Enterococcus qinghaiensis SCFF543 and antioxidant application thereof

Abstract

The application provides a new enterococcus strain Enterococcus qinghaiensis The application discloses an SCFF543 strain and an antioxidant application thereof, and belongs to the technical field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center on June 10, 2025, and the preservation number is CGMCC No. 34843. The strain is obtained from cheese slurry prepared by a herdsman family in Delingha City, Haixi Prefecture, Qinghai Province, has the advantages of strong antioxidant capacity, no vancomycin resistance and strong adhesion, and can be applied to the preparation of probiotic products with antioxidant efficacy.

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, which can be referred to as Enterococcus sp. SCFF543 in Chinese. The strain is isolated from cheese slurry made by a family of herdsmen in Delingha City, Haixi Prefecture, Qinghai Province. The full-length 16S rRNA sequence of the strain is 1546 bp, and the highest alignment values of the 16S rRNA, ANI and dDDH of the strain with those of known Enterococcus sp. model strains are 95.87%, 67.64% and 35.5% respectively, confirming that it is a new species. Experiments have found that the strain has strong antioxidant capacity, no vancomycin resistance and strong adhesion, etc.

[0006] In one aspect, the present application provides an Enterococcus sp. new species Enterococcus qinghaiensis SCFF543, the Enterococcus sp. new species Enterococcus qinghaiensis SCFF543 was deposited with the China General Microbiological Culture Collection Center on June 10, 2025, at an address of No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, with a preservation number of CGMCC No. 34843.

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

[0008] In one embodiment, the Enterococcus sp. new species Enterococcus qinghaiensis SCFF543 is isolated from cheese slurry.

[0009] In another aspect, the present application also provides a culture method of the Enterococcus sp. new species Enterococcus qinghaiensis SCFF543, the method comprising:

[0010] The culture medium inoculated with the Enterococcus sp. new species Enterococcus qinghaiensis SCFF543 strain is cultured in a strictly anaerobic environment.

[0011] In one embodiment, the Enterococcus sp. new species Enterococcus qinghaiensis The inoculation amount of the SCFF543 strain in the culture medium is 0.01% to 10%, preferably 0.5% to 8%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8%.

[0012] In one embodiment, the temperature of the culture is 30 to 40°C, preferably 37°C.

[0013] In one embodiment, the pH of the culture medium is 7 to 11, preferably 7.5.

[0014] In one embodiment, the culture time is 24 to 80 h, preferably 48 to 72 h.

[0015] In another aspect, the present application also provides a bacterial agent comprising the Enterococcus sp. Enterococcus qinghaiensis the SCFF543 strain, and / or secretion, extract, fermentation, bacterial suspension, supernatant of the strain.

[0016] Preferably, the strain is a live strain; the secretion of the strain includes but is not limited to intracellular secretion or extracellular secretion, such as enzymes, polysaccharides, polypeptides, acids, RNA, etc.

[0017] It can be understood that the extract of the strain is a substance obtained by extracting the strain using methods known in the art. The fermentation of the strain includes but is not limited to a product obtained by fermenting the strain itself, or a product obtained by fermenting a substrate using the strain. The bacterial suspension of the strain includes but is not limited to a preparation obtained by suspending the strain using methods known in the art. The supernatant of the strain is a supernatant obtained by removing the bottom precipitate after centrifuging the suspension containing the strain.

[0018] In an embodiment, the dosage form of the bacterial agent is a liquid dosage form or a solid dosage form, such as a bacterial solution or a lyophilized powder.

[0019] In an embodiment, the bacterial agent can further comprise at least one pharmaceutically acceptable excipient, such as water, as required by the dosage form.

[0020] In another aspect, the present application also provides the Enterococcus sp. Enterococcus qinghaiensis SCFF543, and / or use of the bacterial agent in the preparation of a product having any one or more of the following uses:

[0021] I) antioxidant;

[0022] II) producing antioxidant enzymes;

[0023] III) colonizing host intestinal cells;

[0024] IV) regulating intestinal flora balance.

[0025] In an embodiment, the antioxidant enzymes include SOD enzymes and CAT enzymes.

[0026] In an embodiment, the product includes a drug, a health food, and / or a feed additive.

[0027] In an embodiment, the dosage form of the product includes but is not limited to a powder, a tablet, a capsule, a granule, or a liquid dosage form.

[0028] In another aspect, the present application also provides a probiotic product having antioxidant efficacy, wherein the active ingredient of the probiotic product comprises the Enterococcus sp. Enterococcus qinghaiensis SCFF543 strain or the bacterial agent.

[0029] In an embodiment, the content of the strain or bacterial agent in the product is 0.01%-99.99%, the % being mass percentage (w / w), volume percentage (v / v), mass-volume ratio (w / v) or volume-mass ratio (v / w).

[0030] The present application has at least the following beneficial effects:

[0031] 1. The present application first isolates a new Enterococcus species from cheese serum made by herdsmen in Qinghai Province, and names it Enterococcus qinghaiensis SCFF543. The 16S rRNA, ANI value and dDDH value of the strain and known close Enterococcus model strains are 95.87%, 67.64% and 35.5% respectively, confirming that it is a new species;

[0032] 2. The new Enterococcus species Enterococcus qinghaiensis SCFF543 provided by the present application is sensitive to 18 of the 30 antibiotics tested, has no resistance to vancomycin, erythromycin and penicillin, and has good safety. At the same time, the strain is a strict anaerobe, the suitable growth temperature is 37℃, the pH is 7.5, and it can adapt to the human intestinal environment, and has great potential application value in the development of probiotic products;

[0033] 3. The new Enterococcus species Enterococcus qinghaiensis SCFF543 provided by the present application has strong antioxidant activity, and the DPPH scavenging ability of its cell-free supernatant and cell-free extract is 91.8±1.8% and 92.1±0.1% respectively, and the superoxide anion radical scavenging ability is 89.9±0.1% and 69.5±7.8% respectively, and it has outstanding antioxidant defense ability, and its SOD enzyme activity and CAT enzyme activity are 41.0±3.1 U / 10 9 cells and 639.0±35.9 U / 10 9 cells respectively, and can be used as an active ingredient in products with antioxidant efficacy;

[0034] 4. The new Enterococcus species Enterococcus qinghaiensis SCFF543 provided by the present application has strong adhesion to cells, especially intestinal cells, and its hydrophobicity, self-aggregation ability and cell adhesion are 78.4±0.5%, 81.3±1.4% and 70.3±12.9% respectively, and can be used as a carrier targeting intestinal cells, and also has a positive effect on regulating intestinal flora balance. BRIEF DESCRIPTION OF DRAWINGS

[0035] The drawings herein are incorporated into the specification and form part of the specification, show embodiments consistent with the present application, and together with the specification serve to explain the principles of the present application.

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Those skilled in the art can also obtain other drawings according to these drawings without paying any creative effort. In the drawings:

[0037] Figure 1 For the new species of Enterococcus based on the method of adjacent (MEGA 10) Enterococcus qinghaiensis The phylogenetic tree result map constructed by the 16S rRNA sequence of SCFF543;

[0038] Figure 2 For the new species of Enterococcus Enterococcus qinghaiensis The growth curve result map of SCFF543 under aerobic and anaerobic conditions respectively;

[0039] Figure 3 For the new species of Enterococcus Enterococcus qinghaiensis The growth curve result map of SCFF543 under different temperature environments;

[0040] Figure 4 For the new species of Enterococcus Enterococcus qinghaiensis The growth curve result map of SCFF543 under different pH environments;

[0041] Figure 5 For the new species of Enterococcus Enterococcus qinghaiensis The basic morphology result of SCFF543, wherein a is the morphology result after 72h culture on blood plate, b is the morphology result after 72h culture on MRS medium, c is the gram staining result, and d is the electron microscope result;

[0042] Figure 6 For the new species of Enterococcus in Example 5 Enterococcus qinghaiensis The antioxidant capacity test result map of SCFF543;

[0043] Figure 7 For the new species of Enterococcus in Example 5 Enterococcus qinghaiensis The enzyme activity detection result map of SOD and CAT enzymes of SCFF543;

[0044] Figure 8 For the new species of Enterococcus in Example 6 Enterococcus qinghaiensis The adhesion detection result map of SCFF543. DETAILED DESCRIPTION

[0045] The present application is described below by specific embodiments. Unless otherwise specified, the technical means used in the present application are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the present application, the essence and protection scope of the present application are mainly defined by the claims. For those skilled in the art, various changes or modifications to the composition and amount of the materials in these embodiments without departing from the essence and scope of the present application also fall within the protection scope of the present application.

[0046] The materials, reagents, experimental instruments used in the following examples, etc. can be obtained from commercial channels unless otherwise specified.

[0047] The main reagents or instruments involved in the following examples are shown in Table 1:

[0048] Table 1

[0049]

[0050] Example 1 Strain Enterococcus qinghaiensis Obtaining of SCFF543

[0051] 1. Screening of strains

[0052] Aseptically collect the cheese slurry made by two local herdsmen in Delingha City, Haixi Prefecture, Qinghai Province, and add 1 mL of each to 9 mL of sterile PBS, mix well and dilute to 10 -1 ~10 -10 The 10 gradient dilutions are spread on MRS anaerobic solid plates using direct coating method, and after 3 days of anaerobic culture at 37°C, single colonies are picked.

[0053] 2. Identification of strains

[0054] The bacterial whole genome extraction kit is used to extract the genome of the strain, and the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') are used to amplify the 16S rRNA gene by PCR, and the product is sent to Genescript Biotech Co., Ltd. (Beijing) for sequencing. The sequencing results are compared with 16S rRNA in NCBI, and the results show that the sequence similarity with NBRC 100486 is the highest, with a similarity of 95.86%. The limit for dividing new species of microorganisms according to 16S rRNA sequence similarity is 98.65%, and the highest 16S rRNA similarity of SCFF543 is 95.86%, supporting that it is a potential new species of Enterococcus, and it is named Enterococcus faecium SCFF543, which can be called Qinghai Enterococcus in Chinese. Enterococcus qinghaiensis SCFF543, which can be called Qinghai Enterococcus in Chinese. Enterococcus qinghaiensis)SCFF543.

[0055] 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.

[0056] Table 2. Genome characteristics of the new species of Enterococcus SCFF543 from Qinghai.

[0057]

[0058] 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.

[0059] 3. Preservation

[0060] 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.

[0061] 4. Average Nucleotide Identity (ANI) and Digital DNA-DNA Hybridization (dDDH)

[0062] 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 / ).

[0063] At the same time, DNA-DNA hybridization based on genomic information is an important standard for identifying bacterial species. In order to further clarify the similarity of SCFF543 and the model strain of Enterococcus, GGDC (Genome-to-Genome Distance Calculator) was used for online hybridization of dDDH to obtain the dDDH value.

[0064] The above results are shown in Table 3:

[0065] Table 3 16S rRNA similarity, ANI and dDDH values of SCFF543 and 10 Enterococcus model strains

[0066]

[0067] From the results in Table 3, it can be seen that the strain Enterococcus qinghaiensis SCFF543 has the highest ANI and dDDH values with the model strain Enterococcus hirae ATCC9790, which are 67.64% and 35.5%, respectively, which are much lower than the currently recognized same species division threshold, i.e. ANI value is 95%-96% and dDDH value is 70%. From the perspective of genomics, Enterococcus qinghaiensis SCFF543 is significantly different from known Enterococcus strains. ANI and dDDH values further support Enterococcus qinghaiensis SCFF543 is a new species of Enterococcus.

[0068] In summary, the strain Enterococcus qinghaiensis SCFF543 has the highest 16S rRNA, ANI and dDDH values with the closely related Enterococcus, which are 95.87%, 67.64% and 35.5%, respectively, which are significantly lower than the new species division threshold, indicating that Enterococcus qinghaiensis SCFF543 is a new species of Enterococcus.

[0069] Example 2 Physiological characteristics of strain Enterococcus qinghaiensis SCFF543

[0070] The specific experimental process is as follows:

[0071] 1. Growth ability under aerobic and anaerobic conditions

[0072] The overnight culture of Enterococcus qinghaiensis SCFF543 strain was inoculated into aerobic and anaerobic MRS culture medium at an inoculation amount of 3%, and cultured at 37°C for 72 h to observe the growth of the strain. The growth was monitored once every 24 h by spectrophotometer. The results are shown in Table 4. Figure 2 Table 4 Growth ability of SCFF543 under aerobic and anaerobic conditions

[0073] From the results in Table 4, it can be seen that the strain Figure 2The results in Table 1 show that, Enterococcus qinghaiensis SCFF543 grew well in anaerobic MRS, but did not grow in aerobic MRS, thus indicating that, Enterococcus qinghaiensis SCFF543 is a strict anaerobe, and is significantly different from other Enterococcus species which have been reported to grow facultatively anaerobically.

[0074] 2. Optimum growth temperature of the strain

[0075] Overnight culture of SCFF543 Enterococcus qinghaiensis The SCFF543 strain was inoculated into MRS anaerobic liquid medium at an inoculation amount of 3%, and was placed at 10°C, 30°C, 37°C and 45°C for culture for 72 h, and the growth was monitored every 12 h using a spectrophotometer. The results are shown in Table 2. Figure 3

[0076] From the results in Table 2, it can be seen that SCFF543 grew well at 30°C-37°C, and grew best at 37°C. Figure 3 3. pH tolerance of the strain

[0077] Overnight culture of SCFF543

[0078] The SCFF543 strain was inoculated into MRS anaerobic liquid medium at an inoculation amount of 3%, and was placed at 10°C, 30°C, 37°C and 45°C for culture for 72 h, and the growth was monitored every 12 h using a spectrophotometer. The results are shown in Table 2. Enterococcus qinghaiensis Figure 4 From the results in Table 2, it can be seen that SCFF543 grew well at 30°C-37°C, and grew best at 37°C.

[0079] Figure 4 Example 3 Morphological observation of the strain Enterococcus qinghaiensis SCFF543

[0080] Morphological observation of SCFF543 Enterococcus qinghaiensis

[0081] The obtained pure culture of SCFF543 was subjected to blood plate, MRS medium and Gram staining, and was observed under an electron microscope, and the specific process was as follows: Enterococcus qinghaiensis Gram staining experiment: the log phase bacteria smear was fixed, crystal violet staining solution was added, and after 1 min, it was washed with water. Iodine solution was added, the smear was covered for 1 min, 95% alcohol was used for decolorization, and after washing with water, safranine solution was used for re-staining for 1 min, and after washing with water, the cell morphology was observed under a 100-fold oil immersion lens and was photographed.

[0082]

[0083] ​​​​​Transmission electron microscope observation: After the bacteria were cultured on the anaerobic solid plate, the bacteria were gently scraped with a swab into PBS to prepare a suspension; 10 μL of the sample was dropped on a copper mesh, and after 10 min, the excess liquid was absorbed with filter paper; 10 μL of 2% phosphotungstic acid or 3% uranyl acetate was dropped on the copper mesh, and after 1-3 min, the excess liquid was absorbed with filter paper; after drying for about 5 min, the cell morphology was observed by transmission electron microscope.

[0084] The observation results are shown in Table 3. Figure 5 . Figure 5 In Table 3, it can be seen from a that the blood plate result shows that the strain has no hemolytic activity after being cultured on the MRS anaerobic blood plate for 3 days. It can be known from b that the strain has a white, opaque, raised, moist, flat-edged, and shiny colony after being cultured on the MRS medium for 72 h. Figure 5 Figure 5 c that it is a typical gram-positive bacterium, and the bacterial body is spherical. Figure 5 Figure 5 d that SCFF543 is spherical, with a diameter of about 1.1-1.3 μm, and has no special structures such as flagella and cilia.

[0085] Example 4 Strain Enterococcus qinghaiensis SCFF543 Drug Sensitivity Test

[0086] The problem of multiple drug resistance of Enterococcus is more prominent: most Enterococcus strains are resistant to penicillin because they can produce special penicillin-binding proteins; in addition, erythromycin resistance is also common in Enterococcus strains, and Enterococcus strains in animal food have high resistance to erythromycin; and the vancomycin resistance gene can be horizontally transferred through mechanisms such as plasmid mediation, causing polyclonal transmission, which is the focus of clinical attention.

[0087] The specific process of the drug sensitivity experiment is as follows:

[0088] The drug sensitivity test was performed using 30 kinds of antibiotics in the antibacterial drug sensitivity test kit, such as chloramphenicol, erythromycin, ciprofloxacin, ofloxacin, norfloxacin, ampicillin, etc. 200 μL of the overnight culture of the SCFF543 strain was inoculated on the MRS anaerobic solid plate, and the plate was incubated at 37°C for 24 h. Enterococcus qinghaiensis The SCFF543 strain was cultured at 37°C overnight, 200 μL of the overnight culture was inoculated on the MRS anaerobic solid plate, and the plate was incubated at 37°C for 24 h. The diameter of the inhibition zone was measured with a vernier caliper, and the drug resistance was determined according to the diameter of the inhibition zone. The results are shown in Table 4:

[0089] Table 4 Drug sensitivity test of SCFF543 ​​

[0090]

[0091] Drug sensitivity judgment refers to the CLSI standard, and the sensitivity, intermediate and resistance of the tested bacteria to the tested drug are reported according to the diameter value of the inhibition zone.

[0092] From the results in Table 4, it can be seen that the strain Enterococcus qinghaiensis SCFF543 is 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. The results show that Enterococcus qinghaiensis The safety of SCFF543 strain is good, and it has potential application value in the development of probiotic products.

[0093] Example 5 Strain Enterococcus qinghaiensis Antioxidant capacity test of SCFF543

[0094] I. DPPH and superoxide anion radical scavenging capacity detection

[0095] The specific experimental process is as follows:

[0096] Activated Enterococcus qinghaiensis After overnight culture, SCFF543 strain was inoculated into 5 mL anaerobic MRS medium, and incubated at 37°C until OD 600 =1.0, 1 mL of the culture was centrifuged to obtain cell-free supernatant (CFS) and cells. The cells were washed with PBS for 3 times, then 1 mL of pre-cooled cell extract was added, and the mixture was ultrasonicated on ice (power 200 W, ultrasonication for 3 s, interval 10 s, repeated 30 times). The supernatant obtained by centrifugation at 10000 g for 10 min at 4°C was the cell-free extract (CFE). The cell-free supernatant and cell-free extract were detected for DPPH and superoxide anion radical scavenging capacity according to the instructions of DPPH radical scavenging capacity detection kit and superoxide anion scavenging capacity kit (Solabio), and each group had 3 replicates. The specific process is as follows: the extract was used as negative control, and 1 mg / mL vitamin C solution was used as positive control.

[0097] (1) The DPPH addition sequence is shown in Table 5:

[0098] Table 5 DPPH addition sequence

[0099]

[0100] The working solution is reagent 2: reagent 1 in a volume ratio of 4:21, which is prepared and used immediately.

[0101] Vortex mixing, room temperature, light protection, standing for 30 min, measuring absorbance at 515 nm. Respectively, Ablank, Ameasurement, Acontrol, Apositive.

[0102] Positive control calculation formula: [(Ablank-Apositive) ÷ Ablank] x 100%.

[0103] Sample calculation formula: [[Ablank-(Ameasurement-Acontrol)] ÷ Ablank] x 100%.

[0104] (2) The superoxide anion radical addition sequence is shown in Table 6:

[0105] Table 6 Superoxide anion radical addition sequence

[0106]

[0107] Superoxide anion radical scavenging rate calculation formula = (Ablank-Ameasurement) ÷ Ablank x 100%.

[0108] The results obtained above are shown in Figure 6 .

[0109] From the results in Figure 6 , Enterococcus qinghaiensis The cell-free supernatant and cell-free extract of SCFF543 strain have 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. The experimental results show that Enterococcus qinghaiensis SCFF543 has very high antioxidant capacity.

[0110] II. Activity determination of antioxidant enzymes SOD and CAT enzymes

[0111] The specific experimental process is as follows:

[0112] 1. Sample pretreatment for SOD and CAT enzyme activity determination

[0113] Activated Enterococcus qinghaiensis The SCFF543 strain was inoculated into 5 mL anaerobic MRS medium after overnight culture, and incubated at 37°C until the OD 600= 1.0, 4°C centrifugation, 8000 rpm centrifugation for 10 min to collect bacteria, and the bacterial cells were washed twice with PBS buffer. 1 mL of bacterial cells was taken for flow counting, and another 1 mL of bacterial cells was added with 1 mL of gram-positive bacterial protein extraction solution, and incubated at 37°C for 2-3 h. Then the extraction solution was frozen at -20°C for 1 h, and then frozen at 4°C for 2 h. 12000 rpm, 4°C centrifugation for 20 min, and the supernatant was taken for SOD and CAT enzyme activity determination according to the instructions of the superoxide dismutase (SOD) activity detection kit (Solebao) and the catalase (CAT) detection kit (Shanghai Yuanye). Each group had 3 replicates, PBS was used as a negative control, and Pediococcus acidilactici DSM 20284 was used as a positive control.

[0114] 2. The sample addition sequence for SOD enzyme activity determination is shown in Table 7

[0115] Table 7 SOD sample addition sequence

[0116]

[0117] Inhibition percentage = (ΔA blank - ΔA determination) ÷ ΔA blank × 100%

[0118] SOD activity calculation formula = 10 × inhibition percentage ÷ (1 - inhibition percentage) × F ÷ N

[0119] F: sample dilution factor; N: total number of cells or bacteria.

[0120] 3. The sample addition sequence for CAT enzyme activity determination is shown in Table 8

[0121] Table 8 CAT sample addition sequence

[0122]

[0123] CAT activity calculation formula = {(A self control - A determination) / A blank} × 650 × F ÷ N

[0124] F: sample dilution factor; N: total number of cells or bacteria.

[0125] From the results of Figure 7 , Enterococcus qinghaiensis The SOD and CAT enzyme activities of SCFF543 strain were 41.0 ± 3.1 U / 10 9 cells and 639.0 ± 35.9 U / 10 9 cells, respectively. The results showed that Enterococcus qinghaiensis SCFF543 strain has outstanding ability in antioxidant defense.

[0126] Example 6 StrainEnterococcus qinghaiensis In vitro adhesion ability test of SCFF543

[0127] 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.

[0128] The specific experimental procedure is as follows:

[0129] I. In vitro hydrophobicity determination:

[0130] 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:

[0131] Hydrophobicity = (Ao - Ax) / Ao × 100%

[0132] In the formula: Ax represents the measured value; Ao represents the initial value.

[0133] II. In vitro self-aggregation test:

[0134] 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:

[0135] Self-cohesion rate = (Ao - At) / Ao × 100%

[0136] In the formula: At represents the measured value; Ao represents the initial value.

[0137] III. HT-29 Cell Adhesion Ability Test:

[0138] (1) After 3 generations of activated HT-29 culture, 1×10⁶ cells were added to each well. 6Cells 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.

[0139] (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;

[0140] (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.

[0141] (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.

[0142] (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℃ for 72 h, the number of attached bacteria was recorded.

[0143] Calculation formula:

[0144] Adhesion rate = (number of bacteria adhered ÷ initial number of bacteria) × 100%.

[0145] The above experimental results are as follows Figure 8 As shown.

[0146] 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.

[0147] 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 qinghaiensis ( Enterococcus qinghaiensis SCFF543, characterized in that, The new species of Enterococcus qinghaiensis ( Enterococcus qinghaiensis SCFF543 was deposited on June 10, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34843.

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

1.

3. The new species of Enterococcus qinghaiensis according to claim 1 ( Enterococcus qinghaiensis SCFF543, characterized in that, The new species of Enterococcus qinghaiensis ( Enterococcus qinghaiensis SCFF543 was obtained by separating it from casein.

4. The new species of Enterococcus qinghaiensis as described in any one of claims 1-3 ( Enterococcus qinghaiensis The cultivation method of SCFF543 is characterized by, The method includes: inoculating the *Enterococcus qinghaiensis*, a new species of *Enterococcus*, with the inoculated solution. 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°C; and / or the pH of the culture medium is 7-11.

6. The method according to claim 5, characterized in that, The culture temperature is 37°C; and / or the pH of the culture medium is 7.

5.

7. A microbial agent, characterized in that, The bacterial agent contains *Enterococcus qinghaiensis*, a new species of *Enterococcus* as described in any one of claims 1-3. Enterococcus qinghaiensis SCFF543 strain, or a bacterial suspension of the strain.

8. The new species of Enterococcus qinghaiensis as described in any one of claims 1-3 ( Enterococcus qinghaiensis SCFF543, and / or the use of the microbial agent as described in claim 7 in the preparation of products for the production of antioxidant enzymes.

9. A probiotic product with antioxidant effects, characterized in that, The active ingredient of the probiotic product includes *Enterococcus qinghaiensis*, a new species of *Enterococcus* as described in any one of claims 1-3. Enterococcus qinghaiensis ) SCFF543 strain or the bacterial agent as described in claim 7.

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).

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