Breast milk-derived bifidobacterium longum YG4059 and application thereof in improving constipation

Abstract

The invention relates to the field of microorganisms, in particular to Bifidobacterium longum YG4059, a microbial agent containing the Bifidobacterium longum YG4059, a composition containing the Bifidobacterium longum YG4059 and application of the Bifidobacterium longum YG4059 and the microbial agent containing the Bifidobacterium longum YG4059 and the composition containing the Bifidobacterium longum YG4059 in preparing products for preventing and / or treating constipation. The Bifidobacterium longum YG4059 has excellent araban metabolism capacity and gastrointestinal tolerance, intestinal flora and bile acid metabolism can be beneficially regulated, constipation and other intestinal health problems are improved and solved by precisely regulating an intestinal-metabolic axis, constipation can be prevented or treated, intestinal microecological balance can be maintained, and the intestinal health can be improved. And the method has obvious application value and market potential.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Bifidobacterium longum YG4059, a bacterial agent and a composition containing the same, and applications of the same in preparing products for preventing and / or treating constipation. Background Art

[0002] Constipation is a common digestive system disease, and there are various ways to prevent and treat it.

[0003] Drug therapy is a common method for treating constipation. Currently, the commonly used drugs on the market mainly include laxatives, stimulant laxatives and osmotic laxatives. These drugs can quickly relieve constipation symptoms, but it should be noted that long-term use may lead to intestinal dependence and even cause side effects such as electrolyte imbalance. Therefore, when using these drugs, patients should use them rationally under the guidance of a doctor to avoid abuse.

[0004] Improving lifestyle habits is also an important way to prevent and treat constipation. Patients are advised to increase their intake of dietary fiber, such as eating more fruits, vegetables and whole grains; at the same time, ensure adequate water intake to promote intestinal peristalsis. In addition, regular exercise can also help improve constipation. It should be noted that these lifestyle adjustments require long-term adherence by patients, and the effects vary from person to person, with large individual differences.

[0005] For patients with severe constipation who are ineffective with drug treatment, surgery becomes the last option. Surgical treatment includes partial colon resection, rectocele repair and other surgical methods. However, surgical treatment has high risks, a long recovery period after surgery, and possible complications. Therefore, when choosing surgical treatment, patients should fully understand the risks of surgery and make decisions under the evaluation of professional doctors.

[0006] In addition to the above methods, some emerging technologies are being studied and applied, such as biofeedback therapy and intestinal microecological regulation. These methods aim to improve intestinal function through non-drug treatments and provide more treatment options for patients with constipation. Summary of the invention

[0007] Purpose of the Invention

[0008] The present invention aims to provide a strain of human milk-derived Bifidobacterium longum YG4059 having the efficacy of preventing and treating constipation, a bacterial agent and a composition containing the same, and their use in preparing a product for preventing and / or treating constipation.

[0009] Solution

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] In the first aspect, the present invention provides a strain of Bifidobacterium longum YG4059, which is classified and named Bifidobacterium longum 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 July 9, 2024, and the deposit number is CGMCC No.31242.

[0012] The Bifidobacterium longum YG4059 of the present invention is isolated from a healthy breast milk sample and is identified as Bifidobacterium longum through 16S rRNA sequencing.

[0013] In some embodiments, the 16SrRNA gene sequence of Bifidobacterium longum YG4059 is shown in SEQ ID NO:1.

[0014] The colony characteristics of the strain include: after culturing in mMRS agar medium for 24 to 48 hours, the diameter is between 0.1 mm and 1.0 mm, and the colony edge is smooth and milky white.

[0015] The inventors have discovered and experimentally confirmed that the Bifidobacterium longum YG4059 (also referred to as YG4059) of the present invention has superior arabinan metabolism function and gastrointestinal tolerance, and has unique biocompatibility and safety. In addition, it can regulate intestinal flora and bile acid metabolism, and can be used to prevent and treat constipation.

[0016] In a second aspect, the present invention provides a bacterial agent, wherein the active ingredient of the bacterial agent includes Bifidobacterium longum YG4059 as described in the first aspect above.

[0017] In a third aspect, the present invention provides a composition comprising an effective amount of Bifidobacterium longum YG4059 as described in the first aspect or the bacterial agent as described in the second aspect, as a first active component.

[0018] In a feasible embodiment, the composition is a pharmaceutical composition, a food composition or a feed composition.

[0019] 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.

[0020] In a feasible embodiment, the composition further comprises a second active ingredient;

[0021] 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.

[0022] In a fourth aspect, the present invention provides the use of Bifidobacterium longum YG4059 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:

[0023] (1) Regulate intestinal flora;

[0024] (2) Regulate bile acid metabolism;

[0025] (3) Prevent and / or treat constipation.

[0026] Preferably, the regulating intestinal flora includes: increasing flora that is beneficial to relieving constipation, and / or inhibiting harmful flora;

[0027] Preferably, the bacterial flora beneficial for relieving constipation includes: Waltera and Bifidobacterium;

[0028] Preferably, the harmful bacteria include: Aestuariispira, Luxibacter and Enterocioster.

[0029] Preferably, the regulating bile acid metabolism comprises:

[0030] 1) Synthesis of bile acids;

[0031] 2) Conversion of bile acids;

[0032] 3) Optimization of metabolite combination; preferably, the optimization of the metabolite combination includes: increasing the content of lithocholic acid, deoxycholic acid, valproic acid and prostaglandin I2, and / or up-regulating the expression of aquaporin.

[0033] Possibly, the prevention and / or treatment of constipation is achieved by a mechanism selected from:

[0034] (1) Regulate intestinal flora;

[0035] (2) Regulate bile acid metabolism;

[0036] (3) Reduce colonic water reabsorption;

[0037] (4) Increase the content of short-chain fatty acids in the colon.

[0038] Specifically, the Bifidobacterium longum YG4059 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 lithocholic acid, deoxycholic acid, valproic acid and prostaglandin I2), thereby improving constipation; and Bifidobacterium longum YG4059 can target and regulate to increase the bacterial flora combination (Waltera and Bifidobacterium) that is beneficial to relieving constipation, and inhibit harmful bacteria (such as Aestuariispira, Luxibacter and Enterocioster, etc.). This precise regulation ensures the balance of the intestinal microecological environment and has a significant effect on improving constipation.

[0039] 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.

[0040] In a feasible embodiment, the treatment of constipation includes alleviating, relieving, and improving the symptoms of constipation.

[0041] Furthermore, in a possible embodiment, the product is a pharmaceutical product.

[0042] Further practicably, the drug is formulated for nasal, oral or intragastric administration.

[0043] 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.

[0044] 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 Bifidobacterium longum YG4059 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.

[0045] 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.

[0046] In a feasible embodiment, the treatment of constipation includes alleviating, relieving, and improving the symptoms of constipation.

[0047] 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.

[0048] Beneficial Effects

[0049] The Bifidobacterium longum YG4059 of the present invention is a unique strain isolated from breast milk. Compared with the reported strains of the same species, it has the following advantages:

[0050] 1) Superior ability to metabolize arabinan and gastrointestinal tolerance;

[0051] 2) It can beneficially regulate intestinal flora and bile acid metabolism, and improve and solve intestinal health problems such as constipation by accurately regulating the intestinal-metabolic axis. It can not only prevent or treat constipation, but also maintain the balance of intestinal microecology;

[0052] 3) Its unique biocompatibility and safety provide a more natural and gentler treatment option for patients with constipation, especially for patients who are concerned about traditional drug treatments;

[0053] In general, the Bifidobacterium longum YG4059 of the present invention, as an innovative biological treatment, demonstrates its significant clinical value and market potential in treating constipation. 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 1 The results of in vitro phenotype and tolerance screening of Bifidobacterium longum are shown; among them, Figure 1 A shows the OD600nm of each strain tested after growing in a medium with arabinan as the sole carbon source for 48 h. Figure 1 B shows the ratio of the survival rate of each strain to the survival rate of the reference strain BB536 after being treated in simulated gastric fluid for 3 hours. Figure 1 C shows the ratio of the survival rate of each test strain to the survival rate of the reference strain BB536 after 4 hours of treatment in simulated intestinal fluid; data represent SD±mean value (n=3), and statistical analysis was performed using one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.

[0056] Figure 2 The effects of 8 candidate strains on defecation parameters and gastrointestinal transit function in acute FC model mice were shown; among them, Figure 2 A, 2B, and 2C show the test results of whole intestinal transit time, small intestinal propulsion rate, and black stool quantity (5 hours), respectively; data represent SD±mean value (n=6), and statistical analysis was performed using one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0057] Figure 3 The Circos genome diagram of Bifidobacterium longum YG4059 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 GC content.

[0058] Figure 4 The results of the hemolytic activity test of Bifidobacterium longum YG4059 are shown.

[0059] Figure 5 The effects of Bifidobacterium longum YG4059 on defecation parameters and gastrointestinal transit function in chronic FC model mice were shown; Figure 5A, 5B, and 5C show the test results of whole intestinal transit time, small intestinal propulsion rate, and black stool quantity (5 hours), respectively; data represent SD±mean value (n=6), and one-way ANOVA was used for statistical analysis, *p<0.05, **p<0.01, ***p<0.001, #p<0.05, ##p<0.01, ###p<0.001.

[0060] Figure 6 The effect of Bifidobacterium longum YG4059 on the mRNA expression levels of colon tissue and related intestinal factors in chronic FC model mice was shown; Figure 6 A is the result of HE staining to detect pathological damage in the mouse colon, 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 6 B is the normalized colon mucosal thickness; Figure 6 C is the detection result of mRNA level of aquaporin AQP4 and AQP8; Figure 6 D is the detection result of mRNA levels of SCFAs receptors GPR41 and GPR43; data represent SD±mean value (n=6), and statistical analysis was performed using one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.

[0061] Figure 7 The regulatory effect of Bifidobacterium longum YG4059 on intestinal flora was shown; among them, Figure 7 A is the PCoA analysis of the samples, showing the similarities and differences between different samples; Figure 7 B is a Venn diagram reflecting the number of common and unique species in different samples; Figure 7 C shows the bacterial community analysis at the phylum level, showing the relative abundance of bacteria in different phyla. Figure 7 D is the bacterial community difference analysis at the genus level, showing the relative abundance of differential bacteria.

[0062] Figure 8 It showed that Bifidobacterium longum YG4059 improved constipation in mice by regulating metabolism and intestinal flora; Figure 8 A shows the β-diversity analysis of the metabolic profile of colonic contents; Figure 8 B shows the volcano plot of differential metabolites generated by the comparison between Lop group and Con group; Figure 8 C shows the volcano plot of differential metabolites generated by the comparison of YG4059 and Lop groups; Figure 8 D shows the comparison results of KEGG enrichment analysis between YG4059 and Lop groups, identifying the affected metabolic pathways; Figure 8 E shows the differential metabolite species between YG4059 and Lop groups; Figure 8F shows the heat map correlation analysis of typical differential metabolites and intestinal flora; Figure 8 G shows the network diagram analysis of metabolites, constipation markers and intestinal flora.

[0063] Fig. 9 It was shown that Bifidobacterium longum YG4059 could upregulate valproic acid ( Fig. 9 A), prostaglandin I2 ( Fig. 9 B) Lithocholic acid ( Fig. 9 C) and deoxycholic acid ( Fig. 9 D) expression or production.

[0064] The Bifidobacterium longum YG4059 of the present invention is classified and named as Bifidobacterium longum, and is deposited in the General Microbiology Center of the China Culture Collection Administration Committee. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 9, 2024, and the deposit number is CGMCC No.31242. DETAILED DESCRIPTION

[0065] 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.

[0066] 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.

[0067] In the following examples, unless otherwise specified, other reagents or raw materials used are commercially available.

[0068] Example 1: Isolation and identification of Bifidobacterium longum

[0069] (1) Isolation and preliminary screening of strains

[0070] Breast milk was collected from healthy mothers in China. 1 mL of breast milk sample was placed in 9 mL of PBS buffer, vortexed and mixed, and then diluted tenfold with sterile saline. -1 , 10 -2 The diluted liquids were spread on MRS+0.1% cysteine ​​hydrochloride (hereinafter referred to as "mMRS" medium) and 0.5% mupirocin salt agar medium, respectively. Two plates were spread for each dilution. The solid plates were cultured in an anaerobic incubator at 37°C for 48 to 72 hours. The colonies were picked and purified on mMRS agar medium for more than 3 times until the colony morphology on the plates was consistent.

[0071] After rapid identification of the purified colonies by mass spectrometry MALDI-TOF, the strains preliminarily identified as Bifidobacterium longum were selected for subsequent analysis.

[0072] (2) Colony morphology characteristics

[0073] After culturing in mMRS agar medium for 24 to 48 hours, the diameter of the preliminarily screened long bifidobacterium was between 0.1 and 1.0 mm, and the colony edge was smooth and milky white.

[0074] Example 2: In vitro screening of strains with the potential to improve constipation

[0075] (1) Strain preparation & reagent preparation

[0076] a. Recovery of strains: Take out the original strain from -80°C, shake and melt it in a 37°C water bath, then inoculate 5% of the strain into 5 mL of mMRS medium and culture it anaerobically at 37°C for 15-18 hours.

[0077] b. Re-check of strains: When the bacterial solution becomes turbid (OD value is about 0.4-0.8), use an inoculation loop to dip into the bacterial solution, draw three lines on mMRS solid culture medium, culture at 37℃ under anaerobic conditions for 48-72h, and pick a single colony for mass spectrometry identification.

[0078] c. Simulated gastric fluid: Dissolve 3 g of pepsin in 1000 mL of sterile saline (0.9% w / v) to a final enzyme concentration of 3 g / L. Adjust the pH to 3 with hydrochloric acid. Filter the pepsin solution with a 0.22 μm sterile filter membrane and prepare it for use immediately.

[0079] d. Simulated intestinal fluid: Dissolve 1 g of trypsin in 1000 mL of sterile saline (0.9% w / v) to a final enzyme concentration of 1 g / L. Adjust the pH to 8 with NaOH solution (add 1.1 μL of 2M NaOH), filter and sterilize with a 0.22 μm sterile filter membrane, and use immediately after preparation.

[0080] (2) In vitro screening of strains with the potential to improve constipation

[0081] a. Phenotype of abfA gene cluster and detection of its arabinan metabolism function

[0082] Generally speaking, Bifidobacterium longum with α-L-arabinofuranosidase (abfA) gene cluster can relieve functional constipation of the host by improving the utilization of arabinan by intestinal flora. Therefore, in this embodiment, by designing two sets of specific primers for the abfA gene cluster and performing specific amplification, 32 strains carrying the abfA gene cluster were screened out from 108 strains of breast milk-derived Bifidobacterium longum strains, and the function of metabolizing arabinan was further tested.

[0083] Method for detecting the metabolic function of arabinan: Arabinan or glucose was added as the sole carbon source at a ratio of 1% (wt / vol, g / mL) to a carbon source-free MRS medium (10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate [(NH4)2HC6H5O7], 1.0 mL of Tween-80, 5.0 g of sodium acetate (CH3COONa·3H2O), 2.0 g of dipotassium hydrogen phosphate (K2HPO4·3H2O), 0.50 g of magnesium sulfate (MgSO4·7H2O), 0.25 g of manganese sulfate (MnSO4·H2O), and 1000 mL of distilled water). The abfA gene-positive strain and the reference strain BB536 were inoculated into each culture medium, respectively, with an initial OD600 of 0.1. After anaerobic culture at 37°C for 48 h, the OD600 absorbance of the strain culture solution was detected.

[0084] b. Simulated gastrointestinal fluid tolerance test

[0085] The strain was inoculated for three generations for activation, then centrifuged and washed twice (5000 rpm, 5 min), the bacteria were collected, and an equal amount of pH 3.0 simulated gastric fluid to the culture medium was added, and cultured at 37°C in an anaerobic environment for 2 h. The number of viable bacteria was determined by the mMRS agar medium pouring method at 0 h and 2 h of anaerobic culture, and after culture at 37°C for 48 h.

[0086] The bacterial solution treated in simulated gastric fluid for 2 h was washed twice by centrifugation (5000 rpm, 5 min), the bacteria were collected, and the same amount of simulated intestinal fluid as the previous simulated gastric fluid was added to continue anaerobic culture at 37°C. The number of viable bacteria was determined by the mMRS agar medium pouring method at 0 h and 4 h of anaerobic culture. Three replicates were set for each strain, and the number of viable bacteria was determined after culturing at 37°C for 48 h.

[0087] Taking the survival rate of the reference strain BB536 as a reference, the results of the tolerance test in simulated gastric or intestinal fluid are expressed as the ratio of the survival rate of the strain to be tested to the survival rate of BB536, that is, the results are expressed as:

[0088] n=[N1-test strain / N0-test strain] / [N1-BB536 / N0-BB536]

[0089] Among them, N0 represents the number of live bacteria at 0h; N1 represents the number of live bacteria after 2h of digestion in simulated gastric juice or 4h of digestion in simulated intestinal juice.

[0090] The test results of the ability to metabolize arabinose are as follows Figure 1 As shown in A; Figure 1 A shows that in the culture medium with glucose as the only carbon source, 11 strains of Bifidobacterium longum (numbered YG4050, YG4059, YG4040, YG4035, YG4136, YG4234, YG4240, YG4245, YG4227, YG3609 and YG4058) can grow effectively (i.e., OD600 (glucose) ≥ 0.8); after these strains were grown for 48 hours under the condition of arabinose as the only carbon source, the OD600 values ​​were significantly higher than the OD600 values ​​of the reference strain BB536 under the same culture conditions, indicating that these 11 strains of Bifidobacterium longum can efficiently utilize arabinose and perform better than the reference strain BB536.

[0091] The results of tolerance test in artificial simulated gastric fluid are as follows Figure 1 As shown in B; Figure 1 B shows that after 2 hours in simulated gastric fluid, the survival rates of 4 strains of Bifidobacterium longum (numbered YG4059, YG4035, YG4136 and YG4058) were higher than those of the reference strain BB536, and the survival rates of another 4 strains (numbered YG4040, YG4234, YG4240 and YG4227) were also high, reaching 80% of the survival rate of the reference strain BB536. In addition, the results of the tolerance test in artificial simulated intestinal fluid are as follows Figure 1 As shown in C; Figure 1 C shows that the survival rates of the above 8 strains after 4 hours in artificial simulated intestinal fluid are higher than the reference strain BB536 or reach 80% of the survival rate of BB536. Therefore, these 8 strains show excellent gastrointestinal tolerance.

[0092] Therefore, through in vitro screening of Bifidobacterium longum carrying the abfA gene cluster, capable of efficiently utilizing arabinose and having good tolerance to artificial simulated gastrointestinal fluid, we obtained a total of 8 candidate strains, numbered YG4058, YG4059, YG4040, YG4035, YG4136, YG4234, YG4240 and YG4227.

[0093] Next, we will test the efficacy of these strains in relieving constipation in animal models.

[0094] Example 3: Screening of Bifidobacterium longum with the effect of improving constipation

[0095] In this example, we evaluated the ability of the above 8 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:

[0096] Animal experiments

[0097] Eight-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 one week, and then randomly divided into 10 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 8 experimental groups (YG4058 group, YG4059 group, YG4040 group, YG4227 group, YG4035 group, YG4136 group, YG4234 group and YG4240 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 The mice in the NC and Lop groups were fed with standard feed and sterilized water. The whole intestinal transit time (time of discharge of the first black stool) and the number of black stools were observed on the 8th day. The mice were killed on the 9th day to observe the small intestinal propulsion rate (ink propulsion rate).

[0098] Index determination

[0099] (1) Determination of the time of first black stool and the number of black stools

[0100] 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 9Each mouse was transferred to a clean empty cage and provided with feed and water. The time was counted from the gavage of ink, and the time of the first black stool of each mouse and the number of black stools within 5 hours were recorded.

[0101] (2) Determination of small intestinal propulsion rate

[0102] 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 9 5% ink of CFU of Bifidobacterium longum (YG4058 group, YG4059 group, YG4040 group, YG4227 group, YG4035 group, YG4136 group, YG4234 group and YG4240 group). The mice were killed after 17 minutes of free feeding and drinking, 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, 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.

[0103] The formula for calculating the small intestinal propulsion rate is as follows:

[0104]

[0105] The test results of whole intestinal transit time, small intestinal propulsion rate and black stool quantity (5 hours) are as follows: Figure 2 As shown in AC; Figure 2 AC showed that compared with the NC group, the whole intestinal transit time of the Lop group was increased, the number of black stool particles and the small intestinal propulsion rate were reduced, indicating that the acute FC mouse model was established.

[0106] For whole-bowel transit time indicators, such as Figure 2 As shown in A, compared with the Lop group, the whole intestinal transit time was reduced in the YG4059 group, the YG4234 group and the YG4240 group (34.33%, 36.48% and 23.87%).

[0107] For small intestinal propulsion rate indicators, such as Figure 2 As shown in B, compared with the Lop group, only the YG4059 group increased the ink propulsion rate by 10.97%, improving the peristaltic ability of the small intestine.

[0108] For the black stool quantity indicator, such as Figure 2As shown in C, compared with the Lop group, the YG4059 group, the YG4234 group, the YG4240 group and the YG4058 group all increased the number of black stool particles excreted by mice within 5 hours (100.00%, 116.29%, 83.70% and 107.41%).

[0109] Taking into account multiple indicators such as gastrointestinal tolerance and mouse phenotype, strain YG4059 was selected from 8 candidate strains. It has excellent gastrointestinal tolerance and the strongest effect in improving constipation symptoms, and therefore has great potential for preventing and treating acute constipation.

[0110] Example 4: Genome determination and safety evaluation of strain YG4059

[0111] 16SrRNA & Whole Genome Sequencing

[0112] Strain YG4059 was sent to Shanghai Biotech 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 Bifidobacterum longum subsp. longum.

[0113] Furthermore, the whole genome of Bifidobacterium longum YG4059 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 Bifidobacterium longum YG4059 was plasmid-free, with a single circular chromosome of 2,305,776 bp and a G+C content of 59.97%; the genome annotation showed that it contained a total of 1848 coding sequences; in addition, the Bifidobacterium longum YG405 genome contained 59 tRNAs, 2 rRNAs and 1844 CDSs.

[0114] Based on the results of whole genome sequencing, the safety of Bifidobacterium longum YG4059 at the genomic level was also evaluated. The results showed that the genome of Bifidobacterium longum YG4059 did not contain genes related to transferable antibiotic resistance and virulence genes.

[0115] Antibiotic susceptibility testing

[0116] The sensitivity of Bifidobacterium longum YG4059 to 9 antibiotics was determined by using the drug sensitivity paper diffusion method (KB method); specifically, the test bacterial solution was spread on the MRS 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.

[0117] Table 1. Results of antibiotic sensitivity tests

[0118]

[0119] Table 1 shows that Bifidobacterium longum YG4059 was sensitive to antibiotics such as tetracycline, chloramphenicol, clindamycin, metronidazole, and ampicillin-sulbactam, and was resistant to some antibiotics such as imipenem, cefotetan, moxifloxacin, and ampicillin.

[0120] Hemolytic test

[0121] Next, we tested the hemolytic activity of Bifidobacterium longum YG4059. In brief, with Staphylococcus aureus ATCC29213 as the positive control and Listeria innocua ATCC 33090 as the negative control, Bifidobacterium longum YG4059 was cultured on a blood plate at 37°C for 48 hours to observe whether there was a hemolytic zone. If the agar around the colony appears green, it is α-hemolytic; if a transparent zone appears around the colony, it is β-hemolytic; otherwise, it is γ-hemolytic, that is, no hemolysis.

[0122] The results are as follows Figure 4 As shown; Figure 4 The results showed that an obvious hemolysis zone appeared around Staphylococcus aureus ATCC 29213, while no obvious hemolysis zone appeared around Listeria monocytogenes ATCC 33090 and Bifidobacterium longum YG4059. This result indicated that Bifidobacterium longum YG4059 was not hemolytic.

[0123] Example 5: Study on the efficacy and mechanism of strain YG4059 in improving chronic constipation and intestinal epithelial barrier damage

[0124] Animal experiments

[0125] 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), Bifidobacterium longum YG4059 group and YG4040 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, YG4059 group and YG4040 group according to the 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 YG4059 group and the YG4040 group were given 1×10 9 The Lac group was treated with lactulose (2.6 g / kg*bw), while the NC and Lop groups 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 stool discharge 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 portion of the collected colon tissue was fixed with 4% paraformaldehyde and stained with HE, and the other was stored at -80°C for subsequent detection of colon factors. Mouse feces and cecal contents were used to detect the abundance and metabolome of mouse intestinal flora, respectively.

[0126] Index determination

[0127] (1) Determination of the time of first black stool and the number of black stools

[0128] 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, YG4059, and YG4040 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 YG4059 and YG4040 groups were gavaged with 1×10 9 Afterwards, each mouse was transferred to a clean empty cage and provided with feed and water. The timing was started from the gavage of ink, and the time of the first black stool of each mouse and the number of black stools excreted within 5 hours were recorded.

[0129] (2) Determination of small intestinal propulsion rate

[0130] 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 YG4059 and YG4040 groups were gavaged with 1×10 9 The mice were anesthetized and killed after free feeding for 17 min with 5% ink containing CFU of Bifidobacterium longum YG4059 and YG4040. 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.

[0131] The formula for calculating small intestinal propulsion rate is:

[0132]

[0133] (3) Pathological examination

[0134] 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.

[0135] (4) Genetic testing

[0136] 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) and short-chain fatty acid receptors (GPR41 and GPR43). PCR amplification was performed under 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-action 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.

[0137] The test results of whole intestinal transit time, small intestinal propulsion rate and black stool quantity (5 hours) are as follows: Figure 5 As shown in AC; Figure 5AC showed 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 YG4059 group decreased, the number of black stool particles increased, and the small intestinal propulsion rate increased, indicating that the strain YG4059 has an improvement effect on chronic functional constipation. In addition, compared with the YG4059 group, the whole intestinal transit time of the YG4040 group increased, the number of black stool particles decreased, and the small intestinal propulsion rate decreased, and the three indicators between the YG4040 group and the Lop group did not show significant differences (p ≥ 0.05). This result shows that the YG4040 group failed to effectively relieve the mouse constipation model, which suggests that the long bifidobacterium has strain differences in its ability to relieve constipation.

[0138] The HE staining results of colon tissue and the statistical results of colon mucosal thickness are shown in Figure 2. Figure 6 As shown in A and 6B, the results of the transcriptional level detection of aquaporins (AQP4 and AQP8) and short-chain fatty acid receptors (GPR41 and GPR43) are shown in Figure 6 C and 6D.

[0139] Depend on Figure 6 As can be seen from A and 6B, the thickness of the colon mucosal layer of the Lop group mice was reduced, and inflammatory infiltration and the number of goblet cells decreased. Under the intervention of Bifidobacterium longum YG4059, the thickness of the colon mucosal layer of chronic FC mice increased by 24.16%, accompanied by reduced inflammatory infiltration and improved degree of colon tissue damage. That is, histopathological examination further confirmed the effectiveness of strain YG4059 in improving chronic constipation.

[0140] Depend on Figure 6 C and 6D, it can be seen that at the gene level, strain YG4059 can significantly inhibit the expression of water channel proteins AQP4 and AQP8 in chronic FC mice ( Figure 6 C), but significantly promoted the expression of short-chain fatty acid receptors GPR41 and GPR43 ( Figure 6 D). These results suggest that strain YG4059 may improve the symptoms of chronic functional constipation by reducing water reabsorption in the colon and increasing the content of short-chain fatty acids in the colon.

[0141] In addition, we also used 16SrRNA gene sequencing technology to explore the effect of YG4059 on intestinal flora in constipation. Figure 7 As shown. Among them, Figure 7A and 7B are the PCoA and Venn diagram analysis results of the samples, respectively, which show that the NC, Lop and YG4059 groups showed different clustering patterns on the PCoA diagram, indicating that there were significant differences among the three groups; moreover, YG4059 significantly changed the β diversity of the intestinal flora and was closer to the Con group. Figure 7 C is the bacterial community analysis at the phylum level, showing the relative abundance of bacteria in different phyla. Figure 7 D is the bacterial community difference analysis at the genus level, showing the relative abundance of differential bacteria; Figure 7 C and 7D, at the phylum level, compared with the Lop group, YG4059 intervention increased the relative abundance of Firmicutes, Actinobacteria, and Bacteroidetes, while decreasing the relative abundance of Verrucomicrobia and Proteobacteria ( Figure 7 C); at the genus level, YG4059 increased the abundance of beneficial bacteria such as Waltera and Bifidobacterium, and reduced the abundance of harmful bacteria such as Eisenbergiella, Lacrimispora, Luxibacter, Enterocloster, and Corynebacterium ( Figure 7 D). The above results indicate that strain YG4059 may improve constipation symptoms by regulating intestinal flora (including increasing the flora that is beneficial to relieving constipation and inhibiting harmful flora).

[0142] In addition, we also explored the effect of YG4059 on the fecal metabolome of the Lop-induced chronic FC mouse model. Specifically, we analyzed the differences in the fecal metabolome of different groups.

[0143] 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 YG4059 group (see Figure 8 A), indicating that YG4059 intervention had a significant effect on the fecal metabolome of Lop-induced chronic FC mice. By comparison, we found that compared with the NC group, 163 metabolites were increased and 273 metabolites were decreased in the Lop group; while compared with the Lop group, 267 metabolites were increased and 149 metabolites were decreased in the YG4059 group (see Figure 8 BC). In order to further analyze the mechanism of sample metabolic changes, we performed metabolic pathway enrichment analysis based on these differential metabolites. The results showed that the YG4059 group significantly enriched 20 metabolic pathways compared with the Lop group, among which the bile secretion, smooth muscle contraction and secondary bile acid biosynthesis pathways were particularly enriched (see Figure 8D). In addition, we screened out 13 significantly differentially expressed metabolites from these three significantly enriched metabolic pathways (P<0.05, VIP>1, see Figure 8 E). It is noteworthy that compared with the Lop group, the levels of metabolites such as lithocholic acid (P<0.01), deoxycholic acid (P<0.05), valproic acid (P<0.05) and prostaglandin I2 (P<0.05) in the YG4059 group were significantly increased, while there was no significant difference between the YG4040 group and the Lop group; and compared with the Lop group, the levels of metabolites such as glycocholic acid, taurochenodeoxycholic acid, pravastatin and cerivastatin in the YG4059 group were decreased (see Figure 8 E). 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 Waltera and Bifidobacterium at the genus level, but negatively correlated with Aestuariispira, Luxibacter, and Enterocioster (see Figure 8 F) To explore the potential mechanism of YG4059 in alleviating constipation in mice, we performed Pearson correlation analysis. Figure 8 As shown in Figure 1, the results showed that lithocholic acid, deoxycholic acid, valproic acid, and prostaglandin I2 were positively correlated with Waltera and Bifidobacterium, and negatively correlated with whole gut transit time, AQP4, and AQP8, and positively correlated with Small intestine transit time, fecal output, GPR41, and GPR43. Based on these findings, we speculate that lithocholic acid, deoxycholic acid, valproic acid, and prostaglandin I2 may be the key metabolites of YG4059 in improving constipation in mice.

[0144] It is well known that lithocholic acid, deoxycholic acid, valproic acid and prostaglandin I2 are closely related to the process of glucose and lipid metabolism. As mentioned above, Bifidobacterium longum YG4059 can upregulate the expression or production of valproic acid, prostaglandin I2, lithocholic acid and deoxycholic acid (see Fig. 9A-9D), therefore, by upregulating the above key metabolites, YG4059 may show potential in alleviating abnormal glucose and lipid metabolism (e.g., improving insulin resistance, regulating cholesterol metabolism, optimizing glucose utilization and lipid balance).

[0145] 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 Bifidobacterium longum YG4059, characterized in that: The Bifidobacterium longum is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is July 9, 2024, and the deposit number is CGMCC No.31242.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes the Bifidobacterium longum YG4059 as described in claim 1.

3. A composition, characterized in that The composition comprises an effective amount of Bifidobacterium longum YG4059 as claimed in claim 1 or the bacterial agent as claimed in 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 Bifidobacterium longum YG4059 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) Regulate bile acid metabolism; (3) Prevent 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: Waltera and Bifidobacterium; And / or, the harmful bacteria include: Aestuariispira, Luxibacter and Enterocioster.

9. The use according to claim 6, characterized in that: The regulation of bile acid metabolism includes: 1) Synthesis of bile acids; 2) Conversion of bile acids; 3) Optimization of metabolite combination; preferably, the optimization of the metabolite combination includes: increasing the content of lithocholic acid, deoxycholic acid, valproic acid and prostaglandin I2.

10. The use according to claim 6, characterized in that: The mechanisms of constipation prevention and / or treatment include: (1) Regulate intestinal flora; (2) Regulate bile acid metabolism; (3) Reduce colonic water reabsorption; (4) Increase the content of short-chain fatty acids in the colon.

Images