CN112501029A - Armillaria matsutake and method for producing ergothioneine by using same - Google Patents
Armillaria matsutake and method for producing ergothioneine by using same Download PDFInfo
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Abstract
The invention provides agaricus (Tricholoma Matsutake), which has a preservation number of CCTCC No: m2020587. The invention also provides a method for producing ergothioneine by using the agaricus bisporus. The screening method of the agaricus bisporus strains provided by the invention has the advantages of simple process, simple and convenient operation and low cost, can screen strains on a large scale, and is easy to screen high-yield strains. The method for biologically synthesizing ergothioneine by combined fermentation of agaricus and hericium has simple process, not only can retain active ingredients such as polysaccharide, amino acid and the like in agaricus fermentation liquor, but also can improve the yield of the ergothioneine.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to agaricus bisporus and a method for producing ergothioneine by using the agaricus bisporus.
Background
Ergothioneine (2-mercaptophilidine trimethylacetate) is a rare amino acid and has antioxidant, ultraviolet protective and cell repairing effects. Ergothioneine is present in many animals and plants, cannot be synthesized by the animal body itself, and can only be taken in from food. There are three methods for preparing ergothioneine: chemical synthesis, extraction and microbial fermentation. Because the chemical synthesis and extraction methods for preparing ergothioneine have the problems of high cost, insecurity and the like, microbial fermentation methods for preparing ergothioneine products are receiving more and more attention.
The matsutake, also known as matsutake, academia matsutake, also known as matsutake, other name of pine vine, fungus combination and Taijun fungus, belongs to basidiomycotina and tricholoma family, is an ectotrophic mycorrhizal fungus of trees such as quercus robur and the like, has unique and strong fragrance, is rare and rare natural medicinal fungus in the world, and is a secondary endangered protected species in China. Matsutake is good in a dry forest land with few nutrients, is generally generated in autumn, and is usually parasitic on the roots of red pine, elytrigia repens, hemlock and Japanese hemlock. The main antler producing areas in China include Shangri-La antler producing areas, Chuxiong antler producing areas, edge extending antler producing areas and the like. The study proves that the agaricus is rich in protein, and has 18 kinds of amino acids, 14 kinds of essential trace elements for human body, 49 active nutrients, 5 kinds of unsaturated fatty acids, nucleic acid derivatives, peptide substances and other rare elements. In addition, the fungus contains 3 kinds of precious active substances, namely double-chain tricholoma matsutake polysaccharide, tricholoma matsutake polypeptide and unique anti-cancer substance-tricholoma matsutake alcohol all over the world, is the most precious natural medicinal fungus all over the world and is known as the king of the fungus.
Hericium erinaceus is Hericium erinaceus of Hericium of Hydnaceae of Hydnales, belongs to Basidiomycetes, is a saprophytic fungus, and is also a famous edible fungus. It is generally called hedgehog fungus or hericium erinaceus because it is similar to hedgehog fungus or hericium erinaceus. The medicinal value of Hericium erinaceus from Ben Cao gang mu of Li Shizhen is well documented. Recorded in Yuanchao 'Zhengyuan' diet, Hericium erinaceus has the functions of benefiting the five internal organs, helping digestion of intestines and stomach, etc.
Chinese patent CN109939027A discloses a method for preparing a chemical cosmetic stock solution containing ergothioneine by fermenting hericium erinaceus, which is characterized in that the stock solution containing the ergothioneine is obtained by fermenting hericium erinaceus mycelium for 7-15 days, and can be applied to the field of cosmetics. Chinese patent CN107708443A discloses a product containing ergothioneine, a preparation method thereof and application of mushroom extracellular fermentation liquid, which is characterized in that the ergothioneine is provided by extracellular fermentation liquid obtained after mushroom is subjected to liquid fermentation and mycelium in the fermentation liquid is removed and/or concentrate of the extracellular fermentation liquid, and the product containing the ergothioneine comprises food, cosmetics and animal feed. Chinese patent CN103734022A discloses a bacterial strain for producing ergothioneine and a method for preparing the ergothioneine, which is characterized in that the ergothioneine is biosynthesized by pleurotus ostreatus, then the ergothioneine is extracted from mycelium cells, and the whole process from the preparation of seed liquid to the end of fermentation needs 10-20 days.
At present, researches on agaricus blazei are mainly focused on mycelium culture and polysaccharide researches, and the growth and propagation conditions of the agaricus blazei mycelium in a culture medium of several exogenous mycorrhiza fungi are researched in the literature of 'optimization research on the agaricus blazei mycelium culture medium'. The Master thesis 'matsutake high-yield strain breeding, fermentation process optimization and biological activity research' adopts modes of mutagenesis, process optimization and the like to breed strains with high mycelium dry weight, polysaccharide and ergosterol. The Master thesis 'research on extraction, separation and purification of tricholoma matsutake polysaccharides and antioxidant activity' researches and screens out a culture medium which is optimal for growth of tricholoma matsutake mycelia and an optimal extraction process of polysaccharides, and separates and purifies 3 novel polysaccharides.
In summary, the current development and utilization of agaricus bisporus mycelium mainly have the following defects:
1. the study on agaricus bisporus mainly focuses on mycelium culture and polysaccharide research, and less researches on other active ingredients produced by fermentation of agaricus bisporus mycelium;
2. the research on the rare amino acid-ergothioneine contained in the fruiting body and mycelium of matsutake is very rare;
3. in the existing research, ergothioneine and agaricus bisporus polysaccharide are not comprehensively utilized.
Disclosure of Invention
The invention provides agaricus (Tricholoma Matsutake), which has been preserved in China center for type culture Collection (CCTCC for short) in Wuhan university in Wuhan City in 2020, 10, 16 months, with the preservation number of CCTCC No: m2020587. The invention also provides a method for producing ergothioneine by using the agaricus and a method for producing the ergothioneine by using the agaricus and hericium erinaceus through combined fermentation. The technical scheme of the invention is as follows:
1. a Matsutake mushroom (Tricholoma Matsutake) characterized in that the preservation number of the Matsutake mushroom is CCTCC No: m2020587.
2. The Armillaria matsutake of item 1, wherein the 18s rRNA gene sequence of Armillaria matsutake is represented by SEQ ID NO:1 (GTCCCTCTAAGAAGCCAGCGGCCAGCAAAAGCCGGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGTTATCG GAATTAACCAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACCACCCATAAAATCATGAAAGAGCTAT CAATCTGTCAATCCTAGTTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACA CCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCAGAACCCAAAGACTTTG ATTTCTCGTAAGGTGCCGAGTAACACATAAATATTGAGATTACCCGATCCCTAGTCGGCATAGTTTACTGTTAAGA CTACAACGGTATCTGATCGTTTTCGATCCCCTAACCTTCGTTCTTGATTAATGAAAACATCCTTGGCAAATGCTTTC GCAGTAGTTAGTCTTGAGTCAATCCAAGAATTTCACCTCTAGCGACTCAATACCAATGCCCCCAACTATCCCTATT AATCATTACGGCGACTCTAGAAACCAACAAAATAGAACCGCACGTCCTATTTTATTATTCCATGCTAATGTATTCG GGCATAAGCCTGCTTTGAACACTCTAATTTTCTCAAGGTAAAAGTCCTGGTTCCCCCACGCACACCAATAAAGGGC ACGCCGGCTCACCAAGAGGTAAGGCCCAGTCAGACAGTACACACCGTGAGGCGGACCGCCCGACCAGGTCTGAA GTTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCAC CAGACTTGCCCTCCAATTGTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTATAAGACCCGAAAGAGCCC TATATTGTTATTTATTGTCACTACCTCCCCGTGTCGGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTG GTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTTATTCCCCGTTACCCGTTGAAACCATGGTAGGCCTCT ATCCTACCATCGAAAGTTGATAGGGCAGATATTTGAATGAAGCATCGCCGGCACAAGGCCATGCGATTCGAGAAG TTATTATGAATCACCAAGGGAGCGGCGAGCCGCGTTGGTTTTTTATCTAATAAATACACCCCTTCCAGAAGTCGGG GCTTGATTGCATGTATTAGCTCTAGAATTACCACAGTTATCCATGTAGCAAGGTAACATCAAATAAACTATAACTG ATTTAATGAGCCATTCGCAGTTTCACAGTACAAATTGT).
3. The Armillaria matsutake of item 1, wherein the ITS sequence of Armillaria matsutake is as set forth in SEQ ID NO 2
(TCATTATTGAATAAAGCTTGGTTAGGTTGTCGCTGGCTCTCCGGGGCATGTGCACGCCTGACGCCAATCTTTTC ACCACCTGTGCACATTTTGTAGGCTTGGATAAATATGTCTCGAGGAAGCTCGGTTTGAGGACTGCCGTGCTGCAAA AGCCAGGCTTTCCTTGTATTTTTCCAGCCTATGCATTTTATTATACACTCGGTATGTCATGGAATGTTATTTGGTTGG CTTAATTGCCAGTAAACCTTATACAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAA ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTC CGAGGAGCATGCCTGTTTGAGTGTCATGAAATTCTCAACCTTTTCAGCTTTTTGTTGAATAGGCTTGGATTTTGGGA GTTGTTGCAGGCTGCTCAGAAGTCTGCTCTCCTTAAATGTATTAGCGGGGCCCTTGTTGTCTAGCATTTGGTGTGAT AATTATCTACGCCATTGTGAACAATGTAATAGGTCGGCTTCTAATCGTCTCGTAAAGAGACAATCTCTGACATTTT GACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCAT).
4. A method for producing ergothioneine by fermenting agaricus, which is characterized by comprising the following steps:
inoculating Armillaria matsutake mycelium into seed culture medium, and culturing to obtain Armillaria matsutake seed solution;
inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation liquid;
processing the fermentation liquor to obtain ergothioneine;
wherein, precursor substances are supplemented in the fermentation process,
wherein the preservation number of the agaricus bisporus is CCTCC No: m2020587.
5. The method of item 4, wherein the fermentation medium comprises the following components: 20.0-60.0 g/L of carbon source, 10.0-30.0 g/L of nitrogen source, 0.1-5.0 g/L of inorganic salt and 0.001-0.005 g/L of vitamin, and preferably comprises the following components: 30.0-50.0 g/L glucose, 10.0-30.0 g/L wheat peptone, 0.1-2.0 g/L inorganic salt, 0.001-0.005 g/L nicotinic acid, vitamin B1 0.001～0.005g/L。
6. The method according to item 4, wherein the precursor substance comprises one or more of cysteine, histidine, methionine, aspartic acid, glutamine, and betaine.
7. The method according to item 4, wherein the culture conditions of the fermentation culture are: culturing for 20-30 days at 15-30 deg.C under 100-300 rpm shaking condition, wherein the precursor is supplemented in 15-20 days.
8. A method for producing ergothioneine by combined fermentation of agaricus and hericium erinaceus is characterized by comprising the following steps:
inoculating Armillaria matsutake mycelium into Armillaria matsutake seed culture medium, and culturing to obtain Armillaria matsutake seed solution;
inoculating Hericium erinaceus mycelium into Hericium erinaceus seed culture medium, and culturing to obtain Hericium erinaceus seed liquid;
inoculating the agaricus bisporus seed liquid into a fermentation culture medium, and inoculating the hericium erinaceus seed liquid into the fermentation culture medium for culture after a period of culture to obtain a fermentation liquid;
processing the fermentation liquor to obtain ergothioneine;
wherein, precursor substances are supplemented in the fermentation process.
9. The method according to item 8, wherein the preservation number of the hericium erinaceus is CCTCC No: m2018567.
10. The method according to item 8, wherein the agaricus bisporus is deposited in a preservation number of CCTCC No: m2020587.
11. The method of claim 8, wherein the fermentation medium comprises the following components: 30.0-100.0 g/L of carbon source, 10.0-100.0 g/L of organic nitrogen source, 0.1-10.0 g/L of inorganic salt, 0.001-0.01 g/L of trace element and 0.001-0.01 g/L of coenzyme, and preferably comprises the following components: 50.0-80.0 g/L glucose, 10.0-30.0 g/L beef extract, 10.0-30.0 g/L wheat peptone, 0.5-2.0 g/L inorganic salt, 0.001-0.005 g/L zinc chloride, 0.003-0.01 g/L nicotinic acid, vitamin B1 0.001～0.005g/L。
12. The method according to item 8, wherein the culture conditions of the fermentation culture are: inoculating the agaricus bisporus seed liquid to a fermentation culture medium, inoculating the hericium erinaceus seed liquid on the 15 th to 20 th days after the agaricus bisporus seed liquid is cultured at the temperature of 15-30 ℃, supplementing the precursor substance on the 20 th to 25 th days after the agaricus bisporus seed liquid is inoculated to the fermentation culture medium, and continuously culturing for 5-10 days to obtain a fermentation liquid.
13. The method according to claim 8, wherein the precursor substance includes one or more of cysteine, histidine, methionine, aspartic acid, glutamine, and betaine.
14. A fermentation broth comprising ergothioneine and matsutake polysaccharides, wherein the concentration of the ergothioneine in the fermentation broth is 32mg/L or more, preferably 41mg/L or more, and the concentration of the matsutake polysaccharides in the fermentation broth is 1.3g/L or more, preferably 1.9g/L or more.
15. The fermentation broth according to item 14, which is prepared from a microorganism having a preservation number of CCTCC No: m2020587 is obtained by fermenting Armillaria matsutake.
16. A fermentation broth, comprising ergothioneine and matsutake mushroom polysaccharide, wherein the ergothioneine concentration in the fermentation broth is 240mg/L or more, preferably 310mg/L or more, and the matsutake mushroom and hericium erinaceus polysaccharide concentration in the fermentation broth is 3.5g/L or more, preferably 4.4g/L or more.
17. The fermentation broth according to item 16, which is prepared from a microorganism having a preservation number of CCTCC No: the agaricus bisporus of M2020587 and the preservation number are CCTCC No: m2018567 by fermenting Hericium erinaceus.
The agaricus (Tricholoma Matsutake) provided by the invention is easy to culture, and the yield of ergothioneine is high;
the screening method of the agaricus bisporus strains provided by the invention has the advantages of simple process, simple and convenient operation and low cost, can screen strains on a large scale, and is easy to screen high-yield strains.
The method for biologically synthesizing ergothioneine by combined fermentation of agaricus and hericium erinaceus, which is provided by the invention, has a simple process, not only can retain active ingredients such as polysaccharide, amino acid and the like in agaricus fermentation liquor, but also can improve the yield of ergothioneine.
Drawings
FIG. 1 shows Armillaria Matsutake SR-LY CCTCC No: photograph of colony morphology features of M2020587.
FIG. 2 shows Armillaria Matsutake SR-LY CCTCC No: photomicrographs of the M2020587 mycelia.
FIG. 3 is Armillaria Matsutake SR-LY CCTCC No: the 18s rRNA gene sequence of M2020587 was determined.
FIG. 4 shows Armillaria Matsutake SR-LY CCTCC No: ITS sequencing of M2020587.
FIG. 5 is an HPLC chart for ergothioneine content determination, wherein a) is a standard (the concentration of ergothioneine content was 7.05. mu.g/mL), b) is sample S1 obtained in example 4, C) is sample S2 obtained in example 5, and d) is sample C1 obtained in comparative example 1.
Detailed description of the invention
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention will be further described with reference to the following specific examples, which should not be construed as limiting the scope of the invention.
The following experimental methods are all conventional methods unless otherwise specified.
The materials, reagents and the like used below are commercially available unless otherwise specified.
As a specific embodiment of the invention, the growth of pine mushroom (Tricholoma Matsutake) (preservation number is CCTCC No: M2020587) on a potato glucose agar culture medium (namely PDA solid culture medium) containing pine root extract is faster, the growth is carried out for 20 days at 25 ℃, the diameter of a colony reaches 20-25 mm, the colony is white, and aerial hyphae on the surface of the colony are twisted to form a burr-shaped velvet shape; the morphological characteristics of the colonies are shown in FIG. 1. The hyphae of the strain consists of tubular cells, has thin walls and obvious transverse septa, is hollow, and is shown in figure 2.
The Matsutake is Tricholoma Matsutake (S.Ito.ethimai.) Sing of Tricholomataceae of Agaricales of Basidiomycetes, and is originated from pine forest with bud such as cornu Cervi Pantotrichum, so it is named as Matsutake in "mycology" of Song Dynasty Chenren Yu. Wherein, the agaricus is preferably agaricus (Tricholoma Matsutake) CCTCC No: m2020587, which has been preserved in China Center for Type Culture Collection (CCTCC) at 16/10/2020.
The agaricus (Tricholoma Matsutake) SR-LY CCTCC No: the 18s rRNA gene sequence of M2020587 is SEQ ID NO 1, see FIG. 3.
The agaricus (Tricholoma Matsutake) SR-LY CCTCC No: the ITS sequence of M2020587 is SEQ ID NO 2, see FIG. 4.
As a specific embodiment of the present invention, a screening method of agaricus (Tricholoma Matsutake) includes:
placing the agaricus bisporus tissue blocks on a potato glucose agar solid culture medium containing antibiotics and pine root leachate, and culturing for 30-60 days at 15-30 ℃ to obtain agaricus bisporus hyphae; transferring the agaricus bisporus mycelium to a potato glucose agar solid culture medium containing pine root leachate, and culturing at 15-30 ℃ for 20-30 days to obtain agaricus bisporus strain;
inoculating the agaricus bisporus strains to a fermentation culture medium, and culturing for 20-30 days at 15-30 ℃, wherein precursor substances are supplemented in 15-20 days of culture to obtain fermentation liquor;
and centrifuging the fermentation liquor, taking supernatant, filtering, detecting the ergothioneine content of filtrate, and screening bacterial strains producing the ergothioneine.
Preferably, after obtaining the fermentation broth, before centrifuging the fermentation broth, a step of leaching ergothioneine from the mycelium cells to the outside of the cells is further included.
In one embodiment, the step of leaching ergothioneine from the mycelium cells intracellularly to the outside is: and (2) scattering and homogenizing the mycelium fermentation liquor by using a homogenizing emulsifying machine under the condition of 6000-10000 rpm, preferably 8000rpm, homogenizing for 10-60 min, preferably 30min, then heating the fermentation liquor to 60-100 ℃, preferably 80 ℃, heating for 20-100 min, preferably 50min, and leaching the ergothioneine from the inside of the mycelium cell to the outside of the cell.
The antibiotic comprises any one or a combination of more than two of penicillin, streptomycin and chloramphenicol. The concentration of the antibiotic is 0.01-0.1 g/L.
The pine root leachate is prepared by taking 200g of fresh pine roots, cleaning, cutting into small blocks, adding 1L of water, boiling for 20min, filtering by using six layers of gauze, and fixing the volume of the filtrate to 1L. The concentration of the pine root leachate is 50.0-100.0 mL/L.
The precursor material comprises: one or more of cysteine, histidine, methionine, aspartic acid, glutamine and betaine.
The concentration of the precursor substance is 1-15 mM.
The content of ergothioneine is measured by adopting a high performance liquid chromatography, and the HPLC conditions are as follows: a chromatographic column: hypersil ODS C18Column (250 mm. times.4.6 mm, particle size 5 μm); column temperature: 30 ℃; mobile phase: acetonitrile-water (3: 97); flow rate: 1.0 mL/min; detection wavelength: 254 nm; sample introduction amount: 20 μ L.
In one embodiment, the fermentation medium comprises: 20.0-60.0 g/L of carbon source, 10.0-30.0 g/L of nitrogen source, 0.1-5.0 g/L of inorganic salt and 0.001-0.005 g/L of vitamin, preferably, the carbon source comprises the following components: 30.0-50.0 g/L glucose, 10.0-30.0 g/L wheat peptone, 0.1-2.0 g/L inorganic salt, 0.001-0.005 g/L nicotinic acid, vitamin B1 0.001～0.005g/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: glucose and maltose.
The nitrogen source of the fermentation medium comprises any one or a composition of at least two of tryptone, yeast powder, soybean meal, 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 their mixture.
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: vitamin B1Vitamin B2Nicotinic acid, pantothenic acid and vitamin B6Vitamin H, vitamin B12Any one or a combination of at least two of (a);
preferably, the vitamins of the fermentation medium comprise: vitamin B1And nicotinic acid or a combination of both.
The invention also provides a method for producing ergothioneine by fermenting the agaricus bisporus, which comprises the following steps:
inoculating Armillaria matsutake mycelium into seed culture medium, and culturing to obtain Armillaria matsutake seed solution;
inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation liquid;
processing the fermentation liquor to obtain ergothioneine;
wherein, precursor substances are supplemented in the fermentation process.
In a specific embodiment, the fermentation medium comprises the following components: 20.0-60.0 g/L of carbon source, 10.0-30.0 g/L of nitrogen source, 0.1-5.0 g/L of inorganic salt and 0.001-0.005 g/L of vitamin, and preferably comprises the following components: 30.0-50.0 g/L glucose, 10.0-30.0 g/L wheat peptone, 0.1-2.0 g/L inorganic salt, 0.001-0.005 g/L nicotinic acid, vitamin B10.001 to 0.005 g/L. In a specific embodiment, the culture conditions of the fermentation culture are: culturing for 20-30 days at 15-30 deg.C under shaking at 100-300 rpm, wherein precursor is supplemented on 15-20 days.
In a specific embodiment, the precursor material comprises one or two or more of cysteine, histidine, methionine, aspartic acid, glutamine, betaine. .
In a specific embodiment, the fermentation broth is treated by homogenizing the broth, heating, and leaching the ergothioneine inside the mycelial cells into the extracellular fermentation broth.
The invention also provides a fermentation liquid, which comprises ergothioneine and agaricus polysaccharide, wherein the concentration of the ergothioneine in the fermentation liquid is more than 32mg/L, preferably more than 41mg/L, and the concentration of the agaricus polysaccharide in the fermentation liquid is more than 1.3g/L, preferably more than 1.9 g/L.
In a specific embodiment, the culture medium is prepared from a collection number of CCTCC No: m2020587 is produced by fermenting Armillaria matsutake.
The invention also provides a method for producing ergothioneine by combined fermentation of agaricus and hericium erinaceus, which comprises the following steps:
inoculating Armillaria matsutake mycelium into Armillaria matsutake seed culture medium, and culturing to obtain Armillaria matsutake seed solution;
inoculating Hericium erinaceus mycelium into Hericium erinaceus seed culture medium, and culturing to obtain Hericium erinaceus seed liquid;
inoculating the agaricus bisporus seed liquid into a fermentation culture medium, and inoculating the hericium erinaceus seed liquid into the fermentation culture medium for culture after a period of culture to obtain a fermentation liquid;
processing the fermentation liquor to obtain ergothioneine;
wherein, precursor substances are supplemented in the fermentation process.
In a specific embodiment, the preservation number of the hericium erinaceus is CCTCC No: m2018567.
In a specific embodiment, the fermentation medium comprises the following components: 30.0-100.0 g/L of carbon source, 10.0-100.0 g/L of organic nitrogen source, 0.1-10.0 g/L of inorganic salt, 0.001-0.01 g/L of trace element and 0.001-0.01 g/L of coenzyme.
Further, the carbon source of the fermentation medium comprises any one or more of soluble starch, glycerol, glucose, sucrose, fructose, maltose, lactose, mannitol, maltitol, potato starch, galactose and maltodextrin.
Preferably, the carbon source of the fermentation medium comprises one or a combination of two of glucose, sucrose and maltose.
The nitrogen source of the fermentation medium comprises one or more of potato extract powder, malt extract powder, beef extract, peptone, yeast powder, yeast extract, soybean meal, wheat peptone, soybean cake powder, soybean meal, bran, casein peptone, tryptone, corn steep liquor, ammonium sulfate, soybean peptide powder and urea.
Preferably, the nitrogen source of the fermentation medium comprises one or a combination of two of beef extract, peptone, soybean meal and wheat peptone.
The inorganic salt of the fermentation medium comprises 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 microelements of the fermentation medium comprise one or more 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 comprises vitamin B1Biotin, vitamin B6Vitamin B12A combination of any one or more of nicotinic acid, pyridoxal phosphate and riboflavin.
Preferably, the coenzyme of the fermentation medium comprises niacin and vitamin B1。
In a particular embodiment, the precursor material comprises a combination of any one or more of cysteine, histidine, methionine, aspartic acid, glutamine, betaine.
Preferably, the precursor substances include cysteine, methionine and betaine.
In a specific embodiment, the culture conditions of the fermentation culture are: inoculating the agaricus bisporus seed liquid to a fermentation culture medium, inoculating the hericium erinaceus seed liquid on 15-20 days after the agaricus bisporus seed liquid is cultured under the condition of oscillation at 100-300 rpm at 15-30 ℃, supplementing the precursor substance on 20-25 days after the agaricus bisporus seed liquid is inoculated to the fermentation culture medium and is cultured under the condition of oscillation at 100-300 rpm at 15-30 ℃, and continuously culturing for 5-10 days to obtain a fermentation liquid.
The invention also provides a fermentation liquid, which comprises ergothioneine and agaricus polysaccharide, wherein the concentration of the ergothioneine in the fermentation liquid is more than 240mg/L, preferably more than 310mg/L, and the concentration of the agaricus polysaccharide and the hericium polysaccharide in the fermentation liquid is more than 3.5g/L, preferably more than 4.4 g/L.
In a specific embodiment, the fermentation liquid is prepared by fermentation with a preservation number of CCTCC No: the agaricus bisporus of M2020587 and the preservation number are CCTCC No: m2018567 by fermenting Hericium erinaceus.
The agaricus bisporus can produce ergothioneine and agaricus bisporus polysaccharide in high yield, the concentration of the ergothioneine in fermentation liquor is more than 32mg/L when the agaricus bisporus is fermented independently, and the concentration of the agaricus bisporus polysaccharide in the fermentation liquor is more than 1.3 g/L. The effect is better when the agaricus and hericium erinaceus are fermented in a combined manner, wherein the concentration of the ergothioneine in the fermentation liquid can reach over 240mg/L, and the concentration of agaricus and hericium erinaceus polysaccharide in the fermentation liquid can reach over 3.5 g/L. The method for biologically synthesizing ergothioneine by using agaricus bisporus provided by the invention not only can retain active ingredients such as polysaccharide, amino acid and the like in agaricus bisporus fermentation liquid, but also can provide a new way for producing the ergothioneine.
The following examples of the present invention are merely illustrative of specific embodiments for carrying out the present invention and are not to be construed as limiting the invention. Other changes, modifications, substitutions, combinations, and simplifications which may be made without departing from the spirit and principles of the invention are intended to be equivalents thereof and to fall within the scope of the invention.
Examples
The present invention is further described below in conjunction with the following examples, which are intended to be illustrative and explanatory only and are not restrictive of the invention.
The experimental methods used in the following examples are all conventional methods, unless otherwise specified.
Materials, reagents and the like used in the following examples can be obtained from commercial routes unless otherwise specified.
Example 1 selection of Strain of Armillaria ergothioneine-producing Strain
(1) Cutting about 0.5 multiplied by 0.5cm internal tissue blocks at pileus of agaricus bisporus fruiting bodies in different producing areas respectively, placing the tissue blocks on a PDA solid culture medium containing antibiotics and pine root extract, culturing at 24 ℃, transferring obtained hyphae to the PDA solid culture medium containing the pine root extract for purification culture, inoculating the purified strains on a PDA inclined plane containing the pine root extract, and preserving at 3-4 ℃ for later use;
(2) inoculating the solid strain obtained in the step (1) to a fermentation medium, and culturing at 24 ℃ for 30 days to obtain a mycelium fermentation liquid, wherein the fermentation medium comprises 30.0g/L glucose25.0g/L of corn steep liquor, 1.0g/L of dipotassium hydrogen phosphate and 0.003g/L of vitamin B6And the balance of water. The precursor substances cysteine, methionine and betaine were fed at a concentration of 5mM each on day 20 of the fermentation.
(3) After fermentation, homogenizing the mycelium fermentation liquor at 8000rpm for 30min, leaching ergothioneine from the mycelium cells to the extracellular fermentation liquor, centrifuging at 10000rpm for 10min, collecting supernatant, filtering with 0.22 μm microporous filter, and screening the strain with highest ergothioneine content in the filtrate as Tricholoma Matsutake (Tricholoma Matsutake) SR-LY.
Example 2 morphological Observation of Armillaria Matsutake SR-LY
The Matsutake mushroom (Tricholoma Matsutake) has CCTCC No: m2020587 grows faster on a potato glucose agar culture medium (namely a PDA solid culture medium) containing pine root extract, the culture medium grows for 20 days at 25 ℃, the diameter of a bacterial colony reaches 20-25 mm and is white, aerial hyphae on the surface of the bacterial colony are twisted to form a burr-shaped velvety shape; the colony morphology is shown in FIG. 1. The hypha of the strain consists of tubular cells, has thin wall and obvious transverse septa, is hollow, and is shown in figure 2.
Example 3 identification and Classification of Armillaria Matsutake
The Armillaria Matsutake (Tricholoma Matsutake) is sequenced by limited company of Shanghai biological engineering (Shanghai), the strain is identified as Armillaria Matsutake, and its taxonomy is: the gene sequence determination results of 18s rRNA of fungi belonging to the genus Tricholoma, the family Tricholomataceae, the order Agaricales, the class Agaricales are shown in FIG. 3, and the determination results of ITS sequences are shown in FIG. 4.
Example 4 fermentative biosynthesis of ergothioneine from Armillaria matsutake
(1) Matsutake mushroom (Tricholoma Matsutake) CCTCC No: inoculating M2020587 mycelium slant strain into liquid seed culture medium, and culturing at 20 deg.C, pH5.0, and 150rpm under shaking condition for 25 days to obtain Armillaria matsutake seed liquid. The seed culture medium comprises lactose 30.0g/L, potato extract powder 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, sodium sulfate 1.0g/L, and water in balance, has pH of 4.5, and is sterilized at 121 deg.C for 20 min;
(2) inoculating the agaricus bisporus seed liquid obtained in the step (1) to a fermentation culture medium, culturing for 25 days at 23 ℃ under the condition of shaking at 180rpm, and supplementing precursor substances on the 20 th day of culture to obtain a fermentation liquid. The fermentation medium comprises 40.0g/L glucose, 15.0g/L wheat peptone, 0.3g/L sodium dihydrogen phosphate, 0.002g/L nicotinic acid, and 0.001g/L vitamin B1And the balance of water, pH 4.5, sterilizing at 121 deg.C for 20 min. The precursor substances are cysteine, methionine and betaine, and the concentration is 5mM respectively.
(3) After fermentation is finished, homogenizing the mycelium fermentation liquor at 8000rpm for 30min, leaching ergothioneine from the cells of the mycelium into the fermentation liquor outside the cells, centrifuging at 10000rpm for 10min, taking supernatant, filtering by a 0.22-micron microporous filter, leaching the ergothioneine in the cells of the mycelium into the fermentation liquor outside the cells, and obtaining an ergothioneine leaching liquor S1.
Example 5 biosynthesis of ergothioneine by Combined fermentation of Armillaria matsutake and Hericium erinaceum
(1) Matsutake mushroom (Tricholoma Matsutake) CCTCC No: inoculating M2020587 mycelium slant strain into liquid seed culture medium, and culturing at 20 deg.C, pH5.0 and 150rpm under shaking condition for 25 days to obtain Armillaria matsutake seed liquid. The seed culture medium comprises lactose 30.0g/L, potato extract powder 20.0g/L, dipotassium hydrogen phosphate 1.0g/L, sodium sulfate 1.0g/L, and water in balance, has pH of 4.5, and is sterilized at 121 deg.C for 20 min;
(2) hericium erinaceus (Hericium erinaceus) CCTCC No: inoculating M2018567 mycelium slant strain into liquid seed culture medium, and culturing at 20 deg.C, pH5.0 and 150rpm under shaking condition for 7 days to obtain Hericium Erinaceus seed solution. The seed culture medium comprises sucrose 40.0g/L, bean cake powder 15.0g/L, sodium dihydrogen phosphate 2.0g/L, sodium sulfate 1.0g/L, and water in balance, has pH of 4.5, and is sterilized at 121 deg.C for 20 min;
(3) inoculating the agaricus bisporus seed liquid obtained in the step (1) to a fermentation culture medium, culturing for 25 days under the shaking condition of 23 ℃ and 180rpm, inoculating the hericium erinaceus seed liquid obtained in the step (2) on the 15 th day of culture, and supplementing precursor substances on the 20 th day to obtain fermentation liquid. Hair-like deviceThe fermentation medium comprises 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 nicotinic acid, and 0.001g/L vitamin B1And the balance of water, pH 4.5, sterilizing at 121 deg.C for 20 min. The precursor substances are cysteine, methionine and betaine, and the concentration is 5mM respectively.
(4) After fermentation, homogenizing the mycelium fermentation liquor at 8000rpm for 30min, leaching ergothioneine from the cells of the mycelium into the fermentation liquor outside the cells, centrifuging at 10000rpm for 10min, taking the supernatant, filtering through a 0.22-micron microporous filter, leaching the ergothioneine in the cells of the mycelium into the fermentation liquor outside the cells, and obtaining an ergothioneine leaching liquor S2.
Comparative example 1 biosynthesis of ergothioneine by Hericium Erinaceus
(1) Hericium erinaceus (Hericium erinaceus) CCTCC No: inoculating M2018567 mycelium slant strain into liquid seed culture medium, and culturing at 23 deg.C under shaking at pH5.0 and 200rpm for 7 days to obtain Hericium erinaceus seed solution. The seed culture medium comprises sucrose 40.0g/L, bean cake powder 15.0g/L, sodium dihydrogen phosphate 2.0g/L, sodium sulfate 1.0g/L, and water in balance, has pH of 4.5, and is sterilized at 121 deg.C for 20 min;
(2) inoculating the hericium erinaceus seed liquid obtained in the step (1) to a fermentation culture medium, culturing for 10 days at 23 ℃ under the condition of 180rpm oscillation, and supplementing a precursor substance on the 3 rd day of culture to obtain a fermentation liquid. The fermentation medium comprises 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 nicotinic acid, and water in balance, has pH of 4.5, and is sterilized at 121 deg.C for 20 min. The precursor substances are cysteine, methionine and betaine, and the concentration is 5mM respectively.
(3) After fermentation, homogenizing the mycelium fermentation liquor at 8000rpm for 30min, leaching ergothioneine from the mycelium cells to the extracellular fermentation liquor, centrifuging at 10000rpm for 10min, taking the supernatant, filtering through a 0.22 mu m microporous filter, leaching the ergothioneine in the mycelium cells to the extracellular fermentation liquor, and obtaining an ergothioneine leaching liquor C1.
Test example 1 ergothioneine content measurement
The ergothioneine content is determined by adopting a high performance liquid chromatography, and the HPLC conditions are as follows: a chromatographic column: hypersil ODS C18 column (250 mm. times.4.6 mm, particle size 5 μm); column temperature: 30 ℃; mobile phase: acetonitrile-water (3: 97); flow rate: 1.0 mL/min; detection wavelength: 254 nm; sample introduction amount: 20 μ L. The ergothioneine content in the samples S1 and S2 prepared in examples 4 and 5 and the sample C1 prepared in comparative example 1 is shown in Table 1, and the content of the ergothioneine in the fermentation liquid can be greatly increased by inoculating hericium erinaceus in the fermentation process of the agaricus. The chromatogram is shown in FIG. 5, where a) is a standard (ergothioneine concentration of 7.05. mu.g/mL), b) is sample S1 obtained in example 4, C) is sample S2 obtained in example 5, and d) is sample C1 obtained in comparative example 1.
TABLE 1 comparison of ergothioneine content in different samples
| Sample (I) | Ergothioneine content (mg/L) |
| S1 | 48.7 |
| S2 | 423.9 |
| C1 | 403.5 |
As can be seen from Table 1, the agaricus bisporus provided by the present invention can produce ergothioneine at a higher yield than that of the conventional agaricus bisporus, thereby expanding the ways of producing ergothioneine, and the method for the combined fermentation of agaricus bisporus and hericium erinaceus provided by the present invention can greatly increase the yield of ergothioneine compared with the single fermentation of agaricus bisporus.
Test example 2 detection of amino acid content
The free amino acid content of samples S1 and S2 prepared in examples 4 and 5 and sample C1 prepared in comparative example 1 were measured by HPLC pre-column derivatization, and the results are shown in Table 2. As can be seen from the results in the table, a variety of amino acids were produced by fermentation of Armillaria matsutake, and more particularly, amino acids were produced by fermentation in combination with Hericium erinaceus.
TABLE 2 comparison of amino acid content in different samples
| Amino acids | S1(mg/100mL) | S2(mg/100mL) | C1(mg/100mL) |
| Aspartic acid | 0.793 | 1.821 | 0.901 |
| Glutamic acid | 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 amount of | 5.917 | 12.275 | 5.927 |
Test example 3 detection of fungal polysaccharide content
The invention adopts phenol-sulfuric acid method to detect polysaccharide content.
(1) The instrument comprises the following steps: visible-ultraviolet spectrophotometer, analytical balance (0.0001g) precision, vortex mixer
(2) Reagent:
2.1 Standard solution: accurately weighing 100.0mg of dry glucose (analytically pure) with constant weight into a volumetric flask, adding water to a constant volume of 250.0mL, shaking up, accurately absorbing 10.0mL of the solution, and adding water to a constant volume of 100.0 mL.
Preparation of 2.280% phenol solution: accurately transferring 80.0mL of redistilled phenol, adding distilled water to a constant volume of 100.0mL to obtain 80% phenol solution, and storing in a brown bottle in a dark place.
Preparation of 2.36% phenol solution: 80% phenol solution is diluted to 6% by adding water and prepared immediately before use.
2.4 concentrated sulfuric acid (super pure)
(3) And (3) detection:
3.1 preparing a standard curve: 0.0, 0.4, 0.8, 1.2, 1.6 and 2.0mL of glucose standard solution is respectively sucked into a test tube with a plug, and water is added to supplement the glucose standard solution to 2.0 mL. Respectively adding 1.0mL of 6% phenol solution, mixing uniformly, quickly adding 5.0mL of concentrated sulfuric acid (the concentrated sulfuric acid is added in a suspended manner and cannot adhere to the wall), shaking uniformly, reacting at room temperature for 20min, measuring absorbance at 490nm, using a 0-tube as blank control, and obtaining a standard curve with the vertical coordinate of polysaccharide concentration and the horizontal coordinate of absorbance.
3.2 sample preparation: samples S1 and S2 prepared in examples 4 and 5 and sample C1 prepared in comparative example 1, which are 0.2mL respectively, are weighed and placed in a 50.0mL volumetric flask, an appropriate amount of water is added, after complete dissolution, the water is added to a constant volume to be used as stock solution, and the stock solution is shaken up. The stock solution was measured 5.0mL before use, placed in a 50.0mL volumetric flask, added water to the scale and diluted 10-fold in the same manner. 2.0mL of the polysaccharide was put in a test tube with a stopper, and the concentration of the polysaccharide in the sample to be tested was obtained from the standard curve by the same method as described above with "1.0 mL of 6% redistilled phenol added", and the polysaccharide content was calculated from the dilution factor, with the results shown in Table 3. As can be seen from the data in tables 1, 2 and 3, the fermentation liquid obtained by the combined fermentation of agaricus bisporus and hericium erinaceus of the invention not only has high ergothioneine yield, but also can keep high yield of amino acid and polysaccharide. The agaricus bisporus polysaccharide has various effects, and has a report that the agaricus bisporus polysaccharide has a good whitening effect, so that the agaricus bisporus polysaccharide has a good application prospect.
TABLE 3 comparison of polysaccharide content in different samples
| Sample (I) | Polysaccharide content (g/L) |
| S1 | 2.34 |
| S2 | 5.28 |
| C1 | 2.57 |
Test example 4 test of whitening effect of fermentation liquid
Preparing a sample solution: the samples S1 and S2 of examples 4 and 5 and the sample C1 of comparative example 1 were prepared as 5% by volume solutions using serum-containing complete culture medium as a diluent, and sterilized by filtration through a 0.22 μm filter.
Taking melanoma cells of B16 mice in logarithmic growth phase at 2X 104Inoculating to 6-well culture plate at density of 3.0 mL/well, placing in carbon dioxide incubator at 37 deg.C and 5% CO2Culturing for 24 hr, discarding old culture solution, adding 3.0mL serum into normal control group and model group, and culturing3.0mL of sample solution was added to the culture solution, and 60.0. mu.L of forskolin solution (2.0mM) was added to each well of the model and experimental groups to stimulate the production of melanin, and the culture was continued for 72 hours.
The method for measuring the melanin content in the cells comprises the following steps: pancreatin digestion, centrifugation to remove supernatant, adding 1.0 mol/L (containing 10% DMSO) NaOH solution 500uL to lyse cells, heating at 80 deg.C for 30min, centrifuging at 3000rpm for 10min, collecting supernatant, adding into 96-well plate, 100 uL/well, and measuring absorbance at 450 nm.
The total melanin content inhibition rate was calculated as follows.
The results of melanin inhibition of each of the obtained samples are shown in table 4.
TABLE 4 Effect of samples on melanin content
| Sample (I) | Total melanin content inhibition/%) |
| S1 | 20.35 |
| S2 | 25.62 |
| C1 | 6.11 |
As can be seen from the results in the table, the agaricus bisporus fermentation broth and the agaricus bisporus-combined hericium erinaceus fermentation broth have better whitening effect than the hericium erinaceus fermentation broth, and may be active substances produced by agaricus bisporus polysaccharide or other agaricus bisporus mycelium.
Sequence listing
<110> Huaxi Biotechnology Ltd
SHANDONG HUAXI HAIYU BIOLOGICAL MEDICINE Co.,Ltd.
<120> Armillaria matsutake and method for producing ergothioneine using the same
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<141> 2020-11-10
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accacccata aaatcatgaa agagctatca atctgtcaat cctagttatg tctggacctg 180
gtgagtttcc ccgtgttgag tcaaattaag ccgcaggctc cacacctggt ggtgcccttc 240
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cgactctaga aaccaacaaa atagaaccgc acgtcctatt ttattattcc atgctaatgt 600
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cctccaattg ttcctcgtta agggatttaa attgtactca ttccaattat aagacccgaa 900
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Claims (10)
1. A Matsutake mushroom (Tricholoma Matsutake) characterized in that the preservation number of the Matsutake mushroom is CCTCC No: m2020587.
2. The Armillaria matsutake of claim 1, wherein the Armillaria matsutake has 18s rRNA gene sequence shown in SEQ ID NO. 1.
3. The Armillaria matsutake as claimed in claim 1, wherein the ITS sequence of Armillaria matsutake is as shown in SEQ ID NO. 2.
4. A method for producing ergothioneine by fermenting agaricus, which is characterized by comprising the following steps:
inoculating Armillaria matsutake mycelium into seed culture medium, and culturing to obtain Armillaria matsutake seed solution;
inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation liquid;
processing the fermentation liquor to obtain ergothioneine;
wherein, precursor substances are supplemented in the fermentation process,
wherein the preservation number of the agaricus bisporus is CCTCC No: m2020587.
5. The method of claim 4, wherein the fermentation medium comprises the following components: 20.0-60.0 g/L of carbon source, 10.0-30.0 g/L of nitrogen source, 0.1-5.0 g/L of inorganic salt and 0.001-0.005 g/L of vitamin, and preferably comprises the following components: 30.0-50.0 g/L glucose, 10.0-30.0 g/L wheat peptone, 0.1-2.0 g/L inorganic salt, 0.001-0.005 g/L nicotinic acid, vitamin B10.001～0.005g/L。
6. The method according to claim 4, wherein the precursor substance comprises one or more of cysteine, histidine, methionine, aspartic acid, glutamine, and betaine.
7. The method of claim 4, wherein the fermentation culture is carried out under the following culture conditions: culturing for 20-30 days at 15-30 deg.C under shaking at 100-300 rpm, wherein precursor is supplemented on 15-20 days.
8. A method for producing ergothioneine by combined fermentation of agaricus and hericium erinaceus is characterized by comprising the following steps:
inoculating Armillaria matsutake mycelium into Armillaria matsutake seed culture medium, and culturing to obtain Armillaria matsutake seed solution;
inoculating Hericium erinaceus mycelium into Hericium erinaceus seed culture medium, and culturing to obtain Hericium erinaceus seed liquid;
inoculating the agaricus bisporus seed liquid into a fermentation culture medium, and inoculating the hericium erinaceus seed liquid into the fermentation culture medium for culture after a period of culture to obtain a fermentation liquid;
processing the fermentation liquor to obtain ergothioneine;
wherein, precursor substances are supplemented in the fermentation process.
9. The method according to claim 8, wherein the preservation number of the hericium erinaceus is CCTCC No: m2018567.
10. The method of claim 8, wherein the agaricus is deposited under the preservation number of CCTCC No: m2020587.
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| CN112195215A (en) * | 2020-10-24 | 2021-01-08 | 上海加新生物科技有限公司 | Method for producing ergothioneine by joint fermentation of pleurotus citrinopileatus and agaricus blazei mycelium |
| CN114214215A (en) * | 2022-01-17 | 2022-03-22 | 广东塔尖生物科技有限公司 | Preparation method of mushroom fermentation liquor and cosmetics |
| CN114214387A (en) * | 2021-12-27 | 2022-03-22 | 广东丸美生物技术股份有限公司 | Method for preparing schizophyllan and ergothioneine by co-fermentation of two strains |
| WO2022100568A1 (en) * | 2020-11-10 | 2022-05-19 | 华熙生物科技股份有限公司 | Tricholoma matsutake and method for producing ergothioneine thereby |
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| CN114214387A (en) * | 2021-12-27 | 2022-03-22 | 广东丸美生物技术股份有限公司 | Method for preparing schizophyllan and ergothioneine by co-fermentation of two strains |
| CN114214387B (en) * | 2021-12-27 | 2022-09-27 | 广东丸美生物技术股份有限公司 | Method for preparing schizophyllan and ergothioneine by co-fermentation of two strains |
| CN114214215A (en) * | 2022-01-17 | 2022-03-22 | 广东塔尖生物科技有限公司 | Preparation method of mushroom fermentation liquor and cosmetics |
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