Intestinal microecological preparation and application thereof

Abstract

The invention provides an intestinal microecological preparation and application thereof, and belongs to the technical field of microorganisms. According to the technical scheme, lactobacillus jensenii, streptococcus diacetolactis, propionibacterium shermannii, bifidobacterium infantis, pediococcus cerevisiae, leuconostoc mesenteroides and lactobacillus cellobiosus are used for constructing a compound microbial flora, and the compound microbial flora is administrated in an oral manner, so that a remarkable immunoregulation effect can be realized. Clinical tests show that the oral cavity mucosa injury can be relieved when the traditional Chinese medicine composition is used for patients suffering from tongue cancer chemoradiotherapy, and the number of CD3 + and CD4 + positive cells is increased, so that the immunity is improved, and the recovery of the patients is facilitated. In practical application, the strains can be screened according to the characteristics of acid resistance, oxidation resistance, cholate resistance, tumor cell adhesion and the like, and the screened strains are used for constructing the fungicide, so that the efficacy level of the fungicide can be improved. In addition, a compound enzyme and a natural pharmaceutical composition can be further added, so that an improvement effect on regulating the micro-ecological environment of the intestinal tract and promoting the anti-tumor effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to an intestinal microecological preparation and application thereof. Background Art

[0002] Intestinal microecology refers to the microbial environment in the intestine and its interaction with the host, which has a direct impact on human health. First, intestinal microorganisms participate in the digestion of food and nutrient absorption, helping the human body to break down and utilize nutrients in food. Secondly, intestinal microorganisms can stimulate and regulate the development and function of the immune system, enhance the body's resistance, and prevent the occurrence of diseases. In addition, intestinal microorganisms are also involved in regulating the body's metabolic processes, affecting aspects such as energy balance and weight control. However, when the intestinal microecology is unbalanced, it may have adverse effects on human health. For example, intestinal flora imbalance may lead to the occurrence of a variety of diseases such as digestive system diseases, metabolic diseases, and immune system diseases. Therefore, maintaining the balance of intestinal microecology is of great significance for maintaining human health.

[0003] In recent years, a variety of microorganisms have been found to promote the pathogenesis of tumors by harming the intestinal microecology. These species include but are not limited to enterotoxigenic strains of Fusobacterium nucleatum, Streptococcus gallolyticus, Bacteroides fragilis, polyketide synthase-positive (PKS+ve) Escherichia coli strains, Enterococcus faecalis, and Peptostreptococcus anaerobicus. They are associated with tumor proliferation, inducing a proinflammatory state, and evading antitumor immunity. In recent years, studies have found that in addition to direct carcinogenesis of microbial metabolites, intestinal microorganisms affect host immunity and response to cancer treatment through several hallmark mechanisms. These include: (1) effects on other microorganisms in the intestine, leading to changes in the ecosystem; (2) effects on the intestinal wall, including enterocytes (inducing autophagy and apoptosis) and intestinal-associated lymphoid tissue; (3) local or systemic stimulation of pattern recognition receptors that sense auxiliary signals; (4) systemic neuroendocrine effects through intestinal hormone secretion; (5) systemic metabolic effects through polyamine and B vitamin synthesis; and (6) induction of immune responses against microbial antigens that cross-react with tumor-associated antigens. Summary of the invention

[0004] The present invention aims to provide an intestinal microecological preparation and its application in view of the technical defects of the prior art, so as to solve the technical problems that the scope of indications and efficacy of conventional intestinal microecological regulating drugs are relatively limited.

[0005] Another technical problem to be solved by the present invention is how to expand the treatment approach for tongue cancer.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: An intestinal microecological preparation, comprising a bacterial agent, wherein the bacterial agent comprises Lactobacillus jensenii, diacetyllactococcus lactis, Propionibacterium schermeri, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose, wherein the ratio of the number of live bacteria of Lactobacillus jensenii, diacetyllactococcus lactis, Propionibacterium schermeri, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose is 1:1.4-1.6:1.1-1.3:1.9-2.1:1.3-1.5:0.7-0.9:1.2-1.4; in the intestinal microecological preparation, the number of live bacteria of the bacterial agent is 10 9 ~10 10 Pieces / g.

[0007] Preferably, in the bacterial agent, the ratio of the number of live bacteria of Lactobacillus jensenii, Streptococcus diacetyllactic acid, Propionibacterium shermanii, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose is 1:1.5:1.2:2:1.4:0.8:1.3.

[0008] Preferably, the number of live bacteria in the intestinal microecological preparation is 3×10 9 ~8×10 9 Pieces / g.

[0009] Preferably, the intestinal microecological preparation further comprises a complex enzyme, wherein the complex enzyme comprises enteropeptidase, sucrase, lactase, trypsin and chymotrypsin.

[0010] Preferably, in each gram of the complex enzyme, the enzyme activity of enteropeptidase is 15000-20000U, the enzyme activity of sucrase is 30000-35000U, the enzyme activity of lactase is 12000-18000U, the enzyme activity of trypsin is 25000-30000U, and the enzyme activity of chymotrypsin is 30000-40000U; each gram of the intestinal microecological preparation contains 0.1-0.2g of the complex enzyme.

[0011] Preferably, the intestinal probiotic preparation also includes a natural medicine composition, which includes the following ingredients in parts by weight: 8 to 10 parts of Astragalus, 6 to 8 parts of Angelica, 3 to 4 parts of Ginkgo, 1 to 2 parts of Scutellaria barbata, 0.5 to 1 part of Taraxacum, and 0.5 to 1 part of Atractylodes macrocephala; each gram of the intestinal probiotic preparation contains 0.2 to 0.3 g of the natural medicine composition.

[0012] Preferably, the natural medicine composition is composed of the following ingredients in parts by weight: 9 parts of Astragalus, 7 parts of Angelica, 3.5 parts of Ginkgo, 1.5 parts of Scutellaria barbata, 0.8 parts of Taraxacum, and 0.7 parts of Atractylodes macrocephala.

[0013] On the basis of the above technical solutions, the present invention further provides the use of the above intestinal microecological preparation for preparing tumor therapeutic drugs.

[0014] Preferably, the tumor is tongue cancer, laryngeal cancer or thyroid cancer.

[0015] Preferably, the dosage form of the drug is an oral dosage form.

[0016] The present invention provides an intestinal microecological preparation and its application. The technical solution constructs a composite microbial flora with Lactobacillus jensenii, Streptococcus diacetyllacti, Propionibacterium shermanii, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose, and can achieve significant immunomodulatory effects by oral administration. Clinical trials have shown that the use of the present invention in patients with tongue cancer undergoing radiotherapy and chemotherapy can alleviate oral mucosal damage and increase CD3 + , CD4 + The number of positive cells is increased, thereby improving immunity and facilitating the recovery of patients. In practical applications, each strain can be screened according to characteristics such as acid resistance, antioxidant capacity, bile salt resistance, and tumor cell adhesion capacity, and the selected strains can be used to construct a bacterial agent, which is beneficial to improving its efficacy level. In addition, the present invention can further add a composite enzyme and a natural drug composition, which has an improvement effect on regulating the intestinal microecological environment and promoting its anti-tumor effect.

[0017] In addition, the technical advantages of the present invention are also concentrated in the following aspects: 1) The microorganisms used in the present invention are safe and non-toxic to the human body and can be directly used; 2) The present invention not only has anti-tumor effects, but also can reduce the side effects of anti-tumor treatment, such as intestinal microbial imbalance, mucosal inflammation, decreased immunity, malabsorption and diarrhea; 3) The microorganisms of the present invention have specific colonization effects. Compared with gene therapy, they have the characteristics of long duration, high safety, stable hemodynamics, etc., and have certain targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a diagram showing the therapeutic effect of the present invention on the oral mucosa of a tongue cancer patient.

[0019] Figure 2 This figure shows the effect of the present invention on immune cells in patients with tongue cancer. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention will be described in detail below. In order to avoid too much unnecessary details, the well-known structures or functions will not be described in detail in the following examples. The approximate language used in the following examples can be used for quantitative expression, indicating that the quantity can be allowed to have a certain change without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meaning as those generally understood by those skilled in the art to which the present invention belongs.

[0021] 1. Screening of strains: (1) Acid resistance test Commercial Lactobacillus jensenii, Streptococcus diacetyllacti, Propionibacterium shermanii, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose were inoculated at a 2% inoculum into 10 mL of the corresponding appropriate culture medium, cultured at 37°C for 18 h, centrifuged (4000 r / min, 20 min), washed with sterile saline, and the cells were collected. The collected cells were suspended in sterile dipotassium hydrogen phosphate solution (0.1 mol / L, pH adjusted to 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 with lactic acid), cultured at 37°C for 2 h, and made 10-fold gradient dilutions at 0 h and 2 h. The number of viable cells was determined by the plate count method, and the survival rate was calculated.

[0022] (2) Bile salt tolerance test Each strain was inoculated into 10 mL of the corresponding appropriate culture medium at a 2% inoculation rate, cultured at 37°C for 18 h, centrifuged (4000 r / min, 20 min), washed with sterile saline, and the bacteria were collected. The collected bacteria were suspended and centrifuged (4000 r / min, 20 min), washed with sterile saline, and the bacteria were collected. The collected bacteria were suspended in the corresponding culture medium (containing 0.1%, 0.3% and 0.5% ox bile salt), and cultured at 37°C for 4 h. The number of viable bacteria was measured at 0 h and 4 h, respectively, and the survival rate was calculated.

[0023] (3) Experiment on the ability to inhibit pathogenic bacteria The Oxford cup method was used to antagonize the growth of 6 common foodborne pathogens (Shigella dysenteriae, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli, Candida albicans, and Lactobacillus acidophilus). The specific method is as follows: 1) Cultivate the obtained bacteria in liquid culture medium for 1-2 days, centrifuge at 8000 rpm for 10 min, and carefully remove the supernatant under sterile conditions.

[0024] 2) The six pathogenic bacteria were activated using LB plate culture medium and then expanded using LB liquid culture medium.

[0025] 3) Adjust the bacterial concentration of the 6 pathogenic bacteria to 10 6CFU / mL, and spread 200 μL of bacterial solution on LB plates.

[0026] 4) After the bacterial solution is blown dry, carefully place a sterile Oxford cup in the plate, add the bacterial culture supernatant into the Oxford cup, and culture at 37℃ for 6-8 hours.

[0027] 5) Measure the diameter of the inhibition zone in the plate.

[0028] (4) Antioxidant test The isolated and identified bacteria were cultured in the corresponding liquid culture medium (without L-cysteine) for 1-2 days, centrifuged (12000 rpm, 10 min), and the supernatant was carefully collected and stored at 4°C for later use.

[0029] 1) Determination of DPPH free radical scavenging ability Add 1 mL of ethanol solution of DPPH free radical (0.2 mM) to 1 mL of bacterial culture supernatant, react in the dark at room temperature for 30 min, extract with chloroform and centrifuge to obtain the supernatant, and measure the absorbance at 517 nm. Deionized water is used as a blank control.

[0030] DPPH free radical scavenging rate: [1-A517(sample) / A517(blank)]×100% 2) Remove superoxide free radicals (O 2－ )Determination of ability 1 mL of Tris-HCl (150 mmol / L pH = 8.0), 1 mL of diethylenetriaminepentaacetic acid (3 mmol / L) and 1.2 mmol / L were mixed and then added with 0.5 mL of lactic acid bacteria culture supernatant. The mixture was reacted in a constant temperature water bath at 25 °C for 10 min and the absorbance was measured at 325 nm.

[0031] O 2－ Clearance rate (%) = [1-(A11-A10) / (A01-A00)] × 100% Wherein: A00 does not contain sample and pyrogallol; A01 does not contain sample but contains pyrogallol; A10 contains sample but does not contain pyrogallol; A11 contains sample and pyrogallol.

[0032] 3) Remove hydroxyl radicals (HO · ) capacity determination Add 1 mL of O-phenanthroline and 1 mL of FeSO to 1 mL of PBS (pH = 7.4). 4 (concentration of 2.5 mmol / L), H 2 O 2(concentration is 20 mmol / L), then add 0.5 mL of bacterial culture supernatant, react in a 37°C constant temperature water bath for 1.5 h, and measure the absorbance at 536 nm.

[0033] HO · Clearance rate (%) = [(A2-A1) / (A0-A1)] × 100% Where: A0 is the volume without sample and H 2 O 2 ; Al is sample without Al, H is sample with 2 O 2 ; A2 contains sample and H 2 O 2 .

[0034] 4) Fe 2+ Determination of chelating capacity The reaction system is: 0.1 mL ascorbic acid (mass fraction 1%), 0.1 mL FeSO 4 0.5 mL of culture supernatant was added to a mixture of 1 mL of NaOH (mass fraction 0.4%) and 1 mL of NaOH (concentration 0.2 mol / L), mixed well and incubated in a water bath at 37°C for 20 min, 10% trichloroacetic acid (TAC) was added to precipitate protein, centrifuged at 5000 rpm and 4°C for 10 min, 0.2 mL of supernatant was added to 2 mL of O-phenanthroline (mass fraction 0.1%) and reacted for 10 min. The absorbance was measured at 510 nm, and PBS was used as a blank control.

[0035] Fe 2+ Chelating capacity = (A blank - A sample / A blank) × 100% 5) Determination of reducing activity Reaction system: 0.5 mL culture supernatant, 0.5 mL potassium ferrocyanide (mass fraction 1%) and 0.5 mL PBS were mixed and incubated in a 50°C water bath for 20 min. After cooling to room temperature, 10% trichloroacetic acid was added to precipitate protein. The mixture was centrifuged at 4°C and 5000 rpm for 10 min. 1 mL of supernatant was mixed with 1 mL FeCl 3 (mass fraction 0.1%) after reaction, the absorbance was measured at 700 nm, with L-cysteine ​​as the standard.

[0036] (5) Cell attachment assay 1) Inoculate the strains screened in the above steps into the corresponding liquid culture medium and incubate at 37°C in CO 2 Cultivate in an incubator (filled with anaerobic bacteria and sealed) until the OD value is equal to 0.6 and then terminate the cultivation; 2) After the tongue cancer cells have grown all over the cell culture flask, digest them with 1 mL of 0.25% trypsin-EDTA solution for 2 minutes, gently blow to detach the cells from the culture flask, wash the cells with 5 mL of DMEM cell culture medium, and blow evenly with a pipette. Take a six-well cell culture plate and add a sterile coverslip and 1 mL of DMEM culture medium to each well, then add 1 mL of the above cell suspension to each well, and place the cell culture plate in a 37°C, 5% carbon dioxide incubator for culture; when the cells grow to 30% of the coverslip, take 100 µL of the above bacterial culture medium and add it to 2 mL of culture medium containing a small amount of tongue cancer cells, and culture in a 37°C cell culture incubator; 3) After 1-1.5 hours of incubation, the cells were washed five times with cold PBS buffer, fixed with methanol, and stained with Gram staining and observed under an oil immersion microscope; (4) Screen out strains with good adhesion ability to tongue cancer cells.

[0037] D. Compounding of probiotic preparations: 1) Activate and culture the screened Lactobacillus jensenii, Streptococcus diacetyllacti, Propionibacterium shermanii, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose, monitor the pH value of the culture medium every 8 hours, and terminate the culture when the pH value reaches 4.2; 2) Use an ultracentrifuge at 3000r / rpm for 10min to obtain precipitated bacteria, and add 10% skim milk, 6% trehalose, and 2% gelatin in a volume ratio of 1:1 as a protective agent during freeze-drying.

[0038] 3) Place in a vacuum freeze dryer for 24 hours to obtain freeze-dried powder 4) The freeze-dried powders of the bacterial agents were mixed in a ratio of 1:1.5:1.2:2:1.4:0.8:1.3, and then mixed with starch as an auxiliary material to obtain an intestinal microecological preparation, so that the total number of viable bacteria per gram of the intestinal microecological preparation was 5×10 9 .

[0039] 2. Efficacy experiment The therapeutic and recovery effects of the intestinal microecological preparations prepared above on patients with tongue cancer were investigated orally.

[0040] (1) Enrollment of patients with tongue cancer Patients with a clear diagnosis of tongue cancer were selected and randomly divided into two groups (group α and group β), with 30-50 cases in each group. Group α was the group receiving radiotherapy for tongue cancer alone, and group β was the group receiving radiotherapy for tongue cancer plus intestinal microecological preparations. Twenty normal people were selected as normal controls.

[0041] (2) Selection criteria Patients with tongue cancer who have been confirmed as having no metastasis by tongue cancer biopsy and excluded by chest CT, abdominal ultrasound and bone scan and who are undergoing radiotherapy (with or without concurrent chemotherapy).

[0042] (3) Exclusion criteria Those who have used antibiotics in the past 2 months.

[0043] (4) Treatment methods and sample collection Before treatment (before the patient receives any treatment): collect stool specimens from the patient (send to refrigerator for freezing), perform blood tests, microbiochemistry, immunoglobulins (IgA, lgG, lgM), CD3 + , CD4 + , CD8 + . Score the patient's condition.

[0044] During treatment (patients receive treatment or take intestinal microecological preparations after admission): record the time of first dysphagia, pain and need for antibiotics, and send blood routine, microbiochemistry, immunoglobulin (IgA, IgG, IgM), CD3 + , CD4 + , CD8 + . Collect stool specimens from patients (send them to refrigerators for freezing) and score the patients’ conditions.

[0045] Treatment completion (after the patient is admitted to the hospital and receives treatment): collect stool specimens from the patient (send to the refrigerator for freezing), perform blood routine tests, microbiochemistry, immunoglobulins (lgA, lgG, lgM), CD3 + , CD4 + , CD8 + . Score the patient's condition.

[0046] (5) Experimental results like Figure 1 As shown, the intestinal probiotic preparation treatment group can significantly reduce the mucositis caused by radiotherapy and chemotherapy of tongue cancer. Compared with tumor patients who do not use intestinal probiotic preparations, it can significantly reduce their mucosal adverse reactions, which is beneficial to the patient's recovery.

[0047] like Figure 2 As shown in the figure, compared with patients who did not use intestinal microecological preparations for radiotherapy and chemotherapy, the CD3 + , CD4 + The significant increase in positive cells indicates that intestinal microecological preparations can significantly improve the patient's immune ability, help the patient recover and resist external immune stimulation.

[0048] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. An intestinal microecological preparation, characterized in that: The intestinal microecological preparation comprises a bacterial agent, wherein the bacterial agent comprises Lactobacillus jensenii, diacetyllactococcus lactis, Propionibacterium schermeri, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose. In the bacterial agent, the ratio of the number of live bacteria of Lactobacillus jensenii, diacetyllactococcus lactis, Propionibacterium schermeri, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose is 1:1.4-1.6:1.1-1.3:1.9-2.1:1.3-1.5:0.7-0.9:1.2-1.4; in the intestinal microecological preparation, the number of live bacteria of the bacterial agent is 10 9 ~10 10 Pieces / g.

2. An intestinal microecological preparation according to claim 1, characterized in that: In the bacterial agent, the ratio of the number of live bacteria of Lactobacillus jensenii, Streptococcus diacetyllactic acid, Propionibacterium shermanii, Bifidobacterium infantis, Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus cellobiose is 1:1.5:1.2:2:1.4:0.8:1.

3.

3. An intestinal microecological preparation according to claim 1, characterized in that: The number of live bacteria in this intestinal microecological preparation is 3×10 9 ~8×10 9 Pieces / g.

4. The intestinal microecological preparation according to claim 1, characterized in that: The intestinal microecological preparation also includes a composite enzyme, which includes enteropeptidase, sucrase, lactase, trypsin and chymotrypsin.

5. An intestinal microecological preparation according to claim 4, characterized in that: In each gram of the complex enzyme, the enzyme activity of enteropeptidase is 15000-20000U, the enzyme activity of sucrase is 30000-35000U, the enzyme activity of lactase is 12000-18000U, the enzyme activity of trypsin is 25000-30000U, and the enzyme activity of chymotrypsin is 30000-40000U; each gram of the intestinal microecological preparation contains 0.1-0.2g of the complex enzyme.

6. The intestinal microecological preparation according to claim 1, characterized in that: The intestinal microecological preparation also includes a natural medicine composition, which includes the following ingredients in parts by weight: 8-10 parts of astragalus, 6-8 parts of angelica, 3-4 parts of ginkgo, 1-2 parts of barbata, 0.5-1 part of dandelion, and 0.5-1 part of atractylodes; each gram of the intestinal microecological preparation contains 0.2-0.3g of the natural medicine composition.

7. An intestinal microecological preparation according to claim 6, characterized in that: The natural medicine composition consists of the following ingredients in parts by weight: 9 parts of astragalus, 7 parts of angelica, 3.5 parts of ginkgo, 1.5 parts of barbata, 0.8 parts of dandelion and 0.7 parts of atractylodes.

8. Use of the intestinal microecological preparation according to claim 1 for preparing tumor therapeutic drugs.

9. The use according to claim 8, characterized in that: The tumor is tongue cancer, laryngeal cancer or thyroid cancer.

10. The use according to claim 8, characterized in that: The dosage form of the drug is oral dosage.

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