Bifidobacterium longum subsp. infantis NHNK-629 and use thereof in the preparation of a product for promoting hair growth

Abstract

This invention relates to the field of microbial technology, specifically to a subsp. *Bifidobacterium longum* infantile strain NHNK-629 and its application in the preparation of products promoting hair growth. This invention isolates and obtains *Bifidobacterium longum* subsp. *infantileum* (NHNK-629) from the feces of healthy female infants. Bifidobacterium longum subsp. infantis NHNK-629, whose live bacteria or fermentation products can improve the survival rate of human hair papilla cells after oxidative damage and promote hair growth, can therefore be used to prepare products that promote hair growth.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a subspecies of Bifidobacterium longum NHNK-629 and its application in the preparation of products that promote hair growth. Background Technology

[0002] Hair growth is a dynamic, balanced physiological process that depends on the periodic development of hair follicles and the normal function of related cell groups. However, influenced by factors such as modern life stress, changes in dietary structure, and abnormal hormone levels, hair loss has become an increasingly common problem.

[0003] Treatment methods for hair loss mainly include medication and hair transplantation. In the early to mid-stages of hair loss, which is considered mild to moderate, medication is the preferred treatment. However, in the late stages of hair loss, the hair follicles in the balding area are largely dead, resulting in a smooth scalp. In these cases, hair follicles from the back of the head, which are unaffected by hormones, need to be transplanted to the balding area to improve the symptoms. However, even after hair follicle transplantation in late-stage hair loss, adjunctive therapy with medications to prevent further hair loss is still necessary to improve the success rate.

[0004] Currently, approved medications for treating androgenetic alopecia mainly include topical minoxidil and oral finasteride. However, these medications have significant limitations: long-term use of minoxidil may cause local reactions such as skin irritation and inflammation; finasteride, as a 5α-reductase inhibitor, works by systematically inhibiting hormone conversion, and may be accompanied by systemic side effects such as sexual dysfunction. Therefore, there is an urgent need to develop safer and more effective products to promote hair growth. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a Bifidobacterium infantis subsp. NHNK-629 and its application in the preparation of products that promote hair growth.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a subspecies of Bifidobacterium longum (Bifidobacterium infantis) Bifidobacterium longum subsp. infantis NHNK-629 was deposited on March 10, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025431.

[0007] The Bifidobacterium longum infant subspecies described in this invention ( Bifidobacterium longum subsp. infantis NHNK-629, derived from the feces of a healthy female infant, was identified by 16S rDNA as *Bifidobacterium longum* subsp. *infantile*. Bifidobacterium longum subsp. infantisThis strain is Gram-positive, appearing as rod-shaped or club-shaped cells with one end swollen under a microscope. When grown on a Deman-Rogosa-Sharp (MRS) solid plate containing 0.1% (m / v) cysteine, it forms smooth, opaque colonies with neat, milky-white, round, raised edges. In MRS liquid medium containing 0.1% (m / v) cysteine, it exhibits uniform turbidity, and upon prolonged storage, the cells form a white precipitate. The optimal growth temperature is 37°C.

[0008] A second aspect of the present invention provides a microbial fermentation product comprising the *Bifidobacterium longum* subsp. *infantitidis* described in the first aspect (… Bifidobacterium longum subsp. infantis Fermentation product lysate and fermentation product filtrate of NHNK-629.

[0009] A third aspect of the present invention provides a method for preparing the microbial fermentation product described in the second aspect, comprising the following steps: Bifidobacterium longum infantile subspecies ( Bifidobacterium longum subsp. infantis NHNK-629 was inoculated into the culture medium and cultured anaerobically to obtain the fermentation broth; The fermentation broth was centrifuged to collect the precipitate and obtain the bacterial cells; the bacterial cells were ground and crushed to collect the bacterial sludge, resuspended in phosphate buffer (PBS) solution, sonicated, centrifuged to collect the supernatant, and filtered to obtain the fermentation product lysate; The fermentation broth was centrifuged to collect the supernatant, and then filtered to obtain the fermentation product filtrate.

[0010] In one or more embodiments, the culture medium is a Deman-Rogoza-Sharp (MRS) medium containing cysteine; Preferably, the mass-volume percentage (m / v) of cysteine ​​is 0.08% to 0.12%.

[0011] In one or more embodiments, the anaerobic culture temperature is 35~40℃ and the anaerobic culture time is 20~50h.

[0012] In one or more embodiments, liquid nitrogen is added during grinding and crushing.

[0013] In one or more embodiments, the ultrasonic breaking power is 250~400 W, and the ultrasonic breaking time is 15~25 min.

[0014] A fourth aspect of the invention provides the *Bifidobacterium longum* subsp. *infantile* described in the first aspect (… Bifidobacterium longum subsp. infantis The use of the microbial fermentation products described in NHNK-629 or the second aspect in any of the following: (a1) Improves the survival rate of human hair papilla cells after oxidative damage; (a2) Prepare products that improve the survival rate of human hair papilla cells after oxidative damage; (a3) Promotes hair growth; (a4) Prepare products that promote hair growth.

[0015] A fifth aspect of the present invention provides a product for improving the survival rate of human dermal papilla cells after oxidative damage, comprising the *Bifidobacterium longum* subsp. *infantitidis* as described in the first aspect (…). Bifidobacterium longum subsp. infantis NHNK-629 or the microbial fermentation products described in the second aspect.

[0016] A sixth aspect of the present invention provides a product for promoting hair growth, comprising the *Bifidobacterium longum* subsp. *infantitidis* described in the first aspect (… Bifidobacterium longum subsp. infantis NHNK-629 or the microbial fermentation products described in the second aspect.

[0017] In the fourth, fifth, and sixth aspects of the present invention, the product includes a pharmaceutical product and a pharmaceutical composition or a cosmetic product.

[0018] The beneficial effects of this invention are as follows: This invention isolates and obtains *Bifidobacterium longum* subsp. infantis from the feces of healthy female infants. Bifidobacterium longum subsp. infantis NHNK-629, whose live bacteria or fermentation products can improve the survival rate of human hair papilla cells after oxidative damage and promote hair growth, can therefore be used to prepare products that promote hair growth. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 Phylogenetic tree for Bifidobacterium longum infantis subspecies NHNK-629; Figure 2 The figure shows the results of reducing oxidative damage in human dermal papilla cells by Bifidobacterium longum subsp. infantis NHNK-629. In the figure, A represents normal human dermal papilla cells, B represents the control group, and C represents the experimental group. The scale bars in A, B, and C are all 200 μm. Figure 3 The results of promoting hair growth with Bifidobacterium infantis subspecies NHNK-629 are shown in the figure. A is the blank group, B is the model group, C is the first experimental group, D is the second experimental group, E is the grayscale image of A, F is the grayscale image of B, G is the grayscale image of C, and H is the grayscale image of D. Figure 4 To quantify the grayscale of mouse hair, among which, This indicates that P < 0.001.

[0021] Preservation instructions: Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis NHNK-629 was deposited on March 10, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025431, and is classified as *Bifidobacterium longum* subsp. *infantitidis*. Bifidobacterium longum subsp. infantis NHNK-629. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] In the following examples, all reagents and consumables used are commercially available products.

[0025] Example 1 Bifidobacterium longum infant subspecies ( Bifidobacterium longum subsp. infantis Separation of NHNK-629: Samples were collected from the feces of healthy female infants. The samples were placed in physiological saline and shaken to mix. The supernatant was streaked onto MRS solid plates containing 0.1% (m / v) cysteine. After anaerobic incubation at 37°C for 48 h, white colonies were picked and repeatedly inoculated and screened until uniform single colonies were obtained, which were named NHNK-629.

[0026] Gram staining and microscopic examination: A single colony of NHNK-629 was streaked onto an MRS solid plate containing 0.1% (m / v) cysteine. After anaerobic incubation at 37°C for 48 h, a single white colony was picked for Gram staining and microscopic examination. Gram staining and microscopic examination showed that NHNK-629 was Gram positive, appearing as rod-shaped or rod-shaped cells with one end enlarged. When growing on an MRS solid plate containing 0.1% (m / v) cysteine, it forms smooth, opaque colonies with neat edges and milky white, round, raised areas. In MRS liquid medium containing 0.1% (m / v) cysteine, it grows in a uniformly turbid manner, and the cells precipitate white after prolonged exposure.

[0027] 16S rDNA gene sequence analysis: Single colonies of NHNK-629 were picked and cultured anaerobicly at 37°C for 48 h in MRS liquid medium containing 0.1% (m / v) cysteine. The cells were collected by centrifugation (8000 rpm, 1 min) and processed according to the instructions of the Gram-positive bacterial DNA extraction kit. Primers used were the universal 16S sequencing primers 27F (nucleotide sequence as shown in SEQ ID NO. 1: AGAGTTTGATCMTGGCTCAG) and 1492R (nucleotide sequence as shown in SEQ ID NO. 2: GGTTACCTTGTTACGACTT). The polymerase chain reaction (PCR) amplification system was 20 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min, 94°C for 15 s, 57°C for 15 s, 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0028] After sequencing the PCR product, homology comparison (BLASTN) with published standard sequences in the GenBank database revealed that NHNK-629 is a subspecies of Bifidobacterium longum infantis. Bifidobacterium longum subsp. infantis The 16S rDNA gene sequence is shown in SEQ ID NO. 3. Other strains closely related to *Bifidobacterium longum* subsp. *infantii* NHNK-629 were selected from the BLASTN interface to construct a phylogenetic tree, as shown below. Figure 1 As shown in the figure. The results show that although NHNK-629 belongs to the same subspecies of Bifidobacterium longum as strains Bi-26, 3012, and JCM7010, it is not closely related and therefore has strain-specific characteristics.

[0029] Example 2 Preparation of microbial fermentation products: Single colonies of *Bifidobacterium longum* subsp. *infantii* NHNK-629 were picked and cultured in MRS liquid medium containing 0.1% (m / v) cysteine. The culture was then incubated anaerobicly at 37°C for 48 h. The OD was adjusted using Durbeco-modified Eagle Medium (DMEM). 600 =1.0, centrifuged (5000 rpm, 10 min), and filtered through a 0.22 μm filter membrane to obtain the fermentation product filtrate of Bifidobacterium longum subsp. infantis NHNK-629.

[0030] Single colonies of *Bifidobacterium longum* subsp. *infantii* NHNK-629 were picked and cultured in MRS liquid medium containing 0.1% (m / v) cysteine ​​for 24 h at 37°C under anaerobic conditions. The precipitate was collected by centrifugation (5000 rpm, 10 min), and the bacterial cells were washed twice with sterile PBS solution. The culture medium was then adjusted to OD using sterile PBS solution. 600=1.0, to obtain live NHNK-629 bacteria. Add liquid nitrogen to grind and break down the bacteria, collect the broken bacterial sludge, and resuspend it in an equal volume of PBS solution; then sonicate (300 W) for 20 min, centrifuge (5000 rpm, 10 min) to collect the supernatant, and then filter it through a 0.22 μm filter membrane to obtain the NHNK-629 fermentation product lysate.

[0031] Example 3 Bifidobacterium longum infantis subspecies NHNK-629 reduces oxidative damage to human hair papilla cells: Human hair dermal papilla cells were seeded in DMEM medium containing 10% (v / v) fetal bovine serum (FBS) and cultured at 37°C and 5% CO2. When the cells reached 80% confluence, they were collected and seeded into 96-well plates and cultured for another 24 h. 100 μL of DMEM medium containing 1.5 mM H2O2 was added to each well, and the cells were incubated at 37°C and 5% CO2 for 3 h. After incubation, the supernatant was discarded, and 99 μL of DMEM medium was added to each well. The experimental group received 1 μL of NHNK-629 fermentation product filtrate (prepared in Example 2), while the control group received an equal volume of DMEM medium. The cells were cultured at 37°C and 5% CO2 for another 20 h. After culture, the cells were photographed (results are shown in Figure 1). Figure 2 (As shown in Table 1). Add 10 μL of CCK-8 reagent to each well and incubate at 37℃ and 5% CO2 for 2 h. Measure the absorbance at 450 nm using a microplate reader.

[0032] Calculation formula: Relative survival rate (%) = ; Table 1. NHNK-629 fermentation product filtrate reduces oxidative damage in human hair papilla cells.

[0033] In Table 1, the SD value represents the standard deviation.

[0034] There is a clear causal relationship between oxidative damage to human dermal papilla cells and hair growth inhibition. Simply put, oxidative damage is one of the key factors leading to dermal papilla cell dysfunction and consequently hair loss; correspondingly, protecting dermal papilla cells from oxidative damage is an important strategy for promoting hair growth. Figure 2 As shown in Table 1, the filtrate of NHNK-629 fermentation products can improve the survival rate of human dermal papilla cells after oxidative damage and reduce oxidative damage to human dermal papilla cells.

[0035] Example 4 Bifidobacterium longum infantis subspecies NHNK-629 promotes hair growth: 4.1 Weigh 1 g of carbomer, add 99 mL of ultrapure water, and stir rapidly with a glass rod. Let stand at room temperature for 3 h until the carbomer swells. Then, add triethanolamine dropwise to adjust the pH to 7 to obtain carbomer gel. Add 1 mL of NHNK-629 fermentation product lysate (prepared in Example 2) to 99 mL of carbomer gel and mix well to obtain NHNK-629 fermentation product lysate gel.

[0036] 4.2 Mouse experiment: Thirty-two 6-week-old specific pathogen-free (SPF) male C57BL / 6 mice were randomly divided into four groups of eight mice each: a control group, a model group, an experimental group (NHNK-629 fermentation product lysate gel application group), and an experimental group (NHNK-629 live bacteria gavage group). All mice were housed within an SPF-grade barrier and had free access to food and water.

[0037] The experiment lasted four weeks. The first week was the acclimatization period for mice, the second week was the modeling period for disrupting the intestinal flora, and the third and fourth weeks were the NHNK-629 intervention period. After the acclimatization period, mice were administered antibiotics by gavage for 7 days to disrupt the original intestinal flora. Ampicillin 15 mg / kg, metronidazole 15 mg / kg, neomycin 25 mg / kg, and vancomycin 12.5 mg / kg were weighed, mixed, and dissolved in sterile water. Each mouse in the model group and experimental group was gavaged with 10 mL / kg of sterile water, while the control group was gavaged with the same amount of sterile water for 7 consecutive days. The aforementioned mg / kg represents the dosage per kilogram of animal body weight. On the first day of the third week, a 2 cm × 3 cm area of ​​hair was shaved off the backs of mice using an electric epilator. The first experimental group mice received 0.1 mL of NHNK-629 fermentation product lysate gel daily on the shaved backs. The control group, model group, and the second experimental group mice received an equal amount of gel without NHNK-629 fermentation product lysate gel on the shaved backs. The second experimental group mice were administered 0.1 mL of live NHNK-629 bacteria daily by gavage. The control group, model group, and the first experimental group mice were administered an equal amount of PBS by gavage.

[0038] After the experiment, photos were taken of the mouse's back. Figure 3 ), and then to Figure 3 Grayscale analysis was performed to evaluate the hair growth status on the backs of mice. Figure 4 The calculation formulas and results are shown in Table 2.

[0039] Table 2. Bifidobacterium longum infantis subsp. NHNK-629 promotes hair growth.

[0040] In Table 2, the SD value represents the standard deviation.

[0041] Figure 3 The results showed that the hair growth of mice in the model group was slow. Compared with the model group, the hair growth of mice in the first experimental group (NHNK-629 fermentation product lysate gel application group) and the second experimental group (NHNK-629 live bacteria gavage group) was dense. Figure 4 yes Figure 3 The quantitative analysis of hair grayness showed a significant difference between the model group and the blank group, indicating successful modeling. The significant difference between the experimental group and the model group suggests that both application and gavage with Bifidobacterium longum subsp. infantis NHNK-629 can significantly increase hair growth, with a growth rate between 38.44% and 60.10%.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A subspecies of Bifidobacterium longum in infants ( Bifidobacterium longum subsp. infantis NHNK-629 was deposited on March 10, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025431.

2. A microbial fermentation product, characterized in that, Including the Bifidobacterium longum infantile subspecies as described in claim 1 ( Bifidobacterium longum subsp. infantis Fermentation product lysate and fermentation product filtrate of NHNK-629.

3. The method for preparing the microbial fermentation product according to claim 2, characterized in that, Includes the following steps: Bifidobacterium longum infantile subspecies ( Bifidobacterium longum subsp. infantis NHNK-629 was inoculated into the culture medium and cultured anaerobically to obtain the fermentation broth; The fermentation broth was centrifuged to collect the precipitate and obtain the bacterial cells; the bacterial cells were ground and crushed to collect the bacterial sludge, resuspended in phosphate buffer solution, ultrasonically crushed, centrifuged to collect the supernatant, and filtered to obtain the fermentation product lysate; The fermentation broth was centrifuged to collect the supernatant, and then filtered to obtain the fermentation product filtrate.

4. The method for preparing microbial fermentation products as described in claim 3, characterized in that, The culture medium is a Delman-Rogosa-Sharp medium containing cysteine; the mass-volume percentage of cysteine ​​is 0.08%~0.12%. The anaerobic culture temperature is 35~40℃, and the anaerobic culture time is 20~50 h; Liquid nitrogen is added during grinding and crushing; The ultrasonic disruption power is 250~400 W, and the ultrasonic disruption time is 15~25 min.

5. The *Bifidobacterium longum* subsp. *infantitidis* as described in claim 1 (… Bifidobacterium longum subsp. infantis The use of NHNK-629 or the microbial fermentation product of claim 2 in any of the following: (a1) Improves the survival rate of human hair papilla cells after oxidative damage; (a2) Prepare products that improve the survival rate of human hair papilla cells after oxidative damage; (a3) Promotes hair growth; (a4) Prepare products that promote hair growth.

6. The application as described in claim 5, characterized in that, The products include pharmaceuticals and pharmaceutical compositions or cosmetics.

7. A product for improving the survival rate of human hair papilla cells after oxidative damage, characterized in that, Including the Bifidobacterium longum infantile subspecies as described in claim 1 ( Bifidobacterium longum subsp. infantis NHNK-629 or the microbial fermentation product as described in claim 2.

8. The product as described in claim 7, characterized in that, The products include pharmaceuticals and pharmaceutical compositions or cosmetics.

9. A product that promotes hair growth, characterized in that, Including the Bifidobacterium longum infantile subspecies as described in claim 1 ( Bifidobacterium longum subsp. infantis NHNK-629 or the microbial fermentation product as described in claim 2.

10. The product as described in claim 9, characterized in that, The products include pharmaceuticals and pharmaceutical compositions or cosmetics.

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