Cordyceps sinensis strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a cordyceps sinensis strain and application thereof.The cordyceps sinensis strain LT4 is classified and named as Hirsutella sinensis and preserved in the China General Microbiological Culture Collection Center on June 7, 2024, the address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO.41331, and the preservation number is CGMCC NO.41331. The cordyceps sinensis strain LT4 is named as Hirsutella sinensis. The ITS sequence of the strain is as shown in SEQ ID NO. 1. The cordyceps sinensis strain provided by the invention can be used for preparing anti-alopecia and hair growth related medical supplies and cosmetics, and a new technical support is provided for alopecia treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Cordyceps sinensis strain and application thereof in products for preventing or repairing hair loss. Background Art

[0002] With the increasing severity of environmental pollution, the increasing pressure of people's lives, and the stimulation of shampoo products, the proportion of people with sensitive scalp is increasing, and more and more people are troubled by hair loss. Hair loss will affect personal image and mental health, so effective prevention and treatment measures need to be taken in time. At present, the clinical treatment methods for hair loss mainly include topical drug treatment and light therapy, but the effect is not satisfactory and there are certain side effects. Therefore, seeking new treatment methods has become a research hotspot in related fields.

[0003] As a traditional Chinese herbal medicine, Cordyceps sinensis is widely used in immune regulation, anti-oxidation, anti-inflammation and other aspects. Recent studies have shown that Cordyceps sinensis and its metabolites have biological activities such as anti-fungal, anti-inflammatory and regulating oil secretion, and have potential application value in the treatment of hair loss. Summary of the invention

[0004] The purpose of the present invention is to provide a Cordyceps sinensis strain and its application in products for preventing or repairing hair loss.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A Cordyceps sinensis strain, the Cordyceps sinensis strain LT4 is classified and named Hirsutella sinensis, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 7, 2024, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41331. The ITS sequence of the strain is shown in SEQ ID NO.1.

[0007] The above Cordyceps sinensis strain is used in products for preventing or repairing hair loss; specifically, the mycelium, mycelium extract, intracellular polysaccharide and extracellular polysaccharide produced by fermenting the Cordyceps sinensis strain LT4 are used in products for preventing or repairing hair loss.

[0008] Furthermore, the products include but are not limited to medicines and cosmetics.

[0009] The cordyceps sinensis strain provided by the present invention can be used to prepare medical products and cosmetics related to hair loss prevention and hair growth, providing new technical support for hair loss treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1This is a diagram showing the hair growth promoting effect of the Cordyceps sinensis strain LT4 of the present invention on C57BL / 6 mice.

[0011] Figure 2 It is a bar graph of the hairless area of ​​the mice of the present invention at different stages.

[0012] Figure 3 This is a bar graph of hair growth rate analysis of different groups of mice in the present invention. DETAILED DESCRIPTION

[0013] Isolation and purification of Cordyceps sinensis strains

[0014] Under running water, the soil on the surface of wild Cordyceps sinensis collected from Litang County, Sichuan was washed clean, and the surface was disinfected with 75% alcohol before tissue separation. The Cordyceps sinensis was cut into small pieces of about 0.5 cm, and then these small pieces were placed in slant culture medium respectively, and then placed in a constant temperature incubator at 12°C for 50 days.

[0015] When the isolated strains grow to a cake of about 1 cm, select the uncontaminated cake, cut it into small pieces, and re-inoculate it on a new slant culture medium. After culturing at 18°C ​​for 50 days, observe the morphology of mycelium and spore stalks with an inverted microscope; then use a hook to pick out mycelium that may be Chinese Hirsutella serrata, inoculate it on a new slant culture medium, and after culturing at 18°C ​​for 100 days, place the above isolated and cultured strains at 4°C for low-temperature treatment until the primordium grows out, select the strains that can produce fruiting bodies, number them, and transfer them to tubes for expanded culture again.

[0016] Genetic identification of bacterial species

[0017] Using the DNA of strain LT4 as a template, PCR amplification was performed using ITS primers, resulting in a fragment of about 580 bp in length. After sequencing, the sequence was compared with the ITS sequence of Ophiocordyceps sinensis (EU570946) and showed that the sequence was 99.66% identical to that of Ophiocordyceps sinensis. It was determined to be the asexual form of Cordyceps sinensis, Hirsutella sinensis.

[0018] ITS sequence of strain LT4 (SEQ ID NO.1):

[0019] TCCGTAGGTG AACCTGCGGA GGGATCATTA TCGAGTTACC ACTCCCAAAC

[0020] CCCCTGCGAA CACCACAGCA GTTGCCTCGG CGGGACCGCC CCGGCGCCCC

[0021] AGGGCCCGGA CCAGGGCGCC

[0022] CGCCGGAGGA CCCCCAGACC CTCCTGTCGC AGTGGCATCT CTCAGTCAAG

[0023] AAGCAAGCAA

[0024] ATGAATCAAA ACTTTCAACA ACGGATCTCT TGGTTCTGGC ATCGATGAAG

[0025] AACGCAGCGA

[0026] AATGCGATAA GTAATGTGAA TTGCAGAATT CAGTGAACCA TCGAATCTTT

[0027] GAACGCACAT

[0028] TGCGCCCGCC AGCACTCTGG CGGGCATGCC TGTCCGAGCG TCATCTCAAC

[0029] CCTCGAGCCC

[0030] CCCGCCTCGC GGCGGCGGGG CCCGGCCTTG GGGGTCACGG CCCCGCGCCG

[0031] CCCCCTAAAC

[0032] GCAGTGGCGA CCCCGCCGCG GCTCCCCTGC GCAGTAGCTC ACCGAGAACC

[0033] TCGCACCGGA

[0034] AGCGCGGAGG CGGTCACGCC GTGAAACCAC CACGCCTTCC AGTTGACCTC

[0035] GGATCAGGTA

[0036] GGGATACCCG CTGAACTTAA GCATATCAAT AAGCGGAGGA

[0037] Hair loss experiment

[0038] 1. Fermentation of strains

[0039] Preparation of culture medium: culture medium formula: 1% yeast powder, 0.5% peptone, 3% glucose, 0.1% potassium dihydrogen phosphate, and 0.05% magnesium sulfate; prepare liquid culture medium according to the above proportions and divide into 250mL conical flasks, 75mL in each bottle; sterilize at 121℃ for 15min, and place in an ultra-clean workbench after cooling; inoculation and cultivation: inoculate different strains at 10% of the inoculum, place in a constant temperature shaker, and culture at 20℃ and 135r / min for 7 days.

[0040] 2. Collection of fermentation drying liquid and extracellular polysaccharides

[0041] After 7 days of liquid culture, filter, collect the filtrate and mycelium respectively, decompress and concentrate the filtrate to 100-200 ml in a rotary evaporator, freeze-dry at -70°C, and store at -20°C to obtain the fermentation liquid dry product; after concentration, centrifuge at 3600rpm for 3 minutes, add 4-5 times the volume of anhydrous ethanol to precipitate, place in a refrigerator at 4°C for 24-36 hours, remove the supernatant, and wash twice with an equal amount of anhydrous ethanol. The precipitate obtained after alcohol precipitation is dialyzed (molecular weight cutoff 3500D), freeze-dried at -70°C to obtain the extracellular crude polysaccharide, and store at -20°C.

[0042] 3. Collection of mycelium extracts and intracellular polysaccharides

[0043] After 7 days of liquid culture, filter and collect the mycelium. After freeze-drying the mycelium, add RO water or other organic solvents at a solid-liquid ratio of 1:20, and extract with hot water at 60℃ for 4 hours; remove the mycelium after filtration, and concentrate the filtrate to 100-200ml in a rotary evaporator under reduced pressure, freeze-dry at -70℃, and store at -20℃ to obtain the mycelium extract; after concentration, centrifuge at 3600rpm for 3min, add 4-5 times the volume of anhydrous ethanol to precipitate, place in a refrigerator at 4℃ for 24-36h, remove the supernatant, and wash twice with an equal amount of anhydrous ethanol. The precipitate obtained after alcohol precipitation is dialyzed (molecular weight cutoff 3500D), freeze-dried at -70℃ in vacuum to obtain the crude intracellular polysaccharide, and stored at -20℃.

[0044] 4. Animal experiment to explore the effect of LT4 Cordyceps sinensis strain on hair growth in C57BL / 6 mice

[0045] (1) Adaptive feeding: ensure that sufficient food and water are provided to the mice and feed them at 22±1°C and 50±5% humidity for 7 days.

[0046] (2) Modeling:

[0047] Female mice: Apply an appropriate amount of 6% sodium sulfide solution evenly on the back of the mouse for hair removal, the application area is about 3cm×2cm, rinse with water immediately after 2 minutes and wipe dry the back 2×3cm 2Except for the blank group, the mice in other groups were placed under UVB light for 2 to 4 hours at a fixed time every day on a horizontal plane about 35 cm away from the UVB light source for 7 days.

[0048] Male mice: Hair removal was performed in the same manner as above. Except for the normal group, which was smeared with 0.1 mL of saline, the mice in the other groups were smeared with 0.5 mg mL of -1 A seborrheic alopecia model was constructed with 0.1 mL of testosterone solution and the drug was administered continuously for 24 days.

[0049] The criteria for successful model establishment: The skin in the application area is clean, hairless, undamaged, smooth and free of edema. Mice with pink skin and hair in the resting phase are selected.

[0050] (3) Product preparation: The products were divided into base cream (20 ml water, 0.4 g carbomer, 1 ml butylene glycol), LT41% strain cream (20 ml water, 400 mg carbomer, 1 ml butylene glycol, 200 mg dried fermentation broth, mycelium extract, extracellular polysaccharides, intracellular polysaccharides), and LT43% strain cream (20 ml water, 400 g carbomer, 1 ml butylene glycol, 600 mg dried fermentation broth, mycelium extract, extracellular polysaccharides, intracellular polysaccharides).

[0051] (4) Drug administration: The female mice were given drugs by applying the drug to the back skin after modeling for 7 consecutive days. The male mice were given drugs by applying the testosterone solution and the treatment drug at the same time for 24 consecutive days.

[0052] (5) The dorsal skin was observed and photographed, and imageJ was used to calculate the hairless and hairy areas of each mouse.

[0053] like Figures 1 to 3 As shown, after the model was established, the area of ​​non-hair growth in mice that were applied with LT4 3% extracellular polysaccharide cream for three consecutive days was significantly reduced, while the application of the basic cream (control group) failed to significantly improve the hair growth of mice. The hair regeneration effect of mice treated with LT4 3% extracellular polysaccharide cream for six days was significantly better than that of all other groups. By calculating the hair growth rate (the hair growth rate is defined as 1-(the area of ​​non-hair growth at the end of administration / the area of ​​non-hair growth at the end of model establishment)), the LT4 3% extracellular polysaccharide cream group had the highest hair growth rate (76.91%), followed by the model group (11.06%), and the control group had the lowest hair growth rate (5.91%).

[0054] The above description is only a preferred implementation manner of the present invention, but the protection scope of the present invention is not limited thereto, and any modification and replacement based on the technical solution and inventive concept provided by the present invention should be included in the protection scope of the present invention.

Claims

1. A Cordyceps sinensis strain, characterized in that: The Cordyceps sinensis strain LT4 is classified and named Hirsutella sinensis, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on June 7, 2024, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41331. The ITS sequence of the strain is shown in SEQ ID NO.

1.

2. A use of the Cordyceps sinensis strain according to claim 1 in products for preventing or repairing hair loss.

3. The use of the Cordyceps sinensis strain according to claim 2 in the preparation of a skin care product having the function of repairing skin photoaging and preventing or promoting hair growth, characterized in that: The mycelium, mycelium extract, intracellular polysaccharide and extracellular polysaccharide produced by fermenting the Cordyceps sinensis strain LT4 are applied to toiletries products.

4. The use of the Cordyceps sinensis strain according to claim 3 in preparing a skin care product with skin photoaging repair function, characterized in that: The products include but are not limited to medicines and cosmetics.

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