Akumen's bacteria YG2604 and its application in treating constipation

Abstract

The present application relates to the field of microorganisms, in particular to a strain of Akkermansia muciniphila YG2604, a microbial agent comprising the same, a composition, and application thereof in the preparation of products for preventing, improving and / or treating constipation. The Akkermansia muciniphila YG2604 of the present application can maintain the intestinal microecological balance and effectively improve or treat constipation; and the inactivated strain thereof can achieve the above-mentioned effects, and the inactivated strain product has better stability, a longer shelf life, and does not need cold chain transportation and storage, and is easier to commercialize and widely used. Therefore, the Akkermansia muciniphila YG2604 of the present application has a good clinical application prospect and has obvious market potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a strain of Akkermansia muciniphila YG2604, a microbial agent and a composition comprising the same, and their use in the preparation of products for preventing, improving and / or treating constipation. BACKGROUND

[0002] Constipation is a common clinical digestive system disease, mainly manifested as difficulty in defecation, hard stool and other symptoms. According to statistics, the incidence of constipation in adults in China is about 10%-15%, and increases with age. Long-term constipation not only affects the quality of life of patients, but also may cause complications such as anal fissure and hemorrhoids. At present, the methods for treating constipation in clinic mainly include drug treatment, diet conditioning, biofeedback therapy, etc., but these methods have problems such as unstable curative effect, large side effects, easy drug dependence, etc.

[0003] In recent years, with the in-depth study of microecology, the role of probiotics in regulating intestinal flora balance and improving intestinal function has been increasingly valued. Probiotics refer to active microorganisms that can produce beneficial effects on human health after being given in a certain amount. At present, a number of studies have confirmed that probiotics have significant efficacy in treating constipation, and are high in safety and low in side effects. However, the existing probiotic products have certain limitations in strain types, number of live bacteria, stability, etc., resulting in poor effect in clinical application.

[0004] Akkermansia muciniphila is a new type of second-generation probiotic that has been widely studied in recent years, belonging to the Verrucomicrobia family. This bacterium was first isolated from the human intestine and named for its unique physiological characteristic of being able to decompose intestinal mucin. The presence of Akkermansia muciniphila in the intestine is associated with a variety of health benefits, including regulating immune response, improving metabolic syndrome, reducing obesity, and improving symptoms of inflammatory bowel disease.

[0005] Previous studies have mainly focused on the application of Akkermansia muciniphila in immune regulation, metabolic syndrome improvement, and treatment of inflammatory bowel disease (IBD). Studies have found that this strain can increase the thickness of the intestinal mucus layer to enhance the intestinal barrier function, thereby reducing the invasion of harmful substances and pathogens and reducing intestinal inflammation. In addition, Akkermansia muciniphila can also promote the differentiation of T regulatory cells (Tregs) in the intestine, which play a key role in maintaining immune tolerance and suppressing inflammatory response. SUMMARY

[0006] Invention objectives

[0007] The present application aims to provide a strain of Akkermansia muciniphila YG2604 having the effect of preventing, improving and / or treating constipation, a bacterial agent and a composition comprising the same, and use thereof in the preparation of a product for preventing, improving and / or treating constipation.

[0008] Solution

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] In a first aspect, the present application provides a strain of Akkermansia muciniphila YG2604, which is classified as Akkermansia muciniphila and is deposited with the China General Microbiological Culture Collection Center, located at No. 1, Yihuan Road, Beijing, China, on October 16, 2024, under the accession number CGMCC No. 46149.

[0011] The Akkermansia muciniphila YG2604 of the present application is isolated from a healthy adult fecal sample, and is identified as Akkermansia muciniphila by 16S rRNA sequencing.

[0012] In some embodiments, the 16S rRNA gene sequence of the Akkermansia muciniphila YG2604 is shown in SEQ ID NO: 1.

[0013] The colony characteristics of the strain include a diameter of 0.1-0.3 mm, a smooth colony edge, and a light yellow color after 48 h of culture in mBHI agar medium.

[0014] The present inventors have found and experimentally confirmed that the inactivated strain of Akkermansia muciniphila YG2604 (also referred to as YG2604) of the present application can be used to improve constipation, and the effect of improving constipation may be achieved through mechanisms such as regulating intestinal flora, regulating bile acid and prostaglandin metabolism, promoting intestinal peristalsis, reducing water reabsorption in the colon, and increasing fecal water content.

[0015] In a second aspect, the present application provides a bacterial agent, wherein the active ingredient of the bacterial agent comprises a live strain or an inactivated strain of Akkermansia muciniphila YG2604 as described in the first aspect above.

[0016] In an implementable embodiment, the active ingredient of the bacterial agent comprises an inactivated strain of Akkermansia muciniphila YG2604.

[0017] Compared with the live strain, the inactivated Akkermansia muciniphila YG2604 has better stability, longer shelf life, and does not need cold chain transportation and storage, and is easier to commercialize and widely used, thus, it is a preferred embodiment to use the inactivated strain of YG2604 as the active ingredient of the bacterial agent.

[0018] In a third aspect, the present application provides a composition comprising an effective amount of a live strain or an inactivated strain of Akkermansia muciniphila YG2604 as described in the first aspect above or a bacterial agent as described in the second aspect above as a first active component.

[0019] In an implementable embodiment, the composition is a pharmaceutical composition.

[0020] In an implementable embodiment, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier and / or excipient. Further implementably, the pharmaceutical composition is formulated into a dosage form for nasal, oral or digestive administration, for example, in the form of a pill, a tablet, a lozenge, a lyophilized powder, a granule, a capsule, an aqueous solution, an alcoholic solution, an oily solution, a syrup, an emulsion, a suspension, a suppository, a solution for injection or infusion, an ointment, a gel, a tincture, a cream, a patch, a lotion, a spray, an aerosol, a powder spray, an effervescent tablet, a transdermal therapeutic system, a microcapsule, an implant or a stick.

[0021] In an implementable embodiment, the composition further comprises a second active component.

[0022] Optionally, the second active component comprises probiotics, postbiotics, prebiotics, antibacterial agents, immunomodulators, anticancer agents, inflammation treatment agents or a combination thereof.

[0023] In a fourth aspect, the present application provides use of Akkermansia muciniphila YG2604 as described in the first aspect above, a bacterial agent as described in the second aspect above or a composition as described in the third aspect above in the manufacture of a product, preferably a pharmaceutical product, for:

[0024] (1) modulating the gut microbiota;

[0025] (2) modulating bile acid and / or prostaglandin metabolism;

[0026] (3) preventing, ameliorating and / or treating constipation.

[0027] Possibly, the modulating the gut microbiota comprises: increasing the population of bacteria that are beneficial for alleviating constipation, and / or inhibiting the population of bacteria that are harmful;

[0028] Preferably, the population of bacteria that are beneficial for alleviating constipation comprises: Lactobacills, Streptococcus and Anaerofustis;

[0029] Preferably, the population of bacteria that are harmful comprises: Corynebacterium, Hungatella and Aestuariispira.

[0030] Possibly, the modulating bile acid and / or prostaglandin metabolism comprises:

[0031] 1) modulating the synthesis of bile acid;

[0032] 2) modulating the conversion of bile acid;

[0033] 3) optimization of the metabolite combination; preferably, the optimization of the metabolite combination comprises: increasing the content of deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2 and / or prostaglandin I2.

[0034] Possibly, the mechanism of preventing, ameliorating and / or treating constipation comprises:

[0035] (1) modulating the gut microbiota;

[0036] (2) modulating bile acid and / or prostaglandin metabolism;

[0037] (3) promoting intestinal peristalsis;

[0038] (4) reducing water reabsorption in the colon.

[0039] Specifically, the inactivated strain of Akkermansia muciniphila YG2604 can reduce the intestinal transit time by regulating the synthesis and transformation of bile acids, optimizing the combination of metabolites (including increasing the content of beneficial metabolites such as deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2 and / or prostaglandin I2), thereby improving constipation; and the inactivated strain of YG2604 can target the regulation of increasing the combination of bacteria (Lactobacills, Streptococcus and Anaerofustis) beneficial to the relief of constipation, and inhibit harmful bacteria (such as Corynebacterium, Hungatella and Aestuariispira, etc.), which helps to maintain the balance of the intestinal microecological environment and has a significant effect on improving constipation.

[0040] In possible embodiments, the constipation can be caused by any reason, including acute severe constipation, intractable constipation, habitual constipation, constipation caused by improper diet, etc.

[0041] In possible embodiments, the treatment of constipation includes alleviating, relieving or improving the symptoms of constipation.

[0042] In addition, in possible embodiments, the product is a pharmaceutical product.

[0043] Further possible, the pharmaceutical is formulated for nasal, oral or digestive administration.

[0044] Still further possible, the pharmaceutical is in the form of a pill, a tablet, a lozenge, a lyophilized powder, a granule, a capsule, an aqueous solution, an alcoholic solution, an oily solution, a syrup, an emulsion, a suspension, a suppository, a solution for injection or infusion, an ointment, a gel, a tincture, a cream, a patch, a lotion, a spray, an aerosol, a powder spray, an effervescent tablet, a transdermal therapeutic system, a microcapsule, an implant or a stick.

[0045] In a fifth aspect, the present application provides a method for preventing and / or treating constipation, comprising administering to a subject in need a prophylactically and / or therapeutically effective amount of the live strain or inactivated strain (preferably inactivated strain) of Akkermansia muciniphila YG2604 as described in the first aspect above, the bacterial agent as described in the second aspect above or the composition as described in the third aspect above.

[0046] In possible embodiments, the constipation can be caused by any reason, including acute severe constipation, intractable constipation, habitual constipation, constipation caused by improper diet, etc.

[0047] In an embodiment, the treatment of constipation includes alleviating, relieving, or improving symptoms of constipation.

[0048] The "prophylactically and / or therapeutically effective amount" can vary depending on the subject, the subject's organs, symptoms, administration method, etc., and can be determined according to the judgment of a doctor, taking into account the type of dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, etc.

[0049] Beneficial effects

[0050] The Akkermansia muciniphila YG2604 of the present application is a unique strain isolated from a healthy adult fecal sample, which has the following advantages compared to the reported strains:

[0051] 1) can beneficially regulate intestinal flora, bile acid metabolism and prostaglandin metabolism, thereby helping to maintain intestinal microecological balance and can achieve improvement and solution of constipation and other intestinal health problems; in particular, it can prevent, improve and / or treat constipation;

[0052] 2) The inactivated strain of Akkermansia muciniphila YG2604 can achieve the above effects, compared with traditional live probiotic products, the inactivated Akkermansia muciniphila YG2604 product has better stability, longer shelf life, and does not need cold chain transportation and storage, and is easier to commercialize and widely used.

[0053] Overall, the Akkermansia muciniphila YG2604 of the present application as an innovative probiotic strain shows significant efficacy and value in the clinical application of treating constipation, and has obvious market potential; the successful development of the strain effectively fills the gap of new probiotic products in the current constipation treatment field, and provides more diversified and efficient treatment options for patients. BRIEF DESCRIPTION OF DRAWINGS

[0054] One or more embodiments are illustrated by way of example in the figures that form a part of this patent document. This illustration is not to be considered limiting in relation to the embodiments.

[0055] Figure 1Figure 4 shows the effects of the four candidate strains on the defecation parameters and gastrointestinal transit function of acute FC model mice; wherein, A, B, and C show the results of whole intestinal transit time, small intestinal propulsion rate, and the amount of black stool (5 hours), respectively; the data represent SD ± mean (n = 6), statistical analysis was performed using one-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001.

[0056] Figure 2 Figure 5 shows the Circos genome plot of Akkermansia muciniphila YG2604; wherein, the outermost circle is the scale of genome size, the second and third circles are CDS on the positive strand and the negative strand, different colors represent different COG functional classification of CDS, the fourth circle is rRNA and tRNA, and the fifth circle is GC content.

[0057] Figure 3 Figure 6 shows the effects of Akkermansia muciniphila YG2604 on the defecation parameters and gastrointestinal transit function of chronic FC model mice; wherein, A, B, and C show the results of whole intestinal transit time, small intestinal propulsion rate, and the amount of black stool (5 hours), respectively; the data represent SD ± mean (n = 6), statistical analysis was performed using one-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001, #p < 0.05, ##p < 0.01.

[0058] Figure 4 Figure 7 shows the effects of Akkermansia muciniphila YG2604 on the colon tissue and related intestinal factor mRNA expression levels of chronic FC model mice; wherein, A is the result picture of HE staining for detecting the pathological damage of mouse colon, wherein, the black arrow indicates the colon mucosa thickness, the blue arrow indicates the inflammatory infiltration, and the yellow arrow indicates the goblet cell, 10X under the microscope, scale = 100 pm; B is the normalized colon mucosa thickness; C and D are the results of mRNA level detection of water channel proteins AQP4 and AQP8, respectively; the data represent SD ± mean (n = 6), statistical analysis was performed using one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0059] Figure 5 Figure 8 shows the regulatory effect of Akkermansia muciniphila YG2604 on intestinal flora; wherein, A is the PCoA plot analysis of the sample, showing the similarity and difference between different samples; B is a Venn diagram, reflecting the number of common and unique species in different samples; C shows the bacterial community analysis at the door level, showing the relative abundance of different door bacteria; D is the bacterial community difference analysis at the genus level, showing the relative abundance of different bacteria.

[0060] Figure 6 Figure 1 shows that Akkermansia muciniphila YG2604 improves constipation in mice by regulating metabolism and intestinal flora; wherein, A shows the beta diversity analysis of the metabolic profile of the colon contents; B shows the volcano plot of the differential metabolites produced by comparison between the Lop group and the Con group; C shows the volcano plot of the differential metabolites produced by comparison between the YG2604 group and the Lop group; D shows the comparison results of KEGG enrichment analysis of the YG2604 group and the Lop group, identifying the affected metabolic pathways; E shows the types of differential metabolites of the YG2604 group and the Lop group; F shows the heat map correlation analysis of the typical differential metabolites and intestinal flora; G shows the network graph analysis of the metabolites, constipation markers and intestinal flora.

[0061] The Akkermansia muciniphila YG2604 of the present application is classified as Akkermansia muciniphila and is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Beijing, China Institute of Microbiology, on October 16, 2024, with a preservation number of CGMCC No. 46149. DETAILED DESCRIPTION

[0062] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application. Unless otherwise explicitly indicated, in the entire specification and claims, the term “comprise” or its variants such as “include” or “contain” and the like should be understood as including the stated elements or components, without excluding other elements or components.

[0063] In addition, in order to better illustrate the present application, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present application.

[0064] In the following examples, unless otherwise specified, the reagents or raw materials used are commercially available.

[0065] Example 1: Isolation and identification of Akkermansia muciniphila

[0066] (1) Isolation and preliminary screening of strains

[0067] Healthy adult fecal samples were collected, 1-2 g of glycerol (20% concentration) frozen sample was taken, 9 mL of PB was added and mixed evenly, the dilution was 10 -1 Diluted solution, continue to dilute with PBS gradient to obtain 10 -2 Diluted solution. Take the glycerol frozen sample of the sample, the dilution of 10 -1 , 10 -2 Diluted solution was cultured in enrichment medium (mucin 1 g, calcium chloride 0.011 g, magnesium chloride 0.01 g, sodium phosphate dibasic 0.053 g, potassium phosphate monobasic 0.04 g, sodium chloride 0.03 g, sodium hydrogen phosphate 0.02 g, distilled water 100 mL) for 72 h, then plated on BHI agar medium containing 5% (w / v) N-acetylglucosamine (mBHI) and incubated at 37°C in an anaerobic incubator for 48-72 h. Single colonies were selected and purified on mBHI plates for more than 3 times until the colony morphology on the plate was consistent. The purified colonies were identified by MALDI-TOF, and the strains preliminarily identified as mucinophilic Akkermansia were selected for further analysis.

[0068] (2) Colony morphology

[0069] The preliminarily screened mucinophilic Akkermansia was cultured on mBHI agar medium for 48 h, with a diameter of 0.1-0.3 mm, smooth edges, and a light yellow color.

[0070] Example 2: Screening of mucinophilic Akkermansia with improved constipation efficacy

[0071] In this example, we evaluated the ability of 4 candidate strains to improve functional constipation on a loperamide hydrochloride (hereinafter referred to as "Lop") induced acute mouse constipation (FC) model. The specific experimental process is as follows:

[0072] Animal experiments

[0073] SPF level 8-week-old BALB / c wild-type male mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were adaptively fed for 1 week under standard environment (12 hours light / 12 hours dark cycle, temperature 23±3℃, humidity 50%±10%), then randomly grouped according to 6 mice per group, a total of 6 groups, including: blank control group (NC group, gavage with sterile PBS+sterile PBS), model group (Lop group, gavage with loperamide hydrochloride+sterile PBS) and 4 experimental groups (YG2604 group, YG2638 group, YG2645 group and YG2609 group). From the first day after the end of the adaptation period, all groups were gavaged with loperamide hydrochloride (2.5 mg / kg*bw) according to the body weight of the mice, and the NC group was replaced with sterile PBS, and the modeling was continued for 7 days. From the first day of the modeling period, intervention was started, 0.5h after gavage with loperamide hydrochloride, the experimental groups were gavaged with 1×10 9 CFU of inactivated mucinophilic Akkermansia (YG2604, YG2638, YG2645, YG2609) were intervened, and the NC group and the Lop group were replaced with sterile PBS. During the experiment, the mice in each group were fed with standard feed and drank sterilized water. On the 8th day, the whole intestinal transit time (the time of the first black feces) and the number of black feces were observed. On the 9th day, the mice were sacrificed, and the small intestinal propulsion rate (ink propulsion rate) was observed.

[0074] Index determination

[0075] (1) First black feces time determination and black feces particle number

[0076] On the 8th day (after 18h fasting), the NC group mice were gavaged with normal saline, and the other groups were gavaged with loperamide hydrochloride. 0.5h later, the NC group and the Lop group were gavaged with 5% ink, and the experimental groups were gavaged with 5% ink containing 1×10 9 CFU of inactivated mucinophilic Akkermansia (YG2604, YG2638, YG2645, YG2609). Each mouse was transferred to a clean cage and provided with feed and water, and the time of the first black feces and the number of black feces within 5 hours were recorded from the start of gavage with ink.

[0077] (2) Determination of small intestinal propulsion rate

[0078] On the 9th day (after 18h fasting), the NC group mice were gavaged with normal saline, and the other groups were gavaged with loperamide hydrochloride. 0.5h later, the NC group and the Lop group were gavaged with ink, and the experimental groups were gavaged with ink containing 1×10 95% ink from CFU-inactivated Akkermansia myxophilus (YG2604, YG2638, YG2645, YG2609). Mice were sacrificed after 17 minutes of free access to food and water. The abdominal cavity was opened, the mesentery was separated, and a section of the small intestine extending from the pylorus to the ileocecal junction was cut off. The small intestine was gently stretched into a straight line, and the length of the intestinal section was measured as the total length of the small intestine. The distance from the pylorus to the leading edge of the ink was measured as the ink propulsion length.

[0079] The formula for calculating small intestinal propulsion rate is as follows:

[0080]

[0081] The results of the whole intestinal transit time, small intestinal propulsion rate, and amount of black stool (5 hours) are as follows: Figure 1 The diagrams A, B, and C are shown in the image. Figure 1 Figures A, B, and C show that, compared with the NC group, the Lop group had an increased whole intestinal transit time, a decreased number of black fecal pellets, and a reduced small intestinal propulsion rate, indicating that the acute FC mouse model was established.

[0082] For whole intestinal transit time indicators, such as Figure 1 As shown in Figure A, compared to the Lop group, only the YG2604 group reduced the whole intestinal transit time (by 27.03%).

[0083] For small intestinal propulsion rate indicators, such as Figure 1 As shown in Figure B, compared with the Lop group, only the YG2604 group improved the ink propulsion rate by 13.23%, thus enhancing the peristaltic ability of the small intestine.

[0084] For the quantity of black stool, such as Figure 1 As shown in Figure C, compared with the Lop group, both the YG2604 group and the YG2645 group increased the number of black feces excreted by mice within 5 hours (by 109.52% and 90.47%, respectively).

[0085] Based on the test results of the above indicators, strain YG2604 was selected from the four candidate strains. It has the strongest effect in improving constipation symptoms and therefore has great potential for the prevention and treatment of acute constipation.

[0086] Example 3: Genomic determination and safety evaluation of strain YG2604

[0087] 16S rRNA & whole genome sequencing

[0088] The strain YG2604 was sent to Shanghai Shengong for 16S rRNA gene sequencing, and the 16S rRNA gene sequence thereof is shown as SEQ ID NO: 1. The 16S rRNA gene sequence thereof was subjected to BLAST comparison on the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and in combination with the morphological characteristics of the strain, it was further determined to be Akkermansia muciniphila.

[0089] Further, the whole genome of Akkermansia muciniphila YG2604 was subjected to sequence determination by using an illumina HiSeq sequencing platform, and a Circos genome circle chart is shown in Figure 2 Based on the genome analysis of Figure 2 , it was shown that the genome of Akkermansia muciniphila YG2604 had no plasmid, and a single circular chromosome was 2,794,22 bp, and the G+C content was 55.39%; the genome annotation showed that it contained 2370 coding sequences in total; in addition, the genome of Akkermansia muciniphila YG2604 contained 49 tRNAs, 3 rRNAs and 2358 CDSs.

[0090] Based on the whole genome sequencing result, the safety of Akkermansia muciniphila YG2604 at the genome level was also evaluated, and the result showed that the genome of Akkermansia muciniphila YG2604 had no genes related to transferable antibiotic resistance and virulence genes.

[0091] Antibiotic sensitivity test

[0092] The sensitivity of Akkermansia muciniphila YG2604 to 9 kinds of antibiotics was determined by using a drug sensitivity disc diffusion method (K-B method); specifically, the test bacterial liquid was coated on a modified BHI solid culture medium, and after being completely absorbed, antibiotic discs were placed thereon, and the size of the inhibition zone was determined after anaerobic culture at 37°C for 48 h. According to the standard CLSI (2012) of the American Clinical Laboratory Standardization Committee, it was determined that the strain was resistant (R), intermediate (I) or sensitive (S) to antibiotics. The result is shown in Table 1.

[0093] Table 1. Results of antibiotic sensitivity experiment

[0094]

[0095] Table 1 shows that Akkermansia muciniphila YG2604 has sensitivity to antibiotics such as chloramphenicol, metronidazole and ampicillin-sulbactam, and has resistance to some antibiotics, such as imipenem, tetracycline, cefotetan, clindamycin, moxifloxacin and ampicillin.

[0096] Example 4: Study on the efficacy of strain YG2604 in improving chronic constipation and intestinal epithelial barrier damage and its mechanism

[0097] Animal experiments

[0098] SPF level 8-week-old BALB / c wild type male mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were adaptively fed for 1 week in a standard environment (12 hours light / 12 hours dark cycle, temperature 23±3℃, humidity 50%±10%), then randomly divided into 6 mice per group, a total of 5 groups, including: blank control group (NC group, intragastrically administered sterile PBS+sterile PBS), model group (Lop group, intragastrically administered loperamide hydrochloride+sterile PBS), mucinophilic Akkermansia muciniphila YG2604 group and YG2638 group, and lactulose group (Lac group, intragastrically administered loperamide hydrochloride+lactulose) as a positive drug control. From the first day after the end of the adaptation period, the Lop group, the Lac group, the YG2604 group and the YG2638 group were intragastrically administered loperamide hydrochloride (2.5 mg / kg*bw) according to the body weight of the mice, and the NC group was replaced with sterile PBS, for a continuous modeling period of 14 days. From the first day of the modeling period, intervention was started, and 0.5 h after loperamide hydrochloride was administered, the YG2604 group and the YG2638 group were intragastrically administered 1×10 9 The inactivated strains YG2604 and YG2638 were used for intervention, the Lac group was treated with lactulose (2.6 g / kg*bw), and the NC group and the Lop group were replaced with sterile PBS. During the experiment, the mice in each group were fed with standard feed, and on the 15th day, the whole intestinal transit time (the time of the first black fecal pellet) and the number of black fecal pellets were observed, and on the 16th day, the mice were sacrificed after anesthesia, and samples such as the colon, cecal contents and feces were collected and the small intestinal transit time (ink propulsion rate) was observed. One part of the collected colon tissue was fixed with 4% paraformaldehyde and stained with HE, and the other part was stored at -80℃ for subsequent detection of colon factors. The mouse feces and cecal contents were used to detect the abundance and metabolism of the intestinal flora of the mice.

[0099] Index determination

[0100] (1) First black fecal pellet time determination and black fecal pellet number

[0101] On the 15th day of the experiment (after 18 h of fasting), the NC group mice were intragastrically administered normal saline, and the Lac, Lop, YG2604 and YG2638 groups were intragastrically administered loperamide hydrochloride. After 0.5 h, the NC and Lop groups were intragastrically administered 5% ink, the Lac group was intragastrically administered lactulose containing 5% ink, and the YG2604 and YG2638 groups were intragastrically administered 1×10 95% CFU-inactivated strains YG2604 and YG2638 were used as ink. Afterward, each mouse was transferred to a clean, empty cage and provided with food and water. The time of the first black stool and the number of black stools excreted within 5 hours were recorded from the start of the gavage.

[0102] (2) Determination of small intestinal propulsion rate

[0103] On day 16 of the experiment (after 18 hours of fasting), mice in the NC group were administered saline by gavage, while mice in other groups were administered loperamide hydrochloride by gavage. 0.5 hours later, mice in the NC and Lop groups were administered 5% ink by gavage, the Lac group was administered lactulose containing 5% ink by gavage, and the YG2604 and YG2638 groups were administered lactulose containing 1×10⁻⁶ kJ of saline solution. 9 Mice were anesthetized and euthanized after 17 minutes of free feeding with 5% CFU-inactivated strains YG2604 and YG2638 of ink. The abdominal cavity was opened, the mesentery was separated, and the intestinal segment from the upper end to the pylorus to the lower end to the ileocecal junction was cut off. The small intestine was gently pulled into a straight line, and the length of the intestinal segment was measured as the total length of the small intestine. The length from the pylorus to the leading edge of the ink was the ink propulsion length.

[0104] Formula for calculating small intestinal propulsion rate:

[0105]

[0106] (3) Pathological examination

[0107] Fixed colon tissue was embedded in paraffin and sectioned, stained with hematoxylin and eosin (H&E), and the stained slides were observed under an optical microscope to determine the thickness of the colonic mucosa.

[0108] (4) Genetic testing

[0109] Total RNA was extracted from colon samples using the RNAiso Plus kit (TaKaRa, Japan) and the PrimeScript kit was used. TM II. cDNA was synthesized according to the instructions using the 1st Strand cDNA Synthesis Kit (TaKaRa). The transcriptional levels of aquaporins AQP4 and AQP8 were detected using the FastFire quantitative PCR kit. PCR amplification was performed under the following conditions: initial denaturation at 95℃ for 3 min, followed by 40 cycles of 95℃ for 15 s, 55℃ for 15 s, and 72℃ for 20 s. β-actin was used as an internal reference gene, and 2... -ΔΔCT The formula was used to calculate the relative gene transcription levels. The untreated NC group served as a control.

[0110] The results of the whole intestinal transit time, small intestinal propulsion rate, and amount of black stool (5 hours) are as follows: Figure 3 The diagrams A, B, and C are shown in the image. Figure 3Figures A, B, and C show that compared to the NC group, the Lop group had increased whole-intestinal transit time, decreased number of black feces, and decreased small intestinal propulsion rate, indicating successful establishment of the chronic functional constipation (FC) mouse model. Compared to the Lop group, the Lac and YG2604 groups had decreased whole-intestinal transit time, increased number of black feces, and increased small intestinal propulsion rate, indicating that the inactivated strain YG2604 had an ameliorative effect on chronic functional constipation. Compared to the YG2604 group, the YG2638 group had increased whole-intestinal transit time, decreased number of black feces, and decreased small intestinal propulsion rate, and there were no significant differences in the three indicators between the YG2638 group and the Lop group (p≥0.05). This result indicates that the YG2638 group failed to effectively alleviate the mouse constipation model, suggesting that the ability of Akkermansia myxophilus to alleviate constipation varies among strains.

[0111] The HE staining results of colon tissue and the statistical results of colonic mucosal thickness are as follows: Figure 4 As shown in Figures A and B, the transcriptional levels of aquaporins AQP4 and AQP8 were detected as follows: Figure 4 As shown in Figures C and D.

[0112] Depend on Figure 4 Figures A and B show that the colonic mucosal layer thickness, inflammatory infiltration, and goblet cell count were reduced in the Lop group mice. Under the intervention of Akkermansia myxophilus YG2604, the colonic mucosal layer thickness of chronic FC mice increased by 17.50%, accompanied by a reduction in inflammatory infiltration and an improvement in the degree of colonic tissue damage. That is, histopathological examination further confirmed the effectiveness of inactivated strain YG2604 in improving chronic constipation.

[0113] Depend on Figure 4 Figures C and D show that, at the gene level, the inactivated strain YG2604 significantly inhibited the expression of aquaporins AQP4 and AQP8 in chronic functional constipation (FC) mice. This result suggests that the inactivated strain YG2604 may improve the symptoms of chronic functional constipation by reducing colonic water reabsorption.

[0114] In addition, we used 16S rRNA gene sequencing technology to investigate the effects of inactivated strain YG2604 on the gut microbiota of constipation. The results are as follows: Figure 5 As shown. Figure 5 Figures A and B in the figure show the PCoA and Venn diagram analysis results of the samples, respectively. They show that the NC, Lop, and YG2604 groups exhibit different clustering patterns on the PCoA diagram, indicating significant differences among the three groups. Moreover, YG2604 significantly altered the β diversity of the gut microbiota and was closer to the Con group. Figure 5 Figure C in the diagram represents a phylum-level bacterial community analysis, showing the relative abundance of bacteria from different phyla.Figure 5 Figure D in FIG. 7 is a bacterial community difference analysis at genus level, showing the relative abundance of different bacteria; and Figure 5 As can be seen from FIG. 7C and FIG. 7D, at the level of phylum, compared with the Lop group, the intervention of inactivated strain YG2604 increased the relative abundance of Firmicutes, Actinobacteria and Bacteroidetes, and decreased the relative abundance of Proteobacteria (FIG. 7C); at the level of genus, the intervention of inactivated strain YG2604 increased the abundance of beneficial bacteria such as Lactobacills, Streptococcus and Anaerofustis, and decreased the abundance of harmful bacteria such as Corynebacterium, Hungatella and Aestuariispira (FIG. 7D). The above results show that inactivated strain YG2604 can improve the symptoms of constipation by regulating the intestinal flora, including increasing the flora beneficial to the relief of constipation and inhibiting the harmful flora. Figure 5 Figure 5 In addition, we also explored the effect of inactivated strain YG2604 on the fecal metabolome of the Lop-induced chronic FC mouse model. Specifically, we analyzed the differences in the fecal metabolome of different groups.

[0115] Partial least squares discriminant analysis (PLS-DA) is a supervised machine learning algorithm that can effectively distinguish the overall variability of samples. The PLS-DA visualization clearly shows the clustering trend of the Con group, the Lop group and the YG2604 group (see FIG. 8A), indicating that the intervention of inactivated strain YG2604 has a significant effect on the fecal metabolome of the Lop-induced chronic FC mouse. By comparison, we found that compared with the NC group, the Lop group had 163 increased metabolites and 273 decreased metabolites; and compared with the Lop group, the YG2604 group had 198 increased metabolites and 194 decreased metabolites (see FIG. 8B-C). In order to further analyze the mechanism of sample metabolite changes, we performed metabolic pathway enrichment analysis based on these differential metabolites, and the results showed that the YG2604 group was significantly enriched in 20 metabolic pathways compared with the Lop group, among which the enrichment of primary bile acid synthesis, bile secretion and smooth muscle contraction was particularly significant (see FIG. 8D). In addition, we screened 18 significantly different metabolites (P < 0.05, VIP > 1, see FIG. 9) from these three significantly enriched metabolic pathways.

[0116] Partial least squares discriminant analysis (PLS-DA) is a supervised machine learning algorithm that can effectively distinguish the overall variability of samples. The PLS-DA visualization clearly shows the clustering trend of the Con group, the Lop group and the YG2604 group (see FIG. 8A), indicating that the intervention of inactivated strain YG2604 has a significant effect on the fecal metabolome of the Lop-induced chronic FC mouse. By comparison, we found that compared with the NC group, the Lop group had 163 increased metabolites and 273 decreased metabolites; and compared with the Lop group, the YG2604 group had 198 increased metabolites and 194 decreased metabolites (see FIG. 8B-C). In order to further analyze the mechanism of sample metabolite changes, we performed metabolic pathway enrichment analysis based on these differential metabolites, and the results showed that the YG2604 group was significantly enriched in 20 metabolic pathways compared with the Lop group, among which the enrichment of primary bile acid synthesis, bile secretion and smooth muscle contraction was particularly significant (see FIG. 8D). In addition, we screened 18 significantly different metabolites (P < 0.05, VIP > 1, see FIG. 9) from these three significantly enriched metabolic pathways. Figure 6 Figure 6 Figure 6 Figure 6 ​​​​(See Figure E in the table). Notably, compared to the Lop group, the YG2604 group showed significantly increased levels of metabolites such as deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2, and prostaglandin I2. Furthermore, the YG2604 group exhibited decreased levels of metabolites such as pravastatin, taurochenodeoxycholic acid, cerivastatin, and glycocholic acid (see Figure E in the table). Figure 6 (See Figure E in the original text). By analyzing the association between changes in gut metabolite composition and the abundance of microbial OTUs, we found at the genus level that lithocholic acid, deoxycholic acid, valproic acid, and prostaglandin I2 were positively correlated with Lactobacillus, Streptococcus, and Anaerofustis, while they were negatively correlated with Corynebacterium, Hungatella, and Aestuariispira (see Figure E in the original text). Figure 6 (See Figure F in the original text). To investigate the potential mechanism by which inactivated strain YG2604 alleviates constipation in mice, we performed Pearson correlation analysis, and the results are shown in Figure F. Figure 6 As shown in Figure G, the results indicate that deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2, and prostaglandin I2 were positively correlated with *Lactobacillus*, *Streptococcus*, and *Anaerofustis*, while significantly negatively correlated with whole intestinal transit time, AQP4, and AQP8, and positively correlated with small intestinal transit time, fecal excretion, GPR41, and GPR43. Based on these findings, we hypothesize that deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2, and prostaglandin I2 may be key metabolites of inactivated strain YG2604 that improve constipation in mice.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An inoculant, characterized in that, The active ingredient of the bacterial agent comprises an inactivated strain of Akkermansia muciniphila YG2604, wherein the Akkermansia muciniphila YG2604 is deposited with the China General Microbiological Culture Collection Center on October 16, 2024, and has a deposit number of CGMCC No. 46149, and a 16S rRNA gene sequence as shown in SEQ ID NO:

1.

2. A composition characterized in that, The composition comprises an effective amount of the bacterial agent of claim 1 as a first active component.

3. The composition of claim 2, wherein, The composition is a pharmaceutical composition.

4. The composition according to claim 2 or 3, characterized in that, The composition further comprises a second active component.

5. The composition of claim 4, wherein, The second active component comprises probiotics, postbiotics, prebiotics, antibacterial agents, immunomodulators, anticancer agents, inflammation treatment agents, or combinations thereof.

6. Use of the bacterial agent of claim 1 or the composition of any one of claims 2-5 in the preparation of a pharmaceutical product for: (1) modulating the gut microbiota; said modulating the gut microbiota comprises: increasing a beneficial flora for relieving constipation, and / or inhibiting a harmful flora, wherein the beneficial flora for relieving constipation comprises Lactobacills, Streptococcus, and Anaerofustis, and the harmful flora comprises Corynebacterium, Hungatella, and Aestuariispira; (2) preventing, improving, and / or treating constipation.

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