Lactobacillus mucilaginosus and application thereof
By developing the fermented Lactobacillus mucinus ECON-8, which produces high levels of active polysaccharides, the problems of low yield and difficult extraction of probiotic active polysaccharides have been solved. This has enabled rapid colonization and long-term immune regulation on the host mucosa, with significant antioxidant and antitumor effects.
Patent Information
- Application Number
- CN202410987442.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-23
- Publication Date
- 2026-01-27
AI Technical Summary
Existing technologies have low yields of active polysaccharides produced by probiotics, which are difficult to extract and purify, and lack a clear regulatory framework, thus limiting their application in the food, pharmaceutical and other industries.
A high-yield bioactive polysaccharide-producing Lactobacillus fermentum ECON-8 was developed. Its safety was ensured through whole-genome sequencing and culture. Different routes of administration, such as oral, vaginal, or intratumoral injection, were employed. The composition contained Lactobacillus fermentum ECON-8, other bioactive strains, and pharmaceutically acceptable excipients. The culture method was optimized to increase polysaccharide yield.
Fermented Lactobacillus mucinus ECON-8 produces highly active polysaccharides with excellent cell adhesion and antioxidant capabilities. It can rapidly colonize the host mucosa, providing safe and long-term immunomodulatory effects, and synergistically combating tumors and complex diseases with existing immunotherapies.
Smart Images
Figure HDA0004957850630000011 
Figure HDA0004957850630000012 
Figure HDA0004957850630000021
Abstract
Description
Technical Field
[0001] This invention relates to a high-yield, highly active polysaccharide-producing lactobacillus. It is used in the preparation of specific pharmaceutical products, medical devices, cosmetics, or hygiene products, and is suitable for application to the vagina, vulva, or oral administration. It can be used for vaginal and intestinal immune regulation, restoring host immune balance, and is applicable to tumors and precancerous lesions, allergic diseases, autoimmune diseases, etc. Background Technology
[0002] In recent years, numerous research institutions and companies have studied the beneficial effects of postbiotic components on the host, including inactivated bacterial cells, lipoteichoic acid, peptidoglycans, and other bacterial cell components, as well as extracellular polysaccharides and SCFAs. Furthermore, increasing numbers of in vivo and in vitro experiments have demonstrated that postbiotics have significant effects in enhancing immunity, regulating gastrointestinal function, and alleviating obesity. [1] Capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) are natural macromolecular bioactive substances produced by lactic acid bacteria. They possess unique physiological activities and structural characteristics and are widely used in the food and pharmaceutical industries. The active polysaccharides produced by probiotics have a variety of functional properties. [1] (1) Antioxidant activity: Some EPS can maintain its antioxidant activity at high temperatures, which is crucial for its application in the food industry; (2) Antibacterial activity: Its antibacterial mechanism is to block the enzymes responsible for the biosynthesis of peptidoglycan in the cell wall or to prevent the uptake of nutrients by blocking the sugar uptake system, thereby leading to cell death; (3) Anti-inflammatory activity: Studies have shown that EPS intake can increase the concentration of short-chain fatty acids with anti-inflammatory properties; (4) Immunomodulatory activity: EPS regulates innate and adaptive host immunity through different mechanisms, including promoting the proliferation of T and B lymphocytes and activating (5) Anticancer activity: It has been reported that EPS-M4 of marine probiotic Pediococcus pentosaceus M41 can inhibit the proliferation of Caco-2 and MCF-7 tumor cells; (6) Cholesterol-lowering and antidiabetic activity: Studies have shown that EPS derived from Lactobacillus paracasei M7 can reduce cholesterol levels to 70.78% in vitro; (7) Antibacterial membrane activity: It has been reported that EPS extracted from Lactobacillus acidophilus A4 strain is resistant to Escherichia coli, Salmonella, Pseudomonas aeruginosa, Bacillus cereus, etc.; (8) Antiulcer effect; (9) Antitoxin activity.
[0003] At the regulatory level, my country currently lacks relevant standards for the production and management of postbiotics, resulting in a lack of a clear regulatory framework and application basis. Live bacterial preparations, on the other hand, have a long history of application and standardized regulatory pathways. Furthermore, the low yield of active polysaccharides produced by probiotics, coupled with difficulties in extraction and purification, limits their application. The high-yield probiotics of this invention are beneficial for widespread application in the food, pharmaceutical, and livestock industries. Summary of the Invention
[0004] The present invention aims to provide a fermenting Lactobacillus fermentum ECON-8 strain that produces high levels of active polysaccharides. The strain was identified by whole-genome sequencing.
[0005] The present invention provides a composition comprising the aforementioned fermenting Lactobacillus mucinus ECON-8.
[0006] In some specific embodiments, the viable count of *Lactobacillus fermentans* ECON-8 in the aforementioned composition is 1 × 10⁻⁶. 5 CFU ~ 1×10 12 CFU.
[0007] In some specific embodiments, the viable count of *Lactobacillus fermentans* ECON-8 in the aforementioned composition is 1 × 10⁻⁶. 5 CFU ~ 1×10 9 CFU.
[0008] In some specific embodiments, the aforementioned composition can be administered to patients orally, by intratumoral injection, or by vaginal application.
[0009] In some specific embodiments, the aforementioned composition further includes pharmaceutically acceptable excipients or carriers.
[0010] In some specific embodiments, the lactobacilli in the aforementioned composition exist in the form of live cells and / or inactivated, lysed, cultured metabolites, etc.
[0011] In some specific embodiments, the aforementioned composition also contains other active microbial strains.
[0012] The present invention also provides an application of the aforementioned lactobacillus in effectively preventing or repairing oxidative damage in the body.
[0013] The present invention also provides an application of the aforementioned lactobacillus in improving the tumor immunosuppressive microenvironment.
[0014] The present invention also provides an application of the aforementioned Lactobacillus in the production of bioactive polysaccharides.
[0015] The present invention also provides a method for culturing *Lactobacillus fermentatus*, comprising inoculating *Lactobacillus fermentatus* into a culture medium, carrying out proliferation culture, and obtaining proliferated *Lactobacillus fermentatus*.
[0016] The beneficial effects of this invention are:
[0017] The fermented Lactobacillus mucinus of this invention produces 642 mg / L of highly active polysaccharides.
[0018] The fermented *Lactobacillus mucinus* of this invention has excellent cell adhesion ability, which helps the strain to colonize the host mucosa and exert its medicinal effect more quickly and for a longer period of time.
[0019] The fermented *Lactobacillus mucinus* of this invention has excellent antioxidant capacity and can effectively prevent or repair oxidative damage to the body.
[0020] The fermented *Lactobacillus mucinus* of this invention, as a novel immunomodulatory substance, has advantages over commercially available chemical drugs, including better safety, no side effects, and no drug resistance. It can be administered long-term via multiple routes, addressing the long-term treatment of complex tumor-related diseases. This *Lactobacillus*, as a novel immunomodulatory substance, provides numerous pathogen-associated molecular patterns (PAMPs) that can be recognized by the host's innate immune system. Combined with existing immunotherapies, it exhibits a synergistic therapeutic effect.
[0021] The strain preservation information provided by this invention is as follows:
[0022] Limosilactobacillus fermentum ECON-8 was deposited at the China Center for Type Culture Collection (CCTCC) on September 18, 2023, with accession number CCTCC NO: M 20231726, located in Wuhan, China. Attached Figure Description
[0023] Figure 1 The image shows a Gram-stained microscopic image of fermenting Lactobacillus mucinus ECON-8.
[0024] Figure 2 The image shows the capsule staining pattern of Lactobacillus fermentans ECON-8.
[0025] Figure 3 This study demonstrates the restorative effect of fermented Lactobacillus mucinus ECON-8 on cells damaged by oxidative stress at 600 μM H2O2 for 24 h.
[0026] Figure 4 This study demonstrates the restorative effect of fermented Lactobacillus mucinus ECON-8 on cells damaged by oxidative stress at 640 μM H2O2 for 24 h. Detailed Implementation
[0027] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be noted that, unless otherwise specified, all reagents or instruments used in this invention are commercially available. The culture medium preparation methods used in the following embodiments are all well-known methods.
[0028] Example 1: Isolation, Identification and Gene Safety Analysis of Lactobacillus
[0029] Gram staining and microscopic examination of vaginal secretions from healthy female volunteers of childbearing age were performed, and Nugent scores were assessed. Healthy female volunteers with a Nugent score <3 were selected. Vaginal swabs from healthy volunteers were placed in MRS acidic liquid culture medium and incubated overnight at 37°C for bacterial expansion. The enrichment broth was then serially diluted 10-fold to 10⁻⁶. -5 All dilution gradients were plated on 2% calcium carbonate-0.8% MRS agar and incubated at 37°C for 36-48 h. Single colonies with distinct clear zones were picked from the cultured calcium carbonate-MRS agar to isolate single bacteria. The isolated single bacteria were cultured to the late logarithmic growth stage, and DNA was extracted and sequenced using a high-throughput sequencer. The predicted gene sequences were compared with the non-redundant protein database (NCBI-nr) using Blast software. Furthermore, the average nucleotide identity (ANI) value was obtained using FastANI (Version 1.33) software for species identification. The results of strain identification are shown in Table 1.
[0030] Table 1. FastANI analysis results of strains and the reference genome of *Limosilactobacillus fermentum* EFEL6800.
[0031] index numerical values ANI value (%) 98.82 Reference genome length (bp) 2,103,331 Query genome length (bp) 2,010,978 Number of all sequence fragments in the reference genome 670 Query the number of homologous fragments aligned in the genome 602
[0032] Based on molecular identification results, the isolated strain was identified as *Limosilactobacillus fermentum*, and named *Limosilactobacillus fermentum* ECON-8. Figure 1 Gram staining microscopic image of fermenting Lactobacillus mucinus ECON-8.
[0033] To ensure the safety of medication use, a safety assessment of the isolated strains is necessary. This includes determining the presence of potential pathogenic factors and their relative risks; identifying antibiotic resistance genes to assess the potential impact of the strains in antibiotic-using environments, etc. The amino acid sequence of the predicted gene was compared with the Virulence Factors Database (VFDB) using Diamond (Version 2.1.6) software. A set of strict thresholds was used (sequence match ≥80%, sequence length coverage ≥60%, e-value ≤10). -5 ) [3-4] The results showed that the fermenting Lactobacillus mucinus ECON-8 strain did not contain any known pathogenic genes or key genes related to toxin synthesis, nor did it carry drug resistance genes directly related to drug resistance phenotypes. Therefore, it can be determined that the strain used in this invention has no potential safety issues.
[0034] Example 2: The ability of fermenting *Lactobacillus mucinus* ECON-8 to adhere to cells
[0035] Lactobacillus delbrueckii (hereinafter referred to as DJS), isolated from the commercially available microecological preparation Dingjunsheng using the same method, served as the positive control strain in Example 1.
[0036] A model was used to evaluate the cell adhesion ability of various lactobacilli by co-incubating common human cervical cancer HeLa cell lines (ATCC) and human vaginal epithelial cells VK2 / E6E7 (ATCC) with lactobacilli. Cells were seeded in 24-well cell culture plates and allowed to adhere overnight. *Lactobacillus mucinus* ECON-8 was activated and transferred; the resulting bacterial culture was centrifuged, washed, and then co-incubated with the cells. After incubation, viable bacteria were counted on the adherent cells to assess the in vitro adhesion ability of the tested lactobacilli to cells.
[0037] The results are shown in Table 2. The ability of the fermented Lactobacillus mucinus ECON-8 strain to adhere to cells is superior to that of the control strain DJS. This excellent adhesion ability helps the strain colonize in the vagina, enabling it to exert its therapeutic effect more quickly and for a longer period.
[0038] Table 2 Comparison of cell adhesion ability of Lactobacillus fermentum ECON-8
[0039] strain number VK2 single cell adhesion count (CFU / cell) HeLa single cell adhesion count (CFU / cell) ECON-8 15.00 24.89 DJS 13.39 2.40
[0040] Example 3: The ability of fermenting Lactobacillus mucinus ECON-8 to produce bioactive polysaccharides
[0041] The capsule was stained using the ink negative staining method, and the capsular polysaccharides were observed under an optical microscope. In this method, ink only stains the background and cannot stain the bacterial cells and capsule. After fixation with methanol, crystal violet only stains the bacterial cells. Therefore, when observed under a 100× oil immersion microscope, the substance that appears purple on the bacterial cells and is surrounded by a colorless and transparent ring is the capsular polysaccharide. Figure 2 The results showed that fermentation of Lactobacillus mucinus ECON-8 produced a large amount of capsular polysaccharide.
[0042] The *Lactobacillus fermentans* ECON-8 was cultured to the late logarithmic growth stage, and the supernatant was collected by centrifugation. After precipitating the polysaccharides with anhydrous ethanol, the extracellular polysaccharide yield was determined using the sulfuric acid-phenol method. The results showed that the *Lactobacillus fermentans* of this invention produced a high amount of extracellular mucopolysaccharides, reaching 642 mg / L, indicating its potential for various beneficial growth.
[0043] Example 4: Antioxidant capacity of fermented Lactobacillus mucinus ECON-8
[0044] Excessive reactive oxygen species (ROS) can induce oxidative damage to cell membranes and biomolecules, leading to various chronic diseases. *Lactobacillus fermentum* ECON-8 was cultured to the late logarithmic growth stage, and the supernatant was collected by centrifugation. The antioxidant capacity of the *Lactobacillus fermentum* ECON-8 supernatant was assessed using the classic DPPH and ABTS methods. Table 3 shows that the antioxidant capacity of *Lactobacillus fermentum* ECON-8 was superior to that of the control strain DJS.
[0045] Table 3 Antioxidant capacity of *Lactobacillus mucinus* ECON-8
[0046] strain number ABTS free radical scavenging rate DPPH free radical scavenging rate ECON-8 61.89％ 58.41％ DJS 47.92％ 54.80％
[0047] A model of oxidative damage in Caco-2 cells (intestinal epithelial cells, ATCC) induced by H2O2. [5] After 24 hours of damage from 600 μM H2O2 and 640 μM H2O2, the supernatant of fermenting *Lactobacillus mucilaginosus* ECON-8 was treated for 24 hours to investigate its repair effect on oxidative damage to cells. Results are as follows: Figure 3 , 4 The results showed that fermentation of Lactobacillus mucinus ECON-8 can activate the cellular antioxidant defense system, protecting Caco-2 cells from induced oxidative stress in a dose-dependent manner.
[0048] Example 5: Depolarizing ability of fermented Lactobacillus mucinus ECON-8 on M2 macrophages
[0049] M2 macrophages are associated with an immunosuppressive tumor microenvironment, and high recruitment of M2 cells facilitates tumor escape. Mouse RAW264.7 macrophages (Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences) were cultured in DMEM high-glucose medium (Gipco) containing 10% FBS. When they were about to reach confluence, 2 × 10⁶ cells were added... 5 Cells were seeded in 12-well plates. After overnight cell adhesion, the cells were divided into three groups: C group, M2 group, and M2+ECON-8 group.
[0050] Group C: Treated with DMEM antibiotic-free medium for 72 hours;
[0051] M2 group: M2 macrophages were subjected to polarization treatment, that is, 1 μl of 20 μg / ml IL-4 and 20 μg / ml IL-13 were administered per well (1 ml), with a final concentration of 20 ng / ml IL-4 + 20 ng / ml IL-13, and the treatment lasted for 72 h;
[0052] M2+ECON-8 group: Cytokine treatment was performed in the same manner as M2 group for 48 h. In the drug administration group, 10 μL of fermented Lactobacillus mucin ECON-8 lysate sample was administered per well (1 ml) to a final concentration of 1% for 24 h.
[0053] After 72 hours of treatment, samples were collected, washed once with PBS, and 400 μl of Accutase enzyme was added to each well for digestion. After 5 minutes, the samples were centrifuged at 400g for 5 minutes to remove the supernatant. The expression level of CD206, an M2 cell marker molecule on the cell surface, was detected by flow cytometry.
[0054] Table 4 shows that fermented Lactobacillus mucinus ECON-8 can significantly reduce the expression level of the M2 cell marker molecule CD206, suggesting that the strain of the present invention can enhance host resistance, improve the tumor immunosuppressive microenvironment, and promote anti-tumor effects.
[0055] Table 4. Results of flow cytometry analysis of CD206 expression levels on cell surface
[0056] Group TTEST Mean percentage of CD206 positive C 1 0.34％ M2 0.00050703 71.85％ M2+ECON-8 0.002383399 35.62％
[0057] Unless otherwise specified, the terms used in this invention have the meanings commonly understood by those skilled in the art.
[0058] The embodiments described in this invention are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various other substitutions, changes, and improvements within the scope of this invention. Therefore, this invention is not limited to the above embodiments, but is defined only by the claims. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.
[0059] References:
[0060] [1] Pourjafar H, Ansari F, Sadeghi A, Samakkhah SA, Jafari SM. Functionaland health-promoting properties of probiotics' exopolysaccharides; isolation, characterization, and applications in the food industry. Crit Rev Food SciNutr.2023;63(26):8194-8225.doi:10.1080 / 10408398.2022.2047883.Epub 2022 Mar10.PMID:35266799.
[0061] [2]Wang Y,Thakur R,Shen Q,et al.Influences of vaginal microbiota onhuman papillomavirus infection and host immune regulation:What we havelearned?[J].Decoding Infection and Transmission,2023,1:null-null.DOI:10.1016 / j.dcit.2023.07.001.
[0062] [3]Oh,Y.J.et al.Integrated genome-based assessment of safety andprobiotic characteristics of Lactiplantibacillus plantarum PMO 08isolatedfrom kimchi.Plos One 17,doi:10.1371 / journal.pone.0273986(2022).
[0063] [4]Chokesajjawatee,N.et al.Safety Assessment of a Nham StarterCulture Lactobacillus plantarum BCC9546 via Whole-genome Analysis.ScientificReports 10,doi:10.1038 / s41598-020-66857-2(2020).
[0064] [5]Li,C.,Chen,X.,Li,L.,Cheng,J.,Chen,H.,Gao,Q.,Yang,F.,Cai,X.,&Wang,S.(2023).Protective effect of antioxidant peptides from bass(Lateolabraxjaponicus)on oxidative stress injury in Caco-2 cells.Food Frontiers,4,818–830.https: / / doi.org / 10.1002 / fft2.224
[0065] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein in their entirety for all purposes. However, any mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not and should not be construed as an admission or in any way an implication that they constitute valid prior art or are part of common general knowledge in any country of the world.
Claims
1. A fermenting *Limosilactobacillus fermentum* ECON-8, characterized in that, The accession number of the Lactobacillus mentioned is CCTCC NO: M 20231726 at the China Center for Type Culture Collection.
2. A composition, characterized in that, It includes the fermenting lactobacillus ECON-8 as described in claim 1.
3. The composition according to claim 2, characterized in that, The fermenting Lactobacillus fermentum ECON-8 exists in the form of living cells.
4. The composition according to claim 3, characterized in that, The viable count of the fermenting *Limosilactobacillus fermentum* ECON-8 was 1 × 10⁻⁶. 5 CFU ~ 1×10 12 CFU.
5. The composition according to claim 2 or 3, characterized in that, The composition further includes pharmaceutically acceptable excipients or carriers.
6. The composition according to claim 5, characterized in that, The composition contains other active bacterial strains.
7. The application of the fermented Lactobacillus fermentum ECON-8 as described in claim 1 in effectively preventing or repairing oxidative damage in the body.
8. The application of the fermented Lactobacillus fermentum ECON-8 as described in claim 1 in improving the tumor immunosuppressive microenvironment.
9. The application of the fermenting Lactobacillus fermentum ECON-8 as described in claim 1 in the production of active polysaccharides.
10. The method for culturing the *Limosilactobacillus fermentum* ECON-8 strain according to claim 1, comprising inoculating *Limosilactobacillus fermentum* into a culture medium, carrying out proliferation culture, and obtaining proliferated *Limosilactobacillus fermentum*.