Ackermania muciniphila YG2604 and application thereof in treatment of constipation

Abstract

The invention relates to the field of microorganisms, in particular to Akkermansia muciniphila YG2604, a microbial agent containing the Akkermansia muciniphila YG2604, a composition containing the Akkermansia muciniphila YG2604 and application of the Akkermansia muciniphila YG2604 and the microbial agent containing the Akkermansia muciniphila YG2604 and the composition containing the Akkermansia muciniphila YG2604 to preparation of products for preventing, improving and / or treating constipation. The ackermania muciniphila YG2604 disclosed by the invention can be used for maintaining the micro-ecological balance of intestinal tracts and effectively improving or treating constipation; moreover, the inactivated strain can realize the effects, and the inactivated strain product has better stability and longer shelf life, does not need cold-chain transportation and preservation, and is easier to commercialize and widely apply. Therefore, the Ackermania muciniphila YG2604 disclosed by the invention has a relatively good clinical application prospect and an obvious market potential.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Akkermansia muciniphila YG2604, a bacterial agent and a composition containing the same, and applications of the same in preparing products for preventing, improving and / or treating constipation. Background Art

[0002] Constipation is a common digestive system disease in clinical practice, with symptoms such as difficulty in defecation and hard and dry stool. According to statistics, the incidence of constipation in adults in my country is about 10%-15%, and it increases with age. Long-term constipation not only affects the patient's quality of life, but may also cause complications such as anal fissures and hemorrhoids. At present, the main methods for treating constipation in clinical practice include drug therapy, diet conditioning, biofeedback therapy, etc., but these methods have problems such as unstable efficacy, large side effects, and easy drug dependence.

[0003] In recent years, with the deepening of microecological research, the role of probiotics in regulating the balance of intestinal flora and improving intestinal function has been increasingly valued. Probiotics refer to active microorganisms that can have beneficial effects on human health after being given a certain amount. At present, many studies have confirmed that probiotics have significant therapeutic effects in the treatment of constipation, with high safety and few side effects. However, existing probiotic products have certain limitations in terms of strain types, number of live bacteria, stability, etc., resulting in poor results in clinical applications.

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

[0005] Previous studies have focused on the application of Akkermansia muciniphila in immune regulation, improvement of metabolic syndrome and treatment of inflammatory bowel disease (IBD). Studies have found that this strain can enhance the intestinal barrier function by increasing the thickness of the intestinal mucus layer, 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 inhibiting inflammatory responses. Summary of the invention

[0006] Purpose of the Invention

[0007] The object of the present invention is to provide a strain of Akkermansia muciniphila YG2604 having the efficacy of preventing, improving and / or treating constipation, a bacterial agent and a composition containing the same, and their use in preparing a product for preventing, improving and / or treating constipation.

[0008] Solution

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] In the first aspect, the present invention provides a strain of Akkermansia muciniphila YG2604, which is classified and named Akkermansia muciniphila and is deposited in the General Microbiology Center of China Culture Collection Administration. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 16, 2024, and the deposit number is CGMCC No. 46149.

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

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

[0013] The colony characteristics of the strain include: after 48 hours of cultivation on mBHI agar medium, the diameter is between 0.1 and 0.3 mm, the colony edges are smooth and light yellow.

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

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

[0016] In a feasible embodiment, the active ingredient of the bacterial agent includes an inactivated strain of Akkermansia muciniphila YG2604.

[0017] Compared with live strains, the inactivated Akkermansia muciniphila YGMCC2604 has better stability, longer shelf life, and does not require cold chain transportation and storage, making it easier to commercialize and widely use. Therefore, using the inactivated strain of YG2604 as the active ingredient of the microbial agent is a more preferred implementation scheme.

[0018] In a third aspect, the present invention 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 the bacterial agent as described in the second aspect above, as a first active component.

[0019] In a feasible embodiment, the composition is a pharmaceutical composition, a food composition or a feed composition.

[0020] In a feasible specific embodiment, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier and / or excipient. Further feasible, the pharmaceutical composition is formulated into a dosage form for nasal, oral or digestive tract administration, for example, formulated into a pill, tablet, lozenge, lyophilized powder, granules, capsule, aqueous solution, alcohol solution, oil solution, syrup, emulsion, suspension, suppository, injection or infusion solution, ointment, gel, tincture, cream, patch, lotion, spray, aerosol, powder spray, effervescent tablet, transdermal therapeutic system, microcapsule, implant or stick.

[0021] In a feasible embodiment, the composition further comprises a second active ingredient;

[0022] Optionally, the second active component comprises a probiotic, a postbiotic, a prebiotic, an antimicrobial agent, an immunomodulator, an anticancer agent, an inflammatory therapeutic agent, or a combination thereof.

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

[0024] (1) Regulate intestinal flora;

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

[0026] (3) Prevent, improve and / or treat constipation.

[0027] Preferably, the regulating intestinal flora includes: increasing flora that is beneficial to relieving constipation, and / or inhibiting harmful flora;

[0028] Preferably, the flora beneficial for relieving constipation includes: Lactobacills, Streptococcus and Anaerofustis;

[0029] Preferably, the harmful bacteria include: Corynebacterium, Hungatella and Aestuariispira.

[0030] Preferably, the regulating bile acid and / or prostaglandin metabolism comprises:

[0031] 1) Regulate the synthesis of bile acids;

[0032] 2) Regulate the conversion of bile acids;

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

[0034] Possibly, the mechanisms of preventing, improving and / or treating constipation include:

[0035] (1) Regulate intestinal flora;

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

[0037] (3) Promote intestinal motility;

[0038] (4) Reduce the colon's reabsorption of water.

[0039] Specifically, the inactivated strain of Akkermansia muciniphila YG2604 can reduce the intestinal transit time by regulating bile acid synthesis and conversion and optimizing the metabolite combination (including increasing the content of beneficial metabolites such as deoxycholic acid, chenodeoxycholic acid, bile acid, lithocholic acid, prostaglandin B2 and / or prostaglandin I2), thereby improving constipation; and the inactivated strain of YG2604 can target and regulate to increase the bacterial flora combination (Lactobacills, Streptococcus and Anaerofustis) that is beneficial to relieving constipation, and inhibit harmful bacteria (such as Corynebacterium, Hungatella and Aestuariispira, etc.). This targeted regulation helps maintain the balance of the intestinal microecological environment and has a significant effect on improving constipation.

[0040] In a feasible implementation manner, the constipation may be constipation caused by any reason, including: acute and severe constipation, intractable constipation, habitual constipation, constipation caused by improper diet, and the like.

[0041] In a feasible embodiment, the treatment of constipation includes alleviating, relieving, and improving the symptoms of constipation.

[0042] Furthermore, in a possible embodiment, the product is a pharmaceutical product.

[0043] Further practicably, the drug is formulated for nasal, oral or intragastric administration.

[0044] Further preferably, the drug is in the form of pills, tablets, lozenges, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oily solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder sprays, effervescent tablets, transdermal therapeutic systems, microcapsules, implants or sticks.

[0045] In a fifth aspect, the present invention provides a method for preventing and / or treating constipation, comprising administering to a subject in need thereof a preventive and / or therapeutically effective amount of a live strain or an inactivated strain (preferably an 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 a feasible implementation manner, the constipation may be constipation caused by any reason, including: acute and severe constipation, intractable constipation, habitual constipation, constipation caused by improper diet, and the like.

[0047] In a feasible embodiment, the treatment of constipation includes alleviating, relieving, and improving the symptoms of constipation.

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

[0049] Beneficial Effects

[0050] The Akkermansia muciniphila YG2604 of the present invention is a unique strain isolated from a fecal sample of a healthy adult. Compared with the reported strains of the same species, it has the following advantages:

[0051] 1) It can beneficially regulate intestinal flora, bile acid metabolism and prostaglandin metabolism, thereby contributing to the maintenance of intestinal microecological balance and improving and resolving intestinal health problems such as constipation; in particular, it can prevent, improve and / or treat constipation);

[0052] 2) The inactivated strain of Akkermansia muciniphila YG2604 can achieve the above-mentioned effects. Compared with traditional live probiotic products, the inactivated Akkermansia muciniphila YG2604 product has better stability, longer shelf life, and does not require cold chain transportation and storage, making it easier to commercialize and widely use.

[0053] In general, Akkermansia muciniphila YG2604 of the present invention, as an innovative probiotic strain, has shown significant efficacy and value in clinical applications for the treatment of constipation, and has obvious market potential; the successful development of this strain has effectively filled the gap in new probiotic products in the current field of constipation treatment, and provided patients with more diverse and efficient treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0055] Figure 1The effects of the four candidate strains on the defecation parameters and gastrointestinal transit function of acute FC model mice are shown; Figures A, B, and C respectively show the test results of whole intestinal transit time, small intestinal propulsion rate, and black stool quantity (5 hours); Data represent SD±mean (n=6), and one-way ANOVA was used for statistical analysis, *p<0.05, **p<0.01, ****p<0.0001.

[0056] Figure 2 The Circos genome diagram of Akkermansia muciniphila YG2604 is shown; the outermost circle is the marker for genome size, the second and third circles are the CDS on the positive and negative strands, and different colors represent the functional classification of COGs with different CDSs, the fourth circle is rRNA and tRNA, and the fifth circle is the GC content.

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

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

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

[0060] Figure 6 The results show that Akkermansia muciniphila YG2604 improves constipation in mice by regulating metabolism and intestinal flora. Figure A shows the β-diversity analysis of the metabolic profile of colon contents. Figure B shows the volcano plot of differential metabolites generated by the comparison between the Lop group and the Con group. Figure C shows the volcano plot of differential metabolites generated by the comparison between the YG2604 group and the Lop group. Figure D shows the comparison results of the KEGG enrichment analysis between the YG2604 and Lop groups to identify the affected metabolic pathways. Figure E shows the types of differential metabolites between the YG2604 and Lop groups. Figure F shows the heat map correlation analysis of typical differential metabolites and intestinal flora. Figure G shows the network diagram analysis of metabolites, constipation markers and intestinal flora.

[0061] The Akkermansia muciniphila YG2604 of the present invention is classified and named as Akkermansia muciniphila, and is deposited in the General Microbiology Center of the China Culture Collection Administration. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 16, 2024, and the deposit number is CGMCC No.46149. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0063] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0064] In the following examples, unless otherwise specified, other 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] Collect stool samples from healthy adults, take 1-2g of glycerol (20% concentration) and freeze the sample, add 9mL PB and mix well, the dilution is 10 -1 Dilution, continue to dilute with PBS to obtain 10 -2 Dilution. Take the sample glycerol freezing solution and dilute it to 10 -1 , 10 -2 The dilutions were cultured in enrichment medium (mucin 1g, calcium chloride 0.011g, magnesium chloride 0.01g, disodium hydrogen phosphate 0.053g, potassium dihydrogen phosphate 0.04g, sodium chloride 0.03g, sodium hydrogen phosphate 0.02g, distilled water 100mL) for 72h, and then spread on BHI agar medium (mBHI for short) containing 5% (w / v) N-acetylglucosamine, and cultured in an anaerobic incubator at 37°C for 48-72h. Single colonies were selected and purified on mBHI plates for more than 3 times until the colony morphology on the plates was consistent. After rapid identification by MALDI-TOF, the purified colonies were preliminarily identified as mucinotropic Akkermansia strains for subsequent analysis.

[0068] (2) Colony morphology characteristics

[0069] The diameter of the preliminarily screened mucinophilic Akkermansia was between 0.1 and 0.3 mm after being cultured on mBHI agar medium for 48 hours, and the colony edges were smooth and light yellow.

[0070] Example 2: Screening of Akkermansia muciniphila with constipation-improving effects

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

[0072] Animal experiments

[0073] 8-week-old SPF BALB / c wild-type male mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were fed in a standard environment (12-hour light / 12-hour dark cycle, temperature 23±3°C, humidity 50%±10%) for 1 week, and then randomly divided into 6 groups with 6 mice in each group, including a blank control group (NC group, gavage with sterile PBS+bacterial PBS), a model group (Lop group, gavage with loperamide hydrochloride+sterile PBS) and 4 experimental groups (YG2604 group, YG2638 group, YG2645 group and YG2609 group). Modeling began on 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 weight of mice, and the NC group was replaced with sterile PBS, and the modeling continued for 7 days. The intervention started on the first day of the modeling period. 0.5 h after intragastric administration of loperamide hydrochloride, the experimental groups were intragastricly administered with 1×10 9 CFU of inactivated Akkermansia muciniphila (YG2604, YG2638, YG2645, YG2609) were used for intervention, while the NC group and Lop group were replaced with sterile PBS. During the experiment, mice in each group were fed with standard feed and sterilized water. The whole intestinal transit time (time of first black stool discharge) and the number of black stool discharge were observed on the 8th day. The mice were killed on the 9th day to observe the small intestinal propulsion rate (ink propulsion rate).

[0074] Index determination

[0075] (1) Determination of the time of first black stool and the number of black stools

[0076] On the 8th day (after fasting for 18 h), mice in the NC group were gavaged with normal saline, and mice in the other groups were gavaged with loperamide hydrochloride. 0.5 h later, mice in the NC group and the Lop group were gavaged with 5% ink, and mice in the experimental groups were gavaged with 1×10 9 Each mouse was transferred to a clean empty cage and provided with feed and water. The time of the first black stool of each mouse and the number of black stools within 5 hours were recorded from the time of intragastric administration of ink.

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

[0078] On the 9th day (after fasting for 18 h), mice in the NC group were gavaged with normal saline, and mice in the other groups were gavaged with loperamide hydrochloride. 0.5 h later, mice in the NC group and the Lop group were gavaged with ink, and mice in the experimental groups were gavaged with 1×10 9CFU of inactivated muciniphila Akkermansia (YG2604, YG2638, YG2645, YG2609) in 5% ink. After 17 minutes of free feeding and drinking, the mice were killed, the abdominal cavity was opened, the mesentery was separated, the intestinal tube from the upper end to the pylorus and the lower end to the ileum and cecum was cut, the small intestine was gently pulled into a straight line, and the intestinal tube length was measured as the total length of the small intestine, and the length from the pylorus to the front of the ink was the ink advancement length.

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

[0080]

[0081] The test results of whole intestinal transit time, small intestinal propulsion rate and black stool quantity (5 hours) are as follows: Figure 1 As shown in Figures A, B, and C; Figure 1 Figures A, B, and C in the figure show that compared with the NC group, the whole intestinal transit time of the Lop group was increased, the number of black stool particles was reduced, and the small intestinal propulsion rate was reduced, indicating that the acute FC mouse model was established.

[0082] For whole-bowel transit time indicators, such as Figure 1 As shown in Figure A, compared with the Lop group, only the YG2604 group reduced the whole intestinal transit time (reduced 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 increased the ink propulsion rate by 13.23%, improving the peristaltic ability of the small intestine.

[0084] For the black stool quantity indicator, such as Figure 1 As shown in Figure C, compared with the Lop group, the YG2604 group and the YG2645 group increased the number of black stool particles excreted by mice within 5 hours (increased 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, which has the strongest effect on improving constipation symptoms and therefore has great potential for preventing and treating acute constipation.

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

[0087] 16SrRNA & Whole Genome Sequencing

[0088] Strain YG2604 was sent to Shanghai Biotechnology for 16SrRNA gene sequencing, and its 16SrRNA gene sequence is shown in SEQ ID NO: 1. Its 16SrRNA gene sequence was compared with the NCBI database by BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and combined with the morphological characteristics of the strain, it was further determined to be Akkermansia muciniphila.

[0089] Furthermore, the whole genome of Akkermansia muciniphila YG2604 was sequenced using the Illumina HiSeq sequencing platform. The Circos genome map is shown in Figure 2 .based on Figure 2 The genome analysis showed that the genome of Akkermansia muciniphila YG2604 was plasmid-free, with a single circular chromosome of 2,794.22 bp and a G+C content of 55.39%; the genome annotation showed that it contained a total of 2,370 coding sequences; in addition, the Akkermansia muciniphila YG2604 genome contained 49 tRNAs, 3 rRNAs and 2,358 CDSs.

[0090] Based on the results of whole genome sequencing, the safety of Akkermansia muciniphila YG2604 at the genomic level was also evaluated. The results showed that the genome of Akkermansia muciniphila YG2604 did not contain genes related to transferable antibiotic resistance and virulence genes.

[0091] Antibiotic susceptibility testing

[0092] The sensitivity of Akkermansia muciniphila YG2604 to 9 antibiotics was determined by the drug sensitivity paper diffusion method (KB method); specifically, the test bacterial solution was spread on the modified BHI solid medium, and after it was completely absorbed, the antibiotic paper was placed on it, and the size of the inhibition zone was measured after anaerobic culture at 37°C for 48 hours. With reference to the standards of the American Clinical Laboratory Standards Committee CLSI (2012), the strain was determined to be resistant (R), intermediate (I) or sensitive (S) to antibiotics. The results are shown in Table 1.

[0093] Table 1. Results of antibiotic sensitivity tests

[0094]

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

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

[0097] Animal experiments

[0098] 8-week-old SPF BALB / c wild-type male mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were fed in a standard environment (12-hour light / 12-hour dark cycle, temperature 23±3°C, humidity 50%±10%) for 1 week, and then randomly divided into 5 groups with 6 mice in each group, including: blank control group (NC group, gavage with sterile PBS+bacterial PBS), model group (Lop group, gavage with loperamide hydrochloride+sterile PBS), Akkermansia muciniphila YG2604 group and YG2638 group, and lactulose group (Lac group, gavage with loperamide hydrochloride+lactulose) as a positive drug control. Modeling began on the first day after the end of the adaptation period. Loperamide hydrochloride (2.5 mg / kg*bw) was gavaged in the Lop group, Lac group, YG2604 group and YG2638 group according to the body weight of mice, and sterile PBS was used instead in the NC group. Modeling continued for 14 days. The intervention started on the first day of the modeling period. 0.5 h after loperamide hydrochloride was administered orally, the YG2604 group and the YG2638 group were given 1×10 9 The inactivated strains YG2604 and YG2638 with CFU were used for intervention. The Lac group was treated with lactulose (2.6 g / kg*bw), while the NC group and the Lop group were replaced with sterile PBS. During the experiment, mice in each group were fed with standard feed. On the 15th day, the whole intestinal transit time (time of first black stool discharge) and the number of black stools discharged were observed. On the 16th day, the mice were killed after anesthesia, and 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 was stored at -80℃ for subsequent detection of colon factors. Mouse feces and cecal contents were used to detect the abundance and metabolome of mouse intestinal flora, respectively.

[0099] Index determination

[0100] (1) Determination of the time of first black stool and the number of black stools

[0101] On the 15th day of the experiment (after fasting for 18 h), the mice in the NC group were gavaged with normal saline, and the mice in the Lac, Lop, YG2604, and YG2638 groups were gavaged with loperamide hydrochloride. 0.5 h later, the mice in the NC and Lop groups were gavaged with 5% ink, the Lac group was gavaged with lactulose containing 5% ink, and the YG2604 and YG2638 groups were gavaged with 1×10 9CFU of inactivated strains YG2604 and YG2638 were added to 5% ink; then, each mouse was transferred to a clean empty cage and provided with feed and water. The time of the first black stool of each mouse and the number of black stools excreted within 5 hours were recorded starting from the gavage of ink.

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

[0103] On the 16th day of the experiment (after fasting for 18 h), mice in the NC group were gavaged with normal saline, and mice in the other groups were gavaged with loperamide hydrochloride. 0.5 h later, mice in the NC and Lop groups were gavaged with 5% ink, mice in the Lac group were gavaged with lactulose containing 5% ink, and mice in the YG2604 and YG2638 groups were gavaged with 1×10 9 The mice were anesthetized and killed after 17 min of free feeding with 5% ink of inactivated CFU of strains YG2604 and YG2638. The abdominal cavity was opened, the mesentery was separated, and the intestinal tube from the upper end to the pylorus to the lower end to the ileum and cecum was cut. The small intestine was gently pulled into a straight line, and the length of the intestinal tube was measured as the total length of the small intestine, and the length from the pylorus to the front of the ink was the ink advancement length.

[0104] The formula for calculating small intestinal propulsion rate is:

[0105]

[0106] (3) Pathological examination

[0107] The fixed colon tissues were embedded in paraffin and sectioned, stained with hematoxylin and eosin (H&E), and the stained slides were observed under an optical microscope and the thickness of the colon mucosa was counted.

[0108] (4) Genetic testing

[0109] Total RNA from colon samples was extracted using the RNAiso Plus kit (TaKaRa, Japan), and the PrimeScript TM cDNA was synthesized using Ⅱ1st Strand cDNA Synthesis Kit (TaKaRa) according to its instructions. The FastFire fluorescent quantitative PCR kit was used to detect the transcription levels of water channel proteins AQP4 and AQP8. PCR amplification was performed using the following conditions: initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, 55°C for 15 s, and 72°C for 20 s. β-actin was used as the internal reference gene, and 2 -ΔΔCT The relative gene transcription level was calculated by the formula. The untreated NC group was used as the control.

[0110] The test results of whole intestinal transit time, small intestinal propulsion rate and black stool quantity (5 hours) are as follows: Figure 3 As shown in Figures A, B, and C; Figure 3Figures A, B, and C in the figure show that compared with the NC group, the whole intestinal transit time of the Lop group increased, the number of black stool particles decreased, and the small intestinal propulsion rate decreased, indicating that the chronic FC mouse model was successfully established; compared with the Lop group, the whole intestinal transit time of the Lac group and the YG2604 group decreased, the number of black stool particles increased, and the small intestinal propulsion rate increased, indicating that the inactivated strain YG2604 has an improvement effect on chronic functional constipation. Compared with the YG2604 group, the whole intestinal transit time of the YG2638 group increased, the number of black stool particles decreased, and the small intestinal propulsion rate decreased, and the three indicators between the YG2638 group and the Lop group did not show significant differences (p ≥ 0.05). The results show that the YG2638 group failed to effectively relieve the mouse constipation model, which suggests that the ability of Akkermansia muciniphila to relieve constipation has strain differences.

[0111] The HE staining results of colon tissue and the statistical results of colon mucosal thickness are shown in Figure 2. Figure 4 As shown in Figures A and B in Figure 1, the transcriptional level detection results of water channel proteins AQP4 and AQP8 are as follows: Figure 4 As shown in Figures C and D.

[0112] Depend on Figure 4 As can be seen from Figures A and B, the thickness of the colon mucosal layer of the Lop group mice was reduced, inflammatory infiltration and the number of goblet cells decreased. Under the intervention of Akkermansia muciniphila YG2604, the thickness of the colon mucosal layer of chronic FC mice increased by 17.50% and was accompanied by reduced inflammatory infiltration and improved degree of colon tissue damage. That is, histopathological examination further confirmed the effectiveness of the inactivated strain YG2604 in improving chronic constipation.

[0113] Depend on Figure 4 As shown in Figures C and D, at the gene level, the inactivated strain YG2604 can significantly inhibit the expression of water channel proteins AQP4 and AQP8 in chronic FC mice. This result suggests that the inactivated strain YG2604 may improve the symptoms of chronic functional constipation by reducing the reabsorption of water in the colon.

[0114] In addition, we also used 16SrRNA gene sequencing technology to explore the effect of inactivated strain YG2604 on the intestinal flora of constipation. Figure 5 shown. Figure 5 Figures A and B in the figure are the PCoA and Venn diagram analysis results of the samples, respectively, which show that the NC, Lop and YG2604 groups showed different clustering patterns on the PCoA diagram, indicating that there were significant differences among the three groups; moreover, YG2604 significantly changed the β diversity of the intestinal flora and was closer to the Con group. Figure 5 Figure C is a phylum-level bacterial community analysis, showing the relative abundance of bacteria in different phyla. Figure 5 Figure D in the figure is a genus-level bacterial community difference analysis, showing the relative abundance of differentially expressed bacteria; Figure 5 As shown in Figures C and D, at the phylum level, compared with the Lop group, the intervention of the inactivated strain YG2604 increased the relative abundance of Firmicutes, Actinobacteria, and Bacteroidetes, while reducing the relative abundance of Proteobacteria ( Figure 5 At the genus level, the inactivated strain YG2604 increased the abundance of beneficial bacteria such as Lactobacills, Streptococcus and Anaerofustis, and reduced the abundance of harmful bacteria such as Corynebacterium, Hungatella and Aestuariispira ( Figure 5 The above results indicate that the inactivated strain YG2604 may improve constipation symptoms by regulating intestinal flora (including increasing the flora that is beneficial to relieve constipation and inhibiting harmful flora).

[0115] In addition, we also explored the effect of the 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.

[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 results clearly show the clustering trend of the Con group, LOP group, and YG2604 group (see Figure 6 Figure A in Figure 2 shows that the intervention of inactivated strain YG2604 significantly affected the fecal metabolome of Lop-induced chronic FC mice. By comparison, we found that compared with the NC group, 163 metabolites in the Lop group were increased and 273 metabolites were decreased; while compared with the Lop group, 198 metabolites in the YG2604 group were increased and 194 metabolites were decreased (see Figure 6 In order to further analyze the mechanism of metabolic changes in samples, we conducted metabolic pathway enrichment analysis based on these differential metabolites. The results showed that 20 metabolic pathways were significantly enriched in the YG2604 group compared with the Lop group, among which the enrichment of primary bile acid synthesis, bile secretion and smooth muscle contraction was particularly significant (see Figure 6 In addition, we screened out 18 significantly differentially expressed metabolites from these three significantly enriched metabolic pathways (P<0.05, VIP>1, see Figure 6It is noteworthy that, compared with the Lop group, the levels of metabolites such as deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2, and prostaglandin i2 in the YG2604 group were significantly increased, and the levels of metabolites such as pravastatin, taurochenodeoxycholic acid, cerivastatin, and glycocholic acid in the YG2604 group were decreased (see Figure 5). Figure 6 By analyzing the association between changes in intestinal metabolite composition and the abundance of microbial OTUs, we found that lithocholic acid, deoxycholic acid, valproic acid, and prostaglandin I2 were positively correlated with Lactobacillus, Streptococcus, and Anaerofustis at the genus level, but negatively correlated with Corynebacterium, Hungatella, and Aestuariispira (see Figure 6 In order to explore the potential mechanism by which the inactivated strain YG2604 relieved constipation in mice, we performed Pearson correlation analysis. Figure 6 As shown in Figure G, the results showed that deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2 and prostaglandin I2 were positively correlated with Lactobacillus, Streptococcus and Anaerofustis, and were 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 speculate that deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2 and prostaglandin I2 may be the key metabolites of the inactivated strain YG2604 to 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Akkermansia muciniphila YG2604, characterized in that: The Akkermansia muciniphila YG2604 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of October 16, 2024 and a deposit number of CGMCC No.46149.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes a live strain or an inactivated strain of Akkermansia muciniphila YG2604 as claimed in claim 1.

3. A composition, characterized in that The composition comprises an effective amount of the live strain or inactivated strain of Akkermansia muciniphila YG2604 according to claim 1 or the bacterial agent according to claim 2 as the first active component.

4. The composition according to claim 3, characterized in that The composition is a pharmaceutical composition, a food composition or a feed composition.

5. The composition according to claim 3 or 4, characterized in that The composition further comprises a second active ingredient; Optionally, the second active component comprises a probiotic, a postbiotic, a prebiotic, an antimicrobial agent, an immunomodulator, an anticancer agent, an inflammatory therapeutic agent, or a combination thereof.

6. Use of Akkermansia muciniphila YG2604 according to claim 1, the bacterial agent according to claim 2 or the composition according to any one of claims 3 to 5 in the preparation of a product, preferably a pharmaceutical product, for the following purposes: (1) Regulate intestinal flora; (2) regulating bile acid and / or prostaglandin metabolism; (3) Prevent, improve and / or treat constipation.

7. The use according to claim 6, characterized in that: The regulating of intestinal flora includes: increasing flora that is beneficial to relieving constipation, and / or inhibiting harmful flora.

8. The use according to claim 7, characterized in that: The bacteria that are beneficial for relieving constipation include: Lactobacills, Streptococcus and Anaerofustis; And / or, the harmful bacteria include: Corynebacterium, Hungatella and Aestuariispira.

9. The use according to claim 6, characterized in that: The regulation of bile acid and / or prostaglandin metabolism comprises: 1) Regulate the synthesis of bile acids; 2) Regulate the conversion of bile acids; 3) Optimization of metabolite combination; preferably, the optimization of the metabolite combination includes: increasing the content of deoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, prostaglandin b2 and / or prostaglandin I2.

10. The use according to claim 6, characterized in that: The mechanisms for preventing, improving and / or treating constipation include: (1) Regulate intestinal flora; (2) regulating bile acid and / or prostaglandin metabolism; (3) Promote intestinal motility; (4) Reduce the colon's reabsorption of water.

Images