CN114196599A

CN114196599A - Microbial composition for intestinal flora blending and preparation method thereof

Info

Publication number: CN114196599A
Application number: CN202210019012.3A
Authority: CN
Inventors: 张洵; 孙龙
Original assignee: Beijing Haodingrui Micro Ecological Technology Research Institute Co ltd
Current assignee: Beijing Haodingrui Micro Ecological Technology Research Institute Co ltd
Priority date: 2022-01-06
Filing date: 2022-01-06
Publication date: 2022-03-18
Also published as: CN115838649A

Abstract

The present invention relates to a microbial composition for gut flora tempering comprising strains of bifidobacterium, lactobacillus and/or other genera having essentially the same properties, wherein the strains of bifidobacterium are selected from the group comprising bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum. Fecal bacteria transplantation is a powerful means for achieving the purpose of disease treatment by regulating intestinal microecology. The components for the coprophilous fungi transplantation are too complex, and quality control and accurate intervention cannot be realized. Therefore, the invention selects a plurality of core strains playing an important role in the ecology through the data mining and analysis of the intestinal flora to realize the regulation and control of the whole intestinal microecology, thereby realizing the alleviation and treatment of the symptoms of the diseases.

Description

Microbial composition for intestinal flora blending and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a microbial composition for regulating intestinal flora.

Background

Irritable Bowel Syndrome (IBS) is a global functional disease in which abdominal pain, abdominal discomfort, a change in Bowel habits, or a change in stool characteristics are mainly manifested, and the global prevalence of IBS is 10% to 20%, which is considered to be the highest human incidence in recent years. However, irritable bowel syndrome is a multifactorial disease, the etiology and pathophysiology of the irritable bowel syndrome are complex, the pathogenesis factors comprise intestinal flora imbalance, small intestinal bacterial overgrowth, intestinal motility abnormality, intestinal hypersensitiveness, intestinal secretion and absorption function abnormality, intestinal barrier function damage, intestinal mucosa immunity abnormality, brain and intestinal axis abnormality and the like, and an effective treatment method is not available at present. Due to the multi-factor pathogenic mechanism of IBS, the drug selection and alleviation means in the treatment process can bring about differentiation effects due to individual differences. Therefore, it is of great interest to actively search for new treatments for IBS.

A large number of beneficial microorganisms which help the human body digest and absorb nutrition and resist harmful substances exist in the human intestinal tract, and the beneficial microorganisms are named as probiotics. Probiotics are colonised in the human body and mainly adhere to the mucous membranes and villi of the intestinal tract. In recent years, the incidence of IBS is found to be related to the imbalance of intestinal flora, and the clinical application of probiotics to treat IBS achieves a certain curative effect, wherein the bacillus subtilis dual live bacteria preparation and the enterococcus faecium R-026 are taken as common intestinal probiotics, so that the intestinal environment of a human body can be regulated, the growth and the reproduction of normal intestinal flora are promoted, and the growth of intestinal pathogenic bacteria is inhibited, thereby effectively protecting the intestinal tract. With the progress of medical treatment, Fecal Transplantation (FMT) is becoming an emerging means for the treatment of intestinal and parenteral diseases. The FMT method is mainly characterized in that functional flora in excrement of healthy people is transplanted into gastrointestinal tracts of patients, intestinal flora with normal functions is reconstructed, and treatment of intestinal tract and parenteral diseases is achieved. Currently, "fecal transplantation" has become a safe and effective method for treating recurrent Clostridium Difficile Infection (CDI) and has been written in the united states of america at the guideline for clinical use.

Specifically, chinese patent publication No. CN112089736A provides a whole fungus capsule, a preparation method and an application thereof, wherein the preparation method comprises the following steps: (1) screening the fecal strain donor by using high-throughput sequencing; (2) collecting excrement of the excrement donor screened in the step (1) to prepare an excrement bacterial liquid; (3) and (3) mixing the fecal bacteria liquid prepared in the step (2) with a freeze-drying protective agent, cooling, freezing and vacuum-drying to obtain fecal bacteria freeze-dried powder, and filling the obtained fecal bacteria freeze-dried powder into a capsule shell to obtain the whole-bacteria capsule. The whole bacterium capsule is also used for treating diseases caused by intestinal flora imbalance such as clostridium difficile infection, irritable bowel syndrome, inflammatory bowel disease and the like, and carries out the concept of fecal microbe transplantation. The donor screening of whole bacterial capsules involved in this patent is complex, relying on high throughput sequencing for screening and simple processing of feces. On one hand, donor screening only through high-throughput sequencing cannot completely eliminate unhealthy donors due to complex physiological reactions of organisms, and on the other hand, the whole bacteria capsules for simple excrement treatment have complex components, and individual differences among the capsules occur during use, so that differences of individual treatment reactions of patients occur during use.

At present, most of domestic fecal strain transplantation technologies for treating IBS compensate the total amount of microbial populations of patients through screened fecal strain liquid of healthy human bodies, but the fecal strain used for FMT treatment can hardly provide exact quantitative data or quantitative fixed planting indexes for the microbial populations according to current cognition, and samples provided by different donors have large difference, so that the fecal strain is difficult to meet the standard in the aspects of preparation, standard type and quality control.

In the prior art, Chinese patent with publication number CN109008958A uses filtration and other means to screen and transplant the fecal bacteria, but does not perform accurate quantification and screening of the fecal bacteria, so that the current fecal bacteria treatment can not achieve the purpose of accurate treatment.

Intestinal microorganisms are a complex ecosystem, and fecal microbe transplantation for the purpose of disease treatment by regulating intestinal microecology is a powerful means. In the prior art, intestinal micro-ecological regulation is more dependent on transplantation of the microbial environment of healthy individuals, but the screening of the healthy individuals is more complex, and the difference among different individuals can cause additional negative effects on the micro-ecological environment of the healthy individuals during intestinal micro-ecological modification of patients. Meanwhile, the components for the coprophilous fungi transplantation are too complex, and quality control and accurate intervention cannot be realized. Due to uncertainty of the variety and the number of flora, the fecal strain transplantation can not be accurately treated according to individuals during clinical treatment, and thus, the cure rate is reduced. Details can be found in the clinical trial protocol previously provided. Therefore, by analyzing the intestinal flora data, a plurality of strains which play an important role in the ecology are selected to realize the regulation and control of the whole intestinal microecology, thereby realizing the alleviation and treatment of the symptoms of the diseases.

The invention is based on a selective coprophilous bacterium transplanting method, and the microbial composition with good curing or relieving effect on IBS patients is obtained by screening.

Furthermore, on the one hand, due to the differences in understanding to the person skilled in the art; on the other hand, since the applicant has studied a great deal of literature and patents when making the present invention, but the disclosure is not limited thereto and the details and contents thereof are not listed in detail, it is by no means the present invention has these prior art features, but the present invention has all the features of the prior art, and the applicant reserves the right to increase the related prior art in the background.

Disclosure of Invention

In view of the deficiencies of the prior art, the present invention provides a microbial composition for gut flora tempering comprising strains of bifidobacterium, lactobacillus and/or other genera having substantially the same properties.

According to a preferred embodiment, the bifidobacterium strain is selected from the group comprising bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum.

According to a preferred embodiment, the lactobacillus strain is selected from the group comprising lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus helveticus.

According to a preferred embodiment, the microbial composition is capable of alleviating or treating a non-healthy state caused by abnormal intestinal motility, high visceral sensitivity, bacterial intestinal infection and/or food allergy by interacting with the intestinal micro-ecology.

According to a preferred embodiment, the microbial composition is in the form of a lyophilized powder.

According to a preferred embodiment, the composition can also be used for the treatment of irritable bowel syndrome.

Further, the microbial composition proposed by the present invention can have the use for the manufacture of a medicament for the prevention or treatment of clostridium difficile infection (pseudomembranous enteritis), inflammatory bowel disease, irritable bowel syndrome, multiple organ dysfunction syndrome, drug hypersensitivity syndrome, combined intestinal flora dysregulation.

A P-fhw capsule comprising the microbial composition of the present invention. On one hand, the microbial composition provided by the invention has qualitative and quantitative microbial species, can be obtained from excrement of screened healthy people and can also be obtained by in vitro culture and screening, so that the quantitative and qualitative flora component in the capsule is accurate; on the other hand, in order to ensure the activity of the flora entering the intestinal tract, the optimal treatment method of the flora when not being taken is a treatment method in a low-activity freeze-dried state, and the flora is packed in a capsule. The capsule can ensure the stability of the in vitro environment of the freeze-dried flora and increase the comfort level of taking.

A method of administering a microbial composition, comprising the steps of:

providing a microbial composition as set forth herein; is implanted into the human body by oral administration or intestinal tract transplantation.

Preferably, the composition comprising the bifidobacterium strain, the lactobacillus strain and/or other genera or strains having substantially the same properties is encapsulated in the form of a lyophilized powder for administration orally.

Preferably, the composition comprising bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus helveticus is encapsulated in the form of a lyophilized powder for oral administration.

A method of preparing a microbial composition for gut flora tempering comprising the steps of:

providing a fecal inoculum comprising the proposed microbial composition; the protective agent is added into the fecal strain liquid, and can be used for directionally protecting microorganisms in the microorganism composition provided by the invention in a fecal strain liquid treatment environment; and (3) treating the liquid dung with the protective agent so that the treated liquid dung can be applied to a human body.

According to a preferred embodiment, a method for preparing a microbial composition for gut flora tempering comprises the steps of:

detecting the intestinal flora of the patient to obtain whether the intestinal flora of the patient is unbalanced and the unbalance degree of the intestinal flora of the patient;

according to the analysis of the detection data of the intestinal flora of the patient, the targeted deletion species of the intestinal flora is selected;

providing a fecal inoculum comprising microorganisms deficient in the patient's intestinal tract;

the method is characterized in that a protective agent is added into the fecal bacteria liquid, and the protective agent can be used for directionally protecting microorganisms missing in intestinal tracts of patients in the fecal bacteria liquid in a fecal bacteria liquid treatment environment;

treating the liquid dung with the protective agent so that the treated liquid dung can be applied to a human body;

the microbial composition capable of being used for intestinal flora harmonizing is obtained through the steps.

culturing in vitro and obtaining the microorganism lacking in intestinal tract of the patient;

mixing the cultured microorganisms to obtain the microorganism composition for regulating intestinal flora.

According to a preferred embodiment, the detection of the intestinal flora of the patient can be performed in a qualitative or quantitative manner.

According to a preferred embodiment, the resulting fecal inoculum solution can be lyophilized or otherwise processed so that the fecal inoculum solution can be administered to the patient's intestine orally or by intestinal transplantation.

According to a preferred embodiment, the resulting liquid manure can be eliminated or protected from the bacterial species it contains during the treatment, based on specific treatment means or specific protective agents.

Drawings

FIG. 1 is the IBS-SSS rating Scale of example 3 provided herein;

FIG. 2 is a line graph of the IBS-SSS score results of example 7 provided by the present invention.

Detailed Description

The following detailed description is made with reference to the accompanying drawings.

The invention provides a microbial composition for intestinal flora blending, a screening method and a preparation method of the composition.

In the context of the present invention, FMT capsules refer to capsules comprising standard fecal bacteria of a healthy population after treatment, comprising whole strains of the intestinal tract of a healthy population. P-fhw refers to a probiotic composition.

At present, the main treatment medicines for irritable bowel syndrome comprise antidiarrheal medicines, spasmolytic medicines and antidepressant medicines, and the treatment means relates to intestinal micro-ecological treatment and the like.

For example, patients with chronic severe constipation received a colonoscopic fecal transplantation plus a second day fecal enema treatment, of which 40 patients (69%) had relieved symptoms soon after the fecal transplantation, including improved defecation, no bloating and abdominal pain, and 18 patients had returned to normal defecation in the subsequent 9-19 months of follow-up. In 2013, 13 patients who had received the "fecal transplantation" treatment in a study of IBS refractory to the "fecal transplantation" treatment, 50% of the patients had relieved symptoms.

However, in the actual "fecal transplantation" treatment process, the number and type of microorganisms are difficult to control quantitatively and qualitatively. Due to individual differentiation and different reconstruction requirements of flora in intestinal tracts, the technology of 'fecal strain transplantation' has certain inefficiency or even ineffectiveness.

The prior art is concerned with a number of probiotics for gut protection, such as bifidobacteria and lactobacilli. The lactobacillus and the bifidobacterium can regulate the intestinal ecological environment in four action directions. Firstly, secretion products of lactobacillus and bifidobacterium, such as lactic acid, hydrogen peroxide and bacteriocin, have certain effects on intestinal inflammation immune response, external coating ablation of harmful bacteria and the like; secondly, the lactobacillus and the bifidobacterium can compete with harmful bacteria in the aspects of intestinal nutrition and intestinal adhesion sites, so that the living space of the harmful bacteria is reduced; thirdly, the lactobacillus and the bifidobacterium can induce the positive regulation of the effector factors of the intestinal epithelial tissue, thereby mobilizing the barrier function of the intestinal epithelial tissue; fourth, lactobacilli and bifidobacteria are also able to direct nonspecific immunomodulation of the gut by surface ligands or other secreted factors. Based on the action mechanism, the lactobacillus and the bifidobacterium can participate in various physiological regulation pathways of the intestinal tract to improve the antagonism of the intestinal tract to harmful bacteria. However, since there are many varieties of lactobacilli and bifidobacteria, selectivity and ratio values become the key to optimal results when feeding probiotics to the intestinal tract. The lactobacillus is a probiotic which actively promotes intestinal peristalsis and kills harmful bacteria, and the bifidobacterium is more inclined to improve human immune response and promote human tissue development to antagonize the harmful bacteria, so the reasonable collocation between the lactobacillus and the bifidobacterium can better protect the intestinal tract of a human body.

For example, the use of bifidobacterium breve for gut protection is known. The Bifidobacterium breve can maintain intestinal tract immunity stability, maintain stable state of mucosal immune response, and reduce systemic immune response reaction by inhibiting excessive production of inflammatory factors by intestinal mucosa. Bifidobacterium breve is also able to enhance non-specific and specific immune responses by activating macrophages, enhancing killer cell activity and increasing levels of immunoglobulin a, among others. Further, Bifidobacterium breve in intestinal tract is effective in inhibiting growth and reproduction of helicobacter pylori, thereby promoting the treatment of gastritis and gastric ulcer.

Lactobacillus acidophilus exists mainly in small intestine, and can secrete antibiotic substances (acidophilic lactobacillus, acidophilic bacilus, and lactein) to exert antagonistic effect on intestinal pathogenic bacteria, thereby preventing intestinal mucosa from being damaged.

The lactobacillus plantarum can degrade harmful substances such as ammonia nitrogen and nitrite, and the damage of the intestinal canal by the spoiled substances in the intestinal canal is reduced. Lactobacillus plantarum is also capable of reducing intestinal discomfort or stress caused by pathogenic bacteria by survivability competition with the pathogenic bacteria in the mucosal tissues of the large or small intestine.

The invention provides a probiotic composition based on a plurality of probiotics possibly existing in excrement, and the probiotic composition can relieve the stress of the intestinal tract of a human body and/or repair the ecological microenvironment of the intestinal tract of the human body through self-selection, particularly the composition of probiotic species existing in the excrement. Clinical tests show that a plurality of probiotics and compositions thereof have relieving and even treating effects on patients with irritable bowel syndrome, and the compositions can positively promote the repair and construction of intestinal ecological barriers causing the irritable bowel syndrome. The contents of the clinical test comprise: screening patients with irritable bowel syndrome, providing FMT capsules and P-fhw capsules (the capsules comprise a microbial composition consisting of a plurality of probiotics), and obtaining the effect of the FMT capsules and the P-fhw capsules on the patients with irritable bowel syndrome through the IBS-SSS scoring scale of the patients.

Example 1

The microbial composition involved in the P-fhw capsule can comprise: one or more of Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus. Preferably, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus helveticus.

According to a preferred embodiment, the composition ratio selected from the group consisting of bifidobacterium strains, lactobacillus strains and/or other strains of the genera having substantially the same properties can be: bifidobacteria strain: lactobacillus strain ═ 1: 1. Preferably, the bifidobacterium breve, the bifidobacterium infantis, the bifidobacterium longum, the lactobacillus acidophilus, the lactobacillus casei, the lactobacillus plantarum, the rhamnosus, the lactobacillus reuteri and the lactobacillus helveticus are proportioned in equal proportion.

According to a preferred embodiment, the ratio of bifidobacterium strains to lactobacillus strains can be 5:1, 4:1, 3:1, 2:1, 1:1 and any range ratio between 5:1 and 1: 1.

According to a preferred embodiment, the ratio of lactobacillus strains and bifidobacterium strains can be in any range of ratios between 5:1, 4:1, 3:1, 2:1 and 1:1, and between 5:1 and 1: 1.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve and lactobacillus acidophilus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium infantis, lactobacillus casei, lactobacillus plantarum and lactobacillus rhamnosus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, bifidobacterium longum.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus reuteri.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus helveticus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus helveticus, lactobacillus plantarum.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus reuteri, lactobacillus plantarum.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus, lactobacillus plantarum.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus helveticus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus helveticus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium longum and lactobacillus plantarum.

According to a preferred embodiment, the microbial composition comprises bifidobacterium longum and lactobacillus rhamnosus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium longum and lactobacillus casei.

According to a preferred embodiment, the microbial composition comprises bifidobacterium longum and lactobacillus helveticus.

According to a preferred embodiment, the microbial composition comprises bifidobacterium longum and lactobacillus acidophilus.

Example 2

Although the above studies preliminarily suggested the possible therapeutic effect of "fecal transplantation" on IBS, the current knowledge of fecal bacteria used for FMT treatment hardly provides exact quantitative data or quantitative indicators of colonization for this population of microorganisms, and the samples provided by different donors vary greatly, and therefore, fecal bacteria hardly meet the standards in terms of preparation, standard type and quality control. Therefore, there is a need for accurate supplementation of intestinal flora and Selective fecal Transplantation (SMT) for stool alteration of IBS patients, i.e., accurate treatment of IBS patient population by culturing quantitative and qualitative beneficial bacteria and providing the beneficial bacteria to the patients.

The treatment process of fecal bacteria transplantation, i.e. standard fecal bacteria transplantation, cannot control the type and amount of microorganism applied to different patients, and therefore, when aiming at IBS patients induced by different factors, standard fecal bacteria transplantation can only perform fecal bacteria transplantation with unknown species content, thereby leaving the diagnosis and treatment method uncontrolled. The invention provides a selective fecal strain transplantation concept aiming at a patient, so that personalized and precise treatment of the patient is achieved, and the benefits of disease diagnosis and treatment and body health care are improved.

The microbial composition involved in selective fecal transplantation can be present in the form of a lyophilized powder. The freeze-dried powder can be treated in a mode of making the microorganisms in a low-activity state without being influenced by the environment as far as possible.

According to a preferred embodiment, the standard fecal bacteria, or fecal bacteria used for standard fecal bacteria transplantation, can be preserved by a protective agent in the presence of the proposed microbial composition of the invention in the case of artificial treatment.

For example, by adding glutamic acid, skim milk, mannitol, fructose and vitamin C into the protective agent, the protective agent can directionally protect the flora of bifidobacteria, lactic acid bacteria and bacteroides in the coprophila liquid in the freeze-drying process of copra bacteria, so that the survival rate of the bifidobacteria, the lactic acid bacteria and the bacteroides is over 90 percent.

The protective agents with different proportions can directionally eliminate or protect some special strains, so that the purpose of screening the strains in the fecal strain liquid is achieved, and the purpose of selective fecal strain transplantation is achieved.

The preparation method of the selective coprophilous fungi transplantation comprises the following steps: determining a physiological state and an intestinal environment of the patient; screening a patient for a desired microbial composition; the standard fecal bacteria solution is directionally treated and provided to the patient in an oral or intestinal transplantation mode.

The species of the supplemented flora is judged by diagnosing patients with different irritable bowel syndromes or other intestinal tract related diseases, so that accurate beneficial flora supplementation is achieved. As the flora of a plurality of intestinal tracts is neutral flora, the method is characterized in that: when the flora in the human body is unbalanced or other problems occur, the neutral flora possibly has negative influence on the human body, so that the intestinal ecological environment of the human body is influenced by excessive supply of other flora when the standard fecal flora is transplanted to maintain the health of the intestinal flora by supplying the flora.

According to the invention, microbial flora species beneficial to individual intestinal tracts are treated or synthesized/extracted in vitro by the fecal flora, so that the problem of intestinal flora damage caused by additional supplement of redundant neutral flora is avoided, and the intestinal tracts are supplemented to lack flora, so that the real stability of intestinal micro-ecology is achieved.

Meanwhile, the invention is different from the intestinal transplantation method in the prior art, and the precise components and dosage are supplied. Based on individual differences, such as sex, height, weight, age and the like, the composition can realize an administration mode with accurate components and dosage, and achieves a better intestinal healing effect through accurate treatment. Meanwhile, the oral administration method is used, so that the psychological and physiological comfort of the patient is improved, the patient can reach a more positive application state, and the comfort and the efficiency of treatment are improved.

Example 3

FMT capsules and P-fhw capsules formed based on the microbial composition are supplied to screened patients, and clinical observation is performed after taking the capsules to judge the curative effect of the accurate P-fhw capsules on IBS patients and patients with related diseases caused by intestinal micro-ecological disorder.

The test design comprises a process design and a sample size design.

The test process comprises the following steps:

screening the subject; the IBS-D patients meeting the requirements enter into groups, and the patients meeting the requirements enter into the groups randomly; the patients after grouping take FMT capsules and P-fhw capsules according to the grouping requirements, and observe the effective rate of clinical relief of IBS-D patients at baseline and after intervention, and observe the clinical characteristics of the patients, such as change of stool flora, change of anxiety and depression condition, and the like.

This test is a randomized, controlled test with an observation endpoint of IBS remission rate. The remission rates between groups were compared, and the sample size N (per group) was calculated to be 24 using PASS 11.0, with α being 0.05 (bilateral), β being 0.2, and the degree of freedom v being k-1, taking into account factors such as 10% detachment, and the like, to meet statistical requirements.

The biologicals used in this trial included FMT capsules, P-fhw capsules and the corresponding placebo.

According to a preferred embodiment, the P-fhw capsule involved in this test is a microbial composition for gut flora harmonization provided by the present invention. The components of the microbial composition are selected from strains of bifidobacterium, lactobacillus and/or other genera or strains having substantially the same properties.

According to a preferred embodiment, the lactobacillus strain is selected from the group comprising lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus helveticus. According to a preferred embodiment, the bifidobacterium strain: lactobacillus strain ═ 1: 1. specifically, bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri and lactobacillus helveticus are mixed in the microbial composition in a medium proportion, namely, the mixing ratio is 1: 1. in the test, the P-fhw capsule contains bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri and lactobacillus helveticus which are mixed in equal proportion.

Subjects must complete each clinical examination after enrollment to assess tolerance for continued drug use. After completion of each clinical examination, the initial condition of the subject was recorded. After the self-screening is qualified, the medicine is taken.

After administration for 4 weeks as prescribed, subjects can be tracked continuously for a long period of time.

Further, the test process is briefly described. 72 IBS patients were selected and randomized into 2 groups, one group was designated as "FMT Capsule", and the other group was designated as "P-fhw Capsule", and the effective rate of clinical relief was observed to evaluate the gastrointestinal symptoms before and after intervention and the improvement of behavioral symptoms. Based on the progress of the test, the test can be divided into a plurality of progress periods.

The first period was a baseline check, the first week to start the trial. And screening qualified subjects to carry out stool specimen collection, intestinal mucosa observation and related detection filled in a related scoring table.

The second period is the treatment period, which is one year from the start of the trial. Patients at the end of the baseline examination period were randomly assigned to either the "selective fecal bacteria transplant" group or the standard "fecal bacteria transplant" group and received the relevant treatment. During this period, patients will be followed at regular intervals after the first transplant. The follow-up examination method comprises the examination method.

The third period was the initial period when treatment was over and no further medications were provided at the end of the first year since the start of the trial. The subjects received a thorough examination to assess the efficacy and safety of the "selective fecal transplantation".

Data is collected. And selecting a proper statistical analysis method according to the data type of each measurement index. The test is a clinical intervention research of random parallel control, and the description of statistical data quantitative indexes calculates the average number and standard deviation; the classification index will describe the number of instances and percentage of each class, using the SPSS statistical software.

The main efficacy index of the two groups is the comparison of the IBS remission rate, and the hypothesis test adopts a two-side test (two-side test), and alpha is 0.05. The difference is considered to be statistically significant when P on one side is less than or equal to 0.025 and P on the other side is less than or equal to 0.05. The credibility of all credibility intervals is 95%.

The main efficacy index of the test is the IBS remission rate, and the Log-Rank test is adopted for the comparison between the accurate bacterium group and the enterobacter capsule group. Suppose that: HO: the group given FMT capsules had the same IBS remission rate as the group given P-fhw capsules; h1: the group given FMT capsules had a different IBS remission rate than the group given P-fhw capsules. Alpha level: 0.05 double sided.

The results are shown in table 1 below, and the results of the tests demonstrated that the effective rate of the group administered with FMT capsules was 66.7% and the effective rate of the group administered with P-fhw capsules was 68% by performing clinical tests on humans, compared with fecal transplantation.

The IBS-SSS score for the group given FMT capsules dropped from 206 to 114.5. The IBS-SSS score for the group given P-fhw capsules decreased from 197.6 to 93.5.

TABLE 1

Test drug information	High efficiency	Pre-dose IBS-SSS score	Post-dose IBS-SSS score
				FMT capsule	66.7％	206	114.5
P-fhw capsule	68％	197.6	93.5

The IBS-SSS score scale is shown in fig. 1 and encompasses a variety of physiological states in which patients autonomously judge changes in themselves. Through the change trend and the cure/relief effective rate, the superiority of selective fecal bacteria transplantation in curing/relief and comfort for irritable bowel syndrome patients can be shown.

The experiments prove that the curative effective rate of the selective fecal strain transplantation has the same or even better effect than that of the standard fecal strain transplantation when the selective fecal strain transplantation is used for treating irritable bowel syndrome. Meanwhile, for patients with irritable bowel syndrome, the effect and comfort level of selective fecal bacteria transplantation are not weaker or even higher than those of standard fecal bacteria transplantation.

In practical use, the coprophilus used in standard coprophilus transplantation is a whole strain, so the source of the coprophilus can only be a healthy human body, scientific replication cannot be realized, and the application form and the application method are extremely limited. Selective fecal transplantation collects or replicates only a portion of the flora of a healthy human to achieve targeted strain administration. Meanwhile, selective fecal strain transplantation uses partial strains, so the components and the dosage of the selective fecal strain are controllable, and in-vitro replication and culture can be achieved through scientific means. The selective fecal bacteria transplantation also has various administration forms and administration methods, such as oral administration in the form of pills, suspensions, direct application to the vagina, intestinal tract or skin of a human body in the form of spray, powder, suspension or pill.

Standard fecal bacteria transplantation presents a certain risk of complications in use, e.g. pneumonia and death after standard fecal bacteria transplantation. And the selective coprophilous fungi transplantation has extremely low probability of side effects or complications because the components and the dosage are in the control range and no redundant components or strains exist.

The composition designed by selective coprophilous fungi transplantation can realize the in-vitro culture and the accurate treatment of coprophilous fungi transplantation with the effect and the comfort degree which are not weaker than or even stronger than those of standard coprophilous fungi transplantation, thereby reducing the medical cost of medicines and the application process on one hand and reducing the additional complication risk brought by the whole-fungi application on the other hand; on the other hand, the probiotics with accurate dosage and components can be used for oral administration or other multi-selective administration methods, and particularly for children, the accurate ratio of the medicines in the treatment process can greatly improve the possibility of curing intestinal tract problems.

Example 4

The experiment also sets P-fhw capsules mixed by bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum and the like in proportion, and shows that compared with an FMT capsule control group, the P-fhw capsules with the components have better effect of relieving intestinal stress signs of patients.

Example 5

The experiment also provided a P-fhw capsule comprising a mixture of two bifidobacterium breve, one bifidobacterium infantis, two lactobacillus acidophilus, one lactobacillus casei and one lactobacillus plantarum, which showed that the P-fhw capsule with the above ingredients had a better effect of relieving the intestinal stress symptoms of the patients compared to the FMT capsule control group.

Example 6

The traditional fecal bacteria transplantation technology is mainly used for treating irritable bowel syndrome by means of fecal bacteria transplantation of intestinal tracts. However, in practice, the intestinal transplantation method not only increases the time and cost of transplantation, but also brings surgical risks to the treatment.

Therefore, after the microbial composition provided by the invention is subjected to a special freeze-drying treatment, the microbial composition provided by the invention can be stored in a capsule to form a P-fhw capsule containing the microbial composition provided by the invention.

The P-fhw capsule can be used for qualitative and quantitative administration of the microbial composition to patients. When oral administration is selected, the comfort of taking the medicine by a patient can be increased.

Example 7

Patients 60, 30 were co-enrolled in this trial with P-fhw capsule (containing complex probiotics) therapy and 30 with FMT capsule therapy according to IBS clinical diagnostic criteria. The intervention mode of the P-fhw capsule is 1 time per day and the continuous intervention lasts for 4 weeks; FMT capsule intervention 1 time. Follow-up visits were made at weeks 0, 2, 4, 8, and 12, respectively, to understand the effect of treatment.

The IBS-SSS scale shown in figure 2 was obtained by the method described above. Specifically, the IBS-SSS scale shown in fig. 2 includes 5 items, such as abdominal pain level, abdominal pain days, abdominal distension, defecation satisfaction, and life disturbance, each of which is integrated into 100 points and the total points are 500 points. According to the integral level, the IBS is divided into light, medium and heavy, the light degree is 76-175 points, the medium degree is 176-300 points, and the heavy degree is over 300 points.

The results show that the patients' IBS symptoms can be improved from moderate to mild by taking the P-fhw capsule for intestinal micro-ecology adjustment involved in the present invention for two weeks without the aid of chemotherapy. The symptoms are continuously relieved after the medicine is continuously taken for 1 month, and the overall effective rate reaches 64.3 percent. The effect of the P-fhw capsule for intestinal micro-ecological regulation involved in the present invention can continue to maintain the healthy state of the intestine after the 8 th and 12 th weeks after the cessation of the drying, and the effective rates are 69.2% (18/26) and 68% (17/25), respectively.

Compared with FMT, the difference analysis shows that the P value at the end of intervention (4W) is 0.994, the P value at 2 months (12W) after intervention is 0.999, the overall effect of intervention is slightly better than that of FMT in terms of efficiency statistics, and the long-term healing effect can be maintained. Therefore, the microbial composition can be used for equivalently replacing a microbial composition for treating intestinal diseases by coprophilous fungus transplantation. The detailed data are shown in Table 2 below.

TABLE 2

The method for protecting and regulating intestinal flora microecology provided by the invention can be used for regulating the intestinal flora of patients with irritable bowel syndrome in a mode of determining the species and the number of strains by taking the probiotic composition provided by the invention. The intestinal flora imbalance caused irritable bowel syndrome patient needs to supplement additional flora to adjust the flora state of the patient, and is different from indiscriminate supplement of unknown flora in the prior art. The aim of accurate medical treatment is achieved through accurate analysis of the intestinal tract of an individual. Specifically, after the distribution of the intestinal flora of the patient is known in a metagenomic sequencing and sequencing data analysis mode, the species abundance of the probiotic strains in the intestinal tract of the patient is determined, so that the species and the quantity of the probiotic strains to be supplemented are obtained through analysis, and the precise flora is supplemented. For example, after the detection of intestinal flora and the data analysis, the patient determines that his own intestinal tract is deficient in bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri and lactobacillus helveticus, and after the abundance of the deficient species in the intestinal tract is basically the same, the patient performs the equal proportion supply of bifidobacterium breve, bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri and lactobacillus helveticus, thereby maintaining the microecological group of healthy species in the intestinal tract.

It should be noted that the above-mentioned embodiments are exemplary, and that those skilled in the art, having benefit of the present disclosure, may devise various arrangements that are within the scope of the present disclosure and that fall within the scope of the invention. It should be understood by those skilled in the art that the present specification and figures are illustrative only and are not limiting upon the claims. The scope of the invention is defined by the claims and their equivalents. The present description contains several inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", each indicating that the respective paragraph discloses a separate concept, the applicant reserves the right to submit divisional applications according to each inventive concept. Throughout this document, the features referred to as "preferably" are only an optional feature and should not be understood as necessarily requiring that such applicant reserves the right to disclaim or delete the associated preferred feature at any time.

Claims

1. A microbial composition for gut flora tempering, characterized in that the microbial composition comprises a strain of bifidobacterium and/or a strain of lactobacillus.

2. Microbial composition according to claim 1, wherein the strain of Bifidobacterium is selected from the group comprising Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum.

3. The microbial composition according to claim 1 or 2, wherein the lactobacillus strain is selected from the group comprising lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus helveticus.

4. The microbial composition of any one of claims 1 to 3, wherein the microbial composition is capable of alleviating or treating a non-healthy state caused by abnormal intestinal motility, high visceral sensitivity, bacterial intestinal infection and/or food allergy by interacting with the intestinal micro-ecological environment.

5. The microbial composition of any one of claims 1 to 4, wherein said microbial composition is a lyophilized powder.

6. The microbial composition of any one of claims 1 to 5, wherein said composition is further capable of being used for the treatment of irritable bowel syndrome.

7. Use of a microbial composition according to any one of claims 1 to 6 for the prevention or treatment of clostridium difficile infection, inflammatory bowel disease, irritable bowel syndrome, multiple organ dysfunction syndrome, drug hypersensitivity syndrome or combined gut flora dysregulation.

8. A P-fhw capsule, comprising the microbial composition of any one of claims 1-6.

9. A method of administering a microbial composition, comprising the steps of:

providing a microbial composition according to any one of claims 1 to 6;

is implanted into the human body by oral administration or intestinal tract transplantation.

10. A method for preparing a microbial composition for gut flora tempering, comprising the steps of:

providing a liquid manure sample comprising the microbial composition of any one of claims 1-6;

adding a protective agent into the fecal strain liquid, wherein the protective agent can be used for directionally protecting the microorganism related to the claim 1 in a fecal strain liquid processing environment;

and treating the fecal strain liquid added with the protective agent so that the treated fecal strain liquid can be applied to a human body.