A method for increasing the intracellular ergothioneine yield of Pleurotus citrinopileatus

Abstract

The present invention discloses a method for increasing the intracellular ergothioneine yield of Pleurotus citrinopileatus. The Pleurotus citrinopileatus strain is continuously domesticated using hydrogen peroxide, and the domesticated bacteria are used for fermenting to produce ergothioneine. The present invention also optimizes the culture medium for fermenting and culturing Pleurotus citrinopileatus. Substances such as histidine, cysteine, and methionine are added during the fermentation and culture process. A vitamin complex solution is added at 4 - 5 days. Inositol is particularly added to promote nutrient absorption, and at the same time, the rotation speed is increased to strengthen oxygen supply. The highest yield can reach 681.95 mg / L. The consumption rate of corn flour is increased from 3.125 g / d to 4 g / d, with a rate increase of 28%, and the seed vitality is improved. The method of the present invention is simple and easy to control, with a high ergothioneine yield and all in the cells, which is easy to harvest, simplifies the subsequent purification process, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation, and particularly relates to a method for increasing the intracellular ergothioneine yield of Pleurotus citrinopileatus. Background Art

[0002] Ergothioneine (EGT, trimethylbetaine of 2-mercapto-L-histidine) is a natural and rare amino acid derivative, which can directly scavenge free radicals, chelate various divalent metal cations, activate antioxidant enzymes, and inhibit the oxidation of various hemoproteins. In addition, different from other antioxidants, after being absorbed by the human body, ergothioneine does not undergo rapid metabolism, but exists stably in the form of thione and is not prone to auto-oxidation. It is a new type of antioxidant. Currently, it is widely used in the cosmetics and food industries, such as Estée Lauder cream, Clinique essence, Explabs capsule supplements, etc.

[0003] Pleurotus citrinopileatus, also known as Pleurotus citrinopileatus Singer and Pleurotus citrinopileatus, is one of the common edible fungi in China, mainly distributed in regions such as Heilongjiang, Yunnan, and Sichuan. Pleurotus citrinopileatus has extremely high edible value and is rich in various nutrients such as protein, amino acids, minerals, and trace elements, and is deeply loved by consumers. In addition, the mechanism of action of the physiological activity of Pleurotus citrinopileatus, such as antioxidant, anti-inflammatory, and lipid-lowering effects, is gradually being thoroughly studied with the development of technology. Research has confirmed that edible fungi are one of the main sources of ergothioneine. However, the fruiting body of Pleurotus citrinopileatus has a long growth cycle and a high price, and is not suitable as a source for industrial extraction of ergothioneine.

[0004] At present, various submerged fermentation technologies of edible fungi have been used for the production of ergothioneine, but they face problems such as long fermentation cycle, complex process, high production cost, and low yield. Patent CN112195215A requires the co-fermentation of Pleurotus citrinopileatus and Tricholoma matsutake, has high requirements for the synergistic effect of the two fungi, is difficult to control the process, cannot judge the situation of the strains, and Tricholoma matsutake itself has a high price and high cost. Patent CN109939027A uses Hericium erinaceus to ferment and produce ergothioneine. The seed culture takes 10 days, with a long cycle, and the production cost is high using glucose as the carbon source. Patent CN112501029A uses Hericium erinaceus and Tricholoma matsutake to co-ferment and produce ergothioneine. The fermentation cycle is 20 - 30 days, and the yield is only 423.9 mg / L, which is difficult to meet the current production requirements. Therefore, there is an urgent need to develop an ergothioneine production process with high yield, low production cost, short cycle, and easy control. Summary of the Invention

[0005] Aiming at the problems in the existing methods, such as low production capacity of strains, limited fermentation process control methods and insignificant effects, the present technology has developed a method for increasing the intracellular ergothioneine yield of Pleurotus citrinopileatus.

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

[0007] A method for increasing the intracellular ergothioneine yield of Pleurotus citrinopileatus. The Pleurotus citrinopileatus strain is continuously domesticated using hydrogen peroxide, and the domesticated strain is used for fermenting to produce ergothioneine.

[0008] Through the domestication technology, the present invention uses the continuous oxidation stimulation of low-concentration H2O2 to enable the strain to have good antioxidant capacity and improve the performance of producing ergothioneine.

[0009] As a preferred embodiment, after inoculating the Pleurotus citrinopileatus strain onto a plate containing hydrogen peroxide and continuously culturing, the growth of the strain is detected, and a well-growing domesticated strain is selected to ferment the domesticated strain to produce ergothioneine.

[0010] As a preferred embodiment, the method for determining the domestication concentration of hydrogen peroxide is as follows:

[0011] The Pleurotus citrinopileatus strain is inoculated onto plates containing different concentrations of hydrogen peroxide, arranged in ascending order of hydrogen peroxide concentration, and the growth differences are compared with the mycelial diffusion diameter as an index to determine the hydrogen peroxide concentration that causes a mutation in the growth rate of the Pleurotus citrinopileatus strain but still allows normal growth, which is used as the domestication concentration.

[0012] As a preferred embodiment, the domesticated strain is inoculated into a fermentation medium for fermentation culture. The carbon source of the fermentation medium is selected from one or more of glucose, glycerol, and corn flour, preferably 20 - 40 g / L of corn flour or 30 g / L of glucose;

[0013] The nitrogen source is selected from one or more of peptone, soy peptone, tryptone, yeast powder, beef extract, and soybean meal powder; preferably 10 - 15 g / L of soy peptone or 10 g / L of tryptone.

[0014] As a preferred embodiment, when fermenting and culturing the domesticated strain, one or more of histidine, methionine, and cysteine are added to the fermentation medium at a concentration of 1 - 2 g / L; preferably, a combination of 1 - 2 g / L of histidine, 1 - 2 g / L of methionine, and 1 - 2 g / L of cysteine is added.

[0015] As a preferred embodiment, when fermenting and culturing the domesticated strain, the liquid loading amount of the fermentation medium is 50 - 125 mL.

[0016] As a preferred embodiment, after 4 - 5 days of fermenting and culturing the domesticated strain, a vitamin complex solution is added to the fermentation medium, and its components include p-aminobenzoic acid, nicotinic acid, calcium pantothenate, thiamine nicotinate, pyridoxine hydrochloride, and inositol. Ergothioneine is a secondary metabolite. Preferably, the vitamin complex solution is added in the later stage to provide sufficient nutrients. In particular, inositol is added to promote the absorption of other active ingredients, and at the same time, the rotation speed can be increased to strengthen the oxygen supply.

[0017] As a preferred embodiment, ergothioneine is produced by fermenting and domesticating bacteria in a way of forming small-sized bacterial pellets through crushing inoculation. The method of crushing inoculation is adopted to facilitate the formation of small-sized bacterial pellets, which is more conducive to the absorption of nutrients by the bacterial pellets.

[0018] As a preferred embodiment, during the fermentation and culture of the domesticated bacteria, the rotation speed is 150 rpm to 170 rpm.

[0019] As a preferred embodiment, after the domesticated bacteria are activated on a plate and cultured in seeds, they are inoculated into a fermentation medium for fermentation and culture. Among them, the medium used for plate activation is a sucrose solid medium, which is composed of sucrose, soy peptone, and agar powder.

[0020] As a preferred embodiment, the temperature for fermentation and culture is 24 - 27 °C, preferably 25 °C.

[0021] As a preferred embodiment, the steps of fermentation and culture are as follows:

[0022] a. Plate culture;

[0023] b. Crushing and inoculating the plate mycelium into the primary seed medium and culturing until a large amount of mycelium or bacterial pellets are formed;

[0024] c. After the primary seed liquid is crushed, it is inoculated into the secondary seed liquid and cultured for 3 - 4 days;

[0025] d. Crushing and inoculating the cultured seed liquid into the fermentation medium and continuing to culture for 7 - 8 days.

[0026] The present invention has the following beneficial effects:

[0027] (1) By continuously oxidizing and stimulating Pleurotus citrinopileatus Sing. strains with low-concentration H2O2, the domesticated bacteria can accumulate a large amount of antioxidant substances such as ergothioneine to protect the mycelium from oxidative stress damage, improve the antioxidant performance of the strains themselves, enhance the mycelial vitality, and provide an excellent strain for subsequent fermentation.

[0028] (2) The deep fermentation characteristics of the domesticated bacteria are studied, and a complete fermentation process flow is established. The present invention selects corn flour and soy peptone as carbon and nitrogen sources, and on this basis, components such as magnesium sulfate, potassium dihydrogen phosphate, His, Met, Cys, vitamins, and organic acids are added. All fermentation raw materials are easily available, and under the conditions of this rich medium, the yield of ergothioneine is relatively high. The yield can reach 681.95 mg / L after 7 days of fermentation, which is the highest among all single-strain fermentations at present. It greatly improves the fermentation performance of a single strain.

[0029] (3) The liquid submerged fermentation technology provided by the present invention is simple to operate and the raw materials are inexpensive, which is very suitable for the industrial production of ergothioneine. The liquid seeds for subsequent fermentation can be continuously passaged without reducing the production performance, ensuring the stability of production while increasing the yield. Description of the Drawings

[0030] Figure 1 Effect diagram of yield improvement. Detailed Embodiments

[0031] In the examples, the Pleurotus citrinopileatus strain (Pleurotus citrinopileatus 303) was purchased from the Edible Fungi Research Institute of Shouguang City, Shandong Province.

[0032] PDA medium: 26 g / L of potato dextrose water, 2 g / L of soy peptone, 15 - 20 g / L of agar powder.

[0033] Sucrose solid medium: 20 g / L of sucrose, 2 g / L of soy peptone, 15 - 20 g / L of agar powder.

[0034] The steps for the strain to ferment and produce ergothioneine are as follows (the original strain and the domesticated strain adopt the same fermentation conditions):

[0035] Plate culture: Inoculate the original strain / domesticated strain on the sucrose solid medium and culture at 24 - 27°C until the mycelium covers the entire plate.

[0036] Seed culture: Crush the plate mycelium and inoculate it into the primary seed medium, the components of which mainly include 20 - 26 g / L of potato dextrose water, 0.5 - 2 g / L of soy peptone, 0.5 - 2 g / L of magnesium sulfate, and 0.5 - 2 g / L of potassium dihydrogen phosphate. Culture for 4 - 5 days until a large amount of mycelium or mycelial balls grow, continue to crush the mycelial balls, and inoculate them into the secondary seed medium (with the same components as the primary seed medium) at an inoculation amount of 5% (v / v), and use it for fermentation culture after 2 - 3 days of culture.

[0037] Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation amount of 20% (v / v), and culture at 24 - 27°C and 150 - 170 rpm for 7 - 8 days.

[0038] Ergothioneine extraction: Collect the thalli, wash them repeatedly with ultrapure water, extract them with hot water at 95°C for 1 h, centrifuge, and detect the ergothioneine content in the supernatant by passing it through a membrane and using high-performance liquid chromatography.

[0039] The Pleurotus citrinopileatus Sing. strain was inoculated on PDA plates containing different concentrations of hydrogen peroxide, and the growth differences were compared using the hyphal diffusion diameter as an index. A concentration of 10 mmol / L was preferably selected for subsequent domestication experiments. The Pleurotus citrinopileatus Sing. was continuously cultured on PDA plates containing 10 mmol / L H2O2 for 10 generations in a cycle. The growth of the strain was measured, and a well-growing domesticated strain was selected and punched and inoculated on a new PDA plate without H2O2 and a PDA plate containing 10 mmol / L H2O2. At the same time, the original strain was inoculated on a PDA plate without H2O2 and cultured at a constant temperature of 25°C. The results showed that when no H2O2 was added, the growth of the domesticated strain was better than that of the original strain. The domesticated strain and the original strain were both inoculated into the initial fermentation medium (30 g / L glucose, 10 g / L peptone, 2 g / L magnesium sulfate, 2 g / L potassium dihydrogen phosphate), and after fermentation at 150 rpm and 25°C for 7 days, the samples were centrifuged, and the cells were repeatedly washed with ultrapure water, then ultrapure water was added, and ergothioneine was extracted with hot water at 95°C. The intracellular ergothioneine content was measured by passing the extract through a membrane.

[0040] Domestication results: Under the stimulation of 10 mmol / L H2O2, the hyphal growth was slow, but when the domesticated strain was re-inoculated on a normal plate, the domesticated strain showed a better growth rate than the original strain. And it can be seen from three repeated experiments (Table 1) that the improvement effect of this method on the growth rate is stable, and the growth rate of the domesticated strain after H2O2 stimulation is better than that of the original strain. Using the domesticated strain picked from the normal PDA plate - A for the preliminary ergothioneine yield test, it was measured that the ergothioneine yield of the domesticated strain increased from the original 67.46 mg / L to 110.21 mg / L compared with the original strain, an increase of 64.18%. The hyphal growth rate increased by 8.33% compared with the original strain.

[0041] Table 1 Changes in the hyphal growth rate on the plate under domestication with different concentrations of H2O2

[0042]

[0043] Pleurotus citrinopileatus Sing. can grow using a variety of carbon sources, but different carbon sources vary greatly in the accumulation of ergothioneine in Pleurotus citrinopileatus Sing.

[0044] In this example, 30 g / L glucose, glycerol, molasses, sucrose, and corn starch were used as carbon sources respectively, 10 g / L soy peptone was used as the nitrogen source, and 2 g / L potassium dihydrogen phosphate and 2 g / L magnesium sulfate were added at the same time. Fermentation was carried out at 150 rpm and 25°C for 7 days. The samples were centrifuged, the supernatant was discarded, and the cells were repeatedly washed with ultrapure water 2 - 3 times, then ultrapure water was added, and extraction was carried out with hot water at 95°C for 1 h. The supernatant was centrifuged and passed through a 0.22 µm filter membrane, and the ergothioneine content was measured by liquid phase.

[0045] The test results of ergothioneine content are shown in Table 2. The results show that corn flour is most suitable as the carbon source for the production of ergothioneine by Pleurotus citrinopileatus fermentation. Further optimizing the concentration of corn flour, 20, 30, 40, 50, and 60 g / L of corn flour were added as the C source respectively. The test results of ergothioneine content are shown in Table 2. 30 g / L of corn flour was preferably selected as the optimal addition amount, and the yield could reach 220.67 mg / L, and the consumption rate of corn flour was 3.125 g / d.

[0046]

[0047] The accumulation of ergothioneine in Pleurotus citrinopileatus is also very different with different nitrogen sources. Using 30 g / L of corn flour as the carbon source, and using 10 g / L of peptone, soy peptone, tryptone, yeast powder, beef extract, and soybean meal powder as the nitrogen source respectively, while adding 2 g / L of potassium dihydrogen phosphate and 2 g / L of magnesium sulfate. Ferment at 150 rpm and 25 °C for 7 days. Sampling and centrifuging, discarding the supernatant, and repeatedly washing the thalli with ultrapure water 2-3 times, then adding ultrapure water, extracting with hot water at 95 °C for 1 h, centrifuging to take the supernatant and passing it through a 0.22 µm filter membrane, and measuring the ergothioneine content by liquid phase.

[0048] The test results of ergothioneine content are shown in Table 3. The results show that soy peptone is most suitable as the nitrogen source for the production of ergothioneine by Pleurotus citrinopileatus fermentation. Further optimizing the concentration of soy peptone, 5, 10, 15, 20, 25, and 30 g / L of soy peptone were added as the N source respectively. The test results of ergothioneine content are shown in Table 3. 10 g / L of soy peptone was preferably selected as the optimal addition amount, and the yield could reach 236.12 mg / L.

[0049]

[0050] As a sulfur-containing amino acid, ergothioneine requires a large amount of sulfur source supply. In addition, the biosynthesis of ergothioneine also requires histidine to provide the backbone and methionine to provide the methyl group. Therefore, one or more combinations of 2 g / L of histidine, methionine, and cysteine were added respectively. Other nutrients were 30 g / L of corn flour, 10 g / L of soy peptone, 2 g / L of potassium dihydrogen phosphate, and 2 g / L of magnesium sulfate. Ferment at 150 rpm and 25 °C for 7 days. Sampling and centrifuging, discarding the supernatant, and repeatedly washing the thalli with ultrapure water 2-3 times, then adding ultrapure water, extracting with hot water at 95 °C for 1 h, centrifuging to take the supernatant and passing it through a 0.22 µm filter membrane, and measuring the ergothioneine content by liquid phase.

[0051] The test results of ergothioneine content are shown in Table 4. The results show that the synergistic addition of the three amino acids is more conducive to the accumulation of ergothioneine in Pleurotus citrinopileatus, and the yield can reach 318.04 mg / L.

[0052]

[0053] The liquid loading volume can affect oxygen supply and the synthesis of ergothioneine. Therefore, five liquid loading volumes of 50, 75, 100, 125, and 150 mL were set. Fermentation was carried out with 30 g / L corn flour, 10 g / L soy peptone, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and a combination of 2 g / L histidine, methionine, and cysteine. Fermentation was carried out at 150 rpm and 25 °C for 7 days. Samples were taken and centrifuged, the supernatant was discarded, and the cells were washed repeatedly with ultrapure water 2 - 3 times. Then, ultrapure water was added, and the cells were extracted with hot water at 95 °C for 1 h. The supernatant was centrifuged and filtered through a 0.22 µm filter membrane, and the ergothioneine content was measured by liquid chromatography.

[0054] The test results of ergothioneine content are shown in Table 5. The results show that under the condition of a liquid loading volume of 75 mL, the intracellular accumulation amount of ergothioneine can reach 448.87 mg / L.

[0055]

[0056] Fermentation was carried out with 30 g / L corn flour, 10 g / L soy peptone, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and a combination of 2 g / L histidine, methionine, and 1 g / L cysteine. The liquid loading volume was 75 mL, and fermentation was carried out at 150 rpm and 25 °C for 7 days. Samples were taken and centrifuged, the supernatant was discarded, and the cells were washed repeatedly with ultrapure water 2 - 3 times. Then, ultrapure water was added, and the cells were extracted with hot water at 95 °C for 1 h. The supernatant was centrifuged and filtered through a 0.22 µm filter membrane, and the ergothioneine yield was measured by liquid chromatography. The yield could reach 524.27 mg / L.

[0057] Under the same fermentation conditions, the EGT yield of the original strain was 316.10 mg / L.

[0058] Fermentation was carried out with 30 g / L corn flour, 10 g / L soy peptone, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and a combination of 0.5 g / L histidine, 2 g / L methionine, 1 g / L cysteine, 0.2 g / L citric acid, and 0.004 g / L vitamin complex solution (200 mg / L p - aminobenzoic acid, 1 g / L nicotinic acid, 1 g / L calcium pantothenate, 1 g / L thiamine nicotinate, 1 g / L pyridoxine hydrochloride, 2 g / L inositol). The special vitamin complex solution was added on the 4th - 5th day, and the rotation speed was increased from 150 rpm to 170 rpm. The liquid loading volume was 75 mL, and fermentation was carried out at 25 °C for 7 days. Samples were taken and centrifuged, the supernatant was discarded, and the cells were washed repeatedly with ultrapure water 2 - 3 times. Then, ultrapure water was added, and the cells were extracted with hot water at 95 °C for 1 h. The supernatant was centrifuged and filtered through a 0.22 µm filter membrane, and the ergothioneine yield was measured by liquid chromatography. The yield could reach 681.95 mg / L, and the consumption rate of corn flour was 4 g / d.

[0059] Under the same fermentation conditions, the EGT yield of the original strain was 479.33 mg / L.

[0060] The liquid seeds were passaged to the 18th generation, placed in the refrigerator for 25 days, and then taken out. The liquid seeds were inoculated into the seed medium at an inoculation amount of 5% and cultured for 2 - 3 days, repeating Example 6. Samples were taken and centrifuged, the supernatant was discarded, and the cells were washed repeatedly with ultrapure water 2 - 3 times. Then, ultrapure water was added, and hot water extraction was carried out at 95°C for 1 h. After centrifugation, the supernatant was taken and filtered through a 0.22 µm filter membrane, and the ergothioneine content was measured by liquid chromatography. The yield could reach 610.97 mg / L, achieving an effect close to that of Example 6, proving that the seeds had good stability.

Claims

1. A method for increasing the intracellular ergothioneine yield of Pleurotus citrinopileatus, characterized in that, After inoculating the Pleurotus citrinopileatus strain on a plate containing 10 mmol / L hydrogen peroxide and continuously culturing and domesticating it, the growth of the strain was detected, and one domesticated bacterium with good growth was selected to ferment the domesticated bacterium to produce ergothioneine. When the domesticated bacterium was fermentatively cultured, the carbon source of the fermentation medium was selected from 20 - 40 g / L corn flour or 30 g / L glucose; the nitrogen source was selected from 10 - 15 g / L soy peptone or 10 g / L tryptone; 1 - 2 g / L histidine, 1 - 2 g / L methionine and 1 - 2 g / L cysteine were added to the fermentation medium; after 4 - 5 days of fermentative culture of the domesticated bacterium, 0.004 g / L of a vitamin complex solution was added to the fermentation medium, and its components included 200 mg / L p-aminobenzoic acid, 1 g / L nicotinic acid, 1 g / L calcium pantothenate, 1 g / L thiamine nicotinate, 1 g / L pyridoxine hydrochloride and 2 g / L inositol.

2. The method according to claim 1, wherein When the domesticated bacterium was fermentatively cultured, the liquid loading volume of the fermentation medium was 50 - 125 mL.

3. The method according to claim 1, wherein The domesticated bacterium was fermented to produce ergothioneine by a method of forming small particle size bacterial pellets through fragmentation inoculation.

4. The method according to claim 1, wherein When the domesticated bacterium was fermentatively cultured, the rotation speed was 150 rpm - 170 rpm.

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