Enterococcus faecium and application thereof

Abstract

The invention provides enterococcus faecium with excellent probiotic characteristics. The enterococcus faecium is an original strain obtained by separating and purifying healthy infant excrement. The enterococcus faecium strain is identified by the China General Microbiological Culture Collection Center and is classified and named as enterococcus faecium, and the preservation number is CGMCC NO.33919. The enterococcus faecium obtained after separation, purification and subculture of the enterococcus faecium has the characteristics of high gastric acid resistance, high cholate resistance, high adhesion and the like. The enterococcus faecium can effectively improve diarrhea of mice caused by escherichia coli, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of probiotic research, specifically to a type of Enterococcus faecalis and its applications. Background Technology

[0002] Infectious diarrhea is highly prevalent and has a high incidence rate. The medical and economic burden caused by diarrheal diseases is severe in developing countries, making it a significant global public health issue. The World Health Organization (WHO) estimates that tens of millions of people worldwide suffer from diarrheal diseases every day, with 1.7 billion cases annually, resulting in 2.2 million deaths from severe diarrhea. Among bacterial infections causing diarrhea, Escherichia coli is the most frequent cause. Global estimates of the prevalence of specific types of bacterial diarrhea among all causes of diarrhea include Escherichia coli 10%-25%, Shigella 10%, Salmonella 3%, and Campylobacter 3%-6%.

[0003] The principles of diarrhea treatment are to remove and treat the underlying cause, prevent and correct dehydration, provide nutritional support, and use medication rationally. Currently, the traditional treatment for diarrhea is antibiotics. However, long-term, improper use of antibiotics suppresses or kills most sensitive bacteria and normal flora, while drug-resistant bacteria proliferate due to the selective effect of antibiotics. Therefore, long-term, high-dose use of broad-spectrum antibiotics is a common cause of dysbiosis. Antibiotic-associated diarrhea (AAD) is a common complication of clinical antibiotic use. Using probiotics concurrently with antibiotics can significantly reduce the incidence and severity of AAD.

[0004] As normal gut flora, probiotics can effectively regulate the gut microbiota, restore normal microecological balance, and resist the colonization and invasion of pathogens. Therefore, probiotics exhibit good preventive and therapeutic effects in cases of acute and chronic diarrhea, as well as intestinal flora imbalance caused by antibiotics. It is generally believed that the main functions of probiotics are: altering the composition of the gut microbiota, enhancing the intestinal mucosal barrier function, regulating intestinal mucosal immunity, and inhibiting pathogen colonization, thereby improving the body's resistance to diarrhea. Therefore, developing probiotics with high tolerability and high adhesion to treat diarrhea and antibiotic-induced intestinal flora imbalance is very important. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a *Enterococcus faecium* and its applications. This *Enterococcus faecium* is suitable for the treatment of diarrhea and has promising application prospects.

[0006] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0007] This invention provides a self-selected Enterococcus faecium with high tolerance and adhesive activity, classified and named Enterococcus faecium. This strain was deposited at the China General Microbiological Culture Collection Center on March 21, 2025, with accession number CGMCC NO.: 33919.

[0008] The present invention also discloses the application of the above-mentioned Enterococcus faecalis in improving diarrhea caused by Escherichia coli.

[0009] Furthermore, the Enterococcus faecalis includes the aforementioned Enterococcus faecalis or the aforementioned Enterococcus faecalis suspension.

[0010] Furthermore, the method for preparing the Enterococcus faecalis bacterial suspension includes the following steps: inoculating Enterococcus faecalis into a solid slant culture medium for activation, performing seed culture on the activated strain to obtain a primary seed liquid, and inoculating the primary seed liquid into a broth culture medium for fermentation culture to obtain the Enterococcus faecalis bacterial suspension.

[0011] Furthermore, the solid slant culture medium is an MRS solid culture medium, the components of which include: 1% peptone, 0.8% beef extract, 0.4% yeast extract, 2% glucose, 0.2% dipotassium hydrogen phosphate, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.02% magnesium sulfate, 0.004% manganese sulfate, 0.1% Tween 80, and 1.4% agar.

[0012] Furthermore, the components of the expanded culture medium include: 1% peptone, 0.8% beef extract, 0.4% yeast extract, 2% glucose, 0.2% dipotassium hydrogen phosphate, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.02% magnesium sulfate, 0.004% manganese sulfate, and 0.1% Tween 80.

[0013] Furthermore, the culture temperature for the Enterococcus faecalis is 35-37℃.

[0014] Furthermore, the culture time for the Enterococcus faecalis suspension is 20-24 hours.

[0015] Furthermore, the components of the fermentation medium include: 1% peptone, 0.8% beef extract, 0.4% yeast extract, 2% glucose, 0.2% dipotassium hydrogen phosphate, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.02% magnesium sulfate, 0.004% manganese sulfate, and 0.1% Tween 80.

[0016] Furthermore, the Enterococcus faecalis suspension is cultured at 35-37°C for 20-24 hours.

[0017] Furthermore, the primary seed culture was inoculated into shake flask culture medium at an inoculation rate of 2% for fermentation culture.

[0018] The Enterococcus faecalis colonies are milky white, round, opaque, and have a smooth surface. This strain exhibits good acid and heat resistance, and maintains excellent fermentation characteristics even under multiple pressure stresses. It is an important core functional microorganism for brewing, with a growth temperature range of 35-37℃ and an optimal growth pH range of 5.0-7.5.

[0019] The preparation method of the above-mentioned Enterococcus faecalis bacterial suspension includes: inoculating Enterococcus faecalis into a solid slant culture medium for activation, with a culture temperature of 35-37℃ and static culture for 24-48 hours; inoculating the activated solid plate strain into a primary seed culture medium for primary seed expansion culture, with a culture temperature of 35-37℃ and static culture for 20-24 hours; inoculating the primary seed culture into a fermentation medium at an inoculation rate of 2% for expansion culture, with a culture temperature of 35-37℃ and static culture for 20-24 hours, and collecting the bacterial suspension for later use.

[0020] The present invention also provides the application of the above-mentioned Enterococcus faecalis or the above-mentioned Enterococcus faecalis suspension in the treatment of diarrhea.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. High Stress Resistance and Cost-Effectiveness: This strain is not only widely available and inexpensive, but also maintains a high survival rate in extreme environments (such as strong acid and high bile salt conditions), demonstrating excellent adaptability. When the activated strain was inoculated into artificial gastric fluid (pH = 3.0 ± 0.5) and cultured at 37°C for 2 hours, the survival rate still reached 86.2%. When the activated strain was inoculated into a bacterial culture medium solution containing 0.3% bile salts and cultured at 37°C for 4 hours, the survival rate still reached 69.7%.

[0023] 2. Strong adhesion: It exhibits a high adhesion index of 57.3% when co-cultured with Caco-2 cells, confirming that it is a highly adhesive strain with excellent colonization effect, which provides a foundation for its application in biomedicine and environmental science.

[0024] 3. Effectively improves diarrhea in mice: After administering Enterococcus faecalis suspension to diarrhea-prone mice via gavage for 8 days, the survival rate of these mice was observed to be high, and the rate of loose stools and diarrhea index were significantly reduced. Serum inflammatory factor levels were also significantly lower than those in the model group. This strain can also increase the number of beneficial intestinal bacteria and reduce the number of potential pathogens to some extent. Attached Figure Description

[0025] Figure 1 The morphology of Enterococcus faecalis (YF-M-02) of the present invention on a solid culture medium.

[0026] Figure 2Microscopic image of Enterococcus faecalis (YF-M-02) of this invention.

[0027] Figure 3 The survival rate of multiple intestinal strains of the present invention in artificial gastric fluid for 2 hours.

[0028] Figure 4 The survival rate of multiple intestinal strains of the present invention in 0.3% bile salt medium for 4 hours is shown.

[0029] Figure 5 This invention demonstrates the adhesion effect of multiple intestinal strains on Caco-2 cells.

[0030] Figure 6 This image shows the effect of Enterococcus faecalis (YF-M-02) of the present invention in reducing inflammatory factors in diarrheal mice.

[0031] Figure 7 This represents the changes in the gut microbiota of mice at the genus level. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms, or direct or indirect applications in other related technical fields, made by those skilled in the art, shall fall within the scope defined by the appended claims.

[0033] Example 1: Screening of strains

[0034] (1) Collect infant stool samples using sterile test tubes and cotton swabs, transport them back to the laboratory at low temperature, and perform serial dilutions of the infant stool samples using sterile physiological saline: 10 1 10 2 10 3 10 4 10 5 Take 0.1 mL of each serial dilution and spread it on solid culture medium (culture medium formula: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, 14.0 g agar, 1000 mL purified water) and incubate at 37°C for 48 h.

[0035] (2) Select different colonies and streak them onto solid culture medium (culture medium formula: 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, 14.0g agar, 1000mL purified water), and incubate at 37℃ for 48h. Observe whether the isolated colonies are purified. If there are contaminating bacteria, repeat the step until a pure culture is obtained.

[0036] (3) The purified strain was inoculated into liquid culture medium (10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, 1000mL purified water) and incubated at 37℃ for 24h.

[0037] (4) Take 0.2 mL of the above bacterial culture and inoculate it into 10 mL of culture medium, and incubate at 37℃ for 24 h. Subsequently, inoculate it into artificial gastric juice and culture medium containing 0.3% bile salts to detect its survival rate. Then, co-culture it with Caco-2 cells to test its adhesion performance, thereby screening out Enterococcus faecalis with excellent probiotic characteristics. This strain is an excellent probiotic strain, and the strain is further identified.

[0038] The aforementioned Enterococcus faecalis was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 33919.

[0039] Example 2: Identification of bacterial strains

[0040] The strain YF-M-02, exhibiting good probiotic characteristics, was cultured on MRS medium at 37°C for 48 hours. The strain was then identified, and the results are as follows: Gram-positive, no spores or flagella, colonies were milky white, round, opaque, and smooth. Figure 1 The results of the 16S rRNA gene sequence determination of the bacterial strain are shown below. Based on the comprehensive analysis of experimental data such as the culture characteristics, microscopic characteristics, and 16S rRNA gene sequence of the bacterial strain, the strain was identified as Enterococcus faecium.

[0041] The 16S rRNA gene sequence of the *Enterococcus faecalis* is as follows:

[0042] CTTCTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGG

[0043] GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGG

[0044] TGCTAATACCGTATAACAATCAAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGG

[0045] GTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCA

[0046] CCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGA

[0047] GACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACG

[0048] AAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACT

[0049] CTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTA

[0050] ACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA

[0051] AGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGA

[0052] TGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAG

[0053] TGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGA

[0054] GGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAA

[0055] AGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGA

[0056] GTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCAC

[0057] TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCC

[0058] CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCA

[0059] GGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGT

[0060] GACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC

[0061] GCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCAA

[0062] GACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCC

[0063] TTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTCGCGAAGTC

[0064] GCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGCAGGCTGCAACT

[0065] CGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATA

[0066] CGTTCCCGGGCCTTGTACACACCGCCCGTCACACC

[0067] Example 3 Gram staining microscopy

[0068] (1) Picking colonies: Generally, fresh plates are selected to pick colonies.

[0069] (2) Smear: The smear should not be too thick and should be distributed as evenly as possible. Before smearing, add a small amount of sterile water or physiological saline to the slide to facilitate the even distribution of bacteria.

[0070] (3) Fixation: After the smear dries, quickly pass the specimen over an alcohol lamp three times, for a total of about 2 to 3 seconds.

[0071] (4) Initial staining: Stain with crystal violet for 1 min.

[0072] (5) Mordant: Stain with iodine solution for 1 min.

[0073] (6) Decolorization: Add a few drops of 95% ethanol. Once the purple color just stops appearing, stop the decolorization process immediately.

[0074] (7) Re-dyeing: Before re-dyeing, use a paper towel to absorb excess water, then re-dye with phenol red for 30-60 seconds, and rinse with a small stream of water.

[0075] (8) Microscopic examination: First observe with a low-power or high-power microscope. After the target is found, observe with an oil immersion lens. After observing with an oil immersion lens, clean the objective lens with xylene in time.

[0076] The results are as follows Figure 2 As shown, Enterococcus faecalis (YF-M-02) is Gram-positive, round or oval in shape, without spores or capsules, and most of them are arranged in pairs or short chains.

[0077] Example 4

[0078] Research on the characteristics of Enterococcus faecalis probiotics:

[0079] (1) Acid resistance: Enterococcus faecalis was inoculated onto MRS agar medium and cultured at 37°C for 24-48 h. It was then transferred to MRS broth and cultured at 37°C for 24 h. The Enterococcus faecalis culture was centrifuged (5000 rpm, 8 min), and the bacterial pellet was resuspended in artificial gastric fluid. The pellet was then inoculated into artificial gastric fluid and cultured at 37°C. After 2 h, samples were taken for viable cell counting. The viable cell count at 0 h was used as a control to calculate the survival rate. Results are as follows: Figure 3 As shown, the survival rate of Enterococcus faecalis (YF-M-02) after culturing in artificial gastric fluid (pH=3.0±0.5) at 37℃ for 2 hours reached 86.2%, which was significantly higher than that of other strains.

[0080] (2) Bile salt tolerance: Enterococcus faecalis was inoculated onto MRS agar medium and cultured at 37°C for 24-48 h. It was then transferred to MRS broth and cultured at 37°C for 24 h. The Enterococcus faecalis culture was centrifuged (5000 rpm, 8 min), resuspended in bile salt solution, and inoculated into bacterial culture medium containing 0.3% bile salts. After 4 h, samples were taken for viable cell count, with the viable cell count at 0 h as a control. The survival rate was calculated. Results are as follows: Figure 4 As shown, the survival rate of Enterococcus faecalis (YF-M-02) after culturing in a medium containing 0.3% bile salts at 37°C for 4 hours reached 69.7%, which was significantly higher than that of other strains.

[0081] (3) Adhesion: Centrifuge the activated Enterococcus faecalis culture medium, collect the bacterial pellet, wash with PBS, resuspend in DMEM high-glucose medium (without antibiotics), adjust the bacterial concentration, and conduct adhesion experiments. Results are as follows: Figure 5 As shown, the survival rate of Enterococcus faecalis (YF-M-02) after co-culturing with Caco-2 cells for 2 hours reached 69.7%, which was significantly higher than that of other strains.

[0082] Example 5

[0083] A study on the improvement of diarrhea in mice by Enterococcus faecium probiotics:

[0084] (1) Inoculate Enterococcus faecalis onto fresh solid culture medium (culture medium formula: 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, 14.0g agar, 1000mL purified water).

[0085] (2) Pick a single colony from the plate and inoculate it into liquid culture medium (10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, 1000mL purified water) and incubate at 37℃ for 24h.

[0086] (3) Escherichia coli was administered to mice by gavage (10) 7 A mouse model of acute diarrhea was established using CFU / mL / mouse.

[0087] (4) Take the Enterococcus faecalis bacterial suspension, centrifuge at 5000 rpm for 10 min, wash and resuspend, and administer to mice by gavage to observe its therapeutic effect on mice with diarrhea. As shown in Table 1, the saline group was the unmodeled mice (positive control). Enterococcus faecalis (YF-M-02) had a significant effect on diarrhea in mice, and the diarrhea phenomenon was significantly improved. The diarrhea rate and diarrhea index were significantly reduced compared with the model group, and the survival rate was improved. Figure 6 The results showed that Enterococcus faecalis (YF-M-02) could significantly reduce the levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β. Figure 7 The results showed a significant decrease in Lactobacillus spp. in the model group mice, and a significant decrease in Escherichia spp. under YF-M-02 intervention. This suggests that Enterococcus faecalis (YF-M-02) can, to some extent, increase the number of other beneficial bacteria and reduce the number of potential pathogens.

[0088] Table 1: Statistics on diarrhea in mice of each group after 8 days

[0089]

[0090] Note: Diarrhea rate = (Number of loose stools excreted within a certain period of time) / (Total number of stools excreted within a certain period of time) × 100%

[0091] Diarrhea Index = Loose Stool Rate × Loose Stool Grade

Claims

1. A type of Enterococcus faecalis, characterized in that: The strain was classified and named Enterococcus faecium. It was deposited at the China General Microbiological Culture Collection Center on March 21, 2025, with accession number CGMCC NO.: 33919.

2. The use of the Enterococcus faecalis according to claim 1 in improving bacterial diarrhea.

3. The application according to claim 2, characterized in that, The bacteria in question is Escherichia coli.

4. The application according to claim 2, characterized in that, The Enterococcus faecalis includes the Enterococcus faecalis as described in claim 1 or a suspension of Enterococcus faecalis.

5. The application according to claim 2, characterized in that, The method for preparing the Enterococcus faecalis suspension includes the following steps: inoculating Enterococcus faecalis into a solid slant culture medium for activation, performing seed culture on the activated strain to obtain a primary seed liquid, and inoculating the primary seed liquid into a broth culture medium for fermentation culture to obtain the Enterococcus faecalis suspension.

6. The application according to claim 5, characterized in that, The culture temperature for Enterococcus faecalis is 35-37℃.

7. The application according to claim 5, characterized in that, The pH for the expanded culture of Enterococcus faecalis was 5.0-7.

5.

8. The application according to claim 5, characterized in that, The fermentation culture was carried out at 35-37℃.

9. The application according to claim 5, characterized in that, The fermentation culture was a static culture for 20-24 hours.

10. The application according to claim 5, characterized in that, The primary seed culture was inoculated into broth medium at a 2% inoculation rate for expansion culture to obtain a bacterial suspension of Enterococcus faecalis.

Images