WO2022100568A1 - Tricholoma matsutake and method for producing ergothioneine thereby - Google Patents

Abstract

Provided is Tricholoma Matsutake, wherein the deposit number thereof is CCTCC No: M 2020587. Also provided are a method and a fermentation broth for the production of ergothioneine by means of Tricholoma Matsutake alone or in combination with Hericium erinaceus. The method is simple in terms of process, and not only can active ingredients such as polysaccharides and amino acids in the fermentation broth of Tricholoma Matsutake be retained by means of same, but also the yield of ergothioneine can be increased.

Description

Pine mushroom and method for producing ergothioneine technical field

The invention relates to the technical field of biochemical industry, in particular to a pine mushroom and a method for producing ergothioneine.

Background technique

Ergothioneine (2-mercaptohistidine trimethylbetaine) is a rare amino acid with antioxidant, UV protection and cell repair properties. Ergothioneine exists in many animals and plants, and cannot be synthesized by the animal body itself, but can only be ingested from food. There are three methods for preparing ergothioneine: chemical synthesis, extraction and microbial fermentation. Due to the problems of high cost and unsafety in the preparation of ergothioneine by chemical synthesis and extraction methods, the preparation of ergothioneine products by microbial fermentation has attracted more and more attention.

Matsutake, also known as Matsutake, scientific name Matsutake Mushroom, alias Matsutake, Synthetic Fungus, Taiwan Fungus, belonging to Basidiomycota, Trichoderma, is an ectogenetic mycorrhizal fungus of pine oak and other trees, with a unique strong fragrance, is the world's largest. Rare and precious natural medicinal fungi, my country's second-class endangered protected species. Pine mushrooms are best grown in dry woodlands with few nutrients, usually in autumn, and usually parasitic on the roots of red pine, pine, hemlock, and Japanese hemlock. The main velvet producing areas in my country are Shangri-La velvet producing area, Chuxiong velvet producing area and Yanbian velvet producing area. Studies have shown that pine mushrooms are rich in protein, 18 kinds of amino acids, 14 kinds of essential trace elements, 49 kinds of active nutrients, 5 kinds of unsaturated fatty acids, nucleic acid derivatives, peptides and other rare elements. It also contains 3 kinds of precious active substances, namely double-chain matsutake polysaccharide, matsutake polypeptide and the world's unique anti-cancer substance - matsutake alcohol. king".

Hericium erinaceus is a Hericium erinaceus, belonging to the class of Basidiomycetes. It is a saprophytic fungus and a well-known edible fungus. The whole shape resembles a hedgehog or Hericium, so it is commonly known as Hericium erinaceus or Hericium erinaceus. Li Shizhen's "Compendium of Materia Medica" has a clear record of the medicinal value of Hericium. It is recorded in "Yinshan Zhengyao" of the Yuan Dynasty that Hericium is beneficial to the five internal organs and helps the stomach and digestion functions.

Chinese patent CN109939027A discloses "a method for preparing a cosmetic stock solution containing ergothioneine by fermentation of Hericium erinaceus", which is characterized in that the stock solution containing ergothioneine is obtained by fermenting ergothioneine-containing mycelium for 7-15 days, which can be applied to cosmetics field. Chinese patent CN107708443A discloses "products containing ergothioneine and its preparation method and use of mushroom extracellular fermentation broth", which is characterized in that ergothioneine is obtained by liquid fermentation of mushrooms and removing the mycelium in the fermentation broth. The extracellular fermentation broth and/or the concentrate of the extracellular fermentation broth are provided, and products containing ergothioneine include food, cosmetics and animal feed. Chinese patent CN103734022A discloses "a strain producing ergothioneine and a method for preparing ergothioneine", which is characterized in that ergothioneine is biosynthesized by using Pleurotus chinensis, and then ergothioneine is extracted from mycelial cells, and the The whole process from the seed liquor to the end of fermentation takes 10 to 20 days.

At present, the research on Matsutake mainly focuses on the research of mycelium culture and polysaccharide. The literature "Optimization of Matsutake Mycelium Medium" studies the growth of Matsutake mycelium in several ectomycorrhizal cultures. reproductive status. Master's thesis "Breeding of Matsutake High-yielding Strains, Optimization of Fermentation Process and Research on Biological Activity", using mutagenesis, process optimization and other methods to select strains with high production of dry weight of mycelium, polysaccharide and ergosterol. Master's thesis "Extraction, Separation and Purification of Matsutake Polysaccharides and Research on Antioxidant Activity", researched and screened the most suitable medium for the growth of Matsutake mycelium and the most suitable extraction process of polysaccharides, and isolated and purified 3 kinds of novel polysaccharides.

In summary, the current development and utilization of pine mushroom mycelium mainly have the following shortcomings:

1. The research on pine mushroom mainly focuses on the research of mycelium culture and polysaccharide, while the research on other active components produced by the fermentation of pine mushroom mycelium is less;

2. There are very few studies on the rare amino acid-ergothioneine contained in the fruiting body and mycelium of pine mushrooms;

3. Ergothioneine and pine mushroom polysaccharide have not been comprehensively utilized in the existing research.

SUMMARY OF THE INVENTION

The present invention provides a kind of pine mushroom (Tricholoma Matsutake), which has been deposited in the China Center for Type Culture Collection (CCTCC) in Wuhan University (zip code 430072), Wuhan, China on October 16, 2020, and the deposit number is CCTCC No: M 2020587. The present invention also provides a method for producing ergothioneine from the above-mentioned pine mushrooms, and a method for producing ergothioneine by joint fermentation of the above-mentioned pine mushrooms and Hericium erinaceus. The technical scheme of the present invention is as follows:

1. A pine mushroom (Tricholoma Matsutake), characterized in that the deposit number of the pine mushroom is CCTCC No: M 2020587.

2、如项1所述的松蕈，其特征在于，所述松蕈的18s rRNA基因序列如SEQ ID NO:1(GTCCCTCTAAGAAGCCAGCGGCCAGCAAAAGCCGGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGTTATCG GAATTAACCAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACCACCCATAAAATCATGAAAGAGCTATCAATCTGTCAATCCTAGTTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACACCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCAGAACCCAAAGACTTTGATTTCTCGTAAGGTGCCGAGTAACACATAAATATTGAGATTACCCGATCCCTAGTCGGCATAGTTTACTGTTAAGACTACAACGGTATCTGATCGTTTTCGATCCCCTAACCTTCGTTCTTGATTAATGAAAACATCCTTGGCAAATGCTTTCGCAGTAGTTAGTCTTGAGTCAATCCAAGAATTTCACCTCTAGCGACTCAATACCAATGCCCCCAACTATCCCTATTAATCATTACGGCGACTCTAGAAACCAACAAAATAGAACCGCACGTCCTATTTTATTATTCCATGCTAATGTATTCGGGCATAAGCCTGCTTTGAACACTCTAATTTTCTCAAGGTAAAAGTCCTGGTTCCCCCACGCACACCAATAAAGGGCACGCCGGCTCACCAAGAGGTAAGGCCCAGTCAGACAGTACACACCGTGAGGCGGACCGCCCGACCAGGTCTGAAGTTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTATAAGACCCGAAAGAGCCCTATATTGTTATTTATTGTCACTACCTCCCCGTGTCGGGATTGGGT AATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTTATTCCCCGTTACCCGTTGAAACCATGGTAGGCCTCTATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGAAGCATCGCCGGCACAAGGCCATGCGATTCGAGAAGTTATTATGAATCACCAAGGGAGCGGCGAGCCGCGTTGGTTTTTTATCTAATAAATACACCCCTTCCAGAAGTCGGGGCTTGATTGCATGTATTAGCTCTAGAATTACCACAGTTATCCATGTAGCAAGGTAACATCAAATAAACTATAACTGATTTAATGAGCCATTCGCAGTTTCACAGTACAAATTGT)所示。

3, according to the described pine mushroom of item 1, it is characterized in that, the ITS sequence of described pine mushroom is such as SEQ ID NO:2

(TCATTATTGAATAAAGCTTGGTTAGGTTGTCGCTGGCTCTCCGGGGCATGTGCACGCCTGACGCCAATCTTTTCACCACCTGTGCACATTTTGTAGGCTTGGATAAATATGTCTCGAGGAAGCTCGGTTTGAGGACTGCCGTGCTGCAAAAGCCAGGCTTTCCTTGTATTTTTCCAGCCTATGCATTTTATTATACACTCGGTATGTCATGGAATGTTATTTGGTTGGCTTAATTGCCAGTAAACCTTATACAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATGAAATTCTCAACCTTTTCAGCTTTTTGTTGAATAGGCTTGGATTTTGGGAGTTGTTGCAGGCTGCTCAGAAGTCTGCTCTCCTTAAATGTATTAGCGGGGCCCTTGTTGTCTAGCATTTGGTGTGATAATTATCTACGCCATTGTGAACAATGTAATAGGTCGGCTTCTAATCGTCTCGTAAAGAGACAATCTCTGACATTTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCAT)所示。

4, a method for fermenting pine mushrooms to produce ergothioneine, is characterized in that, comprises the following steps:

The pine mushroom mycelium is inoculated into the seed medium and cultivated to obtain the pine mushroom seed liquid;

The seed liquid is inoculated into a fermentation medium and cultivated to obtain a fermentation liquid;

The fermentation broth is processed to obtain ergothioneine;

Among them, the precursor substances are added during the fermentation process,

Wherein the preservation number of the pine mushroom is CCTCC No: M 2020587.

5. The method according to item 4, wherein the fermentation medium comprises the following components: carbon source 20.0-60.0 g/L, nitrogen source 10.0-30.0 g/L, inorganic salt 0.1-5.0 g/L , vitamin 0.001～0.005g/L, preferably including the following components: glucose 30.0～50.0g/L, wheat peptone 10.0～30.0g/L, inorganic salt 0.1～2.0g/L, niacin 0.001～0.005g/L, Vitamin B ₁ 0.001～0.005g/L.

6. The method according to item 4, wherein the precursor substance comprises one of cysteine, histidine, methionine, aspartic acid, glutamine, betaine or two or more.

7. The method according to item 4, characterized in that the culture conditions for the fermentation culture are: culturing for 20-30 days at 15-30° C. and 100-300 rpm shaking conditions, wherein the precursor is supplemented on the 15-20th day substance.

8, a method for the combined fermentation of pine mushroom and Hericium ergothioneine to produce ergothioneine, is characterized in that, comprises the following steps:

The pine mushroom mycelium is inoculated into the pine mushroom seed medium and cultivated to obtain the pine mushroom seed liquid;

The Hericium erinaceus mycelium is inoculated into the Hericium erinaceus seed medium for cultivation to obtain the Hericium erinaceus seed liquid;

The pine mushroom seed liquid is inoculated into a fermentation medium, and after culturing for a period of time, the Hericium erinaceus seed liquid is inoculated into the fermentation medium for cultivation to obtain a fermentation liquid;

The fermentation broth is processed to obtain ergothioneine;

Among them, the precursor substances are added during the fermentation process.

9. The method according to item 8, wherein the deposit number of the Hericium erinaceus is CCTCC No: M 2018567.

10. The method according to item 8, wherein the deposit number of the pine mushroom is CCTCC No: M 2020587.

11. The method according to item 8, wherein the fermentation medium comprises the following components: carbon source 30.0-100.0 g/L, organic nitrogen source 10.0-100.0 g/L, inorganic salt 0.1-10.0 g/L L, trace elements 0.001-0.01g/L and coenzyme 0.001-0.01g/L, preferably including the following components: glucose 50.0-80.0g/L, beef extract 10.0-30.0g/L, wheat peptone 10.0-30.0g/L , inorganic salt 0.5～2.0g/L, zinc chloride 0.001～0.005g/L, niacin 0.003～0.01g/L, vitamin B ₁ 0.001～0.005g/L.

12. The method according to item 8, wherein the culture conditions for the fermentation culture are as follows: after the pine mushroom seed liquid is inoculated into the fermentation medium, the 15th to 20th day after culturing at 15 to 30°C Insert the Hericium erinaceus seed liquid, after the pine mushroom seed liquid is inoculated into the fermentation medium, supplement the precursor substance on the 20th to 25th day after culturing at 15-30°C, and continue to cultivate for 5- Fermentation broth was obtained in 10 days.

13. The method according to item 8, wherein the precursor substance comprises one of cysteine, histidine, methionine, aspartic acid, glutamine, betaine or two or more.

14. A fermented liquid, characterized in that the fermented liquid comprises ergothioneine and pine mushroom polysaccharide, wherein the concentration of the ergothioneine in the fermented liquid is more than 32mg/L, preferably more than 41mg/L , the concentration of the pine mushroom polysaccharide in the fermentation broth is 1.3 g/L or more, preferably 1.9 g/L or more.

15. The fermentation broth according to item 14, which is produced by fermenting pine mushrooms whose deposit number is CCTCC No: M 2020587.

16. A fermented liquid, characterized in that the fermented liquid comprises ergothioneine and pine mushroom polysaccharide, wherein the concentration of the ergothioneine in the fermented liquid is more than 240mg/L, preferably more than 310mg/L , the concentration of the pine mushroom and Hericium erinaceus polysaccharide in the fermentation broth is above 3.5g/L, preferably above 4.4g/L.

17. The fermentation broth according to item 16, which is produced by fermentation of the pine mushroom with the deposit number CCTCC No: M 2020587 and the Hericium erinaceus with the deposit number CCTCC No: M 2018567.

Tricholoma Matsutake provided by the present invention is easy to cultivate and has high ergothioneine yield;

The screening method for pine mushroom strains provided by the invention has the advantages of simple process, simple and convenient operation, low cost, large-scale screening of bacterial species, and easy screening of high-yield bacterial species.

The method for biosynthesizing ergothioneine by combined fermentation of pine mushroom and Hericium ergonium provided by the invention has simple process, can retain active components such as polysaccharides and amino acids in the pine mushroom fermentation liquid, and can improve the yield of ergothioneine.

Description of drawings

Figure 1 is a photograph of the morphological characteristics of the colony of Tricholoma Matsutake SR-LY CCTCC No: M 2020587.

Figure 2 is a photomicrograph of Tricholoma Matsutake SR-LY CCTCC No: M 2020587 mycelium.

Figure 3 is the result of the 18s rRNA gene sequence determination of Tricholoma Matsutake SR-LY CCTCC No: M 2020587.

Figure 4 is the ITS sequence determination result of Tricholoma Matsutake SR-LY CCTCC No: M 2020587.

Fig. 5 is the HPLC collection of illustrative plates of ergothioneine content determination, wherein, a) is a standard product (the ergothioneine content concentration is 7.05 μg/mL), b) is the sample S1 that embodiment 4 makes, c) is embodiment 5 The prepared sample S2, d) is the sample C1 prepared in the comparative example 1.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, related technical and scientific terms in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or identical to those described herein can be used in experiments or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions therein, will control, and otherwise, the materials, methods and examples are illustrative and not restrictive. The present invention is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present invention.

The experimental methods used below are conventional methods unless otherwise required.

The materials and reagents used below can be obtained from commercial sources unless otherwise specified.

As a specific embodiment of the present invention, Tricholoma Matsutake (the preservation number is CCTCC No: M 2020587) grows faster on the potato dextrose agar medium (i.e. PDA solid medium) containing the pine root extract, and is After growing at 25°C for 20 days, the colony diameter reached 20-25mm, white in color, and the aerial hyphae on the colony surface were kinked, forming a burr-like velvet shape; the morphological characteristics of the colony were shown in Figure 1. The hyphae of this strain consisted of tubular cells with thin walls, distinct diaphragms, and hollows, see Figure 2.

Matsutake is a fungus, Basidiomycetes, Agaric, Tricholoma Matsutake (S.Ito.etImai.) Sing, born under the pine forest, the buds are like deer antlers, hence the name Matsutake, in the "Bacteria Spectrum" written by Chen Renyu in the Song Dynasty Call this fungus the pine mushroom. Among them, the pine mushroom is preferably the pine mushroom (Tricholoma Matsutake) CCTCC No: M 2020587, which has been deposited in the Chinese Type Culture Collection (CCTCC) in Wuhan University, Wuhan, China (zip code 430072) on October 16, 2020.

The 18s rRNA gene sequence of the Tricholoma Matsutake SR-LY CCTCC No: M 2020587 is SEQ ID NO: 1, see Figure 3.

The ITS sequence of the Tricholoma Matsutake SR-LY CCTCC No: M 2020587 is SEQ ID NO: 2, see Figure 4.

As a specific embodiment of the present invention, the screening method of Tricholoma Matsutake comprises:

The pine mushroom tissue block was placed on the potato dextrose agar solid medium containing antibiotics and pine root extract, and cultured at 15 to 30°C for 30 to 60 days to obtain the pine mushroom mycelium; On the potato dextrose agar solid medium of the pine root extract, under the condition of 15-30 ℃, cultivate for 20-30 days to obtain the pine mushroom strain;

inoculating the pine mushroom strain into a fermentation medium, and culturing at 15-30° C. for 20-30 days, wherein the precursor substances are supplemented on the 15-20th day of the culturing to obtain a fermentation broth;

The fermentation broth is centrifuged, the supernatant is taken, filtered, the ergothioneine content of the filtrate is detected, and the strains producing ergothioneine are screened.

Preferably, after the fermentation broth is obtained and before centrifugation of the fermentation broth, the step of leaching ergothioneine from the inside of the mycelium cells to the outside of the cells is also included.

In a specific embodiment, the step of leaching ergothioneine from the inside of the mycelium cells to the outside of the cells is as follows: the mycelium fermented liquid is disintegrated under the condition of 6000-10000rpm using a homogenizer emulsifier Homogenization, preferably 8000rpm, homogeneous for 10-60min, preferably 30min, then the fermentation broth is heated to 60-100°C, preferably 80°C, heated for 20-100min, preferably 50min, ergothioneine is removed from mycelium cells Internal leaching to the outside of the cell.

The antibiotics include any one of penicillin, streptomycin, and chloramphenicol, or a combination of two or more. The concentration of the antibiotic is 0.01-0.1 g/L.

The pine root extract was taken as 200 g of fresh pine roots, washed and cut into small pieces, added with 1 L of water, boiled for 20 minutes, filtered through six layers of gauze, and the filtrate was adjusted to 1 L. The concentration of the pine root extract is 50.0-100.0 mL/L.

The precursor substances include: one or more of cysteine, histidine, methionine, aspartic acid, glutamine, and betaine.

The concentrations of the precursor substances are respectively 1-15 mM.

The content of ergothioneine was determined by high performance liquid chromatography. HPLC conditions: chromatographic column: Hypersil ODS C ₁₈ column (250mm×4.6mm, particle size 5μm); column temperature: 30°C; mobile phase: acetonitrile-water (3 : 97); flow rate: 1.0 mL/min; detection wavelength: 254 nm; injection volume: 20 μL.

In a specific embodiment, the fermentation medium comprises: carbon source 20.0-60.0g/L, nitrogen source 10.0-30.0g/L, inorganic salt 0.1-5.0g/L, vitamin 0.001-0.005g/L , preferably, including the following components: glucose 30.0-50.0g/L, wheat peptone 10.0-30.0g/L, inorganic salt 0.1-2.0g/L, niacin 0.001-0.005g/L, vitamin B ₁ 0.001-0.005 g/L.

Further, the carbon source of the fermentation medium comprises any one or a combination of at least two of soluble starch, glucose, sucrose, fructose, maltose, and corn flour;

Preferably, the carbon source of the fermentation medium comprises: any one or a combination of two of glucose and maltose.

The nitrogen source of the fermentation medium comprises any one or a combination of at least two of tryptone, yeast powder, soybean powder, bran, soybean peptide powder, wheat peptone, casein peptone, corn steep liquor, beef extract, and ammonium sulfate ;

Preferably, the nitrogen source of the fermentation medium comprises: beef extract, wheat peptone or a combination of both.

The inorganic salt of the fermentation medium comprises any one or a combination of at least two of sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium sulfate;

Preferably, the inorganic salt of the fermentation medium is sodium dihydrogen phosphate.

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

Preferably, the vitamins of the fermentation medium comprise: any one or a combination of two of vitamin B ₁ and niacin.

The present invention also provides a method for producing ergothioneine by fermentation of the above-mentioned pine mushrooms, the method comprising the following steps:

The pine mushroom mycelium is inoculated into the seed medium and cultivated to obtain the pine mushroom seed liquid;

The seed liquid is inoculated into a fermentation medium and cultivated to obtain a fermentation liquid;

The fermentation broth is processed to obtain ergothioneine;

Among them, the precursor substances are added during the fermentation process.

In a specific embodiment, the fermentation medium comprises the following components: carbon source 20.0-60.0 g/L, nitrogen source 10.0-30.0 g/L, inorganic salt 0.1-5.0 g/L, vitamin 0.001-0.005 g /L, preferably including the following components: glucose 30.0-50.0g/L, wheat peptone 10.0-30.0g/L, inorganic salt 0.1-2.0g/L, niacin 0.001-0.005g/L, vitamin B ₁ 0.001-0.005 g/L. In a specific embodiment, the culture conditions of the fermentation culture are: culturing for 20-30 days at 15-30° C. and 100-300 rpm shaking conditions, wherein the precursor substances are supplemented on the 15-20th day.

In a specific embodiment, the precursor substances include one or two or more of cysteine, histidine, methionine, aspartic acid, glutamine, and betaine. .

In a specific embodiment, the treatment of the fermentation broth includes homogenizing the fermentation broth, heating, and leaching the ergothioneine in the mycelium cells into the extracellular fermentation broth.

The present invention also provides a fermentation broth, the fermentation broth comprises ergothioneine and pine mushroom polysaccharide, wherein the concentration of the ergothioneine in the fermentation broth is above 32 mg/L, preferably above 41 mg/L, so The concentration of the mushroom polysaccharide in the fermentation broth is 1.3 g/L or more, preferably 1.9 g/L or more.

In a specific embodiment, it is produced by the fermentation of pine mushrooms whose deposit number is CCTCC No: M 2020587.

The present invention also provides a method for producing ergothioneine by joint fermentation of above-mentioned pine mushroom and Hericium erinaceus, comprising the following steps:

The pine mushroom mycelium is inoculated into the pine mushroom seed medium and cultivated to obtain the pine mushroom seed liquid;

The Hericium erinaceus mycelium is inoculated into the Hericium erinaceus seed medium for cultivation to obtain the Hericium erinaceus seed liquid;

The pine mushroom seed liquid is inoculated into a fermentation medium, and after culturing for a period of time, the Hericium erinaceus seed liquid is inoculated into the fermentation medium for cultivation to obtain a fermentation liquid;

The fermentation broth is processed to obtain ergothioneine;

Among them, the precursor substances are added during the fermentation process.

In a specific embodiment, the deposit number of the Hericium erinaceus is CCTCC No: M 2018567.

In a specific embodiment, the fermentation medium comprises the following components: carbon source 30.0-100.0g/L, organic nitrogen source 10.0-100.0g/L, inorganic salt 0.1-10.0g/L, trace element 0.001- 0.01g/L and coenzyme 0.001～0.01g/L.

Further, the carbon source of the fermentation medium includes any one or more of soluble starch, glycerol, glucose, sucrose, fructose, maltose, lactose, mannitol, maltitol, potato starch, galactose, and maltodextrin. combination.

Preferably, the carbon source of the fermentation medium includes one or a combination of two of glucose, sucrose, and maltose.

The nitrogen sources of the fermentation medium include potato extract powder, malt extract powder, beef extract, peptone, yeast powder, yeast extract, soybean powder, wheat peptone, soybean meal powder, soybean meal, bran, casein peptone, tryptone, corn steep liquor The composition of any one or more of , ammonium sulfate, soybean peptide powder and urea.

Preferably, the nitrogen source of the fermentation medium includes one or a combination of two of beef extract, peptone, soybean meal and wheat peptone.

The inorganic salts of the fermentation medium include a combination of any one or more of sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium sulfate, potassium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

Preferably, the inorganic salts of the fermentation medium include sodium sulfate and sodium dihydrogen phosphate.

The trace elements of the fermentation medium include any one or more compositions of manganese sulfate, manganese chloride, ferrous chloride, zinc chloride, and ferrous sulfate.

Preferably, the trace element of the fermentation medium is zinc chloride.

The coenzyme of the fermentation medium includes a combination of any one or more of vitamin B ₁ , biotin, vitamin B ₆ , vitamin B ₁₂ , niacin, pyridoxal phosphate, and riboflavin.

Preferably, the coenzymes of the fermentation medium include niacin and vitamin B ₁ .

In a specific embodiment, the precursor substance comprises a combination of any one or more of cysteine, histidine, methionine, aspartic acid, glutamine, and betaine.

Preferably, the precursor substances include cysteine, methionine and betaine.

In a specific embodiment, the culture conditions of the fermentation culture are as follows: after inoculating the pine mushroom seed liquid into the fermentation medium, inoculation on the 15th to 20th day after culturing under shaking conditions of 15-30°C and 100-300rpm into the Hericium erinaceus seed solution, after the pine mushroom seed solution is inoculated into the fermentation medium, the precursor substance is supplemented on the 20th to 25th day after culturing under the shaking conditions of 15-30° C. and 100-300 rpm, Continue to cultivate for 5 to 10 days to obtain a fermentation broth.

The present invention also provides a fermentation broth, the fermentation broth comprises ergothioneine and pine mushroom polysaccharide, wherein the concentration of the ergothioneine in the fermentation broth is above 240 mg/L, preferably above 310 mg/L, so The concentration of the pine mushroom and Hericium erinaceus polysaccharide in the fermentation broth is above 3.5 g/L, preferably above 4.4 g/L.

In a specific embodiment, the fermentation broth is produced by fermentation of pine mushrooms with a deposit number of CCTCC No: M 2020587 and Hericium erinaceus with a deposit number of CCTCC No: M 2018567.

The pine mushroom of the present invention can produce ergothioneine and pine mushroom polysaccharide in high yield, and the concentration of ergothioneine in the fermentation broth is more than 32 mg/L during single fermentation, and the concentration of pine mushroom polysaccharide in the fermentation broth is more than 1.3 g/L. And when the pine mushroom and Hericium erinaceus are fermented together, the effect is better, in which the concentration of ergothioneine in the fermentation broth can reach more than 240mg/L, and the concentration of pine mushroom and Hericium erinaceus polysaccharide in the fermentation broth can reach 3.5 g/L or more. The method for biosynthesizing ergothioneine from pine mushrooms provided by the invention can not only retain active components such as polysaccharides and amino acids in the pine mushroom fermentation broth, but also provide a new way for the production of ergothioneine.

The following examples of the present invention are only used to illustrate specific embodiments for realizing the present invention, and these embodiments should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention are regarded as equivalent substitution methods and fall within the protection scope of the present invention.

Example

The present invention will be further described below in conjunction with the examples, and it should be understood that the examples are only used to further illustrate and illustrate the present invention, and are not intended to limit the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise required.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Screening of ergothioneine-producing pine mushroom strains

(1) Cut out about 0.5 × 0.5 cm of internal tissue blocks from the fungus fruiting body caps of different origins, place them on PDA solid medium containing antibiotics and pine root extract, cultivate at 24°C, and transfer the obtained mycelia to Received on the PDA solid medium containing the pine root extract, purified and cultivated, the purified bacterial species was inoculated on the PDA slant surface containing the pine root extract, and stored at 3 to 4°C for subsequent use;

(2) inoculate the solid bacterial strain obtained in step (1) into fermentation medium, cultivate at 24° C. for 30 days to obtain mycelium fermentation broth, fermentation medium consists of 30.0g/L of glucose, 25.0g/L of glucose Corn steep liquor, 1.0 g/L dipotassium hydrogen phosphate, 0.003 g/L vitamin B ₆ , and the rest are water. On the 20th day of fermentation, the precursor substances cysteine, methionine and betaine were supplemented at a concentration of 5 mM, respectively.

(3) after the fermentation is finished, the mycelium fermentation broth is homogenized at 8000rpm for 30min, ergothioneine is leached into the extracellular fermentation broth from the intracellular mycelium, centrifuged at 10000rpm for 10min, the supernatant is taken, and the The strain with the highest ergothioneine content in the filtrate was filtered through a 0.22 μm microporous filter and named Tricholoma Matsutake SR-LY.

Example 2 Morphological observation of Tricholoma Matsutake SR-LY

The pine mushroom (Tricholoma Matsutake) CCTCC No: M 2020587 grows faster on the potato dextrose agar medium (i.e. PDA solid medium) containing the pine root extract, grows for 20 days at 25°C, and the colony diameter reaches 20-25mm, It is white, and the aerial hyphae on the colony surface are kinked, forming a burr-like velvet shape; the morphological characteristics of the colony are shown in Figure 1. The hyphae of this strain consisted of tubular cells with thin walls, distinct diaphragms, and hollows, see Figure 2.

Example 3 Identification and classification of Tricholoma Matsutake

The Tricholoma Matsutake was sequenced and identified by Shanghai Sangon Bioengineering (Shanghai) Co., Ltd. The species was identified as Tricholoma Matsutake, and its taxonomy was: Fungi, Dikaryon, Basidiomycetes , Agaricus subphylum, Agaricus, Agaricus subclass, Agaricus, Trichoderma, Trichoderma, its 18s rRNA gene sequence determination results are shown in Figure 3, and the ITS sequence determination results are shown in Figure 4.

Example 4 Fermentation and biosynthesis of ergothioneine by pine mushrooms

(1) Inoculate pine mushroom (Tricholoma Matsutake) CCTCC No: M 2020587 mycelium slant strain into the liquid seed medium, and cultivate for 25 days at 20 ° C, pH 5.0, and 150 rpm shaking conditions to obtain pine mushroom seeds liquid. The seed medium is composed of 30.0g/L lactose, 20.0g/L potato extract powder, 1.0g/L dipotassium hydrogen phosphate, 1.0g/L sodium sulfate, and the rest is water, pH 4.5, at 121 ° C Sterilize for 20min;

(2) Inoculate the pine mushroom seed liquid obtained in step (1) into the fermentation medium, culture at 23° C. and 180 rpm shaking conditions for 25 days, and supplement the precursor material on the 20th day of the culture to obtain a fermentation broth. The fermentation medium is composed of 40.0g/L glucose, 15.0g/L wheat peptone, 0.3g/L sodium dihydrogen phosphate, 0.002g/L niacin, 0.001g/L vitamin B ₁ , and the rest is water , pH 4.5, and sterilized at 121 °C for 20 min. The precursor substances were cysteine, methionine and betaine, with a concentration of 5 mM, respectively.

(3) after the fermentation is finished, the mycelium fermentation broth is homogenized at 8000rpm for 30min, ergothioneine is leached into the extracellular fermentation broth from the intracellular mycelium, centrifuged at 10000rpm for 10min, the supernatant is taken, and the Filtration through a 0.22 μm microporous filter to extract thioneine in the mycelial cells into the fermentation broth outside the cells to obtain thioneine extract S1.

Example 5 Combined fermentation and biosynthesis of ergothioneine by Pine mushroom and Hericium erinaceus

(1) Inoculate pine mushroom (Tricholoma Matsutake) CCTCC No: M 2020587 mycelial slant strain into the liquid seed medium, and cultivate for 25 days at 20 ° C, pH 5.0, and 150 rpm shaking conditions to obtain pine mushroom seed liquid . The seed medium is composed of 30.0g/L lactose, 20.0g/L potato extract powder, 1.0g/L dipotassium hydrogen phosphate, 1.0g/L sodium sulfate, and the rest is water, pH 4.5, sterilized at 121°C bacteria 20min;

(2) Hericium erinaceus CCTCC No: M 2018567 mycelium slant strain was inoculated into the liquid seed medium, and cultured for 7 days at 20° C., pH 5.0, and 150 rpm shaking conditions to obtain Hericium erinaceus seed fluid. The seed medium is composed of 40.0g/L sucrose, 15.0g/L soybean meal powder, 2.0g/L sodium dihydrogen phosphate, 1.0g/L sodium sulfate, and the rest is water, pH 4.5, sterilized at 121°C 20min;

(3) inoculate the pine mushroom seed liquid obtained in step (1) into the fermentation medium, and under the condition of shaking at 23° C. and 180 rpm, cultivate for 25 days, wherein the monkey obtained in step (2) is inserted on the 15th day of the cultivation. The head fungus seed liquid was supplemented with precursor substances on the 20th day to obtain a fermentation broth. The fermentation medium consists of 70.0g/L glucose, 20.0g/L beef extract, 15.0g/L wheat peptone, 0.7g/L sodium dihydrogen phosphate, 0.3g/L sodium sulfate, 0.003g/L Zinc chloride, 0.006g/L niacin, 0.001g/L vitamin B ₁ , the rest are water, pH 4.5, sterilized at 121°C for 20min. The precursor substances were cysteine, methionine and betaine, with a concentration of 5 mM, respectively.

(4) after the fermentation ends, the mycelium fermentation broth is homogenized at 8000rpm for 30min, ergothioneine is leached into the extracellular fermentation broth from the intracellular mycelium, centrifuged at 10000rpm for 10min, the supernatant is taken, and the Filtration through a 0.22 μm microporous filter, and leaching the intracellular ergothioneine in the mycelium into the extracellular fermentation broth to obtain the thioneine leaching solution S2.

Comparative Example 1 Ergothioneine biosynthesis by Hericium erinaceus

(1) Inoculate Hericium erinaceus CCTCC No: M 2018567 mycelium slant strain into liquid seed medium, and cultivate for 7 days at 23°C, pH 5.0, 200rpm shaking conditions to obtain Hericium erinaceus seed fluid. The seed medium is composed of 40.0g/L sucrose, 15.0g/L soybean meal powder, 2.0g/L sodium dihydrogen phosphate, 1.0g/L sodium sulfate, and the rest is water, pH 4.5, sterilized at 121°C 20min;

(2) inoculate the Hericium erinaceus seed liquid obtained in step (1) into the fermentation medium, and cultivate for 10 days at 23° C. and 180 rpm shaking conditions, wherein the precursor substances are supplemented on the 3rd day of the cultivation to obtain a fermentation broth . The fermentation medium consists of 40.0g/L glucose, 15.0g/L beef extract, 0.5 g/L sodium dihydrogen phosphate, 0.02g/L sodium sulfate, 0.002g/L zinc chloride, 0.006g/L niacin, the rest is water, pH 4.5, sterilized at 121°C for 20min. The precursor substances were cysteine, methionine and betaine, with a concentration of 5 mM, respectively.

(3) after the fermentation is finished, the mycelium fermentation broth is homogenized at 8000rpm for 30min, ergothioneine is leached into the extracellular fermentation broth from the intracellular mycelium, centrifuged at 10000rpm for 10min, the supernatant is taken, and the Filter through a 0.22 μm microporous filter, and extract thioneine in the mycelial cells into the fermentation broth outside the cells to obtain thioneine extract C1.

Test Example 1 Detection of ergothioneine content

The content of ergothioneine was determined by high performance liquid chromatography. HPLC conditions: chromatographic column: Hypersil ODS C18 column (250mm×4.6mm, particle size 5μm); column temperature: 30℃; mobile phase: acetonitrile-water (3∶97 ); flow rate: 1.0 mL/min; detection wavelength: 254 nm; injection volume: 20 μL. The ergothioneine content in the samples S1 and S2 prepared by Examples 4 and 5, and the ergothioneine content in the sample C1 prepared by Comparative Example 1 are as shown in Table 1. In the fermentation process of pine mushrooms, the combined fermentation of Hericium erinaceus can greatly increase the amount of ergothioneine in the fermentation broth. The content of ergothioneine. The chromatogram is as shown in Figure 5, wherein a) is the standard product (the ergothioneine content concentration is 7.05 μg/mL), b) is the sample S1 prepared in Example 4, and c) is the sample S2 prepared in Example 5 , d) is the sample C1 prepared in Comparative Example 1.

Table 1 Comparison of ergothioneine content in different samples

sample	Ergothioneine content (mg/L)
S1	48.7
S2	423.9
C1	403.5

It can be known from Table 1 that the pine mushroom provided by the invention can high-yield ergothioneine compared with the existing pine mushroom, thereby expanding the approach of producing ergothioneine, and the method for the combined fermentation of the pine mushroom provided by the invention and the Hericium ergonium, the same Compared with single pine mushroom fermentation, the yield of ergothioneine can be greatly improved.

Test example 2 Amino acid content detection

For samples S1 and S2 prepared in Examples 4 and 5, and sample C1 prepared in Comparative Example 1, the content of free amino acids in the samples was determined by HPLC pre-column derivatization method, and the results are shown in Table 2. As can be seen from the results in the table, pine mushrooms can produce a variety of amino acids during fermentation, especially when combined with Hericium erinaceus, the amino acids produced are higher.

Table 2 Comparison of amino acid content in different samples

amino acid	S1(mg/100mL)	S2(mg/100mL)	C1(mg/100mL)
aspartic acid	0.793	1.821	0.901
Glutamate	0.804	1.542	0.714
Serine	0.251	0.489	0.245
Histidine	0.137	0.754	0.618
Glycine	0.332	0.502	0.139
Threonine	0.229	0.519	0.327
Arginine	0.324	0.601	0.228
Alanine	0.553	0.807	0.309
tyrosine	0.201	0.453	0.132
cysteine	-	0.335	0.302
Valine	0.432	0.753	0.311
methionine	0.319	0.774	0.437
Phenylalanine	0.332	0.687	0.317
Isoleucine	0.274	0.621	0.316
Leucine	0.407	0.802	0.369
Lysine	0.324	0.598	0.262
Proline	0.205	0.217	-
total	5.917	12.275	5.927

Test Example 3 Fungal polysaccharide content detection

The invention adopts the phenol-sulfuric acid method to detect the polysaccharide content.

(1) Instruments: Visible-UV spectrophotometer, analytical balance (0.0001g) accuracy, vortex mixer

(2) Reagents:

2.1 Standard solution: Accurately weigh 100.0 mg of dry and constant weight glucose (analytical grade) into a volumetric flask, add water to make up to 250.0 mL, shake well and accurately draw 10.0 mL of the solution, add water to make up to 100.0 mL.

2.2 Preparation of 80% phenol solution: Accurately pipette 80.0mL of redistilled phenol, add distilled water to make up to 100.0mL to obtain 80% phenol solution, and store in a brown bottle away from light.

2.3 Preparation of 6% phenol solution: dilute 80% phenol solution with water to 6%, and prepare it for immediate use.

2.4 Concentrated sulfuric acid (superior pure)

(3) Detection:

3.1 Make a standard curve: Pipette 0.0, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of glucose standard solution into a stoppered test tube, respectively, and add water to make up to 2.0 mL. Add 1.0 mL of 6% phenol solution respectively, and after mixing evenly, quickly add 5.0 mL of concentrated sulfuric acid (when concentrated sulfuric acid is added in the air, it cannot be attached to the wall), shake it up immediately, and measure the absorbance at 490 nm after 20 minutes of reaction at room temperature, and use 0 tube as a blank control. The ordinate is the polysaccharide concentration, the abscissa is the absorbance, and the standard curve is obtained.

3.2 Sample preparation: Weigh the samples S1 and S2 prepared in Examples 4 and 5, respectively, and 0.2 ml of each sample C1 prepared in Comparative Example 1 to be placed in a 50.0 mL volumetric flask, add an appropriate amount of water, and after it is completely dissolved, add water to the mark. As a stock solution, shake well. Measure 5.0mL of the stock solution before use, put it in a 50.0mL volumetric flask, add water to the mark, and then dilute it 10 times in the same way. Take 2.0 mL in a stoppered test tube, and operate in the same way as the above "add 1.0 mL of 6% redistilled phenol" to obtain the polysaccharide concentration of the sample to be tested from the standard curve, and calculate the polysaccharide content according to the dilution ratio. The results are shown in Table 3. . As can be seen from the data in Tables 1, 2 and 3, the fermentation broth of the combined fermentation of pine mushrooms and Hericium erinaceus not only has higher ergothioneine yields, but also maintains high yields of amino acids and polysaccharides. Among them, pine mushroom polysaccharide has various effects. It is reported that pine mushroom polysaccharide has a good whitening effect, so it has a very good application prospect.

Table 3 Comparison of polysaccharide content in different samples

sample	Polysaccharide content (g/L)
S1	2.34
S2	5.28
C1	2.57

Test Example 4 Fermentation liquid whitening effect test

Preparation of the sample solution: using the serum-containing complete culture medium as the diluent, the samples S1 and S2 in Examples 4 and 5 and the sample C1 in Comparative Example 1 were respectively prepared into solutions with a volume concentration of 5%, 0.22 μm filter membrane Filter sterilized.

Take logarithmic growth phase B16 mouse melanoma cells, inoculate 2×10 ⁴ cells/mL in a 6-well culture plate, 3.0 mL per well, incubate in a carbon dioxide incubator at 37°C, 5% CO ² for 24 h, discard the old Culture medium, the normal control group and model group were added with 3.0 mL of serum-containing culture medium, the experimental group was added with 3.0 mL of sample solution, and 60.0 μL of forskolin solution (2.0 mM) was added to each well of the model group and experimental group to stimulate the production of melanin. , continue to cultivate for 72h.

The measurement method of intracellular melanin content is as follows: trypsin digestion, centrifuge to remove the supernatant, add 500uL of 1.0mol/L (containing 10% DMSO) NaOH solution to lyse the cells, heat at 80°C for 30min, centrifuge at 3000rpm for 10min, take the supernatant and add 96-well plate, 100uL/well, measure the absorbance at 450nm.

The calculation method of the inhibition rate of total melanin content is as follows.

The obtained results of melanin inhibition of each sample are shown in Table 4.

Table 4 Effect of each sample on melanin content

sample	Inhibition rate of total melanin content/%
S1	20.35
S2	25.62
C1	6.11

From the results in the table, it can be seen that the fermentation broth of pine mushroom and the fermentation broth of pine mushroom combined with Hericium erinaceus have better whitening effect than the fermentation broth of Hericium erinaceus, which may be the pine mushroom polysaccharide or other pine mushroom mycelium. The resulting active substances work.

Claims (17)

1. A pine mushroom (Tricholoma Matsutake), characterized in that the deposit number of the pine mushroom is CCTCC No: M 2020587.
2. pine mushroom as claimed in claim 1, is characterized in that, the 18s rRNA gene sequence of described pine mushroom is as shown in SEQ ID NO:1.
3. pine mushroom according to claim 1, is characterized in that, the ITS sequence of described pine mushroom is as shown in SEQ ID NO:2.
4. A method for producing ergothioneine by fermentation of pine mushrooms, characterized in that, comprising the following steps:

   The pine mushroom mycelium is inoculated into the seed medium and cultivated to obtain the pine mushroom seed liquid;

   The seed liquid is inoculated into a fermentation medium and cultivated to obtain a fermentation liquid;

   The fermentation broth is processed to obtain ergothioneine;

   Among them, the precursor substances are added during the fermentation process,

   Wherein the preservation number of the pine mushroom is CCTCC No: M 2020587.
5. The method according to claim 4, wherein the fermentation medium comprises the following components: carbon source 20.0-60.0 g/L, nitrogen source 10.0-30.0 g/L, inorganic salt 0.1-5.0 g/L, Vitamin 0.001-0.005g/L, preferably including the following components: glucose 30.0-50.0g/L, wheat peptone 10.0-30.0g/L, inorganic salt 0.1-2.0g/L, niacin 0.001-0.005g/L, vitamin B ₁ 0.001～0.005g/L.
6. The method according to claim 4, wherein the precursor substance comprises one or two of cysteine, histidine, methionine, aspartic acid, glutamine and betaine more than one species.
7. The method according to claim 4, wherein the culture conditions of the fermentation culture are: culturing for 20-30 days under the shaking conditions of 15-30°C and 100-300 rpm, wherein the precursor substances are supplemented on the 15-20th day .
8. A method for producing ergothioneine by combined fermentation of pine mushroom and Hericium ergonium, comprising the following steps:

   The pine mushroom mycelium is inoculated into the pine mushroom seed medium and cultivated to obtain the pine mushroom seed liquid;

   The Hericium erinaceus mycelium is inoculated into the Hericium erinaceus seed medium for cultivation to obtain the Hericium erinaceus seed liquid;

   The pine mushroom seed liquid is inoculated into a fermentation medium, and after culturing for a period of time, the Hericium erinaceus seed liquid is inoculated into the fermentation medium for cultivation to obtain a fermentation liquid;

   The fermentation broth is processed to obtain ergothioneine;

   Among them, the precursor substances are added during the fermentation process.
9. The method according to claim 8, wherein the deposit number of the Hericium erinaceus is CCTCC No: M 2018567.
10. The method according to claim 8, wherein the deposit number of the pine mushroom is CCTCC No: M 2020587.
11. The method according to claim 8, wherein the fermentation medium comprises the following components: carbon source 30.0-100.0 g/L, organic nitrogen source 10.0-100.0 g/L, inorganic salt 0.1-10.0 g/L , trace elements 0.001-0.01g/L and coenzyme 0.001-0.01g/L, preferably including the following components: glucose 50.0-80.0g/L, beef extract 10.0-30.0g/L, wheat peptone 10.0-30.0g/L, Inorganic salt 0.5～2.0g/L, zinc chloride 0.001～0.005g/L, niacin 0.003～0.01g/L, vitamin B ₁ 0.001～0.005g/L.
12. The method according to claim 8, wherein the culturing conditions of the fermentation culture are as follows: after the pine mushroom seed liquid is inoculated into the fermentation medium, the inoculation is carried out on the 15th to 20th day after culturing at 15-30°C. into the Hericium erinaceus seed solution, after the pine mushroom seed solution is inoculated into the fermentation medium, the precursor material is supplemented on the 20th to 25th day after culturing at 15-30°C, and the culture is continued for 5-10 days. day to get the fermentation broth.
13. The method according to claim 8, wherein the precursor substance comprises one or two of cysteine, histidine, methionine, aspartic acid, glutamine and betaine more than one species.
14. A fermented liquid, characterized in that the fermented liquid comprises ergothioneine and pine mushroom polysaccharide, wherein the concentration of the ergothioneine in the fermented liquid is more than 32 mg/L, preferably more than 41 mg/L, so The concentration of the mushroom polysaccharide in the fermentation broth is 1.3 g/L or more, preferably 1.9 g/L or more.
15. The fermented liquid according to claim 14, which is produced by the fermentation of pine mushrooms whose deposit number is CCTCC No: M 2020587.
16. A fermented liquid, characterized in that the fermented liquid comprises ergothioneine and pine mushroom polysaccharide, wherein the concentration of the ergothioneine in the fermented liquid is more than 240mg/L, preferably more than 310mg/L, so The concentration of the pine mushroom and Hericium erinaceus polysaccharide in the fermentation broth is above 3.5 g/L, preferably above 4.4 g/L.
17. The fermented liquid according to claim 16, which is produced by fermentation of the pine mushroom with the deposit number CCTCC No: M 2020587 and the Hericium erinaceus with the deposit number CCTCC No: M 2018567.

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