Propionibacterium acnes bacteriophage, microbial preparation and application

Abstract

The present application relates to the technical field of microorganisms, and particularly relates to a propionibacterium acnes bacteriophage, a microbial preparation and application, the propionibacterium acnes bacteriophage comprises a propionibacterium acnes bacteriophage BZNK009, and / or a mutant strain or a derivative strain with a genomic homology of greater than or equal to 94% with the propionibacterium acnes bacteriophage BZNK009; the taxonomic name of the propionibacterium acnes bacteriophage BZNK009 is Cutibacterium acnes phage , and the preservation number is GDMCC No: 68623-B1. The propionibacterium acnes bacteriophage has a broad-spectrum lysis and a physicochemical stable core performance, can efficiently lyse propionibacterium acnes, has no lysis effect on other species of bacteria, and has specificity; and the propionibacterium acnes bacteriophage has a wide range of physicochemical tolerance, the temperature stable interval is 30 DEG C to 65 DEG C, the pH stable interval is 3.5 to 11.5, is suitable for various skin care product processing and storage; meanwhile, the propionibacterium acnes bacteriophage has a high safety genome, has no lysogeny, drug resistance and virulence related genes, has no risk of gene horizontal transfer, and has a better external use safety than some wild bacteriophages.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Propionibacterium acnes phage, microbial preparations and applications. Background Technology

[0002] Propionibacterium acnes is now classified as follows: Cutibacterium acnes Propionibacterium acnes, formerly known as acne bacteria, is a Gram-positive, non-spore-forming anaerobic bacterium that naturally colonizes the micro-anaerobic environment deep within human hair follicles, serving as a native resident microorganism of the skin. In a healthy state of balanced skin flora, this bacterium maintains a low colonization abundance, participating in the regulation of skin microecological homeostasis and resisting the adhesion and invasion of exogenous harmful microorganisms without causing skin lesions. However, once pores become clogged with keratin, creating a closed, oxygen-deficient environment, the large amount of sebum continuously secreted by the sebaceous glands provides ample nutrition for the bacteria, promoting rapid and massive proliferation of Propionibacterium acnes. The originally harmless symbiotic bacteria can directly transform into conditionally pathogenic bacteria that induce acne. Among clinically isolated strains, the IA1 evolutionary subtype is most closely associated with moderate to severe inflammatory acne and multidrug resistance. Propionibacterium acnes can continuously initiate and amplify local inflammatory responses in the skin through multiple independent pathways. The complete pathogenic process forms a closed loop that is difficult to resolve on its own. The esterases secreted by the strain can continuously decompose triglycerides in sebum to generate free fatty acids, which constantly stimulate the abnormal proliferation and accumulation of keratinocytes in the hair follicle duct, gradually forming microcomedones that clog pores, which are the initial lesions of acne. The bacterial structural proteins and secreted proteases can also activate TLR2 and TLR4 immune receptors on the surface of skin keratinocytes, inducing the body to release large amounts of IL-6, IL-8, and TNF-α pro-inflammatory factors, recruiting neutrophils to aggregate and infiltrate, forming visible red and swollen papules and pustules. In addition, Propionibacterium acnes can build a dense biofilm structure on the inner wall of the hair follicle, greatly enhancing its tolerance to the body's immune cells and topical antibacterial substances, directly causing acne to recur and be difficult to eradicate completely. Porphyrins produced during the metabolic process of the strain generate reactive oxygen species when exposed to light, which continuously damage epithelial tissue and further aggravate post-inflammatory hyperpigmentation and acne scars.

[0003] Currently, existing Propionibacterium phage strains for acne exhibit significant shortcomings in physicochemical tolerance. They have narrow applicable temperature and pH ranges, and their activity is rapidly lost during heat processing in skincare products or long-term storage with formulations at different pH levels, resulting in short shelf-life for finished products. Most strains also have limited host lysis coverage, only capable of lysing laboratory standard strains. They show weak antibacterial effects against clinically isolated multidrug-resistant Propionibacterium acnes and have insufficient ability to improve severe, recurrent acne.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a Propionibacterium acnes phage, a microbial preparation and its application, aiming to solve the problems of poor physicochemical stability, narrow lysis spectrum, low amplification efficiency and insufficient formulation compatibility of existing Propionibacterium acnes phages.

[0006] The technical solution of the present invention is as follows: A Propionibacterium acnes phage, comprising Propionibacterium acnes phage BZNK009, and / or a mutant or derivative strain having ≥94% genomic homology with said Propionibacterium acnes phage BZNK009; The taxonomic name of the Propionibacterium acnes phage BZNK009 is: Cutibacterium acnes phage The accession number is GDMCC No: 68623-B1.

[0007] The aforementioned Propionibacterium acnes phage is used to lyse Propionibacterium acnes; the Propionibacterium acnes includes one or more of BNCC330605, BNCC192214, BNCC336443, BNCC252396, and ATCC6919.

[0008] The *Propionibacterium acnes* phage described herein, wherein the full-length genome of *Propionibacterium acnes* phage BZNK009 is 29620 bp; *Propionibacterium acnes* phage BZNK009 is double-stranded DNA, with a total base ratio of guanine and cytosine of 54.04%, and has 59 coding sequences.

[0009] The use of a Propionibacterium acnes phage in the preparation of drugs that lyse Propionibacterium acnes and / or drugs that inhibit the proliferation of Propionibacterium acnes.

[0010] Application of a Propionibacterium acnes phage in the preparation of acne-removing drugs, skin care solutions, acne creams or masks.

[0011] Application of a Propionibacterium acnes phage in an in vitro detection reagent for Propionibacterium acnes.

[0012] A microbial preparation containing the Propionibacterium acnes phage.

[0013] The microbial preparation, wherein the content of Propionibacterium acnes phage in the microbial preparation is 10. 5 pfu / mL-10 10 pfu / mL.

[0014] The microbial preparation further includes a pharmaceutically acceptable carrier and / or excipients.

[0015] The use of a microbial preparation in the preparation of a medicament for the prevention and / or treatment of Propionibacterium acnes infection.

[0016] Beneficial Effects: This invention provides a Propionibacterium acnes phage, a microbial preparation, and its application. The Propionibacterium acnes phage includes Propionibacterium acnes phage BZNK009, and / or mutant strains or derivatives with ≥94% genomic homology to said Propionibacterium acnes phage BZNK009; the taxonomic name of said Propionibacterium acnes phage BZNK009 is... Cutibacterium acnes phage The accession number is GDMCC No: 68623-B1. The *Propionibacterium acnes* phage BZNK009 and its mutant or derivative strains provided by this invention possess broad-spectrum lysis and physicochemical stability, enabling efficient lysis of *Propionibacterium acnes* and addressing the problem of poor antibacterial activity in existing drug-resistant phage strains. It exhibits no lysis effect on other bacterial species, demonstrating specificity. Furthermore, this *Propionibacterium acnes* phage exhibits broad-range physicochemical tolerance, with a temperature stability range of 30℃~65℃ and a pH stability range of 3.5~11.5, making it suitable for processing and storing various skincare products. Simultaneously, this *Propionibacterium acnes* phage possesses a highly safe genome, lacking lysogens, drug resistance, and virulence-related genes, eliminating the risk of horizontal gene transfer, and demonstrating superior safety for topical application compared to some wild-type phages. In addition, this *Propionibacterium acnes* phage exhibits high amplification efficiency, with an optimal MOI of 10, high burst volume, high liquid amplification titer, and lower industrial production costs. It is effective alone without the need for oil formulation, offering high flexibility in formulation development, and achieving long-lasting antibacterial effects without the addition of excipients such as petroleum jelly. Attached Figure Description

[0017] Figure 1 Morphological image of phage plaque from Propionibacterium acnes phage BZNK009; Figure 2 Electron micrograph of Propionibacterium acnes phage BZNK009; Figure 3 A complete genome loop of Propionibacterium acnes phage BZNK009; Figure 4 The results of phylogenetic analysis of Propionibacterium acnes phage BZNK009; Figure 5 VIRIDIC heatmap of genome similarity for Propionibacterium acnes phage BZNK009; Figure 6 Thermal stability diagram of Propionibacterium acnes phage BZNK009; Figure 7 Acid-base stability diagram of Propionibacterium acnes phage BZNK009. Detailed Implementation

[0018] This invention provides a Propionibacterium acnes phage, a microbial preparation, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0020] Acne is a very common chronic inflammatory skin disease of the hair follicles and sebaceous glands. Lesions are mainly concentrated on the face, chest, and back—areas with abundant sebaceous glands. Clinical manifestations include various forms such as whiteheads, blackheads, inflammatory papules, pustules, subcutaneous nodules, and deep cysts. The severity of the condition varies significantly from person to person. Mild acne only causes cosmetic blemishes, while moderate to severe acne is prone to leaving permanent pigmentation and pitted acne scars after healing, causing irreversible damage to the patient's appearance. At the same time, long-term recurring acne can also lead to negative psychological emotions such as low self-esteem, anxiety, and social avoidance, impacting health on both physiological and psychological levels. The overall pathogenesis of acne is driven by four factors: excessive sebum secretion by androgens, abnormal keratinization of the hair follicle duct, skin microecological imbalance, and local immune inflammatory stimulation. Among these, the excessive proliferation of Propionibacterium acnes is the core trigger for various types of inflammatory acne.

[0021] Currently, the mainstream clinical treatment for acne relies on oral and topical antibiotics. Commonly used antibiotics include clindamycin, erythromycin, and tetracycline derivatives. Long-term improper use has led to widespread and irreversible multidrug resistance. Global multicenter clinical isolate monitoring results show that Propionibacterium acnes has a resistance rate of over 92% to erythromycin, 78% to clindamycin, and an overall resistance rate of over 60% to tetracyclines. The resistance level of strains encased in biofilms will be further increased, and conventional dosages are completely ineffective in inhibiting bacteria. At the same time, antibiotics do not have targeted action characteristics. While inhibiting pathogenic Propionibacterium acnes, they indiscriminately kill beneficial symbiotic bacteria on the skin, such as Staphylococcus epidermidis, directly damaging the skin's microbial diversity and barrier function. After discontinuation of the drug, Propionibacterium acnes will rapidly rebound and proliferate, significantly increasing the probability of acne recurrence. In addition, oral antibiotics can easily cause gastrointestinal discomfort, photosensitivity reactions, and endocrine fluctuations, while topical antibiotics often cause dry, peeling, redness, and sensitivity of the skin. Pregnant women and people with sensitive skin face many strict restrictions on the use of these antibiotics.

[0022] Bacteriophages are naturally occurring, tiny viruses that specifically infect corresponding bacteria. The *Propionibacterium acnes* phage targets and lyses *Propionibacterium acnes*, without affecting other beneficial bacteria in the skin. It is an ideal biological raw material to replace antibiotics in acne treatment, offering multiple unique advantages over traditional chemical antibacterial ingredients. These phages rely solely on specific receptors on the bacterial surface for recognition and attachment, with a pathway completely independent of antibiotics. They can effectively kill various multidrug-resistant clinical strains without the risk of cross-resistance. Their natural microbial properties ensure that topical application leaves no hormone or chemical irritant residues, preventing skin peeling, stinging, or other discomfort, making them suitable for sensitive skin and post-cosmetic procedures. Their tiny particle size allows them to penetrate bacterial biofilms, eliminating pathogens hidden deep within hair follicles and reducing the probability of acne recurrence at its source. Furthermore, the physicochemically stable phages are compatible with various skincare and pharmaceutical formulations such as liquids, creams, and masks, maintaining good activity retention during processing and room-temperature storage, and lowering the operational barriers to industrialization.

[0023] Currently, multiple strains of *Propionibacterium acnes* phages have been disclosed in domestic and international research and patent documents. Representative strains include PCC / QD21122505 as described in patent CN116064419A and LPPA1 as disclosed in patent CN118222517A, but these have performance limitations. Other sporadically reported wild-type phages also generally exhibit performance shortcomings. Furthermore, the vast majority of currently available *Propionibacterium acnes* phage strains have genomic homology values ​​exceeding 95%, and existing phages exhibit poor in vitro proliferation performance, long latency periods, and low single lysis bursts. High-titer stock solutions are difficult to obtain through anaerobic liquid culture, requiring higher costs for large-scale production. Some phages, when used alone, cannot achieve long-lasting antibacterial effects and must be combined with oil-based excipients, limiting their application in mainstream skincare formulations such as refreshing serums and toners, thus restricting product development. Additionally, some wild-type phages carry integrase and bacterial virulence-related genes within their genomes, posing a potential biosafety risk of horizontal gene transfer from long-term topical application.

[0024] Based on this, the present invention provides a Propionibacterium acnes phage, including Propionibacterium acnes phage BZNK009, and / or a mutant strain or derivative strain with ≥94% genomic homology to said Propionibacterium acnes phage BZNK009. The taxonomic name of the Propionibacterium acnes phage BZNK009 is: Cutibacterium acnes phage (Classified as vB_CacP BZNK009), with accession number GDMCC No: 68623-B1, it was deposited on July 15, 2026 at the Guangdong Provincial Center for Microbial Culture Collection (Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou).

[0025] In this embodiment, the provided Propionibacterium acnes phage BZNK009 and its mutant or derivative strains possess broad-spectrum lysis and physicochemical stability, enabling efficient lysis of Propionibacterium acnes and addressing the issue of poor antibacterial activity in existing drug-resistant phage strains. It exhibits no lysis effect on other bacterial species, demonstrating specificity. Furthermore, this Propionibacterium acnes phage exhibits broad-range physicochemical tolerance, with a temperature stability range of 30℃~65℃ and a pH stability range of 3.5~11.5, making it suitable for processing and storing various skincare products. Simultaneously, this Propionibacterium acnes phage possesses a highly safe genome, lacking lysogens, drug resistance, and virulence-related genes, eliminating the risk of horizontal gene transfer, and demonstrating superior safety for topical application compared to some wild-type phages. Additionally, this Propionibacterium acnes phage exhibits high amplification efficiency, with an optimal MOI of 10, high burst volume, high liquid amplification titer, and lower industrial production costs. It is effective alone without the need for oil formulation, offering high flexibility in formulation development and achieving long-lasting antibacterial effects without the addition of excipients such as petroleum jelly.

[0026] Specifically, the Propionibacterium acnes phage BZNK009 and its mutant or derivative strains provided by this invention have a genomic sequence identity of less than 95% with existing known phages; it possesses excellent high and low temperature and acid and alkali tolerance, rapid proliferation rate, high burst volume, non-toxic genome, drug resistance, lysogenous genes, and high biosafety; furthermore, the Propionibacterium acnes phage has strong lytic activity and can be used to prepare topical preparations such as acne-removing drugs, skin care solutions, acne creams, and masks, reducing acne inflammation and providing a novel and safe raw material for antibiotic-free acne treatment and skin care, and clinical acne treatment.

[0027] In some embodiments, the Propionibacterium acnes phage is used to lyse Propionibacterium acnes; the Propionibacterium acnes includes one or more of BNCC330605, BNCC192214, BNCC336443, BNCC252396, and ATCC6919.

[0028] Specifically, a virulent bacteriophage, BZNK009, was isolated and purified from the water bodies of a park in Shenzhen, Guangdong Province, using Propionibacterium acnes BNCC330605 as the host. This bacteriophage exhibited good lytic activity against Propionibacterium acnes BNCC330605, BNCC192214, BNCC336443, BNCC252396, and ATCC6919.

[0029] In some embodiments, the full-length genome of the Propionibacterium acnes phage BZNK009 is 29620 bp; the Propionibacterium acnes phage BZNK009 is a double-stranded DNA with a total base ratio of guanine and cytosine of 54.04% and 59 coding sequences.

[0030] In this embodiment, the bacteriophage BZNK009 is a virulent bacteriophage of *Propionibacterium acnes*, isolated from the water in a park in Shenzhen, Guangdong Province. The host bacterium is *Propionibacterium acnes* BNCC330605. Bacteriophage BZNK009 exhibits a polyhedral head and long tail structure, with a head length of approximately 50 ± 5 nm and a tail length of approximately 250 ± 5 nm. According to the 2025 classification criteria for viruses published by the International Committee on Taxonomy of Viruses, this bacteriophage belongs to the order *Leptophage* and family *Leptophage*. Bacteriophage BZNK009 can form clear, regular plaques with a diameter of 8-10 mm on a double-layer solid culture medium, which are its unique morphological characteristics. The full-length genome of bacteriophage BZNK009 is 29620 bp, which is double-stranded DNA with a GC content of 54.04% and 59 coding sequences. Whole-genome sequencing and whole-genome characterization revealed that bacteriophage BZNK009 does not contain any antibiotic resistance genes or integrase genes, thus avoiding the risk of antibiotic resistance gene spread at the genomic level, and it has no lysogenic properties.

[0031] Specifically, the *Propionibacterium acnes* phage BZNK009 exhibits excellent tolerance at 37–50°C and pH 5–9, a range perfectly suited to the human physiological environment (around 37°C, pH 7.35–7.45), thus solving the problem of poor environmental adaptability of existing phages. Phage BZNK009 can efficiently lyse *Propionibacterium acnes* BNCC330605, BNCC192214, BNCC336443, BNCC252396, and ATCC-6919.

[0032] In some embodiments, the anaerobic amplification preparation method of the Propionibacterium acnes phage includes: providing a sample containing the Propionibacterium acnes phage, culturing it using thioglycolate fluid medium (FT medium) or brain heart infusion medium (BHI medium); simultaneously controlling the culture temperature and anaerobic duration; and purifying the sample by centrifugation and filtration after the process to obtain the Propionibacterium acnes phage.

[0033] In some embodiments, the culture temperature is 30℃~40℃; preferably, the culture temperature is 37℃. The anaerobic duration is: 24~120h for liquid amplification and 48~168h for double-layer plate plaque culture.

[0034] In some embodiments, the centrifugal filtration purification process includes, but is not limited to, gradient centrifugation at 8000~15000 r / min or multi-stage filtration using a 0.22 μm aqueous filter membrane.

[0035] In addition, the present invention also provides the application of the Propionibacterium acnes phage, including: In a first aspect, the present invention also provides the use of the Propionibacterium acnes phage in the preparation of drugs that lyse Propionibacterium acnes and / or drugs that inhibit the proliferation of Propionibacterium acnes.

[0036] Secondly, the present invention also provides the use of the Propionibacterium acnes phage in the preparation of acne-removing drugs, skin care solutions, acne creams or masks.

[0037] Thirdly, the present invention also provides the application of the aforementioned Propionibacterium acnes phage in an in vitro detection reagent for Propionibacterium acnes.

[0038] In this embodiment, the *Propionibacterium acnes* phage can be used to prepare topical medications for acne treatment, and applied in acne-removing skincare products and skin care products; including various dosage forms such as gels, liquids, creams, lyophilized agents, and masks containing the *Propionibacterium acnes* phage. Specifically, application scenarios include, but are not limited to, medical acne creams, home acne-removing skincare, post-medical aesthetic care, and in vitro bacterial detection kits.

[0039] In addition, the present invention also provides a microbial preparation containing the Propionibacterium acnes phage.

[0040] In some embodiments, the content of Propionibacterium acnes phage in the microbial preparation is 10. 5 pfu / mL-10 10 pfu / mL.

[0041] In some embodiments, the microbial preparation further includes a pharmaceutically acceptable carrier and / or excipients. The Propionibacterium acnes phage can be used alone as an active ingredient or in combination with a pharmaceutically acceptable carrier and / or excipients.

[0042] In some embodiments, the Propionibacterium acnes phage can be combined with various skin care or pharmaceutical carriers such as glycerin, propylene glycol, hyaluronic acid, plant soothing extracts, and medical gel matrix.

[0043] In some embodiments, the microbial preparation further comprises, by weight percentage: 0.6%–1.3% carbomer 980, 2.0%–4.0% glycerol, 1.0%–3.0% propylene glycol, 0.3%–0.8% panthenol, 0.1%–0.3% allantoin, 0.05%–0.15% low molecular weight sodium hyaluronate, 0.02%–0.05% disodium EDTA, 0.01%–0.08% citric acid, 0.02%–0.12% sodium citrate, 0.3%–0.6% phenoxyethanol, 0.1%–0.3% ethylhexylglycerin, and 0.05%–0.15% tocopherol polyethylene glycol succinate.

[0044] In some embodiments, the microbial preparation is preserved by various methods such as liquid cryopreservation, spray freeze-drying, vacuum freeze-drying, and microencapsulation.

[0045] In addition, the present invention provides the use of a microbial preparation in the preparation of a medicament for the prevention and / or treatment of Propionibacterium acnes infection.

[0046] In some embodiments, the present invention provides a multiphage cocktail antibacterial composition comprising Propionibacterium acnes phage BZNK009. The Propionibacterium acnes phage BZNK009 is mixed and compounded with other existing Propionibacterium acnes phages at a ratio of (1-2):(1-2) to form a multiphage cocktail antibacterial composition, further enhancing the long-lasting antibacterial effect.

[0047] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0048] Example 1 Isolation and purification of bacteriophages Isolation and purification of bacteriophages: 15 mL of water sample from a park in Shenzhen, Guangdong Province, was filtered through a 0.22 μm pore size membrane and mixed with 15 mL of 2×BHI medium. 1 mL of logarithmic-phase Propionibacterium acnes BNCC330605 (OD600≈0.6-0.8) was added, and the mixture was incubated at 37℃ under anaerobic conditions for 1 day. After incubation, the sample was centrifuged at 10000 r / min for 10 min and filtered through a 0.22 μm pore size membrane to obtain the incubation sample solution. 500 μL of the incubation solution was mixed with 500 μL of logarithmic-phase Propionibacterium acnes BNCC330605 (OD600≈0.6-0.8), then mixed with 10 mL of semi-solid agar and poured onto BHI solid plates to prepare a double-layer agar plate. The plate was then anaerobically cultured at 37℃ for 2 days. Single phage plaques were then selected and cultured using the double-layer agar plate method. The plaque selection and purification process was repeated five times to obtain phage plaques with uniform morphology and transparency for enrichment. The morphology of *Propionibacterium acnes* phage BZNK009 plaques is shown in the image below. Figure 1 As shown. The Propionibacterium acnes phage BZNK009 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located in Guangzhou, with accession number GDMCC No: 68623-B1, and deposit date July 15, 2026.

[0049] Example 2 Morphological identification of Propionibacterium acnes phage BZNK009 Phages were observed by TEM using phosphotungstic acid (PTA) negative staining. Concentrated phage particles were dropped onto a Formvar carbon support membrane and allowed to dry for 10 min. Excess liquid was removed with paper. 10 μL of PTA was added to the membrane for staining for 10 min. Excess PTA was washed away with deionized water, and the sample was dried again for 10 min with paper. The morphology of the phages was observed under TEM. The electron micrograph of Propionibacterium acnes phage BZNK009 is shown below. Figure 2 As shown, Propionibacterium acnes phage BZNK009 has a polyhedral head and long tail structure, with a head length of 50±5 nm and a tail length of about 250±5 nm. It belongs to the order Tail Phages and the family Longtail Phagesidae.

[0050] Example 3 Whole genome sequencing analysis of Propionibacterium acnes phage BZNK001 After amplifying the purified Propionibacterium acnes phage BZNK009 from Example 1, 1 mL of phage (1.0 × 10⁻⁶) was taken. 10 Genomic DNA was extracted using a TIANGEN viral genome extraction kit (pfu / mL). Whole-genome sequencing was performed using an Illumina NextSeq sequencer. Figure 3 The complete genome map of Propionibacterium acnes phage BZNK009 is shown. Genome analysis revealed that the BZNK009 genome is dsDNA, 29620 bp in size, with a GC content of 54.04%, and 59 open reading frames (ORFs) are annotated (as shown in Table 1). The genome contains no antibiotic resistance genes or integrase genes, ensuring biosafety at the genomic level. Furthermore, it lacks lysogenic genes, indicating a highly virulent phage with excellent lysis efficiency.

[0051] Using the BZNK009 whole genome sequence as the query sequence, a preliminary BLASTN search was performed in the NCBI nucleic acid database to screen for potentially closely related phages and determine if any published sequences with a similarity exceeding 95% existed. Subsequently, the whole genome sequences of 100 Propionibacterium acnes-related phages were downloaded from NCBI and compared with BZNK009 using uniform parameters on a local server. Coverage-corrected whole genome similarity was calculated. Based on these results, the 20 phages with the highest similarity to BZNK009 were selected for subsequent pairwise VIRIDIC whole genome comparison analysis.

[0052] BLASTN comparison results showed that BZNK009 had the highest similarity to the published phage *Propionibacterium phage Enochoraptor* (PV105554.1), with a coverage-corrected whole-genome similarity of 93.63%, which is lower than the 95% species classification threshold specified by ICTV. VIRIDIC analysis further showed that the whole-genome similarity between BZNK009 and closely related published phages did not reach the level of the same species, indicating that BZNK009 could represent a new phage species.

[0053] Furthermore, BLASTN results also showed that the genomic similarity of BZNK009 with most published Propionibacterium acnes phages was mainly distributed between 85% and 95%, indicating a close phylogenetic relationship among these phages. Therefore, the core SNP method was used to analyze the phylogenetic relationship between BZNK009 and closely related phages. Parsnp was used to perform core genome alignment of BZNK009 and 100 published phage genomes, and core SNP sites were extracted, ultimately obtaining a core SNP alignment matrix containing 101 sequences and 826 nucleotide sites. Subsequently, a maximum likelihood phylogenetic tree was constructed using IQ-TREE, and the optimal nucleotide substitution model was automatically selected using ModelFinder, followed by 1000 bootstrap tests and 1000 SH-aLRT tests. The optimal substitution model was TIM+F+R4. The phylogenetic tree results were visualized using the R packages ggtree and ggplot2.

[0054] The results of the phylogenetic analysis of Propionibacterium acnes phage BZNK009 are shown in the figure below. Figure 4 As shown, phylogenetic analysis revealed that BZNK009 clustered with several previously published *Propionibacterium acnes* phages in closely related groups. To further illustrate the evolutionary grouping of phages within the phylogenetic tree, hierarchical clustering was performed based on the cophenetic distance matrix of the phylogenetic tree, and the average silhouette width was calculated within the range of K=2-10 to evaluate the clustering effect of different numbers of clusters. The *Propionibacterium acnes* phage BZNK009 genome similarity VIRIDIC heatmap is shown below. Figure 5As shown, the results indicate that the average silhouette coefficient is highest when K=3, therefore it is considered the optimal number of visualization clusters for this dataset. Among them, BZNK009, Propionibacteriumphage Enochoraptor (PV105554.1), Propionibacterium phage Pirate (NC_027623.2), and Propionibacterium phage PHL071N05 (NC_022337.1) all belong to the same evolutionary cluster.

[0055] Based on the comprehensive analysis of BLASTN whole genome similarity, VIRIDIC pairwise comparison results, and core SNP phylogenetic analysis, although BZNK009 is closely related to previously published closely related bacteriophages, its whole genome similarity is below the 95% species classification threshold. Therefore, it can be identified as a novel Propionibacterium acnes bacteriophage.

[0056] Table 1. Complete genome annotation of Propionibacterium acnes phage BZNK009

[0057] Example 4 Thermal stability of Propionibacterium acnes phage BZNK009 Several sterile 2 mL EP tubes were used, and 500 μL of Propionibacterium acnes phage was added to each tube. The tubes were incubated at 4℃, 25℃, 30℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 1 h. The phage titer after incubation at different temperatures was determined using the double-layer agar method. The thermostability diagram of Propionibacterium acnes phage BZNK009 is shown below. Figure 6 As shown, Propionibacterium acnes phage BZNK009 maintains high biological activity within a temperature range of 37℃ to 50℃, with no significant decrease in titer, demonstrating excellent thermal stability and solving the problem of rapid activity drop of existing phages above 45℃.

[0058] Example 5 pH stability of Propionibacterium acnes phage BZNK009 The pH of LB liquid medium was adjusted with HCl and NaOH to obtain solutions with pH values ​​ranging from 3 to 12, which were then filtered through a 0.22 μm microporous membrane. 100 μL of phage was mixed with 900 μL of solutions at different pH values ​​and incubated in a 37°C water bath for 1 h. The titer of the phage at different pH values ​​was determined using the double-layer agar method. The acid-base stability diagram of Propionibacterium acnes phage BZNK009 is shown below. Figure 7As shown, Propionibacterium acnes phage BZNK009 maintains high biological activity and stable titer under acidic and alkaline conditions of pH 5-9.

[0059] Example 6 Host profile determination of Propionibacterium acnes phage BZNK009 Phage lysis profiles were determined using the standard dot assay. 500 µL of *Propionibacterium acnes* in mid-logarithmic growth phase was mixed with BHI semi-solid medium, spread onto double-layer agar plates, and after the agar plates dried and solidified, 5 µL of phage BZNK009 was added to the plate surface. The plates were then anaerobically cultured at 37°C for 2 days. After incubation, the phage plaques were observed to assess phage lysis activity. The results are shown in Table 2. Phage BZNK009 effectively lysed *Propionibacterium acnes* BNCC330605, BNCC192214, BNCC336443, BNCC252396, and ATCC-6919, but showed no lysis effect on *Escherichia coli* ATCC25922, *Pseudomonas aeruginosa* PAO1, *Staphylococcus aureus* USA300, and *Salmonella typhimurium* BNCC392794, demonstrating its specific lysis characteristic against *Propionibacterium acnes*.

[0060] Table 2. Lysis spectrum of Propionibacterium acnes phage BZNK009

[0061] In summary, this invention provides a Propionibacterium acnes phage, a microbial preparation, and its application. The Propionibacterium acnes phage includes Propionibacterium acnes phage BZNK009, and / or mutant strains or derivatives with ≥94% genomic homology to Propionibacterium acnes phage BZNK009; the taxonomic name of Propionibacterium acnes phage BZNK009 is... Cutibacterium acnes phageThe accession number is GDMCC No: 68623-B1. The *Propionibacterium acnes* phage BZNK009 and its mutant or derivative strains provided by this invention possess broad-spectrum lysis and physicochemical stability, enabling efficient lysis of *Propionibacterium acnes* and addressing the problem of poor antibacterial activity in existing drug-resistant phage strains. It exhibits no lysis effect on other bacterial species, demonstrating specificity. Furthermore, this *Propionibacterium acnes* phage exhibits broad-range physicochemical tolerance, with a temperature stability range of 30℃~65℃ and a pH stability range of 3.5~11.5, making it suitable for processing and storing various skincare products. Simultaneously, this *Propionibacterium acnes* phage possesses a highly safe genome, lacking lysogens, drug resistance, and virulence-related genes, eliminating the risk of horizontal gene transfer, and demonstrating superior safety for topical application compared to some wild-type phages. In addition, this *Propionibacterium acnes* phage exhibits high amplification efficiency, with an optimal MOI of 10, high burst volume, high liquid amplification titer, and lower industrial production costs. It is effective alone without the need for oil formulation, offering high flexibility in formulation development and achieving long-lasting antibacterial effects without the addition of excipients such as petroleum jelly.

[0062] Specifically, the strain of Propionibacterium acnes phage exhibits excellent physicochemical stability, is compatible with current mainstream skincare product production lines, requires no special low-temperature processing equipment, has a low production threshold, and can achieve low-cost mass production; products containing the Propionibacterium acnes phage are free of antibiotics, hormones, and irritating chemical antibacterial agents, and have broad market demand; furthermore, the strain of Propionibacterium acnes phage is free of lysogen genes, and there is no risk of microbial gene contamination with long-term topical skin use, and its safety indicators meet the regulatory standards for cosmetics and topical medicines.

[0063] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A Propionibacterium acnes phage, characterized in that, Including Propionibacterium acnes phage BZNK009, and / or mutant strains or derivatives that have ≥94% genomic homology with said Propionibacterium acnes phage BZNK009; The taxonomic name of the Propionibacterium acnes phage BZNK009 is: Cutibacterium acnes phage The accession number is GDMCC No: 68623-B1.

2. The Propionibacterium acnes phage according to claim 1, characterized in that, The phage of Propionibacterium acnes is used to lyse Propionibacterium acnes; the Propionibacterium acnes includes one or more of BNCC330605, BNCC192214, BNCC336443, BNCC252396, and ATCC6919.

3. The Propionibacterium acnes phage according to claim 1, characterized in that, The full-length genome of the Propionibacterium acnes phage BZNK009 is 29620 bp; the Propionibacterium acnes phage BZNK009 is a double-stranded DNA, with a total base ratio of guanine and cytosine of 54.04%, and has 59 coding sequences.

4. The use of a Propionibacterium acnes phage as described in any one of claims 1-3 in the preparation of drugs that lyse Propionibacterium acnes and / or drugs that inhibit the proliferation of Propionibacterium acnes.

5. The use of Propionibacterium acnes phage as described in any one of claims 1-3 in the preparation of acne-removing drugs, skin care solutions, acne creams, or face masks.

6. The use of the Propionibacterium acnes phage as described in any one of claims 1-3 in an in vitro detection reagent for Propionibacterium acnes.

7. A microbial preparation, characterized in that, The microbial preparation contains Propionibacterium acnes phage as described in any one of claims 1-3.

8. The microbial preparation according to claim 7, characterized in that, The content of Propionibacterium acnes phage in the microbial preparation is 10. 5 pfu / mL-10 10 pfu / mL.

9. The microbial preparation according to claim 7, characterized in that, The microbial preparation also includes pharmaceutically acceptable carriers and / or excipients.

10. The use of a microbial preparation as described in any one of claims 7-9 in the preparation of a medicament for the prevention and / or treatment of Propionibacterium acnes infection.

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