New enterococcus strain Enterococcus qinghaiensis SCFF543 and antioxidant application thereof

Abstract

The invention provides a novel enterococcus strain Enterococcus qinghaiensis SCFF543 and an anti-oxidation application of the novel enterococcus strain Enterococcus qinghaiensis SCFF543, and belongs to the technical field of microorganisms. Wherein the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 10, 2025, and the preservation number is CGMCC No.34843. The invention further discloses a preparation method of the strain. The strain is separated from homemade casein of herdsman families in Demaoha City in Haixi, Qinghai province, has the advantages of strong antioxidant capacity, no vancomycin resistance, strong adhesiveness and the like, and can be suitable for preparing probiotic products with antioxidant effects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a new species of Enterococcus. Enterococcus qinghaiensis SCFF543 and its antioxidant applications. Background Technology

[0002] Enterococcus spp. Enterococcus Enterococci (*Enterococcus*) belong to the phylum Firmicutes, class Bacillus, order Lactobacilliles, family Enterococciaceae, and are a type of Gram-positive cocci. Enterococci are a group of commensal bacteria widely found in the intestines of humans and animals, and are also common species in some fermented foods. Enterococci are facultative anaerobic, catalase-negative, chain-arranged Gram-positive cocci, with a G+C content of 37-45 mol% in their DNA. In recent years, with the deepening of research on intestinal flora, specific Enterococcus strains have gradually attracted attention due to their probiotic properties such as regulating intestinal flora and enhancing intestinal barrier function. Some Enterococcus strains, such as *Enterococcus faecium* (…),… Enterococcus faecium ) and Enterococcus faecalis ( Enterococcus faecalis Due to its good tolerability and safety, it has been widely used as a probiotic and silage fermentation agent.

[0003] Buttercream is a traditional fermented dairy product from the pastoral areas of Qinghai Province. It has a long history of consumption, a unique flavor, and is rich in protein and nutrients, characterized by high calcium and low fat. Isolating new strains of bacteria from buttercream and studying their activity can provide new functional ingredients for the food industry.

[0004] Currently, some probiotics have been found to possess certain antioxidant capabilities, such as *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Enterococcus faecalis*. These strains primarily exert their effects by producing antioxidant enzymes or secreting metabolites with antioxidant activity. This allows them to effectively eliminate reactive oxygen species in the intestines, and these antioxidants can also scavenge free radicals in the body, thereby preventing various diseases caused by oxidative stress, including cardiovascular disease, cancer, and neurodegenerative diseases. At the same time, the safety of probiotics and their ability to adhere to intestinal cells are also important considerations for their effective development and utilization. Summary of the Invention

[0005] To address the aforementioned problems, this application provides a novel species of Enterococcus, and names it as follows: Enterococcus qinghaiensisSCFF543, also known as Qinghai Enterococcus SCFF543, was isolated from homemade cheese curd by herders in Delingha City, Haixi Prefecture, Qinghai Province. Analysis revealed that the full-length 16S rRNA sequence of this strain is 1546 bp, and its 16S rRNA, ANI, and dDDH values ​​compared to known closely related Enterococcus type strains are the highest at 95.87%, 67.64%, and 35.5%, respectively, confirming it as a new species. Experiments also showed that this strain possesses advantages such as strong antioxidant capacity, lack of vancomycin resistance, and strong adhesion.

[0006] On the one hand, this application provides a new species of Enterococcus. Enterococcus qinghaiensis SCFF543, the new species of Enterococcus Enterococcus qinghaiensis SCFF543 was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34843.

[0007] In one embodiment, the new species of Enterococcus Enterococcus qinghaiensis The full-length 16S rRNA sequence of SCFF543 is shown in SEQ ID NO:1.

[0008] In one embodiment, the new species of Enterococcus Enterococcus qinghaiensis SCFF543 was obtained by isolating from casein.

[0009] On the other hand, this application also provides the aforementioned new species of Enterococcus. Enterococcus qinghaiensis A method for cultivating SCFF543, the method comprising: Inoculation with the aforementioned new species of Enterococcus Enterococcus qinghaiensis The culture medium for SCFF543 was cultured in a strictly anaerobic environment.

[0010] In one embodiment, the new species of Enterococcus Enterococcus qinghaiensis The inoculum amount of SCFF543 in the culture medium is 0.01% to 10%, preferably 0.5% to 8%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%.

[0011] In one embodiment, the culture temperature is 30~40°C, preferably 37°C.

[0012] In one embodiment, the pH of the culture medium is 7-11, preferably 7.5.

[0013] In one embodiment, the culture time is 24-80 h, preferably 48-72 h.

[0014] On the other hand, this application also provides a microbial agent comprising the aforementioned new species of Enterococcus. Enterococcus qinghaiensis SCFF543 strain, and / or the secretions, extracts, fermentation products, bacterial suspensions, and supernatants of the strain.

[0015] The strain is preferably a live bacterium; the secretions of the strain include, but are not limited to, intracellular or extracellular secretions, such as enzymes, polysaccharides, polypeptides, acids, RNA, etc.

[0016] It is understood that the extract of the strain is a substance obtained by extracting the strain using methods known in the art. The fermentation product of the strain includes, but is not limited to, products obtained by fermenting the strain itself or by fermenting the substrate. The bacterial suspension of the strain includes, but is not limited to, preparations obtained by suspending the strain using methods known in the art. The supernatant of the strain is the clear liquid obtained by centrifuging the suspension containing the strain and removing the bottom precipitate.

[0017] In one embodiment, the bacterial agent is in the form of a liquid or solid, such as bacterial liquid or lyophilized powder.

[0018] In one embodiment, the microbial agent may also contain at least one pharmaceutically acceptable excipient, such as water, depending on the dosage form requirements.

[0019] On the other hand, this application also provides the aforementioned new species of Enterococcus. Enterococcus qinghaiensis SCFF543, and / or the use of the aforementioned microbial agent in the preparation of products having any one or more of the following uses: I) Antioxidant; II) Production of antioxidant enzymes; III) Colonizing host intestinal cells; IV) Regulate the balance of gut microbiota.

[0020] In one embodiment, the antioxidant enzyme includes SOD enzyme and CAT enzyme.

[0021] In one embodiment, the product includes pharmaceuticals, health foods, and / or feed additives.

[0022] In one embodiment, the dosage form of the product includes, but is not limited to, powder, tablet, capsule, granule or liquid dosage form.

[0023] On the other hand, this application also provides a probiotic product with antioxidant effects, wherein the active ingredient of the probiotic product contains the aforementioned new species of Enterococcus. Enterococcus qinghaiensis SCFF543 strain or the aforementioned inoculum.

[0024] In one embodiment, the content of the strain or agent in the product is 0.01% to 99.99%, where % is a mass percentage (w / w), volume percentage (v / v), mass-volume ratio (w / v), or volume-mass ratio (v / w).

[0025] This application has at least the following beneficial effects: 1. This application marks the first isolation of a novel species of Enterococcus from homemade cheese milk from herders in Qinghai Province, named […]. Enterococcus qinghaiensis SCFF543 was identified as a new species because it had the highest 16S rRNA, ANI, and dDDH values ​​(95.87%, 67.64%, and 35.5%, respectively) compared to known closely related Enterococcus type strains. 2. The new species of Enterococcus provided in this application Enterococcus qinghaiensis SCFF543 is sensitive to 18 of the 30 antibiotics tested, and has no resistance to vancomycin, erythromycin, or penicillin, demonstrating good safety. At the same time, this strain is a strict anaerobic bacterium, with an optimal growth temperature of 37°C and a pH of 7.5, which allows it to adapt to the human intestinal environment. It has great potential application value in the development of probiotic products. 3. The new species of Enterococcus provided in this application Enterococcus qinghaiensis SCFF543 exhibits strong antioxidant properties. Its cell-free supernatant and cell-free extract showed DPPH scavenging abilities of 91.8±1.8% and 92.1±0.1%, respectively, and superoxide anion radical scavenging abilities of 89.9±0.1% and 69.5±7.8%, respectively. Furthermore, it demonstrates outstanding antioxidant defense capabilities, with SOD and CAT enzyme activities of 41.0±3.1 U / 10⁻⁶. 9 Cells and 639.0±35.9 U / 10 9 Cells can be used as an active ingredient in products with antioxidant effects; 4. The new species of Enterococcus provided in this application Enterococcus qinghaiensis SCFF543 exhibits strong adhesion to cells, especially intestinal cells, with hydrophobicity, self-aggregation, and cell adhesion rates of 78.4±0.5%, 81.3±1.4%, and 70.3±12.9%, respectively. It can serve as a carrier targeting intestinal cells and also plays a positive role in regulating the balance of intestinal flora. Attached Figure Description

[0026] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 To use the neighbor-joining method (MEGA 10) to identify new species of Enterococcus Enterococcus qinghaiensis Phylogenetic tree diagram constructed from the 16S rRNA sequence of SCFF543; Figure 2 A new species of Enterococcus Enterococcus qinghaiensis Growth curves of SCFF543 under aerobic and anaerobic conditions, respectively. Figure 3 A new species of Enterococcus Enterococcus qinghaiensis Growth curves of SCFF543 under different temperature conditions; Figure 4 A new species of Enterococcus Enterococcus qinghaiensis Growth curves of SCFF543 under different pH conditions; Figure 5 A new species of Enterococcus Enterococcus qinghaiensis Basic morphological results of SCFF543, where a is the morphological result after 72 h of culture on blood agar plates, b is the morphological result after 72 h of culture on MRS medium, c is the Gram staining result, and d is the electron microscopy result. Figure 6 The new species of Enterococcus in Example 5 Enterococcus qinghaiensis The results of the antioxidant capacity test of SCFF543 are shown in the figure. Figure 7 The new species of Enterococcus in Example 5 Enterococcus qinghaiensis The results of SOD and CAT enzyme activity assays in SCFF543 are shown in the figure. Figure 8 The new species of Enterococcus in Example 6 Enterococcus qinghaiensis The adhesion test results of SCFF543 are shown in the figure. Detailed Implementation

[0028] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention. The essence and scope of protection of this invention are primarily defined by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the invention, also fall within the scope of protection of this invention.

[0029] Unless otherwise specified, all materials, reagents, and experimental instruments used in the following examples are commercially available.

[0030] The main sources of reagents or instruments involved in the following examples are shown in Table 1: Table 1

[0031] Example 1 Strains Enterococcus qinghaiensis Acquisition of SCFF543 1. Screening of strains Aseptically collected 1 mL of homemade cheese milk from two herder households in Delingha City, Haixi Prefecture, Qinghai Province. The milk was added to 9 mL of sterile PBS, mixed thoroughly, and then diluted sequentially to 10⁻⁶. -1 ~10 -10 Ten gradient dilutions were spread onto MRS anaerobic solid plates using the direct coating method. After anaerobic incubation at 37°C for 3 days, single colonies were picked.

[0032] 2. Identification of strains The bacterial genome was extracted using a bacterial whole-genome extraction kit. Using this genome as a template, the 16S rRNA gene was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The product was sent to Qingke Biotechnology Co., Ltd. (Beijing) for sequencing. The sequencing results were compared with the 16S rRNA sequence using NCBI, and the results showed that... Enterococcus faecium NBRC 100486 showed the highest sequence similarity, with a similarity rate of 95.86%. The 16S rRNA sequence similarity threshold for classifying new microbial species is 98.65%, and SCFF543 exhibited the highest 16S rRNA similarity of 95.86%, supporting its potential status as a new Enterococcus species. Therefore, it was named... Enterococcus qinghaiensis SCFF543, also known as Qinghai Enterococcus ( Enterococcus qinghaiensis )SCFF543.

[0033] To further determine the phylogenetic and taxonomic position of this strain, the genomic DNA of SCFF543 was sent to Guangzhou Megabio Technology Co., Ltd. for whole-genome sequencing. The full-length 16S rRNA sequence of this strain is shown in SEQ ID No. 1, and the basic genomic characteristics are shown in Table 2.

[0034] Table 2. Genome characteristics of the new species of Enterococcus SCFF543 from Qinghai.

[0035] The full-length 16S rRNA sequence of SCFF543 obtained from whole-genome sequencing was aligned with EzBioCloud, and the results are shown in Table 3. Enterococcus faecium LMG11423 (equivalent to) Enterococcus faecium The highest similarity was found in NBRC100486, with a similarity of 95.87%. The Neighbor-Joining (NJ) algorithm in MEGA10 software was used based on... Enterococcus qinghaiensis A phylogenetic tree was constructed using the 16S rRNA sequences of the SCFF543 and the 10 closest type strains from the Ezbiocloud database. The results are as follows: Figure 1 As shown. By Figure 1 The results show that SCFF543 forms a separate branch.

[0036] 3. Preservation The strain was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34843, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] 4. Average Nucleotide Identity (ANI) and Digital DNA-DNA Hybridization (dDDH) Whole-genome analysis of strains provides more reliable taxonomic determination results. ANI and dDDH values ​​are mainly used to assess the phylogenetic relationships between species at the whole-genome level. The ANI values ​​of SCFF543 with 10 closely related type strains of the genus Enterococcus were calculated using the online analysis platform ANI Calculator (https: / / www.ezbiocloud.net / ).

[0038] Meanwhile, DNA-DNA hybridization based on genomic information is an important standard for identifying bacterial species. In order to further clarify the similarity between SCFF543 and the Enterococcus type strain, dDDH online hybridization was performed using GGDC (Genome-to-Genome Distance Calculator) to obtain dDDH values.

[0039] The results obtained above are shown in Table 3: Table 3. 16S rRNA similarity, ANI, and dDDH values ​​of SCFF543 and 10 closely related Enterococcus strains.

[0040] The results in Table 3 show that the strains Enterococcus qinghaiensis SCFF543 and the type strain Enterococcus hirae ATCC9790 had the highest ANI and dDDH values, at 67.64% and 35.5% respectively, far below the currently accepted thresholds for species classification, namely an ANI of 95%–96% and a dDDH of 70%. From a genomics perspective, Enterococcus qinghaiensis SCFF543 differs significantly from known Enterococcus strains. ANI and dDDH values ​​further support this. Enterococcus qinghaiensis SCFF543 is a new species of Enterococcus.

[0041] In summary, the strains obtained in this application are... Enterococcus qinghaiensis The 16S rRNA, ANI, and dDDH values ​​of SCFF543 and its closely related Enterococcus species were the highest at 95.87%, 67.64%, and 35.5%, respectively, all significantly lower than the threshold for new species classification, indicating that... Enterococcus qinghaiensis SCFF543 is a new species of Enterococcus.

[0042] Example 2 Strains Enterococcus qinghaiensis Physiological characteristics of SCFF543 The specific experimental procedure is as follows: 1. Growth capacity under aerobic and anaerobic conditions overnight cultivation Enterococcus qinghaiensis The SCFF543 strain was inoculated at a 3% inoculum into both aerobic and anaerobic MRS media, and cultured at 37°C for 72 h. Growth was observed and monitored every 24 h using a spectrophotometer. The results are as follows: Figure 2 As shown.

[0043] Depend on Figure 2 The results show that Enterococcus qinghaiensis SCFF543 grows well in anaerobic MRS but not in aerobic MRS, which indicates that... Enterococcus qinghaiensis SCFF543 is a strict anaerobic bacterium, which is significantly different from the facultative anaerobic growth of other reported Enterococcus species.

[0044] 2. Optimal growth temperature of the strain overnight culture Enterococcus qinghaiensis The SCFF543 strain was inoculated at a 3% inoculum into MRS anaerobic liquid medium and incubated at 10℃, 30℃, 37℃, and 45℃ for 72 h, respectively. Growth was monitored every 12 h using a spectrophotometer. The results are as follows: Figure 3 As shown.

[0045] Depend on Figure 3The results show that SCFF543 grows well at temperatures between 30℃ and 37℃, with the best growth at 37℃.

[0046] 3. pH tolerance of the strain overnight culture Enterococcus qinghaiensis The SCFF543 strain was inoculated at a rate of 3% into MRS anaerobic liquid medium with pH values ​​of 3.5, 5, 7.5, and 11, respectively, and cultured at 37°C for 72 h. Growth was monitored every 12 h using a spectrophotometer. The results are as follows: Figure 4 As shown.

[0047] Depend on Figure 4 The results show that Enterococcus qinghaiensis SCFF543 grows well in pH ranges of 7.5-11, with the best growth observed at pH 7.5.

[0048] Example 3 Strains Enterococcus qinghaiensis Morphological observation of SCFF543 For the obtained Enterococcus qinghaiensis The pure culture of SCFF543 was subjected to blood agar plate, MRS medium, Gram staining, and electron microscopy observation. The specific procedures are as follows: Gram staining experiment: Fix the smear of bacteria in the logarithmic growth phase, add crystal violet staining solution, and wash with water after 1 min. Add iodine solution, cover the smear for 1 min, decolorize with 95% alcohol, wash with water, counterstain with safranin solution for 1 min, wash with water, observe the cell morphology under 100x oil immersion and take pictures.

[0049] Transmission electron microscopy observation: After culturing bacteria on anaerobic solid plates, the bacteria were gently scraped into PBS using an inoculation loop to prepare a suspension; 10 μL of sample was dropped onto a copper grid, and after 10 min, the excess liquid was aspirated away with filter paper; 10 μL of 2% phosphotungstic acid or 3% uranium acetate was dropped onto a copper grid, and after 1-3 min, the excess liquid was aspirated away with filter paper; after drying for about 5 min, the cell morphology was observed using a transmission electron microscope.

[0050] Observation results are shown in Figure 5 . Figure 5 In the middle, by Figure 5 As can be seen from point a, the blood agar plate results showed that this strain did not exhibit hemolytic activity after 3 days of culture on MRS anaerobic blood agar plates. Figure 5 b shows that after culturing the strain on MRS medium for 72 h, the colonies are milky white, opaque, raised, moist, with smooth and glossy edges. Figure 5 Gram staining results for c showed that it was a typical Gram-positive bacterium with spherical cells. Figure 5 Electron microscopy results showed that SCFF543 was spherical with a diameter of approximately 1.1-1.3 μm and lacked special structures such as flagella and cilia.

[0051] Example 4 Strains Enterococcus qinghaiensis Drug susceptibility testing of SCFF543 The problem of multidrug resistance in enterococci is quite prominent: most enterococci are resistant to penicillin because they can produce special penicillin-binding proteins; in addition, erythromycin resistance is also common in enterococcal strains, and enterococcal strains in animal food are highly resistant to erythromycin; and vancomycin resistance genes are a key focus of clinical concern because they can be horizontally transferred through mechanisms such as plasmid-mediated transfer, leading to polyclonal transmission.

[0052] The specific procedure for drug sensitivity testing is as follows: Antimicrobial susceptibility testing was performed using 30 antibiotics from an antimicrobial susceptibility testing kit, including chloramphenicol, erythromycin, ciprofloxacin, ofloxacin, norfloxacin, and ampicillin. Three consecutive generations of activated antibiotics were used. Enterococcus qinghaiensis Strain SCFF543 was cultured anaerobically overnight at 37℃. 200 μL of the overnight culture was spread onto an MRS anaerobic solid plate, spread three times from different angles, and then the plate was covered and allowed to dry at room temperature for 3 minutes. After placing a drug susceptibility test strip on sterile forceps, the plate was further anaerobically incubated at 37℃ for 24 h. The diameter of the inhibition zone was measured using calipers, and drug resistance was determined based on the diameter of the inhibition zone. The results are shown in Table 4. Table 4 Drug susceptibility testing of SCFF543

[0053] *Drug susceptibility assessment is based on CLSI standards. The test bacteria are reported as sensitive, intermediate, or resistant to the test drug based on the diameter of the inhibition zone.

[0054] As can be seen from the results in Table 4, the strain Enterococcus qinghaiensis SCFF543 was sensitive to 18 of the 30 antibiotics tested, including cefoperazone, ceftriaxone, cefuroxime sodium, cefazolin, piperacillin, ampicillin, penicillin, azithromycin, erythromycin, minocycline, tetracycline, lincomycin, vancomycin, chloramphenicol, clindamycin, levofloxacin, doxycycline, and florfenicol. This result indicates that... Enterococcus qinghaiensis The SCFF543 strain has good safety profile and has potential application value in the development of probiotic products.

[0055] Example 5 Strain Enterococcus qinghaiensis Antioxidant capacity test of SCFF543 I. Detection of DPPH and superoxide anion free radical scavenging capacity The specific experimental procedure is as follows: Activated Enterococcus qinghaiensisAfter overnight culture, SCFF543 strain was inoculated into 5 mL of anaerobic MRS medium and incubated at 37°C until OD500. 600 =1.0, 1 mL of culture was centrifuged to obtain cell-free supernatant (CFS) and cells. Cells were washed three times with PBS, then 1 mL of pre-chilled cell extraction buffer was added. The mixture was sonicated on ice (200 W, 3 s, 10 s interval, repeated 30 times), and centrifuged at 10000 g for 10 min at 4°C. The supernatant was then collected as cell-free extract (CFE). The DPPH and superoxide anion scavenging abilities of the cell-free supernatant and cell-free extract were tested according to the instructions of the DPPH free radical scavenging kit and the superoxide anion scavenging kit (Solepro), respectively, with three replicates per group. Specifically, the extract served as a negative control, and a 1 mg / mL vitamin C solution served as a positive control.

[0056] (1) The DPPH sample addition sequence is shown in Table 5: Table 5 DPPH Sampling Sequence

[0057] The working solution is prepared fresh and used immediately with a volume ratio of 4:21 for reagent 2 and reagent 1.

[0058] Vortex to mix thoroughly, incubate at room temperature in the dark for 30 min, and measure absorbance at 515 nm. Record the results as A blank, A assay, A control, and A positive, respectively. Each assay tube also includes a control tube.

[0059] The formula for calculating the positive control is: [(A blank - A positive) ÷ A blank] × 100%.

[0060] Sample calculation formula: [[A blank - (A determination - A control)] ÷ A blank] × 100%.

[0061] (2) The order of superoxide anion radical addition is shown in Table 6: Table 6. Order of superoxide anion radical addition

[0062] The formula for calculating superoxide anion scavenging rate is: (A blank - A determination) ÷ A blank × 100%.

[0063] The results obtained above are as follows Figure 6 As shown.

[0064] Depend on Figure 6 The results show that Enterococcus qinghaiensisThe DPPH scavenging abilities of cell-free supernatant and cell-free extract of strain SCFF543 were 91.8±1.8% and 92.1±0.1%, respectively, and their superoxide anion radical scavenging abilities were 89.9±0.1% and 69.5±7.8%, respectively. The experimental results indicate that... Enterococcus qinghaiensis SCFF543 has a very high antioxidant capacity.

[0065] II. Determination of the activity of antioxidant enzymes SOD and CAT The specific experimental procedure is as follows: 1. Sample pretreatment for SOD and CAT enzyme activity assay Activated Enterococcus qinghaiensis After overnight culture, SCFF543 strain was inoculated into 5 mL of anaerobic MRS medium and incubated at 37°C until OD500. 600 =1.0, centrifuged at 4℃ for 10 min at 8000 rpm to collect bacteria, and washed twice with PBS buffer. 1 mL of bacterial cells was used for flow cytometry counting. Another 1 mL of bacterial cells was added to 1 mL of Gram-positive bacterial protein extraction solution and incubated at 37℃ for 2-3 h on a shaker. The extract was then frozen at -20℃ for 1 h and thawed at 4℃ for 2 h. Centrifuged at 12000 rpm for 20 min at 4℃, and the supernatant was used to determine SOD and CAT enzyme activities according to the instructions of the Superoxide Dismutase (SOD) Activity Assay Kit (Solepro) and Catalase (CAT) Assay Kit (Shanghai Yuanye). Each group had three replicates, with PBS as a negative control and *Pediococcus lactis* DSM 20284 as a positive control.

[0066] 2. The order of sample addition for SOD enzyme activity assay is shown in Table 7. Table 7 SOD Sampling Sequence

[0067] Inhibition percentage = (ΔA blank - ΔA determination) ÷ ΔA blank × 100% SOD activity calculation formula = 10 × inhibition percentage ÷ (1 - inhibition percentage) × F ÷ N F: Sample dilution factor; N: Total number of cells or bacteria.

[0068] 3. The order of sample addition for CAT enzyme activity assay is shown in Table 8. Table 8 CAT Sample Addition Sequence

[0069] CAT activity calculation formula = {(A self-control - A assay) / A blank} × 650 × F ÷ N F: Sample dilution factor; N: Total number of cells or bacteria.

[0070] Depend on Figure 7 The results show that Enterococcus qinghaiensis The SOD and CAT enzyme activities of strain SCFF543 were 41.0 ± 3.1 U / 10⁻⁶. 9 Cells and 639.0±35.9 U / 10 9 Cells. This result indicates that, Enterococcus qinghaiensis The SCFF543 strain exhibits outstanding antioxidant defense capabilities.

[0071] Example 6 Strain Enterococcus qinghaiensis In vitro adhesion ability test of SCFF543 Adhesion ability is one of the important indicators for evaluating the potential probiotic properties of probiotics. Adhesion ability not only determines the colonization ability of probiotics in the host gut, but is also closely related to their various health benefits. To systematically evaluate the adhesion ability of bacteria SCFF543, this application measured... Enterococcus qinghaiensis The in vitro hydrophobicity, self-aggregation ability, and adhesion to HT-29 cells of strain SCFF543.

[0072] The specific experimental procedure is as follows: I. In vitro hydrophobicity determination: Activated Enterococcus qinghaiensis The SCFF543 bacterial culture was centrifuged at 4000 rpm for 5 min at 4°C, the supernatant was removed, and the bacterial pellet was washed three times with PBS buffer. The bacterial cells were resuspended in PBS buffer, and the OD concentration of the bacterial culture was adjusted. 600 The absorbance is approximately 0.4 (denoted as Ao). Take 3 mL of bacterial suspension with an absorbance of 0.4 and mix it with 1 mL of chloroform. Vortex for 30 s, let stand at room temperature for 30 min, and measure the absorbance of the upper layer at 600 nm (denoted as Ax). The experiment was repeated in triplicate, and the absorbance was calculated using the following formula: Hydrophobicity = (Ao - Ax) / Ao × 100% In the formula: Ax represents the measured value; Ao represents the initial value.

[0073] II. In vitro self-aggregation test: After centrifugation, discard the supernatant of the overnight cultured bacterial culture. Wash the bacterial pellet three times with PBS buffer, then resuspend in PBS and adjust the bacterial concentration (OD). 600 Approximately 0.4 (denoted as Ao). Take 4 mL of the suspension, vortex for 10 s, and let stand at room temperature for 3 h. Measure the absorbance of the upper suspension at a wavelength of 600 nm (denoted as At). Perform the experiment three times, and calculate using the following formula: Self-cohesion rate = (Ao - At) / Ao × 100% In the formula: At represents the measured value; Ao represents the initial value.

[0074] III. HT-29 Cell Adhesion Ability Test: (1) After 3 generations of activated HT-29 culture, 1×10⁶ cells were added to each well. 6 Cells were seeded at a density of [number] cells per well in 6-well plates. Cells were incubated at 37°C with 5% CO2 for 24 h until 100% confluence was achieved. (2) Culture the bacteria to the logarithmic growth phase and adjust the concentration to OD. 600 =1.0, take 5 mL of bacteria, wash twice with PBS, and then resuspend in 5 mL of DMEM medium for later use; (3) Take 1 mL of resuspended bacteria, dilute it serially with PBS, spread it on MRS anaerobic plates, incubate at 37℃ for 72 h, and count the bacteria. Record the initial bacterial count. (4) Remove DMEM complete medium from HT-29, wash 3 times with PBS, add 1 mL of bacterial resuspension to each well, and co-incubate at 37℃ and 5% CO2 for 2 h. (5) After co-culture, the cells were gently washed three times with PBS to remove unattached bacteria. After digestion with 500 μL of trypsin for 5 min, 1.5 mL of DMEM medium was added to stop the digestion. The cells were then serially diluted with PBS and spread on MRS anaerobic plates. After incubation at 37°C for 72 h, the number of attached bacteria was recorded.

[0075] Calculation formula: Adhesion rate = (number of bacteria adhered ÷ initial number of bacteria) × 100%.

[0076] The above experimental results are as follows Figure 8 As shown.

[0077] Depend on Figure 8 The results show that Enterococcus qinghaiensis The hydrophobicity, self-aggregation, and cell adhesion of strain SCFF543 were 78.4±0.5%, 81.3±1.4%, and 70.3±12.9%, respectively, indicating that it has a good adhesion ability to intestinal cells.

[0078] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A new species of Enterococcus. Enterococcus qinghaiensis SCFF543 is characterized by, The new species of Enterococcus Enterococcus qinghaiensis SCFF543 was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 34843.

2. The new species of Enterococcus according to claim 1 Enterococcus qinghaiensis SCFF543 is characterized by, The new species of Enterococcus Enterococcus qinghaiensis The full-length 16S rRNA sequence of SCFF543 is shown in SEQ ID NO:

1.

3. A new species of Enterococcus according to claim 1 Enterococcus qinghaiensis SCFF543 is characterized by, The new species of Enterococcus Enterococcus qinghaiensis SCFF543 was obtained by isolating from casein.

4. The new species of Enterococcus as described in any one of claims 1-3 Enterococcus qinghaiensis The cultivation method of SCFF543 is characterized by, The method includes: inoculating the novel species of Enterococcus with the aforementioned bacteria. Enterococcus qinghaiensis The culture medium for SCFF543 was cultured in a strictly anaerobic environment.

5. The method according to claim 4, characterized in that, The culture temperature is 30~40℃, preferably 37℃; and / or the pH of the culture medium is 7~11, preferably 7.

5.

6. A microbial agent, characterized in that, The bacterial agent contains a new species of Enterococcus as described in any one of claims 1-3. Enterococcus qinghaiensis SCFF543 strain, and / or the secretions, extracts, fermentation products, bacterial suspensions, and supernatants of the strain.

7. The new species of Enterococcus as described in any one of claims 1-3 Enterococcus qinghaiensis SCFF543, and / or the use of the microbial agent as described in claim 6 in the preparation of products having any one or more of the following uses: I) Antioxidant; II) Production of antioxidant enzymes; III) Colonizing host intestinal cells; IV) Regulate the balance of gut microbiota.

8. The application according to claim 7, characterized in that, The products include pharmaceuticals, health foods, and / or feed additives.

9. A probiotic product with antioxidant effects, characterized in that, The active ingredient of the probiotic product contains the novel species of Enterococcus as described in any one of claims 1-3. Enterococcus qinghaiensis SCFF543 strain or the bacterial agent as described in claim 6.

10. The product according to claim 9, characterized in that, The content of the strain or agent in the product is 0.01% to 99.99%, where % is a mass percentage (w / w), volume percentage (v / v), mass-volume ratio (w / v), or volume-mass ratio (v / w).

Images