Application of L-asparagine and / or L-threonine in promoting cordyceps sinensis germ spores to develop and form hyphae

Abstract

The invention discloses an application of L-asparagine and L-threonine in promoting growth of cordyceps sinensis germ spores to form hyphae. The L-asparagine and / or the L-threonine are / is added into the spores of the cordyceps sinensis, so that the conversion rates of the spores are increased (averagely 45.5% and 49.0% respectively in 6 days) and are obviously higher than the mycelium conversion rate (averagely 24.0% in 6 days) of a control group without adding the L-asparagine and the L-threonine, and the yield of the spores is increased (averagely 45.5% and 49.0% respectively in 6 days) and is obviously higher than that of the control group without adding the L-asparagine and the L-threonine. The L-asparagine and / or the L-threonine are / is added into the cordyceps sinensis germ spores, so that the ratio of converting the cordyceps sinensis germ spores into hyphae can be obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of artificial cultivation technology of Cordyceps sinensis, specifically involving the application of L-asparagine and / or L-threonine in promoting the development of Cordyceps sinensis bud spores and the formation of mycelia. Background Technology

[0002] Cordyceps sinensis is a complex of the cadaver and fruiting body of the ghost moth larvae formed by the fungus *Ophiocordyceps sinensis* infecting them. It has a long history of use in food and medicine and holds significant medicinal and edible value. Cordyceps sinensis is rich in polysaccharides, sterols, amino acids, nucleosides, fatty acids, flavonoids, and various inorganic elements and volatile substances. These chemical components give Cordyceps sinensis significant health benefits and pharmacological effects, exhibiting good efficacy in improving immunity, anti-inflammation, anti-oxidation, anti-aging, and protecting bodily functions. Due to its significant health benefits and pharmacological effects, Cordyceps sinensis is highly favored by consumers. Its enormous commercial value has led to over-harvesting, resulting in a gradual decline in wild resources and damage to the high-altitude ecological environment of its production areas. Furthermore, the huge market demand for Cordyceps sinensis as a traditional Chinese medicine means that the increasingly scarce wild resources can hardly meet market demand. After decades of independent research and development, a comprehensive artificial cultivation technology system for Cordyceps sinensis has been established. This system includes artificial cultivation techniques for Cordyceps sinensis fruiting bodies, large-scale artificial rearing techniques for the host insect, the ghost moth, at low altitudes, and artificial cultivation techniques for obtaining Cordyceps sinensis comparable to wild Cordyceps sinensis by infecting ghost moth larvae with Cordyceps sinensis fungus. This has greatly promoted the effective protection of the plateau ecological environment and precious wild resources, as well as the high-quality development of the bioeconomy. However, the core technology that needs to be overcome in the artificial cultivation of Cordyceps sinensis is improving the efficiency of the conversion of Cordyceps sinensis bud spores into mycelia.

[0003] Fungal dimorphism refers to the ability of some fungi to transform their cell morphology between yeast-like and hyphal forms under the influence of environmental factors. *Cordyceps sinensis* is a typical example of dimorphic fungi (budding spores transform into prehyphae and hyphae). Currently, it is known that N-acetylglucosamine, proline, farnesol, tyrosol, methylfarnesyl ester, ecdysone, mannitol, naftifine hydrochloride, and tryptophan can promote the transformation of *Cordyceps sinensis* budding spores into hyphae to varying degrees in vitro.

[0004] L-Asparagine, one of the 20 most common amino acids, belongs to the α-amino acid family and contains an amide group in its side chain. It is used to treat abnormal liver function, heart disease, and fatigue recovery. Its biological activities, such as its involvement in protein glycosylation and tumor treatment, are currently a hot research topic. However, the effect of L-asparagine at appropriate concentrations on promoting the development of mycelia from budspores of Cordyceps sinensis in vitro has not been reported.

[0005] L-Threonine is an essential amino acid that humans and animals cannot synthesize, but it can be synthesized in plants and microorganisms. L-Threonine is widely used in medicine, food, and animal feed. In the human body, it participates in protein synthesis and plays an important role in maintaining nitrogen balance, delaying aging, enhancing immunity, and promoting growth and development. However, the effect of L-asparagine at appropriate concentrations on promoting the development of mycelia from bud spores of Cordyceps sinensis in vitro has not been reported. Summary of the Invention

[0006] Based on the above problems, the purpose of this invention is to provide an application of L-asparagine and / or L-threonine to promote the development of mycelia from bud spores of Cordyceps sinensis in vitro, thereby solving the problem of low sclerosing rate in artificially cultured Cordyceps sinensis.

[0007] The present invention has found that adding L-asparagine and / or L-threonine to the culture medium of Cordyceps sinensis buds can promote the transformation of buds into hyphae.

[0008] Therefore, the present invention provides the use of L-asparagine and / or L-threonine in the preparation of formulations that promote the transformation of Cordyceps sinensis bud spores into mycelia.

[0009] Preferably, the inoculation dose of Cordyceps sinensis bud spores is 10 × 10⁻⁶ per milliliter. 6 indivual.

[0010] Preferably, the concentration of the L-asparagine and / or L-threonine in the culture medium is 0.002-0.2 mM. More preferably, it is 0.02 mM.

[0011] The present invention also provides an formulation for promoting the transformation of Cordyceps sinensis bud spores into mycelia, which contains L-asparagine and / or L-threonine as active ingredients.

[0012] The formulation may contain available excipients.

[0013] The present invention also provides a method for promoting the transformation of Cordyceps sinensis bud spores into mycelia, which involves adding L-asparagine and / or L-threonine to the culture system of Cordyceps sinensis bud spores.

[0014] This invention addresses the problem of low sclerotization rate of Cordyceps sinensis by adding L-asparagine to the budspores of Cordyceps sinensis, thereby improving the budspore conversion rate (average 45.5% at 6 days). Adding L-threonine to the budspores of Cordyceps sinensis further improves the budspore conversion rate (average 49.0% at 6 days), significantly higher than the mycelial conversion rate of the control group without L-asparagine and L-threonine (average 24.0% at 6 days). This indicates that adding L-asparagine and / or L-threonine to the budspores of Cordyceps sinensis can significantly improve the mycelial conversion rate. Attached Figure Description

[0015] Figure 1 This indicates that L-asparagine and L-threonine promote the conversion of Cordyceps sinensis budspores into prehyphae. Addition of budspores (final concentration 1.0 × 10⁻⁶) 7 The concentrations of L-proline (0.2 mM, 0.02 mM, 0.002 mM), L-asparagine (0.2 mM, 0.02 mM, 0.002 mM), and L-threonine (0.2 mM, 0.02 mM, 0.002 mM) in each well were measured. The control was sterile deionized water. On day 6, the percentage of budding spores converted to hyphae in each well was examined under a microscope. Data are presented as mean ± standard error. Detailed Implementation

[0016] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0017] Example 1:

[0018] The Cordyceps sinensis strain O. sinensis was isolated from wild Cordyceps sinensis in Qinghai (No.: QH1208) by tissue isolation method [Cao, L., Ye, Y., & Han, R. (2015). Fruiting body production of the medicinal Chinese caterpillar mushroom, Ophiocordyceps sinensis (Ascomycetes), in artificial medium. International Journal of Medicinal Mushrooms, 17(11), 1107–1112. The applicant also holds and deposits it for 20 years from the date of the applicant and makes it available to the public]. After molecular identification, it was stored at -80℃. Cordyceps sinensis liquid culture medium (PMG): 200g peeled potato juice, 20g maltose, 10g peptone, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 0.02g vitamin B1, and 5g fresh moth larvae (grinded) are mixed and diluted to 1L with distilled water. The mixture is dispensed into 150mL 250mL Erlenmeyer flasks and autoclaved at 121℃ for 30min. After cooling, it is used. On a clean bench, the solid-cultured Cordyceps sinensis blocks (approximately 0.5cm) are placed... 3 Inoculate the above-mentioned liquid culture medium and incubate on a shaker at 120 rpm and 9-13℃ for 45 days, then collect budding spores. The budding spore collection method is as follows: Filter the liquid culture medium through three layers of sterile lens paper, collect the filtrate into a 50 mL sterile centrifuge tube, centrifuge at 8000 rpm and 10℃ for 15 min, discard the supernatant, resuspend in sterile phosphate-buffered saline (PBS; pH = 6.2), centrifuge again and discard the supernatant, then dilute the collected budding spores with sterile PBS to a concentration of 1.0 × 10⁻⁶. 8 1 / mL was used for subsequent experiments. PM liquid culture medium was also added to the 96-well plate.

[0019] PM liquid culture medium: Boil 200g of peeled potatoes to extract juice, add 20g of maltose, 10g of peptone, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, and 0.02g of vitamin B1. Mix the ingredients and bring the volume to 1L with distilled water. Autoclave at 121℃ for 30 minutes and use after cooling.

[0020] Using sterile deionized water as a solvent, L-asparagine (a product of McLean) was prepared into concentrations of 2mM, 0.2mM, and 0.02mM, and then filtered through a 0.22μm bacterial filter (Millipore) before use.

[0021] Using sterile deionized water as a solvent, L-threonine (a product of McLean) was prepared into concentrations of 2mM, 0.2mM, and 0.02mM, and then filtered through a 0.22μm bacterial filter (Millipore) before use.

[0022] Using sterile deionized water as a solvent, L-proline (a product of McLean) was prepared into concentrations of 2mM, 0.2mM, and 0.02mM, and then filtered through a 0.22μm bacterial filter (Millipore) before use.

[0023] Add 80 μL of sterile PM liquid medium and 10 μL of budding spores (1.0 × 10⁻⁶) of Cordyceps sinensis strain O. sinensis to each well of a 96-well plate. 8 10 μL of L-asparagine, L-threonine, and L-proline solutions (2 mM, 0.2 mM, and 0.002 mM respectively) were added to each well to achieve final concentrations of 0.2 mM, 0.02 mM, and 0.0002 mM for each of the budding spores (per mL). The control was sterile deionized water. The total volume per well was 100 μL. The plates were then shaken for 5 min using a micro-shaker, sealed with sealing film, and placed in a sterile plastic box for incubation at 12–14 °C. Each treatment was replicated in quadruplicate. On day 6, the number of budding spores and prehyphae was accurately counted using a hemocytometer. The entire experiment was repeated three times.

[0024] Figure 1 The results showed that microscopic examination on day 6 revealed the addition of budding spores (final concentration 1.0 × 10⁶). 7 The mycelial conversion rates were compared with those of different concentrations of L-asparagine solutions (number of mycelia / mL). The average mycelial conversion rates with 0.2 mM, 0.02 mM, and 0.0002 mM L-asparagine (45.1%, 45.5%, and 42.7%, respectively) were significantly higher than those with sterile deionized water (24.0%). These results demonstrate that L-asparagine promotes the conversion of Cordyceps sinensis buds into mycelia.

[0025] Figure 1 The results showed that microscopic examination on day 6 revealed the addition of budding spores (final concentration 1.0 × 10⁶). 7 The mycelial conversion rates were compared with those of different concentrations of L-threonine solutions (number of spores / mL). The average mycelial conversion rates with 2 mM, 0.2 mM, and 0.02 mM L-threonine (46.0%, 49.0%, and 44.7%, respectively) were significantly higher than those with sterile deionized water (24.0%). These results demonstrate that L-threonine promotes the conversion of Cordyceps sinensis buds into mycelia.

[0026] Figure 1 The results showed that microscopic examination on day 6 revealed the addition of budding spores (final concentration 1.0 × 10⁶).7 The mycelial conversion rates were compared with those of different concentrations of L-proline solutions (number of mycelia / mL). The average mycelial conversion rates with the addition of 2 mM, 0.2 mM, and 0.02 mM L-proline (38.1%, 33.4%, and 41.1%, respectively) were all higher than those with sterile deionized water (24.0%). These results demonstrate that L-proline promotes the conversion of Cordyceps sinensis buds into mycelia.

[0027] In summary, L-proline also promotes spore-prehyphae transformation, but its effect is not as good as that of L-asparagine and L-threonine. Figure 1 As shown, 0.02 mM L-proline showed no significant difference compared to the control.

Claims

1. Application of L-asparagine and / or L-threonine in promoting the development of mycelium from bud spores of Cordyceps sinensis.

2. Application of L-asparagine and / or L-threonine in the preparation of formulations that promote the transformation of Cordyceps sinensis bud spores into mycelia.

3. The application according to claim 1 or 2, characterized in that, The inoculation dose of Cordyceps sinensis bud spores is 10 × 10⁻⁶ per milliliter. 6 indivual.

4. The application according to claim 1 or 2, characterized in that, The concentration of L-asparagine and / or L-threonine in the budding spore culture system is 0.002-0.2 mM.

5. The application according to claim 4, characterized in that, The concentration of L-asparagine and / or L-threonine in the budding spore culture system is 0.02 mM.

6. A preparation for promoting the transformation of Cordyceps sinensis bud spores into mycelia, characterized in that, It contains L-asparagine and / or L-threonine as active ingredients.

7. The formulation according to claim 5, characterized in that, The formulation contains available excipients.

8. A method for promoting the transformation of Cordyceps sinensis bud spores into mycelia, characterized in that, L-asparagine and / or L-threonine are added to the culture system of Cordyceps sinensis bud spores.

9. The method according to claim 8, characterized in that, The concentration of the L-asparagine and / or L-threonine is 0.002-0.2 mM.

10. The method according to claim 9, characterized in that, The concentration of the L-asparagine and / or L-threonine is 0.02 mM.

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