Plant lactobacillus CCFM1353 and preparation of anti-hair loss and hair care metaplasma and application of transformed cypress leaves

Abstract

The application discloses a plant Lactobacillus plantarum CCFM1353, a metapolybiose prepared from the plant Lactobacillus plantarum CCFM1353 for preventing hair loss and promoting hair growth, and application of the metapolybiose in combination with transformed Chinese arborvitae leaves, and belongs to the technical fields of microorganisms and medicines. The plant Lactobacillus plantarum CCFM1353 and the metapolybiose and symbiotic preparation prepared from the plant Lactobacillus plantarum CCFM1353 can promote the expression of IGF-1, VEGF and TGF-beta 2 of hair follicle dermal papilla cells (HDPC), promote hair growth of mice, and promote the expression of VEGF, IGF-1, Wnt10b, Cyclin D1, Ki67 and Bcl2 in the skin of mice. The plant Lactobacillus plantarum CCFM1353 and the metapolybiose and symbiotic preparation prepared from the plant Lactobacillus plantarum CCFM1353 can be used for preparing products for preventing and / or treating hair loss, and have great application prospects.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus plantarum CCFM1353 and its application as a postgenetic agent for preventing hair loss and protecting hair, and its synergistic effect with transformed Platycladus orientalis leaves, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Hair loss is a common skin condition. It is well known that illness, nutritional deficiencies, aging, hormonal imbalances, and stress can all lead to hair loss. To date, the number of people suffering from hair loss has increased dramatically. Although the U.S. Food and Drug Administration (FDA) has approved two hair loss medications, minoxidil and finasteride, patient adherence is often low due to unpredictable efficacy and side effects. Therefore, there is an urgent need to develop more and better treatment options.

[0003] The hair growth cycle consists of three phases: anagen (growth phase), catagen (transitional phase), and telogen (resting phase). Disruptions in the hair growth cycle have been shown to be associated with the pathogenesis of androgenetic alopecia. Dermal cells (DPCs) play a crucial role in the proliferation and differentiation of hair follicles and in controlling each phase of the hair cycle. Furthermore, DPCs secrete various substances to promote and maintain hair follicle growth and development, such as insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and apoptosis-related proteins Bcl2 and BAX.

[0004] In recent years, natural herbs have gained widespread attention for improving hair loss symptoms due to their multi-component, multi-target, and synergistic effects. Arborvitae leaves, the dried, tender branches and leaves of the cypress family plant *Platycladus orientalis* (L.) Franco, are among the most frequently used ingredients in traditional Chinese medicine prescriptions for treating hair loss. Their hair-growth efficacy is recorded in numerous ancient Chinese medicine classics, including *Zhouhou Fang*, *Compendium of Materia Medica*, and *Chinese Materia Medica*. Current research also indicates that water extracts of arborvitae leaves can promote hair growth through the Akt / GSK3β / β-catenin signaling pathway. The active substances may be flavonoids such as quercetin and isoquercetin; however, flavonoids often have low activity and are difficult to absorb. Therefore, current technology needs further improvement and development, and substances that can enhance the hair loss prevention and hair care effects of arborvitae leaves need to be identified. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a plant lactobacillus ( Lactiplantibacillus plantarum The application of post-genes and the synergistic effect of transformed Platycladus orientalis leaves in the preparation of anti-hair loss and hair care products.

[0006] This invention provides a strain of *Lactobacillus plantarum* CCFM1353 ( Lactiplantibacillus plantarum The plant lactobacillus ( Lactiplantibacillus plantarum It was deposited on October 25, 2023, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCC No: 63923, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The plant lactobacillus ( Lactiplantibacillus plantarum CCFM1353 is isolated from the feces of healthy human beings and has the following characteristics:

[0008] The plant lactobacillus ( Lactiplantibacillus plantarum After inoculating CCFM1353 onto MRS solid medium and culturing for 48 hours, the colonies are generally milky white, smooth, raised, and round with a diameter of 0.5-2 mm.

[0009] The plant lactobacillus ( Lactiplantibacillus plantarum CCFM1353 is a Gram-positive, facultative anaerobic bacterium that thrives in warm temperatures. Its optimal growth temperature is 35-40℃ and its optimal growth pH is 6.0-7.0.

[0010] The present invention provides a composition containing *Lactobacillus plantarum* CCFM1353, or containing a metabiotic prepared from *Lactobacillus plantarum* CCFM1353.

[0011] In one embodiment, the metabiotic includes fermentation supernatant, cell lysate, and / or fermentation broth.

[0012] In one embodiment, the metabiotic is obtained by inoculating the above-mentioned *Lactobacillus plantarum* CCFM1353 into MRS medium, culturing the bacterial solution, and then subjecting it to heat treatment and lysis.

[0013] In one embodiment, the heat treatment is performed at 60-70°C for 25-35 minutes.

[0014] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging the above-mentioned fermentation broth.

[0015] In one embodiment, the method for preparing the cell lysate is to homogenize the heat-treated fermentation broth under high pressure and centrifuge it to obtain the cell lysate.

[0016] In one embodiment, the post-generic agent can be dried into powder or used directly by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, and fluidized bed drying.

[0017] This invention provides the above-mentioned *Lactobacillus plantarum* ( Lactiplantibacillus plantarum Application of CCFM1353 in the preparation of anti-hair loss and hair care products.

[0018] In one embodiment, the application method includes, but is not limited to, topical application or oral administration.

[0019] This invention provides the use of the composition in the preparation of medicaments for the prevention and / or treatment of hair loss.

[0020] The present invention also provides pharmaceuticals or cosmetics containing the said *Lactobacillus plantarum* CCFM1353 postbiotic.

[0021] In one embodiment, the pharmaceutical product comprises the above-described composition, a drug carrier, and / or pharmaceutical excipients.

[0022] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0023] In one embodiment, the cosmetic comprises the above-described composition, matrix ingredients, and / or conventional excipients.

[0024] In one embodiment, the matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gel-based raw materials, coagulants, and surfactants.

[0025] In one embodiment, the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0026] Beneficial effects:

[0027] This invention screened and obtained a strain of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum CCFM1353, this plant lactobacillus ( Lactiplantibacillus plantarum CCFM1353 and its prepared post-natal vitamins have the ability to prevent hair loss and protect hair, and have a synergistic effect with Platycladus orientalis leaves, specifically manifested in:

[0028] (1) Promotes the expression of related genes and proteins in dermal papillary cells (HDPCs):

[0029] Promote IGF-1 The expression, IGF-1 Expression was increased 6.28-fold; VEGF expression was promoted, with secretion upregulated by up to 51.0%; and VEGF secretion was inhibited. [[ID=ll]]TGF-β2 Express, TGF-β2 The expression decreased by 61.7%;

[0030] (2) Promote hair growth in mice. Gavage with both Lactobacillus plantarum CCFM1353 and Platycladus orientalis leaf extract can enhance the hair growth promoting effect of Platycladus orientalis leaf on mice.

[0031] (3) Promotes the expression of related genes and proteins in mouse skin:

[0032] Promoting VEGF expression, compared with oral administration of the same dose of Platycladus orientalis leaf via gavage, the combination of oral administration of Platycladus orientalis leaf aqueous extract and live Lactobacillus plantarum CCFM1353 significantly enhanced VEGF expression. [[ID=l3]]VEGF Expression levels increased by 4.0%;

[0033] Promoting IGF-1 expression, compared with the same dose of Platycladus orientalis leaves administered by gavage, oral administration of a combination of Platycladus orientalis ferment broth, Platycladus orientalis water extract, and live Lactobacillus plantarum CCFM1353 significantly increased IGF-1 expression. IGF-1 Expression levels increased by 23.5% and 47.5%;

[0034] Compared with the same dose of Platycladus orientalis leaves administered by gavage, oral administration of a combination of Platycladus orientalis ferment broth, Platycladus orientalis water extract, and live Lactobacillus plantarum CCFM1353 bacteria increased Wnt10b expression by 2.3 times and 3.1 times, respectively.

[0035] Compared with the same dose of Platycladus orientalis leaves administered by gavage, oral administration of a combination of Platycladus orientalis ferment broth, Platycladus orientalis water extract, and live Bacillus plantarum CCFM1353 increased Cyclin D1 expression by 1.8 times and 2.2 times, respectively.

[0036] Compared with the same dose of Platycladus orientalis leaves administered by gavage, oral administration of a combination of Platycladus orientalis leaf fermentation broth, Platycladus orientalis leaf aqueous extract, and live Lactobacillus plantarum CCFM1353 bacteria increased Ki67 expression by 32.5% and 66.9%, respectively.

[0037] Promoting Bcl2 expression, compared with the same dose of Platycladus orientalis leaves administered by gavage, oral administration of a combination of Platycladus orientalis ferment broth, Platycladus orientalis water extract, and live Lactobacillus plantarum CCFM1353 significantly increased Bcl2 expression. Bcl2 Expression levels increased by 0.8 times and 1.2 times.

[0038] Therefore, *Lactobacillus plantarum* ( Lactiplantibacillus plantarum CCFM1353 and its prepared post-biotic and arborvitae leaf fermentation liquid have great application prospects in the preparation of hair care and anti-hair loss products.

[0039] Preservation of biological materials

[0040] A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum CCFM1353, taxonomically named Lactiplantibacillus plantarum It was deposited on October 25, 2023, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCC No: 63923, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0041] Figure 1 Effects of Platycladus orientalis leaf fermentation supernatant prepared from Lactobacillus plantarum CCFM1353 on HDPC cell proliferation and migration;

[0042] Figure 2 Effects of the post-biotic CCFM1353 on the expression of TGF-β2 mRNA and IGF-1 mRNA in HDPC cells;

[0043] Figure 3 Effects of the post-biotic CCFM1353 on VEGF expression in HDPC cells;

[0044] Figure 4 Effects of Lactobacillus plantarum CCFM1353 and its prepared metagener and synergistic agents on hair growth in mice;

[0045] Figure 5 Lactobacillus plantarum CCFM1353 and its prepared metabiotics and synbiotics have effects on mouse skin. Wnt10b mRNA, Cyclin D1 mRNA and Bcl2 The effect of mRNA expression;

[0046] Figure 6 Effects of Lactobacillus plantarum CCFM1353 and its prepared postbiotic and synbiotic formulations on the expression of VEGF, IGF-1 and Ki67 in mouse skin.

[0047] "*" indicates a statistically significant difference from the control group (P<0.05), "**" indicates a statistically significant difference from the control group (P<0.01), and "***" indicates an extremely statistically significant difference from the control group (P<0.001). Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments.

[0049] The strain *Lactobacillus plantarum* FHNMY13M5 involved in the following examples was a self-screened strain from the Food Biology Center Laboratory of Jiangnan University.

[0050] Terminology Explanation

[0051] Postbiotics of Lactobacillus plantarum CCFM1353: Preparations of non-living microorganisms and / or components thereof that are beneficial to the health of the host, including non-living Lactobacillus plantarum CCFM1353 cells, bacterial metabolites and / or components after bacterial lysis.

[0052] The culture media involved in the following examples are as follows:

[0053] PBS buffer solution ( / L): Sodium chloride 8.0 g, potassium chloride 0.2 g, disodium hydrogen phosphate 1.44 g, potassium dihydrogen phosphate 0.24 g, adjust pH to 7.4. Autoclave at 115℃ for 20 min.

[0054] MRS liquid culture medium ( / L): anhydrous glucose 20.0 g, beef extract 10.0 g, peptone 10.0 g, yeast extract 5.0 g, dipotassium hydrogen phosphate trihydrate 2.6 g, anhydrous sodium acetate 2.0 g, diammonium hydrogen citrate 2.0 g, magnesium sulfate heptahydrate 0.5 g, manganese sulfate monohydrate 0.25 g, Tween 80 1.0 mL. Autoclave at 115℃ for 20 min.

[0055] For MRS solid medium, add 1.5% agar to the prepared liquid medium and autoclave at 115°C for 20 minutes.

[0056] Arborvitae leaf fermentation medium ( / L): 50 g Arborvitae leaf extract, 10 g yeast powder, adjust pH to 6.0-6.2. Autoclave at 115℃ for 20 min.

[0057] Arborvitae leaf aqueous extract ( / L): Dissolve 50g of Arborvitae leaf extract in 0.85% physiological saline and adjust the pH to 6.0-6.2. Autoclave at 115℃ for 20 min.

[0058] Cell culture medium: 89% (v / v) DMEM medium, 10% fetal bovine serum, 1×10 5 U / L penicillin, 100 mg / L streptomycin.

[0059] Example 1: Screening and identification of Lactobacillus plantarum

[0060] 1. Screening

[0061] The samples were derived from feces of healthy individuals. After pretreatment, the samples were stored in 30% glycerol at -80°C. After thawing, 0.2 mL of the sample was diluted in 1.8 mL of sterile physiological saline to obtain 10... -1 Dilute the solution, then take 0.5 mL of 10 -1 The diluent was diluted in 4.5 mL of physiological saline to obtain 10. -2 Diluents were prepared by repeating this process to obtain different concentrations of diluents. Suitable gradient diluents were then plated onto MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus plantarum* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium and incubated at 37°C for 18 h. The culture was preserved in 30% glycerol to obtain *Lactobacillus plantarum* CCFM1353. Typical colonies of *Lactobacillus plantarum* are white, round, smooth, and raised.

[0062] 2. Identification

[0063] The genome of strain CCFM1353 was extracted, and the 16S rDNA of strain CCFM1353 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The nucleotide sequence of the amplified 16S rDNA of CCFM1353 was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was *Lactobacillus plantarum*, and it was named *Lactobacillus plantarum*. Lactiplantibacillus plantarum (CCFM1353)

[0064] Example 2: Lactobacillus plantarum CCFM1353 promotes HDPC cell migration

[0065] 1. Cell resuscitation and culture

[0066] First, retrieve the frozen human dermal papillary cells (HDPCs), rapidly thaw them in a 37°C water bath, then centrifuge at 1000 r / min for 5 min, discard the supernatant, and use a solution containing 10% fetal bovine serum and 1×10⁻⁶... 5 Cells were cultured in DMEM high-glucose medium containing U / L penicillin and 100 mg / L streptomycin at 37°C in a cell culture incubator with 5% CO2. When the cells adhered and reached confluence of more than 80%, they were digested and passaged using trypsin containing 0.25% EDTA, and cells in the logarithmic growth phase were used for experiments.

[0067] 2. Preparation of post-biotics from *Lactobacillus plantarum* CCFM1353

[0068] All strains were preserved in 30% glycerol at -80°C before activation. A small amount of *Lactobacillus plantarum* CCFM1353 bacterial suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h. Single colonies were then picked and inoculated into MRS liquid medium and incubated at 37°C for 18 h. Finally, a 2% inoculation rate was added to 200 ml of MRS liquid medium, and the culture was continued at 37°C for another 18 h to obtain 9.5 × 10⁹ cells / mL. 8 CFU / ml bacterial culture.

[0069] The bacterial culture was heat-treated at 65 °C for 30 min, centrifuged (8000 g, 4 °C, 15 min), and the supernatant was collected and freeze-dried to obtain *Lactobacillus plantarum* CCFM1353 supernatant freeze-dried powder (CCFM1353-S) for later use. The centrifuged bacterial sludge was homogenized under high pressure (800~1200 MPa, 3 times) to obtain bacterial cell lysates, which were then freeze-dried to obtain *Lactobacillus plantarum* CCFM1353 bacterial cell lysate freeze-dried powder (CCFM1353-N) for later use. The preparation methods for *Lactobacillus plantarum* FHNMY13M5 supernatant freeze-dried powder (FHNMY13M5-S) and bacterial cell lysate freeze-dried powder (FHNMY13M5-N) were the same as above.

[0070] During use, the above-mentioned metabiotics were appropriately diluted and added to the cell culture medium. The supernatant of *Lactobacillus plantarum* CCFM1353 and *Lactobacillus plantarum* FHNMY13M5 was resuspended in fresh culture medium. The concentration after resuspending was the same as that after fermentation to 4.75 × 10⁻⁶. 7 The concentration of postbiotics prepared from bacterial suspensions at CFU / ml was equivalent to that obtained from fermentation to 5.93 × 10⁻⁶ CFU / ml. The cell lysates of *Lactobacillus plantarum* CCFM1353 and *Lactobacillus plantarum* FHNMY13M5 were resuspended in fresh culture medium; the concentration of the resuspended cell lysates was equivalent to that obtained from fermentation to 5.93 × 10⁻⁶ CFU / ml. 7 The concentration of metabiotics prepared from bacterial cultures with CFU / ml is equivalent.

[0071] 3. Effects of metabiotics prepared from *Lactobacillus plantarum* CCFM1353 on HDPC cell proliferation

[0072] HDPC cells in the logarithmic growth phase were digested, counted, and the cell suspension was adjusted to 1×10⁶. 5 100 μl of each sample was seeded per well in a 96-well plate, with the outermost ring filled with PBS solution to prevent edge effects. The plates were then incubated in a 5% CO2, 37 °C incubator for 24 h. The culture supernatant was then replaced with 100 μl of the supernatant according to the blank, control and treatment groups described below.

[0073] Control group: Contains only cell culture medium and does not contain HDPC cells;

[0074] Control group: Contains both cell culture medium and HDPC cells, without the addition of post-biotics;

[0075] Yangshen group: contains both cell culture medium and HDPC cells, with 0.1μM minoxidil added.

[0076] Treatment group: Contains both cell culture medium and HDPC cells, with added metabiotics. The amount of metabiotics added should refer to the dilution instructions in step 2 above. The supernatants of *Lactobacillus plantarum* CCFM1353 and *Lactobacillus plantarum* FHNMY13M5 are named CCFM1353-S and FHNMY13M5-S, respectively; the cell lysates of *Lactobacillus plantarum* CCFM1353 and *Lactobacillus plantarum* FHNMY13M5 are named CCFM1353-N and FHNMY13M5-N, respectively.

[0077] Cells treated in each group were incubated overnight at 37°C. After incubation, the supernatant was discarded, and freshly prepared MTT working solution was added. Cells were then incubated at 37°C in the dark. After 4 hours, the supernatant was discarded, and 100 μl of dimethyl sulfoxide was added to each well. The cells were shaken to dissolve and crystallize for 5 minutes. The absorbance (OD value) was read at 490 nm using a microplate reader. The cell proliferation rate was calculated using the following formula: Cell proliferation rate (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0078] Effects on cell proliferation, such as Figure 1 As shown, compared with the control group (cell proliferation rate of 100%), the cell proliferation rates of Minoxidil, Lactobacillus plantarum CCFM1353-S, CCFM1353-N, FHNMY13M5-S, and FHNMY13M5-N were 97%, 95%, 97%, 106%, and 101%, respectively, which had no significant effect on HDPC cell proliferation. This concentration can be selected for subsequent cell experiments.

[0079] 4. Effects of metabiotics prepared from *Lactobacillus plantarum* CCFM1353 on HDPC cell migration

[0080] All strains were preserved in 30% glycerol at -80°C before activation. A small amount of *Lactobacillus plantarum* CCFM1353 bacterial suspension was streaked onto MRS solid medium using a sterile inoculation loop and incubated at 37°C for 48 h. Single colonies were then picked and inoculated into MRS liquid medium and incubated at 37°C for 18 h. Finally, a 2% v / v inoculation was performed into 200 ml of *Platycladus orientalis* leaf fermentation medium, and incubated at 37°C for another 18 h to obtain 2.3 × 10⁻⁶ cells / year. 9 CFU / ml bacterial culture.

[0081] The bacterial culture was heat-treated at 65 °C for 30 min, centrifuged (8000g, 4 °C, 15 min), and the supernatant was collected and freeze-dried to obtain *Lactobacillus plantarum* CCFM1353 *Platycladus orientalis* leaf fermentation supernatant freeze-dried powder (CCFM1353-CP) for later use. The preparation method for *Lactobacillus plantarum* FHNMY13M5 *Platycladus orientalis* leaf fermentation supernatant freeze-dried powder (FHNMY13M5-CP) was the same, and the concentration used was as follows. Logarithmic growth phase cells were digested and counted using HDPC, and the cell suspension was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 2 mL / well in 6-well plates and cultured in a 5% CO2, 37°C incubator until 90% confluence. Cells were then spread in a straight line using a 200 μL pipette tip and washed three times with PBS. Control and treatment groups were established using the same procedure.

[0082] Control group: Contains both cell culture medium and HDPC cells;

[0083] The Platycladus orientalis leaf fermentation medium group (CP) contains both cell culture medium and HDPC cells, with the cell culture medium resuspended in Platycladus orientalis leaf fermentation medium (the Platycladus orientalis leaf fermentation medium was diluted tenfold, and the content of Platycladus orientalis leaf extract after dilution was 5 mg / ml).

[0084] Treatment groups (CCFM1353-CP and FHNMY13M5-CP): The fermentation supernatant of Platycladus orientalis leaves was resuspended in cell culture medium (the resuspended fermentation supernatant of Platycladus orientalis leaves is equivalent to the fermentation supernatant obtained from fermentation medium containing 5 mg / ml Platycladus orientalis leaf extract).

[0085] Following the setup of the control and treatment groups described above, fresh culture medium was used. After incubation for 15 hours, the width of the scratches was photographed using an optical microscope as shown in the image. Figure 1 .

[0086] like Figure 1 As shown, the cell migration rate was 32.63% in the control group, 42.63% in the CP group, 62.36% in the fermentation supernatant prepared from *Lactobacillus plantarum* CCFM1353 (1.91 times that of the control group), and 23.55% in the fermentation supernatant prepared from *Lactobacillus plantarum* FHNMY13M5. Compared with the direct addition of *Lactobacillus plantarum* extract, the fermentation supernatant prepared from the co-fermentation of *Lactobacillus plantarum* CCFM1353 and *Lactobacillus plantarum* extract increased the migration rate of HDPC cells by 1.46 times. Cell migration is crucial in hair follicle development, and the fermentation supernatant prepared from *Lactobacillus plantarum* CCFM1353 significantly promoted the migration ability of HDPC cells and improved wound healing.

[0087] Example 3: Lactobacillus plantarum CCFM1353 promotes the expression of anti-hair loss and hair care-related genes and proteins in HDPC cells.

[0088] 1. The effect of metagenes prepared from *Lactobacillus plantarum* CCFM1353 on HDPC cells TGF-β2 mRNA and IGF-1 Effect of mRNA expression

[0089] The preparation methods and concentrations of *Lactobacillus plantarum* CCFM1353 and *Lactobacillus plantarum* FHNMY13M5 postbiotics are the same as in Example 2.

[0090] HDPC cells in the logarithmic growth phase were digested, counted, and the cell suspension was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 2 mL / well in a 6-well plate and cultured overnight in a 5% CO2 incubator at 37 °C until cell attachment was achieved. The old culture medium was discarded, and the cells were washed three times with PBS. The control and treatment groups were set up as in Example 2.

[0091] Add 2 ml of each of CCFM1353-S, CCFM1353-N, FHNMY13M5-S, and FHNMY13M5-N to a 6-well plate and incubate overnight at 37°C. Perform triple replicates for each sample. For assay, wash cells twice with pre-cooled PBS, add 1 mL of Trizol, and repeatedly pipette. Add 200 μl of chloroform, mix thoroughly, and incubate for 10 min. Centrifuge at 12000 rpm for 15 min at 4°C. Collect the supernatant to a new enzyme-free centrifuge tube, add 0.8 times the volume of isopropanol, invert to mix, and incubate for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C. Discard the supernatant, add 1 mL of 75% ethanol, and centrifuge at 7500 rpm for 5 min at 4°C. Repeat twice. Collect the white precipitate at the bottom of the tube; this is the sample RNA. After synthesizing cDNA using a reverse transcription kit, real-time quantitative polymerase chain reaction (RT-qPCR) was performed according to the instructions for SYBR Green fluorescent dye. The internal control was GAPDH, and the primers are shown in Table 1.

[0092] Table 1 Primer Sequences

[0093]

[0094] Depend on Figure 2 It can be seen that, using the control group TGF-β2 mRNA, IGF-1 The mRNA expression level of *Lactobacillus plantarum* CCFM1353-S was 1. [[ID=3T]]TGF-β2 mRNA, IGF-1 The relative expression levels of mRNA were 1.05 and 6.28, respectively; CCFM1353-N TGF-β2 mRNA, IGF-1The relative expression levels of mRNA were 0.38 and 0.80, respectively; while those of *Lactobacillus plantarum* FHNMY13M5-S... TGF-β2 mRNA, IGF-1 The relative expression levels of mRNA were 0.64 and 0.77, respectively; FHNMY13M5-N TGF-β2 mRNA, IGF-1 The relative expression levels of mRNA were 1.51 and 1.19, respectively; these results indicate that the cell lysate prepared from *Lactobacillus plantarum* CCFM1353 significantly inhibited the expression of mRNA in HDPC cells. TGF-β2 mRNA, and the supernatant prepared from Lactobacillus plantarum CCFM1353 significantly enhanced HDPC cells. IGF-1 mRNA expression.

[0095] Therefore, it can be seen that the metabiotic prepared by *Lactobacillus plantarum* CCFM1353 has the ability to regulate... TGF-β2 mRNA, IGF- 1 The expression of mRNA has the potential to prolong the anagen phase of hair follicles and prevent hair follicles from transitioning to the catagen phase, thereby preventing / improving hair loss.

[0096] 2. Effects of metabiotics prepared from *Lactobacillus plantarum* CCFM1353 on VEGF expression in HDPC cells.

[0097] The preparation methods for *Lactobacillus plantarum* CCFM1353 postbiotic and *Lactobacillus plantarum* FHNMY13M5 postbiotic are the same as in Example 2.

[0098] HDPC cells in the logarithmic growth phase were digested, counted, and the cell suspension was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 2 mL / well in a 6-well plate and cultured overnight in a 5% CO2 incubator at 37 °C until cell attachment was achieved. The old culture medium was discarded, and the cells were washed three times with PBS. The control and treatment groups were set up as in Example 2.

[0099] Add 2 ml of each of CCFM1353-S, CCFM1353-N, FHNMY13M5-S, and FHNMY13M5-N to a 6-well plate and incubate overnight at 37°C. Perform triple replicates for each sample. For assay, determine the VEGF concentration in the culture supernatant according to the ELISA kit instructions.

[0100] The effect of post-genetic agents on VEGF expression in HDPC cells is as follows: Figure 3As shown, compared with the control group (VEGF content 466.24 pg / mL), the VEGF contents of the Yangshen group, CCFM1353-S, and CCFM1353-N were 621.83, 409.71, and 704.21 pg / mL, respectively. Compared with the control group, the addition of *Lactobacillus plantarum* CCFM1353-N promoted VEGF secretion in HDPC cells, and its promoting effect was higher than that of the Yangshen group (increased by 13.25%).

[0101] Therefore, it can be seen that the post-genetic agent prepared from Lactobacillus plantarum CCFM1353 has the potential to promote VEGF secretion, prolong the hair follicle growth phase, and thus prevent / improve hair loss.

[0102] Example 4: Effects of *Lactobacillus plantarum* CCFM1353 and its prepared metabiotics and synergistic agents on hair growth in mice.

[0103] The preparation method of live *Lactobacillus plantarum* CCFM1353 is the same as in Example 2, except that after obtaining the bacterial solution, the bacterial sludge is collected by centrifugation, and no heat treatment or lysis is performed. The preparation method of postbiotics of *Lactobacillus plantarum* CCFM1353 is the same as in Example 2, except that no lysis treatment is performed.

[0104] Extensive literature review has shown that C57BL / 6J mice possess melanocytes only in hair follicles, thus melanin synthesis coincides with the hair growth cycle. Therefore, monitoring the transition of skin color from pink (hairless) to black (fully grown hair) is sufficient to easily characterize the hair growth cycle, and this method has been widely applied in hair loss research.

[0105] Sixty-four male C57BL / 6J mice, 6 weeks old (approximately 20 g), were used for the experiment after a week of acclimatization. First, the backs of all mice were shaved using an animal hair removal device. Then, hair removal cream was applied to remove all hair, covering an area of ​​approximately 3 cm × 3 cm, causing the hair growth of the C57BL / 6J mice to enter the resting phase (resulting in pink skin), thus establishing the C57BL / 6J mouse alopecia model.

[0106] After 24 hours of skin stabilization, mice in the telogen phase were randomly divided into 8 groups of 8 mice each, for a total of 64 mice. The groups were set as follows: the metabiotic group (CCFM1353-D group and CCFM1353-O group): containing CCFM1353 cells and CCFM1353 metabolites; the synbiotic preparation group (CCFM1353-I): containing a combination of Platycladus orientalis leaf aqueous extract and live Lactobacillus plantarum CCFM1353 bacteria.

[0107] Control group: Using physiological saline as a control;

[0108] Yangshen group: Finasteride was used as a control at a dose of 10 mg / kg mouse body weight;

[0109] Low-dose group of Platycladus orientalis leaves (CP-L): Aqueous extract of Platycladus orientalis leaves, calculated as the dose of Platycladus orientalis leaf extract, was 160 mg / kg;

[0110] High-dose group of Platycladus orientalis leaves (CP-H): Aqueous extract of Platycladus orientalis leaves, calculated as the dose of Platycladus orientalis leaf extract, was 320 mg / kg;

[0111] CCFM1353-D group: In vitro fermentation broth obtained by fermenting *Lactobacillus plantarum* CCFM1353 in MRS liquid medium (9.5 × 10⁻⁶ viable cells). 8 The concentration of CFU / ml was calculated based on the lyophilized fermentation broth, at a dose of 500 mg / kg mouse body weight. The in vitro fermentation broth was inactivated before gavage.

[0112] CCFM1353-O group: In vitro fermentation broth obtained by gavage of *Lactobacillus plantarum* CCFM1353 in *Platycladus orientalis* leaf fermentation medium (viable count 9.5 × 10⁻⁶). 8 The concentration of CFU / ml was 160 mg / kg mouse body weight, based on the dosage of the lyophilized fermentation broth. The in vitro fermentation broth was inactivated before gavage.

[0113] CCFM1353-L group: Live Lactobacillus plantarum CCFM1353 was used at a dose of 5 × 10⁻⁶. 9 CFU / kg mouse body weight;

[0114] Group CCFM1353-I: Patchouli leaf aqueous extract and live *Lactobacillus plantarum* CCFM1353 were administered simultaneously by gavage; the dose of the patchouli leaf aqueous extract, calculated as patchouli leaf extract, was 160 mg / kg; the concentration of live *Lactobacillus plantarum* CCFM1353 was 5 × 10⁻⁶. 9 CFU / mL.

[0115] Each mouse was administered 0.2 ml / day by gavage for 3 weeks. The lyophilized postbiotic powders were dissolved in fresh culture medium before being administered by gavage.

[0116] The skin samples from the backs of mice were recorded at 7 days, 14 days, and 21 days, and the results are as follows: Figure 4 As shown.

[0117] Depend on Figure 4It can be seen that after 7 days of administration, compared with the blank group, the skin of mice in all other groups showed varying degrees of graying; after 14 days of administration, hair shafts appeared in all groups. Compared with the blank control group, the black area of ​​the back skin of mice in the intervention group was significantly increased. Among them, the CCFM1353-O group and CCFM1353-L group showed similar effects to the positive drug treatment group, with dense hair growth visible to the naked eye on the back; after 21 days of administration, the back skin of mice in all groups turned black, and hair growth was vigorous. Except for the blank control group and CCFM1353-D group, the hair growth on the back of mice in the other groups was complete and dense, with only a small amount of hair growing slowly at the junction of the head and back in the hair-removed treatment group. Among them, the hair at the junction of the head and back in the positive drug treatment group, the 320 mg / kg Platycladus orientalis leaf treatment group, the CCFM1353-O group, and the CCFM1353-I group had completely grown back.

[0118] Example 5: Lactobacillus plantarum CCFM1353 promotes the expression of genes and proteins related to hair loss prevention and hair care.

[0119] 1. *Lactobacillus plantarum* CCFM1353 and its prepared metabiotics and synbiotics have effects on mouse skin. Wnt10b mRNA, Cyclin D1 mRNA and Bcl2 Effect of mRNA expression

[0120] For specific experimental setup, refer to Example 4. Mice were sacrificed on day 21, and skin tissue from the backs of the mice was harvested and analyzed for [various components]. Wnt10b mRNA, Cyclin D1 mRNA and t The gene expression level of mRNA was measured, with mouse GAPDH as the internal control. The sequence is shown in Table 1.

[0121] Table 2 Primer Sequences

[0122]

[0123] Further analysis of the expression of genes regulating hair follicles revealed that, in the control group... Bcl2 mRNA, Wnt10b mRNA, and Cyclin D1 If the mRNA expression level is 1, then the live bacteria group of *Lactobacillus plantarum* CCFM1353... Bcl2 mRNA, Wnt10b D1 mRNA, CyclinThe expression levels of mRNA were 6.41, 4.95, and 1.94, respectively; the expression levels of the post-biotic group (CCFM1353-D) prepared from *Lactobacillus plantarum* CCFM1353 were 3.98, 2.04, and 1.62, respectively; and the expression levels of the synbiotic preparation group (CCFM1353-I) prepared from *Lactobacillus plantarum* CCFM1353 were 5.99, 1.53, and 1.26, respectively. Compared with gavage administration of *Platycladus orientalis* leaf extract (CP-L), gavage administration of *Lactobacillus plantarum* CCFM1353 prepared with fermentation medium containing *Platycladus orientalis* leaf extract (CCFM1353-O) can... Bcl2 Expression levels increased by 2.3 times. Wnt10b Expression levels increased by 1.8 times. Cyclin D1 The expression level increased by 0.8 times; oral administration of the same dose of Platycladus orientalis leaf aqueous extract and live Lactobacillus plantarum CCFM1353 (CCFM1353-I) could... Bcl2 Expression levels increased by 3.1 times. Wnt10b Expression level increased by 2.2 times. Cyclin D1 The expression level increased by 1.2 times.

[0124] The results above indicate that, compared with the control group, oral administration of live Lactobacillus plantarum CCFM1353 significantly promoted the growth of Lactobacillus plantarum in skin tissue. Bcl2 mRNA, Wnt10b mRNA and Cyclin D1 mRNA expression; compared with gavage administration of Platycladus orientalis leaf extract alone, gavage administration of *Lactobacillus plantarum* CCFM1353 and *Platycladus orientalis* leaf extract in vitro fermentation products, or gavage administration of *Lactobacillus plantarum* CCFM1353 and *Platycladus orientalis* leaf extract in vivo fermentation in mice. Bcl2 mRNA, Wnt10b mRNA and Cyclin D1 mRNA expression; activates the Wnt / β-catenin signaling pathway, inhibits hair follicle cell apoptosis, and promotes hair growth.

[0125] 2. Effects of *Lactobacillus plantarum* CCFM1353 on the expression of VEGF, IGF-1, and Ki67 in mouse skin.

[0126] After the experiment, the mice were euthanized, and 30 mg of skin tissue from the back of the mice was cut off. The tissue was homogenized in 270 μL of PBS, centrifuged for 20 min (3000 rpm / min), and the supernatant was carefully collected. The contents of VEGF, β-catenin, IGF-1 and Ki67 in the skin were detected by ELISA kit.

[0127] pass Bcl2 Figure 6 It should be noted that there may be some inaccuracies in the original text, such as the incorrect tag "ll" which should probably be "11". This translation is based on the provided text as accurately as possible while following the translation rules.It can be seen that, compared with the blank group (VEGF content of 53.39 ng / L), the VEGF content after intervention with live *Lactobacillus plantarum* CCFM1353 was 60.13 ng / L; after intervention with *Lactobacillus plantarum* CCFM1353 followed by a probiotic (CCFM1353-D group) was 57.12 ng / L; and compared with the low-dose *Platycladus orientalis* leaf group (VEGF content of 69.51 ng / L), the VEGF content after intervention with *Lactobacillus plantarum* CCFM1353-I group was 72.27 ng / L.

[0128] Compared with the blank group (IGF-1 content 8.97 μg / L), the IGF-1 content after intervention with live *Lactobacillus plantarum* CCFM1353 was 11.61 μg / L; the IGF-1 content after intervention with *Lactobacillus plantarum* CCFM1353 was 11.60 μg / L; compared with the low-dose *Platycladus orientalis* leaf group (IGF-1 content 9.16 μg / L), the IGF-1 content after intervention with *Lactobacillus plantarum* CCFM1353-I group was 13.52 μg / L (an increase of 47.5%), indicating that simultaneous gavage administration of *Platycladus orientalis* leaf extract and live *Lactobacillus plantarum* CCFM1353, allowing for in vivo fermentation, can enhance the promoting effect of *Platycladus orientalis* leaf on IGF-1 expression.

[0129] Compared with the blank group (Ki67 content 25.01 pg / mL), the Ki67 content after intervention with live *Lactobacillus plantarum* CCFM1353 was 32.93 pg / mL; the Ki67 content after intervention with *Lactobacillus plantarum* CCFM1353 was 30.46 pg / mL; compared with the low-dose *Platycladus orientalis* leaf group (Ki67 content 23.66 pg / mL), the Ki67 content after intervention with *Lactobacillus plantarum* CCFM1353-I group was 36.49 pg / mL (an increase of 66.9%), indicating that simultaneous gavage administration of *Platycladus orientalis* leaf extract and live *Lactobacillus plantarum* CCFM1353, allowing for in vivo fermentation, can enhance the promoting effect of *Platycladus orientalis* leaf on Ki67 expression.

[0130] Ki67 is a nuclear protein involved in the cell cycle. The cell cycle is divided into G1, S, G2, and M phases. After the M phase, i.e., the mitotic phase, some cells enter the G0 phase, i.e., the quiescent phase, and temporarily cease dividing and proliferating. In normal tissues, Ki67 is expressed in the G1, S, G2, and M phases of cell division. However, after the M phase, Ki67 is rapidly degraded, and cells in the G0 phase do not express Ki67. Therefore, Ki67 is an important biomarker for detecting cell proliferation.

[0131] The results above show that the levels of IGF-1 and Ki67 in live *Lactobacillus plantarum* CCFM1353 and postbiotics were significantly higher than those in the control group. Compared with the live bacteria group and the group with the same dose of *Platycladus orientalis*, the levels of IGF-1 and Ki67 in the fermented *Lactobacillus plantarum* CCFM1353 group were significantly higher. This can reduce hair loss by regulating the proliferation and differentiation of epithelial cells to form hair shafts and prolonging the growth phase of hair follicle cells.

[0132] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum CCFM1353, characterized in that, The *Lactobacillus plantarum* CCFM1353 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63923, and the deposit date is October 25, 2023.

2. A microbial preparation containing the *Lactobacillus plantarum* CCFM1353 as described in claim 1.

3. The microbial preparation as claimed in claim 2, characterized in that The microbial preparation is a liquid or solid preparation containing Lactiplantibacillus halli CCFM1353, wherein the viable count of Lactiplantibacillus halli CCFM1353 in the microbial preparation is ≥ 10 6 CFU / mL or ≥ 10 6 CFU / g.

4. Process for the preparation of the microbial preparation according to claim 2 or 3, characterized in that, The *Lactobacillus plantarum* CCFM1353 of claim 1 is fermented in a culture medium.

5. A detangling hair care product characterized in that, The product contains inactivated *Lactobacillus plantarum* CCFM1353 or metabiotics prepared from *Lactobacillus plantarum* CCFM1353. The metabiotics include lysates of *Lactobacillus plantarum* CCFM1353 or fermentation supernatant of *Lactobacillus plantarum* CCFM1353. The *Lactobacillus plantarum* CCFM1353 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63923 and deposit date of October 25, 2023.

6. A method for enhancing the hair retention function of cypri s s us funereus, characterized in that, The *Lactobacillus plantarum* CCFM1353 of claim 1, or the microbial preparation of claim 2 or 3, is fermented in a culture medium containing *Platycladus orientalis* leaf extract.

7. Use of Lactobacillus plantarum CCFM 1353 according to claim 1, or of the microbial preparation according to claim 2 or 3, for the preparation of a hair loss prevention product, characterized in that, The product in question is a pharmaceutical or cosmetic product.

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