A method for improving ergothioneine production efficiency through two-stage regulation

Abstract

This invention discloses a two-stage regulation method to improve ergothioneine production efficiency. Seed culture of *Pleurotus ostreatus* is inoculated onto a fermentation medium supplemented with H2O2 and fermented for 5-7 days. Then, H2O2 is added again to the fermentation medium for 0.5-1.5 hours to stimulate fermentation, followed by the addition of vitamin C. Fermentation is terminated after 1.5-2.5 hours, and the mycelium is collected for ergothioneine extraction. The ergothioneine production efficiency was increased by up to 58.81% compared to the original strain. This invention's production method is simple and easy to control, yields high ergothioneine production, and all ergothioneine is intracellular, making it easy to harvest and simplifying subsequent purification processes, making it suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a method for improving ergothioneine production efficiency through two-stage regulation. Background Technology

[0002] Ergothioneine (EGT) is an essential natural water-soluble sulfur-containing amino acid with various physiological functions, including antioxidant, anti-inflammatory, anti-cancer, and skin-protective effects. In recent years, ergothioneine has been widely used in skincare products, pharmaceuticals, and health supplements, and has significant market potential in the future.

[0003] *Pleurotus ostreatus*, also known as Golden Top Pleurotus, is a fungus belonging to the family Pleurotaceae and the genus *Pleurotus*. The fruiting body is generally medium-sized. The cap is straw-yellow to bright yellow, smooth, funnel-shaped, with an inrolled edge, and 3-10 cm in diameter. The flesh is white, and the gills are white, dense, decurrent, and of unequal length. The stipe is eccentric, white, solid, 2-10 cm long, and 0.5-1.5 cm thick, often connected at the base. *Pleurotus ostreatus* has extremely high nutritional value, rich in polysaccharides, proteins, amino acids, minerals, and trace elements, possessing various physiological functions such as antioxidant, anti-inflammatory, and lipid-lowering effects, making it very popular among consumers. Studies have confirmed that *Pleurotus ostreatus* contains abundant ergothioneine and is a dominant strain for ergothioneine production.

[0004] Currently, various deep fermentation technologies for edible fungi have been used for ergothioneine production, but the fermentation cycle is generally long. Patent CN109939027A utilizes Hericium erinaceus fermentation to produce ergothioneine, requiring 10 days of seed culture, resulting in a long cycle and high production costs due to the use of glucose as a carbon source. Patent CN112501029A uses a combination of Hericium erinaceus and Pine Fungus for ergothioneine production, with a fermentation cycle of 20-30 days and a yield of only 423.9 mg / L, which is insufficient to meet current production demands. Patent CN113293184A uses Boletus edulis liquid fermentation to produce ergothioneine, with a fermentation cycle of 8 days and a yield of 600 mg / L. Although the yield is high, Boletus edulis strains are expensive, and the extraction method uses ultrasonic extraction with ethanol, resulting in high organic solvent consumption. The applicant's earlier patent CN115029398A uses hydrogen peroxide domestication to obtain strains with high antioxidant activity. However, considering that the domestication cycle is long and the effect is slow, there is an urgent need to develop a production process for ergothioneine that is high-yield, fast-acting, low-cost, short-cycle, and easy to control. Summary of the Invention

[0005] To address the problems of low strain production capacity, limited fermentation process control methods, and insignificant effects in existing methods, this invention provides a two-stage control method to improve ergothioneine production efficiency.

[0006] To achieve the above technical objectives, the present invention adopts the following solution:

[0007] A method for improving ergothioneine production efficiency through two-stage regulation includes:

[0008] The seed liquid of *Pleurotus ostreatus* was inoculated onto a fermentation medium supplemented with H2O2 and fermented for 5-7 days. Then, H2O2 was added to the fermentation medium again for 0.5-1.5 hours to stimulate fermentation. After that, vitamin C was added, and fermentation was terminated after 1-3 hours. The mycelium was collected and ergothionein was extracted.

[0009] In a preferred embodiment, the amount of H2O2 added twice is such that the final concentration of H2O2 in the fermentation medium is 5-15 mM, preferably 10 mM.

[0010] In a preferred embodiment, the final concentration of H2O2 added for the second time is the same as the concentration of H2O2 in the initial fermentation medium.

[0011] In a preferred embodiment, the amount of VC added is such that the final concentration of VC in the fermentation medium is 1.5 g / L.

[0012] As a preferred implementation method, H2O2 is added again after 5 days of fermentation.

[0013] As a preferred embodiment, H2O2 was added to the fermentation medium again for 1 hour to stimulate it, followed by the addition of VC.

[0014] As a preferred implementation method, fermentation is terminated 2 hours after the addition of vitamin C.

[0015] In a preferred embodiment, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and precursor substances, wherein the precursor substances are histidine, methionine, and / or cysteine. The carbon source in the medium can be one or more of glucose, glycerol, corn flour, sucrose, and molasses; the nitrogen source can be one or more of peptone, soybean peptone, tryptone, yeast extract, beef extract, and soybean meal. *Pleurotus ostreatus* 303 can absorb and utilize most carbon and nitrogen sources. Preferably, using corn flour as the carbon source and soybean peptone as the nitrogen source achieves maximum absorption and utilization as well as optimal ergothioneine yield.

[0016] The preferred fermentation medium consists of 30 g / L corn flour, 10 g / L soybean peptone, 6 g / L magnesium sulfate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L histidine, 2.5 g / L methionine, and 1.5 g / L cysteine.

[0017] As a preferred embodiment, the elm yellow mushroom spawn is cultured on a plate and then seed cultured, and the seed liquid is inoculated onto a fermentation medium for fermentation culture.

[0018] As a preferred embodiment, the harvested bacterial cells are washed with ultrapure water, mechanically crushed by centrifugation, and then extracted with hot water. The supernatant is collected by centrifugation, and ergothionein is extracted.

[0019] As a preferred implementation method, the fermentation control strategy employs a crushing inoculation method to facilitate the formation of small-diameter mycelial pellets, which is more conducive to the absorption of nutrients by the mycelial pellets.

[0020] The present invention has the following beneficial effects:

[0021] (1) A fermentation method for improving the production efficiency of ergothionein is provided. Through the two-stage oxidation stimulation of low concentration H2O2, elm yellow mushroom can accumulate a large amount of antioxidant substances such as ergothionein to protect the mycelium from oxidative stress damage and improve the antioxidant performance of the strain itself.

[0022] (2) The fermentation process of this invention is simple to operate. It preferably uses corn flour and soybean peptone as carbon and nitrogen sources, and only adds magnesium sulfate, potassium dihydrogen phosphate, His, Met, and Cys. All fermentation raw materials are readily available, the culture medium composition is simple, and under these conditions, ergothioneine yield is high, reaching 641.76 mg / L after 5 days of fermentation, significantly shortening the fermentation cycle and improving the fermentation performance of the strain. Production efficiency increased from 80.82 mg / L / d to 128.35 mg / L / d, an increase of 58.81%. Furthermore, the change in intracellular CAT enzyme levels also reflects the intracellular stress level under this strategy. This invention adds a mechanical crushing step to the traditional hot water extraction method, ensuring the full extraction of intracellular ergothioneine. Attached Figure Description

[0023] Figure 1 The effect of different concentrations of H2O2 stimulation on the ergot thioglycol yield of *Pleurotus ostreatus*; (A) EGT yield; (B) dry weight.

[0024] Figure 2 The effect of short-term H2O2 stimulation time on ergothioneine yield in *Pleurotus ostreatus* seeds; (A) ROS level in mycelial pellets; (B) ergothioneine yield.

[0025] Figure 3 Effects of transient H2O2 stimulation combined with VC mitigation on ROS levels and ergothioneine yield in *Pleurotus ostreatus* seeds; (A) ROS levels in mycelial pellets; (B) Ergothioneine yield.

[0026] Figure 4 The changes in CAT and EGT production under two-stage stimulation conditions on day 5 of fermentation are shown, including:

[0027] Control-1: Blank control group (no stimulation added);

[0028] Control-2: Single-stage stimulation group (only H2O2 stimulation for 1 hour on the fifth day, followed by the addition of VC, and bacterial harvesting 2 hours later);

[0029] H2O2: Two-stage stimulation group (H2O2 was added for stimulation at the beginning and on the fifth day of fermentation, and VC was added 1 hour after stimulation on the fifth day, and the bacteria were harvested 2 hours later). Detailed Implementation

[0030] The *Pleurotus ostreatus* 303 used in this example was purchased from the Shouguang Edible Fungus Research Institute in Shandong.

[0031] Example 1

[0032] This example demonstrates the normal fermentation process for elm yellow mushroom 303.

[0033] The optimal growth temperature for *Pleurotus ostreatus* 303 is 24–27℃. Under conditions of 25℃, the strain can reach peak production of ergothioneine by shaking it in a flask at 150 rpm for 7–8 days.

[0034] The fermentation process for producing EGT in this embodiment is as follows:

[0035] (1) Plate culture: Inoculate the elm yellow mushroom spawn onto sucrose solid medium (sucrose 20g / L, soybean peptone 2g / L, agar powder 1.5-2%) and culture at 25℃ until the mycelium covers the entire plate.

[0036] (2) Seed culture: The mycelia on the plate were broken and inoculated into the primary seed culture medium, the main components of which included 20-26 g / L potato dextrose solution, 0.5-2 g / L soybean peptone, 0.5-2 g / L magnesium sulfate, and 0.5-2 g / L potassium dihydrogen phosphate. In this example, the seed culture medium used had the components of 26 g / L potato dextrose solution, 2 g / L soybean peptone, 2 g / L magnesium sulfate, and 2 g / L potassium dihydrogen phosphate. The components of the secondary seed culture medium were the same as those of the primary seed culture medium. After culturing for 4-5 days until a large number of mycelia or mycelial balls grew, the mycelial balls were broken and inoculated into the secondary seed culture medium. After culturing for 2-3 days, it was used for fermentation culture.

[0037] (3) Fermentation culture: The seed liquid was inoculated into the normal fermentation medium at an inoculation rate of 20%. The medium components included 30 g / L corn flour, 10 g / L soybean peptone, 6 g / L magnesium sulfate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L histidine, 2.5 g / L methionine, and 1.5 g / L cysteine. After culturing at 25°C and 150 rpm in the dark for 7 days, the cells were harvested.

[0038] (4) Ergothionein content detection: After repeatedly washing the harvested bacteria with ultrapure water, add ultrapure water, mechanically crush at 5000 rpm for 30 s, extract with hot water at 95℃ for 1 h, centrifuge, and pass the supernatant through a membrane to detect the ergothionein content.

[0039] Example 2

[0040] This example illustrates the effect of different H2O2 stimulation concentrations on EGT synthesis.

[0041] Different concentrations of H2O2 (0, 5, 10, 15, 20, 25, and 30 mM) were added to the fermentation medium to study the effects of H2O2 on EGT synthesis and cell biomass. EGT production changes were monitored on days 2, 3, 4, 5, 6, and 7. Figure 1 As shown in Figure A, with the increase of H2O2 addition from 0-10 mM, EGT production significantly increased from 420.36 mg / L to 557.28 mg / L, an increase of 32.57%. Further increasing the H2O2 concentration gradually decreased ergothioneine production. This indicates that adding low concentrations of H2O2 to the *Pleurotus ostreatus* fermentation medium can lead to mild oxidative stress, which may increase ergothioneine synthesis, while excessively high concentrations are detrimental to ergothioneine accumulation. Figure 1 As shown in Figure B, within the 0-10 mM H2O2 range, the cell dry weight decreased from 39.45 g / L to 38.68 g / L, a decrease of only 1.95%, indicating that low concentrations of H2O2 have little effect on cell growth. Further increasing the H2O2 concentration to 30 mM resulted in a dry weight content of only 35.02 g / L, a decrease of 11.23% compared to the control group. This result is due to the toxic effect of high-concentration H2O2, leading to cell damage and inhibiting cell growth. Therefore, the reduced synthesis of ergothioneine under high-concentration H2O2 stimulation is due to both a decrease in cell biomass and the fact that EGT, as an antioxidant, is used to combat the oxidative damage caused by H2O2. In conclusion, 10 mM is the optimal concentration for promoting EGT synthesis.

[0042] Example 3

[0043] This embodiment specifically illustrates the effect of the brief stimulation time of H2O2 on EGT synthesis, which is used to determine the harvesting time.

[0044] After culturing in second-generation seed culture medium for 3 days, 10 mM H2O2 was added, and the changes in EGT were observed at stimulation times of 0, 0.5, 1, 2, 3, 4, 5, and 6 hours. Figure 2 As shown in Figure A, the ROS level within the bacteria changes continuously over different time periods, indicating that oxidative balance is a dynamically regulated process. Figure 2As shown in Figure B, EGT production gradually increased during the first hour of H2O2 stimulation, then declined. Although there were fluctuations in the following hours, the overall production was lower than that after 1 hour of stimulation. In the control group, the increase in EGT production within 0-1 hour was 24.56%, while in the experimental group, the increase was 47.20%. This indicates that even a brief 1-hour stimulation with H2O2 is beneficial for EGT biosynthesis. This may be because the bacteria activate their own antioxidant defense system to cope with stress when stimulated, promoting the synthesis of their own antioxidants.

[0045] Example 4

[0046] This embodiment specifically illustrates the effect of transient H2O2 stimulation combined with VC mitigation on EGT synthesis.

[0047] Following the brief H2O2 stimulation for 1 hour in Example 3, vitamin C was added to the culture medium to a final concentration of 1.5 g / L. Figure 3 As shown in Figure A, within two hours of adding VC, the bacterial cells turned white after NBT staining, indicating no oxidative stress and suggesting that the cells were in oxidative equilibrium. Figure 3 As shown in Figure B, after adding vitamin C for 1 hour, the EGT yield reached 148.49 mg / L, which was 1.62 times that of the previous hour. After adding vitamin C for 2 hours, the yield further increased to 153.71 mg / L, but after another hour, the yield showed a downward trend. Figure 3 The results from the study also showed that ROS levels increased again after 3 hours of vitamin C addition, indicating a recurrence of oxidative stress. Therefore, the optimal condition was determined to be adding vitamin C for 2 hours after a brief 1-hour H2O2 stimulation to alleviate the stress.

[0048] Example 5

[0049] Example 5 uses the same batch of seeds as in Example 1 for a comparative experiment, as detailed below:

[0050] Control group: Example 1;

[0051] Experimental Group 1: The only difference from Example 1 was the addition of H2O2 to the fermentation medium at a final concentration of 10 mM;

[0052] Experimental Group 2: The only difference from Experimental Group 1 was that on the fifth day of fermentation, a final concentration of 10 mM H2O2 was added to the fermentation medium for a brief stimulation of 1 hour before harvesting the bacteria. The results were as follows: Figure 4 .

[0053] Experimental Group 3: The only difference from Experimental Group 2 was that on the fifth day of fermentation, a short-term stimulation of 10 mM H2O2 was added to the fermentation medium for 1 hour, followed by the addition of 1.5 g / L VC to alleviate the stress for 2 hours before harvesting the bacteria. The results were as follows: Figure 4.

[0054] Experimental Group 4: The only difference from Experimental Group 1 was that on the sixth day of fermentation, H2O2 with a final concentration of 10mM was added to the fermentation medium for a brief stimulation of 1 hour before the bacteria were harvested.

[0055] Experimental Group 5: The only difference from Experimental Group 3 was that on the sixth day of fermentation, H2O2 with a final concentration of 10mM was added to the fermentation medium for a short-term stimulation of 1 hour, followed by the addition of VC with a final concentration of 1.5g / L to relieve the stress for 2 hours before harvesting the bacteria.

[0056] Experimental Group 6: The only difference from Experimental Group 2 was that on the seventh day of fermentation, H2O2 with a final concentration of 10mM was added to the fermentation medium for a brief stimulation of 1 hour before harvesting the bacteria.

[0057] Experimental Group 7: The only difference from Experimental Group 3 was that on the seventh day of fermentation, H2O2 with a final concentration of 10mM was added to the fermentation medium for a brief stimulation of 1 hour, followed by the addition of VC with a final concentration of 1.5g / L to relieve the stress for 2 hours before harvesting the bacteria.

[0058] The EGT yield results for Examples 1 and 5 are shown in Table 1:

[0059] Table 1. Ergothionein yield under different experimental conditions

[0060]

[0061]

[0062] It can be seen that the proposed solution can be directly used for production using 303 elm mushrooms, and the yield on the 5th day is comparable to that on the 7th day, which greatly improves production efficiency.

Claims

1. A method for improving ergothioneine production efficiency through two-stage regulation, characterized in that, include: The seed liquid of *Pleurotus ostreatus* was inoculated onto a fermentation medium supplemented with H2O2 and fermented for 5-7 days. Then, H2O2 was added to the fermentation medium again for 0.5-1.5 hours to stimulate fermentation. After that, vitamin C was added and fermentation was terminated after 1-3 hours. The mycelium was collected and ergothionein was extracted. The amounts of H2O2 added in the two additions were such that the final concentration of H2O2 in the fermentation medium was 5-15 mM; the final concentration of H2O2 added in the second addition was the same as the initial concentration of H2O2 in the fermentation medium. The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and precursor substances, wherein the precursor substances are histidine, methionine, and / or cysteine.

2. The method according to claim 1, characterized in that, The amount of H2O2 added twice was such that the final concentration of H2O2 in the fermentation medium was 10 mM.

3. The method according to claim 1, characterized in that, The amount of VC added is such that the final concentration of VC in the fermentation medium is 1.5 g / L.

4. The method according to claim 1, characterized in that, H2O2 was added again after 5 days of fermentation.

5. The method according to claim 1, characterized in that, H2O2 was added to the fermentation medium again for 1 hour to stimulate it, followed by the addition of VC.

6. The method according to claim 1, characterized in that, Fermentation was terminated 2 hours after adding vitamin C.

7. The method according to claim 1, characterized in that, After plate culture and seed culture, the elm yellow mushroom spawn was inoculated onto a fermentation medium for fermentation culture.

8. The method according to claim 1, characterized in that, The harvested bacterial cells were washed with ultrapure water, mechanically crushed by centrifugation, and then extracted with hot water. The supernatant was collected by centrifugation and ergothionein was extracted.

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