged, counted, and inoculated into 6-well plates; after the cells in the wells were completely attached to the wall, the old culture medium was discarded and the cells were treated according to the following groups: A (NC, blank control group): blank culture medium was used as negative control; B (blank control + metabolic supernatant): 10% (volume) of probiotic Bacillus cereus extracellular metabolites were added; then the cells in the above groups were placed in a 37°C incubator for further culture for 24 h; the cells were washed twice with PBS, digested with EDTA-free trypsin, centrifuged at 1,000 rpm / min for 5 min, and the resulting precipitate was washed twice with PBS. Then 200 μL BindingBuffer was added to resuspend the cell precipitate; 5 μL Annexin V-FITC staining solution was added and gently mixed, and incubated at 4°C in the dark for 15 min; 5 μL PI staining solution was added and gently mixed, and ice bathed in the dark for 5 min; and the flow cytometer was used for detection immediately.

[0036] The results showed that probiotic Bacillus cereus B. cereus The metabolic supernatant of CGMCC04060.4 can promote apoptosis of esophageal cancer cells ECA109 and gastric cancer cells HGC-27 (e.g. Figure 2 as shown).

[0037] The results showed that probiotics B. cereusThe extracellular metabolites of CMCC63305 can also promote apoptosis of esophageal cancer cell ECA109 and gastric cancer cell HGC-27 (e.g. Figure 3 as shown).

[0038] The results showed that probiotics B. cereus The extracellular metabolites of DM423 can also promote apoptosis of esophageal cancer cell ECA109 and gastric cancer cell HGC-27 (e.g. Figure 4 as shown).

[0039] Example 3. Probiotic Bacillus cereus B. cereus CGMCC 04060.4 inhibits the proliferation of subcutaneous tumors of esophageal cancer 3.1 The preparation method of the probiotic Bacillus cereus live bacteria agent is as follows: Bacillus cereus B. cereus CGMCC 04060.4 was streaked onto TSB solid medium and cultured (37°C, 18 h). A single colony was picked and transferred to TSB liquid medium for further activation culture until OD 600 The value was 0.6-0.8; the rejuvenated bacteria were inoculated into TSB liquid culture medium at a 5% inoculation rate, and cultured in a 37°C shake flask for 20-24 hours until the stable period, and centrifuged at 4°C and 12000 rpm to collect fresh bacteria; the fresh bacteria were washed 3 times with sterile PBS, and then fully suspended with an appropriate amount of PBS to obtain a bacterial suspension, so that the final bacterial density in the bacterial suspension was 2×10 9 cfu / mL, which is the prepared probiotic Bacillus cereus live bacterial agent. Refrigerate at 4°C for subsequent anti-tumor activity determination.

[0040] The method for constructing a mouse esophageal cancer subcutaneous tumor model is as follows: Collect AKR cells with good growth status, wash the cells twice with sterile PBS, and then dilute the cells with sterile PBS to adjust the cell concentration to 2.3 × 10 7 cells / mL, and placed on ice for later use. AKR was inoculated into the middle and posterior part of the axilla at 100 µL / mouse; mice in the oral administration group were inoculated with AKR cells and given oral administration every day. B. cereus CGMCC 04060.4 (abbreviation: B. cereus ) live bacterial agent or its extracellular metabolite (abbreviated as: S-B. cereus ) 100 μL, and the same amount of PBS or TSB was used as control; 7 days after the mice were inoculated with AKR cells, they were injected intratumorally every day B. cereus CGMCC04060.4 (abbreviation: B. cereus ) live bacterial agent or its extracellular metabolite (abbreviated as: S-B. cereus) 50 μL, and an equal amount of PBS or TSB was used as a control. During the experiment, the volume change of subcutaneous tumor tissue was detected every day, and the subcutaneous tumor growth curve was prepared. Mice in the intratumoral injection group were dissected after 19 days, and mice in the gavage group were dissected after 21 days. Tumor tissues were taken for photography, weighing, and tumor-related index analysis. B. cereus Antitumor effect of CGMCC 04060.4 live bacterial agent or its extracellular metabolites.

[0041] The results showed that intratumoral injection and oral gavage B. cereus CGMCC 04060.4 (abbreviation: B. cereus ) live bacteria can inhibit the growth of esophageal cancer subcutaneous tumors; intratumoral injection B. cereus CGMCC 04060.4 Extracellular metabolites (abbreviated as: S-B. cereus ) can also inhibit the growth of esophageal cancer subcutaneous tumors, while oral administration B. cereus CGMCC 04060.4 Extracellular metabolites (abbreviated as: S-B. cereus ) had no significant tumor suppressive effect (e.g. Figure 5 , as shown in 6).

[0042] Example 4. Immunohistochemical staining to detect the expression of proliferation molecule Ki-67 in subcutaneous tumor tissue of esophageal cancer in mice Immunohistochemistry (HE staining) was used to detect the positive rate of Ki-67 proliferation factor in subcutaneous tumor tissue, and the effect of live bacteria on the growth of esophageal cancer cells in subcutaneous tumor tissue was further detected. The specific operation steps are as follows: The tumor tissue sections were placed in an oven at 60°C for 60 min, taken out and soaked in xylene for 10 min to complete dewaxing. Then, the sections were placed in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol in turn, soaked for 5 min each, and then washed with ddH2O 3 times, 5 min each time. The sections were placed in a previously prepared citrate buffer, heated in a microwave for 14 min, taken out and cooled to room temperature, and antigen repair was performed, and then washed with ddH2O and PBST twice, 5 min each time. The sections were placed flat in a black wet box and 25 μL of inactivation enzyme reagent was added. After 15 min, the inactivation enzyme reagent was shaken off and placed in PBST, and washed 3 times / 5 min. 3% BSA was dripped on the sections for blocking for 30 min. After removing the blocking solution, an appropriate amount of Ki-67 antibody (1:200) was added and incubated overnight at 4°C. Take out the next day, rewarm, place in PBST, wash 3 times / 5 min, then add appropriate amount of enzyme-labeled secondary antibody, incubate at 37℃ for 30 min. Wash 3 times / 5 min with PBST, perform DAB color development, and then put the tissue sections into pure water to stop color development. Then use 20 μL hematoxylin to counterstain, then use 1% hydrochloric acid-ethanol for differentiation, and finally put in PBST to turn blue. Soak the sections in 70% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol, respectively, for 5 min each process, and then place in xylene twice, soaking for 10 min each time. Finally, seal the sections with neutral gum and take photos under a microscope.

[0043] Immunohistochemistry results showed that intratumoral injection B. cereus CGMCC 04060.4 live bacteria and their extracellular metabolites, oral administration B. cereus CGMCC 04060.4 live bacteria agent, Ki-67 positive rate significantly reduced, such as Figure 7 This result shows that B. cereus CGMCC 04060.4 can inhibit the proliferation activity of esophageal cancer cells in subcutaneous tumor tissues of mice.

[0044] Example 5. Probiotic Bacillus cereus extracellular protein compound preparation inhibits the proliferation of esophageal cancer cells and gastric cancer cells The preparation method of the probiotic Bacillus cereus extracellular protein complex comprises the following steps: Will B. cereus CGMCC 04060.4 was streaked onto TSB solid medium for culture (37°C, 18 h), and a single colony was picked and transferred to TSB liquid medium for continued activation culture until OD 600The value was 0.6-0.8. The rejuvenated bacteria were inoculated into 200 mTSB liquid culture medium at a 5% inoculation rate, and cultured in a flask at 37℃ for 16-18h to the mid-logarithmic phase to obtain the fermentation seed liquid; the fermentation seed liquid was added to 5L TSB liquid culture medium at a 2% inoculation rate. The culture was shaken at 37℃ and 200 rpm for 4 h, and the supernatant was collected by centrifugation at 4℃ and 12000 rpm in an ultra-high-speed refrigerated centrifuge. The protein in the supernatant was extracted by graded precipitation. First, an equal volume of 40% ammonium sulfate solution was added, and the supernatant was collected by centrifugation after standing at 4℃ overnight. An equal volume of 80% ammonium sulfate solution was added, and the precipitate was collected by centrifugation after standing at 4℃ overnight. The precipitated protein was separated and purified using a G200 dextran gel column, and four eluted protein fractions were obtained after elution with pure water. The protein fractions with inhibitory activity on esophageal cancer proliferation were screened by CCK8 experiment, and the protein concentrate was obtained by filtration through a 50KD ultrafiltration tube. Take the protein concentrate prepared above, add 0.05% soy peptone and 0.05% sodium chloride solution respectively, stir at low speed to mix, and freeze-dry into powder in a vacuum freeze dryer to obtain the lyophilized powder of probiotic Bacillus cereus extracellular protein complex (BcE-pro). The obtained lyophilized powder is quantified and prepared into a 50 mg / mL protein solution for subsequent anti-tumor activity detection.

[0045] Detection of the effect of Bacillus cereus extracellular protein complex on the proliferation of esophageal cancer cells and gastric cancer cells The specific experimental steps are detailed in Example 1. The anti-tumor activity of the probiotic Bacillus cereus extracellular protein complex (BcE-pro) was determined. Esophageal cancer cells ECA109 and gastric cancer cells HGC-27 were selected as the test objects. Figure 8 As shown in the figure, the results showed that the extracellular protein complex BcE-pro could inhibit the proliferation of esophageal cancer cells ECA109 by 62.31% at a concentration of 100 μg / mL; when the final concentration was 200 μg / mL, the inhibition rate of esophageal cancer cell ECA109 proliferation could reach 79.54%, and the inhibition rate of gastric cancer cell HGC-27 proliferation could reach 47.83%. When the concentration of BcE-pro increased to 400 μg / mL, the inhibition rate of gastric cancer cell HGC-27 proliferation could also reach 65.63%. Therefore, the prepared Bacillus cereus extracellular protein complex has significant inhibitory activity on the proliferation of esophageal cancer and gastric cancer cells.

[0046] Experimental study on the effect of Bacillus cereus extracellular protein complex on the proliferation of esophageal cancer cells and gastric cancer cells The specific operation of EdU experiment to detect the anticancer activity of extracellular protein complex is as follows: Esophageal cancer cells ECA109 and gastric cancer cells HGC-27 in good condition were uniformly inoculated into 96-well cell culture plates at 9000 cells / well. After one day of cultivation, 100 μg / mL of probiotic Bacillus cereus extracellular protein complex (BcE-pro) was added. After 24 hours, the culture supernatant was aspirated, and 80 μL of the culture medium in the 50 μM EdU kit (Yisheng Biotechnology (Shanghai) Co., Ltd.) was added to each well. The cells were incubated in a 37°C incubator for 2 hours, and the EdU culture medium was discarded. The cells were washed twice with PBS and shaken at low speed for 5 minutes. This was repeated 2-3 times. Then, 100 μL of fixative solution was added to each well and the cells were fixed at 30 rpm at room temperature for 40 minutes. 100 μL of PBS was added to each well and the cells were washed twice at low speed on a shaker for 5 minutes each time. 100 μL of 0.5% TritonX-100 was added to each well and the cells were incubated at low speed on a shaker for 10 minutes. Then, 100 μL of PBS was added to each well for washing and shaking on a shaker for 10 minutes. Prepare an appropriate amount of 1 × Apollo staining solution according to the instructions, incubate at room temperature in the dark for 0.5 h, add 100 μL PBS to wash the cells, then add 100 μL permeabilization agent, incubate twice, and finally wash once with methanol and PBS. Prepare a certain volume of 1 × Hoechst 33342 reaction solution, incubate at room temperature at 30 rpm on a shaker in the dark for 30 min; add PBS to each well to wash twice, each time for 5 min, and elute the Hoechst 33342 reaction solution; finally, add 100 μL PBS to each well, take pictures under a fluorescence microscope, and count.

[0047] The results of the EdU experiment showed that the probiotic Bacillus cereus extracellular protein complex (BcE-pro) significantly inhibited the percentage of EdU incorporation into cells after acting on esophageal cancer cells ECA109 and gastric cancer cells HGC-27, indicating that the prepared Bacillus cereus extracellular protein complex has good anti-tumor activity. Figure 9 shown.

[0048] Animal experiment to detect the anti-esophageal cancer activity of Bacillus cereus extracellular protein complex in vivo The specific operation of animal experiment to detect the anti-tumor activity of extracellular protein complex is as follows: Subcutaneous tumor mouse models were constructed according to the method of Example 3. After 7 days, 25 mg / mL of B. cereus CGMCC 04060.4 Extracellular protein complex (BcE-pro) or its extracellular metabolites (abbreviated as: S-B. cereus) 50μL, and an equal amount of PBS or TSB was used as a control. During the experiment, the volume change of subcutaneous tumor tissue was detected every day, and the subcutaneous tumor growth curve was prepared. The mice were dissected 19 days after intratumoral injection, and the tumor tissue was taken for photography, weighing, and tumor-related index analysis. B. cereus CGMCC 04060.4 Anti-tumor effect of extracellular protein complex or its extracellular metabolites.

[0049] The results showed that intratumoral injection B. cereus CGMCC 04060.4 Extracellular protein complex (BcE-pro) or its extracellular metabolites (abbreviated as: S-B. cereus ) can significantly inhibit the growth of esophageal cancer cells in subcutaneous tumors of mice. Figure 10 shown.

[0050] 5.4 Immunohistochemical staining was used to detect the expression of esophageal cancer cell proliferation molecule Ki-67 in mouse subcutaneous tumor tissue.

[0051] The specific experimental operation method is shown in Example 4.

[0052] The results showed that intratumoral injection B. cereus CGMCC 04060.4 Extracellular protein complex (BcE-pro) or its extracellular metabolites (abbreviated as: S-B. cereus ), the percentage of Ki-67 positive cells decreased. In summary, the research results of the present invention prove that the probiotic Bacillus cereus B. cereus CGMCC 04060.4 extracellular protein can inhibit the proliferation of esophageal cancer cells in vivo, such as Figure 11 shown.

[0053] Safety testing of probiotic Bacillus cereus extracellular protein complex in mice.

[0054] The specific operation method of HE staining experiment is as follows: Soak the tissue sections of each organ in xylene for 10 min, replace with new xylene and continue soaking for 10 min to dewax, soak in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 5 min in sequence; soak in ddH2O for 5 min, wash 3 times, and then put the tissue sections in hematoxylin staining solution for about 10 min. Rinse with pure water for about 10 min to make the color of the sections blue, put the tissue sections in 1% hydrochloric acid ethanol to fade, rinse with pure water for about 1 min after ten seconds, put in PBS for anti-blueing for 1 min, rinse with pure water again for 1 min, put in 75% ethanol and 85% ethanol for 5 min in sequence, then put in eosin ethanol for counterstaining for 10 min, put in 95% ethanol for 10 s, replace with new 95% ethanol and continue soaking for dehydration. Put in xylene twice again, soak for 3-5 min each time. Finally, seal the sections with neutral gum for preservation and take photos under a microscope.

[0055] The results showed that mice given B. cereus CGMCC 04060.4 Extracellular protein complex (BcE-pro) or its extracellular metabolites (abbreviated as: S-B. cereus The body weight after 4 h was almost the same as that of the normal group; and HE staining showed that the liver, spleen, and kidney images of all mouse groups showed no obvious tissue damage, such as Figure 12 shown.

Claims

1. A probiotic Bacillus cereus live bacterial agent, characterized in that The total number of viable bacteria in the inoculant is not less than 2×10 9 cfu / mL.

2. The probiotic Bacillus cereus viable agent according to claim 1, characterized in that The probiotic Bacillus cereus comprises one or more strains of CGMCC No. 04060.4, CMCC 63305 and DM423.

3. The probiotic Bacillus cereus viable agent according to claim 1, characterized in that Prepared by the following steps: 1) Streak the frozen strain on TSB solid medium, pick a single colony and transfer it to TSB liquid medium for further activation culture; 2) Inoculate the rejuvenated cells into TSB liquid culture medium at a rate of 5-10%, culture in a shake flask at 37±2℃ until the stable phase, centrifuge and collect fresh cells; 3) Wash the fresh bacteria with sterile phosphate buffered saline (PBS, pH 7.0-7.2), and then fully suspend the bacteria with an appropriate amount of PBS to obtain a bacterial suspension, so that the density of the bacteria in the bacterial suspension is not less than 2×10 9 cfu / mL, that’s it.

4. The probiotic Bacillus cereus viable agent according to claim 3, characterized in that In step 1), the streak culture temperature is 37±2℃ for 12-24 h; activation culture is performed until OD 600 The value is 0.6-0.

8.

5. A probiotic Bacillus cereus extracellular metabolite, characterized in that: Prepared by the following steps: 1) Streak the frozen strain on TSB solid medium, pick a single colony and transfer it to TSB liquid medium for further activation culture; the culture temperature is 37±2℃, the time is 12-24 h; activate the culture until OD 600 The value is 0.6-0.8; 2) Inoculate the rejuvenated cells into TSB liquid medium at a rate of 5-10%, culture at 37±2℃ in a shake flask for 16-18h until the end of the logarithmic phase, centrifuge, collect the fresh cells, wash with sterile PBS, and then transfer the cells to the same volume of TSB liquid medium as before centrifugation, and culture at 37±2℃ with shaking for 4-6h; 3) Take the culture medium obtained above, centrifuge it, collect the supernatant, and filter it with a 0.22 μm sterile filter membrane to obtain the product.

6. A probiotic Bacillus cereus extracellular protein complex, characterized in that: Prepared by the following steps: 1) Streak the frozen strain on TSB solid medium, pick a single colony and transfer it to TSB liquid medium for further activation culture; inoculate the rejuvenated bacteria into TSB liquid medium at an inoculum rate of 3-5%, and culture in a shake flask at 37±2℃ for 16-18h until the mid-logarithmic phase to obtain the fermentation seed liquid; 2) Add 5-10% of the inoculum to the sterilized TSB liquid medium and culture with shaking at 37±2℃ for 4-6 hours, centrifuge and collect the supernatant; 3) adding an equal volume of 38-42% ammonium sulfate solution to grade the protein, letting it stand, centrifuging and collecting the supernatant, further adding an equal volume of 75-85% ammonium sulfate solution to grade the protein, letting it stand, centrifuging and collecting the precipitate, purifying the collected precipitated protein by using a dextran gel column filtration chromatography, screening a protein component with anti-tumor activity from the multiple separated protein components by using a CCK8 experiment, collecting the active protein component, further purifying and concentrating it through an ultrafiltration tube to obtain a protein concentrate; 4) Prepare protein complex solution: Take the protein concentrate prepared above, add soy peptone and sodium chloride, mix well, and freeze-dry to obtain the product.

7. The probiotic Bacillus cereus extracellular protein complex according to claim 6, characterized in that: In step 3), the molecular weight cutoff of the ultrafiltration tube is 50-100 KD; in step 4), soy peptone and sodium chloride are added to final concentrations of 0.04-0.06% and 0.04-0.06%, respectively.

8. Use of the probiotic Bacillus cereus live bacteria agent, extracellular metabolites and protein complex agent according to any one of claims 1 to 7 in preventing and treating malignant tumors.

9. The use according to claim 8, characterized in that The malignant tumors include one or more of esophageal cancer, intestinal cancer, nasopharyngeal cancer, gastric cancer, liver cancer, colorectal cancer, non-small cell lung cancer, breast cancer, melanoma, head and neck squamous cell carcinoma, lymphoma and the like.

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