Probiotic composition and application thereof

Abstract

The invention relates to a probiotic composition and application thereof, and belongs to the technical field of compositions. Comprising Ackerman muciniphila ATCC BAA-835 and Bifidobacterium animalis subsp. Lactis BB-12, wherein the CFU (colony forming unit) ratio of the Ackerman muciniphila ATCC BAA-835 to the Bifidobacterium animalis subsp. Lactis BB-12 is 1 The two strains have a synergistic effect, and the prepared probiotic composition can synergistically relieve the phenomena of weight loss, colon shortening, intestinal structure and intestinal barrier injury, proinflammatory factor increase, anti-inflammatory factor reduction, intestinal flora and histidine metabolism disorder and the like of dextran sodium sulfate induced mouse ulcerative colitis. Therefore, the effect of preventing and relieving the severity of the inflammatory bowel disease is achieved.

Description

Technical Field

[0001] This invention belongs to the field of composition technology, specifically relating to a probiotic composition and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Inflammatory bowel disease (IBD), a chronic, relapsing inflammatory disease affecting the intestines, mainly includes two types: Crohn's disease and ulcerative colitis. Its exact cause is not fully understood. Patients often present with a range of symptoms such as abdominal pain, diarrhea, rectal bleeding, and weight loss. The disease course is protracted and treatment is challenging. Current treatments include traditional aminosalicylic acid drugs, glucocorticoids, and novel biologics (such as vedelizumab and anti-TNF-α drugs). While these treatments can significantly improve clinical remission rates, approximately 30% to 50% of patients still face treatment ineffectiveness or severe adverse drug reactions.

[0004] Currently, substantial research evidence indicates that gut microbiota imbalance plays a crucial role in the occurrence and development of IBD. Therefore, microbiota intervention strategies aimed at reshaping the gut microecological balance are gradually becoming an emerging research direction for alleviating and even treating IBD. Among these, *Akkermansia myxophilus* (… Akkermansia muciniphila Akkermansia myxophilus is a gut-symbiotic mucus-degrading bacterium that primarily colonizes the intestinal mucus layer of humans and animals. Multiple studies have observed significant abnormalities in its abundance in IBD patients and animal models. Furthermore, both live and pasteurized cells, as well as specific components (such as outer membrane proteins and extracellular vesicles), have been shown to act on multiple pathological aspects of IBD, including regulating abnormal immune responses, improving disordered gut microbiota structure, influencing host metabolism, and repairing damaged intestinal barrier function. How to safely, stably, and efficiently translate Akkermansia myxophilus or its active components into clinically usable therapeutic agents to maximize its potential in IBD treatment is a pressing challenge that researchers and technicians must overcome. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a probiotic composition and its application. A composition of *Bifidobacterium animalis* subsp. *lactamase* and *Akermansia myxotropicum* exhibits a synergistic effect in the prevention and relief of inflammatory bowel disease.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, a probiotic composition comprising Akkermansia myxophilus ATCC BAA-835 and Bifidobacterium animalis subsp. lactis BB-12 in a CFU ratio of 1:(0.1~10).

[0007] Optionally, the CFU ratio of the *Ackermania glutinis* ATCC BAA-835 and *Bifidobacterium animalis* subsp. *lactobacter* BB-12 is 1:1.

[0008] Optionally, the total bacterial concentration of the probiotic composition is (0.5~2)×10⁻⁶. 9 CFU / mL.

[0009] Secondly, the above-mentioned probiotic composition is used in the preparation of medicaments for the prevention or treatment of IBD.

[0010] Optionally, the specific manifestations of the prevention or treatment of IBD include: (a1) Inhibits weight loss caused by IBD; (a2) Inhibit the increase in disease activity index caused by IBD; (a3) Inhibits colonic shortening caused by IBD; (a4) Inhibits IBD-induced damage to intestinal structure and intestinal barrier; (a5) Inhibits the increase in pro-inflammatory factors and the decrease in anti-inflammatory factors caused by IBD; (a6) Inhibits gut microbiota dysbiosis caused by IBD; (a7) Synergistically regulates the histidine metabolic pathway, inhibiting IBD-induced increases in imidazole propionic acid and N-acetylhistamine, as well as decreases in aminomethylene glutamate.

[0011] Thirdly, a health food or medicine comprising the above-mentioned probiotic composition.

[0012] Optionally, the dosage form of the drug includes, but is not limited to, wettable powder, water-dispersible granules, aqueous suspension, and dispersible oil suspension.

[0013] Optionally, the pharmaceutical product includes pharmaceutically acceptable carriers and other non-toxic, compatible substances used in pharmaceutical formulations.

[0014] Optionally, the pharmaceutical product may contain a wetting agent, an emulsifier, and a lubricant.

[0015] Optionally, the drug may include colorants, release agents, coating agents, sweeteners, flavoring agents and aroma agents, preservatives and antioxidants.

[0016] Optionally, the drug is a mixture containing the probiotic composition, including but not limited to premixed addition, direct addition, and coated addition.

[0017] Optionally, the total bacterial concentration in the mixture is (0.5~2)×10⁻⁶. 9 CFU / mL.

[0018] The beneficial effects of this invention are as follows: This invention provides a probiotic composition comprising *Akermansia myxophilus* ATCC BAA-835 and *Bifidobacterium animalis* subsp. *lactamase* BB-12. Studies have shown that the two strains have a synergistic effect, and the prepared probiotic composition can synergistically alleviate the weight loss, colon shortening, increased pro-inflammatory factors, decreased anti-inflammatory factors, and intestinal flora and histidine metabolism disorders induced by sodium dextran sulfate in mice with ulcerative colitis, thereby playing a role in preventing and alleviating the severity of inflammatory bowel disease. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 This is a daily weight statistics chart of the mice in Example 4 of the present invention; Figure 2 This is a statistical chart of the disease activity index (DAI) of mice in each group in Example 4 of the present invention; Figure 3 This is a statistical chart of colon length in each group of mice in Example 4 of the present invention; Figure 4 These are histopathological images of the colon tissue of mice in each group in Example 4 of this invention; Figure 5 This is a statistical chart of the expression levels of inflammatory factors in the colon tissues of mice in each group in Example 4 of the present invention. Among them, a is the expression level of mRNA of pro-inflammatory factor il-1β, b is the expression level of mRNA of pro-inflammatory factor il-6, c is the expression level of mRNA of pro-inflammatory factor tnf-α, and d is the expression level of mRNA of anti-inflammatory factor il-10. Figure 6 This is a graph showing the results of the intestinal flora diversity index of mice in each group in Example 4 of the present invention. In this graph, a is the Chao index graph of α diversity and b is the principal coordinate analysis result graph representing β diversity. Figure 7 This is a heatmap of histidine metabolism-related products in the feces of mice in Example 4 of the present invention. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions. All raw materials and reagents used in the following examples are commercially available unless otherwise indicated.

[0024] The formulations of this invention can be conveniently available in unit dosage form and can be prepared using methods known in the pharmaceutical field. The amount of active ingredient that can be prepared in a single-dose form, typically in combination with a carrier substance, is the amount of the compound that produces the therapeutic effect.

[0025] Pharmaceutically acceptable carriers, recognized in the art, include pharmaceutically acceptable materials, compositions, or carriers suitable for administering the compounds of the present invention to mammals. These carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that participate in carrying the host substance or transferring it from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of compatibility with other components in the formulation and harmlessness to the patient. Examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate, powdered tragacanth gum, malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; and other non-toxic and compatible substances used in pharmaceutical preparations.

[0026] The composition may also contain wetting agents, emulsifiers and lubricants such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and aroma agents, preservatives and antioxidants.

[0027] The following specific implementation methods mainly involve the following reagents: Dextran sulfate sodium (DSS), also known as sodium dextran sulfate, is mainly used to induce animal colitis models.

[0028] Example 1 A probiotic composition, prepared by a method comprising: S1. The mucin-loving Ackermann strain ATCC BAA-835 was inoculated into BHI liquid medium containing 5 wt% mucin and cultured anaerobically at 37°C for 48 h until it reached the logarithmic phase. The cultured bacterial suspension was centrifuged at 4000 rpm for 5 min at 4°C, and the bacterial cells in the precipitate were collected. The bacterial cells were washed three times with sterile PBS, and the washed bacterial cells were resuspended in sterile PBS. The OD600 value of the bacterial suspension was measured using a spectrophotometer to achieve a final concentration of 1 × 10⁻⁶. 9 CFU / mL was used to obtain a suspension of Akkermansia myxophilus; S2. Inoculate *Bifidobacterium animalis* subsp. *lactamase* BB-12 into MRS liquid medium and culture anaerobicly at 37°C for 18 h until it reaches the logarithmic phase. Centrifuge the cultured bacterial suspension at 3500 rpm for 5 min at 4°C, collect the bacterial cells from the precipitate, wash the cells three times with sterile PBS, and resuspend the bacterial cells in sterile PBS. Measure the OD600 value of the bacterial suspension using a spectrophotometer to achieve a final concentration of 1 × 10⁻⁶. 9 CFU / mL was used to obtain a suspension of Bifidobacterium lactis subsp. animalis.

[0029] S3. The *Ackermania* suspension obtained in S1 and the *Bifidobacterium lactis* suspension obtained in S2 were mixed in an anaerobic environment at a 1:1 ratio, with a total bacterial concentration of 1 × 10⁻⁶. 9 The CFU / mL concentration was used to obtain the probiotic composition, which was then stored in an anaerobic tube for later use.

[0030] Example 2 A probiotic composition is prepared in a method that differs from that in Example 1 in that: in S3, the Akkermansia myxophilus suspension obtained in S1 and the Bifidobacterium lactis suspension obtained in S2 are mixed in an anaerobic environment at a ratio of 1:0.1 to obtain the probiotic composition.

[0031] Example 3 A probiotic composition is prepared in a method that differs from that in Example 1 in that: in S3, the Akkermansia myxophilus suspension obtained in S1 and the Bifidobacterium lactis suspension obtained in S2 are mixed in an anaerobic environment at a ratio of 1:10 to obtain the probiotic composition.

[0032] Example 4 This example is an experiment to investigate the effect of the probiotic composition prepared in Example 1 on clinical symptom-related indicators in mice with dextran sulfate sodium (DSS)-induced colitis.

[0033] Laboratory animals: 25 male SPF-grade C57BL / 6 mice, 7 weeks old, purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (License: SCXK (Lu) 2022 0006).

[0034] The experimental steps include: Step 1: After the purchased experimental animals have been adapted to the laboratory for one week, the mice are randomly divided into 5 groups (n=5): control group, colitis model group, first prevention group, second prevention group and third prevention group.

[0035] Step two: Process the experimental animals, including... The control group was treated by gavage with 200 μL of sterile PBS for 14 consecutive days, with free access to water. The treatment method for the colitis model group (DSS) was as follows: 200 μL of sterile PBS was administered by gavage for 14 consecutive days. Water was allowed freely for the first 7 days and water containing 2 wt% DSS was allowed freely for the next 7 days. The first prevention group (DSS_AK) was the probiotic-only intervention group. The treatment method was: for 14 consecutive days, oral administration of 200 μL of Akkermansia myxophilus suspension prepared in S1 of Example 1 (bacterial concentration of 1×10⁻⁶). 9 (CFU / mL), drink water freely for the first 7 days, and drink water containing 2wt% DSS freely for the next 7 days; The second prevention group (DSS_BB) was a probiotic-only intervention group, treated by gavage for 14 consecutive days with 200 μL of Bifidobacterium lactis suspension prepared in Example 1 (S2 concentration of 1×10⁻⁶). 9 (CFU / mL), drink water freely for the first 7 days, and drink water containing 2wt% DSS freely for the next 7 days; The third prevention group (DSS_AB) was the probiotic composition intervention group. The treatment method was: for 14 consecutive days, 200 μL of the probiotic composition prepared by S3 in Example 1 was administered by gavage. The total bacterial concentration of the two bacteria was 1 × 10⁻⁶. 9 CFU / mL, drink water freely for the first 7 days, and drink water containing 2wt% DSS freely for the next 7 days; Mice in all groups had their body weight, visible fecal consistency, and fecal bleeding recorded daily over 14 days. DAI scores were calculated using a scoring scale to obtain mouse body weight and disease activity index, thus assessing disease severity. Body weight assessment results are as follows: Figure 1As shown, the body weight of mice in the DSS-induced colitis model group (DSS) was significantly lower than that of the healthy control group (Control). The probiotic combination intervention group (i.e., the third prevention group DSS_AB) significantly alleviated the body weight loss in colitis mice, and the effect was superior to the probiotic-only intervention groups (i.e., the first prevention group DSS_AK and the second prevention group DSS_BB). The DAI score assessment results are as follows: Figure 2 As shown, the DAI score of the colitis model group was significantly higher than that of the control group, indicating that DSS successfully induced colitis symptoms. The DAI score of the probiotic composition intervention group (DSS_AB) was significantly lower than that of the DSS group and better than that of the probiotic alone intervention group (i.e., the first prevention group DSS_AK and the second prevention group DSS_BB), indicating that the probiotic composition provided by the present invention can significantly alleviate the symptoms of colitis mice.

[0036] Step 3: After a 14-day intervention period and a 2-day recovery period, all mice in all groups underwent ocular blood sampling and cervical dislocation euthanasia. The mice were then dissected and their blood, colon tissue, and feces were collected and analyzed.

[0037] The analysis includes: The method for analyzing colon length was as follows: Colons were collected from mice, and the colon length of each mouse was measured and recorded using a ruler; the results are as follows: Figure 3 As shown, the colon length in the DSS-induced colitis model group (DSS) was significantly shorter than that in the control group (Control). However, the probiotic combination intervention group (DSS_AB) significantly alleviated the effect of DSS on the colon length of mice, and the intervention effect was significantly better than that of the probiotic alone intervention group (i.e., the first prevention group DSS_AK and the second prevention group DSS_BB).

[0038] The histopathological analysis method for colon tissue was as follows: mouse colons were collected, fixed with 4% paraformaldehyde, and then stained with hematoxylin and eosin (H&E) for observation and evaluation of the histopathological condition of the mouse colon tissue; the results are as follows. Figure 4 As shown, compared with the Control group, the DSS group exhibited typical pathological features of colonic inflammation, including deformed crypt structure, reduced crypt number, epithelial layer destruction, significant inflammatory cell infiltration in the lamina propria, and a significant reduction in the number of goblet cells. This indicates that DSS successfully induced colitis in mice. The probiotic composition intervention group (DSS_AB) significantly alleviated the changes in colonic crypt structure, crypt number, epithelial layer structure, inflammatory cell infiltration, and goblet cell number caused by DSS after probiotic composition intervention, and was superior to the probiotic-only intervention groups (i.e., the first prevention group DSS_AK and the second prevention group DSS_BB). This demonstrates that the probiotic composition provided by this invention can significantly alleviate the damage to colonic structure and intestinal barrier caused by colitis.

[0039] The method for assessing the expression levels of inflammatory factors was as follows: Collected mouse colon tissue was ground in Trizol reagent, and RNA was extracted from the colon tissue; the expression levels of pro-inflammatory factors (IL-1β, IL-6, and TNF-α) and anti-inflammatory factor (IL-10) mRNA were quantified using real-time quantitative PCR; the results are as follows: Figure 5 As shown in a, the mRNA level of the pro-inflammatory factor il-1β was significantly increased in the DSS group compared with the Control group. Figure 5 As shown in b, compared with the Control group, the mRNA level of the pro-inflammatory factor il-6 was significantly increased in the DSS group, as shown in Figure b. Figure 5 As shown in c, compared with the Control group, the mRNA level of the pro-inflammatory factor TNF-α was significantly increased in the DSS group, while... Figure 5 As shown in d, the mRNA level of anti-inflammatory factor il-10 was significantly reduced; after intervention with the probiotic composition (DSS_AB), the levels of all four inflammatory factors were significantly improved, indicating that the probiotic composition provided by the present invention can effectively inhibit the overexpression of pro-inflammatory factors and enhance the secretion of anti-inflammatory factors to regulate immune balance.

[0040] The evaluation method for the effect of the probiotic composition on the intestinal flora was as follows: 16S rRNA high-throughput sequencing analysis was performed on fecal samples collected in step three of Example 4. This included: extracting fecal DNA using a fecal total DNA extraction kit; performing PCR amplification of the V3-V4 variable region of the bacterial 16S rRNA gene using specific primers; homogenizing the amplified products after purification and quantification; constructing a sequencing library; and using QIIME software to comprehensively evaluate the α-diversity and β-diversity of the samples after filtering and denoising purification of the sequences using the DADA2 algorithm. The results are shown below. Figure 6 As shown. Figure 6 From the Chao index diagram of α diversity, we can see that ( Figure 6 (a) Compared to the Control group, the DSS group showed a significant decrease in species richness, indicating that DSS-induced colitis disrupted the balance of the mouse gut microbiota. However, after intervention with the probiotic composition (DSS_AB), the Chao index significantly increased, indicating that the probiotic composition could alleviate the disruption of gut microbiota richness caused by DSS in mice. Principal coordinate analysis (PCoA) representing β-diversity showed that ( Figure 6 (b) There were significant changes in the gut microbiota composition between the DSS group and the Control group. After intervention with the probiotic composition (DSS_AB), the composition was relatively closer to the Control group and significantly different from the DSS group. This indicates that the probiotic composition provided by the present invention is more conducive to restoring the changes in gut microbiota composition induced by DSS.

[0041] The evaluation method for the effect of probiotic compositions on fecal metabolism includes: mixing methanol and water at a volume ratio of 4:1 to obtain an extract; grinding feces in the extract and performing low-temperature ultrasonic extraction; centrifuging and transferring the extract to a sample vial for ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-FFT-MS) for mass spectrometry detection; matching the mass spectrometry information with a database after testing to obtain metabolite information; and obtaining... Figure 7 The histidine metabolite heatmap shown reveals that, compared to the Control group, the DSS group exhibited a significant decrease in formic acid (formiminoglutamic acid), a metabolite of histidine urate catabolism. However, after intervention with the probiotic combination (DSS_AB), formic acid significantly increased, and this increase was significantly greater than that of the probiotic-only intervention group. Furthermore, the two products of the histidine-histamine metabolic pathway, imidazole propionic acid and N-acetylhistamine, were significantly increased in the DSS group compared to the Control group. The probiotic combination (DSS_AB) also significantly increased both products compared to the probiotic-only intervention groups (i.e., the first prevention group DSS_AK and the second prevention group DSS_BB). Specifically, while the first prevention group DSS_AK showed an increase in imidazole propionic acid, the probiotic combination (DSS_AB) significantly reduced it, indicating that the probiotic combination was more synergistically effective in improving DSS-induced histidine metabolic disorders in mice.

[0042] comprehensive Figures 1-7 The data analysis showed that the probiotic combination (DSS_AB) was more effective than the probiotic intervention groups alone (i.e., the first prevention group DSS_AK and the second prevention group DSS_BB) in synergistically improving the weight loss, increased disease activity index, colon shortening, intestinal structure and intestinal barrier damage, increased expression of pro-inflammatory factors, decreased expression of anti-inflammatory factors, and intestinal flora and histidine metabolism disorders in DSS-induced colitis mice.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A probiotic composition, characterized in that, The ratio of the number of CFU of Akkermansia muciniphila ATCC BAA-835 and Bifidobacterium animalis lactis BB-12 is 1: (0.1-10).

2. The probiotic composition of claim 1, wherein The ratio of the number of CFU of Akkermansia muciniphila ATCC BAA-835 and Bifidobacterium animalis lactis BB-12 is 1:

1. or, the total bacterial concentration of the probiotic composition is (0.5~2)×10 9 CFU / mL.

3. Use of the probiotic composition according to any one of claims 1-2 in the manufacture of a medicament for preventing or treating IBD.

4. Use according to claim 3, wherein the compound is ###0002### The specific manifestations of preventing or treating IBD include: (a1) inhibiting the decrease of body weight caused by IBD; (a2) inhibiting the increase of disease activity index caused by IBD; (a3) inhibiting the shortening of colon caused by IBD; (a4) inhibiting the damage of intestinal structure and intestinal barrier caused by IBD; (a5) inhibiting the increase of pro-inflammatory factors and the decrease of anti-inflammatory factors caused by IBD; (a6) inhibiting the intestinal flora disorder caused by IBD; (a7) synergistically regulating the histidine metabolic pathway, inhibiting the increase of imidazole propionic acid and N-acetyl histamine and the decrease of amine methylene glutamic acid caused by IBD.

5. A health food or a medicine comprising the probiotic composition according to any one of claims 1-2.

6. The pharmaceutical product according to claim 5, wherein The dosage form of the medicine includes, but is not limited to, wettable powder, water dispersible granules, water suspension and dispersible oil suspension.

7. The pharmaceutical product according to claim 5, wherein The medicine includes pharmaceutically acceptable carriers and other non-toxic compatible substances used in pharmaceutical preparations.

8. The pharmaceutical product according to claim 5, wherein The medicine includes wetting agents, emulsifying agents and lubricants; Or, the medicine includes coloring agents, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants.

9. The pharmaceutical product according to claim 5, wherein The medicine is a mixed solution containing the probiotic composition, and the addition of the probiotic composition includes pre-mixing, direct addition and coating addition.

10. The pharmaceutical product according to claim 9, characterized in that The total bacterial concentration in the mixed solution is (0.5-2) x 10 9 CFU / mL.

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