Phytobacterium plantarum ZJUIDS24 capable of improving male hormone alopecia and application of phytobacterium plantarum ZJUIDS24

Abstract

The invention belongs to the technical field of food microorganisms, and in particular relates to a plant lactobacillus ZJUIDS24 with a function of improving male hormone alopecia and application of the plant lactobacillus ZJUIDS24. The invention provides a lactobacillus plantarum ZJUIDS24, and the preservation number of the lactobacillus plantarum ZJUIDS24 is CGMCC (China General Microbiological Culture Collection Center) No.33081. The lactobacillus plantarum ZJUIDS24 has the advantages that the preservation number is CGMCC No.33081; the invention also provides an application of the lactobacillus plantarum ZJUIDS24 at the same time, and the application of the lactobacillus plantarum ZJUIDS24 is used for improving the phenomenon of androgenetic alopecia and treating the androgenetic alopecia.

Description

Technical Field

[0001] The invention belongs to the technical field of food microorganisms, and in particular relates to Lactobacillus plantarum ZJUIDS24 with the function of improving androgenic alopecia and applications thereof. Background Art

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

[0003] Minoxidil and finasteride are commonly used drugs for the treatment of AGA and have been approved for marketing by the US Food and Drug Administration (FDA). Minoxidil reportedly increases cell proliferation in hair follicles by enhancing nutrient supply through vasodilation and activation of the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways, thereby inducing cell proliferation of dermal papilla cells. In addition, finasteride alleviates 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 in the case of long-term use. Therefore, it is necessary to develop natural materials that are non-toxic, have few side effects, and effectively improve hair loss during the treatment of AGA.

[0004] In recent years, the use of probiotics to treat AGA has become a research hotspot. Many studies have shown that probiotics regulate intestinal flora, thereby reducing inflammation, oxidative stress, intestinal nutrient metabolism, etc., and play a vital role in the prevention or treatment of AGA. Compared with ordinary drugs, probiotics have the advantages of high safety, no side effects, no drug resistance and low cost. As one of the typical probiotics, Lactobacillus plantarum has a variety of physiological functions that are beneficial to human health, such as anti-inflammatory, anti-tumor and anti-atherosclerosis, based on its antioxidant and immunomodulatory effects.

[0005] The invention of CN202410554959.3, "A plant lactobacillus that helps prevent hair loss, its screening method and application", discloses plant lactobacillus HX-LP93, and also provides a screening method for plant lactobacillus HX-LP93 and freeze-dried bacterial powder of plant lactobacillus HX-LP93. The plant lactobacillus HX-LP93 provided by the invention can metabolize tryptophan in the human body, thereby regulating the metabolic level of hormones in the human body, and can reduce the content of dihydrotestosterone in the epidermis.

[0006] The invention of CN202311564505.6, "A postbiotic prepared by Lactobacillus plantarum CCFM1351 that achieves anti-hair loss and hair care effects based on multiple targets", discloses a postbiotic prepared by Lactobacillus plantarum CCFM1351 (Lactiplantibacillus plantarum) that achieves anti-hair loss and hair care effects based on multiple targets, belonging to the fields of microbial technology and pharmaceutical technology, and the deposit number of the strain is GDMCC No: 63921. The postbiotic prepared by Lactobacillus plantarum CCFM1351 provided by the invention has the function of regulating hair follicle development and inhibiting its apoptosis, can inhibit the expression of BAX, TGF-β2 and DKK-1 of hair follicle dermal papilla cells (HDPC), and promote the expression of VEGF in HDPC.

[0007] The invention of CN117625460A "A strain of Lactobacillus plantarum CCFM1353 and its prepared postbiotics for preventing hair loss and protecting hair, and the application of transformed Platycladus orientalis leaves for synergistic enhancement" discloses Lactobacillus plantarum CCFM1353 (Lactiplantibacillus plantarum), with a preservation number of GDMCC No: 63923, which can promote hair growth in mice. Gavage of Lactobacillus plantarum CCFM1353 and Platycladus orientalis leaf extract together can enhance the promoting effect of Platycladus orientalis leaves on hair growth in mice.

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

[0009] However, there are still few relevant strains that can improve the problem of 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 and application thereof for improving androgenic alopecia.

[0011] In order to solve the above problems, the present invention provides a plant lactobacillus (Lactiplantibacillus plantarum) ZJUIDS24, whose preservation number is CGMCC No.33081.

[0012] The present invention also provides the use of the Lactiplantibacillus plantarum ZJUIDS24: improving the phenomenon of androgenic alopecia and treating androgenic alopecia.

[0013] That is, the application of the present invention in the preparation of drugs for improving the phenomenon of androgenic alopecia and treating androgenic alopecia.

[0014] The present invention also provides application of the Lactiplantibacillus plantarum ZJUIDS24 in preparing a medicine for preventing or assisting in treating hair loss.

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

[0016] Increase hair growth area,

[0017] Increase hair length,

[0018] Delay hair follicles entering the regression phase,

[0019] The phenomenon of shortened growth period, thinning skin, reduced hair bulb size and reduced HF length is alleviated.

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

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

[0022] Preparation of food, health products or dietary supplements for preventing or assisting in the treatment of hair loss and related diseases;

[0023] Preparation of anti-hair loss medicines or cosmetics.

[0024] The deposit information of strain ZJUIDS24 of the present invention is as follows:

[0025] The deposited name is Lactiplantibacillus plantarum, the depository unit is General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC NO.33081, and the deposit date is December 16, 2024.

[0026] The invention screens out a Lactobacillus plantarum ZJUIDS24 from the old yogurt "Tarige" in Inner Mongolia, and identifies the strain through bacterial morphology, physiology and culture characteristics combined with 16S rDNA sequencing.

[0027] The screening method is as follows: fresh Inner Mongolia old yogurt is collected with a sterile tube, cultured in an MRS broth medium for 48 hours, colonies are picked from the MRS medium for streak purification until the colony morphology on the entire plate is consistent, single colonies are picked and cultured in an MRS broth medium for bacterial enrichment, and finally the plant lactobacillus ZJUIDS24 is obtained.

[0028] The Lactobacillus plantarum ZJUIDS24 provided by the present invention has a strong therapeutic effect on androgenic alopecia. Compared with the existing Lactobacillus plantarum, the Lactobacillus plantarum ZJUIDS24 of the present invention has the following advantages: the present invention uses in vivo animal experiments to prove that the strain of the present invention also has a strong effect of improving androgenic alopecia in vivo, including delaying hair follicles from entering the regression phase, promoting hair growth, etc., and also has a strong antioxidant ability and the ability to inhibit pathogens.

[0029] The present invention also provides the use of the plant lactobacillus ZJUIDS24 (Lactiplantibacillus plantarum ZJUIDS24) in preparing foods that can alleviate androgenic alopecia. The foods that can alleviate androgenic alopecia are, for example, fermented yogurt, fruit and vegetable juice, bacterial powder and solid beverages that can alleviate androgenic alopecia.

[0030] The invention can provide excellent bacterial strain resources for the development of local probiotics in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

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

[0033] Figure 2 This is the Gram-stained bacterial morphology of ZJUIDS24.

[0034] Figure 3 This is a general diagram of the effect of ZJUIDS24 on the hair coverage area of ​​androgenic alopecia mice.

[0035] Figure 4 This is a statistical chart showing the effect of ZJUIDS24 on the hair coverage area of ​​androgenic alopecia mice.

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

[0037] Figure 6 This is a cross-sectional image showing the effect of ZJUIDS24 on the back hair follicles of androgenic alopecia mice.

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

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

[0040] Fig. 9 The effect of ZJUIDS24 on the diameter of hair bulb at the base of hair follicles (≥30 HF) in androgenic alopecia mice. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific embodiments, but the protection scope 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 the present invention was isolated from old yogurt (a product called Tazhige) in Inner Mongolia, and a total of 10 samples were collected.

[0046] 1.2 Isolation and purification of strains

[0047] Take about 15g of fresh sample and collect it in a sterile tube, and immediately send it to the laboratory for strain isolation. Take 1g of sample and put it into 9mL of MRS broth medium, vortex and mix it, and then enrich and culture it at 37℃ for 48h; then take 1mL of enrichment solution in the clean bench, dilute it tenfold with sterile saline, and select 10 -6 , 10 -7 , 10 -8Three dilution gradients, 100 μL of bacterial solution from each gradient was applied to MRS agar medium and cultured at 37°C for 48 hours. After the culture was completed, a plate with 30-300 single colonies was selected from the agar medium, and a typical colony (the colony was round, with a diameter of 0.3-1.5 mm, a smooth surface, and a strong yogurt flavor) was picked and purified by streaking on the MRS agar plate several times until the colony morphology on the entire plate was consistent, and a single colony was picked to MRS broth medium for bacterial enrichment culture. The obtained strains were all frozen and stored at -80°C in MRS broth medium containing 40% glycerol.

[0048] 2. Identification of Lactobacillus plantarum ZJUIDS24

[0049] 2.1 Colony characteristics

[0050] After the isolated and purified plant lactobacillus ZJUIDS24 was cultured in MRS agar medium for 48 hours, the colonies were round, with a diameter of 0.3-1.5 mm, a smooth surface, and a strong yogurt flavor. Figure 1 .

[0051] 2.2 Morphology under microscope:

[0052] Colony smear of Lactobacillus plantarum ZJUIDS24: Gram-positive, non-spore-forming, round-ended straight rods, single, paired or short chains, see Figure 2 .

[0053] 2.3 16S rDNA identification

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

[0055] The sequence alignment results of strain ZJUIDS24 were combined with the physiological and biochemical results, and it was determined that the screened lactic acid bacteria ZJUIDS24 was Lactiplantibacillus plantarum ZJUIDS24.

[0056] The deposit information of strain ZJUIDS24 of the present invention is as follows:

[0057] The deposited name is Lactiplantibacillus plantarum, the depository unit is General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, Yard 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 androgenic alopecia

[0059] 1. Establishment of the mouse model of androgenic alopecia

[0060] Six-week-old male C57BL / 6 mice were randomly divided into 4 groups, 10 mice in each group, namely normal group, model group (DHT), Lactobacillus plantarum ZJUIDS24 group (DHT+ZJUIDS04), and positive control group (DHT+minoxidil). The mice were fed with conventional adaptive feeding for one week before the formal experiment. The experimental period was from day 0 to day 29. Three days before the start of the experimental period, DHT modeling was started (5 mg / ml DHT-corn oil solution was injected intraperitoneally every day, 10 ul / g body weight), and 3*3.5 cm hair on the back of the mice was removed with depilatory cream and electric shaver on day 0 (the start day of the experimental period). The mice in the normal group had normal diet and water intake during the entire experimental period (0-29 days), and were gavaged with 10ul / g body weight of phosphate buffered saline (PBS, pH 7.2-7.4) every day as a control; the model group, the Lactobacillus plantarum ZJUIDS24 group, and the positive control group were intraperitoneally injected with 5mg / ml DHT every 2 days and administered with 10ul / g body weight every 1 day. The mice in the model group were gavaged with 10ul / g body weight of PBS every day during the entire experiment as a control, and the number of viable bacteria in the Lactobacillus plantarum ZJUIDS24 group was 1×10 9 CFU / mL bacterial solution, and the positive control group was smeared with 10ul / g body weight of 5% minoxidil solution every day. Other feeding conditions such as temperature, humidity, light, and feed were the same. Animal welfare and experimental procedures were carried out in accordance with the guidelines of the Zhejiang University Laboratory Animal Management Regulations. The experimental animal grouping and treatment methods are shown in Table 1.

[0061] The number of viable bacteria is 1×10 9The preparation method of CFU / mL bacterial solution (fermentation bacterial solution) is as follows: take out the glycerol tube of the preserved strain ZJUIDS24, pick out the strain with a disposable inoculation loop, streak on MRS solid medium, activate three generations, take a single colony and inoculate it in 30mL MRS liquid medium and place it in a 37℃ constant temperature incubator for 18h, then take it out, centrifuge it at 4℃, 8000g for 15min, discard the supernatant and retain the bacteria, add PBS (pH 7.2-7.4) and shake evenly, centrifuge it at 4℃, 8000g for 15min, repeat this twice to wash the bacteria. Finally, according to the OD value, use PBS to adjust the strain concentration to 1×10 9 CFU / mL.

[0062] Table 1 Animal experiment design

[0063]

[0064]

[0065] 2. Improvement of hair growth area in mice by Lactobacillus plantarum ZJUIDS24

[0066] During the experiment, when the mouse back hair grows stubble, it is necessary to take a general picture for observation and record. After the mouse is anesthetized with isoflurane, the mouse is placed on A4 paper with the back skin facing up, the limbs are spread out, a ruler is placed, and a camera is used to take pictures. The observation and photography positions of each mouse are kept consistent. After the hair removal model is established, the mouse is observed and photographed every four days with the stubble, and the color of the mouse back skin and hair coverage are continuously tracked.

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

[0068] ImageJ was used to analyze the hair coverage area on the back of mice on days 19 and 23. Figure 4It can be seen that on the 19th day, the hair coverage area of ​​the ZJUIDS24 treatment group was significantly higher than that of the model group (P < 0.05). On the 23rd day, the hair coverage area of ​​the ZJUIDS24 treatment group was still significantly higher than that of the model group (P < 0.05), and there was no significant difference with the normal group and the minoxidil group (P > 0.05). It can be seen that after the intervention of Lactobacillus plantarum ZJUIDS24, the hair growth of androgenic alopecia mice was improved and the hair growth area was significantly increased.

[0069] 3. Improvement of hair length in mice by Lactobacillus plantarum ZJUIDS24

[0070] On the 19th and 24th days, use flat-tip tweezers to randomly pluck a small handful of hair perpendicular to the same five places on the back skin of the mice. After thoroughly mixing in a plastic bag, select 5 hairs from each mouse, spread the hair flat on A4 paper with tweezers, place a ruler, take pictures of individual hairs with a camera, and use a vernier caliper to measure the hair length under sufficient light.

[0071] The results are as follows Figure 5 As shown in the figure, on the 19th and 24th days, 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 between the normal group and the positive group (P > 0.05). It can be seen that after the intervention of Lactobacillus plantarum ZJUIDS24, the hair length of mice with androgenic alopecia was improved, and the effect was comparable to that of the positive group treated with minoxidil.

[0072] 4. Lactobacillus plantarum ZJUIDS24 alleviated the shortened growth period, thinning skin, reduced hair bulb size, and reduced HF length in AGA mice.

[0073] Mouse hair follicles show a cyclical transition from growth phase to regression phase to resting phase. The following operations were performed on the 10th, 24th and 29th days respectively: skin tissue was obtained from the same part of the back of mice in the four experimental groups, placed in 4% PFA solution, fixed the tissue morphology overnight at 4°C, embedded in paraffin, sectioned at 5 μm intervals, and then stained with hematoxylin and eosin (HE).

[0074] The test results are as follows Figure 6The representative slice results show that on the 10th day, the hair follicles in the normal group entered the growth phase first, the hair follicles in the minoxidil group and the ZIUIDS24 group were in the resting phase or the growth phase I / II, and the hair follicles in the model group were in the resting phase; on the 24th day, the normal group had entered the resting phase, the hair follicles in the model group and the minoxidil group had subsided in the growth phase, and the ZIUIDS24 group was still in the growth phase; until the 29th day, the model group and the minoxidil group had entered the late catagen or the resting phase, and the ZIUIDS24 group began to enter the catagen. The slice results show that Lactobacillus plantarum ZJUIDS24 alleviated the shortening of the growth phase of AGA mice.

[0075] The thickness of the subcutaneous fat layer, the length of the hair follicle, and the diameter of the hair bulb are all important indicators for judging the growth cycle. When the hair follicle enters the growth phase, the thickness of the subcutaneous fat layer, the length of the hair follicle, and the diameter of the hair bulb continue to increase, and gradually become thinner as they transition to the regression phase and the resting phase. Figure 7 , 8 , 9 It can be seen that at the three sampling time points, the three indicators of the ZJUIDS24 group were significantly higher than those of the model group, and were significantly higher than those of the positive control group at 24 days and 29 days. The above results show that Lactobacillus plantarum ZJUIDS24 alleviated the thinning of the skin, the reduction of hair bulb size, and the reduction of HF length in AGA mice.

[0076] 5. Comparison

[0077] Reference was made to relevant literature for comparison, and the results are shown in Table 2. Lactobacillus plantarum CCFM1351, Lactobacillus plantarum CCFM1353, and Lactobacillus plantarum FTCM001 all showed different hair growth promoting effects, including promoting the expression of hair-related genes, promoting epithelial cell proliferation, and increasing the synthesis of melanin, etc.; but did not show its therapeutic effect on androgenic alopecia; Lactobacillus plantarum HX-LP931 has the effect of improving androgenic alopecia, specifically in reducing the oil content of the scalp, promoting hair growth, increasing hair weight, etc., but did not show the effect of improving the hair growth cycle.

[0078] Patients with androgenic alopecia have a short hair growth period and quickly enter the regression phase, which leads to hair loss. Prolonging the hair follicle growth period is of great significance for AGA patients. However, the existing Lactobacillus plantarum does not have this property.

[0079] The Lactobacillus plantarum ZJUIDS24 of the present invention can increase hair area and hair length, prolong the growth period of hair follicles, and has excellent effects of improving the phenomenon of androgenic alopecia and treating androgenic alopecia.

[0080] Table 2 Comparison of the effects of plant lactobacillus 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 stored in the glycerol tube was streaked on the MRS solid medium and incubated at 37°C for 48 hours. A single colony was picked with an inoculation loop and inoculated into 30 mL of sterilized MRS liquid medium and incubated at 37°C for 18-24 hours to obtain a culture solution. The culture solution was adjusted with distilled water to a lactic acid bacteria concentration of 10 10 CFU / mL, centrifuge at 4°C, 8000×g for 20 min, collect the supernatant as the fermentation supernatant. Resuspend and wash the bacterial pellet after centrifugation with 0.02M PBS buffer (pH=7.4), centrifuge at 4°C, 8000×g for 20 min, repeat 3 times. Resuspend the washed bacterial cells in PBS buffer and adjust the bacterial concentration to 10 10 CFU / mL, and the bacterial suspension was obtained.

[0086] The supernatant and cells of Lactobacillus plantarum ZJUIDS24 were used as sample solutions;

[0087] 2. DPPH clearance rate

[0088] 1 mL of sample solution and 1 mL of 0.2 mM DPPH anhydrous ethanol solution were mixed by oscillation for 1 min, and then reacted at room temperature in the dark for 30 min. The supernatant was centrifuged and the absorbance was measured at 517 nm. 95% ethanol was used instead of DPPH as a control, and PBS (pH 7.2-7.4) was used instead of the sample solution as a blank.

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

[0090] Description: 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 Hydroxyl free radical scavenging rate

[0093] 1) Take 1 mL of o-phenanthroline (2.5 mmol / L), add 1 mL of phosphate buffer (PBS concentration is 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 is 2.5 mmol / L), mix well, add 1 mL of hydrogen peroxide (H2O2 mass fraction is 20 mmol / L), and measure its absorbance at 536 nm as Ap after 1.5 hours in a 37°C water bath;

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

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

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

[0098] 4. Restoration capability

[0099] Experimental group: 0.5 mL of sample solution, 0.5 mL of potassium ferrocyanide (mass fraction 1%), and 0.5 mL of phosphate buffer (PBS concentration of 0.2 mol / L, pH = 6.6) were mixed to form a reaction raw material solution; the reaction raw material solution was placed in a 50°C water bath for 20 min, cooled to room temperature after the reaction, and 0.5 mL of trichloroacetic acid (TAC mass fraction of 10%) was added to precipitate protein, and after centrifugation, 1 mL of supernatant was taken to react with 1 mL of ferric chloride (FeCl3 mass fraction of 0.1%), and the absorbance was measured at 700 nm;

[0100] Blank control: PBS or MRS liquid culture medium was used to replace the sample solution in the experimental group, and the rest was the same as the experimental group.

[0101] Reducing power = (As-Ab) / Ab

[0102] As: absorbance of experimental group; Ab: absorbance of 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 strained by the present invention has higher reducing ability and hydroxyl radical scavenging ability, and the fermentation supernatant has higher DPPH scavenging ability. In general, the strain and its metabolites have good antioxidant capacity.

[0108] Example 4: Confirmation of the inhibitory ability of Lactobacillus plantarum ZJUIDS24 on pathogens

[0109] The antibacterial activity of lactic acid bacteria was determined by the internationally accepted agar diffusion method. The four frozen indicator strains (Escherichia coli, Salmonella, Staphylococcus aureus and Listeria monocytogenes) were activated 2 to 3 times on LB solid medium. The activated single colonies were picked out and placed in LB medium and cultured at 37°C for 18 hours. The bacterial cells were collected by centrifugation and resuspended in PBS buffer (pH 7.2-7.4) to a concentration of 10 8 CFU / mL. Add the indicator bacteria suspension at 1% (v / v) to the sterilized LB solid medium cooled to about 55°C, mix well and pour into the culture dish (15mL / dish), and remove the sterile Oxford cup placed in advance after condensation. Inoculate the activated plant lactobacillus ZJUIDS24 in MRS culture medium at 1% (v / v), expand the culture for 18h, collect the supernatant after centrifugation (8000rpm, 5min, 4°C), and discard the bacterial precipitate. Add the fermentation supernatant to the cup hole (200μL / hole), and use the uninoculated MRS culture medium (pH6.2) as a blank control. Culture at 37°C and measure the diameter of the inhibition zone after 24h. Select the strains with obvious inhibition zones around the small holes, measure the diameter of the inhibition zones, and repeat three times for each.

[0110] As shown in Table 4, the metabolites of Lactobacillus plantarum ZJUIDS24 have certain inhibitory effects on Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Listeria monocytogenes. It can be seen that the metabolites of this strain have antibacterial properties.

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

[0112]

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

[0114] 1. Acid resistance test

[0115] A single colony of Lactobacillus plantarum ZJUIDS24 was selected and expanded in MRS liquid medium at 37°C for 18 hours. The expanded bacterial suspension was inoculated into MRS liquid medium at 1% and cultured at 37°C for 18 hours. The culture solution was centrifuged at 8000r / min for 5 minutes at 4°C to collect the bacteria, and then washed twice with PBS buffer (pH7.2-7.4). The bacteria were suspended in MRS liquid medium with pH adjusted to 3.0 in advance, and the initial viable count was corrected to about 10 8 CFU / mL, cultured at 37℃ for 3h. The live bacteria in the samples at 0h and 3h were counted by pouring plate method. The plates were cultured at 37℃ for 48h and then the live bacteria were counted to determine the survival rate. The survival rate calculation formula is as follows:

[0116]

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

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

[0119] 2. Bile salt tolerance test

[0120] The activated and enlarged Lactobacillus plantarum ZJUIDS24 bacterial suspension was inoculated into MRS liquid culture medium at a volume of 1%, cultured at 37°C for 18 h, vortexed and mixed, and the initial viable count was corrected to about 10 9 CFU / mL. The bacteria were suspended in MRS liquid medium containing 0.3% (m / v) ox bile salts, and the initial viable count was corrected to about 108 CFU / mL, and cultured at 37°C for 3h. The viable bacteria in the 0h and 3h samples were counted by pouring plate method, and the poured plates were cultured at 37°C for 48h to determine their survival rate. The survival rate calculation formula is as follows:

[0121]

[0122] In the above formula, N0 is the viable count (CFU / mL) of the test strain at 0h; Nt is 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, the acid and bile salt resistance of Lactobacillus plantarum ZJUIDS24 is high (growth is achieved within 3 hours), and its survival rate in MRS medium with pH 3.0 is as high as 131±0.34%. The survival rate in an environment containing 0.3% ox bile salt is as high as 136±0.42%, indicating that it has good bile salt tolerance. Experiments have shown that Lactobacillus plantarum ZJUIDS24 has a high gastrointestinal tract survival ability.

[0124] Table 5 Results of strain 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 a series of adverse environments such as gastric acid and bile in the gastrointestinal tract to play their probiotic role. The plant lactobacillus ZJUIDS24 provided by the present invention can grow and proliferate under the conditions of pH 3.0 and 0.3% ox bile salt, and can smoothly pass through the acidic environment in the stomach to reach the small intestine. It can survive in the intestine, and can effectively improve the intestinal flora and play a role.

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

[0128] According to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI), the paper diffusion method was used to detect the antibiotic sensitivity of the strain. After centrifugation and washing of the bacteria, the concentration of the bacterial suspension was controlled to reach 10 8 CFU / mL. Take 100μL of bacterial solution and spread it evenly on MRS solid culture medium. Then use sterile tweezers to gently press the antibiotic paper against the surface of the plate and culture it in a 37℃ constant temperature box for 24 hours. Use a vernier caliper to measure and record the diameter of the inhibition zone. The antibiotic sensitivity of the strain is determined according to the diameter of the inhibition zone. According to the "Antimicrobial Susceptibility Test Implementation Standard" (Seventeenth Edition) formulated by CLSI, the resistance of lactic acid bacteria to antibiotics is determined, which is divided into three levels: sensitive (S), intermediate (I) and resistant (R).

[0129] The diameter of the inhibition zone of Lactobacillus plantarum ZJUIDS24 to antibiotic sensitivity is shown in Table 6. It is sensitive to penicillin, ampicillin, ceftriaxone, cefazolin, chloramphenicol, cefoperazone, tetracycline, and erythromycin, and is only resistant to gentamicin. The experimental results show that Lactobacillus plantarum ZJUIDS24 is sensitive to common antibiotics and has high safety.

[0130] Table 6 Results of antibiotic sensitivity of Lactobacillus plantarum ZJUIDS24

[0131]

[0132] Example 7: Preparation of fermented yogurt with the efficacy of alleviating androgenic alopecia using Lactobacillus plantarum ZJUIDS24 1. Yogurt processing process:

[0133] raw material Preheat Homogenization Provisioning Sterilization cool down preparation Vaccination Fermentation After-ripening refrigeration.

[0134] 2. Operation points

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

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

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

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

[0139] (5) Sterilization: Sterilize the sweetened milk in a water bath at 90°C for 10 min;

[0140] (6) Cooling: After sterilization, the milk is cooled to 40-50°C for later use;

[0141] (7) Preparation of starter culture: Lactobacillus plantarum ZJUIDS24 was inoculated into a test tube containing sterilized skim milk (12%, w / v) under a sterile environment and cultured at 37°C for 20 hours. The inoculation volume for each subculture was 2-4% (v / v), and the culture was subcultured 2-3 times to restore vitality and stored in a refrigerator at 4°C;

[0142] (8) Inoculation and fermentation: Under sterile conditions, inoculate the activated Lactobacillus plantarum ZJUIDS24 at an inoculation rate of 2-4% (v / v). Ferment at a constant temperature of 42° C. for 6-10 h;

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

[0144] (10) Filling and refrigeration: After ripening, fill into 250 mL sterilized glass bottles and send to cold storage for refrigeration.

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

[0146] raw material Cleaning Flash Beating Provisioning Homogenization Sterilization cool down Vaccination Closed fermentation After-ripening Filling refrigeration

[0147] 2. Operation points

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

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

[0150] (3) Flash evaporation: Use flash evaporation to inactivate the enzyme for 0.5 to 1 min at 121°C and quickly exhaust the air.

[0151] (4) Beating: According to the ratio of pumpkin to water (weight ratio) = 1:1, gradually put pumpkin and water into a colloid mill and grind them once for coarse grinding and once for fine grinding. After removing the core of the apple, beat it with a beater until the pulp is uniform and free of lumps.

[0152] (5) Mixing and homogenizing: 15% pumpkin juice and 30% apple juice are added, and the soluble solid content is adjusted to 10° Brix with sucrose. 0.2% stabilizer CMC is added and mixed evenly. A two-stage homogenization method is adopted, first low pressure (15 MPa) and then high pressure (25 MPa), so that the diameter of the melon pulp particles is 2 to 3 μm.

[0153] (6) Sterilization and cooling: The prepared composite fruit and vegetable juice is kept at 100°C for 10 min and then cooled to about 40°C.

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

[0155] (8) Post-ripening: After fermentation, place in a 4°C refrigerator for 3 h.

[0156] (9) Filling and refrigeration: After ripening, fill into 250 mL sterilized glass bottles and send to cold storage for refrigeration.

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

[0158] Pick a single colony of plant lactobacillus ZJUIDS24 and inoculate it in 50mL MRS liquid culture medium, and place it in a 37°C incubator to cultivate for 18h. Activate it again in 250mL MRS liquid culture medium at 5% inoculum, and place it in a 37°C incubator to cultivate for 24h. Finally, the activated plant lactobacillus ZJUIDS24 is cultured in a 10L fermenter at a high density anaerobic culture with a 5% inoculum, and cultivated for 18h at 37°C and pH 6.8. Afterwards, centrifuge for 15min at 8000r / min and 4°C, discard the supernatant, collect the bacterial precipitation, and rinse the bacterial body twice with sterile phosphate buffer (pH 7.0). You can get the plant lactobacillus ZJUIDS24 bacterial mud.

[0159] 2. Preparation of Protective Agent

[0160] The freeze-dried protective agent contains 15% skim milk powder, 5% trehalose, 3% sodium glutamate, 1% glycerol, and 0.5% cysteine ​​hydrochloride; water is used as a solvent; and the product is sterilized at 110° C. for use.

[0161] 3. Preparation of Lactobacillus plantarum ZJUIDS24 powder

[0162] The prepared Lactobacillus plantarum ZJUIDS24 bacterial precipitate was thoroughly mixed with the protective agent solution at a ratio of 1:5. It was pre-frozen at -40°C for 5 hours to freeze evenly on the inner wall of the container, and then vacuum-freeze-dried for 18 to 20 hours to obtain Lactobacillus plantarum ZJUIDS24 bacterial powder. After rehydration with physiological saline, it was washed twice, and the number of viable bacteria in the Lactobacillus plantarum ZJUIDS24 bacterial powder was measured to be 1.0×10 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 powder

[0165] The freeze-dried powder of Lactobacillus plantarum ZJUIDS24 was prepared with reference to Example 9, and the number of viable bacteria in the powder was 1.0×10 11 ~1×10 12 CFU / g.

[0166] 2. Preparation of milk formula powder

[0167] Primary selection of raw materials: milk powder, protein powder, sugar compounds, vegetable oil, additives: vitamins, trace elements, functional factors, others;

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

[0169] Crushing: crush the weighed materials through a crusher;

[0170] Mixing: Add vegetable oil and trace elements to the crushed materials and put them into the mixer to mix evenly;

[0171] Puffing: The mixed materials are made into granular materials through the puffing machine;

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

[0173] Grading screening: The logistics is passed through the grading screen and the particles are controlled at 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 in a ratio of 1:100. The live bacteria in the final product are 8 CFU / g or above. After filling, the product is stored in the warehouse for sale.

[0176] Example 11. Preparation of solid beverage using powder of Lactobacillus plantarum ZJUIDS24 for preventing and treating androgenic alopecia 1. Preparation of powder of Lactobacillus plantarum ZJUIDS24: Prepare powder of Lactobacillus plantarum ZJUIDS24 with reference to Example 9.

[0177] 2. The formula of solid beverage is as follows: edible corn starch, white sugar (10%), oligofructose (6%), oligomaltose (9%), citric acid, edible flavor, bacterial powder (2-5%);

[0178] 3. Mixed packaging: Add according to the formula and mix evenly, pack according to 10 / 20g per small package, and pack 10-20 small packages into a large package.

[0179] Example 12. Preparation of hair growth tablets using powder of Lactobacillus plantarum ZJUIDS24 for preventing and treating androgenic alopecia. 1. Preparation of powder of Lactobacillus plantarum ZJUIDS24: Prepare powder of Lactobacillus plantarum ZJUIDS24 with reference to Example 9.

[0180] 2. The Chinese medicine formula is as follows: use herbal plant extract ingredients, such as Polygonum multiflorum, ginseng, angelica, Platycladus orientalis leaves, and starch, mix them evenly and set aside.

[0181] 3. Mixed packaging: Add according to the formula and mix evenly, pack according to 10 / 20g per small package, each small package contains 10 live bacteria 9 above.

[0182] 4. Tablet compression: The mixture is compressed into tablets by a tablet press and then packaged.

[0183] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

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

2. The use of Lactiplantibacillus plantarum ZJUIDS24 as claimed in claim 1, characterized in that: Improve the phenomenon of androgenic alopecia and treat androgenic alopecia.

3. Use of Lactiplantibacillus plantarum ZJUIDS24 as claimed in claim 1 in the preparation of a medicament for preventing or assisting in the treatment of hair loss.

4. The use / application according to claim 2 or 3, characterized in that At least one of the following: Increase hair growth area, Increase hair length, Delay hair follicles entering the regression phase, The phenomenon of shortened growth period, thinning skin, reduced hair bulb size and reduced HF length is alleviated.

5. The use / application according to claim 2 or 3, characterized in that: It is androgenic alopecia caused by dihydrotestosterone.

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