Enterococcus faecalis strain ELB9416, its microecological composition, and its application

Abstract

This invention provides a strain of Enterococcus faecalis ( Enterococcus faecalis The ELB9416 strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026716. This invention also provides a microecological composition containing the aforementioned Enterococcus faecalis ELB9416. The microecological composition contains an ELB9416 cell count (including dead cells) greater than 10. 7 CFU / mL and viable count not higher than 10 5 CFU / mL (or even 0 viable bacteria count). The Enterococcus faecalis ELB9416 or its microecological composition of the present invention can be used to prepare products for improving memory, regulating intestinal flora, improving intestinal dysfunction, scavenging free radicals and / or antioxidation, with the advantages of well-defined composition, controllable quality, convenient administration and good safety.

Description

Technical Field

[0001] This invention belongs to the field of microbial and microecological preparation technology, specifically relating to a strain of Enterococcus faecalis ( Enterococcus faecalis ELB9416, a microecological composition containing the strain, a method for preparing the microecological composition, and the use of the strain or microecological composition in the preparation of products for improving memory, regulating gut microbiota, improving intestinal dysfunction, scavenging free radicals and / or antioxidation. Background Technology

[0002] The gut microbiota is an important component of the human body's micro-ecological system. Currently, various probiotic products on the market are available to regulate gut microbiota or improve gastrointestinal discomfort. These products typically use live bacteria such as lactobacilli, bifidobacteria, and enterococci as their main functional ingredients and generally emphasize a high number of live bacteria. The live bacteria count in current probiotic products typically reaches 10^ ... 7 -10 10 When CFU / mL or CFU / g is above a certain level, some solid powders may enter the small intestine after ingestion, potentially leading to high local concentrations of live bacteria. For individuals with sensitive intestines, abnormal intestinal motility, or a high risk of small intestinal bacterial overgrowth (SIOMB), excessively high live bacteria intake may cause discomfort such as bloating, abdominal pain, diarrhea, and flatulence, and may even increase the risk of SIOMB. Therefore, developing strains and formulations that can still exert their regulatory effects on the microecological system under low live bacteria counts is of great significance for improving the safety and applicability of microecological products.

[0003] On the other hand, traditional probiotic theory generally holds that live bacteria must survive, colonize, or proliferate briefly in the gut in order to play a role in regulating gut microbiota and improving gut function.

[0004] Current probiotic products suffer from limited functionality. Most products are designed primarily for single symptoms such as diarrhea or constipation, often emphasizing unidirectional regulation. For example, some products mainly relieve diarrhea, while others promote bowel movements. However, for mixed intestinal dysfunction characterized by alternating diarrhea and constipation, especially symptoms related to mixed irritable bowel syndrome (IBS), the applicability and stability of existing products remain insufficient. In clinical and real-world scenarios, intestinal dysfunction often manifests as complex symptoms such as abdominal pain, bloating, borborygmus, mucus in stool, bowel discomfort, and alternating diarrhea and constipation. Products targeting only a single symptom are insufficient to meet practical needs. Therefore, screening for strains with bidirectional regulatory effects on intestinal function and promotion of intestinal microecological balance is one of the urgent problems to be solved in this field.

[0005] Besides conventional probiotics, fecal microbiota transplantation (FMT) has become an important technique for reconstructing the gut microbiota in recent years. FMT transfers fecal microbiota from a healthy donor to the recipient's gut to restore the recipient's gut microecological balance, showing promising applications in research on recurrent Clostridium difficile infection, inflammatory bowel disease, and some metabolic and neurological diseases. However, FMT and its derivatives still face several challenges. First, donor sources vary significantly, with substantial differences in microbial composition and metabolic characteristics between different donors, leading to complex product compositions and difficulties in controlling batch-to-batch consistency. Second, fecal-derived products involve complex microbial communities, making it difficult to fully identify the active ingredients and resulting in a somewhat "black box" characteristic of their mechanisms of action. Third, while donor screening and pathogen detection can reduce risks, there are still theoretical safety concerns such as pathogen transmission, transfer of unknown microorganisms, or drug-resistant genes. Furthermore, the preparation, storage, transportation, and administration of FMT products are typically complex, leading to high costs for standardization and industrialization.

[0006] For example, Rebyota, a fecal microbiota transplantation product currently approved by the U.S. Food and Drug Administration. TM and VOWST TM Both are used to prevent recurrence of recurrent Clostridium difficile infection in adults after antimicrobial therapy. Among them, Rebyota... TM This is a broad-spectrum bacterial suspension derived from the feces of healthy donors, administered as a single rectal enema. It typically requires administration by healthcare professionals, leading to poor patient compliance and associated discomfort and procedural risks. VOWST TM The capsules are Firmicutes spores derived from the feces of healthy donors and purified through ethanol treatment. Although they can be administered orally, bowel cleansing is usually required before administration, such as by taking laxatives like magnesium citrate. Some patients have poor tolerance to these medications, and taking them multiple times over several days can also affect adherence.

[0007] Furthermore, while the aforementioned fecal-derived products can reduce the risk of recurrent Clostridium difficile infection to some extent, their active ingredients are all derived from donor feces and obtained through complex processing. This results in drawbacks such as complex composition, difficulty in quality control, potential batch-to-batch variations in microbial composition, high production costs, and stringent storage and transportation requirements. The product instructions also list a certain percentage of adverse reactions, such as abdominal pain, diarrhea, bloating, flatulence, nausea, fatigue, constipation, and chills. Therefore, developing single-strain microecological preparations with clearly defined sources, well-known components, controllable quality, convenient administration, and good safety profiles is of significant application value as a standardized alternative or supplementary solution to fecal microbiota transplantation or complex microbial products.

[0008] Enterococci are a group of Gram-positive bacteria commonly found in the intestines of humans and animals. Different sources and strains of Enterococci exhibit significant differences in safety, drug resistance, metabolic capacity, colonization ability, and functional effects.

[0009] Currently, there is a lack of *Enterococcus faecalis* strains and their microecological compositions that are clearly derived, have a single composition, controllable quality, can still function even in low viable counts or inactivated states, and simultaneously possess the functions of improving memory, regulating gut microbiota, improving intestinal dysfunction, scavenging free radicals, and / or anti-oxidation. Therefore, screening and developing such safe and effective new strains is of great significance for improving the shortcomings of existing probiotics and fecal microbiota transplantation products. Summary of the Invention

[0010] The purpose of this invention is to provide a strain of Enterococcus faecalis ELB9416. This strain has a clear origin, has been preserved, and exhibits good safety and genetic stability, making it suitable for preparing microecological compositions.

[0011] Another object of the present invention is to provide a microecological composition comprising the aforementioned Enterococcus faecalis ELB9416. This microecological composition can be in the form of containing live bacteria, low-viable-bacteria, or no live bacteria, and in particular, it can be prepared with a live bacteria count not exceeding 10. 5 Low-viable-bacteria preparations at CFU / mL, or preparations made into inactivated bacterial preparations with a viable bacterial count of 0, can reduce the safety risks that may arise from the intake of high-viable-bacteria preparations.

[0012] To achieve the above objectives, the present invention adopts the following technical solution.

[0013] In a first aspect, the present invention provides a strain of Enterococcus faecalis, said strain being Enterococcus faecalis (… Enterococcus faecalis ELB9416 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026716.

[0014] In some embodiments, the Enterococcus faecalis ELB9416 is a Gram-positive bacterium.

[0015] In some embodiments, the genome size of the Enterococcus faecalis ELB9416 is approximately 2.7 Mbp.

[0016] In a second aspect, the present invention provides a microecological composition comprising the Enterococcus faecalis ELB9416.

[0017] In some embodiments, the microecological composition further comprises one or more of the following: inactivated cells of Enterococcus faecalis ELB9416, cell lysates, cell wall components, fermentation broth, fermentation supernatant, and metabolites.

[0018] In some embodiments, the cell wall components include lipoteichoic acid, peptidoglycan, or a combination thereof.

[0019] In some embodiments, the microecological composition is an oral preparation.

[0020] In some preferred embodiments, the oral preparation is an oral liquid, drops, suspension, granules, capsules, tablets, lyophilized powder, or powder.

[0021] In some embodiments, the total bacterial count (including dead cells) of Enterococcus faecalis ELB9416 in the microecological composition is greater than 10. 7 CFU / mL and viable count not higher than 10 5 CFU / mL.

[0022] In some embodiments, the Enterococcus faecalis ELB9416 in the microecological composition is an inactivated bacterial cell, and the number of live bacteria in the microecological composition is 0.

[0023] In some embodiments, the inactivated bacterial cells are obtained by heat inactivation, pasteurization, ultraviolet inactivation, autoclaving, spray drying inactivation, or a combination thereof.

[0024] Thirdly, the present invention provides the use of the Enterococcus faecalis ELB9416 or the microecological composition in the preparation of products for improving memory.

[0025] In some implementations, the improvement of memory includes one or more of the following: improving learning memory ability, improving spatial memory ability, improving memory acquisition, improving memory consolidation, and improving memory retrieval.

[0026] Fourthly, the present invention provides the use of the Enterococcus faecalis ELB9416 or the microecological composition in the preparation of products for regulating intestinal flora.

[0027] In some embodiments, the regulation of gut microbiota includes improving gut microbiota imbalance, promoting gut microecological balance, increasing the number of beneficial bacteria, and / or inhibiting the excessive proliferation of conditionally pathogenic bacteria.

[0028] Fifthly, the present invention provides the use of the Enterococcus faecalis ELB9416 or the microecological composition in the preparation of products for regulating gastrointestinal function.

[0029] In some embodiments, the regulation of gastrointestinal function includes improving one or more of the following: constipation, diarrhea, abdominal pain, bloating, borborygmus, abnormal stool consistency, abnormal frequency of defecation, defecation discomfort, tenesmus, post-defecation discomfort, loss of appetite, or poor appetite.

[0030] In a sixth aspect, the present invention provides the use of the Enterococcus faecalis ELB9416 or the microecological composition in the preparation of products for scavenging free radicals and / or for anti-oxidation.

[0031] In some implementations, the product is food.

[0032] In some other preferred embodiments, the product is a pharmaceutical product.

[0033] In a seventh aspect, the present invention provides the use of the Enterococcus faecalis ELB9416 or the microecological composition in the preparation of a medicament for the prevention or treatment of irritable bowel syndrome.

[0034] In some embodiments, the irritable bowel syndrome includes diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable bowel syndrome, mixed irritable bowel syndrome, or unclassified irritable bowel syndrome.

[0035] Eighthly, the present invention provides a method for preparing the microecological composition, comprising the following steps:

[0036] The *Enterococcus faecalis* ELB9416 was inoculated into a culture medium for fermentation, and the fermentation products were collected; and

[0037] The fermentation product is prepared into a microecological composition containing live bacteria, low-live bacteria, inactivated bacteria, bacterial lysates, cell wall components, fermentation broth, fermentation supernatant and / or metabolites.

[0038] In some embodiments, the fermentation product is subjected to one or more of the following treatments: centrifugation, filtration, concentration, inactivation, lysis, homogenization, freeze drying, spray drying, and blending.

[0039] Compared with the prior art, the present invention has at least the following beneficial effects:

[0040] 1. This invention provides a strain of Enterococcus faecalis ELB9416, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026716. The strain has a clear origin and can be obtained repeatedly.

[0041] 2. This invention uses a single preserved bacterial strain as the core component, which is different from the complex bacterial community or spore mixture from donor feces. It has the advantages of clear composition, controllable quality, high batch consistency and high degree of standardization.

[0042] 3. The Enterococcus faecalis ELB9416 of the present invention has been evaluated for safety and has not shown typical pathogenic characteristics. It is hemolytic negative, has not detected typical high-risk virulence factors, has not detected high-risk horizontally transferable acquired drug resistance genes, and drug susceptibility testing has not indicated high-risk transferable drug resistance. Its safety profile is good.

[0043] 4. The Enterococcus faecalis ELB9416 of the present invention can be used to prepare preparations with a viable count not exceeding 10. 5A low-live-bacteria microecological composition with CFU / mL, compared to traditional high-live-bacteria probiotic products, helps reduce bloating, abdominal pain, diarrhea, or small intestinal bacterial overgrowth (i.e., controlling small intestinal bacteria to less than 10 CFU / mL) caused by high live bacteria intake. 3 CFU / g).

[0044] 5. The Enterococcus faecalis ELB9416 of the present invention can also be prepared as inactivated bacterial cells, bacterial lysates, cell wall components, fermentation broth, fermentation supernatant or metabolites. Even when the number of live bacteria is 0, it can still play a role in microecological regulation, breaking through the limitation of traditional probiotics that must rely on live bacteria to exert their effects.

[0045] 6. The microecological composition of the present invention can be used directly as a liquid fermentation broth or as an oral composition after concentration, formulation and addition of a protectant. It does not require intestinal cleansing, pretreatment with a laxative, or rectal enema. It is convenient to use and has good patient compliance.

[0046] 7. The microecological composition of the present invention can be prepared by conventional liquid fermentation process, without the need for donor recruitment, fecal collection and complex purification process. The process is simple, the cost is low, and the storage and transportation are relatively convenient, making it suitable for large-scale production.

[0047] 8. The Enterococcus faecalis ELB9416 or its microecological composition of the present invention is resistant to gastric acid and choline, and has the effects of improving memory, regulating intestinal flora, improving intestinal dysfunction, scavenging free radicals and / or antioxidation. It can be used to improve learning and memory ability, improve spatial memory ability, improve intestinal flora imbalance, promote intestinal microecological balance, and improve symptoms or conditions such as diarrhea, constipation, abdominal pain, abdominal distension, borborygmus, mucus stool, defecation discomfort or alternating diarrhea and constipation.

[0048] 9. The Enterococcus faecalis ELB9416 or its microecological composition of the present invention can exert a comprehensive microecological regulatory effect by inhibiting the excessive proliferation of conditionally pathogenic bacteria (such as Clostridium difficile), competitive exclusion, regulating the structure of intestinal flora, promoting the production of beneficial metabolites, improving the intestinal microecological environment and / or regulating the host immune response.

[0049] 10. The microecological composition of the present invention can be prepared as oral liquid, drops, suspension, granules, capsules, tablets, lyophilized powder or powder, with flexible dosage form selection, suitable for the development of food, health food, special dietary food, pharmaceuticals or other health products, and has good prospects for industrial application.

[0050] The foregoing and other aspects of this disclosure are set forth in more detail in the following description. Attached Figure Description

[0051] Figure 1The image shows a microscopic examination of the bacterial morphology of Enterococcus faecalis ELB9416. The strain is Gram-positive after Gram staining, and the bacteria are spherical or oval in shape, and can be arranged singly, in pairs or in chains.

[0052] Figure 2 This is a Circos genome diagram of Enterococcus faecalis ELB9416.

[0053] Figure 3 This is a photograph of the hemolytic test results for Enterococcus faecalis ELB9416. It shows that after culturing on blood agar plates, no clear hemolytic zone or greenish-yellow hemolytic ring formed around the colonies, indicating that this strain is hemolytic negative. Note: 1. Negative control bacteria: Listeria innocense (… Listeria innocua CICC 10417; 2. Positive control bacteria: Staphylococcus aureus ( Staphylococcus aureus CICC 10473; 3. Sample: ELB9416. Detailed Implementation

[0054] Unless otherwise defined, 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. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0055] Enterococcus faecalis ELB9416 was deposited on April 17, 2026, at the China Center for Type Culture Collection (Wuhan University, Wuhan, China) with accession number CCTCC NO: M 2026716 and classified as Enterococcus faecalis.

[0056] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0057] In this invention, the term "microecological composition" refers to a composition containing Enterococcus faecalis ELB9416 or its related components, which can be used to regulate the host microecology or produce corresponding physiological effects. The related components include, but are not limited to, live, low-viable, inactivated, and cell-lysate ELB9416 cells, cell wall components, fermentation broth, fermentation supernatant, and metabolites.

[0058] In this invention, the term "low viable count" refers to a composition in which live ELB9416 cells can still be detected, but the viable count is controlled at a low level. In some embodiments, the viable count of ELB9416 in the low viable count composition is no higher than 10. 5 CFU / mL.

[0059] In this invention, the term "inactivated bacterial cells" refers to ELB9416 bacterial cells that have lost their ability to be cultured and proliferated after being treated by heat treatment, moist heat treatment, pasteurization, ultraviolet treatment, irradiation treatment, high pressure treatment, or other methods. In some embodiments, the viable bacterial count in the microecological composition containing inactivated bacterial cells is 0.

[0060] In some embodiments, Enterococcus faecalis ELB9416 or its microecological composition of the present invention can inhibit the excessive proliferation of opportunistic pathogens by means of competitive exclusion, regulation of microbial community structure and improvement of intestinal microecological environment, thus providing new strain resources for microecological intervention of intestinal flora disorder related to opportunistic pathogens such as Clostridium difficile.

[0061] In some implementations, depending on the specific product application requirements, the ELB9416 seed culture can be inoculated into a culture medium containing different carbon sources, nitrogen sources, or plant additives, and then statically cultured at 37-39°C for 12-18 hours. When the pH of the culture medium drops below 3.8, the culture is terminated, yielding ELB9416 fermentation stock solution for different applications.

[0062] In some embodiments, the carbon source in the culture medium may be selected from cereals, legumes, tubers, molasses, glucose, fructose, sucrose, maltose, lactose, starch, dextrin, glycerol, mannitol, sorbitol, organic acids and their salts, and any combination thereof.

[0063] In some embodiments, the grains may be selected from one or more of corn, rice, wheat, barley, oats, sorghum, and millet; the legumes may be selected from one or more of soybeans, black beans, and mung beans; and the tubers may be selected from one or more of potatoes, sweet potatoes, and cassava.

[0064] In some embodiments, the nitrogen source in the culture medium may be selected from soybean meal, peanut meal, cottonseed meal, rapeseed meal, corn steep liquor, corn gluten meal, soybean peptone, wheat peptone, wheat bran, rice bran, yeast powder, yeast extract, and any combination thereof.

[0065] In some embodiments, one or more plant extracts or herbal additives may be added to the culture medium. The plant extracts or herbal additives may be selected from wolfberry, jujube, lotus seed, codonopsis, astragalus, angelica, schisandra, artemisia capillaris, lophatherum gracile, rehmannia glutinosa, hawthorn, codonopsis pilosula, gastrodia elata, tartary buckwheat, yam, notoginseng, coix seed, atractylodes macrocephala, tangerine peel, chrysanthemum, polygonatum sibiricum, jujube fruit, cordyceps militaris, kudzu root, mulberry twig, eucommia ulmoides, fig leaf, and any combination thereof; or selected from substances listed in the catalog of food and medicine homologous substances published by the National Health Commission.

[0066] The plant extracts or herbal additives are not used as the main nutrient source of the culture medium, and their addition does not change the basic performance of the culture medium in supporting the growth and fermentation of strain ELB9416.

[0067] In some embodiments, the culture medium may also contain food science, pharmaceutical science or nutritionally acceptable excipients or additives, which are food additives or food processing aids that comply with GB 2760 or other applicable food safety standards and are permitted for use in the corresponding food categories.

[0068] In one specific embodiment, ELB9416 has a MIC of 1 μg / mL against ampicillin, 2 μg / mL against vancomycin, 0.5 μg / mL against tetracycline, 4 μg / mL against chloramphenicol, 1 μg / mL against erythromycin, 16 μg / mL against gentamicin, 64 μg / mL against kanamycin, 128 μg / mL against streptomycin, and a MIC greater than 16 μg / mL against clindamycin.

[0069] In some embodiments, the microecological compositions of the present invention or products containing them are suitable for subjects who require improved memory, regulation of gut microbiota, regulation of gastrointestinal function, scavenging of free radicals and / or antioxidant effects.

[0070] In some implementations, the subjects are infants, children, adolescents, adults, the elderly, people with sensitive intestines, people with gastrointestinal dysfunction, people with constipation, people with diarrhea, people with loss of appetite, people with irritable bowel syndrome-related symptoms, people with elevated levels of oxidative stress, or people at risk of memory decline.

[0071] The present disclosure will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the embodiments that do not specify specific conditions are performed under conventional conditions known in the art or under conditions recommended by the manufacturer. Example

[0072] Example 1: Isolation, purification and preparation of fermentation stock broth of Enterococcus faecalis ELB9416

[0073] Enterococcus faecalis ELB9416 was isolated from fecal samples of healthy infants.

[0074] Laboratory isolation and purification of bacterial strains: Under aseptic conditions, the sample is appropriately diluted and inoculated or plated onto MRS agar plates, and incubated at 36-40℃ for 36-72 hours. After incubation, single colonies are picked and streaked onto MRS agar plates for purification. The purification and incubation are repeated until morphologically consistent single colonies are obtained, and Gram staining and microscopic examination are performed to confirm the purification status of the strain.

[0075] Preparation of slant culture for production: Take a purified single colony of ELB9416, inoculate it into MRS agar slant medium, and incubate at 36-40℃ for 36-72 hours until the bacterial cells cover the slant, thus obtaining the slant culture for production.

[0076] Seed culture for production: The slant culture for production is inoculated into MRS liquid medium and incubated at 36-40℃ for 12-18 hours. The culture is terminated when the pH of the culture medium drops below 3.8 to obtain the seed culture for production.

[0077] Product fermentation: The seed liquid for production is inoculated into MRS liquid medium at an inoculation rate of 2%-5%, and cultured statically at 36-40℃ for 12-18 hours. The culture is ended when the pH of the culture medium drops below 3.8 to obtain ELB9416 fermentation stock solution.

[0078] Example 2: Colony morphology and microscopic morphology of Enterococcus faecalis ELB9416

[0079] Enterococcus faecalis ELB9416 obtained in Example 1 was inoculated onto MRS agar plates and cultured at 36-40℃ for 36-72 hours. Colony morphology was then observed.

[0080] The results showed that ELB9416 formed white colonies on MRS medium. The colonies were round, moist, opaque, and had neat edges.

[0081] Bacterial cells were collected, Gram-stained, and observed under a microscope. The results are as follows: Figure 1 As shown. By Figure 1 As can be seen, ELB9416 is a Gram-positive bacterium with elongated oval cells, arranged singly, in pairs, or in chains. These results indicate that ELB9416 possesses the typical morphological characteristics of Enterococcus faecalis.

[0082] Example 3: 16S rRNA gene sequence and GTDB classification of Enterococcus faecalis ELB9416

[0083] Genomic DNA was extracted from strain ELB9416, and its 16S rRNA gene was amplified and sequenced, yielding a 1500 bp 16S rRNA gene sequence (SEQ ID NO: 1). The obtained 16S rRNA gene sequence was compared with known bacterial 16S rRNA gene sequences in the database.

[0084] The comparison results showed that ELB9416 and *Enterococcus faecalis* had high homology (similarity greater than 99%). To further confirm the taxonomic position of ELB9416, genomic classification and annotation analysis of ELB9416 was performed using the GTDB (Genome Taxonomy Database) and its accompanying classification tool GTDB-Tk, based on the whole-genome sequencing data of this strain. The analysis results showed that ELB9416 belongs to the domain Bacteria, phylum Bacillota, class Bacilli, order Lactobacillales, family Enterococaceae, and genus *Enterococcus* in the GTDB classification system, and the classification result is *Enterococcus faecalis*.

[0085] In summary, based on the 16S rRNA gene sequence alignment results, GTDB genome classification analysis results, colony morphology, microscopic morphology, Gram staining results, and whole genome sequencing analysis results, ELB9416 was identified as Enterococcus faecalis.

[0086] Example 4: Whole genome sequencing analysis of Enterococcus faecalis ELB9416

[0087] Genomic DNA was extracted from strain ELB9416, and whole-genome sequencing was performed using a high-throughput sequencing platform. The sequencing data were then assembled, annotated, and analyzed. Circos diagrams of the sample sequences were drawn using Circos software.

[0088] The genomic analysis results of ELB9416 are as follows: Figure 2 As shown. Notes on the Circos genome diagram: From the outside in, the diagram consists of: (1) the genome backbone; (2–3) the annotation functional elements on the positive (upper) and negative (lower) strands, including protein-coding genes (CDS, gray), rRNA (light red), tRNA (light green), non-coding RNA (ncRNA, cyan) and CRISPR array (purple); (4) the deviation of GC content from the whole genome average (green: above average; red: below average); (5) GC skew (orange: positive; blue: negative), with the origin of replication (oriC) marked.

[0089] The results showed that the genome size of strain ELB9416 was approximately 2.7 Mbp. Genome annotation results indicated that this strain contains genes related to DNA replication, transcription, translation, carbohydrate metabolism, amino acid metabolism, cell wall synthesis, stress response, substance transport, and microecological adaptation.

[0090] Based on the results of 16S rRNA gene sequence alignment, morphological observation, and whole genome sequencing analysis, ELB9416 was identified as belonging to Enterococcus faecalis.

[0091] Example 5: Safety evaluation of Enterococcus faecalis ELB9416

[0092] To evaluate the safety of Enterococcus faecalis ELB9416 as the core strain of the microecological composition, hemolytic activity was detected, whole-genome virulence factor analysis was performed, whole-genome drug resistance genes and transferability were assessed, and antimicrobial susceptibility phenotypes were verified.

[0093] 1. Hemolytic test

[0094] Enterococcus faecalis ELB9416 was inoculated onto blood agar plates and anaerobically cultured at 37°C for 48 hours. The presence of transparent or grass-green hemolytic zones around the colonies was then observed.

[0095] The results are as follows Figure 3 As shown, no transparent hemolytic zone or greenish hemolytic ring was observed around the ELB9416 colony, indicating that it was hemolysis-negative (i.e., γ-hemolysis). This result indicates that ELB9416 did not exhibit typical pathogenic hemolytic activity.

[0096] 2. Genome-wide virulence factor analysis

[0097] Whole genome sequencing was performed on Enterococcus faecalis ELB9416, and the predicted coding genes were compared with the virulence factor database VFDB.

[0098] The results showed that this strain did not contain typical bacterial virulence genes such as hemolysin (hly), enterotoxin, invasivein, and type III / IV secretion system. The annotated homologous sequences mainly consisted of adhesion proteins essential for maintaining intestinal colonization (such as laminin-binding protein lmb, collagen adhesion protein ace, and the pili assembly system ebpABC) and housekeeping proteins related to antioxidant stress (such as superoxide dismutase sodB and molecular chaperones groEL / dnaK). These factors are widely present in non-pathogenic lactic acid bacteria / enterococci and do not possess the function of tissue destruction or systemic infection.

[0099] 3. Assessment of genome-wide drug resistance genes and their transferability

[0100] The ELB9416 genome was scanned for resistance genes and its transferability risk was assessed using the comprehensive antibiotic resistance database CARD.

[0101] The results showed that ELB9416 did not detect any typical acquired drug resistance genes that can be horizontally transferred, including but not limited to macrolide resistance genes ermA, ermB or ermC, tetracycline resistance genes tetM, tetL or tetK, glycopeptide resistance genes vanA, vanB or vanC, aminoglycoside modifying enzyme gene aac(6')-Ie-aph(2''), and chloramphenicol acetyltransferase gene cat, etc.

[0102] The sequences rpoB, gyrA, parC, fusA, murA, liaR, liaS, and liaF detected in the database comparison are mainly housekeeping genes or genes related to basic life activities inherent in bacterial chromosomes, such as DNA gyrase, RNA polymerase, cell wall synthesis-related proteins, and signal regulation-related proteins. The detection of these sequences is due to their homology with wild-type sequences of drug resistance mutation-related proteins in the database, and does not indicate that ELB9416 carries exogenous drug resistance determinants mediated by plasmids, transposons, or other mobile genetic elements.

[0103] Further plasmid sequence analysis revealed no identifiable plasmid sequences in ELB9416. Publicly available genomic information shows that some applied or publicly available Enterococcus faecalis strains can carry plasmids; for example, Enterococcus faecalis EF2001 carries two plasmids. Compared to these plasmid-carrying strains, the absence of identifiable plasmids in the genomic characteristic of ELB9416 suggests that it lacks a clear genomic basis for plasmid-mediated exogenous gene transfer, which is beneficial in reducing the potential risk of horizontal transfer of drug resistance genes or virulence-related genes.

[0104] 4. Antimicrobial susceptibility phenotype verification

[0105] The minimum inhibitory concentration (MIC) of ELB9416 against commonly used antimicrobial drugs was determined using the microbroth dilution method. The MIC results are shown in Table 1.

[0106] Table 1. MIC results of ELB9416 drug susceptibility test

[0107]

[0108] Note: 1. MIC: Minimum inhibitory concentration.

[0109] As shown in Table 1, strain ELB9416 exhibited MIC values ​​within the sensitive range of the CLSI / EUCAST standard for ampicillin (1 μg / mL), vancomycin (2 μg / mL), tetracycline (0.5 μg / mL), chloramphenicol (4 μg / mL), erythromycin (1 μg / mL), gentamicin (16 μg / mL), kanamycin (64 μg / mL), streptomycin (128 μg / mL), and clindamycin (>16 μg / mL). This result confirms that although some endogenous or low-level efflux pump-related genes associated with enterococci (such as lsaA and emeA) can be detected in the genome, they do not lead to a clinically significant drug resistance phenotype.

[0110] Based on the comprehensive results of hemolytic activity detection, whole-genome virulence factor analysis, whole-genome resistance gene and transferability assessment, and antimicrobial susceptibility phenotype verification, ELB9416 showed no typical hemolytic activity, no typical high-risk virulence factors, no high-risk horizontally transferable acquired resistance genes, and no high-risk transferable resistance phenotype.

[0111] The above results indicate that ELB9416 has a good safety profile and is suitable for development as a core strain in microecological compositions.

[0112] Example 6: The effect of Enterococcus faecalis ELB9416 on improving memory

[0113] This embodiment uses animal experiments to evaluate the effects of ELB9416 and its metabolites on memory function.

[0114] Experimental animals were selected and randomly divided into control and experimental groups according to sex and weight. The experimental group was given samples containing ELB9416 and its metabolites, while the control group was given corresponding control samples. After sample administration, the animal memory function was assessed using the dark avoidance method. The latency before the animal received electrical stimulation in the dark room and the number of electrical stimulations received within 5 minutes were recorded. A longer average latency and fewer stimulations indicated a stronger ability to retain electrical stimulation and better memory function.

[0115] The results of the dark avoidance method are shown in Table 2.

[0116] Table 2. Effects of ELB9416 and its metabolites on animal memory function

[0117]

[0118] As shown in Table 2, in female animals, compared with the control group, the average latency of the experimental group increased from 125 seconds to 250 seconds, and the average number of hits decreased from 2.7 to 0.6. In male animals, compared with the control group, the average latency of the experimental group increased from 130 seconds to 240 seconds, and the average number of hits decreased from 3.1 to 1.2.

[0119] The above results indicate that ELB9416 and its metabolites can prolong the latency period in the darkness avoidance test and reduce the number of times the animal is struck within 5 minutes, suggesting that they can improve the animal's learning and memory abilities in response to electrically stimulated stimuli, and enhance memory acquisition, consolidation, and retrieval. Statistical analysis showed that the differences between the experimental group and the control group were statistically significant.

[0120] Example 7: Regulatory effects of Enterococcus faecalis ELB9416 and its metabolites on intestinal flora

[0121] This embodiment uses a mouse intestinal flora imbalance model to evaluate the regulatory effects of ELB9416 and its metabolites on the intestinal flora.

[0122] Male Kunming mice, weighing 18-20g, were randomly divided into a normal control group, a rhubarb model group, and a rhubarb plus ELB9416 group. The rhubarb model group and the rhubarb plus ELB9416 group were administered 1mL of rhubarb solution by gavage daily for 8 consecutive days to establish an animal model of intestinal flora imbalance. During the modeling process, mice exhibited diarrhea, loss of appetite, piloerection, and dull fur, indicating the establishment of the flora imbalance model.

[0123] After the experiment, the mice were euthanized, and the contents of the cecum were collected under aseptic conditions to detect the quantities of major intestinal flora, including Enterococcus, Escherichia coli, Lactobacillus, Bacteroides, and Bifidobacterium. The results are shown in Table 3.

[0124] Table 3. Effects of ELB9416 and its metabolites on intestinal flora imbalance in mice

[0125]

[0126] Note: The values ​​in the table are the logarithmic mean ± standard deviation of bacterial counts in 24 mice.

[0127] As shown in Table 3, compared with the normal control group, the number of Enterococcus, Lactobacillus and Bifidobacterium in the intestine of mice in the rhubarb model group decreased, while the number of Escherichia coli increased, indicating that rhubarb modeling can cause intestinal flora imbalance.

[0128] Compared with the rhubarb model group, the rhubarb + ELB9416 group showed an increase in the number of Enterococci, Lactobacilli, and Bifidobacteria in the intestines of mice, a decrease in the number of Escherichia coli, and a number of Bacteroides approaching the level of the normal control group. These results indicate that ELB9416 and its metabolites can improve intestinal flora imbalance, promote intestinal microecological balance, increase the number of beneficial bacteria, and inhibit the excessive proliferation of opportunistic pathogens or abnormally proliferating bacteria.

[0129] Further investigation was conducted into the effects of ELB9416 and its metabolites on the gut microbiota of normal mice. The results are shown in Table 4.

[0130] Table 4. Effects of ELB9416 and its metabolites on the number of Bifidobacteria in normal mice

[0131]

[0132] As shown in Table 4, compared with the normal control group, the number of Bifidobacteria in the intestine of mice in the ELB9416 group was significantly increased, and the difference was statistically significant (P < 0.01).

[0133] In summary, ELB9416 and its metabolites have a regulatory effect on intestinal flora imbalance and can increase the number of beneficial bacteria in the gut, especially increasing the level of Bifidobacteria.

[0134] Example 8: The effect of Enterococcus faecalis ELB9416 and its metabolites on improving gastrointestinal symptoms associated with irritable bowel syndrome.

[0135] This embodiment evaluates the effect of an oral composition containing ELB9416 and its metabolites on improving gastrointestinal symptoms associated with irritable bowel syndrome through clinical observation.

[0136] Thirteen patients meeting the diagnostic criteria for irritable bowel syndrome (IBS) were selected, including 8 males and 5 females, with an average age of 32 years and an average disease duration of 2-3 years. The included patients presented with symptoms such as abdominal pain, bloating, borborygmus, constipation, diarrhea, or alternating diarrhea and constipation, and organic diseases were ruled out by relevant examinations.

[0137] The patient was given an oral combination containing ELB9416 and its metabolites, 20 mL three times daily, for one month. Antibiotics and other similar probiotics were discontinued during treatment. The frequency and characteristics of bowel movements, the duration of diarrhea relief and cessation, clinical symptoms and signs, and any side effects were observed before and after treatment.

[0138] The criteria for judging the efficacy are as follows:

[0139] Significant effect: Bowel movement frequency returns to normal, stool consistency is normal, there is no obvious discomfort during defecation, no tenesmus or only mild abdominal pain, and routine stool examination is normal.

[0140] Effective: The above indicators reach or improve from their original state within 3-7 days after taking the medication, but do not fully return to normal.

[0141] Ineffective: No significant difference before and after treatment.

[0142] The results of changes in stool characteristics are shown in Table 5.

[0143] Table 5. Effects of ELB9416 and its metabolites on stool characteristics in patients with irritable bowel syndrome.

[0144]

[0145] As shown in Table 5, after taking the oral composition containing ELB9416 and its metabolites, the number of patients with loose stools, watery stools and mucus stools decreased, and the stool characteristics tended to be formed and soft.

[0146] The results of the changes in bowel movement frequency are shown in Table 6.

[0147] Table 6. Effects of ELB9416 and its metabolites on bowel frequency in patients with irritable bowel syndrome

[0148]

[0149] As shown in Table 6, after taking the medication, the number of daily diarrhea episodes decreased from 5.0±2 times to 1.0±1.1 times, and the difference was statistically significant (P<0.001). Among the 4 patients with diarrhea, 3 patients' diarrhea began to improve after 5 days, and the diarrhea stopped after 5±2 days.

[0150] The improvement of symptoms is shown in Table 7.

[0151] Table 7. The effect of ELB9416 and its metabolites on the symptoms of irritable bowel syndrome.

[0152]

[0153] As shown in Table 7, the oral composition containing ELB9416 and its metabolites can improve symptoms such as diarrhea, abdominal pain, bloating, constipation, and alternating diarrhea and constipation. No significant side effects were observed during the treatment.

[0154] The above results indicate that ELB9416 and its metabolites can regulate gastrointestinal function and improve symptoms associated with irritable bowel syndrome, such as diarrhea, constipation, abdominal pain, bloating, abnormal stool characteristics, abnormal defecation frequency, and defecation discomfort.

[0155] Example 9: The ameliorative effect of Enterococcus faecalis ELB9416 and its metabolites on gastrointestinal dysfunction.

[0156] This embodiment evaluates the effect of an oral composition containing ELB9416 and its metabolites on improving gastrointestinal dysfunction through clinical observation.

[0157] Twenty-one critically ill patients with gastrointestinal dysfunction in the general ICU were selected, including 11 males and 10 females, with a mean age of 44.1 years. The patients' gastrointestinal manifestations included abdominal distension, decreased or absent bowel sounds, tympanic percussion sounds in the abdomen, constipation or diarrhea, etc. Among them, 13 patients presented with constipation and 8 patients presented with diarrhea.

[0158] All patients were administered an oral combination containing ELB9416 and its metabolites via gastric tube, 60 mL each time, three times daily. Bowel sounds, abdominal distension, bowel regularity, stool characteristics, and improvement in gastrointestinal symptoms were observed.

[0159] The criteria for judging the efficacy are as follows:

[0160] Significant effect: Bowel sounds return to normal, abdominal distension disappears, bowel movements are smooth and of normal consistency, and gastrointestinal symptoms are significantly improved.

[0161] Effective: The above indicators did not fully meet the criteria for significant effect, but there was improvement.

[0162] Ineffective: Gastrointestinal symptoms do not improve or worsen.

[0163] The results showed that among the 21 patients, 8 showed significant improvement, 10 showed improvement, and 3 showed no improvement, with a total of 18 patients showing improvement and an overall effective rate of 85.7%. No obvious toxic side effects were observed during the treatment.

[0164] The above results indicate that ELB9416 and its metabolites can improve gastrointestinal dysfunction, specifically by improving abdominal distension, decreased bowel sounds, constipation, diarrhea, abnormal stool characteristics, and abnormal defecation function.

[0165] Example 10: The effect of Enterococcus faecalis ELB9416 and its metabolites on improving constipation and loss of appetite.

[0166] This embodiment evaluates the effect of an oral composition containing ELB9416 and its metabolites on improving constipation and loss of appetite in children through clinical observation.

[0167] Thirty children (15 boys and 15 girls) aged 5 months to 8 years who presented with constipation and / or poor appetite were selected. The children were given an oral composition containing ELB9416 and its metabolites, 10-30 mL three times daily, for 7 consecutive days. The efficacy was then observed.

[0168] The observation indicators included daily bowel movement frequency, stool consistency, and appetite. The criteria for judging the therapeutic effect based on bowel movement characteristics are as follows:

[0169] Significant effect: Bowel movement intervals are reduced by more than 2 days compared to before, and the stool consistency changes from hard to soft;

[0170] Effective: Bowel movement intervals are reduced by 1 day compared to before, or the stool consistency changes from hard to soft;

[0171] Ineffective: No improvement in constipation and bowel movements.

[0172] Appetite is categorized as good, moderate, or poor. The criteria for judging the effectiveness of appetite improvement are as follows:

[0173] Significant effect: Appetite improved from poor to good;

[0174] Effective: Appetite improves from poor to moderate, or from moderate to good;

[0175] Ineffective: No change in appetite.

[0176] The results showed that, regarding bowel movements, 13 out of 30 children (43%) showed significant improvement, and 16 (54%) showed improvement, with a total effective rate of 97%. Regarding appetite, 6 out of 30 children (20%) showed significant improvement, and 14 (47%) showed improvement, with a total effective rate of 67%. No significant side effects were observed during the treatment.

[0177] The above results indicate that ELB9416 and its metabolites can improve constipation in children, increasing the frequency of bowel movements, shortening the interval between bowel movements, and changing the stool from hard to soft; at the same time, they can also improve loss of appetite and poor digestion.

[0178] Example 11: Free radical scavenging effects of Enterococcus faecalis ELB9416 and its metabolites

[0179] In this embodiment, the free radical scavenging effect of ELB9416 and its metabolites was evaluated using the xanthine oxidase method.

[0180] Samples containing ELB9416 and its metabolites were used as test samples, and their ability to scavenge oxidative free radicals was detected using the xanthine oxidase method. The inhibition rate of the free radical luminescence signal was calculated using the free radical luminescence number 21128 as a baseline.

[0181] The results showed that ELB9416 and its metabolites had a significant scavenging effect on oxidative free radicals, with a free radical inhibition rate of 76%.

[0182] The above results indicate that ELB9416 and its metabolites have free radical scavenging effects and can be used to prepare free radical scavenging and / or antioxidant products.

[0183] Example 12: Antioxidant-related effects of Enterococcus faecalis ELB9416 and its metabolites

[0184] This embodiment evaluates the antioxidant effects of ELB9416 and its metabolites by detecting the activity of superoxide dismutase in erythrocytes and the content of malondialdehyde in myocardial tissue.

[0185] Experimental animals were randomly divided into a control group and a sample group according to sex and weight. The sample group was given a sample containing ELB9416 and its metabolites, while the control group was given the corresponding control sample. After sample administration, the activity of superoxide dismutase (SOD) in blood erythrocytes and the content of malondialdehyde (MDA) in myocardial tissue were measured.

[0186] The results of the blood red blood cell SOD activity test are shown in Table 8.

[0187] Table 8. Effects of ELB9416 and its metabolites on SOD activity in blood erythrocytes

[0188]

[0189] As shown in Table 8, in female animals, compared with the control group, the SOD activity of erythrocytes in the sample group increased from 156.2±10.7 U / mL to 171.3±11.2 U / mL; in male animals, compared with the control group, the SOD activity of erythrocytes in the sample group increased from 165.3±8.6 U / mL to 181.3±9.3 U / mL. Statistical analysis showed that the difference between the sample group and the control group was statistically significant (P<0.05).

[0190] The results of MDA detection in myocardial tissue are shown in Table 9.

[0191] Table 9. Effects of ELB9416 and its metabolites on MDA content in myocardial tissue

[0192]

[0193] As shown in Table 9, the MDA content in the myocardial tissue of the control group was 37.1 nmol / g, while that of the experimental group was 35.02 nmol / g. Compared with the control group, the MDA content in the myocardial tissue of the experimental group was lower, with a reduction rate of 5.6%. Statistical analysis showed that the difference between the experimental group and the control group was statistically significant (P < 0.05).

[0194] The above results indicate that ELB9416 and its metabolites can increase the SOD activity of blood erythrocytes and reduce the MDA content of myocardial tissue, thus having antioxidant and lipid peroxidation-reducing effects.

[0195] Those skilled in the art will understand that many and various modifications can be made without departing from the spirit of the invention. Therefore, it should be clearly understood that the form of the invention is merely illustrative and is not intended to limit the scope of the invention.

[0196] All publications, patent applications, patents, patent publications, database accession number sequences, and other references mentioned herein are incorporated in their entirety by reference for the teachings relating to the sentences and / or paragraphs presenting those references.

[0197] The foregoing is illustrative of the invention and should not be construed as limiting it. The invention is defined by the appended claims, including their equivalents.

Claims

1. A strain of Enterococcus faecalis, characterized in that, The strain is Enterococcus faecalis ( Enterococcus faecalis ELB9416 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026716.

2. The Enterococcus faecalis strain according to claim 1, characterized in that, The strain is a Gram-positive bacterium.

3. A microecological composition, characterized in that, It contains Enterococcus faecalis ELB9416 as described in claim 1 or 2.

4. The microecological composition according to claim 3, characterized in that, The microecological composition further comprises one or more of the following: inactivated cells of Enterococcus faecalis ELB9416, cell lysates, cell wall components, fermentation broth, fermentation supernatant, and metabolites.

5. The microecological composition according to claim 3 or 4, characterized in that, The microecological composition is an oral preparation, preferably an oral liquid, drops, suspension, granules, capsules, tablets, lyophilized powder, or powder.

6. The microecological composition according to any one of claims 3 to 5, characterized in that, The total bacterial count of Enterococcus faecalis ELB9416 in the microecological composition is higher than 10. 7 CFU / mL and viable count not higher than 10 5 CFU / mL.

7. The microecological composition according to claim 6, characterized in that, The Enterococcus faecalis ELB9416 in the microecological composition is an inactivated bacterial cell, and the number of live bacteria in the microecological composition is 0.

8. The use of Enterococcus faecalis ELB9416 as described in claim 1 or 2, or the microecological composition as described in any one of claims 3 to 7, in the preparation of products for improving memory. Preferably, the improvement of memory includes one or more of the following: improving learning memory ability, improving spatial memory ability, improving memory acquisition, improving memory consolidation, and improving memory retrieval.

9. The use of Enterococcus faecalis ELB9416 as described in claim 1 or 2, or the microecological composition as described in any one of claims 3 to 7, in the preparation of products for regulating intestinal flora. Preferably, the regulation of intestinal flora includes improving intestinal flora imbalance, promoting intestinal microecological balance, increasing the number of beneficial bacteria and / or inhibiting the excessive proliferation of conditionally pathogenic bacteria.

10. The use of Enterococcus faecalis ELB9416 as described in claim 1 or 2, or the microecological composition as described in any one of claims 3 to 7, in the preparation of products for regulating gastrointestinal function. Preferably, the regulation of gastrointestinal function includes improving one or more of the following: constipation, diarrhea, abdominal pain, abdominal distension, borborygmus, abnormal stool characteristics, abnormal defecation frequency, defecation discomfort, tenesmus, post-defecation discomfort, loss of appetite, or poor appetite.

11. The use of Enterococcus faecalis ELB9416 as described in claim 1 or 2, or the microecological composition as described in any one of claims 3 to 7, in the preparation of products for scavenging free radicals and / or for antioxidation.

12. The use of Enterococcus faecalis ELB9416 as described in claim 1 or 2, or the microecological composition as described in any one of claims 3 to 7, in the preparation of a medicament for the prevention or treatment of irritable bowel syndrome.

13. A method for preparing the microecological composition according to any one of claims 3 to 7, characterized in that, Includes the following steps: Enterococcus faecalis ELB9416 as described in claim 1 or 2 was inoculated into a culture medium for fermentation, and the fermentation products were collected. The fermentation product is prepared into a microecological composition containing live bacteria, low-live bacteria, inactivated bacteria, bacterial lysates, cell wall components, fermentation broth, fermentation supernatant and / or metabolites.

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