WO2020134687A1 - Method for preparing ergothioneine by biosynthesis and fermentation medium - Google Patents

Abstract

A method for preparing ergothioneine by biosynthesis. The method comprises the following steps: 1) strain inoculating, on slant, mycelia of Hericium erinaceus into a liquid seed medium for cultivation, so as to obtain a seed liquid; 2) inoculating the obtained seed liquid into a fermentation medium for fermentation, adding a precursor substance for cultivation, and determining the fermentation progress by means of pH till the endpoint of the fermentation, so as to obtain a fermentation broth; and (3) adding enzymes after the fermentation is completed, so as to perform enzymolysis till the endpoint, and performing heating to inactivate the enzymes, so as to leach the ergothioneine from the cells of the mycelia into the fermentation broth outside the cells. Further provided is a method for determining an endpoint of fermentation of ergothioneine and a fermentation medium.

Description

Method for preparing ergothione by biosynthesis and fermentation medium Technical field

The invention relates to a method for biosynthesizing ergothioneine by using Hericium erinaceus mycelia and a fermentation medium used therefor, which belongs to the field of microbial fermentation engineering.

Background technique

Ergothioneine (EGT), whose scientific name is 2-mercapto-L-histidine trimethyl internal salt, its molecular structure contains an imidazole-2-thione group represented by the following general formula (1). Ergothione is a natural amino acid rich in many animals and plants. It cannot be synthesized by the animal body. It can only be ingested from food. It is a rare amino acid.

Formula (1)

Studies have shown that ergothioneine has a strong anti-oxidant effect and can also protect cells from cell damage and even death caused by ultraviolet rays and gamma rays, so it has a broad application prospect in industries such as medicine, food and cosmetics. However, the content of ergothioneine in most organisms is very small, and animals cannot synthesize ergothioneine by themselves, and can only be obtained through food. Further research shows that many microorganisms such as fungi and actinomycetes can synthesize ergothioneine. Therefore, the use of biological fermentation technology to synthesize ergothioneine has attracted more and more attention.

At present, there are more and more researches on ergothioneine. For example, Patent Document 1 discloses "Strains for producing ergothioneine and the method for preparing ergothioneine", which is characterized by synthesizing ergothioneine by CGMCC No.6232 Ergothioneine is leached into the aqueous solution from the mycelium cells by stirring and heating, but this method of leaching may cause inadequate rupture of the mycelium cells and ergothioneine is not completely released outside the cells .

It is mentioned in Non-Patent Document 1 that the liquid deep-layer culture method is used for the cultivation of Pleurotus eryngii mycelium to produce ergothioneine. Discussion on optimization, the final fermentation output can reach 62.20mg/L. Coenzymes are indispensable in biocatalytic reactions. Adding an appropriate amount of coenzyme to the fermentation medium sometimes facilitates the production of the target product. If non-patent document 1 can add suitable coenzymes during the fermentation of Pleurotus eryngii mycelia, it may be Will greatly improve the ergothioneine yield.

It is mentioned in Non-Patent Document 2 that the production of ergothioneine by liquid submerged fermentation of Lentinus edodes mycelium can yield a yield of 3.45 mg/g DW, which is converted into 23.6 mg/L of ergothioneine in the fermentation broth.

Patent Document 2 discloses the "biological synthesis method of ergothioneine", which is characterized in that the mycelium of a large fungus is liquid-fermented in a triangular flask with a culture medium, and then the fermentation liquid is heated and stirred to make the mycelium The ergothioneine in the body was transferred from the cell to the fermentation broth, and the highest yield of ergothioneine obtained from the face-faced mushroom was 51mg/L, the highest yield of the lung-shaped pleurotus was 48mg/L, and the highest yield of wild purple spores was 37mg/L. L.

In summary, the current biosynthetic preparation of ergothioneine mainly has the following problems:

1. The fermentation yield is low, the production cost is high, and large-scale production and utilization are impossible;

2. The method for determining the end point of fermentation is not clear. If periodic sampling is used, after the treatment, the liquid phase is used to determine whether the ergothione content continues to increase. The process is complicated, and the endpoint judgment is not timely enough. If ergot sulfur is to be prolonged due to full synthesis, the fermentation time must be increased, and other fermentation by-products must be added, which brings difficulties to the later purification process;

3. The method of leaching ergothioneine from the inside of the mycelium cell to the outside of the cell mostly uses the method of stirring and heating. The cell breakage may not be complete. Ergothioneine cannot be completely leached, which causes waste, and the energy consumption of stirring and heating is high. , Thereby increasing production costs.

Existing technical literature

Patent Literature

Patent Literature 1: CN103734022A

Patent Literature 2: CN103184246A

Non-patent literature

Non-Patent Literature 1: Huang Lingyi, Submerged cultivation of Pleurotus eryngii mycelia with high ergotine content and its physiological activity [D]. Taiwan: Zhongxing University, 2010.

Non-Patent Literature 2: Pramvadee Tepwong et al. Mycobial enhancements of ergothioneine by submerged cultivation of application edible mushrooms mycelia and applications antioxidative compound [J]. Food Chemistry, 2012, 131:247～

Summary of the invention

In order to solve the above problems, the present invention provides a biosynthetic preparation method of ergothioneine and a fermentation medium thereof. The ergothioneine is prepared by deep fermentation of mycelium liquid using Hericium erinaceus CCTCC No: M2018567.

Specifically, the present invention relates to the following.

1. A biosynthetic preparation method of ergothioneine, the method comprises the following steps:

Inoculate Hericium (ericaceus) mycelium slant strains into liquid seed culture medium to obtain seed liquid;

Inoculate the obtained seed liquid into the fermentation medium for fermentation and add precursor material to culture, and judge the fermentation to the end of fermentation by pH to obtain the fermentation liquid; and

After the fermentation is completed, the enzyme is added, the enzyme is hydrolyzed to the end, the enzyme is heated to kill the enzyme, and the ergothioneine in the cell of the mycelium is leached into the fermentation liquid outside the cell.

2. The preparation method according to item 1, wherein the Hericium erinaceus is Hericium erinaceus CCTCC No: M 2018567.

3. The preparation method according to item 1, characterized in that the seed liquid is obtained by culturing for 5 to 10 days under shaking conditions of 15 to 30°C and 100 to 300 rpm.

4. The preparation method according to item 1, characterized in that the fermentation broth is obtained by culturing for 7 to 15 days under shaking conditions of 15 to 30°C and 100 to 300 rpm.

5. The preparation method according to any one of items 1 to 4, characterized in that the seed culture medium comprises the following components: 1 to 10% (w/v) sucrose, 1 to 5% (w/v ) Of soybean cake powder, 0.01 to 1% (w/v) sodium dihydrogen phosphate, 0.01 to 1% (w/v) sodium sulfate, pH 4.0 to 6.0.

6. The production method according to any one of items 1 to 4, wherein the fermentation medium includes the following components: 2 to 6% (w/v) carbon source, 1 to 3% (w/ v) Organic nitrogen source, 0.01 to 1% (w/v) inorganic salt, 0.0001 to 0.001% (w/v) trace elements, 0.0001 to 0.001% (w/v) coenzyme, pH 4.0 to 6.0 .

7. The preparation method according to any one of items 1 to 4, wherein the precursor substance comprises one or more of the following combinations: cysteine, histidine, methionine, Glutamine, aspartic acid, betaine.

8. The preparation method according to any one of items 1 to 4, characterized in that, at the end of fermentation, the pH of the fermentation broth is ≥ 6.0 and the residual sugar is 0.

9. The preparation method according to any one of items 1 to 4, characterized in that the enzyme comprises one or more of the following combinations: collapse enzyme, snail enzyme, hyaluronidase, wall-solving enzyme, β -Dextranase, protease, cellulase, pectinase, further preferably snail enzyme and collapse enzyme.

10. The preparation method according to item 6, wherein the carbon source of the fermentation medium includes one or a combination of the following: soluble starch, glycerin, glucose, sucrose, fructose, maltose, lactose, mannitol, maltose Alcohol, corn flour, galactose, maltodextrin.

11. The preparation method according to item 6, wherein the nitrogen source of the fermentation medium includes one or a combination of the following: beef extract, peptone, yeast powder, yeast extract, soybean powder, wheat peptone, bean cake powder , Soybean meal, bran, casein peptone, tryptone, corn steep liquor, ammonium sulfate, soybean peptide powder, urea.

12. The preparation method according to item 6, wherein the inorganic salt of the fermentation medium includes one or a combination of the following: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sulfuric acid Sodium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate.

13. The preparation method according to item 6, wherein the trace elements of the fermentation medium include one or a combination of the following: manganese sulfate, ferrous chloride, zinc chloride, manganese chloride, ferrous sulfate .

14. The production method according to item 6, wherein the coenzyme of the fermentation medium includes one or a combination of the following: biotin, niacin, pyridoxal phosphate, vitamin B ₆ , vitamin B ₁₂ , nuclear Flavin, vitamin B ₁ .

15. The preparation method according to item 6, wherein the composition of the fermentation medium is: 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% ( w/v) sodium dihydrogen phosphate, 0.01-0.05% (w/v) sodium sulfate, 0.0001-0.001% (w/v) zinc chloride, 0.0001-0.001% (w/v) niacin, the rest For water.

16. The preparation method according to any one of items 1 to 15, further comprising the steps of centrifuging the enzymolyzed fermentation broth and filtering the supernatant to obtain an ergothioneine extract.

17. The production method according to any one of items 1 to 16, wherein the production of ergothioneine is 240 mg/L or more.

18. A method for judging the end point of ergothioneine fermentation, when the fermentation broth pH ≥ 6.0 and the residual sugar is 0 as the end point of fermentation.

19. A fermentation medium consisting of 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% (w/v) sodium dihydrogen phosphate, 0.01-0.05% (w/v) sodium sulfate, 0.0001-0.001% (w/v) zinc chloride, 0.0001-0.001% (w/v) niacin, and the rest is water.

20. The fermentation medium according to item 19, which is composed of 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02 % (W/v) sodium sulfate, 0.0002% (w/v) zinc chloride, 0.0006% (w/v) niacin, and the rest is water.

21. A composition comprising Hericium erinaceus and a fermentation broth, the fermentation broth comprising ergothioneine, wherein the concentration of ergothioneine in the fermentation broth is 240 mg/L or more.

Among them, the fermentation medium used to produce the fermentation broth includes the following components: 2 to 6% (w/v) carbon source, 1 to 3% (w/v) organic nitrogen source, 0.01 to 1% (w/v ) Inorganic salts, 0.0001 to 0.001% (w/v) trace elements, 0.0001 to 0.001% (w/v) coenzymes, pH 4.0 to 6.0.

Specifically, the present invention provides a method for biosynthesis of ergothioneine, which includes the following steps:

Step 1: Inoculate the mycelium beveled strains of Hericium erinaceus into the liquid seed culture medium to obtain the seed liquid;

Step 2: Inoculate the obtained seed liquid into the fermentation medium to ferment and add precursor material to culture, judge the fermentation to the end of fermentation by pH, and obtain the fermentation liquid;

Step 3: After the fermentation is completed, the enzyme is added, the enzyme is hydrolyzed to the end point, the enzyme is heated to kill the enzyme, and the ergothioneine in the cell of the mycelium is leached into the fermentation liquid outside the cell.

The above-mentioned Hericium erinaceus is preferably Hericium erinaceus CCTCC No: M 2018567.

In step 1, specifically, culturing for 5-10 days under shaking conditions of 15-30°C and 100-300 rpm to obtain a seed solution;

Hericium can be obtained from PDA bevel strains;

The above-mentioned seed medium may include the following components: 1-10% (w/v) sucrose, 1-5% (w/v) soybean cake flour, 0.01-1% (w/v) sodium dihydrogen phosphate, 0.01 ~1% (w/v) sodium sulfate, pH 4.0~6.0;

The seed culture temperature is 15-30°C, preferably 20°C; the rotation speed is 100-300 rpm, preferably 150 rpm; the cultivation time is 5-10 days, preferably 7 days.

In step 2, specifically, in step 2, culture is carried out for 7 to 15 days under shaking conditions of 15 to 30°C and 100 to 300 rpm to obtain a fermentation broth;

The seed liquid may be inoculated into the fermentation medium in an amount of 1-10% (v/v), preferably 5% (v/v);

The above precursors include one or more of the following combinations: cysteine, histidine, methionine, glutamine, aspartic acid, betaine;

The addition amount of the precursor substance in the fermentation broth is 1-20 mM, preferably 5 mM; the addition time is 1-5 days of fermentation, preferably 3 days;

The fermentation temperature is 15-30°C, preferably 23°C; the rotation speed is 100-300 rpm, preferably 180 rpm; the cultivation time is 7-15 days;

The fermentation broth pH ≥ 6.0 and residual sugar is 0 as the fermentation end point. The above pH measurement can be directly detected using a pH meter. The above residual sugar content, specifically the residual glucose content can be determined by commonly used chemical methods such as DNS method, Ferrin reagent method, indirect iodometric method or optical rotation method, or using SBA-40D biosensor analyzer for detection ;

The fermentation medium may include the following components: 2 to 6% (w/v) carbon source, 1 to 3% (w/v) organic nitrogen source, 0.01 to 1% (w/v) inorganic salt, 0.0001 ～0.001%(w/v) of trace elements, 0.0001～0.001%(w/v) of coenzyme, pH 4.0～6.0;

Further, the above carbon source may include one or a combination of the following: soluble starch, glycerin, glucose, sucrose, fructose, maltose, lactose, mannitol, maltitol, corn flour, galactose, maltodextrin, preferably Glucose, maltose and/or sucrose, most preferably glucose;

The nitrogen source may include one or more of the following: beef extract, peptone, yeast powder, yeast extract, soybean powder, wheat peptone, soybean cake powder, soybean meal, bran, casein peptone, tryptone, corn steep liquor, sulfuric acid Ammonium, soybean peptide powder, urea, preferably beef extract and/or tryptone, most preferably beef extract;

The above inorganic salt may include one or a combination of the following: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate;

The above trace elements may include one or more of the following combinations: manganese sulfate, ferrous chloride, zinc chloride, manganese chloride, ferrous sulfate;

The above coenzyme may include one or more of the following combinations: biotin, niacin, pyridoxal phosphate, vitamin B ₆ , vitamin B ₁₂ , riboflavin, vitamin B ₁ , preferably niacin, especially at a concentration of 0.0006% (w/v) niacin;

In a specific embodiment, the composition of the fermentation medium is: 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% (w/v) Sodium dihydrogen phosphate, 0.01-0.05% (w/v) sodium sulfate, 0.0001-0.001% (w/v) zinc chloride, 0.0001-0.001% (w/v) niacin, and the rest is water.

In step 3, specifically, the above enzymes may include one or more of the following combinations: collapse enzymes, snail enzymes, hyaluronidase, lysozyme, β-glucanase, protease, cellulase, fruit Pectinase, preferably snail enzyme and collapse enzyme, enzyme activity is preferably 5000 ~ 10000U/g, the amount of enzyme added is 0.001 ~ 0.01% (w/v) of fermentation broth;

The pH of the enzymolysis is 4.5 to 7.5, preferably 6.5; the enzymolysis temperature is 30 to 60°C, preferably 40°C; the enzymolysis time is 2h to 4h, and the end of enzymolysis is no longer increased as the content of ergothioneine;

The process of temperature inactivation (enzyme inactivation) is to increase the temperature to 90～100℃ and keep it for 10～20min.

The biosynthetic preparation method of ergothioneine of the present invention also includes the steps of centrifuging the enzymolyzed fermentation broth, taking the supernatant and filtering to obtain an ergothioneine extract.

According to the biosynthetic preparation method of ergothioneine, the output of ergothioneine can reach more than 240mg/L.

The invention provides a method for judging the end point of ergothioneine fermentation, in which the fermentation end point is when the fermentation broth pH≥6.0 and the residual sugar is 0.

In addition, the invention also provides a fermentation medium, which is composed of 2-6% (w/v) glucose, 1-3% (w/v) beef extract, and 0.01-0.05% (w/v) phosphoric acid Sodium dihydrogen, 0.01～0.05% (w/v) sodium sulfate, 0.0001～0.001% (w/v) zinc chloride, 0.0001～0.001% (w/v) niacin, the rest is water

In a specific embodiment, the composition of the fermentation medium is 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) Sodium sulfate, 0.0002% (w/v) zinc chloride, 0.0006% (w/v) niacin, and the rest is water.

The invention has the following advantages:

By optimizing the carbon source, nitrogen source, coenzyme and other factors in the fermentation medium, the invention can significantly increase the output of ergothioneine. Under optimized conditions, the output of ergothioneine can reach more than 240mg/L, the highest 412.6mg/L.

By measuring the pH value of the fermentation broth, the invention can quickly and simply judge the fermentation end point, end the fermentation in time, and reduce the production of fermentation by-products.

The invention uses enzymes to break mycelium cells, which can fully break the cells, so that ergothioneine can be more completely leached into the fermentation broth, and the conditions are mild, the high temperature time is short, energy consumption is saved, and other active ingredients in the fermentation broth are reduced Loss.

BRIEF DESCRIPTION

Figure 1 is a schematic diagram of ergothioneine production from different fermentation carbon sources;

Figure 2 is a schematic diagram of ergothioneine production at different glucose concentrations;

Figure 3 is a schematic diagram of ergothioneine production from different fermentable nitrogen sources;

Figure 4 is a schematic diagram of ergothioneine production with different beef extract concentrations;

Figure 5 is a schematic diagram of ergothioneine production of different fermentation coenzymes;

Figure 6 is a schematic diagram of ergothioneine production with different niacin concentrations;

7 is a schematic diagram of ergothioneine production at different initial pH of fermentation;

Figure 8 is a schematic diagram of changes in pH, residual glucose content and ergothioneine content in the fermentation broth;

Fig. 9 is the HPLC chart of ergothioneine content determination, where (a) is the HPLC chart of the standard product (ergothioneine concentration concentration is 9.4 μg/mL), and (b) is the niacin content 0.0004% (w/v) The HPLC profile of the fermentation broth, (c) is the HPLC profile of the fermentation broth with a niacin content of 0.0005% (w/v), and (d) is the HPLC profile of the fermentation broth with a niacin content of 0.0006% (w/v).

Specific embodiments of the invention

The present invention will be described in detail below.

The biosynthetic preparation method of ergothioneine provided by the present invention includes the following steps:

Step 1: Inoculate the mycelium beveled strains of Hericium erinaceus into the liquid seed culture medium to obtain the seed liquid;

Step 2: Inoculate the obtained seed liquid into the fermentation medium to ferment and add precursor material to culture, judge the fermentation to the end of fermentation by pH, and obtain the fermentation liquid;

Step 3: After the fermentation is completed, the enzyme is added, the enzyme is hydrolyzed to the end point, the enzyme is heated to kill the enzyme, and the ergothioneine in the cell of the mycelium is leached into the fermentation liquid outside the cell.

The invention judges the fermentation to the end of fermentation by pH, which can quickly and easily determine the end of fermentation, end the fermentation in time, and reduce the production of fermentation by-products.

The invention uses enzymes to break mycelium cells, which can fully break the cells, so that ergothioneine is completely immersed in the fermentation broth, and the conditions are mild, the high temperature time is short, energy consumption is saved, and the loss of other active components in the fermentation broth is reduced .

step 1

Step 1: Inoculate the mycelium slant strain of Hericium erinaceus into the liquid seed medium for cultivation to obtain the seed solution.

Among them, the monkey mushroom is a fungus of the Basidiomycetes, Polyporaceae, Toothiaceae, Hericium, Heriniumus (Rull, F.) Pers., which is a saprophytic fungus and a well-known edible fungus. It looks like a hedgehog or a monkey head, so it is commonly known as hericium erinaceus or monkey mushroom. Among them, the Hericium erinaceus is preferably Hericium erinaceus CCTCC No: M 2018567, which was deposited on August 23, 2018 at the Chinese Type Culture Collection (CCTCC). This strain is a new one discovered by the applicant. Hericium erinaceus species, and found that it can be used for ergothioneine biosynthesis.

The single silk mesh cells are called hyphae, including vegetative hyphae and aerial hyphae. The hyphae gather together to form a certain macroscopic structure called mycelium.

Regarding the bevel strains, the culture medium is placed in a test tube, and after high temperature sterilization, the culture medium is arranged as a ramp culture medium, and the bacterial species inoculated on the bevel culture medium is called a bevel strain.

In a specific embodiment, the seed liquid is obtained by culturing for 5-10 days under shaking conditions of 15-30°C and 100-300 rpm.

In a specific embodiment, Hericium erinaceus is obtained from the PDA oblique strain, wherein PDA medium (Potato Dexrose Agar Medium) is short for potato dextrose agar medium.

In a specific embodiment, the above-mentioned seed medium includes the following components: 1-10% (w/v) sucrose, 1-5% (w/v) soybean cake powder, 0.01-1% (w/v) Sodium dihydrogen phosphate, 0.01 to 1% (w/v) sodium sulfate, pH 4.0 to 6.0. The seed culture temperature is 15-30°C, preferably 20°C; the rotation speed is 100-300 rpm, preferably 150 rpm; the cultivation time is 5-10 days, preferably 7 days. By cultivating Hericium erinaceus under the above conditions, it is more suitable for the growth of the bacterial species, and a bacterial species with good activity can be obtained.

Step 2

Step 2: Inoculate the obtained seed liquid into the fermentation medium to ferment and add precursor material to culture, judge the fermentation to the end of fermentation by pH, and obtain the fermentation liquid.

Fermentation refers to the process by which people use microbial life activities under aerobic or anaerobic conditions to prepare microbial cells themselves, or direct metabolites or secondary metabolites.

In a specific embodiment, the fermentation broth is obtained by culturing for 7 to 15 days under shaking conditions of 15 to 30°C and 100 to 300 rpm.

In a specific embodiment, the amount of seed liquid inoculated into the fermentation medium is not particularly limited, and may be 1-10% (v/v), preferably 5% (v/v).

The aforementioned precursor substance may include one or more of the following combinations: cysteine, histidine, methionine, glutamine, aspartic acid, and betaine. The addition amount of the precursor substance in the fermentation broth is 1-20 mM, preferably 5 mM; the addition time is 1 to 5 days of fermentation, preferably 3 days of fermentation. By adding precursor substances, it can provide better nutrition to Hericium erinaceus, which is conducive to its growth and metabolism.

Regarding the specific conditions of fermentation, the fermentation temperature may be 5 to 30°C, preferably 23°C; the rotation speed is 100 to 300 rpm, preferably 180 rpm; the cultivation time is 7 to 15 days, which is more suitable for the production of metabolites by Hericium erinaceus.

Regarding the method for determining the fermentation to the end point, the fermentation broth pH ≥ 6.0 and the residual sugar is 0 are regarded as the end point of fermentation. The above pH measurement can be directly detected using a pH meter. The above residual sugar content, specifically the residual glucose content can be determined by commonly used chemical methods such as DNS method, Ferrin reagent method, indirect iodometric method or optical rotation method, or using SBA-40D biosensor analyzer for detection . By measuring the pH value of the fermentation broth, the invention can quickly and simply judge the fermentation end point, end the fermentation in time, and reduce the production of fermentation by-products.

In a specific embodiment, the fermentation medium includes the following components: 2 to 6% (w/v) carbon source, 1 to 3% (w/v) organic nitrogen source, 0.01 to 1% (w/ v) Inorganic salt, 0.0001 to 0.001% (w/v) trace elements, 0.0001 to 0.001% (w/v) coenzyme, pH 4.0 to 6.0.

Further, the above carbon source is not particularly limited, and may include one or more of the following combinations: soluble starch, glycerin, glucose, sucrose, fructose, maltose, lactose, mannitol, maltitol, corn flour, galactose, malt paste Sperm, preferably glucose, maltose and/or sucrose, most preferably glucose.

The nitrogen source is not particularly limited, and may include one or more of the following combinations: beef extract, peptone, yeast powder, yeast extract, soybean powder, wheat peptone, soybean cake powder, soybean meal, bran, casein peptone, tryptone, Corn steep liquor, ammonium sulfate, soybean peptide powder, and urea are preferably beef extract and/or tryptone, and most preferably beef extract.

The above inorganic salt is not particularly limited, and may include one or more of the following combinations of sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

The above trace elements are not particularly limited, and may include one or a combination of the following: manganese sulfate, ferrous chloride, zinc chloride, manganese chloride, and ferrous sulfate.

The above coenzyme is not particularly limited, and may include one or more of the following combinations: biotin, niacin, pyridoxal phosphate, vitamin B ₆ , vitamin B ₁₂ , riboflavin, vitamin B ₁ , preferably niacin, Especially niacin with a concentration of 0.0006% (w/v).

In a specific embodiment, the composition of the fermentation medium is: 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% (w/v) Sodium dihydrogen phosphate, 0.01-0.05% (w/v) sodium sulfate, 0.0001-0.001% (w/v) zinc chloride, 0.0001-0.001% (w/v) niacin, and the rest is water.

By optimizing the carbon source, nitrogen source, coenzyme and other factors in the fermentation medium, the invention can significantly increase the output of ergothioneine. Under optimized conditions, the output of ergothioneine can reach more than 240mg/L, the highest 412.6mg/L.

Step 3

Step 3: After the fermentation is completed, the enzyme is added, the enzyme is hydrolyzed to the end point, the enzyme is heated to kill the enzyme, and the ergothioneine in the cell of the mycelium is leached into the fermentation liquid outside the cell.

An enzyme is a protein or RNA produced by living cells that has a high specificity and a high catalytic efficiency for its substrate. Enzymes are a very important type of biocatalyst. Due to the action of enzymes, chemical reactions in organisms can proceed efficiently and specifically under extremely mild conditions. Enzymatic hydrolysis is the decomposition of substances by the catalytic action of enzymes.

The above enzymes are not particularly limited as long as they can sufficiently disrupt cells, and may include one or more of the following combinations: collapse enzymes, snail enzymes, hyaluronidase, wall-solving enzymes, β-glucanase, protease, fiber Enzymes and pectinases are preferably snail enzymes and collapse enzymes. The enzyme activity is preferably 5,000 to 10,000 U/g, and the amount of enzyme added is 0.001 to 0.01% (w/v) of the fermentation broth.

The specific conditions for enzymolysis are not particularly limited as long as they can sufficiently break cells without destroying ergothioneine. Specifically, the pH of enzymolysis is 4.5 to 7.5, preferably 6.5; the enzymolysis temperature is 30 to 60°C , Preferably 40 ℃; enzymolysis time is 2 ~ 4h, in addition, ergothioneine content is no longer increased as the end point of enzymolysis.

The enzyme inactivation is to inactivate the enzyme. The conditions for inactivating the enzyme are not particularly limited as long as the enzyme is inactivated. Specifically, the process of inactivating the enzyme may be to increase the temperature to 90 to 100° C. and maintain it for 10 to 20 min.

The invention uses enzymes to break mycelium cells, which can fully break the cells, so that ergothioneine is completely immersed in the fermentation broth, and the conditions are mild, the high temperature time is short, energy consumption is saved, and the loss of other active components in the fermentation broth is reduced .

In addition, the biosynthetic preparation method of ergothioneine of the present invention further includes the steps of centrifuging the enzymolyzed fermentation broth, filtering the supernatant, and obtaining an ergothioneine extract. Thus, ergothioneine with higher purity can be obtained.

In addition, the present invention also provides a fermentation medium, the composition of which is 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% (w/v) phosphoric acid Sodium dihydrogen, 0.01-0.05% (w/v) sodium sulfate, 0.0001-0.001% (w/v) zinc chloride, 0.0001-0.001% (w/v) niacin, and the rest is water.

In a specific embodiment, the composition of the fermentation medium is 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) Sodium sulfate, 0.0002% (w/v) zinc chloride, 0.0006% (w/v) niacin, and the rest is water. Under this condition, the production of ergothioneine was the highest, reaching 412.6mg/L.

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

Examples

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Separately weigh 5000-10000U/g of snail enzyme and collapse enzyme to make enzyme liquid, and the enzyme activity is 5000U/mL, which are used in the examples, and snail enzyme and collapse enzyme are all commercially available.

Example 1: Ergotia fermentation by Hericium erinaceus CCTCC NO: M 2018567

Liquid seed medium: 4% (w/v) sucrose, 1.5% (w/v) soybean cake powder, 0.2% (w/v) sodium dihydrogen phosphate, 0.1% (w/v) sodium sulfate, The rest is water, pH 4.0 ~ 4.5, sterilized at 121 ℃ for 20min.

Fermentation medium: 3% (w/v) maltose, 2% (w/v) corn steep liquor, 0.05% (w/v) sodium dihydrogen phosphate, the rest is water, pH 4.0～4.5, at 121℃ Sterilize for 20min.

Each bottle of liquid seed culture medium was inoculated with hericium erinaceus CCTCC No: M 2018567 moss picked from the slant of the bacterial species, and cultured on a shaker at 20 rpm and 150 rpm for 7 days to obtain a seed solution. The seed solution was inserted into the fermentation medium at an inoculum volume of 5% by volume, and cultured on a shaker at 180 rpm at 23°C, and the precursor substances cysteine, betaine, and methionine were supplemented with 5mM each for 3 days after fermentation. The Hericium erinaceus CCTCC No: M 2018567 mycelium fermentation broth was obtained, and ergothioneine accumulated in the cells of the mycelium.

Example 2: ergothione extraction using snail enzymes and collapse enzymes

After the fermentation is completed, adjust the pH of the fermentation broth to 6.5±0.1, increase the temperature to 40±2℃, add 0.005% (w/v) snail enzyme and 0.005%(w/v) collapse enzyme, and keep the temperature at 40±2℃ for 2.5 h. After the enzymolysis is finished, raise the temperature of the fermentation broth to 90°C for 20 minutes to inactivate the enzyme. The enzymolyzed fermentation broth is centrifuged, the supernatant is taken, and then filtered through 1.2 μm fine filter paperboard and 0.22 μm filter element to sterilize to obtain the ergothioneine extract.

Example 3: Effect of different fermentation carbon sources on ergothioneine production

Replace the carbon sources of the fermentation medium with soluble starch, glycerol, glucose, sucrose, fructose, maltose, lactose, mannitol, maltitol, corn flour, galactose, and maltodextrin, as in Example 1. Fermentation culture, after the end of fermentation, the content of ergothioneine (EGT) in the fermentation broth was measured. The results are shown in Table 1 and Figure 1. When glucose is used as the carbon source, the content of ergothioneine in the fermentation broth is 151.7mg/L, and the yield is greatly increased. It can be used as the preferred carbon source for the production of ergothioneine by the liquid fermentation of Hericium erinaceus, followed by maltose (ergot) The thioneine content is 135.7 mg/L) and sucrose (ergothioneine content is 132.4 mg/L).

Table 1

Carbon source	Content of ergothioneine in fermentation broth (mg/L)
Soluble starch	83.7
glycerin	100.2
glucose	151.7
sucrose	132.4
fructose	77.2
maltose	135.7
lactose	52.7
Mannitol	57.3
Maltitol	61.3
corn flour	22.1
Galactose	67.9
Maltodextrin	86.6

Example 4: Effect of different glucose concentrations on ergothioneine production

Add glucose in the fermentation medium to 0% (w/v), 1% (w/v), 2% (w/v), 3% (w/v), 4% (w/v), 5 % (W/v), 6% (w/v), 7% (w/v), 8% (w/v), fermentation culture was carried out according to the method of Example 1, and the ergot sulfur in the fermentation broth was measured after the fermentation Due to the content, the results are shown in Table 2 and Figure 2. When the glucose concentration is 0, no ergothione is produced. With the increase of the addition amount, the production of ergothioneine gradually increases. When the addition amount is 4% (w/v), the yield reaches the highest, and then continue to increase the glucose addition amount. Production no longer increases, and there is a slow downward trend.

Table 2

Glucose addition %(w/v)	Content of ergothioneine in fermentation broth (mg/L)
0	0
1	70.4
2	127.9
3	150.1
4	167.9

5	161.4
6	162.3
7	151.0
8	145.3

Example 5: Effect of different fermentation nitrogen sources on ergothioneine yield

Replace the nitrogen sources of the above fermentation medium with: beef extract, peptone, yeast powder, yeast extract, soybean powder, wheat peptone, soybean cake powder, soybean meal, bran, casein peptone, tryptone, corn steep liquor, ammonium sulfate, soybean The peptide powder and urea were fermented and cultured according to the method of Example 1. After the fermentation, the content of ergothioneine in the fermentation broth was measured. The results are shown in Table 3 and FIG. 3. When beef extract is used as a nitrogen source, the content of ergothioneine in the fermentation broth is 191.3 mg/L, with the highest yield. It can be used as the preferred nitrogen source for the production of ergothioneine by the liquid fermentation of Hericium erinaceus, followed by tryptone (ergot sulfur Because the content is 177.5mg/L).

table 3

Nitrogen source	Content of ergothioneine in fermentation broth (mg/L)
Beef paste	191.3
Peptone	144.6
yeast	120.7
Yeast extract	118.5
Soy flour	75.4
Wheat peptone	110.6
Soybean flour	142.5
Soybean meal	131.2
Bran	92.6
Casein peptone	89.4
Tryptone	177.5
corn syrup	137.5
Ammonium sulfate	50.3
Soy peptide powder	65.3
Urea	45.7

Example 6: Effect of different beef extract concentrations on ergothioneine production

Add beef paste in fermentation medium to 0% (w/v), 0.5% (w/v), 1% (w/v), 1.5% (w/v), 2% (w/v), 2.5% (w/v), 3% (w/v), 3.5% (w/v), 4% (w/v), the amount of glucose added was 4% (w/v), according to the method of Example 1 Fermentation culture was carried out, and the content of ergothioneine in the fermentation broth was measured after the fermentation. The results are shown in Table 4 and Figure 4. When the concentration of beef paste is 0, there is almost no ergothione. With the increase of the amount of ergothione, the output of ergothione increases gradually. When the amount of ergothione is increased by 1.5% (w/v), the yield reaches the highest, and the beef paste is added continuously. The quantity and output have a slow downward trend.

Table 4

Addition of beef paste%(w/v)	Content of ergothioneine in fermentation broth (mg/L)
0	0.3
0.5	100.4
1	153.9
1.5	201.4
2	194.5
2.5	193.7
3	191.5
3.5	183.4
4	172.5

Example 7: Effect of different coenzymes on ergothioneine production

Fermentation medium: 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) sodium sulfate, 0.0002% (w/v) zinc chloride, the rest is water, pH 4.0 ~ 6.0, sterilized at 121 ℃ for 20min.

Add 0.0003% (w/v) of biotin, nicotinic acid, pyridoxal phosphate, vitamin B ₆ , vitamin B ₁₂ , riboflavin, vitamin B _{1 to the} above fermentation medium, and follow the method of Example 1 Fermentation culture. After the fermentation, the content of ergothioneine in the fermentation broth was measured. The results are shown in Table 5 and Figure 5. After the addition of coenzyme, the production of ergothioneine was improved, and the production of niacin was the highest. The content of ergothioneine in the fermentation broth reached 303.2mg/L.

table 5

Coenzyme

Content of ergothioneine in fermentation broth (mg/L)

Biotin	234.1
niacin	303.2
Pyridoxal phosphate	245.3
Vitamin B 6	222.6
Vitamin B 12	217.9
Riboflavin	251.7
Vitamin B 1	268.9

Example 8: Effect of different niacin addition on ergothioneine production

Fermentation medium: 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) sodium sulfate, 0.0002% (w/v) zinc chloride, the rest is water, pH 4.0 ~ 6.0, sterilized at 121 ℃ for 20min.

Add 0.0001 to 0.001% (w/v) niacin to the fermentation medium, and carry out fermentation culture according to the method of Example 1. After the fermentation is completed, the content of ergothioneine in the fermentation broth is measured. The results are shown in Table 6 and figure. 6. When the amount of niacin is 0.0006% (w/v), the ergothioneine yield is the highest, reaching 412.6mg/L.

Table 6

Example 9

The composition of the medium is respectively 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% (w/v) sodium dihydrogen phosphate, 0.01-0.05% (w/v) Sodium sulfate, 0.0001 to 0.001% (w/v) zinc chloride, 0.0001 to 0.001% (w/v) nicotinic acid to prepare a fermentation medium, followed by fermentation and fermentation according to the method of Example 1 After the end, the content of ergothioneine (EGT) in the fermentation broth was measured. The results are shown in Table 7. It can be seen from the results that the optimal fermentation medium is composed of 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) Sodium sulfate, 0.0002% (w/v) zinc chloride, 0.0006% (w/v) niacin, and the rest is water.

Table 7 Optimal fermentation medium composition and ergothioneine production

Comparative Example 1

In the Chinese patent CN105296559A, the fermentation is carried out with the mycelia of Pleurotus ostreatus CGMCC No: 6232, the moss of PDA slant strain CGMCC No: 6232 is picked into the seed culture medium, and cultured on a shaker at 150 rpm at 25°C for 4 days to obtain a seed solution . The seed liquid was inserted into the fermentation medium at an inoculation volume of 5% by volume, and cultured at 25 rpm with a shaker at 150 rpm for 15 days to obtain a mycelial fermentation liquid. The mycelial fermentation broth was placed in a 90°C water bath, leached for 15 min under 200 rpm stirring conditions, filtered, the filtrate was collected, and filtered using an ultrafiltration membrane with a molecular weight cutoff of 4 kDa. The resulting permeate was the ergothioneine content in the fermentation broth The maximum output of the samples to be tested reached 315.7mg/L.

Seed medium: corn flour 30g/L, soybean meal flour 15g/L, α-amylase 80U/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1.5g/L, the rest is water, and sterilized at 121°C for 20min.

Fermentation medium: casein peptone 60g/L, glycerin 75g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1.5g/L, methionine 14mmol/L, cysteine 7.5mmol/L, the rest is water, at 121 Sterilized at ℃ for 20min.

Example 10: Effect of different pH on ergothioneine production

Fermentation medium: 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) sodium sulfate, 0.0002% (w/v) zinc chloride, 0.0006% (w/v) nicotinamide, the rest is water, adjust the initial pH of fermentation to 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 at 121℃ Sterilize for 20min.

After the fermentation, the content of ergothioneine in the fermentation broth was measured. The results are shown in Table 8 and Figure 7. When the initial pH of the fermentation is 4.0-4.5, the yield is the highest. When the initial pH is above 6.0, the cell mass is small and the growth rate is slow, and the yield drops sharply.

Table 8

Initial pH of fermentation	Content of ergothioneine in fermentation broth (mg/L)
3.5	364.5
4.0	411.1
4.5	409.9
5.0	385.4
5.5	367.8
6.0	197.4
6.5	100.4
7.0	23.9

Example 11: Determination of fermentation end point

Fermentation medium: 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) sodium sulfate, 0.0002% (w/v) zinc chloride, 0.0006% (w/v) niacin, the rest is water, pH 4.0-4.5, sterilized at 121°C for 20 min.

Fermentation culture was carried out according to the method of Example 1. After 4 days of fermentation, that is, the next day after the supplementation of precursor amino acids, regular sampling was started, and the changes of pH, residual sugar and ergothione content of the fermentation broth were measured. When the pH of the fermentation broth is relatively stable, the sugar consumption rate is faster, and the mycelium synthesizes ergothioneine in large quantities. As the glucose in the culture medium is about to be depleted, ergothioneine synthesis slows down and the pH gradually rises, and the cell may produce other substances under sugar-free conditions. When the pH of the fermentation broth is ≥ 6.0 and the residual sugar is 0, ergothioneine reaches its maximum output, and the cultivation is continued, and the output no longer increases. Combining the results in Example 4 and Example 10, when the glucose content is 0, no ergothione is produced, and when the pH is greater than 6.0, the growth state of the bacteria is poor. Therefore, the fermentation end point is determined to be the fermentation broth pH ≥ 6.0, the residual Sugar is 0.

Example 12: Detection of glucose content in fermentation broth

After turning on the SBA-40D biosensor analyzer and preheating for half an hour, use 100mg/dL glucose standard for calibration. After the calibration is passed, take the fermentation broth and dilute it to a certain multiple (measurement range is 0～100mg/dL) ), after filtering through a 0.22 μm needle filter, the filtrate is taken for detection using a SBA-40D biosensor analyzer with a sample injection volume of 25 μL.

Example 13: Detection of ergothioneine content

The fermentation broth with the niacin content of Example 9 being 0.0004% (w/v), 0.0005% (w/v), and 0.0006% (w/v) was diluted 10 times as the test sample, and high performance liquid chromatography was used Method for ergothione content determination, HPLC conditions: chromatographic column: Hypersil ODS C18 column (250mm×4.6mm, particle size 5μm); column temperature: 30℃; mobile phase: acetonitrile-water (3:97); flow rate: 1.0 mL/min; detection wavelength: 254nm; injection volume: 20μL. The chromatogram is shown in Figure 9, where a) is the standard product (ergothioneine concentration is 9.4 μg/mL), b) is the fermentation broth with niacin content 0.0004% (w/v), c) is the niacin content 0.0005% (w/v) fermentation broth, d) is a fermentation broth with a niacin content of 0.0006% (w/v).

Claims (21)

1. A biosynthetic preparation method of ergothioneine, the method includes the following steps:

   Inoculate Hericium (ericaceus) mycelium slant strains into liquid seed culture medium to obtain seed liquid;

   Inoculate the obtained seed liquid into the fermentation medium for fermentation and add precursor material to culture, and judge the fermentation to the end of fermentation by pH to obtain the fermentation liquid; and

   After the fermentation is completed, the enzyme is added, the enzyme is hydrolyzed to the end, the enzyme is heated to kill the enzyme, and the ergothioneine in the cell of the mycelium is leached into the fermentation liquid outside the cell.
2. The preparation method according to claim 1, wherein the Hericium erinaceus is Hericium erinaceus CCTCC No: M 2018567.
3. The preparation method according to claim 1, wherein the seed solution is obtained by culturing for 5-10 days under shaking conditions of 15-30°C and 100-300 rpm.
4. The preparation method according to claim 1, characterized in that the fermentation broth is obtained by culturing for 7 to 15 days under shaking conditions of 15 to 30°C and 100 to 300 rpm.
5. The preparation method according to any one of claims 1 to 4, wherein the seed medium includes the following components: 1 to 10% (w/v) sucrose, 1 to 5% (w/v) Soybean cake powder, 0.01-1% (w/v) sodium dihydrogen phosphate, 0.01-1% (w/v) sodium sulfate, pH 4.0-6.0.
6. The preparation method according to any one of claims 1 to 4, wherein the fermentation medium includes the following components: 2 to 6% (w/v) carbon source, 1 to 3% (w/v ) Organic nitrogen source, 0.01 to 1% (w/v) inorganic salt, 0.0001 to 0.001% (w/v) trace elements, 0.0001 to 0.001% (w/v) coenzyme, pH 4.0 to 6.0.
7. The preparation method according to any one of claims 1 to 4, wherein the precursor substance comprises one or more of the following combinations: cysteine, histidine, methionine, gluten Aminoamide, aspartic acid, betaine.
8. The preparation method according to any one of claims 1 to 4, wherein the fermentation broth has a pH ≥ 6.0 and a residual sugar of 0 at the end of fermentation.
9. The preparation method according to any one of claims 1 to 4, wherein the enzyme comprises one or a combination of the following: collapse enzyme, snail enzyme, hyaluronidase, wall-solving enzyme, β- Dextranase, protease, cellulase, pectinase, further preferably snail enzymes and collapse enzymes.
10. The preparation method according to claim 6, wherein the carbon source of the fermentation medium comprises one or a combination of the following: soluble starch, glycerin, glucose, sucrose, fructose, maltose, lactose, mannitol, maltitol , Corn flour, galactose, maltodextrin.
11. The preparation method according to claim 6, wherein the nitrogen source of the fermentation medium comprises one or a combination of the following: beef extract, peptone, yeast powder, yeast extract, soybean powder, wheat peptone, soybean cake powder, Soybean meal, bran, casein peptone, tryptone, corn steep liquor, ammonium sulfate, soybean peptide powder, urea.
12. The preparation method according to claim 6, wherein the inorganic salt of the fermentation medium includes one or a combination of the following: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate , Potassium dihydrogen phosphate, dipotassium hydrogen phosphate.
13. The preparation method according to claim 6, wherein the trace elements of the fermentation medium include one or a combination of the following: manganese sulfate, ferrous chloride, zinc chloride, manganese chloride, ferrous sulfate.
14. The preparation method according to claim 6, wherein the coenzyme of the fermentation medium comprises one or a combination of the following: biotin, niacin, pyridoxal phosphate, vitamin B ₆ , vitamin B ₁₂ , riboflavin Vegetarian, vitamin B ₁ .
15. The preparation method according to claim 6, wherein the composition of the fermentation medium is: 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% (w /v) sodium dihydrogen phosphate, 0.01-0.05% (w/v) sodium sulfate, 0.0001-0.001% (w/v) zinc chloride, 0.0001-0.001% (w/v) nicotinic acid, the rest is water.
16. The preparation method according to any one of claims 1 to 15, further comprising the steps of centrifuging the enzymolyzed fermentation broth, filtering the supernatant, and obtaining an ergothioneine extract.
17. The production method according to any one of claims 1 to 16, wherein the production of ergothioneine is 240 mg/L or more.
18. A method for judging the end point of ergotin fermentation, when the fermentation broth pH≥6.0 and the residual sugar is 0 as the end point of fermentation.
19. A fermentation medium consisting of 2-6% (w/v) glucose, 1-3% (w/v) beef extract, 0.01-0.05% (w/v) sodium dihydrogen phosphate, 0.01- 0.05% (w/v) sodium sulfate, 0.0001 to 0.001% (w/v) zinc chloride, 0.0001 to 0.001% (w/v) niacin, and the rest is water.
20. The fermentation medium according to claim 19, which is composed of 4% (w/v) glucose, 1.5% (w/v) beef extract, 0.05% (w/v) sodium dihydrogen phosphate, 0.02% (w/v) Sodium sulfate, 0.0002% (w/v) zinc chloride, 0.0006% (w/v) niacin, and the rest is water.
21. A composition comprising Hericium erinaceus and a fermentation broth, wherein the fermentation broth contains ergothioneine, wherein the concentration of ergothioneine in the fermentation broth is 240 mg/L or more.

Images