Lactobacillus fermentum 11C source membrane vesicle and application thereof

Abstract

The invention discloses a lactobacillus fermentum 11C source membrane vesicle and application thereof, and belongs to the technical field of microorganisms. The lactobacillus fermentum 11C with excellent probiotic characteristics is separated from fresh excrement of Lichuan horses for the first time, the preservation number of the lactobacillus fermentum 11C is CCTCC NO: M20252050, and meanwhile, the effects of the lactobacillus fermentum 11C and membrane vesicles of the lactobacillus fermentum 11C in reducing the uric acid level are researched. Experiments prove that the lactobacillus fermentum 11C membrane vesicles obtained through separation and screening have the effect of reducing serum uric acid, the uric acid reducing effect is better than that of lactobacillus fermentum 11C, and the body weight can be better kept stable. As the membrane vesicles are prepared by centrifuging bacterial liquid supernatant, the membrane vesicles are safer and have no drug resistance risk, and a new thought and approach are provided for the lactobacillus fermentum membrane vesicles in preparation of uric acid reducing products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a membrane vesicle derived from Lactobacillus fermentum 11C and its application. Background Technology

[0002] Hyperuricemia is a common chronic metabolic disease that has long plagued humans and animals. Excessive intake of high-purine foods cannot be metabolized and excreted from the body in a timely manner, activating purine metabolism. The end product of purine metabolism is uric acid, and uric acid retention leads to hyperuricemia. Hyperuricemia is a condition where serum uric acid levels are higher than normal, and it is closely related to various diseases such as obesity, hypertension, gout, kidney stones, kidney damage, and chronic renal failure. Currently, the treatment of metabolic diseases mainly includes drug therapy and bacterial therapy. However, due to the significant side effects and drug resistance issues of drug therapy, and the poor controllability and low safety of bacterial therapy, their development and application as therapeutic agents are limited. Even probiotics may develop drug resistance.

[0003] Membrane vesicles (MVs) are nanoscale particles composed of a lipid bilayer secreted by prokaryotes such as bacteria and archaea. They can transport various components, including proteins and nucleic acids. They participate in many important physiological processes, such as host interaction, horizontal gene transfer, and interspecies or intraspecies communication. The ability of MVs to transport proteins over long distances makes them a popular novel drug carrier in current research. Compared to bacteria, MVs offer significantly higher controllability and safety, and they maintain biological activity even in the absence of live bacteria. These are excellent characteristics that make MVs a promising candidate for novel therapeutics.

[0004] The Lichuan horse is a subspecies of Chinese horse, geographically belonging to the Southwest horse breed. Lichuan horses are mostly ponies, and their characteristics are closely related to the local natural ecological environment and socio-economic factors. The central production area, Lichuan City, is mostly high-altitude, rainy mountainous terrain with complex topography, many steep slopes and dangerous roads, and uneven pastures. Due to Lichuan City's high altitude, wild pasture is insufficient, and horse farms often feed them bean straw and bean husks as a protein supplement. However, because legumes contain high levels of purines, it is believed that Lichuan horses may have a better ability to adapt to a high-purine diet. Generally, gut microbiota possesses some characteristics of the host, therefore, research focuses on whether there are strains in their gut microbiota capable of lowering uric acid. Currently, no gut microbiota studies have been conducted on Lichuan horses. Meanwhile, there has been no well-suited equine probiotic, mainly for three reasons: (1) research on the equine microbiota is relatively underdeveloped; (2) the price and application value of individual horse probiotics are high, while the development and application of probiotics require long-term clinical trials on the animal itself, making it difficult to achieve the clinical trial stage for equine probiotics; and (3) the mechanism of action of probiotics on the intestinal flora is still unclear and is in the early stages of research. Therefore, research on the application of equine probiotics and their membrane vesicles in lowering uric acid is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a *Lactobacillus fermentum* 11C-derived membrane vesicle and its application, thereby addressing the problems existing in the prior art. This invention aims to explore the intestinal microbiota of Lichuan horses for the first time, isolating *Lactobacillus fermentum* with excellent probiotic properties from fresh feces of Lichuan horses, and studying the role of this *Lactobacillus fermentum* and its vesicles in lowering uric acid levels.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] In a first aspect, the present invention provides a membrane vesicle derived from *Limosilactobacillus fermentum* 11C, wherein the *Limosilactobacillus fermentum* 11C-derived membrane vesicle is secreted by *Limosilactobacillus fermentum* 11C; the *Limosilactobacillus fermentum* 11C was deposited at the China Center for Type Culture Collection on September 15, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252050.

[0008] Secondly, the present invention also provides a method for extracting membrane vesicles derived from *Lactobacillus fermentum* 11C, comprising the following steps:

[0009] The Lactobacillus fermentum 11C was inoculated into MRS broth and cultured in a constant temperature shaker for 12-24 h. The supernatant was collected by centrifugation, filtered, concentrated, centrifuged a second time, the precipitate was collected and resuspended, centrifuged a third time, the precipitate was collected in a tube, and filtered a second time to obtain the membrane vesicles derived from Lactobacillus fermentum 11C.

[0010] Preferably, the temperature of the constant temperature shaker is 37°C and the rotation speed is 120 r / min.

[0011] Thirdly, the present invention also provides a microbial preparation comprising the Lactobacillus fermentum 11C-derived membrane vesicles.

[0012] Fourthly, the present invention also provides the use of the Lactobacillus fermentum 11C-derived membrane vesicles or the microbial preparation in the preparation of products that lower uric acid.

[0013] Preferably, the product is a drug.

[0014] Preferably, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0015] Fifthly, the present invention also provides a product for lowering uric acid, characterized in that the product contains the Lactobacillus fermentum 11C-derived membrane vesicles or the microbial preparation.

[0016] Preferably, the dosage form of the product is selected from one or more of the following: injection, tablet, capsule, oral liquid, granule, powder, pill, and suspension.

[0017] Preferably, the product also includes a pharmaceutically acceptable carrier or excipient.

[0018] The present invention discloses the following technical effects:

[0019] This invention is the first to discover that *Lactobacillus fermentum* 11C membrane vesicles have a serum uric acid-lowering effect, and this effect is superior to that of *Lactobacillus fermentum* 11C, resulting in better weight stability. Since the membrane vesicles are prepared by centrifugation of the bacterial supernatant, they are safer and pose no risk of drug resistance. This provides a new approach and method for using *Lactobacillus fermentum* membrane vesicles in the preparation of uric acid-lowering products.

[0020] Preservation information: Limosilactobacillus fermentum 11C was deposited at the China Center for Type Culture Collection (CCTCC) on September 15, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20252050. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Results of colony morphology (A) and Gram staining identification (B) of the isolated strain;

[0023] Figure 2 The growth curve of the isolated strain;

[0024] Figure 3 Phylogenetic tree;

[0025] Figure 4 Transmission electron microscopy image of membrane vesicles of the isolated bacterial strain;

[0026] Figure 5 The results of nanoparticle tracking analysis of the membrane vesicles of the isolated strain;

[0027] Figure 6 A technical roadmap for animal experiments;

[0028] Figure 7 Weekly changes in serum uric acid in animal experiments (A) and serum uric acid levels at week 5 (B);

[0029] Figure 8 Cell assay grouping (A) and Western blot banding (B);

[0030] Figure 9 These are the results of a Western blot experiment. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1: Isolation and Identification of Strains and Preparation of Membrane Vesicles

[0037] 1. Experimental Methods

[0038] 1.1 Sampling

[0039] From July 16th to July 18th, 2021, the inventor traveled to Lichuan City, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province for a three-day sampling operation. Fresh fecal samples were collected from three racecourses in rotation, labeled, and photographed. The labeled fecal samples were then stored in dry ice containers and brought back to the laboratory, where they were kept in a -80°C freezer.

[0040] 1.2 Isolation and Identification of Strains

[0041] Take 1 g of Lichuan horse manure and place it in sterile PBS buffer. Vortex to mix well and incubate at 120 r / min for 30 min in a 37℃ constant temperature shaker. In a sterile laminar flow hood, take 100 μL of the supernatant and spread it evenly on MRS agar medium. Incubate upside down in a 37℃ constant temperature incubator for 48 h. Collect single colonies for subculturing, repeating three times. Name the obtained single colonies 11C. Collect single colonies, mix them into MRS broth, and incubate at 200 r / min and 37℃ in a shaker for 12 h. After incubation, dilute with 50% (v / v) glycerol 1:1 and store at -80℃.

[0042] Gram staining was performed on the isolated strain 11C: smear, dry and fix, stain with crystal violet for 1-2 minutes, wash with water; rinse with iodine solution to remove residual water, and cover with iodine solution for 1 minute; add 95% alcohol, decolorize for 30 seconds, immediately wash with water, counterstain with safranin solution for 1-2 minutes, wash with water; after drying, observe under oil immersion. Start with low magnification, then high magnification. After using the oil immersion lens, clean the lens promptly with lens paper to avoid cedarwood oil residue. Observe, capture, and process the images using ImageView software.

[0043] Colony morphology identification and Gram staining results are as follows Figure 1 The colonies of strain 11C are milky white, round, smooth, and have regular edges. After Gram staining, microscopic observation shows that they are Gram-positive, with short rod-shaped cells, no capsules, and no spores. This conforms to the basic characteristics of Lactobacillus.

[0044] Figure 2 To obtain the colony growth curve, the OD of the bacterial solution was measured. 600 The value is used to determine its growth curve, OD at 4-8 h 600 The changes are most rapid during this period, which is the logarithmic growth phase at 11°C, and the plateau phase is reached around the 14th hour.

[0045] Bacterial DNA was extracted according to the bacterial DNA extraction kit instructions, and PCR amplification was performed using universal primers for the bacterial 16S rRNA gene. The PCR products were detected by 1% agarose gel electrophoresis, and the bands were observed and analyzed using a gel imaging system. Finally, the PCR products were sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing, and the 16S rRNA sequence of the strain was finally obtained, as shown in SEQ ID NO.1.

[0046] SEQ ID NO.1:

[0047]

[0048] The 16S rRNA sequence of this strain was uploaded to the NCBI database. BLAST alignment analysis was performed between the sequencing results and the NCBI database. The sequence with the highest alignment score was *Lactobacillus fermentum* (also known as *Limosilactobacillus fermentum*). Phylogenetic tree analysis revealed (…). Figure 3 Several known strains with the highest sequence similarity to the isolated strain were all Lactobacillus fermentum. Therefore, the strain obtained by this invention is Lactobacillus fermentum (also known as Limosilactobacillus fermentum), and it is named Lactobacillus fermentum 11C.

[0049] The isolated strain was named *Limosilactobacillus fermentum* 11C and was biopreserved.

[0050] 1.3 Isolation and purification of Lactobacillus fermentum 11C membrane vesicles (MVs)

[0051] The isolated *Lactobacillus fermentum* 11C was inoculated into 800 mL of MRS broth and cultured at 37°C for 12 h in a shaker at 120 rpm. The bacterial cells were removed by centrifugation at 8000 g for 30 min, and the supernatant was collected into a new 500 mL centrifuge bottle. The supernatant was then transferred to a 0.22 μm pore size filter cup, and vacuum was applied to transfer the filtrate into the new centrifuge bottle. The ultrafiltration membrane was connected to a peristaltic pump, and the filtrate was concentrated to 250 mL. The concentrated filtrate was aliquoted into centrifuge tubes compatible with the ultra-high-speed refrigerated centrifuge, and the tubes were balanced. The tubes were transferred to an ultra-high-speed refrigerated centrifuge at 4°C and centrifuged at 120000 g for 2 h. The supernatant was carefully decanted, the precipitate was resuspended, and centrifuged at 120000 g for 2 h. The supernatant was removed, and the precipitate was repeatedly pipetted with PBS and collected in a 1.5 mL ep ep filtrate tube. The collected exosomes were filtered through a 0.22 μm pore size cell filter to remove environmental bacteria that had entered the system during centrifugation. All the above operations were performed at 4°C or on ice. The protein concentration was determined by the BCA method. The average concentration of the protein extracted by this method was 1.05 mg / mL. After aliquoting, the protein was stored at -80°C and diluted with physiological saline at 4°C to the required concentration for subsequent use.

[0052] 1.4 Nanoparticle Tracking Analysis (NAT) for Detecting MVs

[0053] (1) Rinse the sample pool three times with pure water;

[0054] (2) After cleaning, use standard (100 nm PS beads, polystyrene microspheres) to calibrate the instrument. After calibration, perform subsequent testing.

[0055] (3) Clean the sample pool with 1×PBS;

[0056] (4) After diluting the MVs with 1×PBS, add them to the sample pool and observe the real-time dynamic images of the OMVs particles on the computer screen.

[0057] (5) Collect information and issue corresponding test reports;

[0058] (6) After the test, clean the sample cell with 1×PBS and then test the next sample.

[0059] result: Figure 5 The particle size distribution and particle concentration of 11C-MVs are determined by... Figure 5 It can be determined that the concentration of 11C-MVs is approximately 2.5 × 10⁻⁶. 12 The number of particles per ml was 153.7 nm.

[0060] 1.5 Transmission electron microscopy (TEM) detection of MVs

[0061] (1) Adsorption: Use a pipette to take 10 μL of the prepared MVs and drop it onto the prepared Parafilm sealing film (the back of the sealing film is attached to the table). Place the copper mesh of the carrier film face down and let it naturally adsorb the suspension droplets for 10-15 minutes. Then use filter paper strips to remove the excess droplets and let it dry slightly.

[0062] (2) Staining: Pipette 10 μL of 2% phosphotungstic acid solution onto the sealing film, place the copper mesh with the front side facing the staining solution, and invert it to stand for 3-5 min.

[0063] (3) Drying: Use filter paper strips to absorb excess liquid droplets and air dry under an incandescent lamp;

[0064] (4) Observation and photography: Observe and photograph under a transmission electron microscope.

[0065] Results: The results of transmission electron microscopy analysis are shown below. Figure 4 ,Depend on Figure 4 It can be seen that 11C-MVs have a complete shape and structure, are saucer-shaped or elliptical, and have smooth edges. The TEM and NTA results show that the above method for extracting MVs is feasible.

[0066] Example 2 Animal Experiment

[0067] Forty male, 3-week-old SPF-grade KM mice (weighing 16 g ± 1 g) were purchased from the Animal Experiment Center of Huazhong Agricultural University. All mice were provided with purified water and had free access to water. The ambient temperature was set at 25 ± 1℃, the air humidity at 40%, and the day and night light duration was 12 h each. After acclimatization for 7 days, all mice were randomly divided into three groups: a normal control group (Control, C group), a model group (Model, M group), a Lactobacillus fermentum 11C treatment group (11C group), and a Lactobacillus fermentum 11C-MVs treatment group (11C-MVs group), with 10 mice in each group.

[0068] Intervention methods: Group C was provided with standard rodent food (Beijing Botai Hongda Biotechnology, catalog number: HD000) and administered 0.2 mL / mouse of physiological saline via gavage daily; Group M was provided with model rodent food and administered 0.2 mL / mouse of physiological saline via gavage daily; Group 11C was provided with model rodent food (Beijing Botai Hongda Biotechnology, catalog number: HD035C) and administered 0.2 mL / 10 g body weight of 11C bacterial solution (dissolved in physiological saline) via gavage daily, with a bacterial concentration of 1x10⁻⁶. 8 CFU / mL, the 11C-MVs group was provided with model mouse diet, and 0.2 mL of 50 μg 11C-MVs (dissolved in physiological saline) was administered by gavage daily for 5 weeks. The technical roadmap is available. Figure 6 .

[0069] Animal experiments ended at 5 weeks, mice were euthanized, and blood samples were collected to measure serum uric acid levels. During the experiment, the mice's food intake was monitored daily, and their serum uric acid levels were measured weekly.

[0070] Results: Throughout the experiment, the food intake for each group was 6 g / animal. Serum uric acid results were available. Figure 7 ,Depend on Figure 7 The results from the AB study showed that the serum uric acid level in the model group mice was significantly higher than that in the control group, while the treatment with the 11C group and the 11C-MVs group significantly reduced the serum uric acid level. Among them, the serum uric acid level in the 11C-MVs group after treatment was comparable to that in the control group, and it had a better uric acid-lowering effect than the 11C group, suggesting that the uric acid-lowering effect of the 11C-MVs group was superior to that of the 11C group.

[0071] Example 3 Cell Experiment

[0072] Experimental objective:

[0073] To investigate the regulatory effect of 11C-MVs on uric acid transporter proteins in CACO-2 cells.

[0074] Experimental methods:

[0075] (1) Grouping: CACO-2 cells were cultured in T75 cell flasks until they reached approximately 70-80% confluence. They were then seeded into 6-well plates and divided into 4 groups with 3 replicates per group: a normal control group (Control, C group), a model group (Model, M group), and a low-dose treatment group (11C-MVs). low Group 1), high-dose treatment group (11C-MVs) high (Groups). At the start of the experiment, the normal control group was given DMEM complete medium (15% fetal bovine serum), the model group was given DMEM complete medium containing 80 µg / mL uric acid, the low-dose treatment group was given DMEM complete medium containing 20 µg / mL 11C-MVs + 80 µg / mL uric acid, and the high-dose treatment group was given DMEM complete medium containing 50 µg / mL 11C-MVs + 80 µg / mL uric acid; 3 mL was added to each well, and the mixture was incubated for 48 hours.

[0076] (2) After 48 hours, discard the culture medium, wash three times with PBS buffer, place on ice, scrape all cells off the bottom wall with a cell scraper, add 200 µL of lysis buffer to each well, and after complete lysis, collect the supernatant and centrifuge at 12000 rpm for 5 min. Transfer the supernatant to a new EP tube, determine the protein concentration using the BCA method, and add Loading Buffer. Denature at 100℃ for 15 min, and store at -80℃. Perform Western blot experiments according to the cell experiment groups and observe the experimental results.

[0077] Experimental results:

[0078] Figure 8 Cell assay grouping (A) and Western blot assay banding (B). Figure 9 These are the grayscale values ​​from the Western blot experiment. Figure 8 and Figure 9 It can be seen that when incubated with uric acid, the expression level of ABCG2 protein in group M was significantly lower than that in the blank control (p < 0.05). However, after incubation with 11C-MVs, the expression level of ABCG2 protein in the low-dose group increased significantly (p < 0.01), but was still lower than that in the blank control group. The high-dose group (50 µg / mL 11C-MVs) had the highest expression level of ABCG2 protein, which was significantly higher than that in the other three groups (p < 0.001).

[0079] Example 4: A product for lowering uric acid

[0080] The product containing Lactobacillus fermentum 11C membrane vesicles prepared based on Example 1 can be used to prepare drugs.

[0081] In one embodiment, the only effective active ingredient in the drug is Lactobacillus fermentum 11C membrane vesicles.

[0082] It can be combined with other excipients to prepare uric acid-lowering products. When combined with other excipients, it can be used to prepare conventional dosage forms such as injections, tablets, capsules, oral liquids, granules, powders, pills, and suspensions.

[0083] Alternatively, as another implementation, Lactobacillus fermentum 11C membrane vesicles are combined with other active ingredients to prepare uric acid-lowering products.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A membrane vesicle derived from *Limosilactobacillus fermentum* 11C, characterized in that, The Lactobacillus fermentum 11C-derived membrane vesicles are secreted by Lactobacillus fermentum 11C; the Lactobacillus fermentum 11C was deposited at the China Center for Type Culture Collection on September 15, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252050.

2. A method for extracting membrane vesicles derived from *Lactobacillus fermentum* 11C as described in claim 1, characterized in that, Includes the following steps: The Lactobacillus fermentum 11C described in claim 1 was inoculated into MRS broth and cultured in a constant temperature shaker for 12-24 hours. The supernatant was collected by centrifugation, filtered, concentrated, centrifuged a second time, the precipitate was collected and resuspended, centrifuged a third time, the precipitate was collected in a tube, and filtered a second time to obtain the membrane vesicles derived from Lactobacillus fermentum 11C.

3. The preparation method according to claim 2, characterized in that, The temperature of the constant temperature shaker is 37℃ and the rotation speed is 120 r / min.

4. A microbial preparation, characterized in that, The microbial preparation contains membrane vesicles derived from Lactobacillus fermentum 11C as described in claim 1.

5. The use of the Lactobacillus fermentum 11C-derived membrane vesicles as described in claim 1 or the microbial preparation as described in claim 4 in the preparation of products that lower uric acid.

6. The application according to claim 5, characterized in that, The product is a medicine.

7. The application according to claim 6, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

8. A product for lowering uric acid, characterized in that, The product contains either the Lactobacillus fermentum 11C-derived membrane vesicles of claim 1 or the microbial preparation of claim 4.

9. The product according to claim 8, characterized in that, The dosage form of the product is selected from one or more of the following: injection, tablet, capsule, oral liquid, granule, powder, pill, and suspension.

10. The product according to claim 8, characterized in that, The product also includes pharmaceutically acceptable carriers or excipients.

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