RU2788417C1 - Strain producing ergothioneine and its screening method - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: strain of Hericium erinaceus fungus with a depository number CCTCC No. M 2018567 for the production of ergothioneine is proposed. A composition for the production of ergothioneine is also proposed, containing the specified strain of Hericium erinaceus fungus in a fermentable medium containing a carbon source, a nitrogen source, inorganic salts and vitamins.

EFFECT: invention provides high yield of ergothioneine.

5 cl, 5 dwg, 1 tbl, 5 ex

Description

The field of technology to which the invention belongs

The present invention relates to the field of microbiology, and in particular to the HT-3 strain of Hericium erinaceus and to a method for its screening.

Prerequisites for the creation of the invention

Ergothioneine, whose scientific name is trimethyl-2-mercapto-L-histidine internal salt, is a naturally occurring 2-thioimidazole-amino acid. Ergothioneine in solution has two tautomeric isomers, thiol and thione, and exists primarily in the form of thione at physiological pH. Ergothioneine was first isolated by Tanret C. from ergot (a sclerotium formed by a fungus that parasitizes rye, that is, a cereal plant) in 1909. The pure product is white crystals, highly soluble in water up to 0.9 mol/l at room temperature, and is quite stable at physiological pH and in a strong alkaline environment.

Ergothioneine has unique physiological functions such as antioxidant action, free radical scavenging, metal ion chelation, UV damage protection, cancer inhibition, etc., and in some cases, it is superior to natural antioxidants such as glutathione. .

Ergothioneine is present in various plants and animals. However, animals cannot synthesize ergothioneine on their own and can only obtain it from food. Many microorganisms, such as fungi and actinomycetes, can synthesize ergothioneine, but bacteria cannot synthesize this compound on their own. At the moment, even more research has been done on the production of ergothioneine using mushrooms.

As described in Chinese Patent Application CN103184246A, "Method for Biosynthesis of Ergothioneine", ergothioneine is synthesized by inoculating and fermenting the mycelium of macrofungi ( Lepista sordida , Pleurotus plumonarius and Pleurotus sapidus wild type), with the highest content of ergothioneine in the fermented broth being 51 mg/l for Lepista sordida , 48 mg/l for Pleurotus plumonarius and 37 mg/l for wild-type Pleurotus sapidus . The yield of ergothioneine was still low, and the growth cycle of fungi was quite long.

Chinese Patent Application CN103734022A "Ergothioneine Producing Strain and Methods for Producing Ergothionein" discloses the steps for synthesizing ergothioneine by Pleurotus ostreatus fermentation and extracting ergothionein from mycelial cells, but does not disclose a screening method for Pleurotus ostreatus strains.

Chinese patent application CN102978121A "Method for producing ergothioneine by transformation of microorganisms" discloses the production of ergothioneine by transformation of the microorganism Lepista caespitosa . However, biotransformation affects the viability of fungal cells, and the efficiency of such transformation is low.

Hericium erinaceus , also known as the "Lion's Mane" mushroom, is named for its comb-like shape. The hyphae of Hericium erinaceus have thin cell walls with septa and a clasp. Fruiting bodies have the appearance of colonies, a flat-hemispherical or capitate shape with hairy fleshy spines on the surface of the villous part. When fresh, this mushroom is white, but when dried, it is light yellow to light brown in color. This mushroom has a narrow base or a slightly shortened stem, as well as an enlarged upper part with a diameter of 3.5-10 cm. From a distance, it looks like a golden comb, and therefore it is called Hericium erinaceus . Hericium erinaceus is very tasty and the body of the mushroom is fresh, tender and fragrant, and this mushroom is known as "vegetarian meat dish". Hericium erinaceus is not only delicious, but also rich in amino acids, 8 of which are necessary for the human body, and in addition, it is rich in polysaccharides, various vitamins, inorganic salts, peptides and unsaturated fatty acids. Hericium erinaceus is easy to ferment and cultivate, grows quickly, and has a short growth cycle and high yield.

The essence of the invention

In order to solve the problems existing in the field, the present invention was developed relating to the Hericium erinaceus HT-3 strain, which was deposited in the China Type Culture Collection (CCTCC) on August 23, 2018, under the deposit number CCTCC No. M 2018567. The present invention also relates to a composition containing Hericium erinaceus and a fermentable broth and a method for screening Hericium erinaceus . The present invention includes the following embodiments:

1. Mushroom Hericium erinaceus with deposit number CCTCC No. M2018567.

2. The fungus Hericium erinaceus according to item 1, which has the 18s rRNA gene sequence shown in SEQ ID NO: 1

(TTGTACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGATG GTACCTTGCTACATGGATAACTGTGGTAATTCTAGAGCTAATACATGCAATTAAGCC CCGACTTCCGGAAGGGGTGTATTTATTAGATAAAAAACCAACGCGGCTCGCCGCTCC TTTGGTGATTCATAATAACTTCTCGAATCGCATGGCCTTGTGCCGGCGATGCTTCATT CAAATATCTGCCCTATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTTTCAAC GGGTAACGGGGAATAAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACC ACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTA GTGACAATAAATAACAATATAGGGCTCTTTTGGGTCTTATAATTGGAATGAGTACAA TTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGG TAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTG AACTTCAGGCCTGGCCGGGCGGTCTGCCTAACGGTATGTACTGTCTGGCCGGGCCTT ACCTCCTGGTGAGCCGGCATGCCCTTCACTGGGTGTGTCGGGGAACCAGGACTTTTA CCTTGAGAAAATTAGAGTGTTCAAAGCAGGCCTATGCCCGAATACATTAGCATGGA ATAATAAAATAGGACGTGCGGTTCTATTTTGTTGGTTTCTAGAATCGCCGTAATGAT TAATAGGGATAGTTGGGGGCATTAGTATTGCGTTGCTAGAGGTGAAATTCTTGGATT TACGCAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAA CGAAGGTTAGGGGATCGAAAACGATCAGATACCGTTGTAGTCTTAACAGTAAACTA TGCCGACTAGGGATCGGGCGACC TCAATTTAATGTGTCGCTCGGCACCTTACGAGAA ATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAA TTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGG GGAAACTCACCAGGTCCAGACATAACTAGGATTGACAGATTGATAGCTCTTTCTTGA TTTTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAAT TCCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGGCCGGCTTTTGCTGGTCGCTGGCTTCTTAGAGGG).

3. Гриб Hericium erinaceus согласно пункту 1, который имеет последовательность ITS, представленную в SEQ ID NO: 2 (TGCGGAAGGATCATTAATGAATTTGAAAGGAGTTGTTGCTGGCCTGAAACCCAGGC ATGTGCACGCTCCAATCTCATCCATCTTACACCTGTGCACCCTTGCGTGGGTCCGTCG GCTTTGCGGTCGATGGGCTTGCGTTTTTCATAAACTCTTATGTATGTAACAGAATGTC ATAATGCTATAAACGCATCTTATACAACTTTCAACAACGGATCTCTTGGCTCTCGCA TCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAA TCATCGAATCTTTGAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCACGCCTGTT CGAGTGTCGTGAAATTCTCAACTCAATCCTCTTGTTATGAGAGGGCTGGGCTTGGAC TTGGAGGTCTTGCCGGTGCTCCCTCGGGAAGTCGGCTCCTCTTGAATGCATGAGTGG ATCCCTTTTGTAGGGTTTGCCCTTGGTGTGATAATTATCTACGCCGCGGGTAGCCTTG CGTTGGTCTGCTTCTAACCGTCTTCGGACAACTTTCATCTCAACTTGACCTCGAATCA GGCGGGACTACCCGCTGAACTTAAGCATATCA).

4. The fungus Hericium erinaceus according to item 1, which produces ergothioneine in the fermentation broth, wherein the amount of ergothioneine in the fermentation broth is more than 41 mg/l, and where the fermentation broth is obtained from a fermentation medium containing a carbon source, a nitrogen source, inorganic salts and vitamins.

5. A composition containing the fungus Hericium erinaceus and a fermentable broth that contains ergothioneine, where the amount of ergothioneine in the fermentable broth is more than 41 mg/l, and where the fermentable broth is obtained from a fermentation medium containing a carbon source, a nitrogen source, inorganic salts and vitamins.

6. The composition according to paragraph 5, where the fungus Hericium erinaceus is the fungus Hericium erinaceus according to any one of paragraphs 1-4.

7. Method for screening the fungus Hericium erinaceus , comprising the following steps:

(1) placing a tissue block of the fungus Hericium erinaceus on a solid medium with potato dextrose agar containing an antibiotic; cultivation for 5-12 days at 23~28°C to obtain the mycelium of Hericium erinaceus ; inoculation of the mycelium of Hericium erinaceus on a solid medium with potato agar containing dextrose; cultivation for 5-12 days at 23~28°C to obtain a strain of Hericium erinaceus ;

(2) inoculating the Hericium erinaceus strain obtained in step (1) into the fermentation medium; culturing for 12-20 days at 23~26°C, where the precursor substance is fed on days 7-10 during cultivation to obtain a fermentable broth;

(3) centrifuging the fermented broth; removal of the supernatant for filtration; determining the amount of ergothioneine in the filtrate; and screening the strain for ergothioneine production;

preferably, the screening method further comprises, after step (2), the step of extractively soaking the ergothioneine from the mycelial cells outside of them.

8. The screening method according to item 7, wherein the step of extracting ergothioneine from the mycelial cells is carried out by:

homogenizing the fermentable mycelium broth and then heating to extract ergothionein from the mycelial cells beyond them; or

collecting the mycelium by separating the solid and liquid phases in the broth for fermenting the mycelium; obtaining a suspension of mycelium by adding water; homogenization; and subsequent heating to extract ergothioneine from the mycelial cells beyond them.

9. The method for screening the fungus Hericium erinaceus according to item 7, wherein the antibiotic in step (1) comprises any one component or a combination of two or more of these components, namely, penicillin, streptomycin and chloramphenicol.

10. The method for screening the fungus Hericium erinaceus according to item 7, wherein the precursor in step (2) comprises any one component or a combination of two or more of these components, namely cysteine, methionine and histidine.

11. The method for screening the fungus Hericium erinaceus according to item 7, wherein the fermentation medium contains 20 to 60 g/l of carbon source, 10 to 30 g/l of nitrogen source, 2 to 5 g/l of inorganic salts, and 0.001~0.005 g/l l vitamins.

The advantages of the present invention are that the strain of Hericium erinaceus HT-3 CCTCC No.: M 2018567 obtained in accordance with the present invention is easy to cultivate and has a high yield of ergothioneine.

The method for screening the fungus Hericium erinaceus according to the invention is simple, convenient to operate and inexpensive, and it can also be applied to large-scale screening, and can easily select a strain of the fungus with a high yield.

Description of drawings

In FIG. 1 is a photograph illustrating the morphological characteristics of a colony of Hericium erinaceus HT-3 CCTCC NO: M 2018567.

In FIG. 2 is a micrograph of the mycelium of Hericium erinaceus HT-3 CCTCC NO: M 2018567.

In FIG. 3 shows the 18s rRNA gene sequence of Hericium erinaceus HT-3 CCTCC NO: M 2018567.

In FIG. 4 shows the ITS sequence of Hericium erinaceus HT-3 CCTCC NO: M 2018567.

In FIG. 5 is an HPLC chromatogram showing the content of ergothioneine in Hericium erinaceus HT-3 fermentation broth CCTCC NO: M 2018567 determined by HPLC.

Detailed description of the invention

The following description of the present invention is provided only to illustrate specific embodiments thereof, and these variations should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations and simplifications that do not go beyond the essence and principle of the present invention are considered as equivalents and are included in the scope of protection of the invention.

The experimental methods used as described below are conventional unless otherwise noted.

The materials and reagents used below are commercially available unless otherwise noted.

As a specific embodiment of the invention, there is provided a strain of Hericium erinaceus HT-3 CCTCC No: M 2018567 which grows rapidly on dextrose-containing potato agar medium (ie, PDA solid medium). The colony after cultivation at 25°C for 8 days reaches 50-55 mm in diameter, is white, shiny and expands radially, and the base of the colony has a light brown color; see fig. 1 with morphological characteristics of the colony. The hyphae of the strain are strong and thick, composed of tubular cells with thin cell walls, and have septa and branches, as well as buckle-like connections, see FIG. 2.

The 'Lion's Mane' mushroom is Hericium erinaceus (Rull ex F.) and belongs to the genus Hydnaceae, Polyporaceae , Basidiomycetes and Fungi, and is also a saprophyte and a well-known edible mushroom. Its shape resembles that of a hedgehog or a comb, which is why it is commonly referred to as a comb or Lion's Mane mushroom. In the present invention, Hericium erinaceus is preferably Hericium erinaceus CCTCC No. M 2018567, which was deposited with the China Type Culture Collection (CCTCC) on August 23, 2018.

The 18s rRNA gene sequence of Hericium erinaceus HT-3 CCTCC No. M2018567 is shown in SEQ ID NO: 1, see FIG. 3.

ITS sequence Hericium erinaceus HT-3 CCTCC no. M 2018567 is shown in SEQ ID NO: 2, see FIG. four.

As a specific embodiment of the invention, a method for screening an ergothione-producing strain of Hericium ( Hericium erinaceus HT-3) comprises:

(1) excising a tissue block of approximately 0.5×0.5 cm from the fungus Hericium erinaceus and placing on a PDA solid medium containing an antibiotic; culturing for 5-12 days, preferably 7-10 days, at 23~28°C, preferably at 24°C, to obtain Hericium erinaceus mycelium; inoculation of Hericium erinaceus mycelium on solid medium with PDA; cultivation for 5-12 days, preferably for 8 days, at 23~28°C, preferably at 24°C, to obtain the mycelium of Hericium erinaceus ,

where the solid medium with PDA, as usual solid medium, is a semi-synthetic medium and was prepared by leaching a solution obtained from 200 g of potatoes, 20 g of glucose, 15-20 g of agar and 1000 ml of water, without adjusting the pH.

(2) inoculating the Hericium erinaceus strain obtained in step (1) into the fermentation medium; cultivation for 12-20 days, preferably for 15 days, at 23~28°C, preferably at 24°C, where the precursor substance is fed for days 7-10 during cultivation to obtain a fermentable broth;

(3) Centrifuge the fermented broth at 8000~10000 rpm, and preferably 8000 rpm, for 10~20 minutes, preferably for 20 minutes, remove the supernatant to be filtered through a 0.22μm microporous filter, determine the amount ergothioneine in the filtrate and strain screening for ergothioneine production. The tissue block of the fungus Hericium erinaceus in step (1) is a villous part, a junction of the villi and a stem, a middle part and a stem base, and preferably a villous part.

The antibiotic in step (1) comprises any one component or a combination of two or more components, namely penicillin, streptomycin and chloramphenicol, and preferably streptomycin.

The antibiotic concentration is 0.01~0.1g/L.

The precursor substance in step (2) comprises any one component or a combination of two or more components, namely cysteine, methionine and histidine.

The concentration of each of the precursor substances in step (2) is 1~3 mM, respectively.

The determination of the amount of ergothioneine in the filtrate in step (3) is carried out using high performance liquid chromatography. The HPLC conditions can be: chromatographic column: Hypersil ODS C18 column (250 mm×4.6 mm, particle size 5 µm); column temperature: 30°C; mobile phase: acetonitrile-water (3:97); flow rate: 1.0 ml/min; detection wavelength: 254 nm; loading volume: 20 µl.

In a specific embodiment of the invention, the screening method further comprises a step carried out after step (2), namely, extractive soaking of ergothioneine from mycelial cells beyond them. In a specific embodiment, the step of extracting the ergothioneine from the mycelium cells is carried out by homogenizing the fermentable mycelium broth at a speed of from 1000 to 6000 rpm, and preferably at 4000 rpm, for 20-100 minutes, preferably for 60 minutes, followed by by raising the temperature of the fermented broth to 60~100°C, preferably 80°C, and heating for 20~100 minutes, preferably for 50 minutes, followed by extraction of ergothioneine from the mycelial cells beyond them.

In a particular embodiment of the invention, the step of extracting the ergothioneine from the mycelial cells is carried out by collecting the mycelium by separating the solid and liquid phases in the mycelial fermentation broth; obtaining a suspension of mycelium by adding water, followed by homogenization at a speed of 1000 to 6000 rpm, and preferably at 4000 rpm, for 20-100 minutes, and preferably for 60 minutes, followed by increasing the temperature of the fermented broth to 60 ~ 100°C, preferably 80°C, and heating for 20~100 minutes, preferably for 60 minutes, followed by extraction of ergothioneine from mycelial cells beyond them.

The fermentation medium contains 20 to 60 g/L of carbon source, 10 to 30 g/L of nitrogen source, 2 to 5 g/L of inorganic salts, and 0.001~0.005 g/L of vitamins.

In addition, the carbon source contained in the fermentation medium includes any one component or a combination of at least two components, namely, soluble starch, glucose, sucrose, fructose, maltose and cornmeal;

preferably, the carbon source contained in the fermentation medium comprises any one component or a combination of at least two components, namely glucose and maltose.

The nitrogen source contained in the fermentation medium includes any one component or a combination of at least two components, namely, tryptone, yeast powder, soy flour, bran, powdered soy peptide, wheat peptone, casein peptone, liquid corn extract, beef extract and ammonium sulfate.

Preferably, the nitrogen source contained in the fermentation medium includes any one component or combination of at least two components, namely beef extract and tryptone.

The inorganic salt contained in the fermentation medium includes any one of the salts or a combination of at least two of NaH ₂ PO ₄ , K ₂ HPO ₄ , KH ₂ PO ₄ and MgSO ₄ .

Preferably, the inorganic salt contained in the fermentation medium is NaH ₂ PO ₄ .

The vitamins contained in the fermentation medium include any one vitamin or a combination of at least two vitamins, namely vitamin B ₁ , vitamin B ₂ , niacin, pantothenic acid, vitamin B ₆ , vitamin H and vitamin B ₁₂ ;

preferably, the vitamins contained in the fermentation medium include any one vitamin or a combination of two vitamins, namely vitamin B ₁ and niacin.

The present invention also relates to a composition containing Hericium erinaceus HT-3 fermentable broth, wherein said composition contains ergothioneine, the amount of which in the fermentable broth is at least 41 mg/l, preferably at least 60 mg/l, more preferably at least 70 mg/l, even more preferably at least 80 mg/l, and most preferably 90 mg/l, 100 mg/l, 110 mg/l, 120 mg/l, 130 mg/l, 140 mg/l or 150 mg/l. Hericium erinaceus HT-3 is Hericium erinaceus HT-3 CCTCC No. M 2018567 according to the invention.

Examples

Example 1

Screening of Hericium erinaceus strain for ergothioneine production

(1) Tissue blocks of approximately 0.5 x 0.5 cm were excised from the villi of Hericium erinaceus fruiting bodies formed at various sites, placed on PDA solid medium containing antibiotics; cultured at 24°C, and the resulting mycelia were inoculated on solid medium with PDA to purify the culture. Purified strains were inoculated onto a PDA slant and stored at 3~4°C for later use;

(2) The solid strains obtained in step (1) were inoculated into the fermentation medium and cultured at 24° C. for 16 days to obtain a mycelium fermentation broth. The fermentation medium consisted of 35 g/l sucrose, 20 g/l liquid corn extract, 3 g/l KH ₂ PO ₄ , 3 mg/l vitamin B ₁ and water. After a 10 day fermentation, the precursor amino acids cysteine, methionine and histidine were fed, each at a concentration of 2 mM, respectively.

(3) At the end of fermentation, the mycelium fermentation broth was homogenized at 4000 rpm for 60 minutes, and then the homogenized fermentation broth was heated to 80°C for 50 minutes, after which ergothioneine was subjected to soak extraction from the mycelium cells into the fermentation broth, was centrifuged at 8000 rpm for 20 minutes, and then the supernatant was removed for filtration through a microporous 0.22 μm filter, and the strain with the highest content of ergothioneine in the filtrate was selected and named Hericium erinaceus HT-3.

Example 2

Morphological observation of Hericium erinaceus HT-3

Hericium erinaceus HT-3 CCTCC NO: M 2018567 proliferated rapidly on dextrose-containing potato agar media, adhering to the surface of the medium and spreading into the medium from the surface. After cultivation at 25°C for 8 days, the colony reached 50-55 mm in diameter, became white and velvety, and then expanded radially into the environment and had a light brown base. The morphological characteristics of the colony are shown in Fig. 1. The hyphae of the strain were strong and thick, consisted of tubular cells with thin cell walls, and had septa, branches, and buckle joints, see FIG. 2.

Example 3

Identification and classification of Hericium erinaceus HT-3

Hericium erinaceus HT-3 was identified by genome sequencing by Shanghai Shenggong Biological Engineering (Shanghai) Co., Ltd. The strain was identified as Hericium erinaceus and had the following taxonomy: Fungi, Dikarya, Basidiomycota, Agaricomycotina, Agaricomycetidae, Russulales, Hericiaceae, Hericium . The result of 18s rRNA gene sequencing is shown in FIG. 3 and the ITS sequencing result is shown in FIG. four.

Example 4

Ergothioneine detection

The HT-3 fermentation broth of Example 1 was used as a test sample, and the ergothioneine content was determined by high performance liquid chromatography. HPLC conditions: Chromatography column: Hypersil ODS C18 column (250 mm×4.6 mm, particle size 5 µm); column temperature: 30°C; mobile phase: acetonitrile-water (3:97); flow rate: 1.0 ml/min; detection wavelength: 254 nm; loading volume: 20 µl. The chromatogram is shown in Fig. 4, where a) is a standard product (ergothioneine concentration is 9.4 µg/ml) and b) is a fermentable broth for HT-3 fermentation.

Example 5

Fermentation media optimization

In this example, five fermentation media with different components, contents and pH values were selected for the experiment. The composition and pH of each fermentation medium is shown in Table 1 below.

(1) Slanted mycelium of Hericium erinaceus CCTCC NO: M 2018567 was inoculated into liquid seed medium and cultured for 5-10 days at 15~30°C and 100~300 rpm.

Liquid seed medium: 4% (w/v) sucrose, 1.5% (w/v) powdered soy flour, 0.2% (w/v) sodium dihydrogen phosphate, 0.1% (w/v) sodium sulfate and water added to 100%.

(2) The seed broth obtained in step (1) was inoculated into the fermentation medium for fermentation supplemented with precursor substances, and cultured for 7-15 days at 15~30°C and 100~300 rpm. At the end of the fermentation, the fermented broth was collected. Precursor amino acids, cysteine, methionine and histidine, each at a concentration of 2 mm, were added on the 10th day of fermentation.

(3) At the end of fermentation, the mycelium fermentation broth was homogenized at 4000 rpm for 60 minutes, and then the homogenized fermentation broth was heated to 80°C for 50 minutes, ergothionein was extracted from the mycelium cells into the extracellular fermentation broth, centrifuged at 8000 rpm for 20 minutes, after which the supernatant was removed for filtration through a microporous 0.22 μm filter and the content of ergothioneine in the filtrate was determined.

Five different fermentation media were selected and the estimated ergothioneine content in the final filtrate is shown in Table 1.

Table 1

Serial number carbon source Nitrogen source inorganic salts vitamins pH value Ergothioneine content 1 Sucrose, 35 g/l Liquid corn extract, 20 g/l _{KH2PO4 _}
3 g/l Vitamin B ₁
3 mg/l 4.5 132.1 mg/l 2 Soluble starch, 20 g/l Trypton, 30 g/l _{KH2PO4 _}
5 g/l Vitamin _B6
5 mg/l 5.2 41.2 mg/l 3 Glucose, 60 g/l Beef extract, 10 g/l _{K2HPO4 _}
3 g/l Vitamin H
1 mg/l 5.0 150.3 mg/l four Maltose, 40 g/l Casein peptone, 25 g/l _{KH2PO4 _}
2 g/l Niacin
2 mg/l 5.5 115.4 mg/l five Fructose, 40 g/l Wheat peptone, 25 g/l _{NaH2PO4 _}
2 g/l Vitamin _B12
2 mg/l 5.1 83.2 mg/l

Several processes for the production of ergothioneine by biological fermentation have now been described. Thus, for example, ergothion can be obtained, for example, by cultivating the mycelium of Pleurotus eryngii in deep liquid culture with a yield at the end of fermentation reaching 62.20 mg/l; by cultivating the mycelium of Lentinus edodes in deep liquid culture with a yield at the end of fermentation reaching 23.6 mg/l; by liquid fermentation of Lepista sordida with a maximum yield of 51 mg/l; by liquid fermentation of Pleurotus plumonarius with a maximum yield of 48 mg/l; by liquid fermentation of wild-type Pleurotus sapidus with a maximum yield of 37 mg/l. It can be seen that the yields of ergothioneine obtained by such biological fermentation methods are low. However, when using the strain according to the invention, a higher yield of ergothioneine can be obtained under conditions using a slightly optimized medium.

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SEQUENCE LIST

<110> BLOOMAGE BIOTECHNOLOGY CORPORATION LIMITED

SHANDONG BLOOMAGE HYINC BIOPHARM CORPORATION LIMITED

<120> ERGOTHIONEIN-PRODUCING STRAIN AND METHOD FOR ITS SCREENING

<130> PB00324

<141> 2018-12-26

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<170> SIPOSequenceListing 1.0

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cgtaatgatt aatagggata gttgggggca ttagtattgc gttgctagag gtgaaattct

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tggatttacg caagactaac tactgcgaaa gcatttgcca aggatgtttt cattaatcaa

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360

caactcaatc ctcttgttat gagagggctg ggcttggact tggaggtctt gccggtgctc

420

cctcgggaag tcggctcctc ttgaatgcat gagtggatcc cttttgtagg gtttgccctt

480

ggtgtgataa ttatctacgc cgcgggtagc cttgcgttgg tctgcttcta accgtcttcg

540

gacaactttc atctcaactt gacctcgaat caggcgggac tacccgctga acttaagcat

600

atcat

605

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Claims (5)

1. The strain of the fungus Hericium erinaceus with deposit number CCTCC No. M 2018567 for the production of ergothioneine. 2. The strain of the fungus Hericium erinaceus according to claim 1, which has the 18s rRNA gene sequence shown in SEQ ID NO: 1. 3. The strain of the fungus Hericium erinaceus according to claim 1, which has the ITS sequence shown in SEQ ID NO: 2. 4. The strain of Hericium erinaceus according to claim 1, which produces ergothioneine in the fermentation broth, wherein the amount of ergothioneine in the fermentation broth is more than 41 mg/l, and where the fermentation broth is obtained from a fermentation medium containing a carbon source, a nitrogen source, inorganic salts, and vitamins. 5. Composition for the production of ergothioneine, containing a strain of the fungus Hericium erinaceus according to any one of paragraphs. 1-4 in a fermentation medium containing a carbon source, a nitrogen source, inorganic salts and vitamins.

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