WO2018192501A1 - 一种预防肿瘤化疗肠道毒性的微生物菌剂 - Google Patents

一种预防肿瘤化疗肠道毒性的微生物菌剂 Download PDF

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WO2018192501A1
WO2018192501A1 PCT/CN2018/083418 CN2018083418W WO2018192501A1 WO 2018192501 A1 WO2018192501 A1 WO 2018192501A1 CN 2018083418 W CN2018083418 W CN 2018083418W WO 2018192501 A1 WO2018192501 A1 WO 2018192501A1
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selenium
enriched
microorganism
group
intestinal
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PCT/CN2018/083418
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English (en)
French (fr)
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陈代杰
邹振荣
邵雷
邱玉爽
陆惠刚
张骏梁
谭俊
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江苏德禧生物科技有限公司
上海医药工业研究院
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Priority to JP2020507731A priority Critical patent/JP2020516699A/ja
Publication of WO2018192501A1 publication Critical patent/WO2018192501A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention belongs to the field of biotechnology; in particular, the present invention relates to a microbial agent for preventing intestinal toxicity of tumor chemotherapy.
  • Chemotherapy is a treatment that uses chemical drugs to prevent the proliferation, infiltration, and metastasis of cancer cells until they eventually kill cancer cells. It is a systemic treatment, together with surgery, radiotherapy, and is called the three major treatments for cancer. Because the selectivity of chemotherapy drugs is not strong, it will inevitably damage normal cells of the human body while killing cancer cells, and thus adverse drug reactions occur. Therefore, when receiving chemotherapy drugs, on the one hand, we hope to achieve the best anti-tumor effect, on the other hand, we must also pay attention to the prevention and identification of adverse reactions of chemotherapy drugs. With the expansion of high-dose chemotherapy in clinical applications, the incidence and severity of adverse reactions caused by chemotherapy have also increased. Many chemotherapy drugs cause severe intestinal mucosal barrier dysfunction, causing damage to the intestinal mucosal barrier.
  • Irinotecan hydrochloride (CPT-11) is a camptothecin anticancer drug and is a potent topoisomerase-I inhibitor. It was first used clinically as a metastatic colorectal cancer after failure of standard chemotherapy. As a first-line medication for metastatic colorectal cancer. However, the drug has serious toxic and side effects on the gastrointestinal tract, which can kill the intestinal mucosal crypt epithelial cells that rapidly divide and proliferate, leading to destruction of intestinal mucosal structure, eventually leading to the occurrence of chemotherapy-induced intestinal mucositis and chemotherapy-related diarrhea.
  • a selenium-enriched microorganism for the preparation of a medicament for:
  • the intestinal damage is intestinal damage caused by tumor chemotherapy.
  • the medicament is also useful for preventing weight loss caused by tumor chemotherapy.
  • the medicament is also used to prolong the survival of a tumor patient.
  • the medicament is also used to increase the level of immunity in a tumor patient.
  • the intestinal damage comprises: intestinal mucosal damage, diarrhea, and the like.
  • the selenium-enriched microorganism has a selenium content of ⁇ 2 mg per gram of dry cells (e.g., ⁇ 4 mg, preferably ⁇ 5 mg, more preferably ⁇ 10 mg, most preferably ⁇ 20 mg).
  • the selenium-enriched microorganism is a selenium-enriched probiotic.
  • the selenium-enriched microorganism is a selenium-enriched Bifidobacterium.
  • the microorganism is a microorganism capable of colonizing the gut of a mammal.
  • a microbial agent comprising a living selenium-enriched microorganism.
  • the selenium-enriched microorganism is a selenium-enriched probiotic.
  • the selenium-enriched microorganism is a selenium-enriched Bifidobacterium.
  • the microbial agent is a liquid preparation, a powder or a tablet.
  • the content of the selenium-enriched microorganism in the microbial agent is 1*10 ⁇ 7-9*10 ⁇ 11 CFU/g, preferably 1*10 ⁇ 9-9*10 ⁇ 10 CFU/g.
  • the selenium content per 10 8 CFU of the selenium-enriched microorganism is 0.1 to 100 ⁇ g; preferably 1 to 50 ⁇ g, more preferably 2 to 30 ⁇ g, and most preferably 3 to 10 ⁇ g.
  • the microbial agent has a selenium content of 10 to 300 ⁇ g/g, preferably 20 to 200 ⁇ g/g, more preferably 30 to 100 ⁇ g/g.
  • the microbial agent further includes a protective agent comprising: 5 parts by weight of skim milk powder, 2 parts by weight of trehalose, and 0.01 part by weight of L-cysteine hydrochloride.
  • a method of preventing and/or treating intestinal damage comprising the step of administering a prophylactically and/or therapeutically effective amount of a selenium-enriched microorganism to a subject in need thereof.
  • the subject is a tumor patient.
  • the intestinal damage is intestinal damage caused by tumor chemotherapy.
  • the selenium-enriched microorganism is a selenium-enriched Bifidobacterium.
  • the selenium-enriched microorganism is administered at a dose of 1*10 ⁇ 7-9*10 ⁇ 10 CFU/subject, preferably 1*10 ⁇ 8-9*10 ⁇ 8 CFU/subject.
  • the object is a human.
  • the selenium content per 10 8 CFU of the selenium-enriched microorganism is 0.1 to 100 ⁇ g; preferably 1 to 50 ⁇ g, more preferably 2 to 30 ⁇ g, and most preferably 3 to 10 ⁇ g.
  • the method comprises administering a prophylactically and/or therapeutically effective amount of a selenium-enriched microorganism to a subject in need thereof prior to chemotherapy.
  • the subject is administered a prophylactically and/or therapeutically effective amount of a selenium-enriched microorganism 1-8 days prior to chemotherapy; preferably, each dose is 1*10 ⁇ 7-1 *10 ⁇ 10 CFU/subject; more preferably, the dose per administration is 1*10 ⁇ 8-1*10 ⁇ 10 CFU/subject.
  • a selenium-enriched microorganism or a microbial agent according to the second aspect in the preparation of a medicament for preventing or treating alcoholic liver disease.
  • the selenium-enriched microorganism has a selenium content of ⁇ 2 mg per gram of dry bacteria (e.g., ⁇ 4 mg, preferably ⁇ 5 mg, more preferably ⁇ 10 mg, most preferably ⁇ 20 mg).
  • the selenium-enriched microorganism is a selenium-enriched probiotic.
  • the selenium-enriched microorganism is a selenium-enriched Bifidobacterium.
  • the microorganism is a microorganism capable of colonizing the gut of a mammal.
  • a selenium-enriched microorganism or a microbial agent according to the second aspect for the preparation of a medicament or a health care product for enhancing immunity.
  • the selenium-enriched microorganism has a selenium content of ⁇ 2 mg per gram of dry bacteria (e.g., ⁇ 4 mg, preferably ⁇ 5 mg, more preferably ⁇ 10 mg, most preferably ⁇ 20 mg).
  • the selenium-enriched microorganism is a selenium-enriched probiotic.
  • the selenium-enriched microorganism is a selenium-enriched Bifidobacterium.
  • the microorganism is a microorganism capable of colonizing the gut of a mammal.
  • a selenium-enriched microorganism or the microbial agent of the second aspect for the preparation of a medicament or a health care product for regulating an intestinal flora.
  • the regulating intestinal flora refers to improving the intestinal flora, increasing the number of probiotics, and reducing the number of pathogenic bacteria.
  • the selenium-enriched microorganism has a selenium content of ⁇ 2 mg per gram of dry bacteria (e.g., ⁇ 4 mg, preferably ⁇ 5 mg, more preferably ⁇ 10 mg, most preferably ⁇ 20 mg).
  • the selenium-enriched microorganism is a selenium-enriched probiotic.
  • the selenium-enriched microorganism is a selenium-enriched Bifidobacterium.
  • the microorganism is a microorganism capable of colonizing the gut of a mammal.
  • a selenium-enriched microorganism or a water-soluble protein thereof, or a microbial agent according to the second aspect for the preparation of a medicament for use as a protective agent for intestinal epithelial cells.
  • the selenium-enriched microorganism has a selenium content of ⁇ 2 mg per gram of dry bacteria (e.g., ⁇ 4 mg, preferably ⁇ 5 mg, more preferably ⁇ 10 mg, most preferably ⁇ 20 mg).
  • the selenium-enriched microorganism is a selenium-enriched probiotic.
  • the selenium-enriched microorganism is a selenium-enriched Bifidobacterium.
  • the microorganism is a microorganism capable of colonizing the gut of a mammal.
  • Figure 4 is an abdominal anatomy of the mouse; (A) Bla Ctr group (B) Mod Ctr group (C) Sel-BL group.
  • Fig. 5 is a HE staining diagram of small intestine tissues of mice in Mod Ctr group, BL group, Na 2 SO 3 group, Sel-BL group and Phy Mix group. Method of operation: The mice were sacrificed on the 6th day, and paraffin sections were prepared from the small intestine tissue 2 cm away from the ileocecal area for HE staining; (A) blank control group; (B) model control group; (C) common bifidobacteria group (D) sodium selenite group; (E) selenium-rich bifidobacterium group.
  • Figure 9 is a comparison of Bifidobacteria in the intestine of BlaCtr group, BL group, Na 2 SO 3 group, Sel-BL group and Phy Mix group; method: 0 day, the first intraperitoneal injection of CPT-11 Before, the feces of the mice were collected, and the genome of the intestinal flora in the feces was extracted. The changes of the bifidobacteria in the prevention group relative to the BlaCtr mice were detected by Real-Time PCR.
  • GSH-Px glutathione peroxidase
  • Figure 12 shows that selenium-enriched probiotic water-soluble protein enhances IEC6 cell viability.
  • IEC6 cells of rat intestinal epithelial cells were treated with 50 ⁇ g/ml LPS, and water-soluble proteins of selenium-enriched probiotics were added at different concentrations. After incubation for 24 hours, cell viability was detected by MTT assay. *P ⁇ 0.05.
  • Se water-soluble protein of selenium-enriched probiotics.
  • FIG. 13 shows that selenium-enriched probiotic water-soluble protein inhibits LPS-induced apoptosis in IEC6 cells.
  • Rat intestinal epithelial cells IEC6 cells were treated with 50 ⁇ g/ml LPS, and different concentrations of selenium-probiotic-producing water-soluble protein were added. After incubation for 24 hours, IEC6 cells were stained with FITC-conjugated Annexin V and PI, and flowed. The cytometer detects apoptosis. Apoptosis was detected as the result of three independent replicates.
  • Se water-soluble protein of selenium-enriched probiotics.
  • FIG 14 shows that selenium-enriched probiotic water-soluble protein inhibits LPS-induced mitochondrial membrane potential collapse in IEC6 cells.
  • Rat intestinal epithelial cells IEC6 cells were treated with 50 ⁇ g/ml LPS, and different concentrations of selenium-probiotic-producing water-soluble protein were added. After incubation for 24 hours, IEC6 cells were stained with JC-1, and mitochondrial membrane was detected by flow cytometry. Potential. The mitochondrial membrane potential was measured as the result of three independent replicates.
  • Se water-soluble protein of selenium-enriched probiotics.
  • FIG. 15 shows that selenium-enriched probiotic water-soluble protein inhibits LPS-induced IEC6 cell tight junction protein ZO-1 and
  • Occludin levels are reduced.
  • the rat intestinal epithelial cells IEC6 cells were treated with 50 ⁇ g/ml LPS, and different concentrations of selenium-probiotic-producing water-soluble protein were added. After incubation for 24 hours, the mRNA levels of ZO-1 and Occludin were detected by Q-PCR. *P ⁇ 0.05, **P ⁇ 0.01. NS, no significant difference.
  • Se water-soluble protein of selenium-enriched probiotics.
  • DTH delayed type hypersensitivity
  • the present inventors have obtained a microbial agent for preventing intestinal toxicity of tumor chemotherapy through extensive and in-depth research, and the microbial inoculum contains selenium-rich bifidobacteria, and the experimental results show that the selenium-rich bifidobacteria prevent and/or Or the treatment of chemotherapy-induced intestinal mucosal injury has a significant effect, and can significantly prolong the survival time of chemotherapy animals, and thus can be used for adjuvant treatment of tumors, reducing the side effects of chemotherapy.
  • the present invention provides a class of selenium-enriched microorganisms (especially Bifidobacterium-rich Bifidobacteria) having a selenium content of ⁇ 2 mg per gram of dry bacteria (e.g., ⁇ 4 mg, preferably ⁇ 5 mg, more preferably ⁇ 10 mg) Most preferably ⁇ 30 mg).
  • the selenium content described in the present invention refers to the content of organic selenium unless otherwise specified.
  • the method for measuring the selenium content can be carried out by a conventional or standard measurement method in the present invention. For example, the method described in the document "GB 5009.93-2010 Determination of Selenium in Foods".
  • the selenium-enriched microorganism has a selenium content per gram of dry cells of ⁇ 50 mg (preferably ⁇ 100 mg, more preferably ⁇ 200 mg).
  • the selenium-enriched microorganism has a selenium content of ⁇ 500 mg per gram of dry cells.
  • the microorganism is a prokaryotic microorganism, or a eukaryotic microorganism.
  • the microorganism is a probiotic.
  • the microorganism is selected from one or more of the group consisting of Bifidobacterium, Lactobacillus, and E. coli.
  • the selenium-enriched microorganism according to the present invention can effectively prevent and/or treat intestinal damage caused by tumor chemotherapy.
  • probiotic refers to a bacterium that has a beneficial effect in animal tissues, such as the human gastrointestinal tract and vagina.
  • the bacteria most commonly used as probiotics are lactic acid bacteria and bifidobacteria; however, other beneficial bacteria such as S. thermophilis may also be probiotics. After proliferating in the stomach and small intestine, some probiotics survive and temporarily survive in the large intestine, where colonic fermentative capacity is positively modified. See, for example, Roberfroid, AM J CLIN NUTR 71 (SUPPL): 1682S-1687S (2000).
  • the probiotic used in the present invention may be any known probiotics, for example, L. acidophilus, L. bulgaricus, L. casei, Lactobacillus kawaii ( L. paracasei), L. fermentum, L. plantarum, L. rhamnosus, L. salivarius, Bifidobacterium bifidum (B) .bifidum), B. infantis, B. animalis subsp. Lactis, B. longum, S. thermophilis , E. faecalis, and E. faecium.
  • probiotics may also be used in the present invention, for example, any additional known and/or available lactic acid bacteria or bifidobacteria.
  • the probiotic may comprise a kind of active microorganism beneficial to the host, which is colonized in the intestinal tract and reproductive system of the human or animal body, and can produce exact health effects, thereby improving the balance of the host micro-ecology and exerting Beneficial effect.
  • the beneficial bacteria or fungi in the human body and animal body mainly include: Clostridium butyricum, lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, actinomycetes, yeasts and the like.
  • the selenium-enriched microorganism involved in the present invention can be produced by the following method, which comprises the steps of:
  • the concentration of selenium in the medium is C ⁇ 10 ⁇ g/ml (eg ⁇ 20 ⁇ g/ml, preferably ⁇ 30 ⁇ g/ml, more preferably ⁇ 50 ⁇ g/ml, most preferably ⁇ 100 ⁇ g/ml, such as ⁇ 200 ⁇ g/ml, ⁇ 500 ⁇ g/ml).
  • the content of selenium in the medium can be gradually increased by increasing the gradient so that the concentration of selenium in the medium reaches C.
  • the method comprises the steps of:
  • the concentration of selenium in the medium is C ⁇ 10 ⁇ g/ml (eg ⁇ 20 ⁇ g/ml, preferably ⁇ 30 ⁇ g/ml, more preferably ⁇ 50 ⁇ g/ml, most preferably ⁇ 100 ⁇ g/ml, such as ⁇ 200 ⁇ g/ml, ⁇ 500 ⁇ g/ml).
  • the concentration of selenium in the medium is C ⁇ 1000 ⁇ g/ml (preferably, ⁇ 800 ⁇ g/ml).
  • the initially added selenium-containing component is inorganic selenium, preferably H 2 SeO 3 , and/or Na 2 SeO 4 .
  • the method comprises the steps of:
  • the method comprises the steps of:
  • a fresh first selenium-containing medium is continuously added, wherein the concentration of selenium in the first selenium-containing medium is C1, and the addition rate is 0.01 V to 1 V per 10 h to 24 h (preferably a concentration of 0.05V to 0.5V, more preferably 0.1V to 0.4V), while maintaining the total volume of the culture medium is about unchanged, removing part of the culture solution, so that the concentration of selenium in the culture solution reaches about C1;
  • a fresh second selenium-containing medium is continuously added, wherein the concentration of selenium in the second selenium-containing medium is C2, and the addition rate is 0.01 V per 10 h to 24 h. 1V (preferably 0.05V ⁇ 0.5V, more preferably 0.1V ⁇ 0.4V), while maintaining the total volume of the culture medium is about the same, remove part of the culture solution, so that the concentration of selenium in the culture solution reaches about C2;
  • a fresh N-th selenium-containing medium is continuously added, wherein the concentration of selenium in the N-th selenium-containing medium is Cn, and the addition rate is 0.01 per 10 h to 24 h.
  • V ⁇ 1V preferably 0.05V ⁇ 0.5V, more preferably 0.1V ⁇ 0.4V
  • n is a positive integer of 2 to 100 (preferably a positive integer of 3 to 50, more preferably a positive integer of 4 to 30, and most preferably a positive integer of 5 to 20, such as 6, 7, 8, 9, 10).
  • the ratio of the concentration of selenium in the selenium-containing medium used in the previous feeding to the concentration of selenium in the selenium-containing medium used in the next feeding is 1:1 to 10 (preferably 1:1.2). ⁇ 5, more preferably 1:1.5 to 3).
  • the concentration of selenium in the selenium-containing medium used in the next feeding is 0 to 500 ⁇ g/ml (preferably 10 to 200 ⁇ g higher) than the concentration of selenium in the selenium-containing medium used in the previous feeding.
  • M more preferably 20 to 100 ⁇ g/ml, such as 50 ⁇ g/ml.
  • the method further comprises the optional step of: stabilizing the culture for 1 h to 24 h during each feeding or between two feedings, in which neither the culture medium is replenished nor Remove the culture solution.
  • the method further comprises the step (3): drying the selenium-enriched microorganism obtained in the step (2) to obtain a dried microbial cell of the selenium-enriched microorganism.
  • the term “Medium” is a manually formulated nutrient for the growth and maintenance of microorganisms, and generally contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), and vitamins and water. Different media can be added with some conventional substances according to actual needs.
  • the term “medium” as used in the present invention refers to a basal medium in the art unless otherwise specified.
  • One skilled in the art can select a suitable medium depending on the type of microorganism being cultured according to the conventional technical knowledge in the art. For example, the medium can be selected by reference to the literature "Microbiology Experiment" (Higher Education Press, 2007).
  • the microorganism culture temperature is from 20 ° C to 45 ° C, preferably from 35 ° C to 40 ° C.
  • the pH of the microorganism culture is from 3.0 to 9.0, preferably from 5.5 to 8.0, such as 7.5.
  • the microbial agent of the present invention comprises the selenium-enriched microorganism according to the present invention.
  • the microbial agent may be a pharmaceutical composition, a nutraceutical composition, or a food composition.
  • the microbial agents of the invention may comprise a physiologically acceptable carrier.
  • the physiologically acceptable carrier can be a food product or a pharmaceutical carrier.
  • physiologically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when properly administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffering agents, which can be used as a vehicle for pharmaceutically acceptable substances, Excipients, binders, lubricants, gels, surfactants, and the like.
  • the microbial agent of the present invention further comprises a pharmaceutical composition comprising, as an active ingredient, a living selenium-enriched microorganism according to the present invention, and one or more pharmaceutically acceptable carriers.
  • the selenium-enriched microorganisms according to the present invention are generally mixed with excipients, diluted with an excipient or encapsulated in a carrier, and may be in the form of a liquid preparation, a capsule, or a tablet. If the excipient serves as a diluent, it can be a solid, semi-solid or liquid material (e.g., physiological saline) used as a vehicle, carrier or vehicle for the active ingredient.
  • the composition may be in the form of a tablet, a pill, a powder, a lozenge, a pouch, a flat capsule, an elixir, a suspension, an emulsion, a solution, a syrup, an aerosol (solid form or in a liquid medium). , ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and aseptically packaged powders.
  • the type of diluent can vary depending on the desired route of administration.
  • the resulting composition may contain additional agents such as preservatives.
  • the excipient or vehicle is selected depending on the mode of administration and route.
  • Suitable pharmaceutical carriers and some of the pharmaceutical ingredients used in pharmaceutical formulations are as well known in the art as Remington's Pharmaceutical Sciences (EW Martin) and USP/NF (United States Pharmacopoeia and National Formula) Said.
  • suitable excipients include: lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystals Cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose.
  • the formulations may also contain: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl- and propylparabens; sweeteners; and flavoring agents.
  • lubricants such as talc, magnesium stearate and mineral oil
  • wetting agents such as talc, magnesium stearate and mineral oil
  • emulsifying and suspending agents such as methyl- and propylparabens
  • preservatives such as methyl- and propylparabens
  • sweeteners sweeteners
  • flavoring agents such as talc, magnesium stearate and mineral oil
  • the pharmaceutical compositions can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient using methods known in the art.
  • compositions for use in the present methods can be prepared using standard techniques, including those in which the selenium-enriched microorganisms according to the present invention are encapsulated in a colloid for oral delivery.
  • the selenium-enriched microorganism according to the present invention can be placed in a capsule for oral administration by drying.
  • the selenium-enriched microorganism according to the present invention may be mixed with one or more excipients, for example, with a disintegrant, a filler, a glidant, or a preservative.
  • Suitable capsules include hard shell capsules or soft shell capsules. Capsules can be formed from any lipid-based or polymer-based colloid.
  • Exemplary polymers for colloidal preparation include gelatin, planting
  • Polysaccharides or derivatives thereof such as modified forms of carrageenan and starch, and cellulose, such as hypromellose.
  • other ingredients such as plasticizers such as glycerin and/or sorbitol may be added to the gelling agent solution to reduce the hardness of the capsule, colorants, preservatives, disintegrants, lubricants and surface treatments.
  • Agent In some embodiments, the capsule does not comprise gelatin. In other embodiments, the capsule does not comprise a plant polysaccharide or a derivative thereof.
  • compositions may be formulated in unit dosage form, each dosage form comprising a daily dose, for example from about 0.005 mg to 2000 mg, of a selenium-enriched microorganism according to the invention.
  • unit dosage form refers to physically discrete units suitable for use as a single dose in a human subject or other mammal, each unit containing a predetermined amount of active ingredient and a suitable pharmaceutical excipient, the predetermined amount being calculated Can produce the desired therapeutic effect.
  • a solid composition such as a tablet, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition comprising a homogeneous mixture of the compounds of the invention.
  • the active ingredient When these preformed mixtures are homogeneous, the active ingredient will generally be dispersed uniformly in the compositions such that the ingredients can be readily further divided into equivalent effective unit dosage forms such as tablets, pills and capsules.
  • the solid preformed mixture is then divided into unit dosage forms of the type described above containing, for example, from 0.005 mg to about 1000 mg of the selenium-enriched microorganism according to the present invention.
  • compositions may be formulated in unit dosage forms, such as, for example, from about 0.1 mg to about 1000 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg, From about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg to about 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1 mg to About 10 mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, from about 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10 mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mg to about 150
  • the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form that achieves the advantages of prolonged action.
  • a tablet or pill can contain an internal dose and an external dose component, the latter encapsulating the former.
  • the two components can be separated by an enteric layer which prevents disintegration in the stomach and allows the internal components to pass intact through the duodenum or delayed release.
  • enteric layer which prevents disintegration in the stomach and allows the internal components to pass intact through the duodenum or delayed release.
  • materials can be used for such casing layers or coatings, including various polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.
  • Liquid dosage forms which may be incorporated into the compositions for oral administration or by injection include: aqueous solutions, suitably flavored syrups; aqueous or oily suspensions; and flavored emulsions containing edible oils, for example, edible oils, for example Cottonseed oil, sesame oil, coconut oil or peanut oil, as well as tinctures and similar pharmaceutical carriers.
  • compositions of the present invention in a pharmaceutical composition can vary depending on a number of factors, including dosage, chemical nature (e.g., hydrophobicity), and route of administration.
  • a selenium-enriched microorganism according to the present invention or one or more metabolites thereof produced may be provided in a capsule comprising from about 0.005 mg to 1000 mg for oral administration.
  • Bifidobacterium longum (Latin name Bifidobacterium longum, accession number CICC 6187) was purchased from China Center of Industrial Culture Collection (CICC).
  • the main components of the RCM medium are beef extract, peptone, yeast powder, glucose, starch, sodium chloride, sodium acetate, L-cysteine hydrochloride, and the content of each component can be adjusted according to conventional knowledge in the art.
  • every 10 parts of RCM medium is divided into parts by weight: 0.1 part of beef extract, 0.05 part of peptone, 0.03 part of yeast powder, 0.05 part of glucose, 0.01 part of starch, 0.05 part of sodium chloride, 0.03 part of sodium acetate, L- Cysteine hydrochloride 0.005 parts, pH 6.8 ⁇ 0.2, the rest is water.
  • the Bifidobacterium longum was inoculated into the RCM liquid medium, and the volume ratio of the inoculum, that is, the volume ratio of the seed liquid to the RCM liquid medium was 1%, and the anaerobic culture was carried out at 37 ⁇ 1 ° C for 8 hours, which was a primary seed, and each of the seeds was 10
  • the RCM medium is divided into parts by weight: about 0.1 part of beef extract, about 0.05 part of peptone, about 0.03 part of yeast powder, about 0.05 part of glucose, about 0.01 part of starch, about 0.05 part of sodium chloride, about 0.03 part of sodium acetate.
  • L-cysteine hydrochloride is about 0.005 parts and the balance is water.
  • the first-stage seed liquid was inoculated into a 2L continuous culture device according to the inoculation volume ratio of 5%.
  • the medium liquid volume was 50%, and the anaerobic gas was continuously maintained by nitrogen gas.
  • the selenium containing a certain concentration was started.
  • the medium sodium selenite
  • the medium was continuously fed (the mass fraction of Se in Na 2 SeO 3 was 45.7%, that is, the content of Se in the Na 2 SeO 3 medium containing 100 ⁇ g/ml was 45.7 ⁇ g/ml).
  • the feeding cycle is regulated as follows:
  • the total selenium content of the prepared live powder is about 120 ⁇ g / g, 60 ⁇ g / g, 30 ⁇ g / g, 15 ⁇ g. /g, the number of viable cells is about 109-10 CFU/g.
  • the composition of the protective agent in parts by weight is: 5 parts of skim milk powder, 2 parts of trehalose, and 0.01 part of L-cysteine hydrochloride.
  • Bifidobacterium longum can be continuously cultured under different conditions of sodium selenite concentration, and the cells can be harvested according to the amount of total selenium enriched by Bifidobacterium longum.
  • CPT-11 (Irinotecan) is used in the treatment of adult metastatic colorectal cancer and can be used as a second-line treatment for patients who have failed 5-Fu chemotherapy.
  • irinotecan has also shown a certain effect on gastric cancer, esophageal cancer, and extensive small cell lung cancer.
  • the invention supplementally supplements common bifidobacteria (live bacteria number 5*10 ⁇ 8 CFU/only), sodium selenite (elemental selenium dosage 0.3 mg/kg BW) and a physical mixture thereof, selenium-enriched Bifidobacteria (elemental selenium dose 0.3mg/kg BW, viable count 5*10 ⁇ 8CFU/only), observed for CPT-11 (CPT-11 intraperitoneal dose of 75mg/kg BW, continuous four Day) causes weight loss, CID and mortality to alleviate.
  • mice in Bla Ctr group continued to increase.
  • the weight loss rate of mice in Mod Ctr group, BL group, Na2SO3 group, Sel-BL group and Phy Mix group was 23.80 ⁇ 7.88%, 20.37 ⁇ 6.48, respectively. %, 22.76 ⁇ 3.41%, 18.45 ⁇ 3.09%, 22.26 ⁇ 5.61%, among which only Sel-BL group had significant difference in weight loss rate compared with Mod Ctr group (P ⁇ 0.05, as shown in Fig. 1).
  • This result indicates that prophylactic supplementation of Sel-BL can attenuate CPT-11-induced weight loss in mice, but simply supplementing Bifidobacterium or sodium selenite or a physical mixture of the two does not improve CPT-11-induced mice. weight loss.
  • mice in the Mod Ctr group, the BL group, the Na2SO3 group, the Sel-BL group and the Phy Mix group had severe diarrhea during 3 to 6 days (the degree of diarrhea was 2 to 3).
  • the incidence rates were 14%, 11%, 16%, 3%, and 14%, respectively.
  • Only the BL group and the Sel-BL group significantly reduced the incidence of grade 2 to 3 diarrhea in mice (P ⁇ 0.05).
  • the bladder of Bla Ctr group is lustrous, the color is normal (dark yellow) without congestion and fluid retention.
  • the intestinal dilatation and edema are severe, the intestinal wall is thin and light, accompanied by mild congestion, BL
  • the group, the Na 2 SO 3 group and the physical mixed group of the two groups were not significantly different from the Mod Ctr group.
  • the Sel-BL group had significantly less intestinal dilatation and fluid retention than the Mod Ctr group, and the intestinal wall was thicker (eg Figure 4 shows the results of microscopic pathological section observation.
  • the present invention prophylactically uses different doses of Bifidobacterium-rich Bifidobacteria (the dose of elemental selenium is 0.15 mg/kg BW, 0.3 mg/kg BW, 0.6 mg/kg BW, 1.2 mg/kg BW, and the viable counts are all 5*10 ⁇ 8 CFU/only), and observed whether the weight loss, CID and mortality caused by CPT-11 (CPT-11 intraperitoneal injection of 75 mg/kg for four consecutive days) were alleviated.
  • the dose of elemental selenium is 0.15 mg/kg BW, 0.3 mg/kg BW, 0.6 mg/kg BW, 1.2 mg/kg BW, and the viable counts are all 5*10 ⁇ 8 CFU/only
  • the body weight of the Bla Ctr group continued to rise, Mod Ctr group, Sel-BL 0.15 mg/kg group, Sel-BL 0.3 mg/kg group, Sel-BL 0.6 mg/kg group and Sel-BL 1.2 mg/
  • the weight loss rate of mice in the kg group was 32.02 ⁇ 5.10%, 29.22 ⁇ 2.95%, 18.45 ⁇ 3.09%, 14.44 ⁇ 6.38%, and 27.04 ⁇ 9.50%, respectively.
  • the weight loss rate of the Sel-BL 0.3 mg/kg group and the Sel-BL 0.6 mg/kg group was statistically different (P ⁇ 0.05) compared with the Mod Ctr group.
  • the present invention observes whether it inhibits CID caused by CPT-11 chemotherapy by prophylactically using different doses of Bifidobacterium-rich Bifidobacterium. There was no diarrhea in the BlaCtr group during the experiment. The incidence of severe diarrhea in the other groups was 14%, 11%, 0, 3%, and 12% during the 3 to 6 days, respectively. Mild diarrhea (the degree of diarrhea was The incidence of grade 1) was 46%, 48%, 8%, 3%, and 7%, respectively.
  • the present invention can prevent the survival time of mice and reduce the death of mice by prophylactically using different doses of Bifidobacterium-rich Bifidobacterium. Compared with Mod Ctr, only Sel-BL 0.3mg/kg group and Sel-BL 0.6mg/kg group can significantly prolong the survival time of mice, reduce the mortality of mice, and improve the tolerance of mice to CPT-11. (log-rank test p ⁇ 0.05), as shown in Figure 8.
  • the Sel-BL 0.15 mg/kg group did not improve CTT-11-induced weight loss, CID and mouse death in mice;
  • Sel-BL 0.3 mg/kg group and Sel-BL 0.6 mg/kg group It can significantly alleviate the weight loss and CID of mice induced by CPT-11 and prolong the survival time of mice;
  • Sel-BL 1.2mg/kg can alleviate the CID of mice, but it can not alleviate the weight loss of mice caused by CPT-11. Prolonging the survival time of mice, the relief effect is limited.
  • the improvement of CPT-11-induced chemotherapy injury by selenium-enriched Bifidobacterium may be related to improving the intestinal flora structure, enhancing the body's antioxidant capacity, and improving immune function.
  • the present invention observes the activity of GSH-Px enzyme in the intestinal flora and whole blood of mice by supplementing normal mice with common bifidobacteria, sodium selenite and a physical mixture thereof, and selenium-rich bifidobacteria. influences.
  • supplementation of Bifidobacterium-rich Bifidobacterium for 8 days can significantly increase the number of bifidobacteria in the intestine (p ⁇ 0.05); supplementation of Bifidobacterium-rich Bifidobacterium can significantly increase GSH in whole blood.
  • -Px enzyme activity p ⁇ 0.05
  • the spleen is an important immune organ of the body, and the spleen index (spleen weight/body weight) is usually used to reflect the body's immune level.
  • the results of the experiment showed that the intraperitoneal injection of CPT-11 resulted in a significant decrease in the spleen index.
  • the preventive supplementation of common bifidobacteria, sodium selenite and their physical mixture could not alleviate the decline of spleen index, but preventive supplementation. It can significantly alleviate the decrease of spleen index caused by CPT-11 (P ⁇ 0.05), as shown in Figure 11. This result indicates that Bifidobacterium-rich Bifidobacterium is superior to Bifidobacterium and Sodium Selenite in alleviating the reduction of immunity caused by CPT-11 in mice.
  • Table 6 reflects that there is no diarrhea in the Bla Ctr group, and the mice in the Mod Ctr group, the BL group, the Na2SO3 group, the Sel-BL group, and the Phy Mix group have severe diarrhea within 3 to 6 days (the degree of diarrhea is 2 to 3). The incidence rates were 14%, 11%, 16%, 3%, and 14%, respectively. Only the BL group and the Sel-BL group significantly reduced the incidence of grade 2 to 3 diarrhea in mice (P ⁇ 0.05).
  • Table 7 reflects the prophylactic use of different doses of Bifidobacterium-rich Bifidobacteria to see if it inhibits CID induced by CPT-11 chemotherapy.
  • the selenium-enriched Bifidobacterium prepared by the invention is prepared by placing the microbial strain into a medium of inorganic selenium (such as sodium selenite), and the selenium is self-absorbed and transformed by the strain to make the selenium and the cells in the cell.
  • the combination of protein, amino acid and polysaccharide can convert inorganic selenium into organic selenium, which effectively reduces the toxicity of inorganic selenium.
  • Bifidobacteria is also a very important probiotic in the intestine. It is the main component of the biological barrier in the intestinal mucosal barrier. It is one of the main probiotics in the human body and participates in the digestion, nutrition, metabolism, absorption, immunity and resistance of the host.
  • the infection process can alleviate the symptoms of diarrhea in patients caused by chemotherapeutic drugs.
  • the selenium-enriched Bifidobacterium of the present invention achieves a significantly better effect in relieving intestinal damage caused by CPT-11 than ordinary Bifidobacteria, as well as a physical mixture of common bifidobacteria and selenium.
  • Example 5 Protective effect of water-soluble protein of selenium-enriched probiotics on IEC6 injury induced by LPS in rat intestinal epithelial cells
  • LPS was used to induce the IEC6 injury model of rat intestinal epithelial cells, and the selenium-probiotic preparations were investigated for IEC6 cell viability, apoptosis, mitochondrial membrane potential, and expression of intercellular tight junction proteins ZO-1 and Occludin. influences.
  • Methods divided into 5 groups, namely: negative control group (PBS treatment), model group (LPS treatment), selenium-probiotic probiotics water-soluble protein 10, 30, 100 ⁇ g / ml three concentration groups (LPS treatment, plus different concentrations Selenium-rich probiotics water-soluble protein).
  • PBS treatment negative control group
  • LPS treatment model group
  • selenium-probiotic probiotics water-soluble protein 10 30, 100 ⁇ g / ml three concentration groups (LPS treatment, plus different concentrations Selenium-rich probiotics water-soluble protein.
  • the selenium-enriched probiotic water-soluble protein was prepared from the selenium-rich Bifidobacterium described in Example 1 by a conventional method.
  • the water-soluble protein of selenium-enriched probiotics can inhibit LPS-induced intestinal epithelial damage, including inhibition of LPS-induced decrease in intestinal epithelial cell viability, apoptosis and mitochondrial membrane potential collapse.
  • the water-soluble protein of selenium-enriched probiotics can inhibit the tight junctional destruction of intestinal epithelial cells induced by LPS and increase the expression of tight junction proteins in intestinal epithelial cells.
  • the selenium-free probiotic water-soluble protein also inhibits LPS-induced intestinal epithelial cell damage and decreases the expression of tight junction proteins.
  • mice Female C57/BL6 mice were randomly divided into normal control group, model group and high- and low-dose group of selenium-rich bifidobacteria. A model of chronic alcoholic liver injury was established, and the selenium-enriched Bifidobacterium was used for intervention. After 40 days of intragastric administration, liver mass was weighed, liver coefficient was calculated, and serum alanine aminotransferase (ALT) and aspartate were measured.
  • ALT serum alanine aminotransferase
  • AST acid aminotransferase
  • TG triglyceride
  • MDA malondialdehyde
  • SOD superoxide dismutase
  • the high and low doses of Bifidobacterium-rich Bifidobacterium can significantly reduce the liver coefficient of mice with alcoholic liver injury, reduce the levels of ALT and AST in serum, and reduce the MDA content in liver tissue. Increase the SOD content in liver tissue, reduce the inflammation index in liver tissue, and reduce the pathological damage of liver tissue.
  • Selenium-enriched Bifidobacterium has a protective effect on alcoholic liver injury, and its mechanism may be related to anti-oxidation and inhibition of inflammation.
  • Example 7 Test for enhancing the immune function of mice by selenium-enriched Bifidobacterium
  • the phagocytosis rate and phagocytic index of macrophages in the high dose group of Bifidobacterium were significantly increased (P ⁇ 0.01); the delayed type hypersensitivity induced by dinitrofluorobenzene was also significantly increased (P ⁇ 0.01).
  • the ConA-induced spleen transformation ability of the mice in the Yixin probiotic group was significantly increased (P ⁇ 0.01).
  • the macrophage phagocytosis rate and phagocytic index, and dinitrofluorobenzene-induced delayed type hypersensitivity in mice were higher than normal mice, but there was no significant difference.
  • selenium-enriched Bifidobacterium can increase the mononuclear-macrophage immune function and cellular immune function of the body.
  • Example 8 Test of the effect of selenium-enriched Bifidobacterium on the regulation of intestinal flora in mice
  • mice Twelve mice were randomly divided into Bifidobacterium group and normal control group, with 6 rats in each group.
  • the bifidobacteria group was given 0.2 ml of Bifidobacterium at a concentration of 6*10 ⁇ 8 cfu/ml every day, and the normal control group was given 0.2 ml of 0.9% sodium chloride solution every day.
  • the Bifidobacterium in mouse feces was used.
  • the number of Enterobacteriaceae was the detection index, and the changes of intestinal flora in mice after 30 samples were given.
  • Bifidobacterium-rich DD98 can improve the intestinal flora, increase the number of probiotics and reduce the number of pathogenic bacteria.

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Abstract

一种含富硒双岐杆菌的微生物菌剂，用于预防和/或治疗化疗引起的肠黏膜损伤、肿瘤的辅助治疗、减轻肿瘤化疗的副作用、预防或治疗酒精性肝病、增强免疫力、调节肠道菌群以及用作肠上皮细胞保护剂。

Description

一种预防肿瘤化疗肠道毒性的微生物菌剂 技术领域
本发明属于生物技术领域；具体地说，本发明涉及一种预防肿瘤化疗肠道毒性的微生物菌剂。
背景技术
化疗是利用化学药物阻止癌细胞的增殖、浸润、转移，直至最终杀灭癌细胞的一种治疗方式。它是一种全身性治疗手段，和手术、放疗一起，并称为癌症的3大治疗手段。由于化疗药物的选择性不强，在杀灭癌细胞的同时也会不可避免地损伤人体正常的细胞，从而出现药物的不良反应。因此，在接受化疗药物的时候，一方面希望能够达到最佳的抗肿瘤作用，另一方面也要注意预防和识别化疗药物的不良反应。随着大剂量化疗在临床应用范围的扩大，化疗所引起的不良反应发生率和严重程度也随之增加。很多化疗药物会引起严重的肠黏膜屏障功能障碍，造成肠黏膜屏障的损伤。
盐酸伊立替康(CPT-11)属于喜树碱类抗癌药物，是一种强效拓扑异构酶-I抑制剂，临床上最早被用于标准化疗方案失败后的转移性结直肠癌，作为转移性结直肠癌的一线用药。但是，该药对胃肠道具有严重的毒副作用，可杀伤快速分裂增殖的肠黏膜隐窝上皮细胞而导致肠黏膜结构破坏，最终导致化疗性肠黏膜炎及化疗相关性腹泻的发生。
因此，本领域迫切需要开发能够有效预防或治疗化疗引起的肠黏膜损伤的药物。
发明内容
本发明的目的在于提供一种预防肿瘤化疗肠道毒性的微生物菌剂。
本发明的第一方面，提供了富硒微生物在制备药物中的应用，所述药物用于：
(1)预防和/或治疗肠道损伤；
(2)肿瘤的辅助治疗；和/或
(3)减轻肿瘤化疗的副作用。
在另一优选例中，所述肠道损伤为肿瘤化疗引起的肠道损伤。
在另一优选例中，所述药物还用于预防肿瘤化疗引起的体重下降。
在另一优选例中，所述药物还用于延长肿瘤患者的生存时间。
在另一优选例中，所述药物还用于提高肿瘤患者的免疫水平。
在另一优选例中，所述肠道损伤包括：小肠粘膜损伤、腹泻等。
在另一优选例中，所述富硒微生物每克干菌体中的硒含量≥2mg(如≥4mg，优选地≥5mg，更优选地≥10mg，最优选地≥20mg)。
在另一优选例中，所述富硒微生物为富硒益生菌。
在另一优选例中，所述富硒微生物为富硒双歧杆菌。
在另一优选例中，所述微生物为能够定植于哺乳动物肠道的微生物。
本发明的第二方面，提供了一种微生物菌剂，所述微生物菌剂包括活体富硒微生物。
在另一优选例中，所述富硒微生物为富硒益生菌。
在另一优选例中，所述富硒微生物为富硒双歧杆菌。
在另一优选例中，所述菌剂为液体制剂、粉剂或片剂。
在另一优选例中，所述微生物菌剂中富硒微生物的含量为1*10^7-9*10^11CFU/g，优选为1*10^9-9*10^10CFU/g。
在另一优选例中，所述富硒微生物中每10^8CFU菌体中的硒含量为0.1～100μg；优选为1～50μg，更优选为2～30μg，最优选为3～10μg。
在另一优选例中，所述微生物菌剂中硒含量为10-300μg/g，优选为20-200μg/g，更优选为30-100μg/g。
在另一优选例中，所述微生物菌剂中还包括保护剂，所述保护剂包括：脱脂奶粉5重量份，海藻糖2重量份，和L-半胱氨酸盐酸盐0.01重量份。
本发明的第三方面，提供了一种预防和/或治疗肠道损伤的方法，所述方法包括步骤；给需要的对象施用预防和/或治疗有效量的富硒微生物。
在另一优选例中，所述对象为肿瘤患者。
在另一优选例中，所述肠道损伤为肿瘤化疗引起的肠道损伤。
在另一优选例中，所述富硒微生物为富硒双歧杆菌。
在另一优选例中，所述富硒微生物的施用剂量为1*10^7-9*10^10CFU/对象，优选为1*10^8-9*10^8CFU/对象。
在另一优选例中，所述对象为人。
在另一优选例中，所述富硒微生物中每10^8CFU菌体中的硒含量为0.1～100μg；优选为1～50μg，更优选为2～30μg，最优选为3～10μg。
在另一优选例中，所述方法中在化疗前给需要的对象施用预防和/或治疗有效量的富硒微生物。
在另一优选例中，所述方法中在化疗前1-8天给需要的对象施用预防和/或治疗有效量的富硒微生物；优选地，每次施用剂量为1*10^7-1*10^10CFU/对象；更优选地，每次施用剂量为1*10^8-1*10^10CFU/对象。
在本发明的第四方面，提供了富硒微生物或第二方面所述微生物菌剂在制备药物中的应用，所述药物用于预防或治疗酒精性肝病。
在一优选例中，所述富硒微生物每克干菌体中的硒含量≥2mg(如≥4mg，优选地≥5mg，更优选地≥10mg，最优选地≥20mg)。
在一优选例中，所述富硒微生物为富硒益生菌。
在一优选例中，所述富硒微生物为富硒双歧杆菌。
在一优选例中，所述微生物为能够定植于哺乳动物肠道的微生物。
在本发明的第五方面，提供了富硒微生物或第二方面所述微生物菌剂在制备药物或保健品中的应用，所述药物用于增强免疫力。
在一优选例中，所述富硒微生物每克干菌体中的硒含量≥2mg(如≥4mg，优选地≥5mg，更优选地≥10mg，最优选地≥20mg)。
在一优选例中，所述富硒微生物为富硒益生菌。
在一优选例中，所述富硒微生物为富硒双歧杆菌。
在一优选例中，所述微生物为能够定植于哺乳动物肠道的微生物。
在本发明的第六方面，提供了富硒微生物或第二方面所述微生物菌剂在制备药物或保健品中的应用，所述药物用于调节肠道菌群。
在一优选例中，所述调节肠道菌群是指改善肠道菌群，增加益生菌数量，降低致病菌数量。
在一优选例中，所述富硒微生物每克干菌体中的硒含量≥2mg(如≥4mg，优选地≥5mg，更优选地≥10mg，最优选地≥20mg)。
在一优选例中，所述富硒微生物为富硒益生菌。
在一优选例中，所述富硒微生物为富硒双歧杆菌。
在一优选例中，所述微生物为能够定植于哺乳动物肠道的微生物。
在本发明的第七方面，提供了富硒微生物或其水溶性蛋白或第二方面所述微生物菌剂在制备药物中的应用，所述药物用作肠上皮细胞的保护剂。
在一优选例中，所述富硒微生物每克干菌体中的硒含量≥2mg(如≥4mg，优选地≥5mg，更优选地≥10mg，最优选地≥20mg)。
在一优选例中，所述富硒微生物为富硒益生菌。
在一优选例中，所述富硒微生物为富硒双歧杆菌。
在一优选例中，所述微生物为能够定植于哺乳动物肠道的微生物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为BL组、Na 2SO 3组、Sel-BL组、Phy Mix组和Mod Ctr组小鼠在第6天时的体重相对于第0天时体重下降率的对比图(n＝12)；操作方法：每天相同时间称重。
图2为BL组、Na 2SO 3组、Sel-BL和Mod Ctr组小鼠在第3天到第6天不同腹泻程度 发生率的对比图(n＝12)；操作方法：每天观察小鼠腹泻情况2次(早晚各一次)，根据文献报道方法并结合实际情况对小鼠的腹泻情况进行评分，评分标准为：0，无腹泻；1，轻度腹泻(大便可见轻微湿软)；2，中度腹泻(大便较湿且不成形，并且有轻度的肛周着色)；3，重度腹泻(水样便并伴有重度肛周着色)。
图3为Mod Ctr组、BL组、Na 2SO 3组、Sel-BL组和Phy Mix组小鼠生存率对比图(n＝12)；操作方法：每天观察小鼠死亡情况2次(早晚各一次)，持续观察时间距最后一次腹腔注射CPT-11至少14天。
图4为小鼠腹部解剖图；(A)Bla Ctr组(B)Mod Ctr组(C)Sel-BL组。
图5为Mod Ctr组、BL组、Na 2SO 3组、Sel-BL组和Phy Mix组小鼠小肠组织的HE染色图。操作方法：第6天时处死小鼠，取距回盲部2cm处的小肠组织制备石蜡切片，进行HE染色；(A)空白对照组；(B)模型对照组；(C)普通双歧杆菌组；(D)亚硒酸钠组；(E)富硒双歧杆菌组。
图6为不同剂量Sel-BL预防组和Mod Ctr组小鼠在第6天时的体重相对于第0天时体重下降率的对比图(n＝12)；操作方法：每天相同时间称重。
图7为不同剂量Sel-BL预防组和Mod Ctr组小鼠在第3天到第6天不同腹泻程度发生率的对比图(n＝12)。
图8为不同剂量Sel-BL预防组和Mod Ctr组小鼠死亡曲线对比图(n＝12)；操作方法：每天观察小鼠死亡情况2次(早晚各一次)，持续观察时间距最后一次腹腔注射CPT-11至少14天。
图9为BlaCtr组、BL组、Na 2SO 3组、Sel-BL组和Phy Mix组肠道内双歧杆菌对比图(n＝3)；方法：0天时，即第一次腹腔注射CPT-11前，收集小鼠粪便，提取粪便中肠道菌群的基因组，采用Real-Time PCR技术检测预防组相对于BlaCtr组小鼠体内双歧杆菌的变化。
图10为Sel-BL组和BlaCtr组小鼠全血中谷胱甘肽过氧化物酶(GSH-Px)活力对比图(n＝6)；血液样本采集方法：0天时，即第一次腹腔注射CPT-11前，眼眶采血，肝素钠抗凝。
图11为Mod Ctr组、BL组、Na 2SO 3组、Sel-BL组和Phy Mix组小鼠脾脏指数对比图(n＝6)；实验操作：第6天时处死小鼠，取出脾脏，称重。
图12显示了富硒益生菌水溶性蛋白增强IEC6细胞活力。其中，用50μg/ml LPS处理大鼠肠上皮细胞IEC6细胞，同时加入不同浓度的富硒益生菌水溶性蛋白，孵育24小时后，分别用MTT法检测细胞活力。*P<0.05。Se:富硒益生菌水溶性蛋白。
图13显示了富硒益生菌水溶性蛋白抑制LPS诱导的IEC6细胞凋亡。用50μg/ml LPS处理将大鼠肠上皮细胞IEC6细胞，同时加入不同浓度的富硒益生菌水溶性蛋白，孵育24小时后，用偶联了FITC的Annexin V和PI染IEC6细胞，并用流式细胞仪检测细胞凋亡。细胞凋亡检测为3次独立重复的实验结果。Se:富硒益生菌水溶性蛋白。
图14显示富硒益生菌水溶性蛋白抑制LPS诱导的IEC6细胞线粒体膜电位倒塌。用50μg/ml LPS处理将大鼠肠上皮细胞IEC6细胞，同时加入不同浓度的富硒益生菌水溶性蛋白，孵育24小时后，用JC-1染IEC6细胞，并用流式细胞仪检测细胞线粒体膜电位。细胞线粒 体膜电位检测为3次独立重复的实验结果。Se:富硒益生菌水溶性蛋白。
图15显示富硒益生菌水溶性蛋白抑制LPS诱导的IEC6细胞紧密连接蛋白ZO-1和
Occludin水平降低。用50μg/ml LPS处理将大鼠肠上皮细胞IEC6细胞，同时加入不同浓度的富硒益生菌水溶性蛋白，孵育24小时后，Q-PCR检测ZO-1和Occludin的mRNA水平。*P<0.05，**P<0.01。NS，无显著差异。Se:富硒益生菌水溶性蛋白。
图16显示富硒长双歧杆菌降低小鼠肝脏指数。用乙醇处理42小时后，收集小鼠肝脏并称重。计算肝脏指数。数值显示为平均值±SD,n＝6,*P<0.05,**P<0.01,NS,无显著性.(学生双截尾t-测试)。Se-B.L.:富硒长双歧杆菌。
图17显示富硒长双歧杆菌抑制乙醇处理后的小鼠血清ALT和AST增加。用乙醇处理42小时后，收集小鼠血清，并检测ALT和AST水平。数值显示为平均值±SD,n＝6,*P<0.05,**P<0.01,***P<0.001,NS,无显著性.(学生双截尾t-测试)。Se-B.L.:富硒长双歧杆菌。
图18显示富硒长双歧杆菌增加小鼠肝脏SOD水平并抑制小鼠肝脏MDA水平。用乙醇和不同剂量的富硒长双歧杆菌处理小鼠42小时后，检测小鼠肝脏MDA和SOD水平。数值显示为平均值±SD,n＝6,*P<0.05,**P<0.01,***P<0.001,NS,无显著性.(学生双截尾t-测试)。Se-B.L.:富硒长双歧杆菌。
图19显示富硒长双歧杆菌抑制小鼠血清TNF-α水平。用乙醇和不同剂量的富硒长双歧杆菌处理小鼠42小时后，检测小鼠血清TNF-α。数值显示为平均值±SD,n＝6,*P<0.05,**P<0.01,***P<0.001.(学生双截尾t-测试).Se-B.L.:富硒长双歧杆菌。
图20显示富硒长双歧杆菌抑制小鼠肝脏损伤。用乙醇和不同剂量的富硒长双歧杆菌处理小鼠42小时后，检测小鼠肝脏组织病理学评分。数值显示为平均值±SD,n＝6,*P<0.05,***P<0.001.(学生双截尾t-测试).Se-B.L.:富硒长双歧杆菌。
图21小时各组小鼠给药30天后迟发型变态反应(DTH)反应值(n＝6,X±SD)；其中，注，与对照组相比*P<0.05，**P<0.01。
图22显示了各组小鼠给药期间体重的变化情况(n＝6,X±SD)。
图23显示了各组小鼠给药30天后肠道内双歧杆菌数量的变化(n＝6,X±SD)；其中，与对照组相比*P<0.05，**P<0.01。
图24显示了各组小鼠给药30天后肠道内肠杆菌数量的变化(n＝6,X±SD)；其中，与对照组相比*P<0.05，**P<0.01。
具体实施方式
本发明人通过广泛而深入的研究，获得一种预防肿瘤化疗肠道毒性的微生物菌剂，所述微生物菌剂中含有富硒双歧杆菌，实验结果表明，富硒双歧杆菌对预防和/或治疗化疗引起的肠黏膜损伤具有显著的疗效，而且能够显著延长化疗动物的生存时间，因而能够用于肿瘤的辅助治疗，减轻肿瘤化疗的副作用。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意 图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
富硒微生物
本发明提供了一类富硒微生物(尤其是富硒双歧杆菌)，所述富硒微生物每克干菌体中的硒含量≥2mg(如≥4mg，优选地≥5mg，更优选地≥10mg，最优选地≥30mg)。本发明中所述的硒含量，如无特别说明均指有机硒的含量。硒含量的测定方法采用本发明中的常规的或标准的测量方法即可。比如，文献《GB 5009.93-2010食品中硒的测定》中记载的方法。
在本发明的一个优选的实施方式中，所述富硒微生物每克干菌体中的硒含量≥50mg(优选地≥100mg，更优选地≥200mg)。
在本发明的一个优选的实施方式中，所述富硒微生物每克干菌体中的硒含量≤500mg。
在本发明的一个优选地实施方式中，所述的微生物为原核微生物、或真核微生物。
在本发明的一个优选地实施方式中，所述微生物为益生菌。
在另一优选例中，所述微生物选自下组中的一种或多种：双歧杆菌(Bifidobacterium)、乳杆菌(Lactobacillus)、大肠杆菌(E.coli)。
根据本发明的富硒微生物能够有效地预防和/或治疗肿瘤化疗引起的肠道损伤。
益生菌
术语“益生菌”指在动物组织中，如人类胃肠和阴道中具有有益作用的细菌。最常用作益生菌的细菌是乳酸菌和双歧杆菌；然而，其他有益细菌，如嗜热链球菌(S.thermophilis)也可以是益生菌。在胃和小肠中增殖后，一些益生菌存活下来，并且在大肠内暂时生存，其中结肠发酵能力被正向修饰。见，例如，Roberfroid，AM J CLIN NUTR 71(SUPPL)：1682S-1687S(2000)。
本发明使用的益生菌可以是任何已知的益生菌，例如，嗜酸乳杆菌(L.acidophilus)，保加利亚乳杆菌(L.bulgaricus)，干酪乳酸杆菌(L.casei)，拟干酪乳杆菌(L.paracasei)，发酵乳杆菌(L.fermentum)，植物乳杆菌(L.plantarum)，鼠李糖乳杆菌(L.rhamnosus)，唾液乳杆菌(L.salivarius)，两歧双歧杆菌(B.bifidum)，婴儿双歧杆菌(B.infantis)，动物型双歧杆菌乳酸亚型(B.animalis subsp.Lactis)，长双歧杆菌(B.longum)，嗜热链球菌(S.thermophilis)，粪肠球菌(E.faecalis)，和屎肠球菌(E.faecium)。
应当了解上述所列仅处于描述目的，而非关于益生菌限制性说明。对于此方面，任何额外的益生菌也可以用于本发明，例如，任何额外已知的和/或可用的乳酸菌或双歧杆菌。
在本发明一个优选地实施方式中，益生菌可以包括一类对宿主有益的活性微生物，其定植于人体或动物体肠道、生殖系统内，能产生确切健康功效从而改善宿主微生态平衡、发挥有益作用。人体、动物体内有益的细菌或真菌主要有：酪酸梭菌、乳酸菌、双歧杆菌、嗜酸乳杆菌、放线菌、酵母菌等。
富硒微生物制备方法
本发明中涉及的富硒微生物，可通过下述方法制备，所述方法包括步骤：
(I)提供含有硒(外源的人工添加的硒)的培养基，并且在所述培养基中培养微生物；和
(II)分离步骤(I)中培养的微生物，从而制得所述富硒微生物；
其中，所述培养基中硒浓度C≥10μg/ml(如≥20μg/ml，优选地≥30μg/ml，更优选地≥50μg/ml，最优选地≥100μg/ml，如≥200μg/ml、≥500μg/ml)。
在本发明的一个优选的实施方式中，可以通过梯度增加的方式，逐步提高培养基中硒的含量，从而使培养基中硒浓度达到C。
在本发明的一个优选地实施方式中，所述方法包括步骤：
(I)提供含有硒的培养基，并且在所述培养基中培养微生物；和
(II)分离步骤(I)中培养的微生物，从而制得所述富硒微生物；
其中，所述培养基中硒浓度C≥10μg/ml(如≥20μg/ml，优选地≥30μg/ml，更优选地≥50μg/ml，最优选地≥100μg/ml，如≥200μg/ml、≥500μg/ml)。
在另一优选例中，所述培养基中硒浓度C≤1000μg/ml(优选地，≤800μg/ml)。
在另一优选例中，所述含有硒的培养基中，初始添加的含硒成分为无机硒，优选为H 2SeO 3、和/或Na 2SeO 4。
在另一优选例中，所述步骤(I)中，包括步骤：
(I0)在不含硒的培养基中，培养微生物；
(I1)向步骤(I0)的培养液中，连续添加新鲜的含硒浓度为C的培养基；在维持培养液总体积约不变的情况下，移除部分培养液，从而使培养液中硒浓度达到并维持在C，并继续培养。
在本发明的一个优选地实施方式中，所述步骤(I)中，包括步骤：
(I0)在不含硒的培养基中，培养微生物，所得培养物的体积为V；
(I1)第1次补料
向步骤(I0)的培养物中，连续添加新鲜的第一含硒培养基，其中所述第一含硒培养基中硒浓度为C1，添加速度为每10h～24h添加0.01V～1V(优选为0.05V～0.5V，更优选为0.1V～0.4V)，在维持培养液总体积约不变的情况下，移除部分培养液，从而使培养液中硒浓度达到约C1；
(I2)第2次补料
向第1次补料后获得的培养物中，连续添加新鲜的第二含硒培养基，其中所述第二含硒培养基中硒浓度为C2，添加速度为每10h～24h添加0.01V～1V(优选为0.05V～0.5V，更 优选为0.1V～0.4V)，在维持培养液总体积约不变的情况下，移除部分培养液，从而使培养液中硒浓度达到约C2；
……
(In)第n次补料
向第n-1次补料后获得的培养物中，连续添加新鲜的第N含硒培养基，其中所述第N含硒培养基中硒浓度为Cn，添加速度为每10h～24h添加0.01V～1V(优选为0.05V～0.5V，更优选为0.1V～0.4V)，在维持培养液总体积约不变的情况下，移除部分培养液，从而使培养液中硒浓度达到约Cn,Cn＝C；
其中，C1≤C2≤……≤Cn，n为2～100的正整数(优选为3～50的正整数，更优选为4～30的正整数，最优选为5～20的正整数，如6、7、8、9、10)。
在另一优选例中，上一次补料中所用的含硒培养基中硒浓度与下一次补料中所用的含硒培养基中硒浓度的比值为1：1～10(优选为1：1.2～5，更优选为1：1.5～3)。
在另一优选例中，下一次补料中所用的含硒培养基中硒浓度比上一次补料中所用的含硒培养基中硒浓度高0～500μg/ml(优选地高10～200μg/ml，更优选地高20～100μg/ml，如高50μg/ml)。
在另一优选例中，所述方法还包括任选地步骤：在每次补料过程中，或两次补料之间，稳定培养1h～24h，在此过程中既不补充培养液也不移除培养液。
在另一优选例中，所述方法还包括步骤(3)：对步骤(2)中获得所述富硒微生物进行干燥处理，获得富硒微生物的干菌体。
在本发明中，术语“培养基”(Medium)是供微生物生长和维持用的人工配制的养料，一般都含有碳水化合物、含氮物质、无机盐(包括微量元素)以及维生素和水等。不同培养基可根据实际需要，添加一些常规物质。如无特别说明，本发明的术语“培养基”即指本领域中的基础培养基。本领域技术人员可以根据本领域的常规技术知识根据所培养的微生物类型的选择合适的培养基。如可以参照文献《微生物学实验》(高等教育出版社,2007)选择培养基。
在本发明的较佳的实施方式中，微生物培养温度为20℃～45℃，优选为35℃～40℃。
在本发明的较佳的实施方式中，微生物培养的pH为3.0～9.0，优选为5.5～8.0，如7.5。
微生物菌剂和施用方法
本发明的微生物菌剂包含根据本发明的富硒微生物。所述微生物菌剂可以为药物组合物、保健品组合物、或食品组合物。
本发明的微生物菌剂可包括生理学上可接受的运载体。生理学上可接受的载体可以是食物产品或药物学载体。本文用术语“生理学上可接受”(或“药学上可接受”)指分子实体和组合物在恰当地给予动物或人时不产生不利、过敏性或其它不良反应。本文所述的术语“药学上可接受的运载体”包括任何和全部可为药学上可接受物质用作介质的溶剂、分散介质、包衣、抗菌剂、等渗和吸收延迟剂、缓冲剂、赋形剂、粘合剂、润滑剂、凝胶、表面活性剂等。
因此，本发明的微生物菌剂还包括药物组合物，其包含作为活性成分的根据本发明的 活体富硒微生物，以及一种或多种药学上可接受的运载体。为了制备本发明的组合物，根据本发明的富硒微生物一般与赋形剂混合，用赋形剂稀释或包裹在载体内，其形式可以是液体制剂、胶囊、片剂。如果辅料用作稀释剂，它可以是固体、半固体或液体材料(例如生理盐水)，用作活性成分的运载体、载体或介质。因此，组合物可以是片剂、丸剂、粉末剂、锭剂、药袋、扁胶囊、酏剂、混悬剂、乳剂、溶液剂、糖浆剂、气雾剂(固体形式或在液体介质中)、软膏剂、软和硬明胶胶囊、栓剂、无菌注射溶液以及无菌包装的粉末剂。如本领域所知，稀释剂类型可根据所需的给药途径改变。得到的组合物可包含额外试剂，例如防腐剂。赋形剂或运载体根据给药模式和途径选择。合适的药物运载体以及药学配方中使用的一些药学必要物如本领域熟知的教科书《雷明顿药物科学》(Remington's Pharmaceutical Sciences(E.W.Martin))，以及USP/NF(美国药典和国家配方)所述。合适的赋形剂的一些例子包括：乳糖、右旋糖、蔗糖、山梨糖醇、甘露醇、淀粉、阿拉伯胶、磷酸钙、藻酸盐、西黄蓍胶、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆和甲基纤维素。制剂还可包含：润滑剂，例如滑石、硬脂酸镁和矿物油；湿润剂；乳化剂和悬浮剂；防腐剂，例如甲-和丙羟基苯甲酸酯；甜味剂；和调味剂。药物组合物可配制成在使用本领域已知的方法施给病人后，提供活性成分的快速、持续或延迟释放。
可用标准技术制备用于本方法的药物学上可接受的组合物，包括其中根据本发明的富硒微生物被包裹在胶体中用于口腔传递的那些。根据本发明的富硒微生物可经干燥置入用于口服给药的胶囊中。在一些实施方式中，根据本发明的富硒微生物可与一种或多种辅料混合，例如与崩解剂、填充剂、助流剂或防腐剂混合。合适的胶囊包括硬壳胶囊或软壳胶囊。可用任何基于脂质或基于聚合物的胶体形成胶囊。用于胶体制备的示范性聚合物包括明胶、植
物多糖或其衍生物，例如角叉菜胶和淀粉的改性形式和纤维素，如羟丙甲纤维素。可任选的，可在成胶剂溶液中加入其他成分，例如增塑剂，如甘油和/或山梨糖醇来降低胶囊的硬度，着色剂，防腐剂，崩解剂，润滑剂和表面处理剂。在一些实施方式中，胶囊不包含明胶。在其他实施方式中，胶囊不包含植物多糖或其衍生物。
组合物可配制成单位剂型，每种剂型包括每日剂量例如约0.005mg到2000mg的根据本发明的富硒微生物。术语“单位剂量形式”指适合作为单一剂量用于人体对象或其他哺乳动物的物理上离散的单位，每个单位包含预定量的活性成分和合适的药用赋形剂，所述预定量经计算能够产生所需的治疗效果。为制备固体组合物如片剂，将主要的活性成分与药用辅料混合，形成包含本发明化合物均质混合物的固体预制组合物。当这些预制混合物为均匀时，活性成分通常均匀地分散在组合物中，使得组分能容易地进一步分成等同的有效单位剂型，例如片剂、药丸和胶囊。然后将固态预制混合物分成上述类型的单位剂型，包含例如从0.005mg到约1000mg的根据本发明的富硒微生物。
组合物可配制成单位剂型，每剂包含例如从约0.1mg到约1000mg、从约0.1mg到约40mg、从约0.1mg到约20mg、从约0.1mg到约10mg、从约0.2mg到约20mg、从约0.3mg到约15mg、从约0.4mg到约10mg、从约0.5mg到约1mg；从约0.5mg到约100mg、从约0.5mg到约50mg、从约0.5mg到约30mg,、从约0.5mg到约20mg、从约0.5mg到约10mg、 从约0.5mg到约5mg；从约1mg到约50mg、从约1mg到约30mg、从约1mg到约20mg、从约1mg到约10mg、从约1mg到约5mg；从约5mg到约50mg、从约5mg到约20mg、从约5mg到约10mg；从约10mg到约100mg、从约20mg到约200mg、从约30mg到约150mg、从约40mg到约100mg、从约50mg到约100mg活性成分。
在一些实施方式中，可对本发明的片剂或丸剂进行包衣或另行复合，提供能够实现延长作用优点的剂型。例如，片剂或丸剂可包含内部剂量和外部剂量组分，后者包封前者。两种组分可由肠衣层分离，该层阻止胃内崩解并允许内部组分完整地通过十二指肠或延迟释放。多种材料可用于此类肠衣层或包衣，这些材料包括各种聚合酸以及聚合酸与诸如虫胶、十六醇和醋酸纤维素等材料的混合物。
可掺有本发明组合物用于口服给予或通过注射给予的液体剂型包括：水性溶液剂，适当调味的糖浆；水性或油性混悬剂；和含食用油的经调味乳剂，所述食用油例如棉籽油、芝麻油、椰子油或花生油，以及酏剂和类似的药用运载体。
在药物组合物中本发明组合物的比例或浓度可根据许多因素变化，包括剂量、化学性质(例如疏水性)、以及给药途径。例如根据本发明的富硒微生物或其产生的一种或多种代谢物可以包含约0.005mg-1000mg的胶囊提供，用于口服给药。
本发明的主要优点在于：
(1)首次揭示了富硒微生物具有预防和/或治疗化疗引起的肠道损伤的作用；
(2)使用富硒双歧杆菌预防和/或治疗化疗引起的肠道损伤取得了预料不到的技术效果，不仅能够显著的降低肠道损伤，而且能够延长化疗动物的生存时间。
(3)使用富硒双歧杆菌预防和/或治疗化疗引起的肠道损伤取得了预料不到的技术效果，能够提高动物机体抗氧化能力、增强动物的免疫力。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1.富硒双歧杆菌的制备
(1)菌株
益生菌菌株
长双歧杆菌(拉丁名Bifidobacterium longum，保藏号CICC 6187)购自中国工业微生物菌种保藏管理中心(China Center of Industrial Culture Collection,CICC)。
(2)培养基
RCM培养基的主要组成为牛肉膏，蛋白胨，酵母粉，葡萄糖，淀粉，氯化钠，乙酸钠，L-半胱氨酸盐酸盐，各个成分的含量可以依据本领域的常规知识进行调整。
优选地，每10份RCM培养基按重量份数组成为：牛肉膏0.1份，蛋白胨0.05份，酵母粉0.03份，葡萄糖0.05份，淀粉0.01份，氯化钠0.05份，乙酸钠0.03份，L-半胱氨酸盐酸盐0.005份，pH6.8±0.2，其余为水。
培养方法：
将长双歧杆菌接种于RCM液体培养基中，接种量体积比，即种子液与RCM液体培养基的体积比为1％，37±1℃厌氧培养8h，为一级种子，其中每10份RCM培养基按重量份数组成为：牛肉膏约0.1份，蛋白胨约0.05份，酵母粉约0.03份，葡萄糖约0.05份，淀粉约0.01份，氯化钠约0.05份，乙酸钠约0.03份，L-半胱氨酸盐酸盐约0.005份，其余为水。将一级种子液按接种量体积比5％接种于2L连续培养装置中，培养基装液量为50％，连续通氮气保持厌氧，37±1℃培养16h后，开始使用含有一定浓度硒(亚硒酸钠)的培养基进行连续补料(Na 2SeO 3中Se质量分数为45.7％，即含100μg/ml的Na 2SeO 3培养基中折合Se的含量为45.7μg/ml)，补料周期调控如下表：
Figure PCTCN2018083418-appb-000001
在连续培养的过程中，于第20天放出500ml发酵液离心浓缩，弃上清，用无菌的0.9％氯化钠溶液重悬菌泥再离心，至上清液无色澄清、离心后无红色颗粒物质集中沉淀在底部，倒出菌泥，小心勿将底部红色较紧实的菌泥倒出。将收集的菌泥与保护剂分别以重量比1比2、1比4、1比8、1比16混匀后进行真空冷冻干燥，制得活菌粉。根据菌泥(每克菌泥相当于约0.05克干菌体)与保护剂混合的比例不同，最终制得的活菌粉中总硒含量约120μg/g、60μg/g、30μg/g、15μg/g，活菌数在约109-10CFU/g。保护剂按重量份数的组成为：脱脂奶粉5份，海藻糖2份，L-半胱氨酸盐酸盐0.01份。
在本实施例中，可实现长双歧杆菌在不同亚硒酸钠浓度的条件下进行连续培养，并可 根据长双歧杆菌菌体所富集的总硒的量收获菌体。
菌体中硒含量的检测方法参考文献《GB 5009.93-2010食品中硒的测定》。
表1.英文缩写词表
Figure PCTCN2018083418-appb-000002
实施例2
CPT-11(伊立替康)用于成人转移性大肠癌的治疗，对于经含5－Fu化疗失败的患者，可作为二线治疗。同时，伊立替康应对胃癌、食管癌、广泛期小细胞肺癌也表现出了一定的疗效。
表2.图1-5和图11中各组活菌数和元素硒的每次给药剂量(一日两次)
Figure PCTCN2018083418-appb-000003
表3.图6-8各组活菌数和元素硒的每次给药剂量(一日两次)
Figure PCTCN2018083418-appb-000004
表4.图1-8和图11的给药方式
Figure PCTCN2018083418-appb-000005
注：预防组每天的用药剂量如表2和表3所示
使用SPSS 20.0软件对数据进行统计学分析，计量资料数据均以X±S表示，两组之间体重变化、脾脏指数、GSH-Px活力、肠道菌群比较采用Independent-samples T test进行分析，腹泻发生率及严重程度比较采用Non-parametric Mann-Whitney U test进行统计分析，死亡率差异采用Log-rank test and Kaplan-Meier Survival Analysis进行统计分析，两组以上计量数据采用One-Way ANOVA Scheffe进行统计分析。P<0.05表示差异显著。
相比于普通双歧杆菌、亚硒酸钠及其二者的物理混合物，富硒双歧杆菌的预防性用药能够显著改善CPT-11导致的小鼠体重下降、CID以及小鼠的死亡。
本发明通过预防性补充普通双歧杆菌(活菌数5*10^8CFU/只)、亚硒酸钠(元素硒的给药剂量0.3mg/kg BW)及其二者的物理混合物、富硒双歧杆菌(元素硒的给药剂量0.3mg/kg BW，活菌数5*10^8CFU/只)，观察其对CPT-11(CPT-11腹腔注射的剂量为75mg/kg BW，连续四天)导致的体重下降、CID和死亡率是否有缓解作用。
实验期间，Bla Ctr组小鼠体重在持续上升，Mod Ctr组、BL组、Na2SO3组、Sel-BL组和Phy Mix组小鼠在第6天时体重下降率分别为23.80±7.88％，20.37±6.48％，22.76±3.41％，18.45±3.09％，22.26±5.61％，其中有且只有Sel-BL组相比于Mod Ctr组体重下降率具有显著性差异(P<0.05，如图1所示)。该结果表明，预防性补充Sel-BL能够缓解CPT-11导致的小鼠体重下降，但是单纯补充普通双歧杆菌或者亚硒酸钠或者二者的物理混合物都不能改善CPT-11导致的小鼠体重下降。
实验期间，Bla Ctr组小鼠均未出现腹泻，Mod Ctr组、BL组、Na2SO3组、Sel-BL组和Phy Mix组小鼠在3～6天期间严重腹泻(腹泻程度为2～3级)发生率分别为14％、11％、16％、3％、14％，只有BL组和Sel-BL组能够显著降低小鼠2～3级腹泻发生率(P<0.05)。
实验第6天时，处死残余小鼠，收集小鼠的小肠组织。肉眼可见Bla Ctr组小鼠肠管有光泽，颜色正常(深黄色)无充血及液体潴留现象，Mod Ctr组小鼠肠管扩张和水肿严重，肠壁薄且透光，并伴有轻度充血，BL组、Na 2SO 3组及其二者的物理混合组与Mod Ctr组差别不大，Sel-BL组的小鼠肠管扩张和液体潴留程度明显轻于Mod Ctr组，且肠壁较厚(如图4所示)；微观病理切片观察结果如图5所示，由图A可看出，空白对照组小鼠小肠结构完整清晰，绒毛和隐窝的形态明显，肠粘膜上皮细胞排列紧密有序。由图B可看到，Mod Ctr组小鼠肠黏膜组织结构被完全破坏，上皮细胞萎缩，绒毛及隐窝结构基本消失，炎症细胞浸润明显。由图C、D、E可看到，普通双歧杆菌组、亚硒酸钠组和富硒双歧杆菌组小鼠的小肠也都遭到了破坏，但破坏程度不一。富硒双歧杆菌组在黏膜底部可看到一些较完整的隐窝结构，且可以看到较完整的绒毛结构，其肠粘膜受损伤程度明显轻于其他组别。
生存曲线结果表明，虽然普通双歧杆菌对延长小鼠生存时间、降低小鼠死亡率没有改善作用。相比于普通双歧杆菌、亚硒酸钠及其二者的物理混合物，富硒双歧杆菌有效地降低了小鼠的死亡率、延长了小鼠的生存时间(log-rank test p＜0.05)，如图3所示。
以上实验结果表明，相比于普通双歧杆菌、亚硒酸钠及其二者的物理混合物，富硒双歧杆菌的预防性用药在减轻CPT-11导致的小鼠体重下降、CID以及延长小鼠存活时间方面具有明显优势。
实施例3
本发明通过预防性使用不同剂量的富硒双歧杆菌(元素硒的剂量分别为0.15mg/kg BW，0.3mg/kg BW，0.6mg/kg BW，1.2mg/kg BW，活菌数均为5*10^8CFU/只)，观察其对CPT-11(CPT-11腹腔注射的剂量为75mg/kg，连续四天)导致的体重下降、CID和死亡率是否有缓解作用。
实验期间，Bla Ctr组小鼠体重在持续上升，Mod Ctr组、Sel-BL 0.15mg/kg组、Sel-BL0.3mg/kg组、Sel-BL 0.6mg/kg组和Sel-BL 1.2mg/kg组小鼠在第6天时体重下降率分别为32.02±5.10％，29.22±2.95％，18.45±3.09％，14.44±6.38％，27.04±9.50％。如图6所示，与Mod Ctr组相比，Sel-BL 0.3mg/kg组和Sel-BL 0.6mg/kg组的体重下降率具有统计学差异(P<0.05)。
本发明通过预防性使用不同剂量的富硒双歧杆菌，观察其对CPT-11化疗导致的CID是否有抑制作用。BlaCtr组小鼠在实验期间均未出现腹泻，其他组小鼠在3～6天期间严重腹泻的发生率分别为14％、11％、0、3％、12％，轻度腹泻(腹泻程度为1级)的发生率分别为46％、48％、8％、3％、7％。实验结果显示，与Mod Ctr相比，Sel-BL 0.15mg/kg组不能缓解CPT-11导致的CID(P>0.05)，Sel-BL 0.3mg/kg组、Sel-BL 0.6mg/kg组和Sel-BL 1.2mg/kg组能够有效减轻CPT-11导致的CID(P<0.05)。
本发明通过预防性使用不同剂量的富硒双歧杆菌，观察其能否延长小鼠的生存时间、降低小鼠的死亡。与Mod Ctr相比，只有Sel-BL 0.3mg/kg组和Sel-BL 0.6mg/kg组能够显著延长小鼠的生存时间、降低小鼠的死亡率，提高小鼠对CPT-11的耐受性(log-rank test p＜0.05)，如图8所示。
综上所述，Sel-BL 0.15mg/kg组对CPT-11导致的小鼠体重下降、CID和小鼠死亡无改善作用；Sel-BL 0.3mg/kg组和Sel-BL 0.6mg/kg组能够显著缓解CPT-11导致的小鼠体重下降和CID，延长小鼠的存活时间；Sel-BL 1.2mg/kg虽然能缓解小鼠CID，但不能缓解CPT-11导致的小鼠体重下降、不能延长小鼠的生存时间，其缓解作用是有限的。
上述结果表明，Sel-BL以剂量依赖性的方式防治CPT-11导致的小鼠体重下降、CID和小鼠死亡。
实施例4
富硒双歧杆菌改善CPT-11导致的化疗损伤可能与其改善小鼠肠道菌群结构、增强机体 的抗氧化能力、改善免疫功能有关。
本发明通过给正常小鼠补充普通双歧杆菌、亚硒酸钠及其二者的物理混合物、富硒双歧杆菌，观察其对小鼠肠道菌群和全血中GSH-Px酶活力的影响。如图9所示，与BlaCtr相比，补充富硒双歧杆菌8天，可以使肠道中双歧杆菌的数目显著增高(p＜0.05)；补充富硒双歧杆菌能够显著提高全血中GSH-Px的酶活力(p＜0.05)，如图10所示。
脾脏是机体重要的免疫器官，脾脏指数(脾脏重/体重)通常用来反映机体的免疫水平。实验结果显示，小鼠腹腔注射CPT-11后会导致脾脏指数显著下降，预防性补充普通双歧杆菌、亚硒酸钠及其二者的物理混合物都不能缓解脾脏指数的下降，但预防性补充能显著缓解CPT-11导致的脾脏指数下降(P<0.05)，如图11所示。该结果表明，富硒双歧杆菌在缓解CPT-11导致的小鼠免疫力降低方面优于普通双歧杆菌和亚硒酸钠。
以上实验结果表明，在提高肠道中双歧杆菌的数目、提高全血中GSH-Px酶活力、改善小鼠免疫力方面，富硒双歧杆菌相比于普通双歧杆菌和亚硒酸钠及其二者的物理混合物具有明显优势。
表6.双歧杆菌、亚硒酸钠及其二者物理混合物和Sel-BL对CID的防治作用
Figure PCTCN2018083418-appb-000006
表6反映了Bla Ctr组小鼠均未出现腹泻，Mod Ctr组、BL组、Na2SO3组、Sel-BL组和Phy Mix组小鼠在3～6天期间严重腹泻(腹泻程度为2～3级)发生率分别为14％、11％、16％、3％、14％，只有BL组和Sel-BL组能够显著降低小鼠2～3级腹泻发生率(P<0.05)。
表7.不同剂量的Sel-BL对CID的防治作用
Figure PCTCN2018083418-appb-000007
Figure PCTCN2018083418-appb-000008
表7反映了预防性使用不同剂量的富硒双歧杆菌，观察其对CPT-11化疗导致的CID是否有抑制作用。BlaCtr组小鼠在实验期间均未出现腹泻，其他组小鼠在3～6天期间严重腹泻的发生率分别为14％、11％、0、3％、12％，轻度腹泻(腹泻程度为1级)的发生率分别为46％、48％、8％、3％、7％。实验结果显示，与Mod Ctr相比，Sel-BL 0.15mg/kg组不能缓解CPT-11导致的CID(P>0.05)，Sel-BL 0.3mg/kg组、Sel-BL 0.6mg/kg组和Sel-BL 1.2mg/kg组能够有效减轻CPT-11导致的CID(P<0.05)。
本发明制备的富硒双歧杆菌是将微生物菌种放入无机硒(如亚硒酸钠)的培养基中培养，通过菌种对硒进行自主吸收和转化，使硒与菌体细胞内的蛋白质、氨基酸、多糖等结合，从而将无机硒转化为有机硒，有效降低了无机硒的毒性。双歧杆菌也是肠道中的一种非常重要的益生菌，是肠黏膜屏障中生物屏障的主要成分，是人体主要的益生菌之一，参与了宿主的消化、营养、代谢、吸收、免疫及抗感染过程，尤其在维持机体肠黏膜屏障的完整性方面起重要作用，能够减轻由化疗药导致的患者的腹泻症状。实验证明，本发明的富硒双歧杆菌在缓解由CPT-11导致的肠道损伤所取得的效果显著优于普通双歧杆菌，以及普通双歧杆菌和硒的物理混合物。
实施例5.富硒益生菌水溶性蛋白对LPS引起的大鼠肠上皮细胞IEC6损伤的保护作用
在本实施例中，应用LPS诱导大鼠肠上皮细胞IEC6损伤模型，考察富硒益生菌制剂对IEC6细胞活力、细胞凋亡、细胞线粒体膜电位以及细胞间紧密连接蛋白ZO-1和Occludin表达的影响。
方法：分5组，分别为：阴性对照组(PBS处理)，模型组(LPS处理)，富硒益生菌水溶性蛋白10、30、100μg/ml 3个浓度组(LPS处理，加不同浓度的富硒益生菌水溶性蛋白)。其中，富硒益生菌水溶性蛋白采用常规方法从实施例1所述的富硒双歧杆菌制备。
LPS处理24小时后，分别检测细胞活力(MTT法)；细胞凋亡(Annexin-V/FITC双染检测)，线粒体膜电位(JC-1染色)，肠上皮细胞紧密连接蛋白ZO-1和Occludin表达。
结论：如图12-15所示，富硒益生菌水溶性蛋白能抑制LPS诱导的肠上皮损伤，包括抑制LPS引起的肠上皮细胞活力下降，细胞凋亡及线粒体膜电位倒塌。富硒益生菌水溶性蛋白能抑制LPS引起的肠上皮细胞紧密连接破坏，增加肠上皮细胞紧密连接蛋白表达。无硒益生菌水溶性蛋白也能抑制LPS诱导的肠上皮细胞损伤，减少紧密连接蛋白表达下降。
实施例6.富硒益生菌对小鼠酒精性肝病的保护作用
在本实施例中，研究了富硒长双歧杆菌对酒精性肝损伤的保护作用与作用机制。将雌性C57/BL6小鼠随机分为正常对照组、模型组及富硒长双歧杆菌高、低剂量组。建立慢性酒精性肝损伤模型，用富硒长双歧杆菌干预，灌胃给药40d后，称取肝质量，计算肝脏系数， 测定小鼠血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)活力、三酰甘油(TG)、肝脏内丙二醛(MDA)和超氧化物歧化酶(SOD)的含量，HE染色观察小鼠肝脏病理变化，评价炎症指数。
结果如图16-20所示，富硒长双歧杆菌高、低剂量组均能明显降低酒精性肝损伤小鼠的肝脏系数，降低其血清中ALT、AST水平，并降低肝组织中MDA含量，升高肝组织中SOD含量，降低肝组织中炎症指数，减少肝组织病理损伤。富硒长双歧杆菌对酒精性肝损伤有一定保护作用，其机制可能与抗氧化作用及抑制炎症有关。
实施例7.富硒长双歧杆菌增强小鼠免疫力功能的测试
相比正常对照小鼠，双歧杆菌高剂量组小鼠巨噬细胞吞噬率和吞噬指数明显升高(P＜0.01)；二硝基氟苯诱导小鼠迟发型变态反应亦明显增高(P＜0.01)。衡欣益生菌组小鼠ConA诱导脾转化能力明显升高(P＜0.01)。双歧杆菌低剂量组小鼠在巨噬细胞吞噬率和吞噬指数，及二硝基氟苯诱导小鼠迟发型变态反应中，相比正常小鼠有增高，但无显著性差异。
结论如图21所示，富硒长双歧杆菌可以提高机体的单核-巨噬细胞免疫功能、细胞免疫功能。
本研究评估双歧杆菌菌株DD98口服灌喂给健康ICR小鼠后对其机体免疫功能的影响。研究证明这株菌均可以增强迟发型变态反应，提高自然杀伤细胞活性以及腹腔巨噬细胞的吞噬活性。表明菌株能够增强机体免疫力并且较低剂量的菌株(10 6cfu/mL)即可发挥免疫增强功能。
实施例8.富硒长双歧杆菌对调节小鼠肠道菌群影响的测试
在本实施例中，研究了富硒双歧杆菌对小鼠肠道菌群的影响。
方法：12只小鼠，随机分为双歧杆菌组以及正常对照组，每组6只。双歧杆菌组每天灌服0.2ml浓度为6*10^8cfu/ml的双歧杆菌，正常对照组每天灌服0.2ml 0.9％氯化钠溶液,30d后，以小鼠粪便中的双歧杆菌、肠杆菌数量为检测指标，研究给予样品后30后小鼠肠道菌群的变化。
结果如图22-24所示，相比对照组，双歧杆菌组小鼠，肠道内双歧杆菌数量明显增加，肠杆菌数量明显下降。
结论：富硒双歧杆菌DD98可以改善肠道菌群，增加益生菌数量，降低致病菌数量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 富硒微生物在制备药物中的应用，所述药物用于：
    (1)预防和/或治疗肠道损伤；
    (2)肿瘤的辅助治疗；和/或
    (3)减轻肿瘤化疗的副作用。
  2. 如权利要求1所述的应用，其特征在于，所述肠道损伤为肿瘤化疗引起的肠道损伤。
  3. 如权利要求1所述的应用，其特征在于，所述肠道损伤包括：小肠粘膜损伤、腹泻等。
  4. 如权利要求1所述的应用，其特征在于，所述富硒微生物每克干菌体中的硒含量≥2mg(如≥4mg，优选地≥5mg，更优选地≥10mg，最优选地≥20mg)。
  5. 如权利要求1所述的应用，其特征在于，所述富硒微生物为富硒益生菌；优选地，所述富硒微生物为富硒双歧杆菌。
  6. 如权利要求1所述的应用，其特征在于，所述微生物为能够定植于哺乳动物肠道的微生物。
  7. 一种微生物菌剂，其特征在于，所述微生物菌剂包括活体富硒微生物。
  8. 如权利要求7所述的微生物菌剂，其特征在于，所述微生物菌剂中富硒微生物的含量为1*10^7-9*10^11CFU/g，优选为1*10^9-9*10^10CFU/g。
  9. 如权利要求7所述的微生物菌剂，其特征在于，所述富硒微生物中每10^8CFU菌体中的硒含量为0.1～100μg；优选为1～50μg，更优选为2～30μg，最优选为3～10μg。
  10. 一种预防和/或治疗肠道损伤的方法，其特征在于，所述方法包括步骤；给需要的对象施用预防和/或治疗有效量的富硒微生物。
  11. 富硒微生物或权利要求7-9中任一项所述微生物菌剂在制备药物中的应用，所述药物用于预防或治疗酒精性肝病。
  12. 富硒微生物或权利要求7-9中任一项所述微生物菌剂在制备药物或保健品中的应用，所述药物用于增强免疫力。
  13. 富硒微生物或权利要求7-9中任一项所述微生物菌剂在制备药物或保健品中的应用，所述药物用于调节肠道菌群。
  14. 富硒微生物或其水溶性蛋白或权利要求7-9中任一项所述微生物菌剂在制备药物中的应用，所述药物用作肠上皮细胞的保护剂。
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