WHOLE BACTERIA CAPSULE, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

Provided are a whole bacteria capsule, a preparation method therefor and a use thereof. The preparation method comprises the steps of: (1) screening fecal bacteria donors using high-throughput sequencing; (2) collecting feces of the fecal bacteria donors screened in step (1), and preparing a fecal bacterial liquid; and (3) mixing the fecal bacterial liquid prepared in step (2) with a freeze-drying protective agent, cooling, freezing and vacuum-drying the mixture, and loading the freeze-dried feces bacteria powder obtained into a capsule shell to obtain the whole bacteria capsule. By screening the fecal bacteria donors using high-throughput sequencing, fecal bacteria donors can be quickly screened for different fecal bacteria receptors, and the whole bacteria capsule prepared can accurately match with the fecal bacteria receptors.

Claims

1. A preparation method of a whole bacteria capsule, comprising the following steps: (1) screening a fecal bacteria donor through high-throughput sequencing; (2) collecting feces of the fecal bacteria donor screened in step (1) to prepare a fecal bacteria liquid; and (3) mixing the fecal bacteria liquid prepared in step (2) with a lyophilizing protective agent and cooling, freezing and vacuum drying the mixture to obtain lyophilized powder of fecal bacteria which is loaded into a capsule shell to obtain the whole bacteria capsule.

2. The preparation method according to claim 1, wherein a method for screening the fecal bacteria donor through the high-throughput sequencing in step (1) comprises the following steps: (1′) performing second-generation sequencing on DNA and/or RNA extracted from the fecal bacteria donor to obtain original sequencing data; (2′) after removing a host gene from the original sequencing data, comparing the sequencing data with a microbial database for bacterial strains identification and abundance detection; (3′) comparing the sequencing data with a pathogenic bacteria database to confirm no pathogenic bacteria in the fecal bacteria donor; and (4′) comparing the sequencing data with an intestinal flora of a fecal bacteria receptor and screening to obtain a fecal bacteria donor complementary to the fecal bacteria receptor.

3. The preparation method according to claim 1, wherein the microbial database in step (2′) comprises any one or a combination of at least two of a bacterial genome, a fungal genome or a viral genome derived from a public database.

4. The preparation method according to claim 1, wherein the pathogenic bacteria database in step (3′) comprises a pathogenic bacterial genome derived from the public database.

5. The preparation method according to claim 1, wherein screening the fecal bacteria donor through the high-throughput sequencing in step (1) is performed according to a biomarker expressed by the fecal bacteria donor and a diversity index of the biomarker.

6. The preparation method according to claim 1, wherein the biomarker comprises any one or a combination of at least two of Escherichia coli, Clostridium ramosum, Eubacterium cylindroides, Roseburia hominis, Faecalibacterium prausnitzii, Bacteroides fragilis or Bacteroides vulgatus.

7. The preparation method according to claim 1, wherein the diversity index of the biomarker comprises α-diversity index of the biomarker.

8. The preparation method according to claim 1, wherein a method for preparing the fecal bacteria liquid in step (2) comprises the following steps: (1″) soaking the collected feces in sterile saline and filtering to obtain a fecal filtrate; (2″) centrifuging the fecal filtrate and mixing a precipitate with sterile saline to obtain the fecal bacteria liquid; preferably, the sterile saline in step (1″) has a temperature of 3-5° C.; preferably, the filtration in step (1″) is performed using a filter screen; preferably, the filter screen has an aperture of 0.25-2 mm; preferably, the filtration in step (1″) comprises: removing the large particles by using 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm filter screens in sequence and then filtering two to three times with the 0.25 mm filter screen to obtain a liquid phase as the fecal filtrate; preferably, the centrifugation in step (2″) has a rotational speed of 1500-3000 r/min; preferably, the centrifugation in step (2″) is performed for 10-20 min.

9. The preparation method according to claim 1, wherein the lyophilizing protective agent in step (3) comprises skim milk powder, trehalose, sucrose, vitamin C and sterile saline; preferably, the lyophilizing protective agent comprises, in mass percentage, 10% to 20% skim milk powder, 10% to 15% trehalose, 1% to 10% sucrose, 1% to 5% vitamin C and saline as balance; preferably, a volume ratio of the fecal bacteria liquid and the lyophilizing protective agent in step (3) is (2-5):1; preferably, conditions of the cooling and freezing in step (3) are as follows: lowering the temperature from room temperature to 3-6° C. for 10-20 s, lowering the temperature from 3-6° C. to −30-(−50) ° C. at 1-2° C./min and lowering the temperature from −30-(−50) ° C. to −75-(−80) ° C. at 4-5° C./min; preferably, the cooling and freezing in step (3) is performed for 12-24 h; preferably, the vacuum drying in step (3) has a vacuum degree of 5-15 pa; preferably, the vacuum drying in step (3) is performed at −50-(−60) ° C.; preferably, the vacuum drying in step (3) is performed for 24-48 h; preferably, the capsule shell in step (3) comprises an enteric capsule shell; and preferably, the whole bacteria capsule is stored at −75-(−80) ° C.

10. The preparation method according to claim 1, comprising the following steps: (1) performing the second-generation sequencing on the DNA and/or RNA extracted from the fecal bacteria donor to obtain the original sequencing data; after removing the host gene from the original sequencing data, comparing the sequencing data with a National Center for Biotechnology Information (NCBI) microbial database for bacterial strains identification and abundance detection; comparing the sequencing data with a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathogenic bacteria database to confirm no pathogenic bacteria in the fecal bacteria donor; and comparing the sequencing data with the intestinal flora of the fecal bacteria receptor and screening to obtain the fecal bacteria donor complementary to the fecal bacteria receptor; (2) collecting the feces of the fecal bacteria donor screened in step (1), soaking the feces in the sterile saline (3-5° C.), and removing the large particles by using the 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm filter screens in sequence, and then filtering two to three times with the 0.25 mm filter screen to obtain the liquid phase as the fecal filtrate; and centrifuging the fecal filtrate for 10-20 min at 1500-3000 r/min and mixing the precipitate with the sterile saline to obtain the fecal bacteria liquid; and (3) mixing the fecal bacteria liquid prepared in step (2) with the lyophilizing protective agent at a volume ratio of (2-5):1, wherein the lyophilizing protective agent comprises, in mass percentage, 10% to 20% skim milk powder, 10% to 15% trehalose, 1% to 10% sucrose, 1% to 5% vitamin C and the saline as the balance, and then lowering the temperature from room temperature to 3-6° C. within 10-20 s, further lowering the temperature from 3-6° C. to −30-(−50) ° C. at a cooling rate of 1-2° C./min and lowering the temperature from −30-(−50) ° C. to −75-(−80) ° C. at a cooling rate of 4-5° C./min, and after the cooling and freezing, vacuum drying the mixture for 24-48 h under the conditions of a vacuum degree of 5-15 pa and −50-(−60) ° C. to obtain the lyophilized powder of fecal bacteria which is loaded into the enteric capsule shell to obtain the whole bacteria capsule which is stored at −75-(−80) ° C.

11. The preparation method according to claim 1, comprising the following steps: (1) performing the second-generation sequencing on the DNA and/or RNA extracted from the fecal bacteria donor to obtain the original sequencing data; and after removing the host gene from the original sequencing data, comparing the sequencing data with the NCBI microbial database and screening a fecal bacteria donor expressing any one or a combination of at least two of Escherichia coli, Clostridium ramosum, Eubacterium cylindroides, Roseburia hominis, Faecalibacterium prausnitzii, Bacteroides fragilis or Bacteroides vulgatus; (2) collecting the feces of the fecal bacteria donor screened in step (1), soaking the feces in the sterile saline (3-5° C.), and removing the large particles by using the 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm filter screens in sequence, and then filtering two to three times with the 0.25 mm filter screen to obtain the liquid phase as the fecal filtrate; and centrifuging the fecal filtrate for 10-20 min at 1500-3000 r/min and mixing the precipitate with the sterile saline to obtain the fecal bacteria liquid; and (3) mixing the fecal bacteria liquid prepared in step (2) with the lyophilizing protective agent at a volume ratio of (2-5):1, wherein the lyophilizing protective agent comprises, in mass percentage, 10% to 20% skim milk powder, 10% to 15% trehalose, 1% to 10% sucrose, 1% to 5% vitamin C and the saline as the balance, and then lowering the temperature from room temperature to 3-6° C. within 10-20 s, further lowering the temperature from 3-6° C. to −30-(−50) ° C. at a cooling rate of 1-2° C./min and lowering the temperature from −30-(−50) ° C. to −75-(−80) ° C. at a cooling rate of 4-5° C./min, and after the cooling and freezing, vacuum drying the mixture for 24-48 h under the conditions of a vacuum degree of 5-15 pa and −50-(−60) ° C. to obtain the lyophilized powder of fecal bacteria which is loaded into the enteric capsule shell to obtain the whole bacteria capsule which is stored at −75-(−80) ° C.

12. A whole bacteria capsule which is prepared through the preparation method according to claim 1.

13. A pharmaceutical composition, comprising the whole bacteria capsule according to claim 12; preferably, the pharmaceutical composition further comprises a PD-1 inhibitor; preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

14. (canceled)

15. A method for treating a disease, comprising administering an effective amount of the whole bacteria capsule according to claim 12 to subject in need thereof; preferably, the disease comprises any one or a combination of at least two of inflammatory bowel disease, intestinal polyp, adenoma, bowel cancer, hepatic encephalopathy, graft-versus-host disease or Clostridium difficile infection.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1 is a flowchart for screening a fecal bacteria donor based on high-throughput sequencing.

[0067] FIG. 2 is a diagram illustrating a comparison between scores of irritable bowel syndrome scale before and after oral administration of a whole bacteria capsule in a patient with irritable bowel syndrome.

[0068] FIG. 3 is a diagram illustrating a differential flora before and after oral administration of a whole bacteria capsule in a patient with irritable bowel syndrome and a relationship with a donor.

DETAILED DESCRIPTION

[0069] To further elaborate on the technical means adopted and effects achieved in the present application, the present application is described below in conjunction with examples and drawings. It is to be understood that the specific examples set forth below are intended to explain the present application and not to limit the present application.

[0070] Experiments without specific techniques or conditions specified in the examples are conducted according to techniques or conditions described in the literature in the art or a product specification. The reagents or instruments used herein without manufacturers specified are conventional products commercially available from proper channels.

Example 1 Determination Criteria of a Healthy Donor

[0071] Feces were derived from a healthy donor. An inclusion criterion of the healthy donor is as follows: [0072] (1) healthy, unmarried men and women aged 18 to 23; [0073] (2) living a regular life and having no adverse hobbies; [0074] (3) no history of antibiotic use within three months; [0075] (4) no gastrointestinal tract disease; [0076] (5) negative serology and fecal infectious pathogen examination and culture; and [0077] (6) good types and diversity of an intestinal flora: types of a probiotic and the diversity of a flora are determined to be good after high-throughput detection of a 16S rDNA gene of the intestinal flora.

[0078] An exclusion criterion of the healthy donor is as follows: [0079] (1) history of drug administration: those who have used an antibiotic, a laxative or a weight-loss drug within three months or those who are taking an immunosuppressive drug or a chemotherapy drug; [0080] (2) history of virus exposure: those who have been or have recently been exposed to HIV virus, hepatitis A virus, hepatitis B virus or hepatitis C virus (HCV); [0081] (3) history of disease: those with psychiatric disorder, malignant obesity, constipation, diabetes, inflammatory bowel disease, allergy, metabolic syndrome, hypoimmunity, autoimmune disease, intestine-related disorder, chronic fatigue syndrome, history of digestive system surgery, gastrointestinal malignant tumor or polyp, or atopic disease (such as eczema, asthma and gastrointestinal eosinophil-related disorder); [0082] (4) virology and etiology: Helicobacter pylori, Yersinia, Campylobacter, Shigella, Salmonella, enteropathogenic Escherichia coli, rotavirus, adenovirus, astrovirus, Toxoplasma gondii and Giardia are detected in a fecal sample; [0083] (5) human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus, Epstein-Barr virus (EBV), syphilis, Strongyloides, amoeba protozoa and the like are detected through serological detection; and [0084] (6) a history of high-risk sexual behaviour, a history of drug use or illicit drug use, a recent history of imprisonment or a recent history of travel to an epidemic area.

[0085] People who met all the conditions of the above donor inclusion criterion and did not have any one of the donor exclusion criterion were determined as the healthy donor.

Example 2 Screening of a Fecal Bacteria Donor Based on High-Throughput Sequencing

[0086] The people determined as the healthy donor according to Example 1 were used as a fecal bacteria donor and further screened based on a high-throughput sequencing technology. A flowchart is shown in FIG. 1, and steps are described below.

[0087] DNA was extracted from feces of the fecal bacteria donor, and a library was constructed for second-generation sequencing to obtain original sequencing data. After a host gene was removed from the original sequencing data, the sequencing data was compared with an NCBI microbial database (a bacterial genome, a fungal genome and a viral genome) for bacterial strains identification and abundance detection; the sequencing data was compared with a KEGG pathogenic bacteria database to confirm no pathogenic bacteria in the fecal bacteria donor; and the sequencing data was compared with an intestinal flora of a fecal bacteria receptor and screened to obtain a fecal bacteria donor complementary to the fecal bacteria receptor.

[0088] In this example, the screened fecal bacteria donor expressed biomarkers Escherichia coli, Clostridium ramosum, Eubacterium cylindroides, Roseburia hominis, Faecalibacterium prausnitzii, Bacteroides fragilis and Bacteroides vulgatus, and a diversity of the biomarkers was 163804.8. Biomarkers positively correlating to health were Clostridium ramosum, Eubacterium cylindroides, Roseburia hominis, Faecalibacterium prausnitzii and Bacteroides vulgatus, and biomarkers negatively correlating to health were Escherichia coli and Bacteroides fragilis.

[0089] In healthy people, a box plot analysis was performed on each microorganism positively correlating to health to obtain a median value for the each microorganism. In patients, the box plot analysis was performed on each microorganism negatively correlating to health to obtain a median value for the each microorganism. The median value for the each microorganism was used as a standard value for the donor screening.

[0090] A screening criterion of the fecal bacteria donor is as follows: a value of each microorganism positively correlating to health is greater than the standard value of the each microorganism, and a value of each microorganism negatively correlating to health is less than the standard value of the each microorganism.

Example 3 Preparation of a Fecal Bacteria Liquid

[0091] The screened feces of the fecal bacteria donor screened in Example 2 that was specific for the fecal bacteria receptor were collected on-site and sent to a laboratory for information registration, fecal identification, weighing, evaluation and treatment within 1 h to prepare a fecal bacteria liquid in an anaerobic environment. Steps are described below. [0092] (1) The collected feces were soaked in sterile saline (5° C.), and large particles were removed by using 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm filter screens in sequence, and then filtered three times with the 0.25 mm filter screen to obtain a liquid phase as a fecal filtrate. [0093] (2) The fecal filtrate was centrifuged for 10 min at 3000 r/min, and a precipitate was mixed with sterile saline to obtain the fecal bacteria liquid.

Example 4 Preparation of a Fecal Bacteria Liquid

[0094] The screened feces of the fecal bacteria donor screened in Example 2 that was specific for the fecal bacteria receptor were collected on-site and sent to a laboratory for information registration, fecal identification, weighing, evaluation and treatment within 1 h to prepare a fecal bacteria liquid in an anaerobic environment. Steps are described below. [0095] (1) The collected feces were soaked in sterile saline (3° C.), and large particles were removed by using 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm filter screens, and then filtered two times with the 0.25 mm filter screen to obtain a liquid phase as a fecal filtrate. [0096] (2) The fecal filtrate was centrifuged for 20 min at 1500 r/min, and a precipitate was mixed with sterile saline to obtain the fecal bacteria liquid.

Example 5 Preparation of a Whole Bacteria Capsule

[0097] The fecal bacteria liquid prepared in Example 3 was mixed with a lyophilizing protective agent (15% skim milk powder, 15% trehalose, 5% sucrose, 5% vitamin C and saline as balance) according to 3:1 (v/v). The temperature was lowered from room temperature to 4° C. within 10 s, further lowered from 4° C. to −40° C. at a cooling rate of 2° C./min and lowered from −40° C. to −80° C. at a cooling rate of 5° C./min After cooling and freezing, the mixture was vacuum dried for 48 h under conditions of a vacuum degree of 10 pa and −50° C. to obtain lyophilized powder of fecal bacteria which was loaded into an enteric capsule shell to obtain the whole bacteria capsule which was stored at −80° C.

Example 6 Preparation of a Whole Bacteria Capsule

[0098] The fecal bacteria liquid prepared in Example 4 was mixed with a lyophilizing protective agent (10% skim milk powder, 15% trehalose, 1% sucrose, 5% vitamin C and saline as balance). The temperature was lowered from room temperature to 6° C. within 10 s, further lowered from 6° C. to −50° C. at a cooling rate of 2° C./min and lowered from −50° C. to −75° C. at a cooling rate of 5° C./min After cooling and freezing, the mixture was vacuum dried for 48 h under conditions of a vacuum degree of 5 pa and −50° C. to obtain lyophilized powder of fecal bacteria which was loaded into an enteric capsule shell to obtain the whole bacteria capsule which was stored at −80° C.

Example 7 Preparation of a Whole Bacteria Capsule

[0099] The fecal bacteria liquid prepared in Example 4 was mixed with a lyophilizing protective agent (20% skim milk powder, 10% trehalose, 10% sucrose, 1% vitamin C and saline as balance). The temperature was lowered from room temperature to 3° C. within 20 s, further lowered from 3° C. to −30° C. at a cooling rate of 1° C./min and lowered from −30° C. to −80° C. at a cooling rate of 4° C./min After cooling and freezing, the mixture was vacuum dried for 24 h under conditions of a vacuum degree of 15 pa and −60° C. to obtain lyophilized powder of fecal bacteria which was loaded into an enteric capsule shell to obtain the whole bacteria capsule which was stored at −75° C.

Example 8 Therapeutic Effect of the Whole Bacteria Capsule

[0100] Patients with irritable bowel syndrome were administrated three doses of the whole bacteria capsule prepared in Example 5: once a week for three weeks.

[0101] As shown in FIG. 2, there was a significant decrease (p<0.01) in the abdominal pain score, the number of defecation per day and the number of food types of second-grade food intolerance, as well as a corresponding decrease in the number of food types of first-grade food intolerance and the number of food types of third-grade food intolerance.

[0102] As shown in FIG. 3, 16S rRNA detection was performed on fecal samples from patients. Samples showed a significant increase (p<0.01) in the abundance of Firmicutes at a family classification level, which tends to be consistent with the healthy donor.

[0103] To conclude, the fecal bacteria donor is screened through the high-throughput sequencing, accurately screening the fecal bacteria donors that are specific for different fecal bacteria receptors, and the prepared whole bacteria capsule is “matched accurately” with a significant pharmaceutical effect and high safety.

[0104] The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that the implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients, selections of specific manners, etc., all fall within the protection scope and the disclosure scope of the present application.