New Streptomyces gunnii MKL-1 and application thereof

Abstract

The invention discloses a novel Streptomyces gunnii MKL-1 strain and an application thereof. The strain is separated and purified from cordyceps gunnii collected in the field to obtain the novel Streptomyces gunnii, the strain is high in growth speed and rich in bioactive substances, and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M20251296. The strain is inoculated into a solid culture medium of bran, cane sugar and soybean meal, and a solid fermentation product with 40% of hypha can be obtained after 96 hours at the fermentation temperature of 20 DEG C. Mouse experiments prove that the solid fermentation product of the neogunnesi MKL-1 can enhance the immunocompetence of animals and promote the growth of the animals.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and specifically relates to Neogunickia MKL-1 and applications thereof. Background Art

[0002] Cordyceps gunnii is an important medicinal fungus, widely distributed in high-altitude areas of southwestern and central China, such as Yunnan, Sichuan, Guizhou, and Anhui. Its natural habitat is temperate forests at altitudes between 1,500 and 3,000 meters. It often parasitizes the larvae of Lepidoptera insects, forming an insect-fungus complex. Based on morphological and molecular biological classification, Cordyceps gunnii belongs to the phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Clavicipitaceae, and genus Keithomyces.

[0003] Traditionally used in Traditional Chinese Medicine (TCM), Cordyceps sinensis (Guni) is a plant with rich active ingredients such as adenosine, ergosterol, polysaccharides, and proteins, exhibiting anti-fatigue, immunomodulatory, and anti-tumor effects. In recent years, overexploitation of Cordyceps sinensis resources has led to a sharp decline in wild stocks, prompting research into artificial cultivation and submerged fermentation techniques. Summary of the Invention

[0004] In order to fill the gap in the prior art, the present invention provides a Keithomyces neogunnii MKL-1, which has the advantages of fast growth rate, rich bioactive substances, etc. and is suitable for industrial development.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In a first aspect of the present invention, a strain of Keithomyces neogunnii MKL-1 is provided. The strain is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M20251296.

[0007] In one embodiment, the ITS sequence of the strain is shown as SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a fermentation product of Keithomyces neogunnii MKL-1.

[0009] A third aspect of the present invention provides a method for preparing a fermentation product of Keithomyces neogunnii MKL-1, characterized in that the method comprises the following steps:

[0010] (1) inoculating the Keithomyces neogunnii MKL-1 into a shake flask culture medium for proliferation and culture to obtain a seed solution;

[0011] (2) transferring the seed liquid into a fermentation broth for liquid fermentation, and the fermentation broth can be used as a seed liquid for solid fermentation;

[0012] (3) inoculating the fermentation broth into a solid culture medium for solid fermentation to obtain a fermentation product.

[0013] In one embodiment, in step (2), the liquid fermentation medium is: molasses 5%, ammonium sulfate 2%, KH2PO4 1%, MgSO4 0.5%; the fermentation conditions are: inoculation amount 5%, temperature 18-22°C, supplementation of ammonia water to maintain pH 6.0, minute ventilation volume ratio of 1:1, and fermentation time 24-36 hours.

[0014] In one embodiment, in step (3), the solid matrix is ​​composed of the following components: bran, sucrose and soybean meal powder, with a mass ratio of 100:10:10; the inoculation ratio of the solid culture matrix is ​​0.5L:10kg to 1L:10kg, and the final moisture content is 80%; the fermentation conditions are: material temperature 18-22°C, air humidity 80%, and fermentation period 72-96 hours.

[0015] In a preferred embodiment, after the fermentation is completed, the material is dried and crushed at 60° C. to obtain a solid fermentation product.

[0016] A fourth aspect of the present invention provides the use of Keithomyces neogunnii MKL-1, a fermentation product thereof, or a method for preparing a fermentation product thereof in the preparation of animal feed or feed additives.

[0017] According to a fifth aspect of the present invention, an animal feed is provided, wherein the feed comprises Keithomyces neogunnii MKL-1 or a fermentation product thereof.

[0018] A sixth aspect of the present invention provides a feed additive, wherein the feed additive comprises Keithomyces neogunnii MKL-1 or a fermentation product thereof.

[0019] Compared with the prior art, the present invention has the following novelties and beneficial results:

[0020] The new Gunnichesium MKL-1 strain provided by the present invention has significantly shorter liquid and solid fermentation times than the fermentation cycles of Cordyceps fungi reported in the literature, showing a huge advantage of rapid growth; at the same time, its mycelium is rich in bioactive substances and is suitable for industrial development.

[0021] The fermentation product of Neogunickia MKL-1 provided by the present invention can significantly improve the immune balance of animals and promote the growth of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Colony morphology of Neogunickia MKL-1 growing on PDA plates;

[0023] Figure 2 The microscopic morphology of hyphae and spores of Neogunickia MKL-1 of the present invention is shown in FIG. DETAILED DESCRIPTION

[0024] Example 1 Isolation and Identification of Neogunickia MKL-1

[0025] 1 Isolation of Neogunickia MKL-1

[0026] Gunni Cordyceps was collected from the forest land above 1000 meters above sea level in Guniujiang Nature Reserve (Qimen County), Anhui Province.

[0027] The fresh stroma of Cordyceps sinensis was disinfected with 70% alcohol, cut open and took the middle part and placed on a PDA plate for incubation at 20℃ ( Figure 1 ), after growing hyphae, transfer and purify ( Figure 2 Seven strains were isolated and purified from seven Cordyceps sinensis plants. The purified strains were stored in freezers at -80°C and 4°C. One strain, designated MKL-1, showed significantly more vigorous growth.

[0028] Identification by the China Center for Type Culture Collection (CCTCC) and analysis of the ITS rDNA sequence (SEQ ID NO. 1) indicated that the isolated and purified MKL-1 fungus was Keithomyces neogunnii, belonging to the phylum Ascomycota, class Sordariomycetes, orders Hypocreales, family Clavicipitaceae.

[0029] The strain (Keithomyces neogunnii MKL-1) was deposited in the China Center for Type Culture Collection (CCTCC) on June 6, 2025, with the deposit number CCTCC NO: M20251296. Deposit address: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: (027)-68754052.

[0030] Example 2 Preparation of fermentation products of Neogunickia MKL-1

[0031] 1 shake bottle of seed solution

[0032] Take Keithomyces neogunnii MKL-1 stored at 4°C, inoculate 100ul of the seed solution of 0.05g wet bacteria per ml on a PDA plate, and culture at 20°C for 5 days; transfer the plate colony to a 1000ml Erlenmeyer flask containing 200ml of YPD medium, and culture at 20°C and 150r / min for 3 days to obtain the seed solution.

[0033] 2 Liquid fermentation

[0034] In a 50L airlift fermenter, add 30L of fermentation medium and an appropriate amount of bubble-proofing, and sterilize at 115℃ for 20 minutes; when the temperature drops to 20℃, add 600ml of seed liquid with 0.1g of wet bacteria per milliliter; maintain the temperature in the tank at 20℃, the ventilation volume at 30L / min, ferment for 60h, and then release the tank to obtain the fermentation liquid.

[0035] The fermentation liquid can be directly inoculated into a solid matrix for solid fermentation, or used as a seed liquid for secondary amplification fermentation.

[0036] Liquid fermentation medium: molasses 5%, ammonium sulfate 2%, KH2PO4 1%, MgSO4 0.5%); fermentation conditions: inoculum size 5%, temperature 18-20°C, supplementation of ammonia water to maintain pH 6.0; fermentation time 24-36 hours, the fermentation broth can be used to inoculate solid fermentation matrix.

[0037] If fermentation is continued for 48 hours, 30 grams of dry mycelium can be harvested per liter of fermentation liquid. Analysis of the harvested mycelium reveals that the content of ergosterol is 5000 mg / kg or greater, adenosine is 7000 mg / kg or greater, cordycepin is 500 mg / kg or greater, cordycepic acid is 1.5% or greater, cordyceps polysaccharide is 7% or greater, and ergothioneine is 1000 mg / kg or greater.

[0038] 3 Solid fermentation

[0039] Solid culture medium: Wheat bran, sucrose, and soybean meal powder are in proportions of 100, 10, and 10 parts by weight, respectively. The fermentation broth is inoculated at a ratio of 1 L:10 kg, with a final moisture content of 80%. The material temperature is controlled at 20°C, the air humidity at 80%, and the fermentation cycle is 72 hours. Upon completion of fermentation, the solid fermentation product is dried and pulverized at 60°C.

[0040] The ergosterol content of the fermentation product after drying and crushing was measured to be more than 2000 mg / kg. Compared with the ergosterol content in pure mycelium (5000 mg / kg), it means that the mycelium accounts for as much as 40% in the solid culture.

[0041] Example 3 Effects of Neogunickia MKL-1 Fermentation Products on Immunity and Growth in Mice

[0042] Female SPF C57 mice weighing 18-20 g were randomly divided into 20 groups. The control group received their usual diet, while the two experimental groups received daily oral administration of 0.2 and 0.4 g of a liquid fermentation mycelial suspension of Neogunickia MKL-1 (containing 0.15 g of wet mycelial cells per gram). These mice were fed this diet for 21 days, and their growth was observed and recorded. At the end of the experiment, five mice were randomly selected from each group for blood sampling to measure relevant immunological parameters and organ indices. All antibodies, markers, and other reagents used were purchased from Santa Cruz, USA. The experimental results are shown in Table 1.

[0043] Table 1 Effects of fermentation products of Neogunickia MKL-1 on immunity and growth of mice

[0044]

[0045] Note: a, b respectively indicate the significant difference of each immunological index compared with the control group (P<0.05, P<0.01)

[0046] The results in Table 1 show that when mice were fed with the fermentation product of M. neogunniche MKL-1 for 21 consecutive days, the thymus and spleen indexes and CD4 cell counts increased significantly, indicating that the mobilization ability of the mice's cellular immunity and humoral immunity was improved; there was no significant change in the levels of IgG and IFN-γ, and IL-4 showed an increasing trend.

[0047] In particular, mice fed with the fermentation product of Neogunickia MKL-1 for 21 consecutive days showed significant weight gain.

[0048] The results of mouse experiments showed that oral administration of fermented mycelium of Neogunickia MKL-1 can regulate immune balance, promote growth, and can be used as a feed additive.

[0049] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A strain of Keithomyces neogunnii MKL-1, characterized in that: The strain was deposited in China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: M20251296.

2. The neogunickia MKL-1 according to claim 1, characterized in that The ITS sequence of the strain is shown as SEQ ID NO.

1.

3. The fermentation product of Keithomyces neogunnii MKL-1 according to claim 1.

4. The method for preparing a fermentation product according to claim 3, wherein: The method comprises the following steps: (1) inoculating the Keithomyces neogunnii MKL-1 into a shake flask culture medium for proliferation and culture to obtain a seed solution; (2) transferring the seed liquid into a fermentation broth for liquid fermentation, and the fermentation broth can be used as a seed liquid for solid fermentation; (3) inoculating the fermentation broth into a solid culture medium for solid fermentation to obtain a fermentation product.

5. The preparation method according to claim 4, wherein In step (2), the liquid fermentation medium is: molasses 5%, ammonium sulfate 2%, KH2PO4 1%, MgSO4 0.5%; the fermentation conditions are: inoculation amount 5%, temperature 18-22°C, supplementation of ammonia water to maintain pH 6.0, minute ventilation volume ratio of 1:1, and fermentation time 24-36 hours.

6. The preparation method according to claim 4, wherein In step (3), the solid matrix is ​​composed of the following components: bran, sucrose and soybean meal powder, with a mass ratio of 100:10:10; the inoculation ratio of the solid culture matrix is ​​0.5L:10kg to 1L:10kg, and the final moisture content is 80%; the fermentation conditions are: material temperature 18-22°C, air humidity 80%, and fermentation period 72-96 hours.

7. The preparation method according to claim 6, wherein After the fermentation is completed, the material is dried and crushed at 60°C to obtain a solid fermentation product.

8. Use of the Keithomyces neogunnii MKL-1 according to claim 1 or 2, the fermentation product according to claim 3, or the preparation method according to any one of claims 4 to 7 in the preparation of animal feed or feed additives.

9. An animal feed, characterized in that The feed contains the Keithomyces neogunnii MKL-1 according to claim 1 or 2 or the fermentation product according to claim 3.

10. A feed additive, characterized in that The feed additive comprises the Keithomyces neogunnii MKL-1 according to claim 1 or 2 or the fermentation product according to claim 3.

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