Cordyceps sinensis strain with high infection rate and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a cordyceps sinensis strain with a high infection rate and application of the cordyceps sinensis strain, the classification name of the cordyceps sinensis strain KK1 is Hirsutella sinensis, the cordyceps sinensis strain KK1 is preserved in China General Microbiological Culture Collection Center on June 7, 2024, the address is No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.969. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.41330, and the ITS sequence of the strain is shown as SEQ ID NO.1. The strain is applied to artificial cultivation of cordyceps sinensis and has high infectivity; the composition can also be applied to skin care products with a skin photoaging repairing function, is high in antioxidant and anti-inflammatory activity, and can effectively improve skin symptoms related to photoaging. The method provides a new technical support for artificial culture of cordyceps sinensis and prevention and treatment of skin photoaging, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Cordyceps sinensis strain with a high infection rate and application thereof in a skin care product with the function of repairing skin photoaging. Background Art

[0002] Cordyceps sinensis is a rare Chinese herbal medicine with extensive medicinal value. In recent years, with the increasing demand for Cordyceps sinensis, wild resources have gradually been depleted, and artificial cultivation has become an important supplement.

[0003] In the process of artificial cultivation of Cordyceps sinensis, the yield of Cordyceps sinensis is closely related to the infectivity of the Cordyceps sinensis strain. At present, the infection rate of wild Cordyceps sinensis on bat moth larvae in the natural environment is low, generally about 10%. In the artificial cultivation environment, the success rate of isolation of Cordyceps sinensis strains is low. Isolation and cultivation, screening of strains with high infectivity and improving the effective infection rate have become bottlenecks in the actual application of Cordyceps sinensis biological research and industrial large-scale production. In order to improve the efficiency of artificial cultivation of Cordyceps sinensis, an infective strain is urgently needed. Summary of the invention

[0004] The purpose of the present invention is to provide a Cordyceps sinensis strain with a high infection rate and a cultivation method thereof, and to apply the strain to skin care products with skin photoaging repair function.

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

[0006] A Cordyceps sinensis strain with a high infection rate, the Cordyceps sinensis strain KK1 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.41330. The ITS sequence of the strain is shown in SEQ ID NO.1.

[0007] The Cordyceps sinensis strain provided by the present invention grows rapidly, has strong infection ability and can produce a large amount of bioactive substances with potential for treating photoaging, providing new technical support for the treatment of skin photoaging and is expected to significantly increase the artificial cultivation yield of Cordyceps sinensis.

[0008] The cordyceps sinensis strain provided by the invention is separated from wild cordyceps sinensis in Kangding area of ​​Sichuan Province, purified and cultured, and a strain with a high infection rate is screened out.

[0009] The high infection rate cordyceps sinensis strain provided by the present invention has a fast growth rate, a short culture period, a strong infection ability, can effectively infect bat moth larvae, and improve the infection efficiency of host larvae; it has strong antioxidant and anti-inflammatory activities and can effectively improve skin symptoms related to photoaging. It provides technical support for the artificial cultivation of cordyceps sinensis and the prevention and treatment of skin photoaging, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Effects of extracellular polysaccharide treatment on the skin performance status of photoaged mice.

[0011] Figure 2 This is a HE staining microscope image of mice with photoaged skin treated with the strain's extracellular polysaccharide.

[0012] Figure 3 To investigate the effects of Cordyceps exopolysaccharides on the characteristics of collagen fibers in the skin of photoaged mice. DETAILED DESCRIPTION

[0013] Isolation and purification of Cordyceps sinensis strains

[0014] Under running water, the soil on the surface of wild Cordyceps sinensis collected in Kangding and Litang counties in Sichuan was washed clean, and the surface was disinfected with 75% alcohol before tissue separation. First, 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 the culture medium was placed in a constant temperature incubator at 12°C for 50 days.

[0015] When the isolated strain grows to a cake of about 1 cm, select the uncontaminated cake, cut it into small pieces, re-inoculate it on a new slant medium, and culture it in a constant temperature incubator at 18°C ​​for 50 days, and observe the morphology of hyphae and spore stalks with an inverted microscope; then pick out the hyphae of Chinese hairy spores with a hook hook, inoculate it on a new slant medium, and continue to culture it in a constant temperature incubator at 18°C ​​for 100 days, then place the isolated and cultured strains in an environment of 4°C for low temperature treatment until fruiting bodies grow out, select the strains that produce fruiting bodies, and transfer them to expand the culture again. This example isolates 3 strains, numbered KK1, KK2 and LT4 respectively.

[0016] Morphological identification of bacteria

[0017] Microscopic observation of the hyphae suspension prepared for infecting the larvae showed that the hyphae were slender, filamentous, with many branches and vigorous growth. Some aged hyphae containing two nuclei were also observed. The morphology of the fungus in the larvae after infection was observed, which showed that after the hyphae invaded the insect body for a period of time, the hyphae began to break into small single-cell hyphae segments, which gradually formed fusiform spores through differentiation and proliferation, so this period was called the fusiform spore period; then, the fusiform spores continued to differentiate and proliferate, and further formed 3-septate hyphae and 7-septate hyphae, the 7-septate hyphae slowly elongated to form a small multi-cellular hyphae fragment, and "H" binding phenomenon was observed between the hyphae, indicating that the hyphae were fusing at this time, so this period was called the sclerotium period; thereafter, for a period of time, it was observed that the small hyphae fragments continued to undergo cell fusion and the fused hyphae proliferated and elongated again, and the elongated hyphae began to tangle with each other, so this period was called the late sclerotium period; after the sclerotium period was completed, the hyphae tangled and formed hard sclerotia, so the last period was called the sclerotium period.

[0018] After infection, the larvae need to go through the stages of dead insects and primordium differentiation and eventually grow fruiting bodies (cordyceps). About 30 days after the larvae were infected, their body fluids were extracted for microscopic observation, which showed that they were in the fusiform spore stage. It was found that the larvae in this period had normal feeding and mobility, and their body surface was light yellow. There was no obvious difference in morphology from uninfected larvae. After that, the infected larvae continued to grow, and their body surface began to turn light yellow or yellow. Their feeding and mobility began to decline, and their bodies gradually became rigid and hard and eventually died. Therefore, the larvae in this state are called dead insects. The hyphae in their bodies are taken to make temporary slices, and microscopic observation shows that they are in the mycelial mating stage at this time. The mycelium continued to grow and primordium differentiation was observed in the head of the larvae, so the larvae at this stage were called primordium differentiation dead insects. The mycelium in their bodies was prepared for slides, and microscopic observation showed that the mycelium at this time began to elongate and entangle with each other, so it should be in the late stage of plasmodiosis; thereafter, the primordium continued to grow, the insect body became harder, the color of its thorax became lighter to beige, containing 3 pairs of legs, while the color of the abdomen became yellow or dark yellow, containing 4 pairs of legs and about 21 ring patterns. The mycelium in its body was prepared for slides, and microscopic examination showed that the morphology of the mycelium at this time was the same as before infection, thus obtaining the head grass of Cordyceps sinensis.

[0019] Genetic identification of bacterial species

[0020] Using the DNA of strain KK1 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 in the NCBI database, showing that the sequence had a 99.66% identity with the ITS sequence of Ophiocordyceps sinensis (EU570946). This indicated that KK1 belonged to Ophiocordyceps sinensis, and therefore it could be determined to be the asexual form of Cordyceps sinensis, Hirsutella sinensis.

[0021] ITS sequence of strain KK1 (SEQ ID NO.1):

[0022] TCCGTAGGTG AACCTGCGGA GGGATCATTA TCGAGTTACC ACTCCCAAAC CCCCTGCGAA

[0023] CACCACAGCA GTTGCCTCGG CGGGACCGCC CCGGCGCCCC AGGGCCCGGA CCAGGGCGCC

[0024] CGCCGGAGGA CCCCCAGACC CTCCTGTCGC AGTGGCATCT CTCAGTCAAG AAGCAAGCAA

[0025] ATGAATCAAA ACTTTCAACA ACGGATCTCT TGGTTCTGGC ATCGATGAAG AACGCAGCGA

[0026] AATGCGATAA GTAATGTGAA TTGCAGAATT CAGTGAACCA TCGAATCTTT GAACGCACAT

[0027] TGCGCCCGCC AGCACTCTGG CGGGCATGCC TGTCCGAGCG TCATCTCAAC CCTCGAGCCC

[0028] CCCGCCTCGC GGCGGCGGGG CCCGGCCTTG GGGGTCACGG CCCCGCGCCG CCCCCTAAAC

[0029] GCAGTGGCGA CCCCGCCGCG GCTCCCCTGC GCAGTAGCTC ACCGAGAACC TCGCACCGGA

[0030] AGCGCGGAGG CGGTCACGCC GTGAAACCAC CACGCCTTCC AGTTGACCTC GGATCAGGTA

[0031] GGGATACCCG CTGAACTTAA GCATATCAAT AAGCGGAGGA

[0032] Determination of bacterial infection rate

[0033] In this example, the infection of three isolated strains, KK1, KK2 and LT4, on the larvae of the small golden bat moth was studied by feeding.

[0034] As shown in Table 1, the results show that: after feeding the larvae of the small golden bat moth, the three strains can all produce dead insects, indicating that they all have a certain infection ability; among them, the percentages of dead insects formed by strains KK1 and KK2 are 24.0% and 24.6% respectively, which are much higher than the percentage of dead insects formed by LT4, which is only 0.8%, indicating that LT4 has the worst ability to infect the larvae of the small golden bat moth; in terms of grass emergence rate, although the number of dead insects formed by strains KK1 and KK2 after infecting larvae is similar, the grass emergence rate of the former is 13.4%, which is much higher than the latter's 2.6%, and the 4 dead insects formed by strain LT4 did not emerge. The research results show that strain KK1 is most suitable for artificial cultivation of Cordyceps sinensis.

[0035] Table 1 Infection of different strains to bat moth larvae

[0036]

[0037] Anti-skin photoaging experiment

[0038] 1. Fermentation of strains

[0039] Culture medium: 1% yeast powder, 0.5% peptone, 3% glucose, 0.1% potassium dihydrogen phosphate, and 0.05% magnesium sulfate; prepare the liquid culture medium according to the above proportions and dispense it into 250mL Erlenmeyer flasks, 80mL per bottle; sterilize at 121℃ for 15min, and place it on a clean bench after cooling.

[0040] Inoculation and culture: After inoculating KK1 strain at 10% of the inoculum, place it in a constant temperature shaker and culture it at 18°C ​​and 135 r / min for 7 days.

[0041] 2. Collection of fermentation drying liquid and extracellular polysaccharides

[0042] After 7 days of liquid culture, filter, collect the filtrate and mycelium respectively, concentrate the filtrate to 100-200mL in a rotary evaporator under reduced pressure, freeze-dry at -70℃, and store at -20℃ to obtain the fermentation liquid dry product; centrifuge the concentrate 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℃ to obtain the crude extracellular polysaccharide, and stored at -20℃.

[0043] 3. Collection of mycelium extracts and intracellular polysaccharides

[0044] After 7 days of liquid culture, filter and collect the mycelia, freeze-dry the mycelia, add RO water or other organic solvents at a solid-liquid ratio of 1:20, and extract with hot water at 60℃ for 4h; remove the mycelia after filtration, decompress and concentrate the filtrate to 100-200mL in a rotary evaporator, freeze-dry at -70℃, and store at -20℃, which is the mycelium extract; centrifuge at 3600rpm for 3min after concentration, 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, and the intracellular crude polysaccharide is obtained, which is stored at -20℃.

[0045] 4. Animal experiments to explore the effect of Cordyceps sinensis strains on skin photoaging repair

[0046] 1) Adaptive feeding (ensure sufficient food and water are provided to ICR mice and feed them at 22±1°C and 50±5% humidity for 7 days).

[0047] 2) The experiment used UVB light to irradiate the model, and the 2×3 cm 2 Except 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 light source for 7 days.

[0048] 3) The groups were divided into base cream (20 mL water, 0.4 g carbomer, 1 mL butylene glycol), KK11% strain cream (20 mL water, 400 mg carbomer, 1 mL butylene glycol, 200 mg fermentation liquid dry matter, mycelium extract, extracellular polysaccharide, intracellular polysaccharide), KK13% strain cream (20 mL water, 400 g carbomer, 1 mL butylene glycol, 600 mg fermentation liquid dry matter, mycelium extract, extracellular polysaccharide, intracellular polysaccharide). The mice after modeling were smeared on the back skin for 7 consecutive days.

[0049] 4) Observe the surface of the back skin, such as Figure 1shown.

[0050] 5) Collect skin from mice after treatment. Disconnect the neck of the mice and take 2×3 cm skin from the back. 2 , HE staining and picrosirius red staining were performed respectively.

[0051] HE staining of mouse skin: dewax the paraffin sections to water, soak the paraffin sections in xylene for 20 minutes, repeat twice; transfer the sections to anhydrous ethanol, 5 minutes each time, repeat twice; place the sections in 75% ethanol solution for 5 minutes to wash away residual substances. Use hematoxylin staining solution to stain the skin sections for 3 to 5 minutes, then wash to remove excess dye, differentiate with differentiation solution, wash, blue with blueing solution, and rinse with running water. Dehydrate the skin sections with 85% ethanol and 95% ethanol for 5 minutes each, and then stain with eosin staining solution for 5 minutes. Finally, dehydrate and seal the sections. After staining is completed, dehydrate with anhydrous ethanol again for 5 minutes each time, repeat 3 times, then place in xylene for 5 minutes, repeat 2 times, and seal the sections with neutral gum. Place the skin sections under an optical microscope for observation and collect images, such as Figure 2 shown.

[0052] Staining of mouse skin with picrosirius red: Place paraffin sections in xylene for 20 minutes to remove paraffin, repeat twice; transfer sections to anhydrous ethanol for 5 minutes, repeat twice; place sections in 75% ethanol for 5 minutes and wash. Stain skin sections with picrosirius red dye for 8 minutes, dehydrate in three cylinders of anhydrous ethanol. Transparently seal the sections, place skin sections in xylene for 5 minutes, and seal with neutral gum. Observe the skin sections under a microscope and collect images, such as Figure 3 shown.

[0053] like Figure 1 to Figure 3 As shown in the figure, after UVB radiation, the mice in the model group showed obvious changes in skin structure. Optical microscopy showed that the boundary between the epidermis and dermis of the skin was not obvious, and the epidermis showed uneven thickness increase. In addition, the collagen fibers in the dermis were arranged in a disordered and loose manner, broken or even broken fibers were seen everywhere, and the density distribution was abnormal. The number of collagen fibers in the dermis decreased, a large number of breaks and fragments occurred, and their arrangement became chaotic and the distribution was no longer uniform.

[0054] The KK11% group, which was treated with low-concentration cordyceps extracellular polysaccharides, showed some improvement. The structure of the skin tissue was relatively clear, the boundary between the epidermis and the dermis was clearly discernible, the thickening of the epidermis was alleviated, and the fragmentation, breakage and disorder of the collagen fibers in the dermis were also reduced. The number of collagen fibers in the skin increased, the breakage decreased, and the fiber arrangement began to return to normal.

[0055] The skin condition of mice in the KK13% group coated with high-concentration Cordyceps polysaccharides was significantly improved. The skin structure of each layer became clearer, the connection between the epidermis and the dermis was tighter, the boundary was clearer, the thickening of the epidermis was controlled, there was basically no obvious degeneration of the dermal collagen fibers, the fibers were arranged neatly and orderly, and the subcutaneous hair follicles and glandular structures were normal.

[0056] 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 with a high infection rate, characterized in that: The Cordyceps sinensis strain KK1 is classified and named Hirsutella sinensis, and was deposited on June 7, 2024 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCCNO.41330. The ITS sequence of the strain is shown in SEQ ID NO.

1.

2. Use of the Cordyceps sinensis strain according to claim 1 in artificial cultivation of Cordyceps sinensis infection.

3. Use of the Cordyceps sinensis strain with a high infection rate according to claim 1 in preparing a skin care product with skin photoaging repair function.

4. The use of the Cordyceps sinensis strain with a high infection rate according to claim 3 in the preparation of a skin care product with a skin photoaging repair function, characterized in that: The mycelium, mycelium extract, intracellular polysaccharide and extracellular polysaccharide produced by fermenting the Cordyceps sinensis strain KK1 are applied to skin care products with skin photoaging repair function.

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