Pleurotus citrinopileatus strain for producing ergothioneine, and preparation method and application of ergothioneine

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a pleurotus citrinopileatus strain for producing ergothioneine and a preparation method and application of ergothioneine. The pleurotus citrinopileatus strain is preserved in China General Microbiological Culture Collection Center (CGMCC). According to the preparation method of the ergothioneine, lactose and casein peptone are used as a carbon source and a nitrogen source of a liquid fermentation culture medium. The pleurotus citrinopileatus strain is used for high yield of ergothioneine, and the ergothioneine produced by fermentation is used for relieving oxidative stress damage of hepatoma carcinoma cells HepG2.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a Pleurotus citrinopileatus strain for producing ergothioneine, a preparation method of ergothioneine and application thereof. Background Art

[0002] Ergothioneine (EGT) is an amino acid analogue usually synthesized in fungi and a natural antioxidant. When the concentration of ergothioneine in tissues reaches the millimolar level, it can stimulate the natural antioxidant defense system of cells, chelate heavy metal ions, and protect red blood cells in the body from oxidative damage by free radicals. Servillo et al. explored its antioxidant mechanism and found that EGT is more stable than other antioxidants under physiological pH conditions and is not easily spontaneously oxidized. Researchers at University College Cork found that [L-(+)-ergothioneine significantly improved the clinical characteristics of preeclampsia in rats. Salama Samir A et al. found that ergothioneine can effectively reduce cisplatin-induced nephrotoxicity, increase GFR in rats, and reduce serum creatinine and serum urea nitrogen. It was also found that ergothioneine can inhibit γ-glutamyl transpeptidase, which plays a key role in the conversion of cisplatin into toxic metabolites. Qian Xiaochen et al. studied the effects of ergothioneine on the postharvest quality of Agaricus bisporus and the possible browning mechanism. After treatment with ergothioneine, the decrease in the brightness and hardness of Agaricus bisporus was effectively delayed, the total phenols and ascorbic acid were kept at a high level, and the browning degree, malondialdehyde content and electrolyte leakage rate of Agaricus bisporus were reduced. A large number of studies have shown that ergothioneine contains a variety of active ingredients and has great application potential in food additives, health products, cosmetics and other fields.

[0003] Scholars have confirmed that edible fungi contain EGT and can be obtained by different preparation methods. Xue Tiankai et al. used distilled water as solvent and refluxed at 100°C to extract ergothioneine from the waste materials of Pleurotus eryngii; Zhang Cui et al. used deionized water as the extraction solvent and heated to reflux at 100°C to extract ergothioneine from a variety of edible fungi, and the ergothioneine content in Flammulina velutipes was measured to be 0.186%. Mo Yuli et al. used ethanol as solvent and used an ultrasonic-microwave combined method to extract ergothioneine from the fruiting bodies of Pleurotus eryngii. LINSD et al. found that both the mycelium and the fermentation broth contained a certain amount of EGT when studying the quality characteristics of the deep fermentation broth of edible fungi mycelium, and then proposed that the fermentation products should be fully utilized to increase the EGT yield of deep fermentation of edible fungi. STAMPFLI AR et al., Liu Qi et al. and et al. pointed out through comparison of different preparation methods of EGT that bio-fermentation synthesis is the main mode of low-cost, large-scale production of EGT. Preparation of EGT by liquid fermentation of edible fungus mycelium is a better industrial production method. However, it is still necessary to further study how to increase the EGT content in the deep fermentation broth of edible fungus mycelium. Strains with high ergothioneine production also need to be further developed. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a Pleurotus citrinum strain for producing ergothioneine, a preparation method of ergothioneine and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A Pleurotus citrinopileatus strain producing ergothioneine. The Pleurotus citrinopileatus strain was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration CGMCC on June 26, 2024, with a deposit number of CGMCC No.41349 and a classification name of Pleurotus citrinopileatus.

[0007] Another object of the present invention is to provide a method for preparing thioneine, which comprises fermenting and producing thioneine using the aforementioned Pleurotus citrinum strain.

[0008] Preferably, lactose and casein peptone are used as carbon source and nitrogen source of the liquid fermentation medium.

[0009] Furthermore, the precursor substance methionine is fed during the fermentation process.

[0010] Preferably, after the fermentation production of ergothioneine, heat extraction is assisted by power ultrasound or repeated freezing and thawing.

[0011] The precursor substance methionine is added on the 3rd to 5th day (more preferably on the 4th day) of fermentation, and the mass volume ratio of the added methionine to the fermentation liquid is 1 g: 500-1500 mL.

[0012] Further, after adding the precursor substance methionine, the ergothioneine production was 223.638 ug / ml.

[0013] Preferably, the fermentation time is 6-20 days.

[0014] Another object of the present invention is to disclose the application of the aforementioned Pleurotus citrinum strain producing ergothioneine in high-yield ergothioneine.

[0015] The last object of the present invention is to provide the use of the aforementioned Pleurotus citrinum strain producing ergothioneine in the preparation of a drug for alleviating oxidative stress damage of liver cancer cells HepG2.

[0016] Beneficial effects of the present invention: The present invention mainly uses ergothioneine content as an indicator, and comprehensively screens out excellent strains of Pleurotus citrinopileatus with high ergothioneine production and carbon and nitrogen sources of the fermentation medium by comprehensive mycelial biomass. The results show that among the 4 strains tested, the ergothioneine content in Pleurotus citrinopileatus No. 1 and No. 3 is generally higher than that in Pleurotus citrinopileatus No. 2 and No. 83520, and when lactose and casein peptone are used as the carbon source and nitrogen source of the liquid fermentation medium, respectively, Pleurotus citrinopileatus No. 1 has the highest ergothioneine content, reaching 106.81ug / ml, and its mycelial biomass is the highest, so Pleurotus citrinopileatus No. 1 is selected as the dominant strain with high ergothioneine production, and lactose and casein peptone are the most suitable carbon and nitrogen sources in its fermentation medium.

[0017] After the precursor substance methionine was added, the ergothioneine production during the fermentation production of Pleurotus citrinum No. 1 was greatly increased to 223.638ug / ml.

[0018] Ergothioneine obtained by fermentation of Pleurotus citrinopileatus No. 1 is used to alleviate oxidative stress damage in liver cancer cells HepG2. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of different carbon and nitrogen source culture media on the mycelial biomass of Pleurotus citrinopileatus;

[0020] Figure 2 Effects of different types of carbon and nitrogen sources on the ergothioneine content of Pleurotus citrinopileatus;

[0021] Figure 3 Optimization of culture medium for high-yield ergothioneine of Pleurotus citrinopileatus

[0022] Figure 4 Schematic diagram of the precursor substance methionine increasing the production of ergothioneine;

[0023] Figure 5 Ergothioneine toxicity test, * indicates significant differences among different sample groups (P<0.05);

[0024] Figure 6 Effects of different concentrations of H2O2 on cell viability. Letters (ae) indicate significant differences among different sample groups (P<0.05);

[0025] Figure 7 The protective effect of ergothioneine on HepG2 cells damaged by H2O2. (ac) indicates significant differences among different sample groups (P<0.05);

[0026] Figure 8 Effects of ergothioneine on ROS content in HepG2 cells damaged by H2O2. Letters (ad) indicate significant differences among different sample groups (P<0.05). A) is the blank group; B) is the model group; C-E) are low, medium and high concentration test groups, respectively.

[0027] Fig. 9 Effects of ergothioneine on the MDA content of HepG2 cells damaged by H2O2. Letters (ae) indicate significant differences among different sample groups (P<0.05);

[0028] Fig.10 Effects of ergothioneine on the SOD content of HepG2 cells damaged by H2O2. Letters (ae) indicate significant differences among different sample groups (P<0.05);

[0029] Fig.11 Effects of ergothioneine on CAT content in HepG2 cells damaged by H2O2. Letters (ae) indicate significant differences among different sample groups (P<0.05);

[0030] Fig.12 Effects of ergothioneine on GSH content in HepG2 cells damaged by H2O2. Letters (ae) indicate significant differences among different sample groups (P<0.05);

[0031] Fig.13 Effects of different concentrations of EGT on the total protein level of Nrf2 in whole cells, with β-actin as the internal control;

[0032] Fig.14 Effects of different concentrations of EGT on the transcription levels of cellular Nrf2 and target genes. Blank is the blank group, Con is the model group, Low, Medium, and High are the low, medium, and high concentrations of EGT treatment groups, respectively; ** indicates that the difference between the groups is extremely significant (P<0.01), *** indicates that the difference between the groups is extremely significant (P<0.001), and **** indicates that the difference between the groups is extremely significant (P<0.0001);

[0033] Fig.15 Schematic diagram of mycelium produced by fermentation of four bacteria;

[0034] Yuhuang No. 1 (abbreviated as Yuhuang No. 1) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration CGMCC on June 26, 2024, with the deposit number CGMCC No.41349 and the classification name Pleurotus citrinopileatus. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention.

[0036] Unless otherwise specified, the test materials or reagents used in the following examples are all existing and can be obtained from commercial channels. If no specific techniques or conditions are specified in the examples, they can be carried out according to conventional techniques or conditions disclosed in the art.

[0037] Example 1

[0038] The fungus species of the present invention: Pleurotus citrinum No. 1 was collected from Yabuli Hufeng Forest Farm, Shangzhi City, Harbin City, Heilongjiang Province in 2022. The Pleurotus citrinum fruiting bodies were collected and separated to obtain Pleurotus citrinum No. 1.

[0039] The specific process is: Use a shovel to carefully dig the surrounding soil and take out the citrus mushrooms and their connected mycelium. Immediately label the collected citrus mushrooms with the collection location, collection time, general morphological characteristics of the citrus mushrooms, soil moisture and other information. Transport the container containing the citrus mushrooms and their connected mycelium in a cool, ventilated insulated box to avoid direct sunlight and severe vibration to prevent the citrus mushrooms from being damaged or deteriorating. After arriving at the laboratory, place the citrus mushrooms in the refrigerator (temperature is about 2-4°C) and maintain appropriate humidity. Before the strain separation experiment, gently rinse the surface of the citrus mushrooms with sterile saline to remove dirt and impurities. Under sterile conditions, use a scalpel or tweezers to cut the fruiting body open, and select the junction of the cap and the stipe or the internal tissues such as the gills as the separation material. The selected tissue block (about 3-5mm in size) 3 ) Use an inoculation needle or tweezers to quickly transfer to a PDA medium plate or a test tube slant medium, and gently press to make the tissue block fully contact with the medium. Inoculate one plate or one test tube for each tissue. After inoculation, place the plate or test tube in a constant temperature incubator and culture at a suitable temperature. During the culture process, observe the growth of mycelium, such as growth rate, color, texture, etc., and judge it to be normal mycelium of Pleurotus citrinopileatus. It was identified as Pleurotus citrinopileatus.

[0040] Comparative strains: Pleurotus citrinopileatus No. 2, No. 3, and No. 83520. All comparative strains were provided by Heilongjiang Institute of Microbiology.

[0041] Culture medium: The carbon source, nitrogen source, amino acid and other reagents required for the experiment were purchased from a reagent company, and all chemical reagents were of analytical grade.

[0042] The instruments and equipment used include: electronic analytical balance (ALC-310.3): Beijing Liduo Instrument Co., Ltd.; high shear mixing emulsifier (IKA T25): IKA (Guangzhou) Instrument Equipment Co., Ltd.; ultrasonic equipment (YPS11B-HB): Hangzhou Chenggong Ultrasonic Equipment Co., Ltd.; refrigerated centrifuge (H2100R): Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; full temperature shaking incubator (HZQ-Q): Harbin Donglian Electronic Technology Development Co., Ltd.; high performance liquid chromatograph (Waters e2695): Beijing Keyi Hengda Technology Co., Ltd.

[0043] 1) Activation and fermentation of Pleurotus citrinopileatus strains

[0044] The basic culture medium for solid slant culture is potato complex culture medium (CPDA medium): 200g peeled potatoes, 20g glucose, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 0.05mg vitamin B1, 20g agar, 1000mL water, natural pH. After the culture medium is prepared, it is divided into test tubes and sterilized under high pressure at 121℃ for 20min.

[0045] Liquid seed medium: 25g / l glucose, 2g / l yeast extract, 1g / l glutamic acid, 0.5mg / l biotin, 0.1g / l thiamine, 2g / l potassium dihydrogen phosphate, 0.5g / l magnesium sulfate, 5ml 0.1M FeCl3, and 5ml 0.1M manganese sulfate, the rest is water, the liquid volume in a 250ml Erlenmeyer flask is 100ml. Autoclave at 115℃ for 20min.

[0046] Fermentation medium: a mixture of 45g / L carbon source, 20g / L nitrogen source, 4g / L potassium dihydrogen phosphate, 2g / L magnesium sulfate heptahydrate, 1g / L ammonium dihydrogen phosphate, 1g / L cysteine, 1g / L methionine and 1mg / L vitamin B1. 45g / L different carbon sources: glycerol, glucose, lactose, maltose, dextrin, starch. 20g / L different nitrogen sources: casein peptone, yeast tryptone, beef extract, soybean cake powder, skimmed milk powder. Autoclave at 121℃ for 20min.

[0047] Activation of strains: Place a wooden stick with a thickness of about 1 cm on the laboratory table. After the culture medium is sterilized, place the head of the test tube on the wooden stick to make the culture medium in the tube tilt naturally. After cooling and solidifying, inoculate the citrus agaricus blocks (citrus agaricus No. 1, No. 2, No. 3, and No. 83520 respectively), place in a 25℃ incubator with shaded culture for 9 days, and activate when the mycelium fills the test tube. Each treatment design is repeated 3 times.

[0048] Seed liquid culture: inoculate solid bacterial strains (CPDA medium slant bacterial strains) into liquid seed culture medium, culture at 26.5°C and 110 rpm for 4 days to prepare seed liquid.

[0049] Fermentation culture: 5 ml of seed liquid was inoculated into the fermentation medium. The volume of the fermentation medium in a 500 ml Erlenmeyer flask was 150 ml, and the culture was carried out at 25-28°C and 100-200 rpm for 10 days to obtain the fermentation liquid.

[0050] 2) Determination of mycelium biomass

[0051] The vacuum freeze drying method is used. The specific operation method is: collect the mycelium from the culture solution after the fermentation is completed, pre-cool the collected mycelium in a glass container at -40℃ for 5-10 hours, freeze-dry it in a vacuum freeze dryer for 24 hours, and weigh the freeze-dried mycelium using an electronic balance and record the result. Figure 1 shown.

[0052] High biomass means more mycelium produced by fermentation. Ergothioneine exists in mycelium, and high biomass means high ergothioneine content. Fig.15 It can be seen that after 10 days of fermentation, the biomass of Pleurotus citrinopileatus No. 1 was higher than that of the control strain.

[0053] 3) Extraction of ergothioneine from Pleurotus citrinopileatus by different methods

[0054] (1) Power ultrasound-assisted thermal extraction: Take the freeze-dried fermented product of Pleurotus citrinopileatus (i.e., the mycelium obtained in step 2), dilute with water to the same concentration (the mass ratio of mycelium to water is 1:100). Extract at 90°C for 30 minutes, stir evenly, and then treat with 300W power ultrasound for 10 minutes, twice, with a 1-minute interval in between. After ultrasound treatment, centrifuge at 10,000 rpm for 10 minutes.

[0055] (2) Repeated freeze-thaw assisted heat extraction: Take the freeze-dried fermentation product of Pleurotus citrinopileatus (i.e., the mycelium obtained in step 2), dilute it with water to the same concentration (the mass ratio of mycelium to water is 1:100), extract it at 90°C for 30 minutes, stir it evenly, freeze-thaw it three times, and centrifuge it at 10,000 rpm for 10 minutes.

[0056] Among them, the power ultrasound assisted heat extraction method has a better extraction effect. The ergothioneine content in this specification is determined by (1) the power ultrasound assisted heat extraction method.

[0057] The content (yield) of ergothioneine in this specification sheet in units of ug / ml refers to how many ug ergothioneine is contained in 1 ml of the extract obtained by the power ultrasound-assisted heat extraction method.

[0058] 4) Determination of ergothioneine content

[0059] Chromatographic analysis conditions: 4.6 mm × 250 mm, 10 um inner diameter liquid chromatography column, column temperature 35 ° C. Detection wavelength 257 nm, flow rate 1 ml / min, mobile phase (acetonitrile: 5 mmol ammonium acetate solution = 80:20, v / v), single sample analysis collection time 30 min, injection volume 10 ul.

[0060] 5) Data processing and analysis

[0061] The experiment was repeated three times, and SPSS software was used for analysis of variance and LSD multiple comparisons. The significance level was set at 0.05, and Origin software was used to draw graphs.

[0062] 6) Effects of different carbon and nitrogen source culture media on the mycelial biomass of Pleurotus citrinopileatus:

[0063] When screening the nitrogen source in the fermentation medium, glucose was fixed as the carbon source, and when screening the carbon source in the fermentation medium, yeast extract powder was fixed as the nitrogen source. This study was conducted to study the effects of different carbon and nitrogen source culture media on the mycelial biomass of Pleurotus citrinopileatus. The results are as follows Figure 1 .

[0064] Carbon and nitrogen sources are essential nutrients for mycelial cells and the main energy source for the growth and development of microbial mycelium. During liquid fermentation, the choice of the initial carbon source type of the culture medium will have a great impact on mycelial growth and the yield of microbial secondary metabolites. Comprehensive comparison of different carbon and nitrogen sources showed that the mycelial biomass of Pleurotus citrinopileatus strain No. 1 was higher.

[0065] Depend on Figure 1 It can be seen that when yeast extract powder is used as nitrogen source, the mycelial biomass of strains 1, 2, 3, and 83520 all change with the type of tested carbon source, but when soluble starch is used as carbon source, the mycelial biomass is the highest, which is 2.24g, 1.88g, 1.51g, and 1.81g respectively. Then, when lactose is used as carbon source, the mycelial biomass of Pleurotus citrinum No. 1 is 0.88g, Pleurotus citrinum No. 2 is 0.77g, Pleurotus citrinum No. 83520 is 0.46g, and Pleurotus citrinum No. 3 uses dextrin as carbon source, and the mycelial biomass is 0.69g. It can be seen that although Pleurotus citrinum No. 1, 2, 3, and 83520 strains can utilize all tested carbon sources, their preferences and utilization abilities for carbon sources are different. When the mycelial biomass is used as reference standard, the best carbon sources for the four strains are soluble starch, lactose, and dextrin, respectively.

[0066] In the experiment of optimizing nitrogen source culture medium, when the strains of Pleurotus citrinum No. 1, 3, and 83520 all used soybean powder as nitrogen source, the mycelial biomass was the highest, which was 1.45g, 1.21g, and 1g respectively. Then, Pleurotus citrinum No. 1 and 3 used casein peptone as nitrogen source, the mycelial biomass was 1.33g and 0.98g respectively, and Pleurotus citrinum No. 83520 used yeast extract powder as nitrogen source, the mycelial biomass was 0.96g. Pleurotus citrinum No. 2 used yeast extract powder as nitrogen source, the mycelial biomass was the highest, which was 1.33g. It can be seen that Pleurotus citrinum No. 1, 2, 3, and 83520 strains can utilize all the tested nitrogen sources, and the best nitrogen sources for the four strains are soybean powder, casein peptone, and yeast extract powder respectively. However, during the experiment, it was found that when soybean powder was used as the nitrogen source and soluble starch was used as the carbon source, there was a lot of sediment in the fermentation broth. This was because the protein and starch in the culture medium were denatured and gelatinized during the fermentation process to prepare mycelium containing ergothioneine, resulting in great uncertainty in the mycelium content in the extracted solids. Therefore, lactose and casein peptone were selected as the best carbon and nitrogen sources.

[0067] 7) Effects of different carbon and nitrogen sources on ergothioneine production in Pleurotus citrinopileatus

[0068] When screening the nitrogen source in the fermentation medium, glucose was fixed as the carbon source, and when screening the carbon source in the fermentation medium, yeast extract powder was fixed as the nitrogen source, so as to study the effects of different carbon and nitrogen sources on the ergothioneine production of Pleurotus citrinopileatus. The results are as follows Figure 2 .

[0069] Depend on Figure 2 It can be seen that different carbon and nitrogen source culture media have a certain effect on the production of ergothioneine by Pleurotus citrinum 1, 2, 3, and 83520 strains. Comprehensive comparison of different carbon and nitrogen sources shows that the ergothioneine content of Pleurotus citrinum 1 and 3 strains is generally higher than that of Pleurotus citrinum 2 and 83520 strains.

[0070] The results of screening the nitrogen source for the production of ergothioneine by Pleurotus citrinopileatus showed that the culture medium using casein peptone as the nitrogen source produced the highest ergothioneine content, Pleurotus citrinopileatus No. 1 could reach 90.56ug / ml, Pleurotus citrinopileatus No. 3 could reach 82.52ug / ml, and yeast extract powder was second. Pleurotus citrinopileatus No. 2 used beef extract as the nitrogen source, and produced the highest ergothioneine content of 85.36ug / ml, followed by yeast extract powder, which was 79.86ug / ml. When Pleurotus citrinopileatus No. 83520 used yeast extract powder as the nitrogen source, the highest ergothioneine production was 55.16ug / ml, followed by casein peptone, which was 36.86ug / ml. Considering the impact on the production of ergothioneine by Pleurotus citrinopileatus, casein peptone was selected as the nitrogen source for subsequent optimization experiments.

[0071] The results of screening the carbon sources for the production of ergothioneine by Pleurotus citrinopileatus showed that the production of ergothioneine showed a relatively obvious change with different carbon sources. When lactose was used as the carbon source in the fermentation medium of the four strains, the ergothioneine content in their mycelium was the highest, with the contents of Pleurotus citrinopileatus No. 1 being 101.18ug / ml, Pleurotus citrinopileatus No. 2 being 90.65ug / ml, Pleurotus citrinopileatus No. 3 being 95.5ug / ml, and Pleurotus citrinopileatus No. 83520 being 68.4ug / ml.

[0072] Different carbon and nitrogen source culture media have a certain influence on the content of ergothioneine synthesized by Pleurotus citrinopileatus. This may be because specific Pleurotus citrinopileatus strains have different degrees of absorption and utilization of different carbon and nitrogen sources, resulting in different abilities to synthesize ergothioneine. Taking all factors into consideration, lactose and casein peptone are the best carbon and nitrogen sources for the fermentation culture medium formula.

[0073] Usually the best culture medium for bacterial strains is glucose, but the data obtained through experiments in the present invention show that lactose is the best carbon source, which is related to the characteristics of the bacteria of the present invention.

[0074] 8) Optimization of culture medium for high-yield ergothioneine of Pleurotus citrinopileatus

[0075] By the above screening of carbon and nitrogen source in the bacterial fermentation medium formula, 10d fermentation culture (same as 1) and 2) in Example 1) was carried out to 4 strains of bacteria using lactose and casein peptone as the carbon and nitrogen source of the fermentation medium, 3) power ultrasound-assisted thermal extraction of Example 1 was used to extract, and its thioneine content was measured. The result was as shown in Table 1. Figure 3 .

[0076] Depend on Figure 3 It can be seen that after optimizing the culture medium, the ergothioneine production of Pleurotus citrinopileatus No. 1 strain was the highest, reaching 106.81ug / ml, followed by Pleurotus citrinopileatus No. 3, 104.93ug / ml, Pleurotus citrinopileatus No. 2, 97.17ug / ml, and Pleurotus citrinopileatus No. 83520, 72.21ug / ml, all of which were higher than the ergothioneine production before optimization. It can be seen that the optimized fermentation medium is more suitable for Pleurotus citrinopileatus to produce ergothioneine. This experimental result is also higher than the content of L-ergothioneine in 23 edible fungi such as Pleurotus ostreatus, Pleurotus eryngii, Pleurotus eryngii, and Boletus, which was determined by ultra-high performance liquid chromatography by E Hengchao et al. Among them, Boletus edulis had the highest content of L-ergothioneine among the tested edible fungi, which was 10.2 mg / g (E Hengchao, Feng Lulu, Zhao Xiaoyan, et al., Determination of L-ergothioneine in Edible Fungi by Ultra-High Performance Liquid Chromatography and Mass Spectrometry Confirmation [J]. Analytical Laboratory, 2021. DOI: 10.13595 / j.cnki.issn1000-0720.2021.061901).

[0077] The present invention takes thioneine content as the final detection index, and the optimal carbon and nitrogen sources of the four strains of Pleurotus citrinopileatus are inconsistent, which is due to the differences in genetic characteristics and gene expression caused by the different test strains. In the final optimized fermentation medium, with casein peptone and lactose as carbon and nitrogen sources, the thioneine output of Pleurotus citrinopileatus No. 1 is higher than that of Pleurotus citrinopileatus No. 2, 3, and 83520 strains, reaching 106.81ug / ml.

[0078] In the above 1)-8), no precursor substances were added during the experiments.

[0079] 9) Effects of precursor substances on ergothioneine production in Pleurotus citrinopileatus

[0080] Ergothioneine is a secondary metabolite. The precursor amino acids are often important nutrients and regulatory factors for the biosynthesis of secondary metabolites. They can enhance the fermentation and accumulation process of ergothioneine and promote the production of ergothioneine.

[0081] Preparation process is basically the same as 1) activation and fermentation of Pleurotus citrinopileatus strains; the difference is only that on the 4th day of fermentation culture, the precursor methionine is added to the fermentation medium (using the aforementioned preferred medium, with lactose and casein peptone as the strain fermentation medium formula); the liquid volume of the fermentation medium in a 250ml triangular flask is 100ml; the seed liquid added to the fermentation medium is 3.4ml, the precursor addition amount is 1g / L, and each bottle of fermentation medium is 100mL, that is, the conversion addition amount is 0.1g / bottle. Weigh 3g of methionine, add 60mL of water, sterilize at 115°C for 20min after fully dissolving, add 2mL to each bottle, continue to culture to the 8th day, and measure the ergothioneine content. Comparing Table 1 and Table 2, it can be seen that the addition of precursors not only increases mycelium yield, but also increases the ergothioneine content in mycelium.

[0082] The ergothioneine production of the No. 1 strain of Pleurotus citrinum without the addition of the precursor substance methionine was 106.81ug / ml, and the ergothioneine production of the No. 1 strain of Pleurotus citrinum with the addition of the precursor substance methionine was 223.638ug / ml, which was more than doubled. Figure 4 As shown, this indicates that methionine can greatly promote the synthesis and accumulation of ergothioneine by Pleurotus citrinum No. 1, achieving unexpected technical effects.

[0083] In this experiment, the effects of different precursors on the ergothioneine content of Elm Yellow No. 1 were also verified. The amount of precursor added in different treatments was 1g / L. When the precursor was a mixture of two substances, the mass ratio of the two substances was 1:1. The results are shown in Table 1, where the mass in Table 1 is the mass of mycelium. It can be seen that the treatment of methionine addition was significantly better than other treatments, and betaine and cysteine ​​would reduce the ergothioneine content of Elm Yellow No. 1.

[0084] Table 1 Effects of different precursors on the ergothioneine content in Yuhuang No. 1

[0085]

[0086] 10) four strains of bacteria are carried out growth cycle mycelium biomass and thioneine content correlation analysis, and the results are shown in Table 2. 1) step in Example 1 is basically adopted to carry out thioneine production, without adding precursor substances, and the liquid amount of fermentation medium in 250ml triangular flask is 100ml; the seed liquor added to the fermentation medium is 3.4ml, and the fermentation culture time is extended to 16d (the process of fermentation culture time 8d, 10d, 12d, 14d, 16d is respectively set), and the carbon source and nitrogen source adopt lactose and casein peptone. In Table 2 and Table 3, thioneine content refers to: after lyophilization, the mycelium obtained by fermentation is diluted with water (the mass ratio of mycelium to water is 1:100), and the extraction is shaken at 90 DEG C for 30min, and 300W power is ultrasonically treated for 10min, 2 times, and 1min is separated in the middle after stirring. After ultrasonic treatment, 10000rpm is centrifuged for 10min, and thioneine content in mycelium aqueous solution is finally obtained. As can be seen from Table 2 and Table 3, the mycelial biomass and thioneine content of Yuhuang No. 1 under the same fermentation time are higher than other comparison strains. And the thioneine production of the comparison strain no longer increases or even decreases after fermentation for 10d, while the thioneine production of Yuhuang No. 1 (i.e. Yuhuang Mushroom No. 1) of the present invention still increases rapidly between 10d-16d of fermentation.

[0087] Table 2 Correlation analysis between mycelial biomass and ergothioneine content during the growth period of Yuhuang No. 1

[0088]

[0089] Table 3 Correlation analysis between mycelial biomass and ergothioneine content during the growth period of Yuhuang No. 2, No. 3 and No. 83520

[0090]

[0091] The present invention mainly carries out ergothioneine yield as the basis for screening of ergothioneine bacterial strains and optimization of carbon and nitrogen sources of culture medium, provides new strain selection for production to improve ergothioneine content, and also provides excellent microbial source for the development of next step ergothioneine, and the multiple active ingredients in ergothioneine bacterial strains are the basis of its clinical efficacy and pharmacological action. The method is simple to operate, has low production cost, is suitable for large-scale production, provides safe raw materials for the development of functional foods rich in ergothioneine, and has a large application prospect.

[0092] Example 2: Effect of Yuhuang No. 1 high-yield ergothioneine on the alleviation of oxidative stress damage in liver cancer cells HepG2

[0093] The ergothioneine obtained by the biological fermentation method is highly safe, and the ergothioneine obtained by the biological fermentation method has excellent function. The ergothioneine of the present embodiment is obtained by fermentation, extraction and purification of edible fungus-Yuhuang No. 1, and the purity reaches 90%.

[0094] According to the activation and fermentation steps of 1) of Example 1, the mycelium was fermented for 10 days. Then, according to 3) of Example 1, the ergothioneine extract was obtained by the power ultrasound-assisted heat extraction method. The purification process is as follows: resin pretreatment: the dry resin is placed in a saturated NaCl solution to swell for 24 hours and washed with distilled water, then treated with anion and cation exchange resins with acid and alkali twice the volume of the resin, and then washed with distilled water until the resin effluent is neutral for standby use; loading, eluting and drying: the HCN-5 resin and TSF-1 resin are wet-packed and used in series, and the total resin dosage is controlled to be 25 mL, the temperature is 40°C, and the volume of the cation exchange resin and the anion exchange resin is 1:2; the extract is loaded at 2BV / h, the loading concentration is 20 mg / mL, and the loading volume is 50 mL. After loading, the resin is washed with pure water twice the volume of the filler, and then 20 times the pure water is used to elute the ergothioneine adsorbed on the resin, and the ergothioneine eluate is collected; the collected eluate is vacuum freeze-dried, and the freeze-dried powder is collected for quantitative and qualitative analysis by UPLC-Q-TOF-MSE.

[0095] HepG2 cell culture: HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum and the density was adjusted to 5×10 4 The cells were cultured in a 37°C, 5% CO2 incubator for 48 h, with the medium replaced every 24 h.

[0096] Ergothioneine toxicity test on HepG2 cells: HepG2 cells were subcultured according to the culture conditions until the density reached 80%-90%, and the density was adjusted to 5×10 4 Each well was inoculated in a 96-well plate. After 24 h of culture, 100 μL of 0.1, 0.5, 1, 2, and 10 mmol / L of EGT was added to the model group, and the blank group was added with an equal amount of basal medium. After 24 h of culture, the cell viability was determined using a CCK-8 kit. The concentration of ergothioneine on HepG2 cells was determined by comparing cell viability.

[0097] Establishment of oxidative stress model: H2O2 was used to induce human liver cancer HepG2 cells to construct an oxidative stress cell model. HepG2 cells were passaged according to the culture conditions until the density reached 80%-90%, and the density was adjusted to 5×10 4Each well was inoculated in a 96-well plate. After 24 hours of culture, 100 μL of different concentrations of H2O2 (0 μmol / L, 200 μmol / L, 400 μmol / L, 600 μmol / L, 800 μmol / L, 1000 μmol / L, 1200 μmol / L) was added to the model group, and the same amount of basal medium was added to the blank group. After 2 hours of culture, the cell viability was determined using the CCK-8 kit. The optimal induction concentration of H2O2 was determined by comparing the cell viability.

[0098] Determination of cell activity: Cell viability was determined using the CCK-8 method. 4 The density of each well was inoculated into a 96-well plate, and 100 μL of cell suspension was inoculated into each well and placed in an incubator for 24 h. Then each well was added with different concentrations of H2O2 or thioneine to treat HepG2 cells for incubation. After the culture was completed, 10 μL of CCK-8 solution was added to each well. After 2 h of cultivation in an incubator, the absorbance at 450 nm was determined by an ELISA instrument. Cell viability was calculated according to the following formula (5):

[0099]

[0100] Wherein: OD(drug added) is the absorbance value of the wells with cells, culture medium, CCK-8 solution and drug solution, OD(blank) is the absorbance value of the wells with culture medium, CCK-8 solution and no cells, OD(0 drug added) is the absorbance value of the wells with cells, culture medium, CCK-8 solution and no drug solution.

[0101] The protective effect of ergothioneine on H2O2-induced HepG2 cell damage: HepG2 cells were passaged according to the culture conditions until the density reached 80%-90%, and the density was adjusted to 5×10 4 The cells were inoculated in 96-well plates and cultured for 24 hours. Experimental grouping: The cells were divided into 5 groups, blank group (normal control, NC, cultured in DEME medium without H2O2), model group (H2O2, 600μmol / L), high-dose group (600μmol / L H2O2+10mmol / L EGT), medium-dose group (600μmol / L H2O2+1mmol / LEGT), and low-dose group (600μmol / L H2O2+0.1mmol / L EGT). After incubation for 2 hours, the cell viability was determined using the CCK-8 kit. The protective effect of ergothioneine on Hep G2 cells induced by H2O2 was compared by comparing cell viability.

[0102] Effect of ergothioneine on ROS content in HepG2 cells induced by H2O2: The cells were treated according to the above grouping, incubated for 2 hours, and the supernatant was discarded. The cells were washed twice with PBS. DCFH-DA was added according to the detection kit method, and the cells were treated at 37°C without light for 30 minutes and washed twice with PBS. The ROS fluorescence intensity of the cells was detected by a fluorescence microplate reader, with an excitation wavelength of 485nm and an emission wavelength of 530nm. At the same time, cell fluorescence images were taken using an inverted fluorescence microscope.

[0103] Effect of ergothioneine on the MDA content in HepG2 cells damaged by H2O2: The cells were treated according to the above grouping and incubated for 2 hours. The MDA content of each group of cells was detected using the MDA kit.

[0104] Effect of ergothioneine on the activity of antioxidant enzymes in HepG2 cells induced by H2O2: The cells were treated according to the above grouping, incubated for 2 hours, and the supernatant was discarded. The cells were washed twice with PBS. The cells were lysed with cell lysis buffer. The BCA kit was used to detect the cell protein concentration, and the CAT, SOD, and GSH-Px kit detection methods were used to detect the CAT, SOD, and GSH-Px contents of each group of cells.

[0105] Figure 5 The effect of ergothioneine on the viability of HepG2 liver cancer cells was shown. Figure 5 As shown, within the concentration range of 0.1-10mM, the effect of ergothioneine on cell viability showed a trend of first increasing and then decreasing with the increase of concentration. As the concentration of EGT increased from 0.1mM to 1mM, the cell viability increased from 112.72% to 122.69%; when the concentration of EGT was 10mM, the viability of HepG2 cells began to decline, which was 117.81%. Compared with the control group, the cell viability of the group with ergothioneine was significantly increased (P<0.05), but the results of the groups with different concentrations of ergothioneine showed that the concentration of EGT had no significant effect on the viability of HepG2 cells (P>0.05). The results showed that the addition of an appropriate amount of ergothioneine had a certain proliferation effect on the growth of HepG2 cells and improved cell viability. Overall, when the concentration of ergothioneine was less than 10mM, the cell viability was more than 90% and there was no cytotoxicity. In summary, 0.1mM, 1mM, and 10mM ergothioneine were selected for subsequent experiments.

[0106] The effects of different concentrations of H2O2 on cell viability are shown in the following table. Figure 6As shown in the figure, the results showed that the oxidative toxicity of H2O2 on Hep G2 cells depended on its concentration. With the increase of H2O2 concentration, the viability of HepG2 cells decreased significantly (P<0.05). Compared with the control group, the cell viability of HepG2 cells treated with 200 μM H2O2 was 85.68±0.54%, a loss of about 14.32%; the cell viability of HepG2 cells treated with 400 μM H2O2 was 59.10±3.15%, a loss of about 40.90%; the cell viability of HepG2 cells treated with 600 μM H2O2 was 53.08±1.51%, a loss of about 46.92%; the cell viability of HepG2 cells treated with 800 μM H2O2 was 41.72±3.55%, a loss of about 58.28%; the cell viability of HepG2 cells treated with 1000 μM H2O2 was 39.40±3.50%, a loss of about 60.60%; the cell viability of HepG2 cells treated with 1200 μM H2O2 was 37.43±2.75%, a loss of about 62.57%. It shows that within the concentration range of 200-800μM, the effect of H2O2 concentration on cell viability is significantly different (P<0.05), and it is dose-dependent. When the concentration exceeds 800μM, the effect on cell viability is no longer significant. Too high cell survival rate will not cause obvious oxidative damage to HepG2 cells, and too low cell survival rate will cause irreversible damage to cells, both of which are not conducive to the construction of oxidative stress model. When HepG2 cells were treated with 600μM H2O2, the cell viability was 53.08±1.51%. The cells were in a damaged state, but not completely dead, and it was possible to repair the damage, which was an ideal modeling concentration. Therefore, 600μM was selected as the induction concentration of the oxidative stress model in subsequent experiments.

[0107] The present invention uses single HepG2 cells as a blank group, uses only 600 μM H2O2 to induce damage as a model group, and uses 600 μM H2O2 and different concentrations of EGT (0.1, 1 and 10 mM) together as an experimental group to explore the protective effect of ergothioneine on HepG2 cells induced by H2O2. The results are as follows Figure 7 As shown, EGT and H2O2 acted together on HepG2 cells, and the viability of H2O2-treated HepG2 cells was enhanced in an EGT dose-dependent manner.

[0108] The cell viability of HepG2 cells under the action of H2O2 was 64.78±2.03%, which was 35.22% lower than that of the blank group. After the action of EGT at different concentrations (0.1mM, 1mM and 10mM), compared with the group treated with the same dose of H2O2, the cell viability increased and gradually returned to normal, and the inhibitory effect of H2O2 on the viability of HepG2 cells was effectively improved. Among them, the effect of 0.1mM EGT treatment on the cells was not significant (P>0.05), and its cell viability was 72.72±9.05%. However, when the concentration of EGT increased to 1mM, the cell viability was significantly increased (P<0.05), reaching 85.23±3.29%. When the concentration of EGT increased to 10mM, its cell viability returned to normal levels close to the blank group, which was 107.77±8.07%. Therefore, our results show that EGT has a significant protective effect on H2O2-induced HepG2 cell toxicity and has significant antioxidant capacity. On the one hand, it protects cells from oxidative stress and inhibits apoptosis of HepG2 cells by destroying the H2O2-mediated cellular oxidation system. On the other hand, EGT has a small molecular weight and is more likely to pass through the cell membrane and enter the cell to directly and effectively remove intracellular free radicals. At the same time, this is consistent with the results of the in vitro antioxidant activity test. EGT scavenges free radicals to play an antioxidant role, and it depends on its dose.

[0109] The present invention uses a single HepG2 cell as a blank group, uses only 600 μM H2O2 to induce damage as a model group, and uses 600 μM H2O2 and different concentrations of EGT (0.1 mM, 1 mM and 10 mM) as a test group to explore the effect of ergothioneine on the ROS content of HepG2 cells induced by H2O2. The results are as follows Figure 8 As shown, EGT acted together with H2O2 on HepG2 cells and reduced the ROS content of H2O2-treated HepG2 cells in an EGT dose-dependent manner.

[0110] Under the action of H2O2, the cellular ROS content of HepG2 cells was 80.57±2.10%, which was about 4.65 times that of the blank group. After the action of EGT at different concentrations (0.1mM, 1mM and 10mM), the ROS content of these cells decreased significantly compared with the group treated with the same dose of H2O2 (P<0.05), and the oxidative effect of H2O2 on HepG2 cells was effectively improved. Among them, the cellular ROS content was 64.20±2.55% after 0.1mM EGT treatment, which was 20.31% lower than that of the model group; when the EGT concentration was increased to 1mM, the cellular ROS content was 43.23±1.56%, which was 46.34% lower than that of the model group; when the EGT concentration was increased to 10mM, the cellular ROS content was 31.41±1.84%, which was 61.02% lower than that of the model group. Therefore, our results show that EGT has a significant inhibitory effect on the formation of ROS in HepG2 cells, thereby reducing oxidative damage, lowering the level of cellular oxidative stress, and protecting cells. This is consistent with the results of the protective effect of ergothioneine on HepG2 cells induced by H2O2.

[0111] The present invention uses single HepG2 cells as a blank group, uses only 600 μM H2O2 to induce damage as a model group, and uses 600 μM H2O2 and different concentrations of EGT (0.1 mM, 1 mM and 10 mM) as a test group to explore the effect of ergothioneine on the MDA content of HepG2 cells induced by H2O2. The results are as follows Fig. 9 As shown, EGT and H2O2 acted together on HepG2 cells, and the MDA content of HepG2 cells treated with H2O2 decreased depending on the EGT dose.

[0112] Under the action of H2O2, the cell MDA content of HepG2 cells was 25.74±0.79U / mg Prot, which was about 2.41 times that of the blank group. After the action of different concentrations of EGT (0.1mM, 1mM and 10mM), the cell MDA content was significantly decreased compared with the group treated with the same dose of H2O2 (P<0.05), and the effect of H2O2 on cell membrane lipid peroxidation was effectively improved. Among them, the cell MDA content was 19.60±1.27% after 0.1mM EGT treatment, which was 23.85% lower than that of the model group; when the EGT concentration was increased to 1mM, the cell MDA content was 12.51±1.08%, which was 51.39% lower than that of the model group; when the EGT concentration was increased to 10mM, the cell MDA content was 10.54±0.57%, which was 59.05% lower than that of the model group. HepG2 cells produced a large amount of MDA due to the action of H2O2, but the action of EGT could inhibit the production of MDA to varying degrees, indicating that EGT alleviated free radical attacks and reduced the degree of cell damage.

[0113] The present invention uses single HepG2 cells as a blank group, uses only 600 μM H2O2 to induce damage as a model group, and uses 600 μM H2O2 and different concentrations of EGT (0.1 mM, 1 mM and 10 mM) together as an experimental group to explore the effect of ergothioneine on the antioxidant enzyme content of HepG2 cells damaged by H2O2.

[0114] The results of the effect of ergothioneine on the SOD content of HepG2 cells damaged by H2O2 are as follows Fig.10As shown. Under the action of H2O2, the cellular SOD content of HepG2 cells decreased significantly to 32.54±1.73U / mg Prot (P<0.05), which was about 88% lower than that of the blank group (282.39±4.46U / mg Prot). After the action of different concentrations of EGT (0.1mM, 1mM and 10mM), the cellular SOD content increased significantly compared with the group treated with the same dose of H2O2 (P<0.05). H2O2 effectively increased the cellular superoxide dismutase content. Among them, the SOD content of cells treated with 0.1mM EGT was 96.27±1.23U / mg Prot, which was about 1.95 times higher than that of the model group; when the EGT concentration increased to 1mM, the SOD content of cells was 104.91±2.08U / mg Prot, which was about 2.22 times higher than that of the model group; when the EGT concentration increased to 10mM, the SOD content of cells was 172.60±2.04U / mgProt, which was about 4.30 times higher than that of the model group. The results showed that the effect of EGT increased the SOD content in H2O2-damaged HepG2 cells in a dose-dependent manner and activated the SOD in H2O2-damaged HepG2 cells.

[0115] The results of the effect of ergothioneine on CAT content in HepG2 cells damaged by H2O2 are shown in Fig.11 As shown in the figure, under the action of H2O2, the CAT content of HepG2 cells decreased significantly to 15.15±1.79U / mg Prot (P<0.05), which was about 74% lower than that of the blank group (60.68±0.87U / mg Prot). After the action of different concentrations of EGT (0.1mM, 1mM and 10mM), the cellular CAT content increased significantly compared with the group treated with the same dose of H2O2 (P<0.05). Among them, the cellular CAT content after 0.1mM EGT treatment was 47.43±1.86U / mg Prot, which was about 2.13 times higher than that of the model group; when the EGT concentration increased to 1mM, the cellular CAT content was 51.74±1.21U / mg Prot, which was about 2.41 times higher than that of the model group; when the EGT concentration increased to 10mM, the cellular CAT content was 57.15±1.86U / mg Prot, which was about 2.77 times higher than that of the model group. The results showed that with the increase of EGT concentration, the CAT content in HepG2 cells damaged by H2O2 continued to increase, and approached the CAT content of the blank group at 10mM. The main function of CAT is to decompose H2O2 into H2O and O2, and it can effectively prevent O2 from generating hydroxyl radicals under the action of metal chelates, thereby reducing and blocking the primary initiation of lipid peroxidation.

[0116] The results of the effect of ergothioneine on the GSH-Px content in HepG2 cells damaged by H2O2 are shown in Fig.12 As shown. Under the action of H2O2, the cellular GSH-Px content of HepG2 cells decreased significantly to 73.06±2.30U / mg Prot (P<0.05), which was 77% lower than that of the blank group (311.58±2.53U / mg Prot). After the action of different concentrations of EGT (0.1, 1 and 10mM), the cellular GSH-Px content increased significantly compared with the group treated with the same dose of H2O2 (P<0.05). H2O2 effectively increased the cellular glutathione reductase content. Among them, the cellular GSH-Px content was 176.12±2.70U / mg Prot after 0.1mM EGT treatment, which was about 1.41 times higher than that of the model group; when the EGT concentration increased to 1mM, the cellular GSH-Px content was 186.53±3.03U / mg Prot, which was about 1.55 times higher than that of the model group; when the EGT concentration increased to 10mM, the cellular GSH-Px content was 252.50±2.70U / mg Prot, which was about 2.45 times higher than that of the model group. The results showed that the GSH-Px content in HepG2 cells damaged by H2O2 increased positively with the increase of EGT concentration.

[0117] In summary, the antioxidant enzymes of HepG2 cells were significantly reduced after being damaged by H2O2, and different concentrations of EGT could activate the antioxidant enzymes in HepG2 cells to varying degrees, thereby removing harmful free radicals and showing strong antioxidant properties.

[0118] Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine zipper protein that functions as a sensor of oxidative or electrophilic stress and prevents genomic instability. It regulates cellular defense against toxic and oxidative damage by expressing genes involved in oxidative stress response and drug detoxification. Nrf2 activity is tightly regulated through a complex transcriptional and post-translational network, enabling it to coordinate cellular responses and adaptations to various pathological stressors to maintain homeostasis. Therefore, we hypothesized that EGT could regulate the expression of antioxidant genes in H2O2-damaged HepG2 cells through the Nrf2 pathway. Fig.13Western blot and protein expression data showed that EGT increased the expression of total Nrf2 in HepG2 cells in a dose-dependent manner. When HepG2 cells were exposed to exogenous H2O2, the protein band of Nrf2 became significantly weaker, indicating that the expression of Nrf2 was significantly reduced (P<0.05). When the concentration of EGT was 0.1mM, the protein band of Nrf2 was close to that of the blank group, indicating that under the action of EGT, the protein expression of Nrf2 was upregulated and was no different from that of undamaged cells. When the concentration of EGT increased to 1 and 10mM, the protein band of Nrf2 became darker and thicker, and the protein expression of Nrf2 increased.

[0119] The present invention explores the antioxidant capacity of EGT on HepG2 cells treated with H2O2, including the reduction of ROS, and its direct relationship with the Nrf2, NQO1, and HO-1 antioxidant genes.

[0120] The protective effect of EGT on oxidative stress is believed to be the result of the induction of antioxidant genes in HepG2 cells damaged by H2O2, that is, the nuclear activation of Nrf2 is mediated by the induction of antioxidant genes. Therefore, the present invention uses a single HepG2 cell as a blank group, only 600μM H2O2 induced damage as a model group, and 600μM H2O2 and different concentrations of EGT (0.1, 1 and 10mM) as a test group. The results show that ( Fig.14 ), compared with the blank group, the transcription levels of Nrf2 protein and NQO-1 and HO-1 antioxidant proteins in HepG2 cells damaged by H2O2 in the model group were significantly decreased (P<0.05). After pretreatment with different concentrations of EGT, the transcription levels of Nrf2 protein and NQO-1 and HO-1 antioxidant proteins increased significantly to varying degrees (P<0.05), and the degree was positively correlated with the EGT concentration. Among them, when the EGT concentration was 0.1mM, the transcription levels of Nrf2 protein and NQO-1 antioxidant protein were close to the blank group, but still lower than the blank group, while the transcription level of HO-1 antioxidant protein increased significantly and was greater than the blank group. When the EGT concentration increased to 1 and 10mM, the transcription levels of Nrf2 protein and NQO-1 and HO-1 antioxidant proteins increased significantly, far exceeding the blank group and the model group. These data suggest that EGT has potent antioxidant properties, activating the nuclear factor Nrf2 and the downstream expression of NQO-1 and HO-1 antioxidant proteins, thereby helping to deal with H2O2-induced oxidative stress.

[0121] In summary, H2O2 can induce HepG2 cell damage. The EGT produced by the Pleurotus citrinum No. 1 of the present invention can dose-dependently reduce MDA generation and ROS levels, restore SOD, CAT and GS H-Px activities, activate nuclear factor Nrf2, and upregulate the transcription levels of Nrf2 protein and downstream NQO-1 and HO-1 antioxidant proteins, thereby improving H2O2-induced HepG2 cell oxidative stress.

[0122] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A Pleurotus citrinum strain producing ergothioneine, characterized in that, The elm yellow mushroom strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration CGMCC on June 26, 2024, with the deposit number CGMCC No.41349 and the classification name Pleurotus eryngii Pleurotus citrinopileatus .

2. the preparation method of thioneine, is characterized in that, The Pleurotus citrinopileatus strain according to claim 1 is used to ferment and produce ergothioneine.

3. the preparation method of thioneine according to claim 2, is characterized in that, Lactose and casein peptone were used as carbon and nitrogen sources in the liquid fermentation medium.

4. the preparation method of thioneine according to claim 2, is characterized in that, During the fermentation process, the precursor substance methionine was fed.

5. the preparation method of thioneine according to claim 2, is characterized in that, After fermentation to produce ergothioneine, power ultrasound or repeated freeze-thaw methods are used to assist heat extraction.

6. the preparation method of thioneine according to claim 4, is characterized in that, On the 3rd to 5th day of fermentation, the precursor substance methionine was fed into the fermentation liquid, and the mass volume ratio of methionine to fermentation liquid was 1 g: 500-1500 mL.

7. the preparation method of thioneine according to claim 6, is characterized in that, After adding the precursor methionine, the ergothioneine production was 223.638 ug / ml.

8. the preparation method of thioneine according to claim 6, is characterized in that, The fermentation time is 6-20 days.

9. Application of a Pleurotus citrinum strain producing thioneine according to claim 1 in high-yield thioneine.

10. Use of the Pleurotus citrinum strain producing ergothioneine according to claim 1 in preparing a drug for alleviating oxidative stress damage in liver cancer cells HepG2.

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