Compositions for promoting hair growth, uses thereof and edible compositions comprising the same

Abstract

The present application provides a composition for promoting hair growth, uses thereof, and an edible composition comprising the same, which comprises active substances of Lactobacillus plantarum GKM3, Lactobacillus plantarum GKD7, and Lactobacillus plantarum GKK1. The composition can improve the hair growth rate and hair distribution rate.

Description

Technical Field

[0001] This invention relates to a composition that promotes hair growth. Specifically, it relates to a composition containing *Lactobacillus plantarum* active substances capable of promoting hair growth, its uses, and an edible composition. Background Technology

[0002] Hair growth is mainly produced by hair follicles. The growth of hair follicles on the scalp includes three phases: germination, catagen, and telogen. These three phases of hair follicle growth cycle coexist. The hair growth phase lasts about 2 to 5 years. Under normal circumstances, about 85 to 90% of hair follicles are in the growth phase and about 10% are in the telogen phase.

[0003] During the growth phase, hair follicle cells continuously divide, causing hair to grow longer and thicker, resulting in long, strong hair that won't fall out. After the growth phase ends, hair enters the regression phase, hair follicle cells gradually shrink, and hair growth stops. During the resting phase, hair begins to fall out. After the resting phase ends, new hair will grow in the same location on a healthy scalp, restarting the hair growth cycle. Since the growth cycles of each hair follicle are not synchronized, it's normal for each person to lose about 50 to 100 hairs per day.

[0004] Persistent hair loss and baldness may be due to familial hair loss, also known as androgenetic alopecia, or it may be caused by long-term nutritional deficiencies, excessive stress, the use of certain medications, or hormonal imbalances. Improper scalp and hair care methods, such as excessive dyeing and perming, and long-term use of hair styling products, can also damage hair follicles and affect scalp health. Abnormal hair loss mainly indicates a problem with the hair follicles. When hair follicles atrophy, hair becomes thin and soft, and falls out easily, increasing hair loss. If the hair follicles atrophy to the point of pore closure, hair will not grow, and the condition of the hair follicles cannot return to normal. Except for androgenetic alopecia (hereditary baldness) which ultimately leads to the death of hair follicles and is difficult to recover from, hair follicle atrophy caused by other reasons can be improved through the dilation of microvessels around the hair follicles to increase blood flow and cell permeability. This includes increasing nitric oxide secretion through autologous or exogenous means or additionally reducing vascular resistance, thereby improving survival and restoring the hair follicles to normal, thus preventing them from eventually closing and dying.

[0005] Most hair growth promotion products on the market are for external use. If there were oral products that could promote hair growth, they would likely provide even better results. Summary of the Invention

[0006] In accordance with the above-mentioned objective of the present invention, a composition for promoting hair growth is provided, comprising: active substances of Lactobacillus plantarum GKM3, Lactobacillus plantarum GKD7, and Lactobacillus plantarum GKK1. GKM3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 14565; GKD7 is deposited at the Patent Microbial Collection Center of Japan (NITE) with accession number NITE BP-03313; and GKK1 is deposited at the Patent Microbial Collection Center of Japan (NITE) with accession number NITE BP-03702.

[0007] Preferably, the active substances of *Lactobacillus plantarum* GKM3, *Lactobacillus plantarum* GKD7, and *Lactobacillus plantarum* GKK1 are prepared by the following method:

[0008] (a) Take the bacterial cells of the plant lactobacillus and inoculate them into a solid culture medium for solid culture to form colonies;

[0009] (b) Inoculate the colony cultured in step (a) into a liquid culture medium for liquid culture to obtain a liquid culture medium containing bacterial cells;

[0010] (c) The liquid culture medium containing bacteria from step (b) is inoculated into a fermentation tank for liquid scale-up to obtain bacterial solution;

[0011] (d) Concentrate the bacterial solution from step (c) to obtain a concentrated bacterial solution.

[0012] Preferably, the ratio of the active substances GKM3, GKD7, and GKK1 is 1:1:1 to 2:1:1.

[0013] To achieve another objective of the present invention, a use is provided for a *Lactobacillus plantarum* active substance in the preparation of a composition promoting hair growth, wherein the *Lactobacillus plantarum* active substance is a fermentation product of *Lactobacillus plantarum* GKM3, *Lactobacillus plantarum* GKD7, and *Lactobacillus plantarum* GKK1, and wherein the *Lactobacillus plantarum* active substance is obtained in the following manner:

[0014] (a) Take the bacterial cells of the plant lactobacillus and inoculate them into a solid culture medium for solid culture to form colonies;

[0015] (b) Inoculate the colony cultured in step (a) into a liquid culture medium for liquid culture to obtain a liquid culture medium containing bacterial cells;

[0016] (c) The liquid culture medium containing bacteria from step (b) is inoculated into a fermentation tank for liquid scale-up to obtain bacterial solution;

[0017] (d) Concentrate the bacterial solution from step (c) to obtain a concentrated bacterial solution.

[0018] Preferably, the ratio of the active substances GKM3, GKD7, and GKK1 is 1:1:1 to 2:1:1.

[0019] Preferably, promoting hair growth means increasing hair length or increasing hair distribution rate.

[0020] Preferably, the concentrated bacterial solution in step (d) is concentrated 2 to 10 times.

[0021] Preferably, the concentrated bacterial solution in step (d) is concentrated 10 times.

[0022] To achieve another objective of the present invention, an edible composition is provided, comprising the hair growth promoting composition as described above.

[0023] Preferably, the dosage form of the edible composition includes tablets, capsules, powders, or granules.

[0024] Preferably, the content of the hair growth promoting composition is 20% by weight relative to the total weight of the composition.

[0025] Through the above-mentioned technical features, the hair growth promoting composition of the present invention can effectively promote hair growth and effectively improve hair distribution rate, and has a significant effect on people with hair growth needs. Attached Figure Description

[0026] Figure 1 To illustrate the hair growth distribution rate (%) of each group on day 8 of the experiment, it was shown that the mixture of Lactobacillus plantarum fermentation concentrate (GKM3:GKD7:GKK1 = 2:1:1) significantly improved the hair growth distribution rate in mice.

[0027] Figure 2 To illustrate the hair growth length (mm) of each group on day 12 of the experiment, it was shown that the mixture of Lactobacillus plantarum fermentation concentrate (GKM3:GKD7:GKK1 = 2:1:1) significantly increased the hair growth length in mice.

[0028] Figure 3 To illustrate the hair growth distribution rate (%) of each group on day 12 of the experiment, it was shown that the mixture of Lactobacillus plantarum fermentation concentrate (GKM3:GKD7:GKK1 = 1:1:1) significantly improved the hair growth distribution rate in mice.

[0029] Figure 4To illustrate hair growth (mm) in each group on day 12 of the experiment, it was shown that neither individual Lactobacillus plantarum fermentation concentrates nor mixtures of Lactobacillus plantarum fermentation concentrates (GKM3:GKD7:GKK1 = 1:1:1) could increase hair growth in mice.

[0030] Figure 5 These are photographs of mouse hair at different days according to embodiments of the present invention.

[0031] Preservation of biological materials for patent procedures:

[0032] Lactobacillus plantarum GKM3 strain;

[0033] Date of deposit: August 25, 2017;

[0034] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0035] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China;

[0036] Accession number: CGMCC No. 14565;

[0037] Classification and nomenclature: Lactobacillus plantarum.

[0038] Lactobacillus plantarum GKD7 strain;

[0039] Date of deposit: November 6, 2020;

[0040] Depository: National Institute of Technology and Evaluation (NPMD), Japan's independent administrative agency for product evaluation and technical bases;

[0041] Address of the depositary: Chiba, Japan (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan);

[0042] Accession number: NITE BP-03313;

[0043] Classification and nomenclature: Lactobacillus plantarum.

[0044] Lactobacillus plantarum GKK1 strain;

[0045] Date of deposit: August 5, 2022;

[0046] Depository: National Institute of Technology and Evaluation (NPMD), Japan's independent administrative agency for product evaluation and technical bases;

[0047] Address of the depositary: Chiba, Japan (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan);

[0048] Accession number: NITE BP-03702;

[0049] Classification and nomenclature: Lactobacillus plantarum. Detailed Implementation

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, the definitions contained herein shall prevail.

[0051] When referring to percentages in this case, the terms "weight%" and "%" used in this article can refer to mass fractions, which are defined as: the percentage of the mass of a substance divided by the total mass of the mixture.

[0052] As used in this case, the terms "produced from" and "comprising" are synonymous. As used herein, the terms "includes," "including," "comprises," "has," "having," "contains," or any other variation thereof are intended to cover a non-exclusive scope. For example, a composition, process, method, article, or apparatus containing a plurality of elements listed is not necessarily limited to those listed, but may include other elements not expressly listed but inherent to the composition, process, method, article, or apparatus. The term "comprising" is generally used in the sense of inclusion, that is, allowing the presence of one or more other features or components.

[0053] The indefinite articles “a” and “one” preceding an element or component in this case are intended to indicate, in a non-restrictive way, the number of instances (i.e., the number of occurrences) of that element or component. Therefore, “a” or “one” should be understood to include one or at least one, and the singular form of that element or component also includes the plural, unless the number clearly refers to the singular.

[0054] The term "cell" as used herein refers to the structure of the lactobacillus itself at various culture stages used in this disclosure. In a specific context, a cell refers to a complete or partial lactobacillus structure. In a specific context, a cell refers to a colony of lactobacillus formed by the division and reproduction of a single bacterium. In a specific context, a culture stage refers to solid-state culture, liquid culture, or liquid-scale inoculation in a fermenter.

[0055] The term "active substance" as used herein refers to substances screened from Lactobacillus cells used in this disclosure after treatment with specific experimental procedures, or mixtures of cells with culture medium or broth. In a specific state, active substance refers to centrifuged bacterial sludge, freeze-dried bacterial powder, or solvent-extracted extract. In a specific state, active substance refers to solid culture medium containing cells, liquid culture medium containing cells, or bacterial broth scaled up in a fermenter.

[0056] Experimental material culture

[0057] Regarding solid-state culture of the bacterial strain, *Lactobacillus plantarum* cells are inoculated onto a solid culture medium for solid-state culture to activate the cells and form colonies. In a preferred embodiment, the solid-state culture temperature is 30 to 45°C, more preferably 32 to 40°C. In a preferred embodiment, the solid-state culture time is 0.5 to 3 days, more preferably 1 to 2.5 days. In a preferred embodiment, the solid culture medium is MRS agar.

[0058] Regarding liquid culture, after colony growth on the solid medium is complete, a single colony is picked up and inoculated into a test tube containing MRS liquid medium for activation via liquid culture. In a preferred embodiment, the temperature for MRS liquid culture is 30 to 55°C, more preferably 32 to 45°C. In a preferred embodiment, the MRS liquid culture time is 12 to 25 hours, more preferably 20 hours. In a preferred embodiment, the pH of the liquid culture is 5.0 to 7.0, more preferably 5.5 to 6.5. In a preferred embodiment, the formulation of the liquid culture medium is shown in Table 1 below.

[0059] Table 1

[0060]

[0061]

[0062] For fermentation culture, the bacterial cells in MRS liquid medium are inoculated into an Erlenmeyer flask containing 1 L of liquid culture medium. After the bacterial cells in the liquid medium have grown, the bacterial cells, along with the liquid medium, are inoculated into a fermentation tank for liquid-scale production to obtain a bacterial culture. In a preferred embodiment, the fermentation tank temperature is 20 to 40°C, more preferably 25 to 37°C. In a preferred embodiment, the fermentation tank time is 16 to 20 hours, more preferably 17 to 19 hours.

[0063] Carbon sources that can be used in the culture medium for *Lactobacillus plantarum* include, but are not limited to, glucose, sucrose, lactose, fructose, mannose, sorbitol, glycerol, molasses, or combinations thereof. The weight percentage and composition of the aforementioned components in the culture medium can be adjusted as needed. In one specific embodiment, the carbon source is glucose. Nitrogen sources that can be used in the culture medium for *Lactobacillus plantarum* include, but are not limited to, soy protein, yeast extract, beef extract, casein powder, whey protein powder, fish protein hydrolysate, plant protein extract, or combinations thereof. The weight percentage and composition of the aforementioned components in the culture medium can be adjusted as needed. In one specific embodiment, the nitrogen source is yeast extract. The addition ratio of carbon source and nitrogen source relative to the total weight of the culture medium has varying effects on the culture efficiency, ranging from 1 to 10 wt%, preferably 3 wt% to 7 wt%. In one specific embodiment, the culture medium contains glucose and yeast extract, and the addition ratio of glucose and yeast extract relative to the total weight of the culture medium is 1 wt% to 10 wt%, preferably 3 wt% to 7 wt%. In one specific embodiment, the culture medium contains only glucose or only yeast extract, and the proportion of glucose or yeast extract added relative to the total weight of the culture medium is from 1 wt% to 10 wt%, preferably from 3 wt% to 7 wt%. *Lactobacillus plantarum* GKM3 can be cultured in solid or liquid media at a temperature between 32°C and 42°C, preferably between 35°C and 40°C, and more preferably 37°C.

[0064] The composition of the liquid culture medium for fermentation is shown in Table 2 below.

[0065] Table 2

[0066] Element Ratio (by weight percentage) carbon source 0 to 10% nitrogen source 0 to 10% ion 0.01 to 2% water The remaining part

[0067] This disclosure discloses compositions containing the active substance of *Lactobacillus plantarum*, further comprising additives. In a preferred embodiment, the additive may be an excipient, preservative, diluent, filler, absorption enhancer, or a combination thereof. The excipient may be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose, or a combination thereof. The preservative, which extends the shelf life of the composition, may be benzyl alcohol or parabens. The diluent may be selected from water, ethanol, propylene glycol, glycerin, or a combination thereof. The filler may be selected from lactose, molasses, high molecular weight polyethylene glycol, or a combination thereof. The absorption enhancer may be selected from dimethyl sulfoxide (DMSO), laurocapram, propylene glycol, glycerin, polyethylene glycol, or a combination thereof. The sweetener may be selected from acesulfame K, aspartame, saccharin, sucralose, neotame, or a combination thereof. In addition to the additives listed above, other suitable additives may be selected as needed, provided that the pharmaceutical efficacy of the composition is not affected.

[0068] This composition can be developed into various products in the pharmaceutical and food additive fields. In a preferred embodiment, the composition is a pharmaceutical, feed, beverage, nutritional supplement, fermentation broth, concentrate, dairy product, yogurt, food, or health food.

[0069] The composition may be in different forms depending on the needs of the recipient. In a preferred embodiment, the composition is in the form of powder, drops, tablets, granules, suppositories, microcapsules, ampoules, liquid sprays, or plugs.

[0070] The compositions disclosed herein can be used in animals or humans. Compositions containing the active substance of *Lactobacillus plantarum* can be formulated into any pharmaceutical form without affecting the efficacy of the active substance, and administered to the animal or human via an appropriate route according to the pharmaceutical form. The compositions disclosed herein, depending on the form of the composition, are administered to the subject at predetermined time points via an appropriate route in a suitable dose.

[0071] In in vitro cell culture experiments, the effective dose is defined as the total volume of cell culture medium used in each culture, expressed in μg / ml. The effective dose obtained from in vitro cell culture experiments can be converted to an effective dose for animal use using the following formula. The effective dose for animal use can then be further converted to an effective dose for humans based on metabolic rate.

[0072] The preparation method and effects of the Lactobacillus plantarum composition provided in this case will be described below with reference to specific embodiments and figures.

[0073] Example 1

[0074] Preparation of concentrated fermentation broth

[0075] bacterial culture

[0076] Lactobacillus plantarum GKM3, GKD7, and GKK1 cells were inoculated onto a solid culture medium (MRS agar) at 30–45°C for 1–2.5 days. After colony growth on the solid medium was complete, single colonies were picked up and inoculated into test tubes containing MRS liquid culture medium for activation. The MRS liquid culture was conducted at 32–45°C for 20 hours at a pH of 5.5–6.5. The formulation of the liquid culture medium is shown in Table 3 below.

[0077] Table 3

[0078] Element Ratio (by weight percentage) glucose 5％ yeast extract 1.5％ ion 0.5％ water 93％

[0079] The bacterial cells in MRS liquid medium were inoculated into an Erlenmeyer flask containing 1 L of liquid culture medium. After the bacterial cells in the liquid medium had grown, the bacterial cells and liquid medium were inoculated into a fermentation tank for liquid scale-up to obtain a bacterial culture. The fermentation tank was incubated at a temperature of 25 to 37°C for 17 to 19 hours.

[0080] In one embodiment, the bacterial solution obtained by the aforementioned method can be centrifuged to obtain bacterial sludge, and the bacterial sludge can be freeze-dried to produce bacterial powder. In another embodiment, the obtained bacterial solution can be concentrated to obtain concentrated bacterial solution, and the concentration ratio can be 2, 3, 5, 8, 10 times, or any other arbitrary ratio as needed.

[0081] Example 2

[0082] Preparation of fermentation concentrate of experimental strain (GKM3:GKD7:GKK1 = 2:1:1)

[0083] Lactobacillus plantarum GKM3, Lactobacillus plantarum GKD7, and Lactobacillus plantarum GKK1 were fermented according to the method described in Example 1 above. The fermentation broths produced were concentrated 10 times, and the three fermentation concentrates were mixed in the ratio GKM3:GKD7:GKK1 = 2:1:1 as the subsequent test material (i.e., MDK group).

[0084] laboratory animals

[0085] Twelve male C57BL / 6Narl strain mice, weighing between 22 and 26 g and aged 6 to 8 weeks, were purchased from the laboratory animal center. The animals were housed in the GLP animal laboratory – mouse area, with 12hr day / 12hr lighting, positive pressure environment, temperature maintained at 22±4℃, and humidity maintained at 30%–70%. LabDiet cat#5058 sterilized mouse feed was used, and the animals were allowed free access to food. The animals were fasted for 16±2 hours the day before hair removal, and feeding was resumed after hair removal was completed. All mouse housing practices complied with the regulations of the Institutional Animal Care and Use Committee of Agricultural Technology Research Institute.

[0086] Experimental Design

[0087] Twelve mice were randomly divided into two groups of six each, with three mice per cage: a saline group (control group) and an MDK group (control group).

[0088] 1. Establishing a hair removal mode

[0089] Animals were induced to be anesthetized with 4% isoflurane, and then maintained anesthesia was maintained at a concentration of 1-2.5%. The hair on the back of the animals was removed with paraffin to establish a hair removal pattern.

[0090] 2. Dosage, method of administration, and frequency of administration of the test substance

[0091] The test substance was administered to mice at a dose of 102.499 mg / kg. Day 0 was the day of hair removal. During the experiment, the hair-removed area on the back was orally administered either saline or the test substance once daily for 12 days. Photos were taken on days 0, 4, 8, and 12 to assess hair growth in the hair-removed area.

[0092] Analysis Project

[0093] 1. Clinical symptom observation

[0094] Observe once a day, noting the patient's mental state, behavior, breathing, mouth, nose and eyes, appearance and skin.

[0095] 2. Weight measurement

[0096] The body weight of mice was monitored and recorded using an electronic balance before administration of the test substance (day 0) and on days 4, 8, and 12 after administration.

[0097] 3. Hair length

[0098] On day 12 after administration of the test substance, the hair removal area was roughly divided into four small sections. Several hairs were plucked using stainless steel tweezers and placed into 1.5 mL microcentrifuge tubes. The hair length was measured in millimeters (mm) using calipers. Three hairs were randomly selected from each section, for a total of 12 hairs from each mouse.

[0099] 4. Hair growth distribution rate

[0100] On days 0, 4, 8, and 12, photos were taken of the treated area on the back using a camera (with a scale sticker attached for correction next to the treated area). ImageJ software was used to analyze and calculate the percentage of hair growth distribution. Hair growth distribution percentage % = (Hair distribution area on day N / Observation area 4cm²) 2 )x 100%.

[0101] 5. Statistical Analysis

[0102] The data was processed using the built-in Excel program in Microsoft Office to calculate the mean and standard deviation (SD), expressed as Mean ± SD. Statistical methods used SPSS 12.0 software with One-Way ANOVA and Duncan's Test to compare differences between groups were employed. A p-value < 0.05 was considered statistically significant between groups.

[0103] Experimental results

[0104] The mice exhibited normal clinical symptoms and no adverse reactions during the administration of the test substance. Before administration of the test substance, the body weights of the mice in each group were similar, and there were no significant differences in body weight among the groups during the experiment and until its conclusion.

[0105] On day 8 of the experiment, the hair growth distribution rate of mice in each group was observed, and the results are as follows: Figure 1 Compared with the saline group, the MDK group mice showed better hair growth distribution on day 8, and the difference was statistically significant (p<0.05).

[0106] On day 12 of the experiment, the hair growth length of mice in each group was measured and statistically analyzed. The results are as follows: Figure 2 The results showed that the hair growth length of mice in the MDK group was better than that in the saline group, and the difference was statistically significant (p<0.05).

[0107] Example 3

[0108] Preparation of experimental strains and fermentation concentrate MDK (GKM3:GKD7:GKK1 = 1:1:1)

[0109] Lactobacillus plantarum GKM3, Lactobacillus plantarum GKD7, and Lactobacillus plantarum GKK1 were fermented according to the method described in Example 1 above. The fermentation broths produced were concentrated 10 times, and the individual concentrated broths and the three fermentation concentrated broths were mixed in a ratio of GKM3:GKD7:GKK1 = 1:1:1 to serve as the subsequent test material (i.e., MDK group).

[0110] laboratory animals

[0111] Twenty-five male C57BL / 6Narl mice, weighing between 22 and 26 g and aged 6 to 8 weeks, were purchased from the laboratory animal center. They were housed in the GLP-certified animal laboratory's mouse section, with 12hr day / 12hr lighting, positive pressure, a temperature maintained at 22±4℃, and humidity at 30%–70%. LabDiet cat#5058 sterilized mouse feed was used, and the animals were allowed free access to food. They were fasted for 16±2 hours the day before hair removal, and were fed again after hair removal was completed. All mouse housing practices complied with the regulations of the Institutional Animal Care and Use Committee of Agricultural Technology Research Institute.

[0112] Experimental Design

[0113] Twenty-five mice were randomly divided into groups of five, with five mice per cage. The groups included: saline group, GKM3 group, GKD7 group, GKK1 group, and MDK group.

[0114] 1. Establishing a hair removal mode

[0115] Animals were induced to be anesthetized with 4% isoflurane, and then maintained anesthesia was maintained with a concentration of 1-2.5%. The hair on the back of the animals was removed with paraffin to establish a hair removal pattern.

[0116] 2. Dosage, method of administration, and frequency of administration of the test substance

[0117] The test substance was administered to mice at a dose of 102.499 mg / kg. Day 0 was the day of hair removal. During the experiment, the hair-removed area on the back was orally administered either saline or the test substance once daily for 12 days. Photos were taken on days 0, 4, 8, and 12 to assess hair growth in the hair-removed area.

[0118] Analysis Project

[0119] 1. Clinical symptom observation

[0120] Observe once a day, noting the patient's mental state, behavior, breathing, mouth, nose and eyes, appearance and skin.

[0121] 2. Weight measurement

[0122] The body weight of mice was monitored and recorded using an electronic balance before administration of the test substance (day 0) and on days 4, 8, and 12 after administration.

[0123] 3. Hair length

[0124] On day 12 after administration of the test substance, the hair removal area was roughly divided into four small sections. Several hairs were plucked using stainless steel tweezers and placed into 1.5 mL microcentrifuge tubes. The hair length was measured in millimeters (mm) using calipers. Three hairs were randomly selected from each section, for a total of 12 hairs from each mouse.

[0125] 4. Hair growth distribution rate

[0126] On days 0, 4, 8, and 12, photos were taken of the hair removal area on the back using a camera (with a scale sticker attached next to the hair removal area for correction). ImageJ software was used to analyze and calculate the percentage of hair growth distribution.

[0127] Hair growth distribution percentage % = (hair distribution area on day N / observation area 4cm) 2 )x 100%.

[0128] 5. Statistical Analysis

[0129] The data was processed using the built-in Excel program in Microsoft Office to calculate the mean and standard deviation (SD), expressed as Mean ± SD. Statistical methods used SPSS 12.0 software with One-Way ANOVA and Duncan's Test to compare differences between groups were employed. A p-value < 0.05 was considered statistically significant between groups.

[0130] Experimental results

[0131] The mice exhibited normal clinical symptoms and no adverse reactions during the administration of the test substance. Before administration of the test substance, the body weights of the mice in each group were similar, and there were no significant differences in body weight among the groups during the experiment and until its conclusion.

[0132] On day 12 of the experiment, the hair growth distribution rate of mice in each group was observed, and the results are as follows: Figure 3 The hair growth distribution rate of mice in the GKM3, GKD7, and GKK1 groups was not significantly different from that in the saline group, but the hair growth distribution rate of mice in the MDK group was better and the difference was significant (p<0.05).

[0133] On day 12 of the experiment, the hair growth length of mice in each group was measured and statistically analyzed. The results are as follows: Figure 4 The results showed that there was no significant difference in hair growth length between the mice in all groups and the physiological saline group.

[0134] Figure 5 These are actual photographs of hair growth in mice from various embodiments of this case. The photographs show that on day 8, the MDK (1:1:1, 2:1:1) groups showed more noticeable hair growth and better hair distribution. On day 12, the hair length in the MDK (2:1:1) group was significantly longer.

[0135] Example 4: Preparation of Edible Composition

[0136] If the Lactobacillus plantarum GKM3, Lactobacillus plantarum GKD7, and Lactobacillus plantarum GKK1 of the present invention are applied to food use, the following composition 1 is provided as an exemplary example.

[0137] Composition 1: Take a concentrated extract (20 wt%) of the Lactobacillus plantarum composition, mix it thoroughly with benzyl alcohol (8 wt%) as a preservative and glycerol (7 wt%) as a diluent, and dissolve it in pure water (65 wt%). Store at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

[0138] If the *Lactobacillus plantarum* GKM3, *Lactobacillus plantarum* GKD7, and *Lactobacillus plantarum* GKK1 of the present invention are used in pharmaceutical applications in liquid dosage form, the following sample of composition 2 is provided as an illustrative example.

[0139] Composition 2: Take a concentrated extract (20 wt%) of the Lactobacillus plantarum composition, mix it thoroughly with benzyl alcohol (8 wt%) as a preservative, glycerol (7 wt%) as a diluent, and sucrose (10 wt%) as a diluent, and dissolve it in pure water (55 wt%). Store at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

[0140] The proportion of the fermentation concentrate disclosed in this invention in the edible composition is not limited thereto, and may contain any proportion of the dose effective for the target of application, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0141] In summary, the above embodiments demonstrate that the composition of Lactobacillus plantarum GKM3, Lactobacillus plantarum GKD7, and Lactobacillus plantarum GKK1 contained in this invention helps promote hair growth and effectively improves hair distribution rate, showing significant effects for individuals with hair growth needs.

[0142] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the invention. Those skilled in the art, upon referring to the above teachings, can make appropriate modifications to the above embodiments while still achieving the effects claimed in this case. Therefore, the scope of protection of the present invention should be determined by its scope.

Claims

1. A composition for promoting hair growth, comprising: active substances of Lactobacillus plantarum GKM3, Lactobacillus plantarum GKD7, and Lactobacillus plantarum GKK1, wherein GKM3 is deposited with the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 14565, GKD7 is deposited with the Patent Microbial Collection Center of the Japan Institute of Product Evaluation and Research (NITE BP-03313) under accession number NITE BP-03702 under accession number NITE BP-03702 under the Patent Microbial Collection Center of the Japan Institute of Product Evaluation and Research (NITE BP-03702).

2. The composition according to claim 1, wherein the active substances of *Lactobacillus plantarum* GKM3, *Lactobacillus plantarum* GKD7, and *Lactobacillus plantarum* GKK1 are prepared by the following method: (a) Take the bacterial cells of the plant lactobacillus and inoculate them into a solid culture medium for solid culture to form colonies; (b) Inoculate the colony cultured in step (a) into a liquid culture medium for liquid culture to obtain a liquid culture medium containing bacterial cells; (c) The liquid culture medium containing bacteria from step (b) is inoculated into a fermentation tank for liquid scale-up to obtain bacterial solution; (d) Concentrate the bacterial solution from step (c) to obtain a concentrated bacterial solution.

3. The composition according to claim 1, wherein the ratio of the active substances GKM3, GKD7, and GKK1 is from 1:1:1 to 2:1:

1.

4. Use of a *Lactobacillus plantarum* active substance in the preparation of a composition for promoting hair growth, wherein the *Lactobacillus plantarum* active substance is a fermentation product of *Lactobacillus plantarum* GKM3, *Lactobacillus plantarum* GKD7, and *Lactobacillus plantarum* GKK1, wherein the *Lactobacillus plantarum* active substance is obtained in the following manner: (a) Take the bacterial cells of the plant lactobacillus and inoculate them into a solid culture medium for solid culture to form colonies; (b) Inoculate the colony cultured in step (a) into a liquid culture medium for liquid culture to obtain a liquid culture medium containing bacterial cells; (c) The liquid culture medium containing bacteria from step (b) is inoculated into a fermentation tank for liquid scale-up to obtain bacterial solution; (d) Concentrate the bacterial solution from step (c) to obtain a concentrated bacterial solution.

5. The use according to claim 4, wherein the ratio of the active substances GKM3, GKD7, and GKK1 is 1:1:1 to 2:1:

1.

6. The use according to claim 4, wherein the promotion of hair growth is to increase hair length or increase hair distribution rate.

7. The use according to claim 4, wherein the concentrated bacterial solution in step (d) is concentrated 2 to 10 times.

8. The use according to claim 4, wherein the concentrated bacterial solution in step (d) is concentrated 10 times.

9. An edible composition comprising the hair growth promoting composition according to claim 1.

10. The edible composition according to claim 9, wherein the dosage form includes fermentation broth, concentrated broth, freeze-dried powder, dairy product, yogurt, drops, capsules, tablets, and granules.

11. The edible composition according to claim 9, wherein the content of the hair growth promoting composition is 20% by weight relative to the total weight of the edible composition.

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