Preparation method and application of viable bacteria enema

Abstract

The invention discloses a viable bacteria enema as well as a preparation method and application thereof, and belongs to the field of medicine preparation. The enema liquid provided by the invention can be individually designed according to intestinal flora detection results of different patients, and has the advantages of accurate matching, good curative effect, quick response and no side effect. The treatment and prevention effects of viable bacteria and prebiotics as main raw materials of the enema liquid on intestinal related diseases are mediated by changing microbiota and / or functions of the microbiota, and related diseases with beneficial effects such as ulcerative colitis and constipation are involved.

Description

Technical Field

[0001] The present invention relates to a live bacteria enema solution, its preparation method and application, belonging to the field of drug preparation. Background Art

[0002] Live bacteria enema solution is a new treatment method, mainly used to regulate the intestinal flora and treat related diseases. A number of studies have shown that live bacteria enema solution shows potential in the treatment of inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, and can relieve symptoms and promote mucosal healing. Fecal microbiota transplantation (FMT) by enema is highly effective in the treatment of recurrent Clostridioides difficile infection, which has promoted the application of live bacteria enema solution.

[0003] Although live bacteria enema solution (such as fecal microbiota transplantation or probiotic enema) shows potential in intestinal flora regulation and related disease treatment, its technology still has some key defects and challenges. Since the live bacteria enema solution applied in current clinical research depends on fresh donor feces, the microbial species and proportions of each batch of preparations vary significantly, the preparation operation is difficult, time-consuming, the loss rate of live bacteria is high, and the product stability is poor. It is difficult to achieve standardization, and there are safety and pollution risks. To solve this problem, not only the production process of the preparation needs to be improved, but also systematic breakthroughs need to be achieved from formula research and development to colonization efficiency, etc., so as to lay a foundation for the development and application of products related to microbiota transplantation. However, the existing technology lacks a standardized enema solution that can efficiently and targetedly improve the intestinal microecology by fecal microbiota transplantation with live bacteria. Summary of the Invention

[0004] In view of the above deficiencies of the prior art, the present invention provides a live bacteria enema solution / suppository that can efficiently and targetedly change the intestinal microecology, aiming to solve the technical problem that the existing technology lacks an enema solution that can efficiently and targetedly improve the intestinal microecology by fecal microbiota transplantation with live bacteria.

[0005] The first technical solution provided by the present invention is a live bacteria enema solution, comprising the following components:

[0006] Component of Bottle A: Phosphate buffer solution with pH = 7.2 - 7.4;

[0007] Component of Bottle B: 1% - 40% of live bacteria powder and 60% - 99% of excipients.

[0008] In some embodiments, the live bacteria powder is the freeze-dried powder of live bacteria used in fecal microbiota transplantation therapy, and the source is the human feces of qualified donors who have passed the test and screening for transmissible pathogens. Among them, the criteria for qualified donors refer to the "Chinese Expert Consensus on the Screening and Management of Donors for Intestinal Microbiota Transplantation".

[0009] In some embodiments, the adjuvants are one or more of pharmaceutical-grade disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), poloxamer, sodium dodecyl sulfate, sodium alginate, fructooligosaccharide, galactooligosaccharide, inulin, resistant dextrin, and stachyose.

[0010] The second technical solution provided by the present invention is a method for preparing the live bacteria enema solution described in the first technical solution, including the following steps:

[0011] S1. Buffer preparation: Prepare a phosphate buffer solution with a pH of 7.2 - 7.4, adjust the osmotic pressure to 280 - 320 mOsm / L using NaCl, bottle and sterilize, and store at 4°C;

[0012] S2. Preparation of live bacteria freeze-dried powder:

[0013] Collect the donor feces using a dedicated sterile fecal bacteria collection device. The weight of the feces should be no less than 200 g, and the feces should be qualified only when the Bristol score is 3 - 5. Immediately enter the bacteria solution production process, filter step by step through a 50 - 800 mesh filter, centrifuge at 2000 rpm / min for 3 min for fecal bacteria separation and washing. Add a freeze-drying protectant to the obtained bacteria solution, mix well, and place it in a vacuum freeze-dryer for drying to obtain the live bacteria freeze-dried powder;

[0014] S3. Preparation of live bacteria - prebiotic freeze-dried powder:

[0015] Crush the live bacteria freeze-dried powder raw material and the adjuvants in step S2 through a 60 - 100 mesh sieve, add them to a V-type mixer and mix evenly, then bottle.

[0016] In some embodiments, during use, mix the buffer solution in step S1 with the live bacteria - prebiotic freeze-dried powder in step S3 so that the total number of live bacteria in the final enema solution is not less than 10 9 CFU / mL (the REBYOTA standard approved by the FDA is 1x10 8 CFU / mL - 5x10 10 CFU / mL).

[0017] In some embodiments, in step S2, the freeze-drying protectant is one or more of pharmaceutical-grade sucrose, trehalose, glucose, lactose, glycerol, mannitol, sorbitol, glycine, Tween 80, sodium hyaluronate, and polyvinylpyrrolidone (PVP).

[0018] In some embodiments, in step S3, the weight percentage of the live bacteria freeze-dried powder in the live bacteria - prebiotic freeze-dried powder is 1% - 40%.

[0019] In some embodiments, the adjuvants are one or more of pharmaceutical-grade disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), poloxamer, sodium dodecyl sulfate, sodium alginate, fructooligosaccharide, galactooligosaccharide, inulin, resistant dextrin, and stachyose.

[0020] In some embodiments, the adjuvants account for 60% to 99% by weight of the live bacteria-prebiotic lyophilized powder.

[0021] In some embodiments, the weight percentage of microcrystalline cellulose (MCC) is 1% - 30%, the weight percentage of carboxymethyl cellulose (CMC) is 1% - 30%, the weight percentage of poloxamer is 0.1% - 5%, and the weight percentages of fructooligosaccharide, galactooligosaccharide, inulin, and stachyose are 1% - 50%.

[0022] In some embodiments,

[0023] The third technical solution provided by the present invention is the application of the enema solution described in the first technical solution in the preparation of a drug for improving intestinal microecology.

[0024] The fourth technical solution provided by the present invention is the application of the enema solution described in the first technical solution in the preparation of a drug for relieving and / or treating ulcerative colitis.

[0025] In some embodiments, the application includes down-regulating the levels of pro-inflammatory cytokines and / or the oxidative stress biomarker MDA in individuals with colitis.

[0026] In some embodiments, the pro-inflammatory cytokines include TNF-α, IL-1β, and IL-6.

[0027] The fifth technical solution provided by the present invention is the application of the enema solution described in the first technical solution in the preparation of a drug for relieving and / or treating constipation.

[0028] The technical effects of the present invention are as follows:

[0029] The enema solution can be personalized designed and precisely matched according to the intestinal flora test results of different patients, with good curative effect, quick onset, and no side effects. Live bacteria and prebiotics, as the main raw materials of the enema solution, mediate the treatment and prevention of intestinal-related diseases by changing the microbiota and / or its functions, and the diseases involved in producing beneficial effects include ulcerative colitis, constipation, etc. Description of the Drawings

[0030] Figure 1 It shows the effects of the live bacteria enema solution on the body weight and disease activity index of ulcerative colitis mice in Example 2 of the present invention; (a) Body weight change (b) DAI score change.

[0031] Figure 2 For the effects of the live bacteria enema solution in Example 2 of the present invention on intestinal inflammation and oxidative stress in DSS-induced colitis mice; (a) Protein level of inflammatory factor TNF-α in the colon of mice; (b) Protein level of inflammatory factor IL-1β in the colon of mice; (c) MDA level in the colon; (d) Expression of TNF-α mRNA in the colon; (e) Expression of IL-1β mRNA in the colon; (f) Expression of IL-6 mRNA in the colon.

[0032] Table 3 shows the changes in the time of the first black stool (min) and fecal water content (%) before and after intervention in SD rats after successful modeling in Example 3 of the present invention.

[0033] Figure 3 For the intestinal propulsion ratio of each group of SD rats in Example 3 of the present invention.

[0034] Figure 4 For the effects of the live bacteria enema solution in Example 3 of the present invention on the intestinal flora diversity in constipation mice induced by loperamide hydrochloride; (a) Shannon index of α diversity; (b) Simpson index of α diversity; (c) Principal component analysis (PCA) indicating β diversity. Detailed implementation manners

[0035] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0036] Technical terms

[0037] The "live bacteria freeze-dried powder" in the present invention refers to a powder product made from healthy human fecal bacteria through processes such as filtration, purification, concentration, and freeze-drying. The water in the bacterial liquid forms ice crystals through pre-freezing, and then in a vacuum environment, the ice crystals are directly converted into water vapor using the sublimation principle to achieve drying, obtaining the live bacteria freeze-dried powder.

[0038] The "enema solution" in the present invention refers to a liquid injected into the rectum or colon through the anus, mainly used for cleaning the intestine, stimulating defecation, exhausting gas, or supplying drugs and nutrients to achieve the purpose of treatment or diagnosis.

[0039] The "remission" in the present invention refers to a certain degree of alleviation or improvement of one or more symptoms, signs, or pathological states after the onset of a disease.

[0040] The "treatment" in the present invention refers to preventing, curing, reversing, weakening, alleviating, minimizing, inhibiting, stopping, and / or halting one or more clinical symptoms of a disease after the onset of the disease.

[0041] "Colitis" in the present invention refers to a disease in which the mucosa of the colon (large intestine) and its underlying tissues become inflamed. It is a common digestive system disease that may be caused by various factors, including infection, autoimmune reactions, genetic factors, etc.

[0042] "Constipation" in the present invention refers to a symptom characterized by reduced frequency of defecation, decreased stool volume, dry and hard stools, and difficulty in defecation.

[0043] "Intestinal microbiota" in the present invention refers to the ecosystem formed by the microbial community in the human intestine and the interactions between them and the host. It is a complex and dynamically balanced ecosystem that plays an important role in maintaining human health.

[0044] "Disease activity index" in the present invention refers to a quantitative index used to evaluate the activity of certain chronic diseases (i.e., the current degree of inflammation or severity of the disease). It gives a numerical value to reflect the activity state of the disease at a specific time point by comprehensively considering various clinical manifestations, laboratory test results, and other factors.

[0045] "Time to first black stool" in the present invention refers to the time required for an animal (such as a mouse or rat) to excrete the first black stool after receiving a certain stimulus (such as intragastric administration of ink) or drug treatment.

[0046] "Intestinal propulsion ratio" in the present invention refers to an index of intestinal peristalsis efficiency, which may represent the ratio of the distance that the intestine propels the contents forward per unit time to the total length of the intestine.

[0047] Raw materials used in the examples:

[0048] 1. Healthy C57BL / 6J mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0049] 2. SD rats were obtained from the Experimental Animal Center of Xi'an Jiaotong University.

[0050] Example 1

[0051] 1. Prepare a phosphate buffer solution with pH = 7.2 - 7.4 as follows:

[0052] Prepare the stock solution 0.2M Na2HPO4: Weigh 71.60 g of Na2HPO4·12H2O and dissolve it in 1 L of purified water.

[0053] Prepare the stock solution 0.2M NaH2PO4: Weigh 31.20 g of NaH2PO4·2H2O and dissolve it in 1 L of purified water.

[0054] Table 1 Buffer preparation system

[0055] pH <![CDATA[0.2M Na2HPO4(mL)]]> <![CDATA[0.2M Na2HPO4(mL)]]> 7.2 28 72 7.3 23 77 7.4 19 81

[0056] Prepare working solutions of buffers with different pH values according to the above table, and adjust the osmotic pressure to 280 - 320 mOsm / L using NaCl.

[0057] Aliquot the prepared buffer into 200 mL / vial (Vial A), sterilize it, and store it at 4°C.

[0058] 2. Preparation of live bacteria freeze-dried powder

[0059] Collect fecal samples provided by qualified donors using a dedicated sterile collection bag, with the fecal weight not less than 200 g. Add the collected feces to a fully automatic fecal bacteria separator to obtain a fecal bacteria solution. Add 10% freeze-drying protective agent excipients (the ratio of trehalose, mannitol, and sorbitol is 4:1:1) to the fecal bacteria solution, mix evenly, and place it in a vacuum freeze-dryer for drying to obtain live bacteria freeze-dried powder.

[0060] 3. Preparation of live bacteria - prebiotic freeze-dried powder:

[0061] Weigh the live bacteria freeze-dried powder and excipients, crush them through a 60 - 100 mesh sieve, add them to a V-type mixer and mix evenly to obtain a fecal bacteria mixed powder, and fill it into vials as Vial B according to the prescription amount. Ensure that the total number of viable fecal bacteria in the final enema solution is not less than 1×10 8 CFU / mL.

[0062] Among them, the weight percentage of the live bacteria freeze-dried powder is 40%, the weight percentage of MCC is 20%, the weight percentage of sodium alginate is 5%, the weight percentage of CMC is 10%, the weight percentage of sodium dodecyl sulfate is 0.15%, the weight percentage of poloxamer is 3%, the weight percentage of fructooligosaccharide is 5%, the weight percentage of galactooligosaccharide is 5%, the weight percentage of inulin is 5%, and the weight percentage of resistant dextrin is 5%.

[0063] Aliquot the above evenly mixed fecal bacteria mixed powder into 10 g / vial (Vial B), fill it with CO2, and store it at 4°C.

[0064] 4. Use of live bacteria enema solution

[0065] Add the live bacteria - prebiotic freeze-dried powder in Vial B to Vial A, shake it vigorously for 3 minutes until it is dispersed into a uniform suspension. After the foam of the suspension disappears, use a disposable enema device to perform enema on the patient.

[0066] Example 2 Influence of live bacteria enema solution on ulcerative colitis

[0067] 1. Experimental animals: 8-week-old healthy C57BL / 6J mice weighing 20 - 22 g, 50 males.

[0068] Construction of animal model:

[0069] After 1 week of adaptive feeding of healthy male C57BL / 6J mice, they were randomly divided into a blank group, a model group, a prebiotic group, a fecal bacteria solution group, and a live bacteria enema solution group, a total of 5 groups, with 10 mice in each group. The blank group was not treated, and the remaining 4 groups freely drank pure water containing 2.5% DSS for 5 days and were fed with ordinary feed. The body weight, stool characteristics, and blood in the stool were detected every day, and the disease activity index of the mice was evaluated according to the Murano standard. After 5 days of feeding, the model of acute ulcerative colitis was successfully established with positive fecal occult blood.

[0070] Drug administration plan: Subsequently, the 2.5% DSS was continued to be drunk for 3 days to strengthen the model, and at the same time, the prebiotic group, the fecal bacteria solution group, and the live bacteria enema solution group were given enema treatment (10 8 CFU / g, quantified according to body weight); the blank group was given an enema of the same volume of phosphate buffer solution. Before enema treatment, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.3 mL / 100 g body weight. The anesthetized rats were fixed on the rat board in the supine position. After lubricating a rubber hose with a diameter of 2 mm with liquid paraffin, it was slowly and gently inserted into the rectum of the rat about 8 cm through the anus. A previously prepared solution of 1 mL / 200 g body weight was slowly injected at one time. After successful enema, the rats were kept in the supine position with their buttocks up for 15 min, and after natural awakening, they were put back into the cage and raised routinely. Each group was given enema administration once a day for 7 consecutive days.

[0071] 2. Detection indicators

[0072] (1) General condition and body weight change of mice

[0073] The results showed that the mice in the blank group had smooth hair, normal stools, and no obvious fluctuations in body weight. From day 0 to day 5, in addition to the blank group, the other four groups of mice showed varying degrees of reduced food intake, slow movement, dull and lusterless hair, loose stools accompanied by blood in the stool, etc. From day 6, the stools of the mice in the prebiotic group, the fecal bacteria solution group, and the live bacteria enema solution group improved, their movement became gradually sensitive, their food intake increased, and their hair became smooth and shiny. On day 12, according to the overall recovery degree of the three groups, the order was live bacteria enema solution group > fecal bacteria solution group > prebiotic group. As Figure 1 a, the body weight of the mice in the blank group gradually increased, while the body weight of the mice in the model group gradually decreased. The body weight of the mice in the model group decreased significantly by 16.2% on day 12 (P < 0.01). The body weights of the mice in the prebiotic group, the fecal bacteria solution group, and the live bacteria enema solution group gradually decreased before day 5. The body weight of the prebiotic group remained stable from day 7, the body weight of the fecal bacteria solution group gradually began to increase from day 7, and the body weight of the live bacteria enema solution group gradually began to increase from day 5. Until day 12, the body weight of the mice in the live bacteria enema solution group had recovered to the initial normal weight, while the body weights of the mice in the prebiotic group and the fecal bacteria solution group had not yet recovered to the normal level.

[0074] (2) The Disease Activity Index (DAI)

[0075] During the experiment, the body weight attenuation rate, stool characteristics, and fecal blood of each group of mice were monitored daily, and the disease activity index DAI of the mice was evaluated according to the Murano standard. The scoring criteria are shown in Table 2.

[0076] Table 2 Disease Activity Index (DAI Scoring Table)

[0077]

[0078] The results were as Figure 1 shown in Fig. b. After DSS treatment, the DAI value of the mice was significantly increased (P < 0.01), while enema with fecal microbiota solution and viable bacteria enema solution could reduce the DAI score of colitis mice. However, compared with the prebiotic group and the fecal microbiota solution group, the recovery effect of the viable bacteria enema solution group was better.

[0079] (3) Detection of Related Pro-inflammatory Cytokines and MDA in Colonic Tissues

[0080] The mRNA expression of related pro-inflammatory cytokines in the colonic tissues of mice was detected by qRT-PCR. The contents of TNF-α and IL-1β in the colonic tissues of mice were measured according to the instructions of the ELISA kit (Shanghai Xinle Company, China). The measurement method of MDA content was measured according to the instructions of the kit (Nanjing Jiancheng Bioengineering Institute, China).

[0081] As Figure 2 shown, after DSS treatment, the protein expressions of TNF-α, IL-1β, and MDA in the colons of mice (p < 0.01) and the mRNA expressions of TNF-α, IL-1β, and IL-6 (P < 0.01) were significantly increased. However, enema with prebiotics, fecal microbiota solution, and viable bacteria enema solution could down-regulate the levels of the above-mentioned pro-inflammatory cytokines and the oxidative stress biomarker MDA in colitis mice to a certain extent. Compared with the prebiotic group and the fecal microbiota solution group, the levels of pro-inflammatory factors and MDA in the viable bacteria enema solution group were lower.

[0082] Example 3 Effect of Viable Bacteria Enema Solution on Constipation

[0083] 1. Experimental animals: 50 male healthy SD rats, 6-8 weeks old, weighing 200-250 g

[0084] Construction of animal model:

[0085] After adaptively raising healthy SD rats for 1 week, they were randomly divided into a blank group, a model group, a prebiotic group, a fecal microbiota solution group, and a live bacteria enema solution group, a total of 5 groups, with 10 rats in each group. The blank group was not treated. After 2 days of balanced feeding for the remaining 4 groups of SD rats, the experimental animals were gavaged with loperamide hydrochloride saline solution for 8 consecutive days, and each SD rat was gavaged with 1.0 mL according to a dose of 20 mg / kg body weight. Criteria for establishing a constipation model: After modeling, the time (min) of the first black feces and the water content (%) of the feces in SD rats were significantly different from those in normal group SD rats.

[0086] Drug administration plan: After the SD rats for modeling reached the criteria for establishing a constipation model, the successfully modeled SD rats were given enema treatment with phosphate buffer solution or live bacteria enema solution for 8 consecutive days. Blank group: Enema with an equal volume of phosphate buffer solution; Model group: Enema with an equal volume of phosphate buffer solution; Prebiotic group: Enema with a prebiotic solution with the same composition and dose as the prebiotic in the live bacteria enema solution; Fecal microbiota solution group: Directly use fecal microbiota solution for enema treatment; Live bacteria enema solution group: Use live bacteria enema solution for enema treatment. Before enema treatment, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL / 100 g body weight. The anesthetized rats were fixed supine on a rat board. After lubricating a rubber hose with a diameter of 2 mm with liquid paraffin, it was slowly and gently inserted into the rectum of the rat about 8 cm through the anus. A previously prepared solution of 1 mL / 200 g body weight was slowly injected at one time. After successful enema, the rats were kept supine with their buttocks up for 15 min, and after natural awakening, they were put back into the cage and reared conventionally. Each group was given enema administration once a day for 7 consecutive days.

[0087] 2. Detection indexes

[0088] (1) Time of the first black feces (min): The experimental animals were gavaged with activated carbon aqueous solution (1.0 ml / rat), and the time required from gavaging the activated carbon aqueous solution to the experimental animals excreting the first black feces was recorded;

[0089] Wet feces weight (g): The feces quality of rats was measured for 2 hours. Fresh feces were collected every 30 minutes and weighed, and the quality of the fresh feces collected within 2 hours was accumulated in this way;

[0090] Dry feces weight (g): After measuring the wet feces weight of rats for 2 hours, the feces were heated in a microwave oven at high power for 5 minutes and then weighed to obtain the dry feces weight;

[0091] Feces water content (%): Dry feces weight (g) / Wet feces weight (g) × 100% = Feces water content (%);

[0092] Table 4 Time of the first black feces and feces water content of SD rats before and after intervention after successful modeling

[0093]

[0094] As shown in Table 4, after 8 days of enema intervention with prebiotic solution, fecal microbiota solution, and live bacteria enema solution, the time to the first black stool in all three groups was significantly shorter than that in the model group, and the water content in feces after intervention was significantly higher than that in the model group (P < 0.01). However, the time to the first black stool and the water content in feces in the live bacteria enema solution group were closer to the normal levels of rats in the blank group, and the recovery effect was better than that in the prebiotic group and fecal microbiota solution group.

[0095] (2) Intestinal propulsion ratio

[0096] After 8 days of enema intervention with enema solution in SD rats after modeling, the SD rats were sacrificed and the intestinal propulsion ratio was measured. The rats were fasted but given water overnight before sacrifice. 45 minutes before sacrificing the rats, 1.0 ml of activated carbon arabic gum aqueous solution was intragastrically administered to the rats. After sacrificing the rats, the rectal segment was pulled out, and the segment from the pylorus to the cecum was laid flat, the whole intestinal segment was measured, and the distance (cm) from the fundus of the stomach to the pylorus to the black aqueous solution was measured. Intestinal propulsion ratio (%) = distance from the fundus of the stomach to the pylorus to the black aqueous solution (cm) / whole intestinal segment (cm).

[0097] The intestinal propulsion ratio was as Figure 3 shown. After intervention, the intestinal propulsion ratio in the prebiotic group was 45.2%, that in the fecal microbiota solution group was 48.8%, and that in the live bacteria enema solution group was 58.7%, which were all significantly higher than those in the model group (P < 0.01). However, there was no significant difference between the live bacteria enema solution group and the blank group after recovery, and the recovery effect was better than that in the prebiotic group and fecal microbiota solution group.

[0098] On the last day before sacrificing the SD rats, feces were collected, and 16S rDNA was used to analyze the microorganisms in the feces of the mice. In R language, the diversity() function in the vegan package can be used (set index = "shannon" to calculate the Shannon index and set index = "simpson" to calculate the Simpson index). According to the results of the Shannon index (the Shannon index is obtained by calculating the negative sum of the product of the relative abundance of each species in the community and the natural logarithm, reflecting the overall diversity of the community and paying attention to the evenness) and the Simpson index (obtained by subtracting the sum of the squares of the relative abundances of each species from 1 (or calculating the relationship between the probability of different species combinations and the total number of combinations), and paying more attention to the dominant species when measuring community diversity), it can be seen that the Shannon and Simpson indices of the rats in the prebiotic, fecal microbiota solution, and live bacteria enema solution enema treatment groups were all higher than those in the model group, indicating that the intervention with prebiotic, fecal microbiota solution, and live bacteria enema solution successfully reversed the decrease in α-diversity caused by loperamide hydrochloride ( Figure 4 a, b). As Figure 4As shown in Figure c, PCA showed that the microbial compositions among groups presented obvious aggregation. The overlap degree between the live bacteria enema solution group and the blank group was significantly higher than that between the prebiotic group / fecal bacteria solution group and the blank group, suggesting that compared with prebiotic / fecal bacteria solution enema, the enema treatment with live bacteria enema solution had a more obvious effect on the transformation of the intestinal flora of constipated rats into that of normal rats.

[0099] (3) Safety

[0100] In addition, we conducted a safety assessment after transplantation of the two FMT preparations. By analyzing the safety data of 100 constipated rats receiving enema, 50 of them were given fecal bacteria solution enema, and the remaining 50 were given live bacteria enema solution enema. Among them, the statistical results of the incidence of common adverse events (including fever, diarrhea with loose stools, nausea / vomiting, and metabolic abnormalities) are shown in Table 5.

[0101] Table 5 Incidence of common adverse reactions

[0102]

[0103]

[0104] The results showed that the total incidence of adverse events in the fecal bacteria solution group was 19.0%, and that in the live bacteria enema solution group was 8.1%. Compared with direct fecal microbiota transplantation, the incidence of adverse events of the standard enema solution preparation obtained by the present invention was lower.

[0105] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A preparation method of a live bacteria enema solution, characterized in that, It includes the following steps: S1. Buffer preparation: Prepare a phosphate buffer with a pH of 7.2 - 7.4, adjust the osmotic pressure to 280 - 320 mOsm / L using NaCl, bottle and sterilize it, and store it at 4°C; S2. Preparation of live bacteria freeze-dried powder: Collect donor feces. The feces weight should be no less than 200 g, and the feces should be qualified with a Bristol score of 3 - 5. Then enter the bacterial liquid production process. Filter it through a 50 - 800 mesh sieve step by step, centrifuge it at 2000 rpm / min for 3 min for fecal bacteria separation and washing. Add a freeze-drying protectant to the obtained bacterial liquid, mix well, and place it in a vacuum freeze-dryer for drying to obtain live bacteria freeze-dried powder; S3. Preparation of live bacteria - prebiotic freeze-dried powder: Crush the raw materials of the live bacteria freeze-dried powder and excipients in step S2 through a 60 - 100 mesh sieve, add them to a V-type mixer and mix evenly, then bottle them.

2. The method according to claim 1, characterized in that, In step S2, the freeze-drying protectant is one or more of sucrose, trehalose, glucose, lactose, glycerol, mannitol, sorbitol, glycine, Tween 80, sodium hyaluronate, and polyvinylpyrrolidone.

3. The method according to claim 1, characterized in that In step S3, the weight percentage of the live bacteria freeze-dried powder in the live bacteria - prebiotic freeze-dried powder is 1% - 40%; the weight percentage of the excipients in the live bacteria - prebiotic freeze-dried powder is 60% - 99%.

4. The method according to claim 1, characterized in that In step S3, the excipients are one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, microcrystalline cellulose, carboxymethyl cellulose, poloxamer, sodium dodecyl sulfate, sodium alginate, fructooligosaccharide, galactooligosaccharide, inulin, resistant dextrin, and stachyose; 5. The method according to claim 4, wherein The weight percentage of microcrystalline cellulose is 1% - 30%, the weight percentage of carboxymethyl cellulose is 1% - 30%, the weight percentage of poloxamer is 0.1% - 5%, and the weight percentages of fructooligosaccharide, galactooligosaccharide, inulin, and stachyose are 1% - 50% respectively.

6. The method according to claim 4 or 5, characterized in that In step S3, the weight percentage of the live bacteria freeze-dried powder is 40%, the weight percentage of MCC is 20%, the weight percentage of sodium alginate is 5%, the weight percentage of CMC is 10%, the weight percentage of sodium dodecyl sulfate is 0.15%, the weight percentage of poloxamer is 3%, the weight percentage of fructooligosaccharide is 5%, the weight percentage of galactooligosaccharide is 5%, the weight percentage of inulin is 5%, and the weight percentage of resistant dextrin is 5%.

7. A live bacteria enema solution prepared by the method according to any one of claims 1 to 6, characterized in that, It includes the following components: Component in bottle A: Phosphate buffer with a pH of 7.2 - 7.4; Component in bottle B: 1% - 40% live bacteria powder, 60% - 99% excipients.

8. Use of the enema solution according to claim 7 in the preparation of a medicament for improving intestinal microecology.

9. Use of the enema solution according to claim 7 in the preparation of a medicament for relieving and / or treating ulcerative colitis or constipation.

10. The application according to claim 9, characterized in that, The said use includes down-regulating the levels of pro-inflammatory cytokines and / or the oxidative stress biomarker MDA in individuals with colitis; Optionally, the pro-inflammatory cytokines include TNF-α, IL-1β, and IL-6.

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