Propionibacterium acnes bacteriophage y2-3, preparation and application thereof

Abstract

This invention relates to a strain of Propionibacterium acnes phage Y2-3, its formulation, and its applications, belonging to the field of biotechnology. This invention provides a strain of Propionibacterium acnes phage Y2-3, which is a novel species capable of rapidly acting on the host bacterium, specifically killing Propionibacterium acnes. It exhibits highly efficient and broad-spectrum lytic activity against Propionibacterium acnes, while being less prone to inducing drug resistance. It is also skin-microecologically friendly, safer to use, and exhibits good stability, providing a new technical means for the prevention and treatment of acne.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a strain of Propionibacterium acnes phage Y2-3, its preparation and application. Background Technology

[0002] Acne is a common chronic inflammatory skin disease that primarily occurs on the face, back, and chest. It has a high incidence rate and seriously affects the physical and mental health of patients. Propionibacterium acnes (…) Cutibacterium acnes Excessive proliferation of acne cells is considered one of the core elements in the pathogenesis of acne.

[0003] Currently, clinical treatment of acne mainly relies on antibiotics (such as clindamycin and erythromycin) and benzoyl peroxide. However, existing technologies have significant drawbacks: (1) Drug resistance: Long-term use of antibiotics has led to the widespread emergence of drug-resistant strains of Propionibacterium acnes, resulting in a significant decrease in the efficacy of antibiotics. (2) Disruption of the skin flora: While broad-spectrum antibiotics kill Propionibacterium acnes, they also indiscriminately kill beneficial bacteria on the skin surface, disrupting the skin's microecological balance and potentially causing skin sensitivity or other infections. (3) Irritation / side effects: Drugs such as benzoyl peroxide have a strong irritant effect on the skin of some patients, leading to adverse reactions such as redness, swelling, and desquamation.

[0004] Therefore, developing a novel antibacterial agent that can specifically kill Propionibacterium acnes, is not prone to drug resistance, and is friendly to the skin microecology has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a Propionibacterium acnes phage Y2-3, a preparation thereof, and its application. The phage Y2-3 can rapidly act on the host bacteria and specifically kill Propionibacterium acnes. It has a highly efficient and broad-spectrum lysis effect, is not prone to drug resistance, and is friendly to the skin microecology.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Propionibacterium acnes phage Y2-3, the preservation number of which is CGMCC No.47222.

[0007] The present invention provides a formulation for preventing or improving acne, the formulation comprising the aforementioned bacteriophage Y2-3.

[0008] Preferably, the formulation further includes excipients.

[0009] Preferably, the excipients include one or more of the following: oily matrix, aqueous matrix, emulsifier, preservative, pH adjuster, humectant, thickener, and buffer.

[0010] Preferably, the bacteriophage content in the formulation is 1×10⁻⁶. 5 ~1×10 10 PFU / mL or 1×10 5 ~1×10 10 PFU / g.

[0011] Preferably, the pH of the preparation is 5.0 to 9.0.

[0012] The present invention also provides the application of the bacteriophage Y2-3 and the preparation thereof in the preparation of products for preventing and treating Propionibacterium acnes infection.

[0013] The present invention also provides the application of the phage Y2-3 and the preparation thereof in the preparation of products for the prevention or improvement of acne.

[0014] Preferably, the pathogen causing the acne includes Propionibacterium acnes.

[0015] Preferably, the Propionibacterium acnes includes antibiotic-resistant Propionibacterium acnes.

[0016] The beneficial effects of this invention are: This invention provides a phage Y2-3 that lyses Propionibacterium acnes, belonging to a new phage species. This phage has the following characteristics: (1) Strong source targeting: The phage Y2-3 of this invention is isolated from acne lesions (skin sebaceous granules). Compared with phages isolated from sewage or soil, it has a stronger ability to recognize and lyse Propionibacterium acnes and is more adaptable to the microenvironment of skin lesions. (2) Highly efficient and broad-spectrum lysis: The unique tail fimbriae structure of Y2-3 enables it to specifically recognize and widely lyse Propionibacterium acnes. It has a highly efficient lysis effect on 30 clinically isolated Propionibacterium acnes (lysis rate >99.99%), with a wide coverage, and its mechanism of action (lysis of bacterial cell wall) is different from that of traditional antibiotics, making it less likely to induce antibiotic resistance in bacteria. (3) High safety: The phage itself is a bacterial virus and is non-toxic to eukaryotic cells. Furthermore, the genome sequencing of the phage Y2-3 of this invention has confirmed that it does not contain virulence factors or antibiotic resistance genes, ensuring its high-efficiency bactericidal ability and biosafety as a virulent phage. (4) Good environmental tolerance: The protein coat structure of the phage Y2-3 itself makes it resistant to ultraviolet light, which is convenient for short-term exposure in the environment during daily use. It is not sensitive to chloroform, indicating that it has no lipid envelope and its structure is stable. It has good stability in the temperature range of -20℃ to 37℃ and the pH range of 5.0 to 9.0, which can meet the pH requirements of daily skin preparations. The stability of the phage can ensure that the phage maintains a high titer in daily life and skin environment, which is convenient for it to play a role in the microenvironment of skin lesions. (5) Fast action: The tail filament adsorption kinetics of the phage Y2-3 of this invention enable it to complete adsorption in a short time, indicating that the phage Y2-3 of this invention can act rapidly on the host bacteria to achieve a rapid bactericidal effect.

[0017] Biological Preservation Information Propionibacterium acnes phage Y2-3, taxonomically named Cutibacterium acnes phage It was deposited on July 22, 2026 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47222. Attached Figure Description

[0018] Figure 1 This image shows the results of phage plaques Y2-3 obtained by isolation and purification using the double-layer agar plate method.

[0019] Figure 2 This is an electron micrograph of bacteriophage Y2-3, scale bar 100 nm.

[0020] Figure 3 This is a circumscribed map of the genome of bacteriophage Y2-3.

[0021] Figure 4 This is a diagram showing the phylogenetic tree analysis results for bacteriophage Y2-3.

[0022] Figure 5 The temperature stability curve of bacteriophage Y2-3 is shown.

[0023] Figure 6 The pH stability curve of bacteriophage Y2-3 is shown.

[0024] Figure 7 The UV inactivation curve of bacteriophage Y2-3 is shown.

[0025] Figure 8 The chloroform stability curve of bacteriophage Y2-3 is shown.

[0026] Figure 9 This is a partial schematic diagram showing the results of specific lysis of 30 strains of Propionibacterium acnes by phage Y2-3.

[0027] Figure 10 This is a lysis curve (viable cell count) of bacteriophage Y2-3.

[0028] Figure 11 This is a lysis curve (OD value) of bacteriophage Y2-3. Detailed Implementation

[0029] This invention provides a Propionibacterium acnes phage Y2-3, the preservation number of which is CGMCC No.47222.

[0030] The bacteriophage Y2-3 of this invention was isolated from acne lesions (skin sebaceous granules). Under a transmission electron microscope, this bacteriophage exhibits a regular polyhedral head with a diameter ranging from 50 to 70 nm (e.g., 60 nm); it also possesses a retractable tail with a length ranging from 100 to 150 nm (e.g., 120 nm). According to the International Committee on Taxonomy of Viruses (ICTV) classification, this bacteriophage belongs to the order Tailed Phages. The genome of bacteriophage Y2-3 is double-stranded DNA (dsDNA), with a full length of 29,420 bp; the G+C content is 56.12% (sequencing results show that the GC content of the positive strand is 56.14%, the GC content of the antisense strand is 56.10%, and the average GC content is 56.12%). The genome does not contain antibiotic resistance genes or virulence factor genes. Whole-genome sequencing and alignment showed less than 90% homology with known Propionibacterium acnes bacteriophages, proving it to be a newly discovered bacteriophage belonging to a new species.

[0031] The bacteriophage Y2-3 of this invention can rapidly act on host bacteria, reaching adsorption saturation (adsorption rate >95%) within 15 minutes. It can efficiently and broadly lyse *Propionibacterium acnes*, with a multiplicity of infection ranging from 0.001 to 0.1. It exhibits highly efficient lysis of 30 clinically isolated *Propionibacterium acnes* strains (lysis rate >99.99%), demonstrating broad coverage. Furthermore, its mechanism of action (lysis of the bacterial cell wall) differs from traditional antibiotics, making it less likely to induce bacterial resistance. The bacteriophage Y2-3 of this invention is skin-microenvironment-friendly, exhibiting high safety, and demonstrates tolerance to changes in ultraviolet light, pH, and temperature. It is insensitive to chloroform and exhibits good stability. Compared to bacteriophages isolated from environments such as sewage or soil, it possesses stronger recognition and lysis capabilities for *Propionibacterium acnes* and is better adapted to the skin lesion microenvironment.

[0032] This invention provides a formulation for preventing or improving acne, the formulation comprising the aforementioned bacteriophage Y2-3. The formulation prepared using bacteriophage Y2-3 utilizes the highly efficient lysis of *Propionibacterium acnes* by bacteriophage Y2-3 to kill *Propionibacterium acnes*, thereby preventing (preventing and / or treating) *Propionibacterium acnes* infection and achieving the effect of preventing or improving acne.

[0033] In this invention, the bacteriophage content in the formulation is preferably 1×10⁻⁶. 5 ~1×10 10 In some embodiments, the PFU / mL can be 1×10⁻⁶. 5 PFU / mL, 1×10 6 PFU / mL, 1×10 7 PFU / mL, 1×10 8 PFU / mL, 1×10 9 PFU / mL or 1×10 10 PFU / mL, or preferably 1×10 5 ~1×10 10 In some embodiments, the PFU / g can be 1×10⁻⁶. 5 PFU / g, 1×10 6 PFU / g, 1×10 7 PFU / g, 1×10 8 PFU / g, 1×10 9 PFU / g or 1×10 10 PFU / g. The pH of the formulation is preferably 5.0 to 9.0, and can be selected from 5.0, 6.0, 7.0, 7.5, 8.0, 8.5 or 9.0.

[0034] In one embodiment, the active ingredient of the formulation is preferably bacteriophage Y2-3 as the sole active ingredient. In some embodiments, the active ingredient of the formulation may also preferably include other components with antibacterial activity against Propionibacterium acnes, wherein the antibacterial activity against Propionibacterium acnes is preferably the activity of inhibiting and / or killing Propionibacterium acnes. The other components with antibacterial activity against Propionibacterium acnes are preferably able to exert an additive or synergistic effect with bacteriophage Y2-3 of the present invention, thereby achieving a better anti-Propionibacterium acnes infection effect and improving the prevention, treatment, or improvement of acne. The present invention does not specifically limit the other components with antibacterial activity against Propionibacterium acnes; they may be bacteriophages or non-phage components. Other components with antibacterial activity against Propionibacterium acnes preferably also include lyases and / or recombinant lyase proteins, wherein the lyases or recombinant lyase proteins are preferably obtained by extracting bacteriophage Y2-3 lyase from bacteriophage Y2-3, or by extracting the bacteriophage Y2-3 lyase gene to construct and prepare recombinant lyase proteins.

[0035] In one embodiment, the formulation preferably further includes excipients, which preferably include one or more of oily matrix, aqueous matrix, emulsifier, preservative, pH adjuster, humectant, thickener, and buffer. In some embodiments, the excipients preferably include matrix components suitable for the preservation of bacteriophage Y2-3. This invention does not specifically limit the matrix components suitable for bacteriophage preservation; conventional components suitable for bacteriophage preservation can be used, specifically SM buffer solution.

[0036] This invention does not impose any particular limitation on the dosage form of the formulation; conventional dosage forms in the art may be used. In some embodiments, the dosage form includes, but is not limited to, solutions, lotions, ointments, emulsions, pastes, gels, or powders. The formulation preferably includes an antibacterial agent or a drug.

[0037] The present invention also provides the application of the bacteriophage Y2-3 and the preparation thereof in the preparation of products for preventing and treating Propionibacterium acnes infection.

[0038] The bacteriophage Y2-3 described in this invention, and the bacteriophage Y2-3 in the preparation, can kill Propionibacterium acnes by lysing it, thereby preventing or treating infections caused by Propionibacterium acnes.

[0039] This invention also provides the application of the aforementioned bacteriophage Y2-3 and the aforementioned formulation in the preparation of products for the prevention or improvement of acne. The pathogens causing acne preferably include Propionibacterium acnes.

[0040] The bacteriophage Y2-3 described in this invention, and the bacteriophage Y2-3 in the preparation, can kill Propionibacterium acnes by lysing it, thereby preventing or improving acne caused by Propionibacterium acnes.

[0041] The present invention does not specifically limit the type of product, but the product preferably includes a drug.

[0042] In this invention, there is no particular limitation on the dosage form of the product; conventional dosage forms in the art can be used. In some embodiments, the dosage form includes, but is not limited to, solutions, lotions, ointments, emulsions, pastes, gels, or powders.

[0043] In this invention, the Propionibacterium acnes preferably includes antibiotic-resistant Propionibacterium acnes, and also includes antibiotic-resistant Propionibacterium acnes.

[0044] The preferred bacteriophage Y2-3 is capable of lysing Propionibacterium acnes. Due to its different antibacterial mechanism from antibiotics, it can still efficiently lyse antibiotic-resistant Propionibacterium acnes, thereby achieving the prevention and treatment of infection caused by antibiotic-resistant Propionibacterium acnes, or the prevention, treatment, or improvement of acne caused by infection with antibiotic-resistant Propionibacterium acnes.

[0045] In this invention, the following terms are explained: Bacteriophages: a type of virus that specifically infects bacteria, can lyse bacteria, and has host specificity.

[0046] Propionibacterium acnes: a Gram-positive anaerobic bacillus, one of the normal flora of the skin, whose excessive proliferation is closely related to acne inflammation.

[0047] PFU (Plaque Forming Unit): A unit used to measure the number of infectious phage particles.

[0048] CFU (Colony Forming Unit): A unit used to measure the number of viable bacteria.

[0049] MOI (Multiplicity of Infection): The ratio of the number of bacteriophages to the number of host bacteria at the time of infection.

[0050] OD 600 Optical density at a wavelength of 600 nm is used to indirectly measure the concentration of bacterial suspension.

[0051] Double-layer agar plate method: a culture method commonly used for the isolation, purification and counting of bacteriophages, consisting of a nutrient agar bottom layer and a semi-solid agar top layer containing host bacteria.

[0052] Adsorption rate: The percentage of bacteriophages that can bind to the surface of the host bacteria after the bacteriophage is mixed with the host bacteria, relative to the initial bacteriophage.

[0053] Chloroform stability: Used to detect whether a bacteriophage has a lipid envelope. Encapsulated bacteriophages are sensitive to chloroform, while those without an envelope are not.

[0054] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Unless otherwise specified, the following embodiments are all conventional methods.

[0056] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0057] Example 1 1. Types and sources of raw materials: Sebaceous granules from the face of acne patients.

[0058] 2. Process flow and parameters: (1) Separation a. Sample processing: Sebaceous granules were collected from the faces of 5 acne patients. The collected sebaceous granules were placed in brain and heart infusion broth culture medium, shaken vigorously for 30 minutes, centrifuged at 4℃ and 8000rpm for 10 minutes, the supernatant was collected, and filtered through a 0.22μm filter membrane for sterilization to obtain the sample filtrate.

[0059] b. Enrichment culture: Take 500 μL of sample filtrate and add 5 mL of Propionibacterium acnes bacterial culture in the logarithmic growth phase (OD200). 600 =0.6), and anaerobic culture at 37℃ for 48 hours yielded an enriched culture medium.

[0060] c. Separation and Purification: Centrifuge the enriched culture medium (4℃, 10000 rpm, 10 minutes), collect the supernatant, filter, and then perform a 10-fold serial dilution of the filtrate using the double-layer agar plate method for separation. The upper agar layer contains the host bacteria (Propionibacterium acnes) and phages, while the lower layer is basic agar. Incubate anaerobically at 37℃ for 48 hours and observe the phage plaques. Using a 200 μL sterile pipette tip, pick the largest, clear phage plaque and place it in SM buffer. Incubate at 4℃ for at least 4 hours or overnight to allow the phages to release into the buffer. The next day, perform a 10-fold serial dilution. Mix 10 μL of the diluted solution with 200 μL of the bacterial culture (Propionibacterium acnes) and perform double-layer agar plate incubation again. Repeat the double-layer agar plate method 10 times to obtain phage plaques with uniform morphology, thus obtaining purified phages.

[0061] (2) Phage culture: The purified phage is cultured with phages in the logarithmic growth phase (OD2). 600The Propionibacterium acnes bacterial suspension (=0.6) was mixed at an MOI of 0.1. The suspension was anaerobically cultured at 37°C for 48 hours until the suspension was clear (the bacteria were completely lysed). The lysate was centrifuged at 4°C and 10,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter to remove bacteria, and a high-titer phage stock solution was obtained using the polyethylene glycol precipitation method for subsequent detection.

[0062] (3) Take the purified phage stock solution from step (2), drop it onto a copper grid, negatively stain with 2% phosphotungstic acid (PTA) for 5 minutes, allow it to air dry, and then observe and photograph it under a transmission electron microscope. Perform genomic analysis on the phage.

[0063] 3. Experimental Results: (1) A bacteriophage was successfully isolated, forming a clear, circular phage plaque with a diameter of about 2-3 mm and a clear edge on a double-layer plate. Figure 1 ).

[0064] (2) Electron microscopy results showed that the phage had a regular polyhedral head (approximately 60 nm in diameter) and a retractable tail (approximately 120 nm), which is consistent with the typical morphological characteristics of tailed phages. Figure 2 ).

[0065] (3) Genome characteristics: See genome circle map Figure 3 See developmental tree Figure 4 The genome is double-stranded DNA (dsDNA), with a full length of 29,420 bp; the G+C content is 56.12% (sequencing results show that the GC content of the sense strand is 56.14%, the GC content of the antisense strand is 56.10%, and the average GC content is 56.12%). Whole-genome sequencing and alignment showed less than 90% homology with known *Propionibacterium acnes* phages, proving it to be a newly discovered phage belonging to a new species. The genome does not contain antibiotic resistance genes or virulence factor genes. Therefore, the *Propionibacterium acnes* phage isolated in this invention is named Y2-3.

[0066] Example 2 Biological characteristics of bacteriophage Y2-3 The following biological characteristics of phage Y2-3 obtained in Example 1 were detected: 1. Optimal Multiple of Infection (MOI, the ratio of phage count to host bacteria count): Select a single, well-defined colony from a host bacterium (Propionibacterium acnes) plate and inoculate it into a test tube containing 5 mL of BHI liquid medium. Prepare two tubes and incubate them in an incubator until the logarithmic growth phase. To ensure more accurate bacterial titers, use the plate count method to measure the bacterial titer. Plate count method procedure: Use a pipette to draw 100 μL of bacterial suspension and perform serial dilutions of 10-fold to a final volume of 10.-7 Next, mix the bacterial suspensions diluted at different ratios with semi-solid BHI medium at 45℃, and quickly pour the mixture into disposable sterile petri dishes while shaking the dishes to level them. After solidification, incubate in an incubator for 36 hours. Select plates with colony counts ranging from 30 to 300 for counting, using the formula: Bacterial titer = Colony count on plate × Dilution factor × 10 (volume 100 μL).

[0067] The host bacterial titer was adjusted to 1 × 10⁻⁶ by adding liquid BHI medium. 8 CFU / mL, phage Y2-3 adjusted to 1×10 8 PFU / mL; 1 mL of bacterial culture was dispensed into 6 sterile test tubes, and different proportions of equal volume of phage were added to each tube (phage number / bacterial number = 10.000, 1.0000, 0.1000, 0.0100, 0.0010, 0.0001), followed by 2 mL of BHI liquid medium. The tubes were then incubated at 37°C in a shaker for 24 hours. The co-culture solution was removed, centrifuged, and filtered to obtain phage Y2-3 solution. The phage titer was measured using the double-layer agar plate method. This experiment was repeated three times. The MOI group that could detect the phage titer after culture and whose post-culture titer was significantly higher than the initial phage titer was determined to be an effective amplified MOI; the minimum to maximum value of the MOI is the effective multiple of infection range of the phage. The optimal MOI is the MOI value at which the phage titer is highest.

[0068] The results show that the infection multiplicity of the bacteriophage Y2-3 of the present invention ranges from 0.001 to 0.1, with the best effect observed at 0.1.

[0069] 2. Temperature stability: The titer was measured after incubating the phage solution at -20℃, 4℃, 37℃, 50℃, 60℃, 70℃, 80℃ and 90℃ for 1 hour.

[0070] Results: Temperature stability curves are shown in the figure. Figure 5 The phage exhibits good stability within the temperature range of -20℃ to 37℃, with no significant decrease in titer, meeting the daily storage and use requirements of 4℃ to 37℃. After treatment at 50℃ for 1 hour, the titer begins to decrease; after treatment at 60℃ or above for 1 hour, the phage activity decreases significantly, even leading to inactivation. The optimal storage temperature is -20℃ to 37℃.

[0071] 3. pH stability: Mix the phage fluid with a buffer solution with a pH value of 1-13 at a volume ratio of 1:9, incubate at 37°C for 1 hour, and then measure the titer.

[0072] Results: The pH stability curve is shown in the figure. Figure 6The phage exhibits good stability within a pH range of 5.0–9.0, with no significant decrease in titer. However, the titer drops sharply or even becomes completely inactive at pH 4.0 and below or pH 10.0 and above. The optimal pH range is 7.0–8.0, which meets the pH requirements of conventional skin preparations.

[0073] 4. UV Sensitivity: Spread 300 μL of phage solution evenly on the bottom of a 16-well plate (approximately 2 mm thick), remove the lid, place in a biosafety cabinet, and apply UV light (wavelength 253.7 nm, intensity ≥135 uW / cm²). 2 The titer was measured immediately after irradiation for 5, 10, 30 and 60 minutes.

[0074] Results: The ultraviolet inactivation curve is shown in the figure. Figure 7 Within 5 minutes of irradiation, the titer decreased by no more than 1 log unit; after 10 minutes of irradiation, the titer decreased by more than 3 log units; after 30 minutes of irradiation, the titer decreased by approximately 4.6 log units; and after 60 minutes of irradiation, the titer decreased by more than 6 log units. This indicates that the bacteriophage has a certain tolerance to short-term ultraviolet exposure, but will be inactivated by prolonged irradiation.

[0075] 5. Chloroform stability: Take 100 μL of phage fluid, add 10 μL, 20 μL, and 50 μL of chloroform respectively, incubate at 37°C for 1 hour and then measure the titer.

[0076] Results: The chloroform stability curve is shown in the figure. Figure 8 After incubation with different volumes of chloroform, the phage titer showed no significant difference from the control group (a decrease of <0.5 log units), indicating that the phage was insensitive to chloroform and belonged to a non-enveloped phage.

[0077] 6. Optimal adsorption time: Take the host bacteria (Propionibacterium acnes) in the logarithmic phase, count them using the plate count method, and adjust the concentration to 1×10⁻⁶. 8 CFU / mL. Simultaneously, the phage titer was adjusted to 1×10⁻⁶. 8 PFU / mL. Take three sterile test tubes, add 4mL of bacterial solution, mix the bacteriophage and bacteria evenly according to the measured optimal MOI (0.1), and put the mixture into an incubator to incubate at 37°C for 5 minutes, 10 minutes and 15 minutes respectively.

[0078] Set the centrifugal force of the low-temperature centrifuge to 10000×g, centrifuge for 10 minutes, filter, and keep the supernatant for later use. Measure the phage titer in each supernatant using the double-layer agar plate method. The optimal adsorption time is defined as the time when the phage titer is lowest. Repeat the experiment three times and take the average of the results.

[0079] The results showed that the adsorption rate could reach over 90% within 10 minutes, and adsorption saturation (adsorption rate > 95%) was reached within 15 minutes. This indicates that the bacteriophage adsorbs host bacteria relatively quickly, and 15 minutes is the optimal adsorption time for bacteriophage Y2-3.

[0080] Example 3 Host range determination of bacteriophage Y2-3 Test method: 1. Test strains: 30 clinically isolated Propionibacterium acnes strains were selected, covering skin lesion samples from different acne patients.

[0081] 2. Host range determination was performed using the double-layer agar plate method: a. Preparation of bottom plates: Prepare solid culture medium plates in advance. The solid culture medium is prepared using brain heart infusion agar (DouDian Biotechnology Co., Ltd.) for later use.

[0082] b. Preparation of the upper layer of bacterial soft agar: Culture each test strain to the logarithmic growth phase (OD). 600 =0.6), take 100μL of bacterial suspension and mix it quickly with 5mL of semi-solid culture medium (keep warm at 45℃). The semi-solid culture medium was prepared using brain heart infusion broth (DouDian Biotechnology Co., Ltd.) and agar powder (Solebo Biotechnology Co., Ltd.), with agar powder added at a ratio of 1.5g / 100mL of brain heart infusion broth.

[0083] c. Plate spreading: Immediately pour the mixed semi-solid culture medium onto the bottom plate, shake evenly, and let it stand to solidify to make a double-layer plate containing bacteria.

[0084] d. Sample addition: After the plate has solidified, take 10 μL of the phage stock solution (1×10⁻⁶). 9 PFU / mL was added dropwise to the surface of the plate.

[0085] e. Culture and observation: Place the plate in an anaerobic incubator at 37℃ and incubate for 24-48 hours. Observe whether transparent plaques appear.

[0086] 3. The test results are shown in Table 1: Table 1. Lysis effect of phage Y2-3 on 30 strains of Propionibacterium acnes

[0087] Note: ++++ Plaques are clear and transparent, +++ plaques are slightly turbid, ++ plaques are moderately turbid, and + plaques are severely turbid.

[0088] (1) Results showed that phage plaques appeared at positions Y2-3 on all 30 strains of Propionibacterium acnes plates. Most plaques were clear and transparent, with only one strain showing slight turbidity. Some examples are as follows: Figure 9As shown.

[0089] (2) Conclusion: The phage showed specific lysis activity against all 30 strains of Propionibacterium acnes tested, indicating that it has a broad lysis spectrum covering different clinical strains.

[0090] Example 4 Determination of lysis efficiency of bacteriophage Y2-3 1. Test method: (1) Take a representative clinical strain of Propionibacterium acnes and culture it to the logarithmic growth phase (OD). 600 =1), at which point the bacterial concentration is greater than 1×10 9 CFU / mL.

[0091] (2) Add bacteriophage Y2-3 purified in Example 1 at the optimal multiplicity of infection (MOI) of 0.1, mix well, and incubate anaerobically at 37°C. Set up a control group without adding bacteriophage.

[0092] (3) Samples were taken at 0h, 2h, 4h, 6h, 8h, 12h and 24h of incubation and OD was measured. 600 The value was calculated, and the viable count (CFU / mL) was simultaneously determined after serial dilution and plate counting.

[0093] 2. Experimental Results: (1) Viable bacteria count ( Figure 10 The viable bacterial count in the control group continued to increase; the viable bacterial count in the experimental group continued to decrease, from an initial value greater than 1×10⁻⁶. 9 CFU / mL decreased to 1×10 3 The concentration of CFU / mL is below 99.99%, and the lysis rate is greater than 99.99%.

[0094] (2) Changes in OD value ( Figure 11 ): The OD value of the control group gradually increased over time; the OD value of the experimental group increased slightly from 0 to 2 hours, then decreased rapidly from 2 to 12 hours, and remained at a low level after 12 hours.

[0095] (3) Conclusion: This phage has a highly efficient and rapid lysis effect on Propionibacterium acnes.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Propionibacterium acnes phage Y2-3, characterized in that, The preservation number of the bacteriophage Y2-3 is CGMCC No. 47222.

2. A preparation for preventing or improving acne, characterized in that, The formulation includes the bacteriophage Y2-3.

3. The formulation according to claim 2, characterized in that, The formulation also includes excipients.

4. The formulation according to claim 3, characterized in that, The excipients include one or more of the following: oil-based matrix, water-based matrix, emulsifier, preservative, pH adjuster, humectant, thickener, and buffer.

5. The formulation according to claim 2, characterized in that, The bacteriophage content in the formulation is 1×10⁻⁶. 5 ~1×10 10 PFU / mL or 1×10 5 ~1×10 10 PFU / g.

6. The formulation according to claim 2, characterized in that, The pH of the preparation is 5.0 to 9.

0.

7. The use of the bacteriophage Y2-3 according to claim 1 and the preparation according to any one of claims 2 to 6 in the preparation of products for preventing and treating Propionibacterium acnes infection.

8. The use of the phage Y2-3 according to claim 1 and the preparation according to any one of claims 2 to 6 in the preparation of products for the prevention or improvement of acne.

9. The application according to claim 8, characterized in that, The pathogens causing acne include Propionibacterium acnes.

10. The application according to claim 7 or 9, characterized in that, The Propionibacterium acnes includes antibiotic-resistant Propionibacterium acnes.

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