Application of Tremella fuciformis in the preparation of ergothioneine

Abstract

The present invention relates to the technical field of microbial fermentation and discloses the use of Tremella fuciformis spores in the preparation of ergothioneine. The present invention discloses for the first time the use of Tremella fuciformis spores in the preparation of ergothioneine, which can be used to prepare foods, cosmetics and / or medicines with ergothioneine as a main active ingredient and having antioxidant, anti-inflammatory and / or anti-aging functions.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to application of Tremella fuciformis in preparing ergothioneine. Background Art

[0002] Ergothioneine is an important natural antioxidant, widely present in organisms. It possesses multiple physiological functions, including antioxidant, anti-inflammatory, and anti-aging properties, and is closely related to human health. Its safety makes it suitable for market development as a new food ingredient, and it currently shows promising application prospects in areas such as food and cosmetics. The human body cannot synthesize ergothioneine on its own and must obtain it from external sources. Research has shown that ergothioneine is most abundant in edible fungi, but the long cultivation cycle and high labor, space, and equipment requirements for fruiting bodies make commercial-scale production impossible. Currently, biosynthesis is a hot topic in the research of ergothioneine production, primarily through fermentation of natural edible mushrooms. The highest reported yield of ergothioneine produced by fermentation of natural mushrooms is approximately 500 mg / L, with a fermentation cycle typically lasting 10 to 20 days. Engineered bacteria can achieve fermentation cycles of 3 to 9 days, with a maximum yield of 1.3 g / L. However, the product cannot be used directly in the food industry and requires purification.

[0003] Tremella fuciformis spores are yeast-like conidia produced by the budding of Tremella fuciformis basidiospores. They can reproduce indefinitely through budding, and grow rapidly, are easy to culture and transform, and have the potential to be used as a bioreactor. Since the ergothioneine content in Tremella fuciformis is lower than that in Pleurotus edible fungi (the literature reports that the ergothioneine content detected in Tremella fuciformis fruiting bodies is only 0.0006%), there are currently no related research reports on the production of ergothioneine using Tremella fuciformis spore fermentation metabolism at home and abroad. Summary of the Invention

[0004] In order to overcome the deficiencies in the prior art, the purpose of a first aspect of the present invention is to provide an application of Tremella fuciformis in preparing thioneine.

[0005] The purpose of the second aspect of the present invention is to provide the use of Tremella fuciformis in preparing products.

[0006] The third aspect of the present invention aims to provide a method.

[0007] The fourth aspect of the present invention aims to provide a culture medium.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides the use of Tremella fuciformis in the preparation of ergothioneine.

[0010] Preferably, the Tremella fungus comprises Tremella fuciformis Tr21.

[0011] Preferably, the Tremella fuciformis comprises at least one of Tremella fuciformis hyphae and Tremella fuciformis spores; further comprising Tremella fuciformis spores.

[0012] Ergothioneine (EGT), scientifically known as 2-mercapto-L-histidine trimethylol salt, has a molecular formula of C9H 15 N3O2S, molecular weight is 229.3, CAS number is 497-30-3.

[0013] A second aspect of the present invention provides the use of Tremella fuciformis in preparing a product, wherein the active ingredient of the product comprises ergothioneine.

[0014] Preferably, the product has at least one of the functions (a1) to (a3): (a1) anti-oxidation; (a2) anti-inflammatory; (a3) anti-aging.

[0015] Preferably, the product comprises at least one of food, cosmetics, and medicine.

[0016] Preferably, the food includes at least one of beverages, biscuits, candies, and cakes.

[0017] Preferably, the candy is a compressed candy.

[0018] Preferably, the beverage is a solid beverage.

[0019] Preferably, the cosmetics include at least one of cleansing milk, toner, lotion, cream, and essence.

[0020] Preferably, the dosage form of the drug includes at least one of capsules, tablets, granules, mixtures, decoctions, pills, granules, dripping pills, teas, tinctures, and wines.

[0021] Preferably, the Tremella fungus comprises Tremella fuciformis Tr21.

[0022] Preferably, the Tremella fuciformis comprises at least one of Tremella fuciformis hyphae and Tremella fuciformis spores; further comprising Tremella fuciformis spores.

[0023] Ergothioneine (EGT), scientifically known as 2-mercapto-L-histidine trimethylol salt, has a molecular formula of C9H 15 N3O2S, molecular weight is 229.3, CAS number is 497-30-3.

[0024] The third aspect of the present invention provides a method for inoculating Tremella fuciformis into a fermentation medium for cultivation; the method is any one of (b1) to (b2):

[0025] (b1) prepare the method for thioneine;

[0026] (b2) A method for preparing a product, the active ingredient of which comprises ergothioneine.

[0027] Preferably, the product has at least one of the functions (a1) to (a3): (a1) anti-oxidation; (a2) anti-inflammatory; (a3) anti-aging.

[0028] Preferably, the product comprises at least one of food, cosmetics, and medicine.

[0029] Preferably, the food includes at least one of beverages, biscuits, candies, and cakes.

[0030] Preferably, the candy is a compressed candy.

[0031] Preferably, the beverage is a solid beverage.

[0032] Preferably, the cosmetics include at least one of cleansing milk, toner, lotion, cream, and essence.

[0033] Preferably, the dosage form of the drug includes at least one of capsules, tablets, granules, mixtures, decoctions, pills, granules, dripping pills, teas, tinctures, and wines.

[0034] Preferably, the fermentation medium comprises: a carbon source, a nitrogen source and vitamins.

[0035] Preferably, the carbon source comprises at least one of glucose, sucrose, fructose and maltose.

[0036] Preferably, the carbon source comprises sugarcane; further comprises sugarcane juice.

[0037] Preferably, the nitrogen source comprises at least one of peptone, beef extract, yeast powder, corn flour, tryptone, and wheat bran; further preferably, the nitrogen source comprises at least two of peptone, beef extract, yeast powder, corn flour, tryptone, and wheat bran; even more preferably, the nitrogen source comprises at least one of corn flour and wheat bran and peptone; even more preferably, the nitrogen source comprises wheat bran and peptone.

[0038] Preferably, the mass ratio of the wheat bran to peptone is 1:(0.5-1.5).

[0039] Preferably, the vitamins include at least one of vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, and folic acid; further preferably, the vitamins include at least one of vitamin B1 and vitamin B6; even further preferably, the vitamins include vitamin B1.

[0040] Preferably, the concentration of the carbon source in the fermentation medium is 3-12% (Bix) based on its soluble solid content; further preferably, the concentration of the carbon source in the fermentation medium is 5-10% (Bix) based on its soluble solid content; even further preferably, the concentration of the carbon source in the fermentation medium is 5-8% (Bix) based on its soluble solid content.

[0041] Preferably, the concentration of the nitrogen source in the fermentation culture is 0.1 to 1.2% by mass volume ratio; further preferably, the concentration of the nitrogen source in the fermentation culture medium is 0.2 to 1.0% by mass volume ratio.

[0042] Preferably, the concentration of the vitamin in the fermentation medium is 0.005-0.15% by mass volume ratio; further preferably, the concentration of the vitamin in the fermentation medium is 0.01-0.1% by mass volume ratio; even more preferably, the concentration of the vitamin in the fermentation medium is 0.01-0.05% by mass volume ratio.

[0043] Preferably, the pH of the fermentation medium is 5-7; further 5.5-6.5; further 5.5-6.

[0044] Preferably, when the fermentation medium does not contain amino acids, the culture conditions are any one of (c1) to (c3):

[0045] (c1) fermenting at 150-400 rpm and 23-35° C. for 5-9 days; further at 200-300 rpm and 25-30° C. for 6-8 days; further at 200-300 rpm and 25-30° C. for 7 days;

[0046] (c2) fermenting at 150-400 r / min and 23-35° C. for 1.5-4.5 days, then adding amino acids, and fermenting at 150-400 r / min and 23-35° C. for 2.5-5.5 days; further fermenting at 200-300 r / min and 25-30° C. for 2-4 days, then adding amino acids, and fermenting at 200-300 r / min and 25-30° C. for 3-5 days; further fermenting at 200-300 r / min and 25-30° C. for 3 days, then adding amino acids, and fermenting at 200-300 r / min and 25-30° C. for 4 days;

[0047] (c3) Fermentation at 150-400 r / min and 23-35°C for 1.5-3.5 days, then adding amino acids, inducing at 150-400 r / min and 8-18°C for 6-28 hours, and finally fermenting at 150-400 r / min and 23-35°C for 0.5-3.5 days; further fermentation at 200-300 r / min and 25-30°C for 2-3 days, then adding amino acids, 200-3 00r / min, 10-15℃ for induction 8-24h, and finally fermentation at 200-300r / min, 25-30℃ for 1-3 days; further: fermentation at 200-300r / min, 25-28℃ for 2-3 days, then add amino acids, induce at 200-300r / min, 10-12℃ for 8-16h, and finally fermentation at 200-300r / min, 25-28℃ for 2 days.

[0048] Preferably, the amino acids include at least one of aspartic acid, cysteine, histidine, methionine, arginine, and glutamate; further preferably, the amino acids include at least two of aspartic acid, cysteine, histidine, methionine, arginine, and glutamate; even further preferably, the amino acids include cysteine and methionine.

[0049] Preferably, the mass ratio of cysteine to methionine is 1:(0.5-1.5).

[0050] Preferably, the final concentration of the amino acid is 0.03-0.20% by mass volume; further preferably, the final concentration of the amino acid is 0.05-0.15% by mass volume; further preferably, the final concentration of the amino acid is 0.1-0.15% by mass volume.

[0051] Preferably, the inoculation amount of Tremella fuciformis is 2-20 v / v% of the fermentation medium; further, 5-15 v / v% of the fermentation medium.

[0052] Preferably, the Tremella fuciformis is Tremella fuciformis seed liquid.

[0053] Preferably, the preparation method of the Tremella fuciformis seed liquid is as follows: taking Tremella fuciformis slant strains and inoculating them into a seed culture medium, culturing, and obtaining.

[0054] Preferably, the seed culture medium is potato glucose water culture medium.

[0055] Preferably, the culture conditions are 100-200 r / min, 23-35° C., and fermentation for 3-7 days.

[0056] Preferably, the Tremella fungus comprises Tremella fuciformis Tr21.

[0057] Preferably, the Tremella fuciformis comprises at least one of Tremella fuciformis hyphae and Tremella fuciformis spores; further comprising Tremella fuciformis spores.

[0058] Preferably, the fermentation medium further comprises amino acids.

[0059] Preferably, the amino acids include at least one of aspartic acid, cysteine, histidine, methionine, arginine, and glutamate; further preferably, the amino acids include at least two of aspartic acid, cysteine, histidine, methionine, arginine, and glutamate; even further preferably, the amino acids include cysteine and methionine.

[0060] Preferably, the mass ratio of cysteine to methionine is 1:(0.5-1.5).

[0061] Preferably, the concentration of the amino acid in the fermentation medium is 0.03 to 0.20% by mass volume ratio; further preferably, the concentration of the amino acid in the fermentation medium is 0.05 to 0.15% by mass volume ratio; even further preferably, the concentration of the amino acid in the fermentation medium is 0.1 to 0.15% by mass volume ratio.

[0062] Preferably, when the fermentation medium contains amino acids, the culture conditions are: fermentation at 150-400 r / min and 23-35°C for 5-9 days; further, fermentation at 200-300 r / min and 25-30°C for 6-8 days; further, fermentation at 200-300 r / min and 25-30°C for 7 days.

[0063] Preferably, the culture further comprises the following steps: homogenization and centrifugation.

[0064] Preferably, the homogenization condition is a homogenization treatment at 15 to 40 MPa for 2 to 10 minutes.

[0065] Preferably, the centrifugal condition is 3000-5000 r / min for 5-15 min.

[0066] The fourth aspect of the present invention provides a reagent comprising: a mixture of a carbon source, a nitrogen source and vitamins; the carbon source comprises at least one of glucose, sucrose, fructose and maltose; the nitrogen source comprises at least one of peptone, beef extract, yeast powder, corn flour, tryptone and wheat bran; the vitamins comprise at least one of vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12 and folic acid; the concentration of the carbon source in the mixture is 3 to 12% (Bix) based on its soluble solid content; the concentration of the nitrogen source in the mixture is 0.1 to 1.2% by mass volume ratio; the concentration of the vitamins in the mixture is 0.005 to 0.15% by mass volume ratio.

[0067] Preferably, the reagent further comprises: amino acids.

[0068] Preferably, the amino acid and the mixture exist independently or are mixed.

[0069] Preferably, the amino acids include at least one of aspartic acid, cysteine, histidine, methionine, arginine, and glutamate; further preferably, the amino acids include at least two of aspartic acid, cysteine, histidine, methionine, arginine, and glutamate; even further preferably, the amino acids include cysteine and methionine.

[0070] Preferably, the mass ratio of cysteine to methionine is 1:(0.5-1.5).

[0071] Preferably, when the amino acid is mixed with the mixture, the final concentration of the amino acid is 0.03 to 0.20% by mass volume ratio; further preferably, the final concentration of the amino acid is 0.05 to 0.15% by mass volume ratio; further preferably, the final concentration of the amino acid is 0.1 to 0.15% by mass volume ratio.

[0072] Preferably, when the amino acid and the mixture exist independently, the working concentration of the amino acid is 0.03 to 0.20% by mass volume ratio; further preferably, the working concentration of the amino acid is 0.05 to 0.15% by mass volume ratio; further preferably, the working concentration of the amino acid is 0.1 to 0.15% by mass volume ratio.

[0073] Preferably, the carbon source comprises sugarcane; further preferably, the carbon source comprises sugarcane juice.

[0074] Preferably, the nitrogen source comprises at least two of peptone, beef extract, yeast powder, corn flour, tryptone, and wheat bran; further preferably, the nitrogen source comprises at least one of corn flour and wheat bran and peptone; even more preferably, the nitrogen source comprises wheat bran and peptone.

[0075] Preferably, the mass ratio of the wheat bran to peptone is 1:(0.5-1.5).

[0076] Preferably, the vitamins include at least one of vitamin B1 and vitamin B6; further preferably, the vitamins include vitamin B1.

[0077] Preferably, the concentration of the carbon source in the mixture is 5-10% (Bix) based on the soluble solid content; further preferably, the concentration of the carbon source in the mixture is 5-8% (Bix) based on the soluble solid content.

[0078] Preferably, the concentration of the nitrogen source in the mixture is 0.2-1.0% by mass volume ratio.

[0079] Preferably, the concentration of the vitamin in the mixture is 0.01 to 0.1% by mass volume ratio; further preferably, the concentration of the vitamin in the mixture is 0.01 to 0.05% by mass volume ratio.

[0080] Preferably, the pH of the mixture is 5-7; further 5.5-6.5; further 5.5-6.

[0081] The beneficial effects of the present invention are:

[0082] The present invention discloses for the first time the use of Tremella fuciformis in the preparation of ergothioneine, which can be used to prepare foods, cosmetics and / or medicines with ergothioneine as a main active ingredient and having antioxidant, anti-inflammatory and / or anti-aging functions.

[0083] Furthermore, the present invention can increase the content of ergothioneine in the fermentation product by adding amino acids in the fermentation medium; especially, after adding cysteine, the content of ergothioneine in the fermentation product can reach 87.36 mg / L.

[0084] Furthermore, the present invention can further improve the content of thioneine in fermentation product by adding amino acid in fermentation mid-term; Wherein, adding amino acid (cysteine+methionine) in fermentation mid-term can further improve the content of thioneine in fermentation product, and its content is up to 181.41mg / L.

[0085] Further, the present invention adopts segmented temperature-controlled fermentation, and the content of thioneine in fermentation product can be further improved; The content of thioneine in fermentation product is as high as 621.05mg / L, which is at least 3.42 times of non-segmented temperature-controlled fermentation, and fermentation time is shortened by 40h. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1Result diagram of the content of ergothioneine in the fermentation products of Examples 1 to 8; the absence of the same letter indicates a significant difference.

[0087] Figure 2 Result graphs showing the content of ergothioneine in the fermentation products of Examples 9 to 13; the absence of identical letters indicates significant differences.

[0088] Figure 3 Result graphs showing the content of ergothioneine in the fermentation products of Examples 14 to 19; the absence of identical letters indicates significant differences.

[0089] Figure 4 This is the liquid chromatogram of ergothioneine standard product.

[0090] Figure 5 is a liquid chromatogram of the fermentation product of Example 4.

[0091] Figure 6 is a liquid chromatogram of the fermentation product of Example 8.

[0092] Figure 7 is a liquid chromatogram of the fermentation product of Example 9.

[0093] Figure 8 is a liquid chromatogram of the fermentation product of Example 27.

[0094] Figure 9 is a liquid chromatogram of the fermentation product of Example 38.

[0095] Figure 10 is a liquid chromatogram of the fermentation product of Example 49. DETAILED DESCRIPTION

[0096] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0097] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0098] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0099] The Tremella fuciformis spores in the following examples are spores of Tremella fuciformis Tr21, which were donated by the Fujian Branch of the National Center for Edible Fungi Variety Improvement and have been disclosed in the literature (Comparison of Physiological and Biochemical Characteristics and Production Agronomic Traits of Tremella fuciformis Tr01 and Tr21, Kong Xuqiang et al., Journal of Edible Fungi, 2019).

[0100] The sugarcane juice described in the following examples was prepared as follows: fresh sugarcane was washed and juiced, diluted with distilled water to an initial sugar content (soluble solids content) of 5%, 8%, and 10% (Bix), and sterilized at 121°C for 20 minutes. Clarification was not required after juicing, as this helps preserve the sugarcane juice's original active ingredients, such as dietary fiber and polyphenols, which are beneficial for the growth of Tremella fuciformis spores.

[0101] Embodiment 1A kind of method for preparing thioneine

[0102] A method for preparing thioneine comprises the steps:

[0103] (1) Tremella fuciformis spore fermentation seed culture: cut off two pieces (0.5 cm wide) of Tremella fuciformis spore slant and inoculate them into potato glucose water culture medium, place them on a shaker, and culture them at 25°C, 150 r / min, and shake for 5 days to obtain Tremella fuciformis spore liquid seeds;

[0104] (2) Tremella fuciformis spore fermentation: Tremella fuciformis spore liquid seeds were inoculated at an inoculum rate of 10 v / v% into a fermentation tank containing an initial fermentation medium, stirred at a speed of 200 r / min, and fermented at 25°C for 7 days. The fermentation product was homogenized at 25 MPa for 5 minutes and centrifuged at 4000 r / min for 10 minutes to obtain a fermentation product; the initial fermentation medium was sugarcane juice containing peptone and vitamin C; wherein the soluble solids content in the sugarcane juice was 5% (Bix), the final concentration of peptone was 0.2% (w / v, g / 100 mL), the final concentration of vitamin C was 0.01% (w / v, g / 100 mL), and the pH was 6.0.

[0105] Embodiment 2A kind of method for preparing thioneine

[0106] A method for preparing thioneine is identical to the method of Example 1, except that the peptone in the initial culture medium is replaced with beef extract.

[0107] Embodiment 3A kind of method for preparing thioneine

[0108] A method for preparing thioneine is identical to the method in Example 1, except that the peptone in the initial culture medium is replaced by yeast powder.

[0109] Embodiment 4A kind of method for preparing thioneine

[0110] A method for preparing thioneine is identical to the method in Example 1, except that the peptone in the initial culture medium is replaced with corn flour.

[0111] Embodiment 5A kind of method for preparing thioneine

[0112] A method for preparing thioneine is identical with the method for Example 1, except that the peptone in the initial culture medium is replaced by tryptone.

[0113] Embodiment 6A kind of method for preparing thioneine

[0114] A method for preparing thioneine is identical to the method of Example 1, except that the peptone in the initial culture medium is replaced by wheat bran.

[0115] Embodiment 7A kind of method for preparing thioneine

[0116] A method for preparing thioneine is the same as the method in Example 1, except that the peptone in the initial culture medium is replaced by a mixture of peptone and corn flour, wherein the mass ratio of peptone to corn flour is 1:1.

[0117] Embodiment 8A kind of method for preparing thioneine

[0118] A method for preparing ergothioneine is the same as the method in Example 1, except that the peptone in the initial culture medium is replaced by a mixture of peptone and wheat bran, wherein the mass ratio of peptone to wheat bran is 1:1.

[0119] Embodiment 9A kind of method for preparing thioneine

[0120] A method for preparing thioneine is identical to the method for Example 8, except that the vitamin C in the initial culture medium is replaced by vitamin B1.

[0121] Embodiment 10A kind of method for preparing thioneine

[0122] A method for preparing thioneine is identical to the method for Example 8, except that the vitamin C in the initial culture medium is replaced by vitamin B2.

[0123] Embodiment 11A kind of method for preparing thioneine

[0124] A method for preparing thioneine is identical to the method for Example 8, except that the vitamin C in the initial culture medium is replaced by vitamin B6.

[0125] Embodiment 12 A kind of method of preparing thioneine

[0126] A method for preparing thioneine is identical to the method for Example 8, except that the vitamin C in the initial culture medium is replaced by vitamin B12.

[0127] Embodiment 13A kind of method of preparing thioneine

[0128] A method for preparing thioneine is identical to the method of Example 8, except that the vitamin C in the initial culture medium is replaced by folic acid.

[0129] Embodiment 14 A kind of method of preparing thioneine

[0130] A method for preparing thioneine is identical to the method for Example 1, except that: initial fermentation medium is different, and the initial fermentation medium in the present embodiment is a mixture comprising peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1 and sugarcane juice of aspartic acid; wherein, in sugarcane juice, soluble solids content is 5% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.2% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.01% (w / v, g / 100mL), and the final concentration of aspartic acid is 0.1% (w / v, g / 100mL), pH 6.0.

[0131] Embodiment 15 A kind of method of preparing thioneine

[0132] A method for preparing thioneine is identical to the method of Example 14, except that aspartic acid is replaced by cysteine.

[0133] Example 16 A method for preparing thioneine

[0134] A method for preparing thioneine is identical to the method of Example 14, except that aspartic acid is replaced by histidine.

[0135] Example 17 A method for preparing thioneine

[0136] A method for preparing thioneine is identical to the method for Example 14, except that aspartic acid is replaced by methionine.

[0137] Example 18 A method for preparing thioneine

[0138] A method for preparing thioneine is identical to the method for Example 14, except that aspartic acid is replaced by arginine.

[0139] Example 19 A method for preparing thioneine

[0140] A method for preparing thioneine is identical to the method for Example 14, except that aspartic acid is replaced by glutamic acid.

[0141] Embodiment 20 A kind of method of preparing thioneine

[0142] A method for preparing ergothioneine is identical to the method for Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 5v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine and methionine (Cys+Met, the mass ratio of cysteine and methionine is 1:1); wherein, in the sugarcane juice, soluble solids content is 5% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.2% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.01% (w / v, g / 100mL), and the final concentration of the mixture of cysteine and methionine is 0.05% (w / v, g / 100mL), and the pH is 5.5.

[0143] Example 21 A method for preparing thioneine

[0144] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), the initial fermentation medium is different: in the present embodiment, the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine and methionine (the mass ratio of cysteine and methionine is 1:1); wherein the soluble solids content in the sugarcane juice is 8% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.6% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.05% (w / v, g / 100mL), and the final concentration of the mixture of cysteine and methionine is 0.1% (w / v, g / 100mL), and the pH is 6.0.

[0145] Example 22 A method for preparing thioneine

[0146] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 15v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine and methionine (the mass ratio of cysteine and methionine is 1:1); wherein the soluble solids content in the sugarcane juice is 10% (Bix), the final concentration of the mixture of peptone and wheat bran is 1.0% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.1% (w / v, g / 100mL), and the final concentration of the mixture of cysteine and methionine is 0.15% (w / v, g / 100mL), and the pH is 6.5.

[0147] Example 23 A method for preparing thioneine

[0148] A method for preparing ergothioneine is identical to the method for Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 5v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine and histidine (Cys+His, the mass ratio of cysteine and histidine is 1:1); wherein, in the sugarcane juice, soluble solids content is 5% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.6% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.05% (w / v, g / 100mL), and the final concentration of the mixture of cysteine and histidine is 0.15% (w / v, g / 100mL), and the pH is 6.5.

[0149] Example 24 A method for preparing thioneine

[0150] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), the initial fermentation medium is different: in the present embodiment, the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine and histidine (the mass ratio of cysteine and histidine is 1:1); wherein the soluble solids content in the sugarcane juice is 8% (Bix), the final concentration of the mixture of peptone and wheat bran is 1.0% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.1% (w / v, g / 100mL), and the final concentration of the mixture of cysteine and histidine is 0.05% (w / v, g / 100mL), and the pH is 5.5.

[0151] Example 25 A method for preparing thioneine

[0152] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), the inoculum size and the initial fermentation medium are different: in the present embodiment, the inoculum size is 15v / v%, and the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine and histidine (the mass ratio of cysteine and histidine is 1:1); wherein the soluble solids content in the sugarcane juice is 10% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.2% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.01% (w / v, g / 100mL), and the final concentration of the mixture of cysteine and histidine is 0.1% (w / v, g / 100mL), and the pH is 6.0.

[0153] Example 26 A method for preparing thioneine

[0154] A method for preparing ergothioneine is identical to the method in Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 5v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and histidine (Cys+Met+His, the mass ratio of cysteine, methionine and histidine is 1:1:1); wherein the sugarcane juice has a soluble solid content of 8% (Bix), a final concentration of the mixture of peptone and wheat bran of 0.2% (w / v, g / 100mL), a final concentration of vitamin B1 of 0.1% (w / v, g / 100mL), and a final concentration of the mixture of cysteine, methionine and histidine of 0.1% (w / v, g / 100mL), and a pH of 6.5.

[0155] Example 27 A method for preparing thioneine

[0156] A method for preparing ergothioneine is the same as the method in Example 1, except that: in step (2), the initial fermentation medium is different: in the present embodiment, the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and histidine (the mass ratio of cysteine, methionine and histidine is 1:1:1); wherein the soluble solids content in the sugarcane juice is 10% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.6% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.01% (w / v, g / 100mL), the final concentration of the mixture of cysteine, methionine and histidine is 0.15% (w / v, g / 100mL), and the pH is 5.5.

[0157] Example 28 A method for preparing thioneine

[0158] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 15v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and histidine (the mass ratio of cysteine, methionine and histidine is 1:1:1); wherein the soluble solids content in the sugarcane juice is 5% (Bix), the final concentration of the mixture of peptone and wheat bran is 1.0% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.05% (w / v, g / 100mL), and the final concentration of the mixture of cysteine, methionine and histidine is 0.05% (w / v, g / 100mL), and the pH value is 6.0.

[0159] Example 29 A method for preparing thioneine

[0160] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 5v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and aspartic acid (Cys+Met+Asp, the mass ratio of cysteine, methionine and aspartic acid is 1:1:1); wherein the sugarcane juice has a soluble solid content of 10% (Bix), a final concentration of the mixture of peptone and wheat bran of 1.0% (w / v, g / 100mL), a final concentration of vitamin B1 of 0.05% (w / v, g / 100mL), and a final concentration of the mixture of cysteine, methionine and aspartic acid of 0.1% (w / v, g / 100mL), and a pH of 5.5.

[0161] Embodiment 30 A kind of method of preparing thioneine

[0162] A method for preparing ergothioneine is the same as the method in Example 1, except that: in step (2), the initial fermentation medium is different: in the present embodiment, the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and aspartic acid (the mass ratio of cysteine, methionine and aspartic acid is 1:1:1); wherein the soluble solids content in the sugarcane juice is 5% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.2% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.1% (w / v, g / 100mL), the final concentration of the mixture of cysteine, methionine and aspartic acid is 0.15% (w / v, g / 100mL), and the pH is 6.0.

[0163] Example 31 A method for preparing thioneine

[0164] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 15v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and aspartic acid (the mass ratio of cysteine, methionine and aspartic acid is 1:1:1); wherein the soluble solids content in the sugarcane juice is 8% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.6% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.01% (w / v, g / 100mL), and the final concentration of the mixture of cysteine, methionine and aspartic acid is 0.05% (w / v, g / 100mL), and the pH is 6.5.

[0165] Example 32 A method for preparing thioneine

[0166] A method for preparing ergothioneine is identical to the method for Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 5v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and glutamic acid (Cys+Met+Glu, the mass ratio of cysteine, methionine and glutamic acid is 1:1:1); wherein, in the sugarcane juice, soluble solids content is 8% (Bix), the final concentration of the mixture of peptone and wheat bran is 1.0% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.01% (w / v, g / 100mL), and the final concentration of the mixture of cysteine, methionine and glutamic acid is 0.15% (w / v, g / 100mL), and the pH is 6.0.

[0167] Example 33 A method for preparing thioneine

[0168] A method for preparing ergothioneine is the same as the method in Example 1, except that: in step (2), the initial fermentation medium is different: in the present embodiment, the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and glutamic acid (the mass ratio of cysteine, methionine and glutamic acid is 1:1:1); wherein the soluble solids content in the sugarcane juice is 10% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.2% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.05% (w / v, g / 100mL), and the final concentration of the mixture of cysteine, methionine and glutamic acid is 0.05% (w / v, g / 100mL), and the pH is 6.5.

[0169] Example 34 A method for preparing thioneine

[0170] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 15v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and glutamic acid (the mass ratio of cysteine, methionine and glutamic acid is 1:1:1); wherein the sugarcane juice has a soluble solid content of 5% (Bix), a final concentration of the mixture of peptone and wheat bran of 0.6% (w / v, g / 100mL), a final concentration of vitamin B1 of 0.1% (w / v, g / 100mL), and a final concentration of the mixture of cysteine, methionine and glutamic acid of 0.1% (w / v, g / 100mL), and a pH of 5.5.

[0171] Example 35 A method for preparing thioneine

[0172] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), the inoculum size and the initial fermentation medium are different: in the present embodiment, the inoculum size is 5v / v%, and the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and arginine (Cys+Met+Arg, the mass ratio of cysteine, methionine and arginine is 1:1:1); wherein the soluble solids content in the sugarcane juice is 10% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.6% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.1% (w / v, g / 100mL), and the final concentration of the mixture of cysteine, methionine and arginine is 0.05% (w / v, g / 100mL), and the pH is 6.0.

[0173] Example 36 A method for preparing thioneine

[0174] A method for preparing ergothioneine is the same as the method in Example 1, except that: the initial fermentation medium in step (2) is different: in the present embodiment, the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and arginine (the mass ratio of cysteine, methionine and arginine is 1:1:1); wherein the soluble solids content in the sugarcane juice is 5% (Bix), the final concentration of the mixture of peptone and wheat bran is 1.0% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.01% (w / v, g / 100mL), the final concentration of the mixture of cysteine, methionine and arginine is 0.1% (w / v, g / 100mL), and the pH is 6.5.

[0175] Example 37 A method for preparing thioneine

[0176] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), inoculum size and initial fermentation medium are different: in the present embodiment, inoculum size is 15v / v%, and initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine, methionine and arginine (the mass ratio of cysteine, methionine and arginine is 1:1:1); wherein the soluble solids content in the sugarcane juice is 8% (Bix), the final concentration of the mixture of peptone and wheat bran is 0.2% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.05% (w / v, g / 100mL), and the final concentration of the mixture of cysteine, methionine and arginine is 0.15% (w / v, g / 100mL), and the pH is 5.5.

[0177] Example 38 A method for preparing thioneine

[0178] A method for preparing ergothioneine is identical to the method of Example 1, except that: in step (2), the initial fermentation medium is different: in the present embodiment, the initial fermentation medium is sugarcane juice comprising a mixture of peptone and wheat bran (the mass ratio of peptone and wheat bran is 1:1), vitamin B1, and a mixture of cysteine and methionine (the mass ratio of cysteine and methionine is 1:1); wherein the soluble solids content in the sugarcane juice is 8% (Bix), the final concentration of the mixture of peptone and wheat bran is 1.0% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.05% (w / v, g / 100mL), and the final concentration of the mixture of cysteine and methionine is 0.15% (w / v, g / 100mL), and the pH is 5.5.

[0179] Example 39 A method for preparing thioneine

[0180] A method for preparing thioneine comprises the steps:

[0181] (1) Tremella fuciformis spore fermentation seed culture: cut off two pieces (0.5 cm wide) of Tremella fuciformis spore slant and inoculate them into potato glucose water culture medium, place them on a shaker, and culture them at 25°C, 150 r / min, and shake for 5 days to obtain Tremella fuciformis spore liquid seeds;

[0182] (2) Tremella fuciformis spore fermentation: Tremella fuciformis spore liquid seeds were inoculated into a fermentation tank filled with initial fermentation medium at an inoculum rate of 10 v / v%, stirred at a speed of 200 r / min, and fermented at 25°C for 3 days. Then, a mixture of cysteine and methionine (the mass ratio of cysteine to methionine was 1:1) was added to make the final concentration of the mixture 0.15% (w / v, g / 100 mL), stirred at a speed of 200 r / min, and fermented at 25°C for 4 days; the fermented product was homogenized under 25 MPa. The mixture was treated for 5 minutes and centrifuged at 4000 r / min for 10 minutes to obtain a fermentation product; the initial fermentation medium was sugarcane juice containing a mixture of peptone and wheat bran (the mass ratio of peptone to wheat bran was 1:1) and vitamin B1; the soluble solids content in the sugarcane juice was 8% (Bix), the final concentration of the mixture of peptone and wheat bran was 1.0% (w / v, g / 100 mL), the final concentration of vitamin B1 was 0.05% (w / v, g / 100 mL), and the pH was 5.5.

[0183] Example 40 A method for preparing thioneine

[0184] A method for preparing thioneine comprises the steps:

[0185] (1) Tremella fuciformis spore fermentation seed culture: cut off two pieces (0.5 cm wide) of Tremella fuciformis spore slant and inoculate them into potato glucose water culture medium, place them on a shaker, and culture them at 25°C, 150 r / min, and shake for 5 days to obtain Tremella fuciformis spore liquid seeds;

[0186] (2) Tremella fuciformis spore fermentation: Tremella fuciformis spore liquid seeds were inoculated into a fermentation tank containing an initial fermentation medium at an inoculation rate of 10 v / v%, and a first-stage fermentation was performed: a stirring speed of 200 r / min, and fermentation was performed at 25°C for 3 days; then, a mixture of cysteine and methionine (the mass ratio of cysteine to methionine was 1:1) was added to make the final concentration of the mixture 0.15% (w / v, g / 100 mL), and low-temperature induction was performed: fermentation was performed at a stirring speed of 200 r / min and 10°C for 8 h; finally, a second-stage fermentation was performed: a stirring speed of 200 r / min, and fermentation was performed at 10°C for 8 h. / min and fermentation at 25°C for 3 days; the fermentation product is homogenized at 25MP for 5 minutes and centrifuged at 4000r / min for 10 minutes to obtain a fermentation product; the initial fermentation medium is sugarcane juice containing a mixture of peptone and wheat bran (the mass ratio of peptone to wheat bran is 1:1) and vitamin B1; wherein the soluble solids content in the sugarcane juice is 8% (Bix), the final concentration of the peptone and wheat bran mixture is 1.0% (w / v, g / 100mL), the final concentration of vitamin B1 is 0.05% (w / v, g / 100mL), and the pH is 5.5.

[0187] Example 41 A method for preparing thioneine

[0188] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 250r / min, 25 DEG C of fermentations for 3 days; the condition of low-temperature induction is: at agitator speed 250r / min, 12 DEG C of bottom fermentations for 16h; the two-stage fermentation condition is: at agitator speed 250r / min, 28 DEG C of bottom fermentations for 2 days.

[0189] Example 42 A method for preparing thioneine

[0190] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 300r / min, 25 DEG C of fermentations for 3 days; the condition of low-temperature induction is: at agitator speed 300r / min, 15 DEG C of lower fermentations for 24h; the two-stage fermentation condition is: at agitator speed 300r / min, 30 DEG C of lower fermentations for 1 day.

[0191] Example 43 A method for preparing thioneine

[0192] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 300r / min, 28 DEG C of fermentations 2 days; the condition of low-temperature induction is: at agitator speed 300r / min, 10 DEG C of lower fermentations 8h; the two-stage fermentation condition is: at agitator speed 300r / min, 28 DEG C of lower fermentations 2 days.

[0193] Example 44 A method for preparing thioneine

[0194] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 200r / min, 28 DEG C of fermentations 2 days; the condition of low-temperature induction is: at agitator speed 200r / min, 12 DEG C of lower fermentations 16h; the two-stage fermentation condition is: at agitator speed 200r / min, 30 DEG C of lower fermentations 1 day.

[0195] Example 45 A method for preparing thioneine

[0196] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 250r / min, 28 DEG C of fermentations 2 days; the condition of low-temperature induction is: at agitator speed 250r / min, 15 DEG C of lower fermentations 24h; the two-stage fermentation condition is: at agitator speed 250r / min, 25 DEG C of lower fermentations 3 days.

[0197] Example 46 A method for preparing thioneine

[0198] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 250r / min, 30 DEG C of fermentations 2 days; the condition of low-temperature induction is: at agitator speed 250r / min, 10 DEG C of lower fermentations 8h; the two-stage fermentation condition is: at agitator speed 250r / min, 30 DEG C of lower fermentations 1 day.

[0199] Example 47 A method for preparing thioneine

[0200] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 300r / min, 30 DEG C of fermentations for 2 days; the condition of low-temperature induction is: at agitator speed 300r / min, 12 DEG C of lower fermentations for 16h; the two-stage fermentation condition is: at agitator speed 300r / min, 25 DEG C of lower fermentations for 3 days.

[0201] Example 48 A method for preparing thioneine

[0202] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 200r / min, 30 DEG C of fermentations 2 days; the condition of low-temperature induction is: at agitator speed 200r / min, 15 DEG C of lower fermentations 24h; the two-stage fermentation condition is: at agitator speed 200r / min, 28 DEG C of lower fermentations 2 days.

[0203] Example 49 A method for preparing thioneine

[0204] A method for preparing thioneine is identical to the method for Example 39, except that: one-stage fermentation, low-temperature induction, and two-stage fermentation conditions are different: the condition of one-stage fermentation in the present embodiment is: at agitator speed 300r / min, 25 DEG C of fermentations for 3 days; the condition of low-temperature induction is: at agitator speed 300r / min, 10 DEG C of lower fermentations for 8h; the two-stage fermentation condition is: at agitator speed 300r / min, 28 DEG C of lower fermentations for 2 days.

[0205] Effect embodiment

[0206] 1. take respectively the fermentation product of embodiment 1~8 (each sample repeats 3 times), with reference to the mensuration (ultra-high performance liquid chromatography) of L-ergothioneine in agricultural industry standard NY / T3872-2021 edible fungus, detect the content of ergothioneine in fermentation product (wherein (wherein, the standard product (50mg / L) of ergothioneine, embodiment 4, the liquid chromatogram of the fermentation product of 8 is as shown in Figure 2). Figure 4 、 5 , 6), the results are as follows Figure 1 As shown in the figure: When the mixture of peptone and wheat bran was used as the nitrogen source, the ergothioneine production was the highest, reaching 45.53 mg / L.

[0207] 2. take respectively the fermentation product of embodiment 9~13 (each sample repeats 3 times), with reference to the mensuration (ultra-high performance liquid chromatography) of L-ergothioneine in agricultural industry standard NY / T3872-2021 edible fungus, detect the content of ergothioneine in fermentation product (wherein, the liquid chromatogram of the fermentation product of embodiment 9 is as shown in Figure 2). Figure 7 The result is as shown in Figure 2 As shown: When vitamin B1 and vitamin B6 are used as vitamins, the ergothioneine production is the highest, reaching 58.67 and 58.16 mg / L respectively.

[0208] 3. take respectively the fermentation product of embodiment 14~19 (each sample repeats 3 times), with reference to the mensuration (ultra-performance liquid chromatography) of L-ergothioneine in agricultural industry standard NY / T3872-2021 edible fungus, detect the content of ergothioneine in fermentation product, result is as shown in Figure 3 As shown in the results, after adding amino acids to the initial fermentation medium, the content of ergothioneine in the fermentation product can be increased; among them, after adding cysteine, methionine and histidine, the content of ergothioneine in the fermentation product is significantly increased, especially after adding cysteine, the content of ergothioneine in the fermentation product can reach 87.36 mg / L.

[0209] 4. take the fermentation product of embodiment 19~38 respectively, with reference to the mensuration (ultra-high performance liquid chromatography) of L-ergothioneine in agricultural industry standard NY / T3872-2021 edible fungus, detect the content of ergothioneine in fermentation product (wherein the liquid chromatogram of the fermentation product of embodiment 27,38 is as shown in Figure 2). Figure 8 、 9 , result is as shown in Table 1: adopt embodiment 27,29,33,37,38 method to significantly improve the content of thioneine in fermentation (all reaching more than 100mg / L), especially, the content of thioneine in fermentation that the method for embodiment 38 prepares is up to 142.12mg / L.

[0210] 5. take the zymation of embodiment 39, with reference to the mensuration (ultra-performance liquid chromatography) of L-ergothioneine in agricultural industry standard NY / T3872-2021 edible fungi, detect the content of thioneine in zymation, result show: fermentation mid-term adds amino acid (cysteine+methionine) and can further improve the content of thioneine in zymation, its content is up to 181.41mg / L.

[0211] 6. get the fermentation product of embodiment 40~49, with reference to the mensuration (ultra-high performance liquid chromatography) of L-ergothioneine in agricultural industry standard NY / T3872-2021 edible fungus, detect the content of ergothioneine in fermentation product (wherein the liquid chromatogram of the fermentation product of embodiment 49 is as shown in Figure 5). Figure 10 3.42 times of Example 39, and its fermentation time has shortened by 40h.

[0212] Table 1 Parameters and results of the preparation of thioneine of Examples 20 to 38

[0213]

[0214]

[0215] Table 2 Parameters and results of the preparation of thioneine of Examples 40 to 49

[0216]

[0217] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method of inoculating Tremella fuciformis Tr21 spores into a fermentation medium for cultivation; the method is any one of (b1) to (b2): (b1) prepares the method for thioneine by biological fermentation method; (b2) a method for preparing a product by a biological fermentation process, wherein the active ingredient of the product comprises ergothioneine; The fermentation medium consists of sugarcane juice, nitrogen source, vitamin B1 and amino acids; The nitrogen sources are peptone and wheat bran; The concentration of the sugarcane juice in the fermentation medium is 8% based on its soluble solid content; the concentration of the nitrogen source in the fermentation medium is 1% based on a mass-to-volume ratio; the mass ratio of the wheat bran to the peptone is 1:1; the concentration of the vitamin B1 in the fermentation medium is 0.05% based on a mass-to-volume ratio; and the pH of the fermentation medium is 5.5; The amino acids are composed of cysteine and methionine in a mass ratio of 1:1; the concentration of the amino acids in the fermentation medium is 0.15% by mass volume ratio; The culture conditions are: 200-300 r / min, 25-30° C., and fermentation for 7 days.

2. A method of inoculating Tremella fuciformis Tr21 spores into a fermentation medium for cultivation; the method is any one of (b1) to (b2): (b1) prepares the method for thioneine by biological fermentation method; (b2) a method for preparing a product by a biological fermentation process, wherein the active ingredient of the product comprises ergothioneine; The fermentation medium consists of sugarcane juice, nitrogen source and vitamin B1; The nitrogen sources are peptone and wheat bran; The concentration of the sugarcane juice in the fermentation medium is 8% based on its soluble solid content; the concentration of the nitrogen source in the fermentation medium is 1% based on a mass-to-volume ratio; the mass ratio of the wheat bran to the peptone is 1:1; the concentration of the vitamin B1 in the fermentation medium is 0.05% based on a mass-to-volume ratio; and the pH of the fermentation medium is 5.5; The culture conditions are any one of (c2) to (c3): (c2) fermenting at 150-400 rpm and 23-35° C. for 1.5-4.5 days, then adding amino acids, and fermenting at 150-400 rpm and 23-35° C. for 2.5-5.5 days; (c3) fermenting at 150-400 rpm and 23-35° C. for 1.5-3.5 days, then adding amino acids, inducing at 150-400 rpm and 8-18° C. for 6-28 hours, and finally fermenting at 150-400 rpm and 23-35° C. for 0.5-3.5 days; The amino acids are composed of cysteine and methionine in a mass ratio of 1:1; the concentration of the amino acids in the fermentation medium is 0.15% in a mass volume ratio.

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