Plant lactobacillus zjuids24 with improved androgenetic alopecia and application thereof

Abstract

This invention belongs to the field of food microbiology technology, specifically relating to *Lactiplantibacillus plantarum* ZJUIDS24, which has the function of improving androgenetic alopecia, and its applications. This invention provides *Lactiplantibacillus plantarum* ZJUIDS24, with accession number CGMCC No. 33081. This invention also provides the uses of the above-mentioned *Lactiplantibacillus plantarum* ZJUIDS24: improving androgenetic alopecia and treating androgenetic alopecia.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology technology, specifically relating to Lactobacillus plantarum ZJUIDS24, which has the function of improving androgenetic alopecia and its application. Background Technology

[0002] In contemporary society, hair plays a vital role in protecting the skin from harmful environmental factors, maintaining body temperature, and promoting individual personality. Androgenetic alopecia (AGA) can affect both men and women. The psychological and emotional distress caused by AGA can negatively impact the overall quality of life of affected individuals. The underlying causes of AGA are genetic and hormonal, with dihydrotestosterone (DHT) being the male hormone associated with the condition. DHT is a testosterone derivative formed by 5α-reductase, which binds to androgen receptors in hair follicles, leading to hair loss and inhibiting hair regrowth.

[0003] Minoxidil and finasteride are commonly used drugs for treating alopecia areata (AGA) and have been approved for marketing by the U.S. Food and Drug Administration (FDA). Minoxidil is reported to enhance nutrient supply by vasodilation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways, thereby increasing cell proliferation in hair follicles and inducing dermal papilla cell proliferation. In addition, finasteride reduces hair loss by inhibiting 5α-reductase. However, these drugs may have specific adverse side effects, including itching, erythema, male sexual dysfunction, female infertility, and allergic contact dermatitis, especially with long-term use. Therefore, there is a need to develop non-toxic, low-side-effect, and effective natural materials to improve hair loss during AGA treatment.

[0004] In recent years, the use of probiotics to treat AGA has become a research hotspot. Numerous studies have shown that probiotics regulate the gut microbiota, thereby reducing inflammation, oxidative stress, and intestinal nutrient metabolism, playing a crucial role in the prevention or treatment of AGA. Compared with ordinary drugs, probiotics have advantages such as high safety, no side effects, no drug resistance, and low cost. *Lactobacillus plantarum*, as a typical probiotic, possesses various physiological functions beneficial to human health based on its antioxidant and immunomodulatory effects, such as anti-inflammatory, anti-tumor, and anti-atherosclerotic effects.

[0005] Invention CN202410554959.3, entitled "A Lactobacillus plantarum that helps prevent hair loss and its screening method and application," discloses Lactobacillus plantarum HX-LP93, and also provides a screening method for Lactobacillus plantarum HX-LP93 and freeze-dried Lactobacillus plantarum HX-LP93 powder. The Lactobacillus plantarum HX-LP93 provided by this invention can metabolize tryptophan in the human body, thereby regulating the body's hormone metabolism levels, and can reduce the content of dihydrotestosterone in the epidermis.

[0006] The invention CN202311564505.6, entitled "An epigenetic agent prepared from Lactiplantibacillus plantarum (CCFM1351) based on multi-target anti-hair loss and hair care effects," discloses an epigenetic agent prepared from Lactiplantibacillus plantarum (CCFM1351) based on multi-target anti-hair loss and hair care effects. It belongs to the fields of microbial technology and pharmaceutical technology, and the strain's preservation number is GDMCC No: 63921. The epigenetic agent prepared from Lactiplantibacillus plantarum (CCFM1351) provided by this invention has the effect of regulating hair follicle development and inhibiting apoptosis. It can inhibit the expression of BAX, TGF-β2, and DKK-1 in dermal papillary cells (HDPCs) of hair follicles and promote the expression of VEGF in HDPCs.

[0007] The invention CN117625460A, entitled "A strain of Lactiplantibacillus plantarum and its application as an anti-hair loss and hair care post-genetic agent and synergistic effect of transformed Platycladus orientalis leaf", discloses that Lactiplantibacillus plantarum (GDMCC No. 63923) can promote hair growth in mice. Gavage administration of Lactiplantibacillus plantarum and Platycladus orientalis leaf extract together can enhance the hair growth promoting effect of Platycladus orientalis leaf on mice.

[0008] The invention CN118975670A, "Application of Lactobacillus plantarum FTCM001 and its products in hair growth and darkening", discloses a Lactobacillus plantarum FTCM001 (accession number CGMCC No. 26813) with hair growth and darkening effects; this strain can activate tyrosinase activity, promote hair growth in mice, and has the effect of hair growth and darkening.

[0009] However, there are still very few strains that can improve the shortened growth phase of AGA hair follicles, thereby alleviating or treating AGA. Summary of the Invention

[0010] The problem to be solved by the present invention is to provide *Lactobacillus plantarum* ZJUIDS24, which has the effect of improving androgenetic alopecia, and its application.

[0011] To address the aforementioned problems, this invention provides a plant lactobacillus (Lactiplantibacillus plantarum) ZJUIDS24, with accession number CGMCC No.33081.

[0012] The present invention also provides the use of the above-mentioned Lactiplantibacillus plantarum ZJUIDS24: to improve androgenetic alopecia and treat androgenetic alopecia.

[0013] That is, its application in the preparation of drugs to improve androgenetic alopecia and treat androgenetic hair loss.

[0014] This invention also provides the application of the above-mentioned Lactiplantibacillus plantarum ZJUIDS24 in the preparation of drugs for the prevention or adjunctive treatment of hair loss.

[0015] As an improvement to the use / application of the present invention, at least one of the following is possible:

[0016] Increase the area for hair growth.

[0017] Increase hair length

[0018] Delaying the entry of hair follicles into the regression phase,

[0019] It alleviates the phenomena of shortened growth period, thinning skin, reduced hairball size, and reduced HF length.

[0020] As a further improvement to the use / application of the present invention: for androgenetic alopecia caused by dihydrotestosterone (DHT).

[0021] The Lactiplantibacillus plantarum ZJUIDS24 of the present invention also has the following uses:

[0022] Prepare foods, health products, or dietary supplements for the prevention or adjunctive treatment of hair loss and related diseases;

[0023] Prepare anti-hair loss drugs or cosmetics.

[0024] The preservation information of strain ZJUIDS24 of this invention is as follows:

[0025] The deposit name is Lactiplantibacillus plantarum, the depositary institution is China General Microbiological Culture Collection Center (CGMCC), the depositary address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC NO.33081, and the deposit date is December 16, 2024.

[0026] This invention screened a strain of Lactiplantibacillus plantarum (ZJUIDS24) from the traditional yogurt "Tarige" in Inner Mongolia. The strain was identified through bacterial morphology, physiology, and culture characteristics, combined with 16S rDNA sequencing.

[0027] The screening method is as follows: Fresh Inner Mongolian yogurt is collected in sterile tubes and cultured in MRS broth medium for 48 hours. Colonies are picked from the MRS medium and streaked for purification until the colony morphology on the entire plate is uniform. Single colonies are picked and cultured in MRS broth medium to obtain the plant lactobacillus ZJUIDS24.

[0028] The *Lactiplantibacillus plantarum* ZJUIDS24 provided by this invention has a strong therapeutic effect on androgenetic alopecia. Compared with existing *Lactiplantibacillus* strains, the *Lactiplantibacillus plantarum* ZJUIDS24 of this invention has the following advantages: In vivo animal experiments have demonstrated that the strain of this invention also has a strong effect on improving androgenetic alopecia in vivo, including delaying the entry of hair follicles into the regression phase and promoting hair growth. In addition, it also has strong antioxidant capacity and the ability to inhibit pathogens.

[0029] This invention also provides the application of *Lactiplantibacillus plantarum* ZJUIDS24 in the preparation of foods that alleviate androgenetic alopecia. Examples of foods that alleviate androgenetic alopecia include fermented yogurt, fruit and vegetable juices, bacterial powders, and solid beverages.

[0030] This invention can provide excellent strain resources for the development of native probiotics in my country. Attached Figure Description

[0031] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0032] Figure 1 This is a colony morphology diagram of ZJUIDS24.

[0033] Figure 2 The image shows the morphology of bacteria stained with ZJUIDS24 Gram stain.

[0034] Figure 3 This is a gross figure showing the effect of ZJUIDS24 on hair coverage area in androgen-induced alopecia mice.

[0035] Figure 4 This is a statistical graph showing the effect of ZJUIDS24 on hair coverage area in androgen-induced alopecia mice.

[0036] Figure 5 The effect of ZJUIDS24 on hair length in androgenetic alopecia mice.

[0037] Figure 6 This is a slice image showing the effect of ZJUIDS24 on hair follicles on the back of mice with androgenetic alopecia.

[0038] Figure 7 The effect of ZJUIDS24 on the thickness of subcutaneous fat layer in androgenetic alopecia mice.

[0039] Figure 8 The effect of ZJUIDS24 on hair follicle length (≥30 HFs) in androgenetic alopecia mice.

[0040] Figure 9 The effect of ZJUIDS24 on the diameter of hair bulbs (≥30 HFs) at the base of hair follicles in mice with androgenetic alopecia. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0042] Example 1: Screening and identification of Lactobacillus plantarum ZJUIDS24:

[0043] 1. Screening of Lactobacillus plantarum ZJUIDS24

[0044] 1.1 Sample Source

[0045] The strain used in this invention was isolated from traditional yogurt (a product called Tarig) from Inner Mongolia, and a total of 10 samples were collected.

[0046] 1.2 Isolation and purification of strains

[0047] Approximately 15g of fresh sample was collected in a sterile tube and immediately sent to the laboratory for bacterial isolation. 1g of sample was placed in 9mL of MRS broth, vortexed, and incubated at 37°C for 48h for enrichment. Then, 1mL of the enrichment solution was extracted in a laminar flow hood and serially diluted tenfold with sterile physiological saline. 10... -6 10 -7 10 -8Three dilution gradients were used, with 100 μL of bacterial culture from each gradient plated onto MRS agar medium and incubated at 37°C for 48 h. After incubation, plates with 30-300 single colonies were selected from the agar medium. Typical colonies (spherical, 0.3-1.5 mm in diameter, smooth surface, and a strong sour milk odor) were picked and streaked multiple times on MRS agar plates for purification until the colonies on the entire plate had a uniform morphology. Single colonies were then picked and enriched in MRS broth medium. All obtained strains were stored frozen at -80°C in MRS broth medium containing 40% glycerol.

[0048] 2. Identification of Lactobacillus plantarum ZJUIDS24

[0049] 2.1 Colony characteristics

[0050] After culturing the isolated and purified *Lactobacillus plantarum* ZJUIDS24 on MRS agar medium for 48 hours, the colonies were round, with a diameter between 0.3 and 1.5 mm, smooth surface, and a strong sour milk odor. Figure 1 .

[0051] 2.2 Microscopic morphology:

[0052] Lactobacillus plantarum ZJUIDS24 colony smear: Gram-positive, non-spore-forming, round-terminated straight bacilli, single, paired, or in short chains, see Figure 2 .

[0053] 2.3 16S rDNA Identification

[0054] Genomic DNA of the target strain was extracted using the Ezup column-based bacterial genomic DNA extraction kit. The extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification. PCR experiments on 16S rDNA were performed using universal bacterial primers 27F and 1492R. After the PCR reaction was completed, the PCR product was sent to BGI Genomics Co., Ltd. for sequencing, and BLAST sequence alignment was performed on the NCBI website. The results showed that the sequence had more than 99% homology with the identified 16S rDNA sequence of *Lactobacillus plantarum*.

[0055] By combining the sequence alignment results and physiological and biochemical results of strain ZJUIDS24, the selected lactic acid bacterium ZJUIDS24 was identified as Lactiplantibacillus plantarum ZJUIDS24.

[0056] The preservation information of strain ZJUIDS24 of this invention is as follows:

[0057] The deposit name is Lactiplantibacillus plantarum, the depositary institution is China General Microbiological Culture Collection Center (CGMCC), the depositary address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC NO.33081, and the deposit date is December 16, 2024.

[0058] Example 2: Lactobacillus plantarum ZJUIDS24 improves androgenetic alopecia

[0059] 1. Establishment of a mouse model of androgenetic alopecia

[0060] Six-week-old male C57BL / 6 mice were randomly divided into four groups of 10 mice each: a normal group, a model group (DHT), a *Lactobacillus plantarum* ZJUIDS24 group (DHT + ZJUIDS04), and a positive control group (DHT + minoxidil). Mice were routinely acclimatized for one week before the formal experiment, which lasted from day 0 to day 29. Three days before the start of the experimental period, DHT injections were initiated to establish the model (5 mg / ml DHT-corn oil solution, 10 μL / g body weight, injected intraperitoneally daily). On day 0 (the start of the experimental period), 3*3.5 cm of hair was removed from the backs of the mice using depilatory cream and an electric shaver. Mice in the normal control group received normal food and water throughout the experimental period (0-29 days) and were administered 10 μL / g body weight of phosphate-buffered saline (PBS, pH 7.2-7.4) daily via gavage as a control. The model group, the *Lactobacillus plantarum* ZJUIDS24 group, and the positive control group received intraperitoneal injections of 10 μL / g body weight of 5 mg / ml DHT every 2 days, with an interval of 1 day between injections. Mice in the model group received 10 μL / g body weight of PBS daily via gavage throughout the experiment as a control, while the *Lactobacillus plantarum* ZJUIDS24 group received 1 × 10⁻⁶ live bacteria daily via gavage. 9 CFU / mL bacterial suspension; the positive control group was treated daily with 10 μL / g body weight of 5% minoxidil solution. All other housing conditions, such as temperature, humidity, light, and feed, were consistent. Animal welfare and experimental procedures were conducted in accordance with the guidelines of Zhejiang University's Laboratory Animal Management Regulations. The grouping and treatment methods for experimental animals are shown in Table 1.

[0061] The number of viable bacteria is 1×10 9The preparation method of CFU / mL bacterial culture (fermentation culture) is as follows: Take the glycerol tube of the preserved strain ZJUIDS24, pick out the strain using a disposable inoculation loop, streak it on MRS solid medium, activate it for three generations, take a single colony and inoculate it into 30 mL of MRS liquid medium. Incubate at 37℃ for 18 h, then remove it and centrifuge at 8000g for 15 min at 4℃. Discard the supernatant and retain the bacterial cells. Add PBS (pH 7.2-7.4), vortex well, and centrifuge at 8000g for 15 min at 4℃. Repeat this washing process twice. Finally, adjust the bacterial concentration to 1×10⁻⁶ using PBS based on the OD value. 9 CFU / mL.

[0062] Table 1 Animal Experiment Design

[0063]

[0064]

[0065] 2. The effect of Lactobacillus plantarum ZJUIDS24 on improving hair growth area in mice.

[0066] During the experiment, when stubble grew back on the backs of the mice, macroscopic images were taken for observation and recording. After anesthetizing the mice with isoflurane, they were placed back-side up on an A4 sheet of paper with their limbs outstretched. A ruler was placed on the paper, and a camera was used to take pictures. The observation and photography positions were kept consistent for each mouse. After the hair removal model was established and stubble appeared, the mice were observed and photographed every four days to continuously track the color of the back skin and the extent of hair coverage.

[0067] Experimental results are as follows Figure 3 As shown, on day 11, the positive group, the ZJUIDS24 treatment group, and the normal group all partially turned black, while the skin on the backs of the model group mice still showed a large area of ​​pink. From the gross images on days 15 and 19, it can be seen that the hair growth in the ZJUIDS24 treatment group was rapid, while that in the model group was relatively slow. By day 23, from the gross images, it can be seen that the hair coverage area in the ZJUIDS24 treatment group was similar to that in the normal group and the positive group, with the backs of the mice almost completely covered, while the coverage area in the model group was smaller.

[0068] The hair coverage area on the backs of mice on days 19 and 23 was statistically analyzed using ImageJ. Figure 4It was observed that on day 19, the hair coverage area in the ZJUIDS24 treatment group was significantly higher than that in the model group (P < 0.05). On day 23, the hair coverage area in the ZJUIDS24 treatment group remained significantly higher than that in the model group (P < 0.05), and there was no significant difference compared to the normal group and the minoxidil group (P > 0.05). Therefore, intervention with *Lactobacillus plantarum* ZJUIDS24 improved hair growth in androgenic alopecia mice and significantly increased their hair growth area.

[0069] 3. The effect of Lactobacillus plantarum ZJUIDS24 on improving hair length in mice.

[0070] On days 19 and 24, a small tuft of hair was randomly plucked from five locations on the back of the mouse using flat-tipped tweezers perpendicular to the skin. After being thoroughly mixed in a plastic bag, five hairs were selected from each mouse. The hairs were laid flat on an A4 sheet of paper with tweezers, a measuring ruler was placed, and a single hair was photographed and recorded. The hair length was measured using calipers under sufficient light.

[0071] The results are as follows Figure 5 As shown, on days 19 and 24, the hair length of mice in the *Lactobacillus plantarum* ZJUIDS24 treatment group was significantly higher than that in the model group (P < 0.05), and there was no significant difference compared with the normal group and the positive group (P > 0.05). Therefore, *Lactobacillus plantarum* ZJUIDS24 intervention improved hair length in mice with androgenetic alopecia, and the effect was comparable to that in the positive group treated with minoxidil.

[0072] 4. Lactobacillus plantarum ZJUIDS24 reduced the shortened growth period, thinner skin, smaller hairball size, and shorter hairline length in AGA mice.

[0073] Mouse hair follicles exhibit a cyclical transition from the anagen phase to the catagen phase to the telogen phase. On days 10, 24, and 29, the following procedures were performed: skin tissue was obtained from the same area on the back of mice in the four experimental groups, placed in 4% PFA solution, fixed at 4°C overnight, embedded in paraffin, sectioned at 5 μm intervals, and then stained with hematoxylin and eosin (HE).

[0074] The test results are as follows Figure 6Representative section results showed that on day 10, hair follicles in the normal group entered the anagen phase first, while those in the minoxidil and ZIUIDS24 groups were in the telogen or anagen phase I / II, and those in the model group were in the telogen phase. By day 24, the normal group had entered the telogen phase, the anagen phase of the model and minoxidil groups had ended, while the ZIUIDS24 group remained in the anagen phase. It wasn't until day 29, when the model and minoxidil groups had entered the late catagen or telogen phase, that the ZIUIDS24 group began to enter the catagen phase. The section results indicate that *Lactobacillus plantarum* ZIUIDS24 alleviated the shortened anagen phase in AGA mice.

[0075] Subcutaneous fat layer thickness, hair follicle length, and hair bulb diameter are all important indicators for judging the growth cycle. When a hair follicle enters the anagen (growth) phase, the subcutaneous fat layer thickness, hair follicle length, and hair bulb diameter continuously increase, gradually thinning as it transitions to the catagen (regression) and telogen (resting) phases. Figure 7 , 8 As shown in Figures 9 and 1, at all three sampling time points, the three indicators in the ZJUIDS24 group were significantly higher than those in the model group, and significantly higher than those in the positive control group at days 24 and 29. These results indicate that *Lactobacillus plantarum* ZJUIDS24 alleviated the thinning of skin, reduction in hairball size, and reduction in HF length in AGA mice.

[0076] 5. Comparison

[0077] A comparative study was conducted based on relevant literature, and the results are shown in Table 2 below. *Lactobacillus plantarum* CCFM1351, CCFM1353, and FTCM001 all demonstrated different hair growth-promoting effects, including promoting the expression of hair-related genes, promoting epithelial cell proliferation, and increasing melanin synthesis; however, they did not show any therapeutic effect on androgenetic alopecia. *Lactobacillus plantarum* HX-LP931 showed an effect in improving androgenetic alopecia, specifically by reducing scalp oil content, promoting hair growth, and increasing hair weight, but it did not show any improvement effect on the hair growth cycle.

[0078] Androgenetic alopecia (AGA) patients have a short hair growth phase, rapidly entering the regression phase, leading to hair loss. Prolonging the hair follicle growth phase is crucial for AGA patients. However, the existing *Lactobacillus plantarum* strains mentioned above do not possess this property.

[0079] The *Lactobacillus plantarum* ZJUIDS24 of this invention can increase hair area and hair length, prolong the hair follicle growth phase, and has excellent effects in improving androgenetic alopecia and treating androgenetic alopecia.

[0080] Table 2 Comparison of experimental effects of Lactobacillus plantarum on improving hair loss

[0081]

[0082]

[0083] Example 3: Confirmation of the antioxidant capacity of Lactobacillus plantarum ZJUIDS24

[0084] 1. Sample preparation:

[0085] The ZJUIDS24 strain, preserved in glycerol tubes, was streaked onto MRS solid medium and incubated upside down at 37°C for 48 hours. A single colony was picked with an inoculation loop and inoculated into 30 mL of sterile MRS liquid medium, and incubated statically at 37°C for 18-24 hours to obtain the culture medium. The culture medium was adjusted to a lactic acid bacteria concentration of 10% with distilled water. 10 Centrifuge at 4℃, 8000×g for 20 min, and collect the supernatant as the fermentation supernatant. Resuspend the centrifuged bacterial pellet in 0.02M PBS buffer (pH=7.4), wash, and centrifuge at 4℃, 8000×g for 20 min, repeating 3 times. Resuspend the washed bacterial cells in PBS buffer and adjust the bacterial concentration to 10. 10 CFU / mL yields the bacterial suspension.

[0086] The supernatant and bacterial cells of Lactobacillus plantarum ZJUIDS24 were used as sample solutions, respectively.

[0087] 2. DPPH removal rate

[0088] Mix 1 mL of sample solution with 1 mL of 0.2 mM DPPH anhydrous ethanol solution by shaking for 1 min, and react at room temperature in the dark for 30 min. Centrifuge and take the supernatant to measure the absorbance at 517 nm. Use 95% ethanol instead of DPPH as a control and PBS (pH 7.2-7.4) instead of sample solution as a blank.

[0089] DPPH clearance rate = 1 - (Ai - Aj) / Ac

[0090] Explanation: Ai: Absorbance of sample solution + DPPH; Aj: Absorbance of sample solution + 95% ethanol; Ac: Absorbance of PBS + DPPH.

[0091] The results are shown in Table 3 below.

[0092] 3-hydroxy radical scavenging rate

[0093] 1) Take 1 mL of o-phenanthroline (2.5 mmol / L), add 1 mL of phosphate buffer (PBS concentration of 0.02 mol / L, pH = 7.4) and 1 mL of distilled water in sequence, mix thoroughly, add 1 mL of ferrous sulfate (FeSO4 concentration of 2.5 mmol / L), mix well, add 1 mL of hydrogen peroxide (H2O2 mass fraction of 20 mmol / L), and measure its absorbance at 536 nm after incubating in a water bath at 37℃ for 1.5 h.

[0094] 2) Replace 1 mL of hydrogen peroxide in step 1) with 1 mL of distilled water, and keep the rest the same. The absorbance measured is Ab.

[0095] 3) Replace 1 mL of distilled water in step 1) with 1 mL of sample, and keep the rest the same. The absorbance is measured as As.

[0096] Hydroxyl radical scavenging rate = (As-Ap) / (Ab-Ap)

[0097] The results are shown in Table 3 below.

[0098] 4. Restoration ability

[0099] Experimental group: A reaction raw material solution was prepared by mixing 0.5 mL of sample solution, 0.5 mL of potassium ferricyanide (1% by mass), and 0.5 mL of phosphate buffer (PBS concentration of 0.2 mol / L, pH=6.6). The reaction raw material solution was incubated in a water bath at 50℃ for 20 min. After the reaction, it was cooled to room temperature and 0.5 mL of trichloroacetic acid (TAC mass fraction of 10%) was added to precipitate the protein. After centrifugation, 1 mL of the supernatant was taken and reacted with 1 mL of ferric chloride (FeCl3 mass fraction of 0.1%). The absorbance was measured at 700 nm.

[0100] Blank control: Replace the sample solution in the experimental group with PBS or MRS liquid culture medium, otherwise the same as the experimental group.

[0101] Reduction ability = (As - Ab) / Ab

[0102] As: Absorbance of the experimental group; Ab: Absorbance of the blank group

[0103] The results are shown in Table 3 below.

[0104] Table 3 Antioxidant activity of Lactobacillus plantarum ZJUIDS24

[0105]

[0106]

[0107] As shown in Table 3, the *Lactobacillus plantarum* ZJUIDS24 cells screened in this invention exhibit high reducing power and hydroxyl radical scavenging ability, while the fermentation supernatant shows even higher DPPH scavenging ability. Overall, this strain and its metabolites possess good antioxidant capacity.

[0108] Example 4: Confirmation of the inhibitory ability of *Lactobacillus plantarum* ZJUIDS24 against pathogens.

[0109] The antibacterial activity of lactic acid bacteria was determined using the internationally accepted agar diffusion method. Four frozen indicator strains (Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes) were activated 2–3 times on LB agar. Single colonies of each activated strain were picked and incubated in LB agar at 37°C for 18 h. Bacterial cells were collected by centrifugation and resuspended in PBS buffer (pH 7.2–7.4) to achieve a concentration of 10⁻⁶. 8 CFU / mL. The indicator bacterial suspension was added at 1% (v / v) to sterilized LB solid medium cooled to approximately 55°C. After thorough mixing, the mixture was poured into petri dishes (15 mL / dish), and the pre-placed sterile Oxford cups were removed after cooling. Activated *Lactobacillus plantarum* ZJUIDS24 was inoculated into MRS medium at 1% (v / v). After 18 h of culture, the supernatant was collected by centrifugation (8000 rpm, 5 min, 4°C), and the bacterial precipitate was discarded. The fermentation supernatant was added to the wells (200 μL / well), with uninoculated MRS medium (pH 6.2) used as a blank control. The culture was incubated at 37°C, and the diameter of the inhibition zone was measured after 24 h. Strains with a clear inhibition zone around the well were selected, and the diameter of the inhibition zone was measured, with each measurement repeated three times.

[0110] As shown in Table 4, the metabolites of *Lactobacillus plantarum* ZJUIDS24 exhibited inhibitory effects against *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, and *Listeria monocytogenes*. This demonstrates that the metabolites of this bacterium possess antibacterial properties.

[0111] Table 4. Antibacterial activity of the strains against four common pathogenic bacteria.

[0112]

[0113] Example 5: Confirmation of the acid and bile salt resistance of *Lactobacillus plantarum* ZJUIDS24

[0114] 1. Acid resistance test

[0115] Single colonies of *Lactobacillus plantarum* ZJUIDS24 were picked and cultured in MRS liquid medium at 37°C for 18 h. The expanded bacterial suspension was then inoculated into MRS liquid medium at a rate of 1% and cultured at 37°C for another 18 h. The culture was then centrifuged at 8000 rpm for 5 min at 4°C to collect the cells. The cells were washed twice with PBS buffer (pH 7.2-7.4). The cells were then resuspended in MRS liquid medium pre-adjusted to pH 3.0, and the initial viable count was corrected to approximately 10⁻⁶. 8 CFU / mL, incubated at 37℃ for 3 h. Viable bacteria in the 0 h and 3 h samples were counted using the pour plate method. After pouring, the plates were incubated at 37℃ for 48 h, and viable bacteria were counted again to determine the survival rate. The survival rate was calculated using the following formula:

[0116]

[0117] In the above formula, N0 represents the viable count (CFU / mL) of the test strain at 0h; N t The number of viable bacteria (CFU / mL) of the test strain after 3 hours.

[0118] The results are shown in Table 5 below.

[0119] 2. Bile salt tolerance test

[0120] The activated and expanded *Lactobacillus plantarum* ZJUIDS24 bacterial suspension was inoculated into MRS liquid medium at a rate of 1%, incubated at 37°C for 18 hours, vortexed to mix, and the initial viable count was corrected to approximately 10⁻⁶. 9 CFU / mL. Bacterial cells were suspended in MRS liquid medium containing 0.3% (m / v) ox bile salts, and the initial viable count was corrected to approximately 10⁸ CFU / mL. The culture was then incubated at 37°C for 3 h. Viable bacteria in the 0 h and 3 h samples were counted using the pour plate method. The poured plates were then incubated at 37°C for 48 h, and the viability was determined using the following formula:

[0121]

[0122] In the above formula, N0 represents the viable count (CFU / mL) of the test strain at 0h; Nt represents the viable count (CFU / mL) of the test strain at 3h. The results are shown in Table 5 below.

[0123] 3. As shown in Table 5, *Lactobacillus plantarum* ZJUIDS24 exhibits high acid and bile salt tolerance (achieving growth within 3 hours), with a survival rate as high as 131 ± 0.34% in MRS medium at pH 3.0. Its survival rate in an environment containing 0.3% ox bile salts is as high as 136 ± 0.42%, indicating excellent bile salt tolerance. Experiments demonstrate that *Lactobacillus plantarum* ZJUIDS24 possesses high gastrointestinal survival ability.

[0124] Table 5 Results of strains' tolerance to acid and bile salts

[0125] strain pH3 survival rate (%) 0.3% bile salt survival rate (%) Lactobacillus plantarum ZJUIDS24 131±0.34 136±0.42

[0126] Probiotics must be able to survive in the adverse environment of the gastrointestinal tract, including stomach acid and bile, in order to exert their beneficial effects. The *Lactobacillus plantarum* ZJUIDS24 provided by this invention can grow and proliferate under conditions of pH 3.0 and 0.3% bovine bile salts. It can successfully pass through the acidic environment of the stomach to reach the small intestine, where it can survive and effectively improve the intestinal flora, thus exerting its beneficial effects.

[0127] Example 6: Confirmation of antibiotic susceptibility of Lactobacillus plantarum ZJUIDS24

[0128] According to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI), the disk diffusion method was used to determine the antibiotic susceptibility of bacterial strains. After centrifugation and washing of the bacterial cells, the concentration of the bacterial suspension was controlled to reach 10. 8 CFU / mL. Spread 100 μL of bacterial suspension evenly onto MRS solid medium. Then, using sterile forceps, gently press the antibiotic disc onto the plate surface and incubate at 37°C for 24 hours. Measure and record the diameter of the inhibition zone using calipers. The antibiotic susceptibility of the strain is determined based on the diameter of the inhibition zone. According to the CLSI Standards for Antimicrobial Susceptibility Testing (17th Edition), antibiotic resistance of lactic acid bacteria is determined, divided into three levels: sensitive (S), intermediate (I), and resistant (R).

[0129] The diameters of the inhibition zones of *Lactobacillus plantarum* ZJUIDS24 to antibiotic susceptibility are shown in Table 6. It exhibits sensitivity to penicillin, ampicillin, ceftriaxone, cefazolin, chloramphenicol, cefoperazone, tetracycline, and erythromycin, but is resistant only to gentamicin. The experimental results indicate that *Lactobacillus plantarum* ZJUIDS24 is sensitive to common antibiotics and has a high safety profile.

[0130] Table 6. Antibiotic susceptibility results of Lactobacillus plantarum ZJUIDS24

[0131]

[0132] Example 7: Preparation of fermented yogurt with the effect of alleviating androgenetic alopecia using Lactobacillus plantarum ZJUIDS24 1. Yogurt processing flow:

[0133] Raw materials are preheated, homogenized, mixed, sterilized, cooled, prepared, inoculated, fermented, matured, and refrigerated.

[0134] 2. Key Operating Points

[0135] (1) Raw materials: 2L of whole milk UHT sterilized milk or fresh milk;

[0136] (2) Preheating: Place in a container and heat to 63°C;

[0137] (3) Homogenization: Pour into a homogenizer for homogenization (pressure 15-25MPa), pour the mixture into an iron can, add 100g of white sugar, and sterilize in a 90℃ water bath for 10min.

[0138] (4) Preparation: Add the ingredients to the milk and dissolve them;

[0139] (5) Sterilization: Sterilize sweetened milk in a 90℃ water bath for 10 minutes;

[0140] (6) Cooling: After sterilization, cool the milk to 40-50℃ for later use;

[0141] (7) Preparation of starter culture: Under aseptic conditions, *Lactobacillus plantarum* ZJUIDS24 strain was inoculated into test tubes containing sterilized skim milk (12%, w / v) and cultured at 37°C for 20 hours. The inoculum amount for each subculture was 2-4% (v / v), and the culture was subcultured 2-3 times to restore viability. The culture was then stored in a refrigerator at 4°C.

[0142] (8) Inoculation and fermentation: Under aseptic conditions, inoculate with activated Lactobacillus plantarum ZJUIDS24 at an inoculation rate of 2-4% (v / v). Ferment at a constant temperature of 42℃ for 6-10 hours.

[0143] (9) Post-fermentation: After fermentation, place it in a 4℃ refrigerator for 12-24 hours for post-fermentation.

[0144] (10) Filling and refrigeration: After the post-ripening is completed, the contents are filled into 250mL sterile glass bottles and sent to the cold storage for refrigeration.

[0145] Example 8: Preparation of fermented fruit and vegetable juice with the effect of alleviating androgenetic alopecia using Lactobacillus plantarum ZJUIDS24 1. Processing flow of fermented fruit and vegetable juice

[0146] Raw material cleaning, flash evaporation, pulping, blending, homogenization, sterilization, cooling, inoculation, sealed fermentation, post-fermentation, ripening, filling, and refrigeration.

[0147] 2. Key Operating Points

[0148] (1) Ingredients: Select fresh pumpkin and apple.

[0149] (2) Wash and cut into pieces: Wash, peel (remove the seeds from the pumpkin), and cut into small pieces.

[0150] (3) Flash evaporation: The enzyme is inactivated by flash evaporation for 0.5 to 1 min at 121°C and the gas is exhausted quickly.

[0151] (4) Pulping: According to the ratio of pumpkin to water (by weight) = 1:1, gradually put the pumpkin and water into the colloid mill and grind them, performing coarse grinding and fine grinding once each. After removing the core from the apple, use a pulper to pulp until the pulp is uniform and free of lumps.

[0152] (5) Mixing and homogenization: Mix 15% pumpkin juice and 30% apple juice, then adjust the soluble solids content to 10°Brix with sucrose, add 0.2% of stabilizer CMC and mix evenly. Use a two-stage homogenization method, first low pressure (15MPa) and then high pressure (25MPa) to make the diameter of the melon pulp particles 2-3μm.

[0153] (6) Sterilization and cooling: The prepared compound fruit and vegetable juice is kept at 100℃ for 10 minutes and then cooled to about 40℃.

[0154] (7) Inoculation and fermentation: Under aseptic conditions, inoculate with activated Lactobacillus plantarum ZJUIDS24, with the initial bacterial count controlled at 10. 7 CFU / mL. Fermented at 37℃ for 24 hours.

[0155] (8) Post-fermentation: After fermentation, place in a 4℃ refrigerator for 3 hours.

[0156] (9) Filling and refrigeration: After the post-ripening is completed, the contents are filled into 250mL sterilized glass bottles and sent to the cold storage for refrigeration.

[0157] Example 9: Preparation of a bacterial powder with the effect of alleviating androgenetic alopecia using *Lactobacillus plantarum* ZJUIDS24 1. Preparation of *Lactobacillus plantarum* ZJUIDS24 bacterial mud

[0158] Single colonies of *Lactobacillus plantarum* ZJUIDS24 were inoculated into 50 mL of MRS liquid medium and incubated at 37°C for 18 h. The colonies were then activated again by inoculating 5% of the colonies into 250 mL of MRS liquid medium and incubated at 37°C for 24 h. Finally, the activated *Lactobacillus plantarum* ZJUIDS24 was inoculated at 5% of the colonies in a 10 L fermenter for high-density anaerobic culture at 37°C and pH 6.8 for 18 h. Afterward, the colonies were centrifuged at 8000 rpm and 4°C for 15 min, the supernatant was discarded, and the bacterial precipitate was collected. The precipitate was washed twice with sterile phosphate buffer (pH 7.0). This yielded *Lactobacillus plantarum* ZJUIDS24 bacterial sludge.

[0159] 2. Preparation of Protective Agent

[0160] The freeze-drying protectant contains 15% skim milk powder, 5% trehalose, 3% monosodium glutamate, 1% glycerol, and 0.5% cysteine ​​hydrochloride; water is used as the solvent; it is sterilized at 110°C for later use.

[0161] 3. Preparation of Lactobacillus plantarum ZJUIDS24 bacterial powder

[0162] The *Lactobacillus plantarum* ZJUIDS24 bacterial precipitate prepared above was thoroughly mixed with a cryoprotectant solution at a ratio of 1:5. The mixture was pre-frozen at -40℃ for 5 hours to ensure uniform freezing onto the inner wall of the container. Then, it was freeze-dried under vacuum for 18–20 hours to obtain *Lactobacillus plantarum* ZJUIDS24 bacterial powder. After rehydration with physiological saline and washing twice, the viable count of *Lactobacillus plantarum* ZJUIDS24 bacterial powder was determined to be 1.0 × 10⁻⁶. 11 ~1×10 12 CFU / g.

[0163] Example 10: Preparation of probiotic milk powder using Lactobacillus plantarum ZJUIDS24

[0164] 1. Preparation of Lactobacillus plantarum ZJUIDS24 bacterial powder

[0165] The *Lactobacillus plantarum* ZJUIDS24 lyophilized bacterial powder was prepared according to Example 9, with a viable count of 1.0 × 10⁻⁶ cells. 11 ~1×10 12 CFU / g.

[0166] 2. Preparation of infant formula

[0167] Initial raw material selection: milk powder, protein powder, saccharides, vegetable oil; additives: vitamins, trace elements, functional factors, others;

[0168] Automatic batching: The raw materials obtained according to the formula are put into the material silo;

[0169] Crushing: Crushing the weighed material through a crusher;

[0170] Mixing: Add vegetable oil and trace elements to the pulverized materials and mix them evenly in a mixer;

[0171] Extrusion: Well-mixed materials are processed into granular materials through an extrusion machine;

[0172] Drying: The mixed materials are dried in a dryer at a temperature controlled at 65-70℃;

[0173] Grading and screening: The material is passed through a grading sieve, and the particle size is controlled to be 2.5-5 mm.

[0174] 3. Preparation of probiotic formula powder

[0175] The bacterial powder prepared in step 1 and the formula milk powder prepared in step 2 are mixed evenly at a ratio of 1:100. The final product contains live bacteria at a concentration of 10... 8CFU / g or higher. After filling, the product is stored in a warehouse for sale.

[0176] Example 11: Preparation of solid beverage using Lactobacillus plantarum ZJUIDS24 bacterial powder which has the effect of preventing androgenetic alopecia. 1. Preparation of Lactobacillus plantarum ZJUIDS24 bacterial powder: Lactobacillus plantarum ZJUIDS24 bacterial powder was prepared according to Example 9.

[0177] 2. The solid beverage formula is as follows: edible corn starch, white sugar (10%), fructooligosaccharides (6%), isomaltooligosaccharides (9%), citric acid, edible flavoring, and starter culture powder (2-5%);

[0178] 3. Mixing and Packaging: After adding according to the formula, mix evenly and package into 10 / 20g packets per small bag. Pack 10-20 small bags into one large bag.

[0179] Example 12: Preparation of hair growth tablets using Lactobacillus plantarum ZJUIDS24 bacterial powder which has the effect of preventing androgenetic alopecia. 1. Preparation of Lactobacillus plantarum ZJUIDS24 bacterial powder: Lactobacillus plantarum ZJUIDS24 bacterial powder was prepared according to Example 9.

[0180] 2. The Chinese medicine formula is as follows: It uses herbal extracts such as Polygonum multiflorum, ginseng, angelica, arborvitae leaves, and starch, which are mixed evenly before use.

[0181] 3. Mixing and Packaging: After adding according to the formula, mix well and package into 10 / 20g packets, each packet containing 10 live bacteria. 9 above.

[0182] 4. Tableting: The mixture is compressed into tablets using a tableting machine and then packaged.

[0183] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Lactobacillus plantarum ( Lactiplantibacillus plantarum ZJUIDS24, characterized in that The accession number is CGMCC No. 33081.

2. The *Lactobacillus plantarum* as described in claim 1 (Lactiplantibacillus plantarum Application of ZJUIDS24 in the preparation of drugs for the prevention or adjunctive treatment of androgenetic alopecia.

3. The application according to claim 2, characterized in that... It is effective for at least one of the following: increasing hair growth area, increasing hair length, delaying the entry of hair follicles into the regression phase, and alleviating the phenomena of shortened growth phase, thinning skin, reduced hair bulb size, and reduced HF length.

4. The application according to claim 2, characterized in that: This is androgenetic alopecia caused by dihydrotestosterone.

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