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WO2022062496A1 - 一种全菌胶囊及其制备方法和应用 - Google Patents

一种全菌胶囊及其制备方法和应用 Download PDF

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Publication number
WO2022062496A1
WO2022062496A1 PCT/CN2021/100784 CN2021100784W WO2022062496A1 WO 2022062496 A1 WO2022062496 A1 WO 2022062496A1 CN 2021100784 W CN2021100784 W CN 2021100784W WO 2022062496 A1 WO2022062496 A1 WO 2022062496A1
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bacteria
fecal
fecal bacteria
preparation
freeze
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PCT/CN2021/100784
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English (en)
French (fr)
Inventor
陆敏
卜雯丽
刘丹
张水龙
金红梅
朱永亮
朱蒙蒙
穆晓静
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苏州普瑞森生物科技有限公司
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Priority to US18/005,380 priority Critical patent/US20230277605A1/en
Priority to JP2023521812A priority patent/JP2023531096A/ja
Priority to EP21870878.2A priority patent/EP4151225A1/en
Publication of WO2022062496A1 publication Critical patent/WO2022062496A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/24—Mucus; Mucous glands; Bursa; Synovial fluid; Arthral fluid; Excreta; Spinal fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Definitions

  • the application belongs to the technical field of fecal bacteria transplantation, and relates to a whole bacteria capsule and a preparation method and application thereof.
  • Microbiota transplantation is a technique for transplanting functional flora from healthy feces into the patient's intestine, establishing a new intestinal flora in the patient's intestine, and treating both intestinal and extra-intestinal diseases.
  • FMT has made breakthroughs in the treatment of intestinal and extra-intestinal diseases, and clinically high hopes are placed on FMT for the treatment of difficult diseases in and out of the intestine.
  • fecal microbiota transplantation has a good effect in the treatment of diseases caused by intestinal flora imbalance, such as Clostridium difficile infection, irritable bowel syndrome, and inflammatory bowel disease.
  • diseases caused by intestinal flora imbalance such as Clostridium difficile infection, irritable bowel syndrome, and inflammatory bowel disease.
  • bacterial flora transplantation is mainly performed through enema, nasogastric tube, duodenum, jejunum, and colonoscopy, but these methods all involve invasive transplantation, and the patient experience is poor.
  • CN209967402U discloses a fecal bacteria transplantation bolus catheter through gastrointestinal endoscope, comprising a hollow handle and a catheter for bolus injection of fecal bacteria, one end of the handle is provided with a connecting part for connecting the catheter, and a side of the handle away from the catheter is provided There are syringes for injecting fecal bacteria.
  • Using the catheter for fecal bacteria transplantation can make the entire bolus injection process under the condition of visualization, and can avoid the fecal bacteria from polluting the inspection instrument, and strengthen the standardization of the operation under the endoscope of fecal bacteria transplantation.
  • the device needs to be used in conjunction with enema treatment, which is likely to cause secondary injury to patients with weak constitution or intestinal erosion damage.
  • CN110638783A discloses a preparation method of capsules.
  • the healthy and high-quality feces of fecal bacteria transplant donors are used to simulate the enzymatic hydrolysis of Codonopsis saponins by intestinal microorganisms in vitro, and through appropriate fermentation conditions, Codonopsis saponins are degraded and converted into secondary saponins. Active small molecule substances, and then exert their efficacy.
  • the fecal bacteria fermentation liquid containing active small molecular substances is put into enteric capsule shells and used by patients, which can improve the intestinal flora and at the same time enable patients to better absorb active substances, which is beneficial to health.
  • this scheme does not carry out operations such as preservation of beneficial bacteria, which is easy to inactivate and needs to be used as soon as possible in a short period of time, and is not suitable for long-term preservation.
  • CN109010380A discloses a process of intestinal flora preparation in the clinical treatment of fecal bacteria transplantation, including three steps of feces collection, preparation of intestinal flora preparation and filling of intestinal flora preparation, wherein the preparation of intestinal flora preparation adopts A new separation method of "aerobic + anaerobic" was developed to maximize the protection of the flora diversity of "aerobic bacteria” and “anaerobic bacteria” in the intestinal tract, and glycerol protective agent was added to maintain the activity of bacteria.
  • the process needs to mix a certain volume of the suspension with the glycerol solution during operation, and the prepared intestinal flora preparation has low bacterial concentration and low efficacy.
  • fecal bacteria transplantation is mainly through mechanical intervention, the process is complicated, and patients have certain pain.
  • the present application provides a whole-bacteria capsule, a preparation method and application thereof, wherein the whole-bacteria capsule is filled with lyophilized powder of fecal bacteria donors screened by high-throughput sequencing, which improves the efficiency of bacterial colony transplantation and comfort, it has a wide range of clinical application prospects.
  • the application provides a preparation method of a whole bacterial capsule, the preparation method comprising the following steps:
  • step (3) after mixing the fecal bacteria liquid prepared in step (2) with the freeze-drying protective agent, cooling and freezing and vacuum drying are carried out, and the obtained fecal bacteria freeze-dried powder is loaded into the capsule shell to obtain the whole bacteria capsule.
  • the use of high-throughput sequencing to screen fecal bacteria donors is conducive to accurately identifying the flora composition of fecal bacteria donors, and has advantages in discovering unknown species and pathogens of fecal bacteria donors, and the detection resolution reaches strains. level, to achieve the effect of detecting multiple fecal bacteria donors at one time.
  • the method for screening fecal bacteria donors by high-throughput sequencing described in step (1) comprises the following steps:
  • high-throughput sequencing is used to identify the fecal bacteria composition of the fecal bacteria donor, and compare it with the intestinal flora of the fecal bacteria recipient (patient), which is conducive to screening different fecal bacteria for different fecal bacteria receptors Donors can achieve "accurate matching" and improve drug efficacy; high-throughput sequencing can also identify pathogenic bacteria, which is conducive to excluding potentially risky fecal bacteria donors for quality monitoring.
  • the microorganism database in step (2') includes any one or a combination of at least two of bacterial genomes, fungal genomes or viral genomes derived from public databases.
  • the pathogenic bacteria database in step (3') includes pathogenic bacteria genomes derived from public databases.
  • public databases such as NCBI and KEGG are used to construct a microbial database and a pathogenic bacteria database, and the sequencing data of fecal bacteria donors are compared with the above-mentioned databases, so as to realize the identification of microorganisms at the bacterial level, and eliminate the fecal bacteria carrying pathogenic bacteria. Bacteria donors, reducing health risks.
  • high-throughput sequencing is used to screen the fecal bacteria donors according to the biomarkers expressed by the fecal bacteria donors and the diversity index of the biomarkers.
  • the biomarkers include Escherichia coli, Clostridium ramosum, Eubacterium cylindroides, Roseburia hominis, Faecalibacterium prausnitzii), Bacteroides fragilis, or Bacteroides vulgatus, or a combination of at least two.
  • the diversity index of the biomarker comprises an alpha diversity index of the biomarker.
  • the preparation method of the described fecal bacteria liquid in step (2) comprises the following steps:
  • the feces of fecal bacteria donors are collected on-site using sterile containers.
  • the collected feces are sent to the laboratory for information registration, feces identification, weighing, evaluation and processing within 1 hour.
  • the feces are donated within 6 hours in an anaerobic environment.
  • the preparation of fecal bacteria solution is completed.
  • the temperature of the sterile physiological saline in step (1") is 3 to 5°C, for example, 3°C, 4°C or 5°C.
  • the filtering in step (1") is performed by using a filter screen.
  • the aperture of the filter screen is 0.25-2 mm, for example, it may be 0.25 mm, 0.5 mm, 1.0 mm or 2.0 mm.
  • the filtration in step (1") includes sequentially using 2.0mm, 1.0mm, 0.5mm and 0.25mm filter screens to remove large particles, and then using 0.25mm filter screen for 2 to 3 times of filtering, and the obtained liquid phase is Fecal filtrate.
  • the rotational speed of the centrifugal treatment in step (2") is 1500-3000r/min, such as 1500r/min, 1600r/min, 1700r/min, 1800r/min, 1900r/min, 2000r/min, 2100r/min min, 2200r/min, 2300r/min, 2400r/min, 2500r/min, 2600r/min, 2700r/min, 2800r/min, 2900r/min or 3000r/min.
  • the time of the centrifugation in step (2") is 10-20 min, such as 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.
  • the lyophilized protective agent in step (3) includes skim milk powder, trehalose, sucrose, vitamin C and sterile physiological saline.
  • the freeze-drying protective agent comprises by mass percentage: skimmed milk powder 10%-20%, trehalose 10%-15%, sucrose 1%-10%, vitamin C 1%-5%, and the balance is physiological brine.
  • the freeze-drying protective agent can effectively prolong the survival time of the flora and improve the colonization effect of the flora.
  • the volume ratio of the fecal bacteria liquid and the freeze-drying protective agent in step (3) is (2 ⁇ 5):1, such as 2:1, 3:1, 4:1 or 5:1, Preferably 3:1.
  • the cooling and freezing conditions in step (3) are as follows: 10-20s from room temperature to 3-6°C, 1-2°C/min from 3-6°C to -30--50°C, 4-5°C /min from -30 ⁇ -50°C to -75 ⁇ -80°C.
  • the cooling and freezing time in step (3) is 12-24h, for example, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h.
  • the vacuum degree of the vacuum drying in step (3) is 5-15pa, for example, it can be 5pa, 6pa, 7pa, 8pa, 9pa, 10pa, 11pa, 12pa, 13pa, 14pa or 15pa.
  • the temperature of the vacuum drying in step (3) is -50 to -60°C, for example, it can be -50°C, -51°C, -52°C, -53°C, -54°C, -55°C, -56°C °C, -57°C, -58°C, -59°C or -60°C.
  • the vacuum drying time in step (3) is 24-48h, for example, it can be 24h, 30h, 36h, 42h or 48h.
  • the capsule shell in step (3) comprises an enteric-coated capsule shell.
  • the enteric-coated capsule shell can effectively resist the decomposition of gastric juice, and the effective flora in the capsule will be released only under the condition of intestinal pH value, which can effectively protect the flora from reaching the intestine, prevent the premature release of the flora, and reduce the number of bacteria in the capsule.
  • the loss of activity of the colony prolongs the release time of the colony, and achieves a specific colonization effect, which is conducive to the realization of oral administration.
  • the whole bacteria capsules are stored at a temperature of -75 to -80°C.
  • the application provides a preparation method of whole bacteria capsules, and the preparation method comprises the following steps:
  • step (2) Collect the feces of the fecal bacteria donors screened in step (1), soak them in sterile saline at 3 to 5°C, and use 2.0mm, 1.0mm, 0.5mm and 0.25mm filters in order to remove large particles, and then Using a 0.25mm filter screen for 2 to 3 times, the obtained liquid phase is fecal filtrate; centrifuge the fecal filtrate at 1500 to 3000 r/min for 10 to 20 minutes, and mix the precipitate with sterile physiological saline to obtain fecal bacterial liquid; and
  • the fecal bacteria liquid prepared in step (2) and the freeze-drying protective agent are mixed in a volume ratio of (2 ⁇ 5): 1, and the freeze-drying protective agent comprises by mass percentage: skim milk powder 10% ⁇ 20%, trehalose 10% to 15%, sucrose 1% to 10%, vitamin C 1% to 5%, the balance is normal saline, and then within 10 to 20s from room temperature to 3 to 6 °C, and further The cooling rate of 1 ⁇ 2°C/min is reduced from 3 ⁇ 6°C to -30 ⁇ -50°C, and the cooling rate of 4 ⁇ 5°C/min is reduced from -30 ⁇ -50°C to -75 ⁇ -80°C.
  • the obtained fecal bacteria freeze-dried powder is put into the enteric-coated capsule shell to obtain the whole bacteria capsule, which is stored at -75 ⁇ -80°C.
  • the application provides a preparation method of whole bacteria capsules, and the preparation method comprises the following steps:
  • step (2) Collect the feces of the fecal bacteria donors screened in step (1), soak them in sterile saline at 3 to 5°C, and use 2.0mm, 1.0mm, 0.5mm and 0.25mm filters in order to remove large particles, and then Using a 0.25mm filter screen for 2 to 3 times, the obtained liquid phase is fecal filtrate; centrifuge the fecal filtrate at 1500 to 3000 r/min for 10 to 20 minutes, and mix the precipitate with sterile physiological saline to obtain fecal bacterial liquid; and
  • the fecal bacteria liquid prepared in step (2) and the freeze-drying protective agent are mixed in a volume ratio of (2 ⁇ 5): 1, and the freeze-drying protective agent comprises by mass percentage: skim milk powder 10% ⁇ 20%, trehalose 10% to 15%, sucrose 1% to 10%, vitamin C 1% to 5%, the balance is normal saline, and then within 10 to 20s from room temperature to 3 to 6 °C, and further The cooling rate of 1 ⁇ 2°C/min is reduced from 3 ⁇ 6°C to -30 ⁇ -50°C, and the cooling rate of 4 ⁇ 5°C/min is reduced from -30 ⁇ -50°C to -75 ⁇ -80°C.
  • the obtained fecal bacteria freeze-dried powder is put into the enteric-coated capsule shell to obtain the whole bacteria capsule, which is stored at -75 ⁇ -80°C.
  • the present application provides a whole-bacteria capsule prepared by the preparation method described in the first aspect.
  • the present application provides a pharmaceutical composition comprising the whole bacterial capsule described in the second aspect.
  • the pharmaceutical composition further includes a PD-1 inhibitor.
  • the pharmaceutical composition further comprises any one or a combination of at least two pharmaceutically acceptable carriers, diluents or excipients.
  • the present application provides the application of the whole bacterial capsule described in the second aspect and/or the pharmaceutical composition described in the third aspect in the preparation of a drug for the treatment of diseases.
  • the disease comprises any one or a combination of at least two of inflammatory bowel disease, intestinal polyps, adenoma, intestinal cancer, hepatic encephalopathy, graft versus host disease, or Clostridium difficile infection.
  • the application utilizes high-throughput sequencing to screen fecal bacteria donors, compares the sequencing data of the fecal bacteria donors to a public database, and compares with the intestinal flora of the fecal bacteria recipients (patients), The rapid screening of fecal bacteria donors for different fecal bacteria recipients has been achieved, and the identification level has reached the level of strains.
  • high-throughput sequencing can also identify pathogenic bacteria, and eliminate potential risk of fecal bacteria donors. quality control;
  • the fecal bacteria flora of the screening of the present application is mixed with freeze-drying protective agent and freeze-drying, effectively prolongs the survival time of flora, improves the colonization effect of flora;
  • Fig. 1 is the flow chart of screening fecal bacteria donors based on high-throughput sequencing
  • Figure 2 is a comparison chart of irritable bowel syndrome scale scores before and after oral administration of Quanjian Capsules in patients with irritable bowel syndrome;
  • Figure 3 shows the differential flora of patients with irritable bowel syndrome before and after oral administration of Quanjian Capsules and the relationship with donors.
  • Feces were obtained from healthy donors, and the inclusion criteria for healthy donors were as follows:
  • the exclusion criteria for healthy donors are as follows:
  • Virology and etiology Fecal samples detected Helicobacter pylori, Yersinia, Campylobacter, Shigella, Salmonella, enteropathogenic Escherichia coli, rotavirus, adenovirus, stellate Viruses, Toxoplasma and Giardia;
  • HIV human immunodeficiency virus
  • HAV human T-cell virus
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • cytomegalovirus Epstein-Barr virus (EBV)
  • EBV Epstein-Barr virus
  • the crowd judged as a healthy donor according to Example 1 is used as a fecal bacteria donor, and is further screened based on high-throughput sequencing technology.
  • the flow chart is shown in Figure 1, and the steps are as follows:
  • Extract DNA from the feces of fecal bacteria donors construct a library for next-generation sequencing, and obtain the original sequencing data; after removing the host genes of the original sequencing data, compare with the NCBI microbial database (bacterial genome, fungal genome, virus genome), Perform bacterial species identification and abundance detection; compare with the KEGG pathogenic bacteria database to confirm that there are no pathogenic bacteria in the fecal bacteria donor; fecal bacteria donor.
  • NCBI microbial database bacterial genome, fungal genome, virus genome
  • Perform bacterial species identification and abundance detection compare with the KEGG pathogenic bacteria database to confirm that there are no pathogenic bacteria in the fecal bacteria donor; fecal bacteria donor.
  • the screened fecal bacteria donors expressed the biomarkers Escherichia coli, Clostridium ramosum, Eubacterium cylindroides, Roseburia hominis, For Faecalibacterium prausnitzii, Bacteroides fragilis and Bacteroides vulgatus, the alpha diversity of the biomarkers was 163804.8.
  • biomarkers that are positively related to health are Clostridium ramosum, Eubacterium cylindroides, Roseburia hominis, Faecalibacterium prausnitzii, and Bacteroides vulgaris (Bacteroides vulgatus), negatively correlated biomarkers of health are Escherichia coli and Bacteroides fragilis.
  • boxplot analysis was performed on each microorganism positively correlated with health to obtain the median value of each microorganism; in patients, boxplot analysis was performed on each negatively correlated microorganism with health to obtain the median value of each microorganism. median value; the median value of each microorganism was used as the standard value for donor screening.
  • the screening criteria for fecal bacteria donors are: the value of each positively correlated microorganism is greater than the standard value of the microorganism, and the value of each negatively correlated microorganism is less than the standard value of the microorganism.
  • the feces of the fecal bacteria donors screened in Example 2 were collected on site, and sent to the laboratory within 1 hour for information registration, fecal identification, weighing, evaluation and treatment, and the fecal bacteria were prepared in an anaerobic environment. liquid, the steps are as follows:
  • the feces of the fecal bacteria donors screened in Example 2 were collected on site, and sent to the laboratory within 1 hour for information registration, fecal identification, weighing, evaluation and treatment, and the fecal bacteria were prepared in an anaerobic environment. liquid, the steps are as follows:
  • the fecal bacteria liquid prepared in Example 3 and the freeze-drying protective agent (skim milk powder 15%, trehalose 15%, sucrose 5%, vitamin C 5%, and the balance is normal saline) according to 3:1 (v/v) Mixing, then drop from room temperature to 4°C within 10s, and further drop from 4°C to -40°C with a cooling rate of 2°C/min, and drop from -40°C to -80°C with a cooling rate of 5°C/min.
  • the obtained fecal bacteria freeze-dried powder is put into an enteric-coated capsule shell to obtain the whole bacteria capsule, which is stored at -80°C.
  • the fecal bacteria liquid prepared in Example 4 was mixed with a freeze-drying protective agent (skim milk powder 10%, trehalose 15%, sucrose 1%, vitamin C 5%, and the balance was physiological saline), and then the temperature was lowered from room temperature within 10s. to 6°C, and further decreased from 6°C to -50°C at a cooling rate of 2°C/min, and decreased from -50°C to -75°C at a rate of 5°C/min. Under the condition of °C, vacuum-drying for 48 hours, the obtained fecal bacteria freeze-dried powder is put into the enteric-coated capsule shell to obtain the whole bacteria capsule, which is stored at -80 °C.
  • a freeze-drying protective agent skim milk powder 10%, trehalose 15%, sucrose 1%, vitamin C 5%, and the balance was physiological saline
  • the fecal bacteria liquid prepared in Example 4 was mixed with a freeze-dried protective agent (skim milk powder 20%, trehalose 10%, sucrose 10%, vitamin C 1%, and the balance was physiological saline), and then the temperature was lowered from room temperature within 20s. to 3°C, and further decreased from 3°C to -30°C at a cooling rate of 1°C/min, and decreased from -30°C to -80°C at a rate of 4°C/min. Under the condition of °C, vacuum-drying for 24 hours, the obtained fecal bacteria freeze-dried powder is put into the enteric-coated capsule shell to obtain the whole bacteria capsule, which is stored at -75 °C.
  • a freeze-dried protective agent skim milk powder 20%, trehalose 10%, sucrose 10%, vitamin C 1%, and the balance was physiological saline
  • the patients with irritable bowel syndrome took the whole bacteria capsules prepared in Example 5 three times, once a week, for a total of three weeks.
  • 16S rRNA was detected in the fecal samples of the patients.
  • the abundance of Firmicutes increased significantly (p ⁇ 0.01), which was consistent with healthy donors.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

提供了一种全菌胶囊及其制备方法和应用。制备方法包括步骤：(1)利用高通量测序筛选粪菌供体；(2)收集步骤(1)筛选的粪菌供体的粪便，制备粪菌液；(3)将步骤(2)制备的粪菌液和冻干保护剂混合后进行降温冷冻和真空干燥，得到的粪菌冻干粉装入胶囊壳内，得到所述全菌胶囊。利用了高通量测序对粪菌供体进行筛选，实现了快速筛选出针对不同的粪菌受体的粪菌供体，由此制备的全菌胶囊与粪菌受体能精确匹配。

Description

一种全菌胶囊及其制备方法和应用 技术领域
本申请属于粪菌移植技术领域，涉及一种全菌胶囊及其制备方法和应用。
背景技术
菌群移植(FMT)是将健康人粪便中的功能菌群移植到患者肠道内，在患者肠道内建立新的肠道菌群，治疗肠道内和肠道外疾病的技术。近10年来，FMT在治疗肠道内和肠道外疾病方面取得了突破性进展，临床上对FMT治疗肠道内外疑难疾病寄予厚望。但是，由于FMT方法学的建立较为复杂，国内外尚无统一的标准，导致了各类研究疗效的异质性较大，极大地限制了FMT的临床推广应用。目前，粪菌移植在治疗难辨性梭状芽孢杆菌感染、肠易激综合征、炎症性肠病等肠道菌群失调引起的疾病方面具有良好的效果。临床上主要通过灌肠、鼻胃管、十二指肠、空肠和结肠镜等途径进行菌群移植，但是这些途径都涉及侵入性移植，患者体验差。
CN209967402U公开了一种经胃肠镜粪菌移植推注导管，包括中空设置的手柄及用于推注粪菌的导管，手柄的一端设置有用于连接导管的连接部，手柄远离导管的一侧设置有用于注射粪菌的注射管。使用该导管进行粪菌移植，可以使整个推注过程在可视化条件下进行，并能避免粪菌对检查仪器造成污染，加强了粪菌移植内镜下操作的规范化。然而该装置需要与灌肠处理配合使用，对体质较弱或肠道有糜烂损伤的患者容易造成二次伤害。
CN110638783A公开了一种胶囊的制备方法，利用粪菌移植供体的健康优质粪便在体外模拟肠道微生物对党参皂苷的酶解作用，通过适当的发酵条件，将党参皂苷降解转化为次级皂甙类活性小分子物质，继而发挥药效。将含有活性小分子物质的粪菌发酵液装入肠溶性胶囊壳，给患者使用，在改善肠道菌群的同时，能使患者更好地吸收活性物质，有益健康。然而，该方案未对有益菌进行保存等操作，容易失活，需在短时间内尽快使用，不宜长期保存。
CN109010380A公开了一种粪菌移植临床治疗中的肠道菌群制剂的工艺，包括粪便采集、肠道菌群制剂制备和肠道菌群制剂灌装三个步骤，其中肠道菌群制剂制备采用了“有氧+厌氧”的新型的分离方法，最大程度地保护肠道内“需 氧菌”和“厌氧菌”的菌群多样性，并加入甘油保护剂来维持细菌的活性。然而，该工艺在操作时需要将一定体积的悬浊液与甘油溶液混合，制成的肠道菌群制剂中菌群浓度低，药效低。
目前，粪菌移植主要通过机械介入的方法，过程复杂，患者有一定的痛苦。如何提供一种快速、高效的FMT新技术，不仅能够减轻患者的痛苦，还能够提供数量充足的有益菌用于疾病治疗或症状改善，已成为亟待解决的问题。
发明内容
本申请提供了一种全菌胶囊及其制备方法和应用，所述全菌胶囊中装有经高通量测序筛选的粪菌供体的菌群冻干粉，提高了菌群移植的有效率和舒适度，在临床上具有广泛的应用前景。
第一方面，本申请提供了一种全菌胶囊的制备方法，所述制备方法包括以下步骤：
(1)利用高通量测序筛选粪菌供体；
(2)收集步骤(1)筛选的粪菌供体的粪便，制备粪菌液；以及
(3)将步骤(2)制备的粪菌液和冻干保护剂混合后进行降温冷冻和真空干燥，得到的粪菌冻干粉装入胶囊壳内，得到所述全菌胶囊。
本申请中，利用高通量测序对粪菌供体进行筛选，有利于准确鉴别粪菌供体的菌群组成，在发现粪菌供体的未知物种、病原体方面具有优势，检测分辨率达到菌株水平，实现了一次检测多个粪菌供体的效果。
优选地，步骤(1)所述利用高通量测序筛选粪菌供体的方法包括以下步骤：
(1’)将提取自粪菌供体的DNA和/或RNA进行二代测序，获得原始测序数据；
(2’)去除原始测序数据的宿主基因后，与微生物数据库进行比对，进行菌种鉴定和丰度检测；
(3’)与致病菌数据库进行比对，确认粪菌供体中无致病菌；以及
(4’)与粪菌受体的肠道菌群进行比较，筛选得到与粪菌受体互补的粪菌供体。
本申请中，采用高通量测序鉴定粪菌供体的粪菌组成，并与粪菌受体(患者)的肠道菌群进行比较，有利于针对不同的粪菌受体筛选不同的粪菌供体， 实现“精准匹配”，提高药效；高通量测序还能鉴定出致病菌，有利于排除有潜在风险的粪菌供体，进行质量监控。
优选地，步骤(2’)所述微生物数据库包括来源于公共数据库的细菌基因组、真菌基因组或病毒基因组中的任意一种或至少两种的组合。
优选地，步骤(3’)所述致病菌数据库包括来源于公共数据库的致病菌基因组。
本申请中，利用NCBI、KEGG等公共数据库构建微生物数据库和致病菌数据库，将粪菌供体的测序数据比对到上述数据库中，实现菌株水平的微生物鉴定，排除了携带致病菌的粪菌供体，减少了健康风险。
优选地，步骤(1)所述利用高通量测序筛选粪菌供体根据粪菌供体表达的生物标志物和所述生物标志物的多样性指数。
优选地，所述生物标志物包括大肠杆菌(Escherichia coli)、多支梭菌(Clostridium ramosum)、柱状真杆菌(Eubacterium cylindroides)、人罗斯拜瑞氏菌(Roseburia hominis)、普拉梭菌(Faecalibacterium prausnitzii)、脆弱拟杆菌(Bacteroides fragilis)或普通拟杆菌(Bacteroides vulgatus)中的任意一种或至少两种的组合。
优选地，所述生物标志物的多样性指数包括所述生物标志物的α多样性指数。
本申请中，利用高通量测序结合机器学习模型，筛选出生物标志物大肠杆菌(Escherichia coli)、多支梭菌(Clostridium ramosum)、柱状真杆菌(Eubacterium cylindroides)、人罗斯拜瑞氏菌(Roseburia hominis)、普拉梭菌(Faecalibacterium prausnitzii)、脆弱拟杆菌(Bacteroides fragilis)和普通拟杆菌(Bacteroides vulgatus)，以及生物标志物的α多样性指数，作为筛选粪菌供体的指标。
优选地，步骤(2)所述粪菌液的制备方法包括以下步骤：
(1”)将收集的粪便浸泡于无菌生理盐水中，过滤后得到粪便滤液；
(2”)将粪便滤液离心处理，取沉淀与无菌生理盐水混合，得到所述粪菌液。
本申请中，粪菌供体的粪便使用无菌容器在现场收集，收集的粪便1h内送至实验室进行信息登记、粪便鉴定、称重、评估和处理，粪便捐献6h内，在厌氧环境中完成粪菌液的制备。
优选地，步骤(1”)所述无菌生理盐水的温度为3～5℃，例如可以是3℃、4℃或5℃。
优选地，步骤(1”)所述过滤采用滤网进行。
优选地，所述滤网的孔径为0.25～2mm，例如可以是0.25mm、0.5mm、1.0mm或2.0mm。
优选地，步骤(1”)所述过滤包括依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤2～3次，得到的液相为粪便滤液。
优选地，步骤(2”)所述离心处理的转速为1500～3000r/min，例如可以是1500r/min、1600r/min、1700r/min、1800r/min、1900r/min、2000r/min、2100r/min、2200r/min、2300r/min、2400r/min、2500r/min、2600r/min、2700r/min、2800r/min、2900r/min或3000r/min。
优选地，步骤(2”)所述离心处理的时间为10～20min，例如可以是10min、11min、12min、13min、14min、15min、16min、17min、18min、19min或20min。
优选地，步骤(3)所述冻干保护剂包括脱脂乳粉、海藻糖、蔗糖、维生素C和无菌生理盐水。
优选地，所述冻干保护剂按质量百分比包括：脱脂乳粉10％～20％、海藻糖10％～15％、蔗糖1％～10％、维生素C 1％～5％，余量为生理盐水。
本申请中，冻干保护剂可以有效延长菌群的存活时间，提高菌群的定植效果。
优选地，步骤(3)所述粪菌液和所述冻干保护剂的体积比为(2～5):1，例如可以是2:1、3:1、4:1或5:1，优选为3:1。
优选地，步骤(3)所述降温冷冻的条件为10～20s由室温降至3～6℃，1～2℃/min由3～6℃降至-30～-50℃，4～5℃/min由-30～-50℃降至-75～-80℃。
优选地，步骤(3)所述降温冷冻的时间为12～24h，例如可以是12h、13h、14h、15h、16h、17h、18h、19h、20h、21h、22h、23h或24h。
优选地，步骤(3)所述真空干燥的真空度为5～15pa，例如可以是5pa、6pa、7pa、8pa、9pa、10pa、11pa、12pa、13pa、14pa或15pa。
优选地，步骤(3)所述真空干燥的温度为-50～-60℃，例如可以是-50℃、-51℃、 -52℃、-53℃、-54℃、-55℃、-56℃、-57℃、-58℃、-59℃或-60℃。
优选地，步骤(3)所述真空干燥的时间为24～48h，例如可以是24h、30h、36h、42h或48h。
优选地，步骤(3)所述胶囊壳包括肠溶胶囊壳。
本申请中，肠溶胶囊壳可以有效抵御胃液的分解，在肠道pH值条件下才会释放胶囊中的有效菌群，可以有效保护菌群抵达肠道，防止菌群过早释放，减少菌群的活性损失，延长了菌群的释放时长，达到特定的定植效果，有利于实现口服途径给药。
优选地，所述全菌胶囊保存于-75～-80℃温度下。
作为优选技术方案，本申请提供了一种全菌胶囊的制备方法，所述制备方法包括以下步骤：
(1)将提取自粪菌供体的DNA和/或RNA进行二代测序，获得原始测序数据；去除原始测序数据的宿主基因后，与NCBI微生物数据库进行比对，进行菌种鉴定和丰度检测；与KEGG致病菌数据库进行比对，确认粪菌供体中无致病菌；与粪菌受体的肠道菌群进行比较，筛选得到与粪菌受体互补的粪菌供体；
(2)收集步骤(1)筛选的粪菌供体的粪便，浸泡于3～5℃无菌生理盐水中，依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤2～3次，得到的液相为粪便滤液；将粪便滤液在1500～3000r/min离心处理10～20min，取沉淀与无菌生理盐水混合，得到粪菌液；以及
(3)将步骤(2)制备的粪菌液和冻干保护剂按照体积比为(2～5):1的比例混合，所述冻干保护剂按质量百分比包括：脱脂乳粉10％～20％、海藻糖10％～15％、蔗糖1％～10％、维生素C 1％～5％，余量为生理盐水，随后在10～20s内由室温降至3～6℃，并进一步以1～2℃/min的降温速度由3～6℃降至-30～-50℃，4～5℃/min的降温速度由-30～-50℃降至-75～-80℃，降温冷冻后在5～15pa真空度和-50～-60℃的条件下真空干燥24～48h，得到的粪菌冻干粉装入肠溶胶囊壳内，得到所述全菌胶囊，保存于-75～-80℃中。
作为优选技术方案，本申请提供了一种全菌胶囊的制备方法，所述制备方法包括以下步骤：
(1)将提取自粪菌供体的DNA和/或RNA进行二代测序，获得原始测序数据；去除原始测序数据的宿主基因后，与NCBI微生物数据库进行比对，筛选 出表达大肠杆菌、多支梭菌、柱状真杆菌、人罗斯拜瑞氏菌、普拉梭菌、脆弱拟杆菌或普通拟杆菌中的任意一种或至少两种的组合的粪菌供体；
(2)收集步骤(1)筛选的粪菌供体的粪便，浸泡于3～5℃无菌生理盐水中，依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤2～3次，得到的液相为粪便滤液；将粪便滤液在1500～3000r/min离心处理10～20min，取沉淀与无菌生理盐水混合，得到粪菌液；以及
(3)将步骤(2)制备的粪菌液和冻干保护剂按照体积比为(2～5):1的比例混合，所述冻干保护剂按质量百分比包括：脱脂乳粉10％～20％、海藻糖10％～15％、蔗糖1％～10％、维生素C 1％～5％，余量为生理盐水，随后在10～20s内由室温降至3～6℃，并进一步以1～2℃/min的降温速度由3～6℃降至-30～-50℃，4～5℃/min的降温速度由-30～-50℃降至-75～-80℃，降温冷冻后在5～15pa真空度和-50～-60℃的条件下真空干燥24～48h，得到的粪菌冻干粉装入肠溶胶囊壳内，得到所述全菌胶囊，保存于-75～-80℃中。
第二方面，本申请提供了一种全菌胶囊，所述全菌胶囊由第一方面所述的制备方法制备得到。
第三方面，本申请提供了一种药物组合物，所述药物组合物包括第二方面所述的全菌胶囊。
优选地，所述药物组合物还包括PD-1抑制剂。
优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
第四方面，本申请提供了第二方面所述的全菌胶囊和/或第三方面所述的药物组合物在制备疾病治疗药物中的应用。
优选地，所述疾病包括炎症性肠病、肠息肉、腺瘤、肠癌、肝脑病、移植物抗宿主病或艰难梭菌感染中的任意一种或至少两种的组合。
与现有技术相比，本申请具有如下有益效果：
(1)本申请利用高通量测序对粪菌供体进行筛选，将粪菌供体的测序数据比对到公共数据库中，并与粪菌受体(患者)的肠道菌群进行比较，实现了快速筛选出针对不同的粪菌受体的粪菌供体，鉴定水平达到菌株水平，同时，高通量测序还能鉴定出致病菌，排除了有潜在风险的粪菌供体，进行质量监控；
(2)本申请的筛选的粪菌菌群与冻干保护剂混合后进行冻干处理，有效延 长了菌群的存活时间，提高了菌群的定植效果；以及
(3)本申请的全菌胶囊实现了针对粪菌受体的“精准匹配”，药效显著，患者服用三次后，腹痛评分、每日排便次数、以及2级食物不耐受的食物种类数量均显著降低，厚壁菌门丰度显著上升，与健康供体趋于一致。
附图说明
图1为基于高通量测序筛选粪菌供体的流程图；
图2为肠易激综合征患者口服全菌胶囊前后肠易激综合征量表评分对比图；
图3为肠易激综合征患者口服全菌胶囊前后的差异菌群以及与供体的关系。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 健康供体的判断标准
粪便来源于健康供体，健康供体的入选标准如下：
(1)年龄18～23岁，身体健康，未婚男女；
(2)生活规律，无不良嗜好；
(3)三个月内无抗生素使用史；
(4)无胃肠道疾病；
(5)血清学和粪便传染性病原体检查及培养均为阴性；
(6)肠道菌群种类及多样性良好：经高通量肠道菌群16s rDNA基因检测后判断为益生菌种类及菌群的多样性好。
健康供体的排除标准如下：
(1)药物服用史：三个月内使用过抗生素、泻药、减肥药或正在服用免疫抑制剂、化疗药物者；
(2)病毒接触史：曾经或近期暴露于HIV病毒、甲肝病毒、乙肝病毒、丙肝病毒者；
(3)疾病史：精神性疾病、恶性肥胖、便秘、糖尿病、炎症性肠病、过敏、代谢综合征、免疫力低下、自身免疫性疾病、肠道相关疾病、慢性疲劳综合症、消化系统手术史、胃肠道恶性肿瘤或息肉、特异反应性疾病(如湿疹、哮喘和胃肠道嗜酸性细胞相关疾病)者；
(4)病毒学和病原学：粪便样本检出幽门螺杆菌、耶尔森菌、弯曲杆菌、志贺菌属、沙门菌属、肠致病性大肠杆菌、轮状病毒、腺病毒、星状病毒、弓形虫和贾第虫者；
(5)血清学检测出人类免疫缺陷病毒(HIV)、人类嗜T细胞病毒(HTLV)、甲肝病毒(HAV)、乙肝病毒(HBV)、丙肝病毒(HCV)、巨细胞病毒、EB病毒(EBV)、梅毒、类圆线虫和阿米巴原虫等；
(6)存在高风险性行为史、吸毒或违禁药物使用史、近期有监禁史或近期有疫区旅游史。
满足上述供体入选标准的全部条件，并且不存在供体排除标准中的任意一项的人群，即判断为健康供体。
实施例2 基于高通量测序筛选粪菌供体
根据实施例1判断为健康供体的人群作为粪菌供体，基于高通量测序技术进一步筛选，流程图如图1所示，步骤如下：
从粪菌供体的粪便中提取DNA，构建文库进行二代测序，获得原始测序数据；去除原始测序数据的宿主基因后，与NCBI微生物数据库(细菌基因组、真菌基因组、病毒基因组)进行比对，进行菌种鉴定和丰度检测；与KEGG致病菌数据库进行比对，确认粪菌供体中无致病菌；与粪菌受体的肠道菌群进行比较，筛选与粪菌受体互补的粪菌供体。
本实施例中，筛选的粪菌供体表达生物标志物大肠杆菌(Escherichia coli)、多支梭菌(Clostridium ramosum)、柱状真杆菌(Eubacterium cylindroides)、人罗斯拜瑞氏菌(Roseburia hominis)、普拉梭菌(Faecalibacterium prausnitzii)、脆弱拟杆菌(Bacteroides fragilis)和普通拟杆菌(Bacteroides vulgatus)，生物标志物的α多样性为163804.8。其中，健康正相关的生物标志物为多支梭菌(Clostridium ramosum)、柱状真杆菌(Eubacterium cylindroides)、人罗斯拜瑞氏菌(Roseburia hominis)、普拉梭菌(Faecalibacterium prausnitzii)和普通拟杆菌(Bacteroides vulgatus)，健康负相关的生物标志物为大肠杆菌(Escherichia  coli)和脆弱拟杆菌(Bacteroides fragilis)。
在健康人群中，对每个健康正相关的微生物进行boxplot分析，得出每个微生物的中位值；在患者中，对每个健康负相关的微生物进行boxplot分析，得出每个微生物的中位值；将每个微生物的中位值作为供体筛选的标准值。
粪菌供体的筛选标准为：每个健康正相关的微生物的值大于该微生物的标准值，每个健康负相关的微生物的值小于该微生物的标准值。
实施例3 粪菌液的制备
现场收集实施例2筛选的特异性针对粪菌受体的粪菌供体的粪便，1h内送至实验室进行信息登记、粪便鉴定、称重、评估和处理，在厌氧环境中制备粪菌液，步骤如下：
(1)将收集的粪便浸泡于5℃无菌生理盐水中，依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤3次，得到的液相为粪便滤液；
(2)将粪便滤液在3000r/min离心处理10min，取沉淀与无菌生理盐水混合，得到粪菌液。
实施例4 粪菌液的制备
现场收集实施例2筛选的特异性针对粪菌受体的粪菌供体的粪便，1h内送至实验室进行信息登记、粪便鉴定、称重、评估和处理，在厌氧环境中制备粪菌液，步骤如下：
(1)将收集的粪便浸泡于3℃无菌生理盐水中，依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤2次，得到的液相为粪便滤液；
(2)将粪便滤液在1500r/min离心处理20min，取沉淀与无菌生理盐水混合，得到粪菌液。
实施例5 全菌胶囊的制备
将实施例3制备的粪菌液和冻干保护剂(脱脂乳粉15％、海藻糖15％、蔗糖5％、维生素C 5％，余量为生理盐水)按照3:1(v/v)混合，随后在10s内由室温降至4℃，并进一步以2℃/min的降温速度由4℃降至-40℃，5℃/min的降温速度由-40℃降至-80℃，降温冷冻后在10pa真空度和-50℃的条件下真空干燥48h，得到的粪菌冻干粉装入肠溶胶囊壳内，得到所述全菌胶囊，保存于-80℃ 中。
实施例6 全菌胶囊的制备
将实施例4制备的粪菌液和冻干保护剂(脱脂乳粉10％、海藻糖15％、蔗糖1％、维生素C 5％，余量为生理盐水)混合，随后在10s内由室温降至6℃，并进一步以2℃/min的降温速度由6℃降至-50℃，5℃/min的降温速度由-50℃降至-75℃，降温冷冻后在5pa真空度和-50℃的条件下真空干燥48h，得到的粪菌冻干粉装入肠溶胶囊壳内，得到所述全菌胶囊，保存于-80℃中。
实施例7 全菌胶囊的制备
将实施例4制备的粪菌液和冻干保护剂(脱脂乳粉20％、海藻糖10％、蔗糖10％、维生素C 1％，余量为生理盐水)混合，随后在20s内由室温降至3℃，并进一步以1℃/min的降温速度由3℃降至-30℃，4℃/min的降温速度由-30℃降至-80℃，降温冷冻后在15pa真空度和-60℃的条件下真空干燥24h，得到的粪菌冻干粉装入肠溶胶囊壳内，得到所述全菌胶囊，保存于-75℃中。
实施例8 全菌胶囊的治疗效果
肠易激综合征患者服用三次实施例5制备的全菌胶囊，每周一次，共服用三周。
如图2所示，患者的腹痛评分、每日排便次数、以及2级食物不耐受的食物种类数量均显著降低(p＜0.01)，1级食物不耐受和3级食物不耐受的食物种类数量也相应降低。
如图3所示，对患者的粪便样本进行16S rRNA检测，样本在科分类水平上，厚壁菌门丰度显著上升(p<0.01)，与健康供体趋于一致。
综上所述，利用高通量测序对粪菌供体进行筛选，准确筛选出特异性针对不同粪菌受体的粪菌供体，制备的全菌胶囊“精准匹配”，药效显著，安全性高。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (14)

  1. 一种全菌胶囊的制备方法，其包括以下步骤：
    (1)利用高通量测序筛选粪菌供体；
    (2)收集步骤(1)筛选的粪菌供体的粪便，制备粪菌液；以及
    (3)将步骤(2)制备的粪菌液和冻干保护剂混合后进行降温冷冻和真空干燥，得到的粪菌冻干粉装入胶囊壳内，得到所述全菌胶囊。
  2. 根据权利要求1所述的制备方法，其中，步骤(1)所述利用高通量测序筛选粪菌供体的方法包括以下步骤：
    (1’)将提取自粪菌供体的DNA和/或RNA进行二代测序，获得原始测序数据；
    (2’)去除原始测序数据的宿主基因后，与微生物数据库进行比对，进行菌种鉴定和丰度检测；
    (3’)与致病菌数据库进行比对，确认粪菌供体中无致病菌；以及
    (4’)与粪菌受体的肠道菌群进行比较，筛选得到与粪菌受体互补的粪菌供体。
  3. 根据权利要求1或2所述的制备方法，其中，步骤(2’)所述微生物数据库包括来源于公共数据库的细菌基因组、真菌基因组或病毒基因组中的任意一种或至少两种的组合。
  4. 根据权利要求1-3任一项所述的制备方法，其中，步骤(3’)所述致病菌数据库包括来源于公共数据库的致病菌基因组。
  5. 根据权利要求1-4任一项所述的制备方法，其中，步骤(1)所述利用高通量测序筛选粪菌供体根据粪菌供体表达的生物标志物和所述生物标志物的多样性指数。
  6. 根据权利要求1-5任一项所述的制备方法，其中，所述生物标志物包括大肠杆菌、多支梭菌、柱状真杆菌、人罗斯拜瑞氏菌、普拉梭菌、脆弱拟杆菌或普通拟杆菌中的任意一种或至少两种的组合。
  7. 根据权利要求1-6任一项所述的制备方法，其中，所述生物标志物的多样性指数包括所述生物标志物的α多样性指数。
  8. 根据权利要求1-7任一项所述的制备方法，其中，步骤(2)所述粪菌液的制备方法包括以下步骤：
    (1”)将收集的粪便浸泡于无菌生理盐水中，过滤后得到粪便滤液；
    (2”)将粪便滤液离心处理，取沉淀与无菌生理盐水混合，得到所述粪菌液；
    优选地，步骤(1”)所述无菌生理盐水的温度为3～5℃；
    优选地，步骤(1”)所述过滤采用滤网进行；
    优选地，所述滤网的孔径为0.25～2mm；
    优选地，步骤(1”)所述过滤包括依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤2～3次，得到的液相为粪便滤液；
    优选地，步骤(2”)所述离心处理的转速为1500～3000r/min；
    优选地，步骤(2”)所述离心处理的时间为10～20min。
  9. 根据权利要求1-8任一项所述的制备方法，其中，步骤(3)所述冻干保护剂包括脱脂乳粉、海藻糖、蔗糖、维生素C和无菌生理盐水；
    优选地，所述冻干保护剂按质量百分比包括：脱脂乳粉10％～20％、海藻糖10％～15％、蔗糖1％～10％、维生素C1％～5％，余量为生理盐水；
    优选地，步骤(3)所述粪菌液和所述冻干保护剂的体积比为(2～5):1；
    优选地，步骤(3)所述降温冷冻的条件为10～20s由室温降至3～6℃，1～2℃/min由3～6℃降至-30～-50℃，4～5℃/min由-30～-50℃降至-75～-80℃；
    优选地，步骤(3)所述降温冷冻的时间为12～24h；
    优选地，步骤(3)所述真空干燥的真空度为5～15pa；
    优选地，步骤(3)所述真空干燥的温度为-50～-60℃；
    优选地，步骤(3)所述真空干燥的时间为24～48h；
    优选地，步骤(3)所述胶囊壳包括肠溶胶囊壳；
    优选地，所述全菌胶囊保存于-75～-80℃温度下。
  10. 根据权利要求1-9任一项所述的制备方法，其中，所述制备方法包括以下步骤：
    (1)将提取自粪菌供体的DNA和/或RNA进行二代测序，获得原始测序数据；去除原始测序数据的宿主基因后，与NCBI微生物数据库进行比对，进行菌种鉴定和丰度检测；与KEGG致病菌数据库进行比对，确认粪菌供体中无致病菌；与粪菌受体的肠道菌群进行比较，筛选得到与粪菌受体互补的粪菌供体；
    (2)收集步骤(1)筛选的粪菌供体的粪便，浸泡于3～5℃无菌生理盐水中， 依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤2～3次，得到的液相为粪便滤液；将粪便滤液在1500～3000r/min离心处理10～20min，取沉淀与无菌生理盐水混合，得到粪菌液；以及
    (3)将步骤(2)制备的粪菌液和冻干保护剂按照体积比为(2～5):1的比例混合，所述冻干保护剂按质量百分比包括：脱脂乳粉10％～20％、海藻糖10％～15％、蔗糖1％～10％、维生素C 1％～5％，余量为生理盐水，随后在10～20s内由室温降至3～6℃，并进一步以1～2℃/min的降温速度由3～6℃降至-30～-50℃，4～5℃/min的降温速度由-30～-50℃降至-75～-80℃，降温冷冻后在5～15pa真空度和-50～-60℃的条件下真空干燥24～48h，得到的粪菌冻干粉装入肠溶胶囊壳内，得到所述全菌胶囊，保存于-75～-80℃中。
  11. 根据权利要求1-10任一项所述的制备方法，其中，所述制备方法包括以下步骤：
    (1)将提取自粪菌供体的DNA和/或RNA进行二代测序，获得原始测序数据；去除原始测序数据的宿主基因后，与NCBI微生物数据库进行比对，筛选出表达大肠杆菌、多支梭菌、柱状真杆菌、人罗斯拜瑞氏菌、普拉梭菌、脆弱拟杆菌或普通拟杆菌中的任意一种或至少两种的组合的粪菌供体；
    (2)收集步骤(1)筛选的粪菌供体的粪便，浸泡于3～5℃无菌生理盐水中，依次使用2.0mm、1.0mm、0.5mm和0.25mm的滤网去除大颗粒物，随后使用0.25mm的滤网过滤2～3次，得到的液相为粪便滤液；将粪便滤液在1500～3000r/min离心处理10～20min，取沉淀与无菌生理盐水混合，得到粪菌液；以及
    (3)将步骤(2)制备的粪菌液和冻干保护剂按照体积比为(2～5):1的比例混合，所述冻干保护剂按质量百分比包括：脱脂乳粉10％～20％、海藻糖10％～15％、蔗糖1％～10％、维生素C1％～5％，余量为生理盐水，随后在10～20s内由室温降至3～6℃，并进一步以1～2℃/min的降温速度由3～6℃降至-30～-50℃，4～5℃/min的降温速度由-30～-50℃降至-75～-80℃，降温冷冻后在5～15pa真空度和-50～-60℃的条件下真空干燥24～48h，得到的粪菌冻干粉装入肠溶胶囊壳内，得到所述全菌胶囊，保存于-75～-80℃中。
  12. 一种全菌胶囊，其由权利要求1-11任一项所述的制备方法制备得到。
  13. 一种药物组合物，其包括权利要求12所述的全菌胶囊；
    优选地，所述药物组合物还包括PD-1抑制剂；
    优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
  14. 权利要求12所述的全菌胶囊和/或权利要求13所述的药物组合物在制备疾病治疗药物中的应用；
    优选地，所述疾病包括炎症性肠病、肠息肉、腺瘤、肠癌、肝脑病、移植物抗宿主病或艰难梭菌感染中的任意一种或至少两种的组合。
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