Fecal bacteriophage, separation and enrichment method thereof and application of fecal bacteriophage in preparation of medicine for treating inflammatory bowel disease

Abstract

The invention relates to faeces bacteriophage, a separation and enrichment method thereof and application of the faeces bacteriophage in preparation of drugs for treating inflammatory bowel diseases, and belongs to the technical field of biological medicines. The invention provides a method for separating and enriching faeces bacteriophages, which comprises the following steps: S1, mixing collected faeces with an SM buffer solution, centrifuging at 500-900 * g for 3-5 minutes, and retaining supernate; s2, centrifuging the supernate obtained in the step S1 at 3000-3400 * g for 25-30 minutes, and retaining the supernate; and S3, filtering the supernate obtained in the step S2, transferring the supernate into an ultrafiltration tube, and centrifuging for 25-30 minutes under 3000-3400 * g to obtain trapped fluid which is the enriched fecal bacteriophage. According to the method, the excrement solution is centrifuged, filtered and ultra-filtered twice, so that large organic particles, bacteria and metabolites in the excrement can be effectively removed, and enrichment of bacteriophages is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a fecal bacteriophage and a separation and enrichment method thereof, and an application of the fecal bacteriophage in the preparation of a drug for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic autoimmune disease of the intestine characterized by intermittent episodes of intestinal inflammation and a progressive course of remission. Crohn's disease (CD) and ulcerative colitis (UC) are the two main subtypes of IBD that affect different areas of the intestine. UC occurs in the colon, while CD occurs anywhere in the intestine, especially the ileum, which leads to more complex and severe disease manifestations and course. Although the exact cause of IBD is still unclear, a disturbed gut microbiome has been shown to be an important influencing factor.

[0003] At present, intestinal microecological transplantation has emerged. One of the components of the intestinal microecology is bacteria. Fecal microbiota transplantation (FMT) is a treatment method that transplants fecal microbiota from healthy donors into patients to restore or improve the intestinal microbial balance of patients. However, FMT has some disadvantages, including the instability of the source of the microbiota, the cumbersome operation, the potential risk of contamination of fecal samples, and sensitivity to the external environment, such as sensitivity to pH and temperature, which leads to easy inactivation of the microbiota and short shelf life. These factors restrict the specificity and sensitivity of FMT to a certain extent. At the same time, the infection risk of the recipient is difficult to control, the preparation and washing of the microbiota is long, the cost is high, and the clinical effect is limited, which limits its extensiveness and effectiveness in clinical applications. Therefore, the development of a new treatment strategy, fecal phage transplantation (FPT), may provide a safer and more effective method for the treatment of inflammatory bowel disease (IBD). However, the preparation of phages and their research in the treatment of IBD have not been reported at home and abroad. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a fecal phage separated and enriched from a healthy organism and a method for separating and enriching the fecal phage. The fecal phage has a certain therapeutic effect on inflammatory bowel disease and can be used in the preparation of drugs for treating inflammatory bowel disease.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for isolating and enriching fecal bacteriophages, comprising the following steps:

[0007] S1. Mix the collected feces with SM buffer, centrifuge at 500-900×g for 3-5 min, and retain the supernatant;

[0008] S2, centrifuging the supernatant obtained in step S1 at 3000-3400×g for 25-30 min, and retaining the supernatant;

[0009] S3. Filter the supernatant obtained in step S2 and transfer it into an ultrafiltration tube, centrifuge it at 3000-3400×g for 25-30 min, and the retentate obtained is the enriched fecal phage.

[0010] The present invention can effectively remove large organic particles, bacteria and metabolites in feces by centrifuging, filtering and ultrafiltration twice on the feces solution, which is beneficial to the enrichment of bacteriophages. In the enrichment method of the present invention, the first centrifugation (centrifugal force of 500-700×g) can effectively remove large organic particles in feces; the second centrifugation (centrifugal force of 3000-3400×g) can effectively remove most of the bacteria in feces; filtration can remove the remaining bacteria in feces; ultrafiltration can remove metabolites in feces and enrich fecal bacteriophages.

[0011] In the method for separating and enriching fecal phages of the present invention, in step S1, the SM buffer is a commonly used buffer in the art, usually containing sodium chloride (NaCl), magnesium chloride (MgCl2), gelatin and Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) etc. For example, in a common formula, the sodium chloride concentration may be around 0.1M, the magnesium chloride concentration is roughly around 5mM, the gelatin content is about 0.01% (mass volume ratio), and Tris-HCl is used to adjust the appropriate pH value (generally pH=about 7.5). For example, the formula of the SM buffer includes but is not limited to: 100mM NaCl, 8mM MgSO4, 50mMTris (Ph=7.5) and 0.002wt% gelatin per 1L, and is filtered into a sterile liquid using a filter with a membrane pore size of 0.22μm before use.

[0012] As a preferred embodiment of the method for separating and enriching fecal phages of the present invention, in step S1, the feces are collected from a healthy organism, and the types of phages in the feces include at least one of Lactobacillus prophage, Valbvirus ValB1MD2, Lagaffevirus lagaffe, Anaposvirus socalone, Chaphamaparvovirus galliform3, Tunavirus JMPW1, Natevirus nate and Erskinevirusasesino.

[0013] Another component in the intestinal microecology is a large number of bacteriophages, which play an important role in the intestinal microecology. They can regulate the composition and function of the intestinal microbiota by infecting and killing specific bacteria, thereby affecting the health of the host. The structure and function of bacteriophages are diverse. They can be DNA viruses or RNA viruses with different life cycles. Phages can be lytic or lysogenic, and the latter can lurk in bacteria to form a lysogenic state, which has little effect on host bacteria. The present invention has carried out virus group sequencing on the collected samples, and found that there are a variety of bacteriophages in the feces, which have played a certain IBD therapeutic effect when transplanted into IBD model mice.

[0014] As a preferred embodiment of the method for separating and enriching fecal phages of the present invention, the species abundance ratio of the fecal phages is Lactobacillus prophage Lj965: Valbvirus ValB1MD2: Lagaffeviruslagaffe: Lactobacillus prophage Lj77: Anaposvirus socalone: ​​Chaphamaparvovirusgalliform3: Tunavirus JMPW1: Natevirus nate: Erskinevirus asesino = (12.00-14.00): (7.00-9.00): (4.00-6.00): (1.50-2.50): (6.50-8.00): (4.00-6.00): (0.05-0.20): (4.50-6.00): (0.50-2.00).

[0015] As a preferred embodiment of the method for isolating and enriching fecal phages of the present invention, the species abundance ratio of the fecal phages is Lactobacillus prophage Lj965: Valbvirus ValB1MD2: Lagaffeviruslagaffe: Lactobacillus prophage Lj77: Anaposvirus socalone: ​​Chaphamaparvovirusgalliform3: Tunavirus JMPW1: Natevirus nate: Erskinevirus asesino=13.53:8.65:5.57:2.20:7.44:4.93:0.12:5.40:1.17.

[0016] As a preferred embodiment of the method for separating and enriching fecal phages of the present invention, in step S1, the ratio of feces to SM buffer is feces: SM buffer = 5-10 g: 10-20 mL.

[0017] As a preferred embodiment of the method for separating and enriching fecal phages of the present invention, in step S1, the centrifugation condition is centrifugation at 700×g for 3 min.

[0018] As a preferred embodiment of the method for separating and enriching fecal phages of the present invention, in step S2, the centrifugation condition is centrifugation at 3200×g for 30 min.

[0019] As a preferred embodiment of the method for separating and enriching fecal bacteriophages of the present invention, in step S3, the pore size of the filter membrane used in the filtration is 0.45 μm and / or 0.22 μm.

[0020] As a preferred embodiment of the method for separating and enriching fecal phages of the present invention, in step S3, the molecular weight cutoff of the ultrafiltration tube is 30-50 kDa.

[0021] As a preferred embodiment of the method for separating and enriching fecal phages of the present invention, in step S3, the centrifugation condition is centrifugation at 3200×g for 30 min.

[0022] In a second aspect, the present invention provides a fecal phage prepared according to the above separation and enrichment method.

[0023] In a third aspect, the present invention provides the use of the fecal phage in the preparation of a drug for treating inflammatory bowel disease.

[0024] Fecal phage transplantation (FPT) is an emerging treatment method that removes harmful substances such as bacteria from the feces of healthy people to screen for phages with therapeutic functions. Compared with FMT, FPT has many advantages, including high specificity, high efficiency and low side effects. By accurately identifying and attacking specific bacteria, phage therapy can treat diseases more accurately and reduce damage to normal cells. In addition, the process of phage infection of bacteria is fast and efficient, and can achieve therapeutic effects in a short time, saving treatment time. Compared with traditional drug therapy, phage therapy has a lower risk of side effects because it acts directly on pathogens instead of affecting the entire body system like many drugs. More importantly, it is easy to obtain phages with therapeutic functions isolated and purified from the feces of healthy people. Once a suitable donor is screened, the same phage can be quickly and continuously isolated and purified, and there is almost no batch error in phage preparation, truly realizing efficient, fast and low-cost synthesis. Therefore, phage therapy provides new ideas and methods for disease treatment and has broad application prospects. The present invention confirms through animal experiments that transplanting fecal phages into IBD model mice can effectively alleviate colon shortening, inflammatory infiltration of colon tissue, and changes in intestinal microbial structure caused by DSS, and has a certain therapeutic effect on IBD.

[0025] As a preferred embodiment of the application of the present invention, the inflammatory bowel disease drug is a drug for alleviating intestinal inflammation.

[0026] As a preferred embodiment of the application of the present invention, the dosage form of the drug includes at least one of tablets, solutions, suspensions, emulsions, microcapsules, microspheres, injections, liposomes and aerosols.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides a method for fecal phage and its separation and enrichment, which ensures the high purity of phage through precise centrifugation and filtration steps. At the same time, the method provided by the present invention is standardized and easy to operate, which is conducive to repeatability in laboratory environment and feasibility in clinical application. In addition, the standard reagents and equipment used in the present invention are cost-effective, which makes the method of the present invention more economically attractive.

[0029] 2. The fecal phages of the present invention have shown potential effects in the treatment of IBD by transplanting them during the remission phase of the IBD mouse model. This method is more effective than transplanting them during the attack phase. This provides a new strategy for the treatment of IBD and may help improve the long-term prognosis of IBD patients and help reduce the recurrence of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1This is a flow chart of the fecal phage separation and enrichment method of the present invention;

[0031] Figure 2 This is a statistical diagram of the relative abundance of different virus types contained in fecal phages obtained in Example 1 of the effect example 1 of the present invention;

[0032] Figure 3 This is the experimental flow chart of Example 2 of the present invention;

[0033] Figure 4 This is a statistical chart of weight changes of mice in different treatment groups in Example 2 of the present invention;

[0034] Figure 5 This is a statistical diagram of the colon length of mice in different treatment groups in Example 2 of the present invention;

[0035] Figure 6 This is a statistical diagram of colon histological scores of mice in different treatment groups in Example 2 of the present invention;

[0036] Figure 7 This is a statistical diagram of the expression levels of pro-inflammatory cytokines in colon tissue of mice in different treatment groups in Example 2 of the present invention;

[0037] Figure 8 This is a graph showing the results of 16S rDNA sequencing analysis of feces of mice in different treatment groups and at different time points in Example 2 of the present invention;

[0038] Fig. 9 The 16S rDNA sequencing analysis results and correlation analysis results of feces of mice in different treatment groups and at different time points in Example 2 of the present invention are shown;

[0039] In the above figures, the "*" mark indicates that there are significant differences between the two groups, where "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001. DETAILED DESCRIPTION

[0040] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0041] Unless otherwise specified, the reagents and consumables used in the following embodiments, comparative examples and effect examples can be obtained through commercial channels.

[0042] The SPF grade C57BL / 6J mice used in the following examples, comparative examples and effect examples were purchased from Guangdong Yaokang Company, and the experiments were conducted in the animal rooms of Sun Yat-sen University and Guangdong Institute of Microbiology. The mice were kept in a standard animal room with a temperature of 23±2°C and a humidity of 50±10%, a 12h / 12h (lighting on at 8:00) circadian rhythm, and free access to food.

[0043] The formula of SM buffer is as follows: each 1L contains 100mM NaCl, 8mM MgSO4, 50mM Tris (Ph=7.5) and 0.002wt% gelatin, and is filtered through a filter with a pore size of 0.22μm to obtain a sterile liquid before use.

[0044] The reverse transcription kit was HiScript III RT SuperMix, purchased from Nanjing Novozymes Biotech Co., Ltd., with the catalog number R323-01.

[0045] qPCR reagents are 2× qPCR SYBR Green Master Mix (No Rox) was purchased from Shanghai Yisheng Biotechnology Co., Ltd. with the catalog number 11201ES08.

[0046] BashingBead TM The buffer solution was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd. (GenStar) with the catalog number AR1112.

[0047] Zymo-Spin TM The III-F filter was purchased from ZYMO RESEARCH, USA, with the item number D4015.

[0048] The genomic lysis buffer was purchased from ZYMO RESEARCH, USA, with the product number D3004-1-100.

[0049] Zymo-Spin TM The IICR column was purchased from ZYMO RESEARCH, USA, with the product number C1078-50.

[0050] The pre-wash buffer used for DNA extraction was purchased from ZYMO RESEARCH, USA, with the product number D3004-5-15.

[0051] g-DNA washing buffer was purchased from ZYMO RESEARCH, USA, with the product number D3004-2-50.

[0052] The qPCR primer sequences for IL-1β, IL-6, IL-17, TNF-α, and β-actin (internal reference) are as follows:

[0053] IL-1β-F: 5-TGGACCTTCCAGGATGAGGACA-3;

[0054] IL-1β-R: 5-GTTCATCTCGGAGCCTGTAGTG-3;

[0055] IL-6-F: 5-TACCACTTCACAAGTCGGAGGC-3;

[0056] IL-6-R: 5-CTGCAAGTGCATCATCGTTGTTC-3;

[0057] IL-17-F: 5-CAGACTACCTCAACCGTTCCAC-3;

[0058] IL-17-R: 5-TCCAGCTTTTCCCTCCGCATTGA-3;

[0059] TNF-α-F: 5-GGTGCCTATGTCTCAGCCTCTT-3;

[0060] TNF-α-R: 5-GCCATAGAACTGATGAGAGGGAG-3;

[0061] β-actin-F: 5-CATTGCTGACAGGATGCAGAAGG-3;

[0062] β-actin-R: 5-TGCTGGAAGGTGGACAGTGAGG-3.

[0063] The experimental methods without specific description of the steps in the following embodiments, comparative examples and effect examples are conventional techniques in the art, and reference may be made to "Molecular Biology Experiments" (2021, Zhejiang University Press), "Biochemistry Experiments" (2020, Science Press), etc.

[0064] Example 1

[0065] This embodiment provides a fecal phage and a method for separating and enriching the same. The separation and enrichment method is based on Figure 1 The process includes the following steps:

[0066] 1. Use a sterile tube to collect fresh feces from healthy C57BL / 6J mice. Add 10 mL of sterile SM buffer to every 5 g of fecal sample, mix, and centrifuge at 700 × g for 3 min. Keep the supernatant.

[0067] 2. Centrifuge the supernatant obtained in step 1 at 3200×g for 30 min and retain the supernatant;

[0068] 3. Filter the supernatant obtained in step 2 through a filter membrane with a pore size of 0.45 μm, and then filter through a filter membrane with a pore size of 0.22 μm to obtain a filtrate;

[0069] 4. The filtrate obtained in step 3 was transferred to a 50 kDa ultrafiltration tube and centrifuged at 3200 × g for 30 min. The retentate obtained was the enriched fecal phage.

[0070] Example 2

[0071] This embodiment provides a fecal phage and a method for separating and enriching the fecal phage, wherein the separation and enrichment method comprises the following steps:

[0072] 1. Use a sterile tube to collect fresh feces from healthy C57BL / 6J mice. Add 10 mL of sterile SM buffer to every 5 g of fecal sample, mix, and centrifuge at 500 × g for 5 min. Keep the supernatant.

[0073] 2. Centrifuge the supernatant obtained in step 1 at 3000×g for 30 min and retain the supernatant;

[0074] 3. Filter the supernatant obtained in step 2 through a filter membrane with a pore size of 0.45 μm, and then filter through a filter membrane with a pore size of 0.22 μm to obtain a filtrate;

[0075] 4. The filtrate obtained in step 3 was transferred to a 50 kDa ultrafiltration tube and centrifuged at 3000 × g for 30 min. The retentate obtained was the enriched fecal phage.

[0076] Example 3

[0077] This embodiment provides a fecal phage and a method for separating and enriching the fecal phage, wherein the separation and enrichment method comprises the following steps:

[0078] 1. Use a sterile tube to collect fresh feces from healthy C57BL / 6J mice. Add 10 mL of sterile SM buffer to every 5 g of fecal sample, mix, and centrifuge at 900 × g for 3 min. Keep the supernatant.

[0079] 2. Centrifuge the supernatant obtained in step 1 at 3400×g for 25 min and retain the supernatant;

[0080] 3. Filter the supernatant obtained in step 2 through a filter membrane with a pore size of 0.45 μm, and then filter through a filter membrane with a pore size of 0.22 μm to obtain a filtrate;

[0081] 4. The filtrate obtained in step 3 was transferred to a 30 kDa ultrafiltration tube and centrifuged at 3400 × g for 25 min. The retentate obtained was the enriched fecal phage.

[0082] Comparative Example 1

[0083] This comparative example provides a fecal phage and a method for separating and enriching the same. The separation and enrichment method is similar to that in Example 1, except that in step 1, the centrifugation conditions are 1200×g and 5 min, and the remaining steps and parameter conditions remain unchanged.

[0084] Comparative Example 2

[0085] This comparative example provides a fecal phage and a method for separating and enriching the same. The separation and enrichment method is similar to that in Example 1, except that in step 2, the centrifugation conditions are 5000×g and 50 min, and the remaining steps and parameter conditions remain unchanged.

[0086] Comparative Example 3

[0087] This comparative example provides a fecal phage and a method for separating and enriching the same. The separation and enrichment method is similar to that in Example 1, except that in step 4, the ultrafiltration tube specification is set to 100 kDa, and the remaining steps and parameter conditions remain unchanged.

[0088] Effect Example 1

[0089] In order to verify whether the separation and enrichment method of Example 1 can enrich phages, the fecal phages obtained in Example 1 were subjected to virome sequencing. The virome sequencing was commissioned to Guangdong Meige Gene Technology Co., Ltd. The results of the virome sequencing are shown in Tables 1 and Figure 2 .

[0090] Table 1 Results of fecal viral group sequencing

[0091] Virus Type Relative abundance (%) Lactobacillus prophage Lj965 13.53 Valbvirus ValB1MD2 8.65 Lagaffevirus lagaffe 5.57 Lactobacillus prophage Lj771 2.20 Anaposvirus socalone 7.44 Chaphamaparvovirus galliform3 4.93 Tunavirus JMPW1 0.12 Natevirus nate 5.40 Erskine virus asesino 1.17 Others 50.99

[0092] like Figure 1-2 As shown in Table 1, the method of Example 1 can enrich a large number of phages, and the enriched fecal phages contain 9 known phages, indicating that the enrichment method of the present invention can effectively separate and enrich phages in feces, and transplanting them into the intestine of an organism with inflammatory bowel disease may cause changes in the intestinal microecology of the organism.

[0093] Effect Example 2

[0094] In order to verify the efficacy of the fecal phage obtained in the present invention on inflammatory bowel disease, the fecal phage was transplanted into inflammatory bowel disease model mice, and the corresponding indicators were detected. The specific scheme is as follows:

[0095] according to Figure 3All mice were housed together for 1 week to balance the microbial community, and then divided into 3 groups: control group (no FPT intervention, control), FPT-f group (FPT given during the onset of inflammatory bowel disease) and FPT-r group (FPT given during the remission of inflammatory bowel disease), with 8 mice in each group. Figure 3 The IBD mouse model (which can simulate the relapse and remission course of IBD) was established by intermittent oral administration of 2-3.5wt% dextran sulfate sodium (DSS) according to the schedule. Figure 3 The FPT-f group and the FPT-r group were gavaged with 200 μL of fecal phage (FPT) obtained in Example 1 according to the time schedule (i.e., the remission period and the attack period during the first round of DSS administration), and 100 μL of 1M NaHCO3 solution was gavaged before FPT to neutralize gastric acid. The weight changes of mice were detected daily. After 12 weeks of the experiment, the mice were killed and the colon and feces samples were collected.

[0096] After the collected colon tissue was measured, a portion was fixed with 10 v / v% formalin, embedded in paraffin and prepared into 5 μm thick sections, stained with hematoxylin and eosin, and pathological scores were performed based on inflammatory cell infiltration and tissue damage. The other portion was extracted with TRIzol reagent for RNA, synthesized with a reverse transcription kit, and qPCR reagents were used on a QuantStudio 7Flex instrument (Applied Biosystems) to perform qPCR reactions to detect pro-inflammatory cytokines (IL-1β, IL-6, IL-17, TNF-α) in colon tissue. The statistical results of weight changes are shown in Figure 4 , colon length measurement results are shown in Figure 5 The histological scoring results of colon tissue are shown in Figure 6 The results of pro-inflammatory cytokine expression level detection are shown in Figure 7 .

[0097] Inflammatory cell infiltration score:

[0098] 0 points: no inflammatory cell infiltration;

[0099] 1 point: mild inflammatory cell infiltration, such as less than 2 inflammatory cells / 20x field of view;

[0100] 2 points: moderate inflammatory cell infiltration, such as 2-4 inflammatory cells / 20x field of view;

[0101] 3 points: severe inflammatory cell infiltration, for example, more than 4 inflammatory cells / 20x field of view.

[0102] Tissue damage score:

[0103] 0 points: no tissue damage;

[0104] 1 point: mild tissue damage, which may be manifested as less than 50% damage;

[0105] 2 points: moderate tissue damage, which may be manifested as 50%-75% damage;

[0106] 3 points: Severe tissue damage, which may be manifested as damage greater than 75%.

[0107] 100 mg of fecal sample was collected and mixed with 750 μL of BashingBead TM The buffer was mixed, vortexed to ensure thorough mixing, and then centrifuged at 10,000 × g for 1 min. The resulting supernatant was transferred to a Zymo-Spin TM III-F filter collection tube and further centrifuge at 8,000 × g for 1 min. After all the supernatant has passed through the filter, add 1200 μL of genomic lysis buffer (Zymo Research, D3004-1-100) to the filtrate and mix thoroughly. Transfer the mixed solution to a Zymo-Spin TM IICR column and centrifuged at 10,000 × g for 1 minute to collect DNA. Then, the DNA collected on the Zymo-Spin was washed with pre-wash buffer (Zymo Research, D3004-5-15) and g-DNA wash buffer (Zymo Research, D3004-2-50). TM The DNA on the IICR column was washed and finally eluted with DNA elution buffer. The extracted DNA was used for subsequent library construction, 16S rDNA metagenomic sequencing and analysis. The experimental results are shown in Figure 8-9 .

[0108] like Figure 4 As shown in the figure, the weight changes of mice receiving FPT-r intervention were not significant in the second and third rounds of DSS, indicating that they were tolerant to repeated DSS exposure. In contrast, the weight of mice in the FPT-f group and the control group (Control, without FPT intervention) decreased significantly in the subsequent DSS. It is particularly noteworthy that the weight loss of mice treated with FPT-f was more significant in the third round of DSS, indicating that phage transplantation during the inflammatory attack period may exacerbate the inflammatory response.

[0109] like Figure 5-7As shown, colon length measurement, histological scores, and expression levels of proinflammatory cytokines all confirmed that FPT-r intervention significantly reduced intestinal inflammation. In contrast, FPT-f intervention did not reduce intestinal inflammation, but may have worsened the inflammatory condition. Taking these data together, we conclude that FPT intervention is effective in treating IBD inflammation during remission, while intervention during the inflammatory attack period is ineffective. These findings provide a new strategy for the treatment of IBD, that is, phage transplantation during the inflammatory remission period may be more helpful in controlling the disease. The colon length of mice in the FPT-r group was significantly longer than that of the control group and the FPT-f group, indicating that administration of FPT during remission can more effectively improve the phenomenon of shortened colon length caused by DSS.

[0110] like Figure 8-9 The results of the analysis of the gut microbiota showed that the FPT-r intervention significantly promoted the relative abundance of bacterial members from the Muribaculaceae and Lachnospiraceae families, which are considered beneficial bacteria and persisted throughout the course of the disease (7 common bacterial species at both T1 and T2 time points). FPT-r led to an increase in Lactobacillus prophages, which in turn led to an increase in its host bacteria Lactobacillus (beneficial bacteria). Lactobacillus can coexist with Lachnospiraceae and Muribaculaceae, suggesting a cross-feeding relationship between them. At the same time, the species abundance of Lachnospiraceae and Lactobacllaceae was positively correlated. These findings emphasize that the FPT-r intervention is beneficial for reconstructing beneficial intestinal bacterial communities and increasing the species abundance of beneficial bacteria, thereby continuously resisting repeated pro-inflammatory stimuli.

[0111] In summary, the fecal phages prepared by the separation and enrichment method provided by the present invention showed resistance to intermittent enteritis when administered to inflammatory bowel disease model mice, could effectively reduce intestinal inflammation, and had a certain anti-inflammatory effect, and had a high potential for treating inflammatory bowel disease.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for separating and enriching fecal bacteriophages, characterized in that: The following steps are involved: S1. Mix the collected feces with SM buffer, centrifuge at 500-900×g for 3-5 min, and retain the supernatant; S2, centrifuging the supernatant obtained in step S1 at 3000-3400×g for 25-30 min, and retaining the supernatant; S3. Filter the supernatant obtained in step S2 and transfer it into an ultrafiltration tube, centrifuge it at 3000-3400×g for 25-30 min, and the retentate obtained is the enriched fecal phage.

2. The method for separating and enriching fecal bacteriophages according to claim 1, characterized in that: In step S1, the feces is collected from a healthy organism, and the types of bacteriophages in the feces include at least one of Lactobacillus prophage, ValbvirusValB1MD2, Lagaffevirus lagaffe, Anaposvirus socalone, Chaphamaparvovirusgalliform3, Tunavirus JMPW1, Natevirus nate and Erskinevirus asesino.

3. The method for separating and enriching fecal phages according to claim 1, characterized in that: The species abundance ratio of the fecal phages is Lactobacillus prophage Lj965: Valbvirus ValB1MD2: Lagaffeviruslagaffe: Lactobacillus prophage Lj77: Anaposvirus socalone: ​​Chaphamaparvovirusgalliform3: Tunavirus JMPW1: Natevirus nate: Erskinevirus asesino = (12.00-14.00): (7.00-9.00): (4.00-6.00): (1.50-2.50): (6.50-8.00): (4.00-6.00): (0.05-0.20): (4.50-6.00): (0.50-2.00).

4. The method for separating and enriching fecal bacteriophages according to claim 3, characterized in that: The species abundance ratio of the fecal phages is Lactobacillus prophage Lj965: Valbvirus ValB1MD2: Lagaffeviruslagaffe: Lactobacillus prophage Lj77: Anaposvirus socalone: ​​Chaphamaparvovirusgalliform3: Tunavirus JMPW1: Natevirus nate: Erskinevirus asesino=13.53:8.65:5.57:2.20:7.44:4.93:0.12:5.40:1.

17.

5. The method for separating and enriching fecal bacteriophages according to claim 1, characterized in that: In step S1, the ratio of feces to SM buffer is feces:SM buffer=5-10 g:10-20 mL.

6. The method for separating and enriching fecal bacteriophages according to claim 1, characterized in that: In step S3, the pore size of the filter membrane used in the filtration is 0.45 μm and / or 0.22 μm.

7. The method for separating and enriching fecal bacteriophages according to claim 1, characterized in that: In step S3, the molecular weight cut-off of the ultrafiltration tube is 30-50 kDa.

8. The method for separating and enriching fecal bacteriophages according to claim 1, characterized in that: Including at least one of the following (I) to (III): (I) In step S1, the centrifugation condition is centrifugation at 700×g for 3 min; (II) In step S2, the centrifugation condition is centrifugation at 3200×g for 30 min; (III) In step S3, the centrifugation condition is centrifugation at 3200×g for 30 min.

9. A fecal phage, characterized in that It is prepared according to the separation and enrichment method according to any one of claims 1 to 7.

10. Use of the fecal phage according to claim 9 in the preparation of a drug for treating inflammatory bowel disease.

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