Space breeding animal bifidobacterium ZK-77-S and application thereof

Abstract

The present application relates to the technical field of microorganisms, and particularly relates to a space-bred animal bifidobacterium ZK-77-S and application thereof; the space-bred animal bifidobacterium ZK-77-S has been preserved in the China General Microbiological Culture Collection Center on December 24, 2024, and the preservation number is CGMCC No.33182, and the preservation address is No.3, Xili, Beichen, Chaoyang District, Beijing; the space-bred animal bifidobacterium ZK-77-S has inhibitory effects on escherichia coli, pseudomonas aeruginosa, fusiform bacterium nucleatum, porphyromonas gingivalis, staphylococcus aureus and candida albicans, and can be used for preparation of related drugs.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of Bifidobacterium for space-bred animals, ZK-77-S, and its applications. Background Technology

[0002] Bifidobacterium is a group of Gram-positive, non-spore-forming, obligate anaerobic bacteria that are widely distributed in the intestines of humans and animals and play an important role in host health.

[0003] Currently, various types of Bifidobacteria have been discovered, the most common being Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium adolescentis. The distribution and function of different species in the intestine vary slightly.

[0004] Applications of Bifidobacterium:

[0005] (1) Pharmaceutical field

[0006] Regulating the balance of gut microbiota: It can improve the gut microecological environment and prevent and treat intestinal dysfunction diseases such as diarrhea and constipation by inhibiting the growth of harmful bacteria, competing for intestinal adhesion sites, and producing antibacterial substances.

[0007] Enhances immune function: It can activate the intestinal immune system, promote the proliferation and differentiation of immune cells, improve the body's immune defense capabilities, help prevent infectious diseases, and may also have a certain inhibitory effect on the occurrence and development of tumors.

[0008] Lowering cholesterol: Some studies have shown that Bifidobacteria can lower blood cholesterol levels through mechanisms such as absorbing cholesterol and interfering with the enterohepatic circulation of cholesterol, which is of certain significance in preventing cardiovascular diseases.

[0009] Relieving lactose intolerance: Bifidobacteria can produce lactase, which helps lactose-intolerant individuals break down lactose and reduce symptoms such as bloating and diarrhea after drinking milk and other dairy products.

[0010] (2) Food industry sector

[0011] Fermented dairy products: widely used in the production of yogurt, fermented milk beverages and other products, which can not only improve the flavor and taste of the products, but also enhance their nutritional value.

[0012] Functional food additives: As probiotic additives, they can be added to various foods such as bread, biscuits, and beverages to meet consumers' demand for healthy foods.

[0013] (3) Animal breeding sector

[0014] Improving animal production performance: In livestock and poultry farming, adding Bifidobacterium preparations can improve animal intestinal health, increase feed utilization, promote animal growth and development, and increase farming efficiency.

[0015] Prevention of animal diseases: Regulates the balance of animal intestinal flora, enhances animal immunity, reduces the incidence of diarrhea, respiratory diseases, etc., reduces the use of antibiotics, and improves the quality and safety of animal products.

[0016] Different types of Bifidobacteria have different applications, and research and development of Bifidobacteria remains crucial in order to obtain more drugs and other applications. Summary of the Invention

[0017] To address the shortcomings of the existing technology, this invention aims to provide a Bifidobacterium ZK-77-S strain for space-bred animals and its applications, which has an inhibitory effect on a variety of pathogenic bacteria.

[0018] To solve the above problems, the present invention adopts the following technical solution:

[0019] In a first aspect, the present invention provides a space-bred animal Bifidobacterium animalis ZK-77-S, which was deposited on December 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33182, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0020] Secondly, this invention provides the application of the aforementioned space-bred animal Bifidobacterium animalis ZK-77-S in the preparation of oral treatment, oral cleaning drugs, and daily necessities.

[0021] Thirdly, the present invention provides the application of the aforementioned space-bred animal Bifidobacterium animalis ZK-77-S in the preparation of an inhibitor or remover of biofilm colonization on the surface of oral medical devices.

[0022] As one possible implementation, the space-bred animal Bifidobacterium animalis ZK-77-S inhibits and resists Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Fusobacterium nucleatum, or Porphyromonas gingivalis.

[0023] As one possible implementation, the space-bred animal Bifidobacterium animalis ZK-77-S inhibits Streptococcus mutans or Streptococcus Gordonii.

[0024] Fourthly, the present invention provides a medicine or daily necessities, wherein the medicine or daily necessities comprise the aforementioned Bifidobacterium animalis ZK-77-S and / or its metabolites.

[0025] The beneficial effects of this invention are as follows: The Bifidobacterium animalis ZK-77-S obtained by space breeding has an inhibitory effect on Escherichia coli, Pseudomonas aeruginosa, Fusobacterium nucleatum, Porphyromonas gingivalis, Staphylococcus aureus, Candida albicans, etc., and can be used in the preparation of related drugs. Attached Figure Description

[0026] Figure 1 This is a microscopic image of the 0th generation of Bifidobacterium animalis ZK-77-S, a species of animal bred in space according to the present invention.

[0027] Figure 2 This is a microscopic image of the first generation of Bifidobacterium animalis ZK-77-S, a strain of the space-bred animal of this invention.

[0028] Figure 3 This is a microscopic image of the second generation of Bifidobacterium animalis ZK-77-S, a species of animal bred in space according to the present invention. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments.

[0030] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0031] The present invention relates to Bifidobacterium animalis ZK-77-S, which was deposited on December 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33182, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] The space-bred Bifidobacterium animalis ZK-77-S of this invention was obtained by space breeding of Bifidobacterium animalis ZK-77 via the Shijian-19 satellite. The subculture method of Bifidobacterium animalis ZK-77-S of this invention is described below. Figures 1-3 .

[0033] The Shijian-19 satellite was launched from the Jiuquan Satellite Launch Center on September 27, 2024, and successfully recovered at the Dongfeng Landing Site on October 11, 2024.

[0034] I. Isolation and Identification of Strains

[0035] 1. Method

[0036] Take 100 μL of Bifidobacterium animalis ZK-77-S sample returned from space and serially dilute it to 10⁻⁶. -1 -10 -4 Double the volume to prepare a diluent;

[0037] MRS medium containing mupirocin lithium salt was used as a selective medium, and a dilution was spread on plates to isolate Bifidobacteria.

[0038] The plate was placed at 37°C and anaerobic overnight. Single colonies were picked, purified, and identified and preserved using 16S culture.

[0039] 16S Identification: 16S PCR fragments were generated using universal primers and sent to a sequencing company for sequencing. For Bifidobacterium animalis, universal primers were used for sequencing, and the amplified target fragments were gene fragments representing different strains and genera. After sequencing, the sequencing results were analyzed using BLAST on the National Center for Biotechnology Information (NCBI) website to determine the bacterial species.

[0040] 2. Results

[0041] The sequencing results of Bifidobacterium animalis ZK-77-S are shown in SEQ ID No. 1:

[0042]

[0043] The DNA identification results of Bifidobacterium animalis ZK-77-S of this invention are 99% consistent with those of Bifidobacterium animalis.

[0044] II. Evaluation of the genetic stability of Bifidobacterium animalis ZK-77-S

[0045] 1. Method:

[0046] 1) Strains Preparation: Two days before the experiment, the bacterial suspensions of Bifidobacterium animalis ZK-77 and the space-cultured ZK-77-S strain were streaked onto plates to obtain ZK-77 plates and ZK-77-S plates. These plates were placed in anaerobic culture bags and incubated at 37°C for 36 hours to obtain plates with different ZK-77 and ZK-77-S colonies. Single colonies of ZK-77 and ZK-77-S were picked from the plates using a pipette tip. The picked single colonies were placed in 1 ml LMR broth and thoroughly mixed to ensure sufficient contact and reproduction of the bacteria. The plates were then placed in anaerobic culture bags and incubated at 37°C for 10-12 hours before detection.

[0047] 2) Culture medium: Modified MRS liquid medium (containing 0.05% L-cysteine ​​hydrochloride), pH 6.2.

[0048] 3) Culture conditions: Anaerobic environment (85% N2, 10% CO2, 5% H2), constant temperature culture at 37℃.

[0049] 4) Subculture: Take the activated bacterial culture and transfer it to fresh culture medium at an inoculum of 1%. Each 5 generations constitutes one testing cycle, and the culture is continuously subcultured up to the 30th generation. Every 5 subcultures (i.e., the 5th, 10th, 15th, 20th, 25th, and 30th generations), take samples to determine the oxygen tolerance coefficient.

[0050] 5) Method for determining oxygen tolerance coefficient: Aerobic conditions: Dilute the bacterial culture to 10... 6 CFU / mL, inoculated onto modified MRS agar plates, incubated aerobically at 37°C for 48 hours, and viable count (A).

[0051] Anaerobic conditions: After inoculation with bacterial solutions of the same concentration, the cultures were placed in an anaerobic tank and cultured for the same period of time. The number of viable bacteria (B) was then counted.

[0052] Oxygen tolerance coefficient = (number of viable bacteria A under aerobic conditions / number of viable bacteria B under anaerobic conditions) × 100%.

[0053] 2. Results:

[0054] Original strain ZK-77: Its oxygen tolerance coefficient decreases significantly with increasing passage number. For example:

[0055] 5th generation: Oxygen tolerance coefficient is 85%;

[0056] 30th generation: Oxygen tolerance coefficient drops to 72%.

[0057] Space-bred strain ZK-77-S: exhibits a small fluctuation range in oxygen tolerance and maintains a high level.

[0058] 5th generation: Oxygen tolerance coefficient is 88%;

[0059] 30th generation: The oxygen tolerance coefficient remains at 83%.

[0060] Conclusion: After 30 consecutive passages, the oxygen tolerance coefficient of ZK-77-S induced by space mutagenesis decreased by only 5%, while that of the original strain ZK-77 decreased by 13%, indicating that the genetic stability of ZK-77-S is significantly better than that of the original strain.

[0061] III. Evaluation of the genetic stability of acid resistance in Bifidobacterium animalis ZK-77-S

[0062] 1. Method:

[0063] 1) Culture medium: simulated gastric juice culture medium (containing 0.3% pepsin, pH 2.5) and recovery culture medium (modified MRS + 0.1% ox bile salt, pH 6.8).

[0064] 2) Cultivation conditions:

[0065] Acid stress culture: simulate the gastric environment by aerobic shaking (150 rpm) at 37°C for 1 hour.

[0066] Recovery culture: The bacterial culture after stress was transferred to recovery culture medium and anaerobic incubated at 37°C for 18 hours.

[0067] 3) Adaptive propagation:

[0068] The activated bacterial solution was divided into two groups for treatment:

[0069] Conventional group: passaged in standard MRS medium.

[0070] Acid stress group: cultured under acid stress first, then transferred to recovery medium for subculture.

[0071] Two culture modes are used alternately every 5 generations to simulate the alternating exposure of the strain to processing and storage (routine) and the digestive tract environment (stress).

[0072] 4) Acid resistance test method:

[0073] Take the bacterial cultures from generations 5 / 10 / 15 / 20 / 25 / 30 for gradient testing:

[0074] Adjust the bacterial concentration to 10. 8 CFU / mL.

[0075] Mix 1 mL of bacterial culture with 9 mL of simulated gastric fluid (final pH 2.5).

[0076] After incubating at 37°C with shaking for 2 hours, the reaction was immediately terminated with PBS buffer (pH 7.4).

[0077] After serial dilution, the samples were plated on MRS agar plates and anaerobically cultured for 48 hours to count the surviving colonies.

[0078] 5) Survival rate calculation:

[0079] Survival rate (%) = (number of viable bacteria after stress / initial number of viable bacteria) × 100%.

[0080] Establish a survival rate decay model: Y = Y0 × e^(-kX), where X is the number of generations and k is the decay coefficient.

[0081] 2. Results:

[0082] 1) Original strain ZK-77:

[0083] In the standard group, the survival rate of the 30th generation decreased from 78% to 55% (k=0.015 / generation).

[0084] Stressed group: Survival rate decline accelerated, with only 42% surviving by the 30th generation (k=0.023 / generation).

[0085] 2) Space strain ZK-77-S:

[0086] Conventional group: Survival rate remained at 82%-79% (k=0.002 / generation).

[0087] Stress group: Survival rate decreased from 84% to 80% (k=0.003 / generation).

[0088] 3) Observation using scanning electron microscopy revealed:

[0089] ZK-77-S retains its intact cell wall structure after passage, and its surface proteins are relatively evenly distributed.

[0090] ZK-77 showed obvious cell wall indentation, and the shedding rate of surface adhesive material reached 20%.

[0091] IV. Growth curves and viable count determination of Bifidobacterium animalis ZK-77-S under different oxygen concentrations

[0092] To investigate the differences in growth characteristics of strain ZK-77 and its space-mutated ZK-77-S under different oxygen concentrations, this example sets up six oxygen gradients of 0%, 5%, 10%, 15%, 20%, and 25%. The effect of oxygen concentration on the growth of the strain is analyzed by measuring the number of viable bacteria and the growth curve.

[0093] 1. Method:

[0094] 1) Culture medium: LB liquid medium (pH 7.0, containing 1% glucose).

[0095] 2) Equipment: Anaerobic workstation (oxygen concentration adjustable, error ±0.5%), spectrophotometer, constant temperature shaker (temperature control ±0.5℃), plate counting device.

[0096] 3) Oxygen concentration groups: 0% (pure nitrogen environment), 5%, 10%, 15%, 20%, 25% (normal oxygen control).

[0097] 4) Activation and pre-culture of microbial strains

[0098] ZK-77 and ZK-77-S were inoculated into LB liquid medium and pre-cultured for 12 hours under normal oxygen conditions (20% oxygen), 37°C, and 180 rpm until the logarithmic growth phase (OD600≈0.6).

[0099] 5) Oxygen concentration grouping and inoculation

[0100] The pre-cultured bacterial solution was transferred to LB medium in 6 anaerobic workstations at an inoculation rate of 1%, with 3 parallel samples in each group.

[0101] The oxygen concentration in the workstation is adjusted to the target value (0%, 5%, 10%, 15%, 20%, 25%) using a nitrogen / oxygen mixture, the temperature is kept constant at 37℃, and the shaking speed is 180 rpm.

[0102] 6) Growth curve determination

[0103] Sampling time point: Sampling is carried out every 6 hours for a period of 72 hours.

[0104] 7) Viable bacteria count determination

[0105] Take 1 mL of bacterial culture and dilute it serially with sterile physiological saline to 10^6 times.

[0106] Spread 100 μL of the diluted solution onto LB agar plates and incubate at 37°C for 24 hours. Count the colony forming units (CFU / mL).

[0107] OD600 value determination: The absorbance of the bacterial solution at 600 nm was measured using a spectrophotometer, and the changes in bacterial density were recorded.

[0108] 8) Data Processing

[0109] Calculate the maximum specific growth rate (μ_max), lag phase duration, and peak viable cell count during the stationary phase.

[0110] 2. Results:

[0111] As shown in Table 1, the original strain ZK-77 could not grow under 0% oxygen conditions, while the space-mutated ZK-77-S showed significant hypoxia tolerance, with a viable count of 2.5 × 10⁻⁶. 8 The CFU / mL level indicates that space-induced mutagenesis enhanced its anaerobic metabolic capacity. ZK-77 exhibited the highest growth rate (μ_max = 0.40 h⁻¹) under 20% oxygen conditions. -1 ZK-77-S reached its maximum viable count (1.2 × 10⁻⁶) under 10% oxygen conditions. 9 The CFU / mL indicates that the optimal oxygen concentration of the strain decreased after space breeding.

[0112] Table 1 Comparison of bacterial counts of ZK-77 and ZK-77-S under different oxygen concentrations

[0113]

[0114] V. Detection of the ability of Bifidobacterium animalis ZK-77-S to produce small molecule fatty acids

[0115] 1. Method

[0116] 1) Sample preparation:

[0117] 100 μL of the bacterial strain suspension was pipetted into 96-well plates, and its absorbance at 600 nm was measured using a spectrophotometer. Based on the preliminary experimental results, the OD values ​​of the six bacterial suspensions were adjusted to be between 0.2 and 0.5 using a concentration gradient dilution method to ensure the accuracy of subsequent experimental measurements.

[0118] Pre-cool the centrifuge to 4°C, centrifuge 700 μL of diluted bacterial solution at 10000g for 2 min to obtain bacterial precipitate; wash the precipitate with 300 μL of PBS reagent pre-cooled at 4°C, and centrifuge again at 10000g for 2 min to obtain bacterial precipitate.

[0119] Add 500 μL of extraction buffer pre-cooled at 4 °C and sonicate in an ice bath for 5 min (power 20%, sonication for 3 s, interval 7 s, repeated 30 times). Centrifuge at 12000 g for 5 min and place the supernatant on ice for analysis.

[0120] 2) Gas chromatography conditions:

[0121] Chromatographic column: VF-WAXms capillary column (30m×0.25mm×0.25μm).

[0122] 3) Heating program: Initial temperature 100℃, increase to 150℃ at 5℃ / min, then increase to 240℃ at 10℃ / min, and hold for 3min.

[0123] 4) Detection: FID detector, injection port temperature 230℃, carrier gas (N2) flow rate 2mL / min.

[0124] 2. Results: Space breeding improved acid production capacity, especially acetic acid, as shown in Table 2.

[0125] Table 2 Comparison of Acid Production Capacity

[0126]

[0127] VI. Antibiotic Resistance Detection of Bifidobacterium animalis ZK-77-S

[0128] 1. Method:

[0129] 1) Drug sensitivity tablet selection: Tetracycline (Tet, 30μg), Chloramphenicol (Cm, 30μg), Erythromycin (Ery, 30μg), Ampicillin (Amp, 30μg), Neomycin (Neo, 30μg), Rifampin (Rif, 30μg).

[0130] 2) Double-layer flat plate method:

[0131] Bottom layer: 1.5% agar nutrient medium.

[0132] Top layer: 0.5% soft agar + 1% bacterial solution (OD) 600 =0.5). After the plate has cured and dried, commercially available antibiotic susceptibility testing tablets are placed inside.

[0133] 3) Result determination:

[0134] An inhibition zone diameter ≥15mm indicates sensitivity, 10~14mm indicates moderate sensitivity, and <10mm indicates resistance.

[0135] 2. Results: Resistance to some antibiotics was enhanced, as shown in Table 3.

[0136] Table 3 Antibiotic Resistance Test Table

[0137]

[0138] VII. In vitro inhibitory effect of Bifidobacterium animalis ZK-77-S on oral pathogenic bacteria

[0139] Method 1:

[0140] 1) Target pathogens: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 27853), Fusobacterium nucleatum (ATCC 25586), Candida albicans (ATCC 10231), Porphyromonas gingivalis (ATCC 33277).

[0141] 2) Culture medium: MRS liquid medium (containing 0.05% L-cysteine) was used for Bifidobacterium; brain heart extract (BHI) medium was used for pathogenic bacteria.

[0142] 3) Preparation of bacterial suspension: ZK-77 and ZK-77-S were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 24 hours. After centrifugation, the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL.

[0143] 4) Antibacterial activity test:

[0144] The agar diffusion method was used: the target pathogenic bacteria were evenly spread on BHI agar plates, and sterile filter paper discs (6 mm in diameter) were placed on the surface. 10 μL of ZK-77 and ZK-77-S bacterial suspensions were added separately. A blank control (sterile saline) and a positive control (0.2% chlorhexidine solution) were set up. After incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured (unit: mm).

[0145] 5) Minimum inhibitory concentration (MIC) determination:

[0146] The ZK-77 and ZK-77-S bacterial suspensions were serially diluted (1×10⁻⁶) using the micro-broth dilution method. 6 ~1×10 9 The concentration of CFU / mL was co-cultured with the target bacteria for 48 hours to observe the lowest concentration that completely inhibited growth.

[0147] 2. Results:

[0148] The inhibition zone diameter of ZK-77-S against all 6 oral pathogens was significantly higher than that of the original strain ZK-77 (p<0.01), with an increase of 24.1% in inhibition against Staphylococcus aureus and 31.0% in inhibition against Candida albicans (see Table 4).

[0149] The MIC value of ZK-77-S is 50%~40% lower than that of ZK-77, indicating that its antibacterial activity is effective at low concentrations, especially against Fusobacterium nucleatum (MIC reduced by 50%) and Candida albicans (MIC reduced by 40%), as shown in Table 5.

[0150] Space mutagenesis enhances the broad-spectrum antibacterial properties of ZK-77-S by increasing the secretion of bacterial metabolites (such as acetic acid and propionic acid), making it suitable for the development of oral care products.

[0151] Table 4 Comparison of inhibition zone diameters between ZK-77 and ZK-77-S against oral pathogens

[0152]

[0153] Note: ** indicates that ZK-77-S is significantly different from ZK-77 (p<0.01, t-test).

[0154] Table 5 Comparison of Minimum Inhibitory Concentrations (MICs) of ZK-77 and ZK-77-S

[0155]

[0156] VIII. Verification of the targeted removal effect of Bifidobacterium animalis ZK-77-S metabolites on dental plaque biofilm.

[0157] 1. Method

[0158] 1) Target strains: Streptococcus mutans (ATCC 25175) and Streptococcus grosvenorii (ATCC 10558).

[0159] 2) Culture medium:

[0160] Bifidobacterium culture: MRS liquid medium (supplemented with 0.1% glucose + 0.02% taurine).

[0161] Biofilm culture: Artificial saliva culture medium (containing 2% sucrose and 0.2% urea).

[0162] 3) Key reagents:

[0163] Crystal violet staining kit (Sigma-Aldrich), live / dead bacteria double staining reagent (L7012, ThermoFisher)

[0164] Scanning electron microscopy fixative (2.5% glutaraldehyde + 4% paraformaldehyde mixture).

[0165] 4) Extraction of metabolites

[0166] ZK-77 and ZK-77-S were cultured in MRS medium until the late logarithmic phase (OD600 = 1.2 ± 0.1, approximately 18 hours). Cell-free supernatant (CFS) was obtained by filtration through a 0.22 μm sterile filter, aliquoted, and stored at -80°C. Before use, it was diluted with PBS buffer to three concentration gradients: 25%, 50%, and 100%.

[0167] 5) Construction of in vitro biomembrane models

[0168] In 96-well U-shaped culture plates, a mixed bacterial suspension of *Streptococcus mutans* and *Streptococcus Gordonii* (1:1 ratio, total bacterial count 1 × 10^6 CFU / well) was inoculated. Artificial saliva culture medium was added to a total volume of 200 μL, and the plates were incubated at 37°C for 48 hours under microaerophilic conditions (5% CO2). The culture medium was replaced with fresh medium every 24 hours to simulate the dynamic oral environment.

[0169] 6) Biomembrane intervention experiment

[0170] After removing the old culture medium, add the following:

[0171] Experimental group: CFS (three concentration gradients) of ZK-77 / ZK-77-S.

[0172] Control group: blank MRS culture medium, 0.12% chlorhexidine solution.

[0173] Continue culturing for 24 hours before testing.

[0174] 7) Quantitative analysis of biofilms

[0175] Crystal violet staining method: Discard the culture medium, gently wash three times with PBS to remove airborne bacteria; add 200 μL of 0.1% crystal violet solution and stain at room temperature for 15 minutes; after desorption with 33% glacial acetic acid, measure the absorbance at 570 nm using an ELISA reader.

[0176] Viable cell count method: The biofilm was disrupted by ultrasonication (40 kHz, 5 min) and then serially diluted; it was spread on BHI agar plates and incubated for 48 hours to count the colonies.

[0177] 8) Microstructure observation

[0178] A biofilm was constructed on a silicon wafer substrate, fixed with 2.5% glutaraldehyde, and then dehydrated in a gradient manner. After gold sputtering, the surface structure was observed using a field emission scanning electron microscope (FE-SEM, Hitachi SU8010).

[0179] 2. Results:

[0180] The ZK-77-S metabolite, at a 100% concentration, exhibited a clearance rate of 86.3% against mixed biofilms, approaching that of chlorhexidine (98.7%) (see Table 6). A 2.3-fold increase in butyric acid concentration was detected in the metabolite (HPLC determination). This substance achieves dual antibacterial effects by disrupting cell membrane integrity and inhibiting gtfB gene expression.

[0181] This embodiment demonstrates that ZK-77-S has clinical application potential in preventing dental plaque formation and is suitable for inhibiting biofilm colonization on the surfaces of orthodontic brackets, dentures, and other instruments.

[0182] Table 6. Inhibitory effects of different concentrations of CFS on mixed biofilms

[0183]

[0184] Note: *p<0.05, **p<0.01 (one-way ANOVA compared with blank control).

[0185] Electron microscopy observation results:

[0186] The biofilm in the blank control group exhibited a typical honeycomb structure, with cells tightly connected by extracellular polysaccharides. The ZK-77 treatment group showed localized cavities, but the overall structure remained largely intact. The ZK-77-S treatment group (100% concentration) showed that most of the biofilm disintegrated, with only scattered individual cells remaining.

[0187] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A type of Bifidobacterium animalis (Bifidobacterium animalis) ZK-77-S, which was deposited at the China General Microbiological Culture Collection Center on December 24, 2024, with accession number CGMCC No. 33182.

2. The use of the space-bred animal Bifidobacterium animalis ZK-77-S as described in claim 1 in the preparation of oral treatment drugs or oral hygiene daily necessities; The oral treatment or oral cleaning is achieved by inhibiting Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Fusobacterium nucleatum, or Porphyromonas gingivalis using Bifidobacterium animalis ZK-77-S bred in space.

3. The use of the space-bred animal Bifidobacterium animalis ZK-77-S as described in claim 1 in the preparation of an inhibitor or remover for biofilm colonization on the surface of oral medical devices; The biofilm colonization is caused by Streptococcus mutans or Streptococcus Gordonii.

4. A medicine or daily necessities, characterized in that, The medicine or daily necessities described herein contain Bifidobacterium animalis ZK-77-S as described in claim 1.

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