Ergothioneine-hydroxytyrosol mixture as well as preparation method and application thereof

Abstract

The invention relates to the technical field of active ingredient extraction, in particular to an ergothioneine-hydroxytyrosol mixture and a preparation method and application thereof, and optimizes a culture medium formula for producing ergothioneine through submerged liquid fermentation of pleurotus citrinopileatus mycelium. In the later stage of the process of producing ergothioneine by mycelium fermentation, a culture medium formula is designed according to nutritional ingredients required by hydroxytyrosol biosynthesis to supplement a sterile culture medium, and meanwhile, secondary metabolites and residual nutritional ingredients generated by fermentation of pleurotus citrinopileatus mycelium are fully utilized; the ergothioneine-hydroxytyrosol mixture is obtained through biosynthesis fermentation of hydroxytyrosol, the production period is short, the efficiency is high, the energy consumption is low, the ergothioneine-hydroxytyrosol mixture is green and safe, nutritional ingredients of a culture solution can be fully utilized, the overall production cost is reduced, the comprehensive production efficiency and productivity are improved, no waste liquid is discharged, and the subsequent application requirements are better met.

Description

Technical Field

[0001] The present invention relates to the technical field of active ingredient extraction, and particularly to an ergothioneine-hydroxytyrosol mixture, a preparation method thereof, and an application thereof. Background Art

[0002] Ergothioneine (EGT) is a natural antioxidant with strong antioxidant and anti-inflammatory effects, and has effects such as protecting nerve cells, anti-aging, and beauty care, and has been widely used in fields such as food, cosmetics, and pharmaceuticals. With the continuous analysis of its physiological functions, the demand for ergothioneine has also increased rapidly. Hydroxytyrosol (HT) has high antioxidant properties and can be applied to multiple industries such as nutritional health products, feeds, cosmetics, and pharmaceuticals, and also has broad application prospects in fields such as food preservatives and additives. However, the existing microbial synthesis of ergothioneine has a long production cycle and high cost, and it is difficult to meet the requirements of industrial production; the preparation yield of hydroxytyrosol in the existing technology is low, the product quality is poor, and it is not suitable for industrial production. In particular, the chemical synthesis method is harmful to the environment, has many by-products, and the product form is not ideal. For example, Chinese Patent (authorized publication number CN101631758B) discloses a method and equipment for preparing an extract containing hydroxytyrosol from solid residues extracted from olives and olive oil, but the extraction of hydroxytyrosol from olive oil and its wastewater is limited by natural materials and the extraction efficiency is low, far from meeting the market demand. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides an ergothioneine-hydroxytyrosol mixture, a preparation method thereof, and an application thereof, and optimizes the medium formula for the submerged liquid fermentation of Pleurotus citrinopileatus mycelium to produce ergothioneine; in the later stage of the process of mycelium fermentation to produce ergothioneine, according to the nutrient components required for the biosynthesis of hydroxytyrosol, a sterile medium is designed to supplement the medium formula, and at the same time, the secondary metabolites and remaining nutrient components produced by the fermentation of Pleurotus citrinopileatus mycelium are fully utilized to carry out the biosynthesis fermentation of hydroxytyrosol to obtain an ergothioneine-hydroxytyrosol mixture, which better meets the subsequent application requirements.

[0004] The present invention provides an ergothioneine-hydroxytyrosol mixture, a preparation method thereof, and an application thereof, which at least include the following steps:

[0005] (1) Submerged liquid fermentation of Pleurotus citrinopileatus mycelium to produce an ergothioneine fermentation filtrate, including: (1.1) Activating the strain to obtain an activated strain; (1.2) Preparing a shake flask medium; (1.3) Inoculating the activated strain into the shake flask medium to culture a shake flask liquid strain; (1.4) Preparing a fermenter medium; (1.5) Inoculating the shake flask liquid strain into the fermenter medium for fermenter culture to obtain a fermentation broth; (1.6) Post-treating the fermentation broth to obtain an ergothioneine fermentation filtrate;

[0006] (2) The ergothioneine-hydroxytyrosol mixed fermentation broth is obtained by co-fermentation culture of hydroxytyrosol, including: (2.1) activating the strain to obtain an activated strain; (2.2) preparing a shake flask seed medium; (2.3) inoculating the activated strain in the shake flask seed medium to culture a shake flask seed solution; (2.4) preparing a fermentation medium; (2.5) inoculating the shake flask seed solution into a fermentation tank medium supplemented with the fermentation medium for fermentation culture to obtain an ergothioneine-hydroxytyrosol mixed fermentation broth;

[0007] (3) The ergothioneine-hydroxytyrosol mixture is prepared by extracting and purifying the ergothioneine-hydroxytyrosol mixed fermentation broth.

[0008] The method provided by the present invention synthesizes ergothioneine through submerged liquid fermentation by preferably using Pleurotus citrinopileatus Singer, and creatively adds a strain capable of synthesizing hydroxytyrosol in the late stage of Pleurotus citrinopileatus mycelium fermentation, and uses the Pleurotus citrinopileatus mycelium fermentation broth for co-fermentation culture. Further, an ergothioneine-hydroxytyrosol mixture is prepared by enzymatic hydrolysis extraction method. The production cycle is short, the efficiency is high, the energy consumption is low, it is green and safe, and it can make full use of the nutrients in the culture solution, reduce the overall production cost, improve the comprehensive production efficiency and production capacity, and there is no waste liquid discharge.

[0009] In one embodiment, the steps of strain activation include: preparing a strain activation medium, inoculating Pleurotus citrinopileatus Singer under sterile conditions, culturing for 7-10 days at 24-26°C under light-shielded conditions, and using the activated strain after passing the detection.

[0010] In one embodiment, by weight, the formula of the strain activation medium includes: 150-250 parts of potato, 15-20 parts of glucose, 5-7 parts of peptone, 0.5-1 part of potassium dihydrogen phosphate, 0.2-0.5 part of magnesium sulfate, 18-20 parts of agar powder, and 1000 parts of water.

[0011] In one embodiment, by weight, the formula of the shake flask medium includes: 200 parts of potato, 18-20 parts of glucose, 6-7 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.2-0.5 part of magnesium sulfate, 18 parts of agar powder, and 1000 parts of water.

[0012] In one embodiment, the steps of preparing the shake flask medium include: preparing the shake flask medium according to the shake flask medium formula and performing autoclaving; by weight, the shake flask medium formula includes: 150-250 parts of potato, 15-20 parts of glucose, 5-7 parts of peptone, 0.5-1 part of potassium dihydrogen phosphate, 0.2-0.5 part of magnesium sulfate, 0.1-0.3 part of agar powder, and 1000 parts of water.

[0013] In one embodiment, by weight, the shake flask culture medium formula comprises: 200 parts of potato, 17 - 20 parts of glucose, 6 - 7 parts of peptone, 0.5 - 1 part of potassium dihydrogen phosphate, 0.2 - 0.5 part of magnesium sulfate, 0.1 part of agar powder, and 1000 parts of water.

[0014] By optimizing the addition amount of potato in the shake flask culture medium formula and using peptone as the nitrogen source, the provided shake flask culture medium is more conducive to the germination and growth of mycelium in the initial stage of strain activation. Moreover, natural culture components such as soybean cake powder and corn flour are not used, which is more conducive to later extraction and reduces interfering substances.

[0015] In one embodiment, the steps of autoclaving include: filling the prepared shake flask culture medium into Erlenmeyer flasks, sealing with cotton plugs or double - layer polypropylene films; placing them in an autoclave and sterilizing at 115 - 125 °C for 15 - 30 min.

[0016] In one embodiment, the steps of culturing shake flask liquid strains include: inoculating the activated strains obtained in step (1.1) into the shake flask culture medium prepared in step (1.2), and performing shaking culture at a rotation speed of 120 - 140 r / min and a temperature of 24 - 26 °C for 5 - 7 days to obtain shake flask liquid strains.

[0017] In one embodiment, the steps of preparing the fermenter culture medium include: adding the fermenter culture medium to the ergothioneine fermenter according to the formula to prepare the fermenter culture medium, and using it after high - temperature sterilization.

[0018] In one embodiment, by weight, the formula of the fermenter culture medium comprises: 5 - 10 parts of starch, 5 - 10 parts of wheat bran, 15 - 20 parts of glucose, 5 - 7 parts of peptone, 0.5 - 1 part of potassium dihydrogen phosphate, 0.2 - 0.5 part of magnesium sulfate, and 1000 parts of water.

[0019] In one embodiment, by weight, the formula of the fermenter culture medium comprises: 7 - 10 parts of starch, 7 - 10 parts of wheat bran, 20 parts of glucose, 5 - 6 parts of peptone, 0.5 - 1 part of potassium dihydrogen phosphate, 0.2 - 0.5 part of magnesium sulfate, and 1000 parts of water.

[0020] In one embodiment, the steps of fermenter culture include: inoculating the shake flask liquid strains cultured in step (1.3) into the ergothioneine fermenter in step (1.4) under sterile conditions for aerated fermentation culture to obtain the fermentation broth, and the temperature of the fermentation culture is 24 - 26 °C.

[0021] Furthermore, the present invention optimizes the design of the fermentation tank culture medium formula. The formula has a reasonable component structure and rich nutrients, which is conducive to the synthesis and accumulation of ergothioneine and facilitates large-scale industrial production. In particular, when combined with a subsequent fermentation culture temperature of 24-26 °C, low-temperature fermentation is achieved while ensuring the fermentation effect.

[0022] In one embodiment, the steps of the post-treatment include: filtering half of the total volume of the fermentation broth in the ergothioneine fermentation tank in step (1.5) to obtain a filtrate; adding activated carbon to the filtrate, where the mass of the added activated carbon is 1.5-3% of the volume of the filtrate, and stirring and adsorbing for 40-60 min at 30-40 °C and 300-400 rpm to obtain a mixture; filtering the mixture successively with 100-mesh, 200-mesh, and 400-mesh filter cloths to obtain a pale yellow to colorless ergothioneine fermentation filtrate.

[0023] In one embodiment, the steps of strain activation include: preparing an activation medium (modified solid LB), inoculating the strain (E. coli TOP10) by streaking under sterile conditions, culturing at 35-38 °C in the dark for 48 h, and using the activated strain after passing the detection.

[0024] In one embodiment, by weight, the formula of the activation medium (modified solid LB) includes: 15-20 parts of glucose, 6-10 parts of peptone, 5-8 parts of yeast powder, 7-10 parts of sodium chloride, 18-20 parts of agar powder, and 1000 parts of water.

[0025] In one embodiment, by weight, the formula of the activation medium (modified solid LB) includes: 15-20 parts of glucose, 6-7 parts of peptone, 5-6 parts of yeast powder, 7-10 parts of sodium chloride, 18 parts of agar powder, and 1000 parts of water.

[0026] In one embodiment, the steps of preparing the shake flask seed medium include: preparing the shake flask seed medium according to the shake flask seed medium formula, filling the prepared shake flask seed medium into Erlenmeyer flasks, and sealing with cotton plugs or double-layer polypropylene films; placing in an autoclave and sterilizing at 115-125 °C for 15-30 min.

[0027] In one embodiment, the shake flask seed medium includes a modified LB medium and a modified M9 fermentation medium.

[0028] In one embodiment, by weight, the formula of the modified LB (lysogeny broth medium) medium includes: 15-20 parts of glucose, 7-10 parts of peptone, 5-8 parts of yeast powder, 7-10 parts of sodium chloride, and 1000 parts of water.

[0029] In one embodiment, by weight, the formula of the improved LB medium comprises: 20 parts of glucose, 8 - 10 parts of peptone, 7 - 8 parts of yeast powder, 7 - 10 parts of sodium chloride, and 1000 parts of water.

[0030] In one embodiment, by weight, the formula of the improved M9 (E. coli medium) fermentation medium comprises: 1 - 5 parts of yeast powder, 15 - 20 parts of glucose, 1 - 5 parts of peptone, 1 - 3 parts of potassium dihydrogen phosphate, 0.3 - 0.5 parts of sodium chloride, 7 - 10 parts of disodium hydrogen phosphate, 0.5 - 1 part of ammonium chloride, and 1000 parts of water.

[0031] In one embodiment, by weight, the formula of the improved M9 fermentation medium comprises: 3 - 5 parts of yeast powder, 20 parts of glucose, 3 - 5 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 parts of sodium chloride, 10 parts of disodium hydrogen phosphate, 0.5 part of ammonium chloride, and 1000 parts of water.

[0032] In one embodiment, the steps of culturing the shake flask seed liquid include: inoculating the activated strain (single colony) obtained in step (2.1) into the improved LB medium prepared in step (2.2), culturing with shaking at a rotation speed of 180 - 220 r / min and a temperature of 35 - 38 °C for 10 - 12 h, and then collecting the thalli; inoculating the thalli into the improved M9 fermentation medium prepared in step (2.2), culturing with shaking at a rotation speed of 180 - 220 r / min and a temperature of 28 - 32 °C for 48 - 50 h, and adding 1 mmol / L of IPTG (isopropyl - β - D - thiogalactoside) for induction when the cell concentration OD600 reaches 0.5 to obtain the shake flask seed liquid.

[0033] In one embodiment, the steps of preparing the fermentation medium include: preparing the improved M9 fermentation medium and performing high - temperature sterilization (both the formula of the improved M9 fermentation medium and the conditions of high - temperature sterilization are the same as those in step 2.2).

[0034] In the present invention, while fermenting and producing ergothioneine with Pleurotus citrinopileatus mycelium, in the second fermentation stage, fresh medium (improved M9 fermentation medium) is supplemented in batches for the fermentation of hydroxytyrosol, which improves production efficiency, reduces production energy consumption, and reduces environmental pollution. In particular, adding the improved M9 fermentation medium after high - temperature sterilization as the feeding liquid can avoid fermentation failure.

[0035] In one embodiment, the steps of fermentation culture include: inoculating the shake flask seed liquid cultured in step (2.3) into the ergothioneine fermentation tank in step (1.5) added with the fermentation medium prepared in step (2.4), and continuing the fermentation culture. During the fermentation culture process, the fermentation medium is supplemented in batches until the fermentation is completed.

[0036] In one embodiment, the conditions for fermentation culture include: the fermentation aeration ratio is 1.4 - 1.6; two-stage temperature-controlled fermentation is adopted, the fermentation temperature is 35 - 37 °C for 0 - 14 h, and the fermentation temperature is 28 - 32 °C for 14 - 48 h.

[0037] In the present invention, the potato is used after being peeled and boiled, specifically the water in which the peeled potato has been boiled.

[0038] In one embodiment, the steps of extraction and purification include the following steps: the ergothioneine-hydroxytyrosol mixed fermentation broth is concentrated under reduced pressure to 40 - 60% of the original volume, a mixed enzyme composition of dextranase, chitinase and cellulase is added, the pH is adjusted to 4.5 - 5.5, the enzymolysis temperature is 20 - 30 °C, and the enzymolysis time is 20 - 24 h to obtain a hydrolyzate; the hydrolyzate is heated to 55 - 65 °C, the pH is adjusted to 8 - 9, protease hydrolysis is carried out, and the supernatant is taken after centrifugation; the supernatant is concentrated to 10 - 30% of the original volume, 3 - 5 times the volume of 90 - 98 wt% ethanol aqueous solution is added under stirring, and it is left standing for 20 - 24 h at 0 - 5 °C, and the extract is obtained by centrifugation; the extract is subjected to adsorption, elution and concentration under reduced pressure to obtain the ergothioneine-hydroxytyrosol mixture.

[0039] In the present invention, the obtained ergothioneine-hydroxytyrosol mixed fermentation broth is extracted by an enzymatic extraction method, which destroys components such as the cell wall and intercellular substance of the raw material, so that ergothioneine and hydroxytyrosol are fully released into the extract, improving the extraction rate.

[0040] In one embodiment, based on the volume of the ergothioneine-hydroxytyrosol mixed fermentation broth after concentration under reduced pressure, the addition amount of dextranase is 0.8 - 0.9% (w / v - mass / volume ratio), the addition amount of chitinase is 0.5 - 0.6% (w / v), and the addition amount of cellulase is 0.3 - 0.4% (w / v).

[0041] In one embodiment, based on the volume of the hydrolyzate, the addition amount of protease is 0.5 - 1% (w / v).

[0042] In one embodiment, based on the volume of the hydrolyzate, the addition amount of protease is 0.8% (w / v).

[0043] In one embodiment, the step of adsorption includes: mixing the extract with D-101 macroporous resin and shaking for adsorption for at least 24 h.

[0044] In one embodiment, the step of elution includes: eluting with deionized water until neutral, and then eluting with 50 - 70 wt% ethanol aqueous solution.

[0045] The present invention enriches and purifies natural active chemical components by macroporous adsorption with D-101 macroporous resin and elution with an ethanol aqueous solution of 50-70 wt% for the extract obtained by enzymatic extraction, significantly increasing their content in the ergothioneine-hydroxytyrosol mixture, which is beneficial for subsequent applications.

[0046] On the other hand, the present invention provides an ergothioneine-hydroxytyrosol mixture, which at least includes the following components: ergothioneine and hydroxytyrosol; the content of hydroxytyrosol in the ergothioneine-hydroxytyrosol mixture ≥ 130 mg / L; the content of ergothioneine in the ergothioneine-hydroxytyrosol mixture ≥ 16 mg / L.

[0047] On the third aspect, the present invention provides an application of an ergothioneine-hydroxytyrosol mixture in the preparation of foods, cosmetics and pharmaceuticals.

[0048] Beneficial effects

[0049] 1. The method provided by the present invention synthesizes ergothioneine by deep liquid fermentation with Pleurotus citrinopileatus Singer strain as the preferred strain, and creatively adds a strain capable of synthesizing hydroxytyrosol in the later stage of Pleurotus citrinopileatus mycelium fermentation, and co-ferments and cultures with Pleurotus citrinopileatus mycelium fermentation broth. Further, an ergothioneine-hydroxytyrosol mixture is prepared by an enzymatic extraction method, which has a short production cycle, high efficiency, low energy consumption, is green and safe, can make full use of the nutrient components of the culture solution, reduces the overall production cost, improves the comprehensive production efficiency and production capacity, and has no waste liquid discharge.

[0050] 2. By optimizing the addition amount of potato in the shake flask culture medium formula and using peptone as the nitrogen source, the shake flask culture medium provided by the present invention is more conducive to the germination and growth of mycelium at the initial stage of strain startup, and does not use natural culture components such as soybean cake powder and corn flour, which is more conducive to subsequent extraction and reduces interfering substances.

[0051] 3. The present invention optimizes the design of the fermentation tank culture medium formula, which has a reasonable composition structure and rich nutrients, is conducive to the synthesis and accumulation of ergothioneine, and is convenient for large-scale industrial production. Especially when combined with a subsequent fermentation culture temperature of 24-26 °C, it can achieve low-temperature fermentation while ensuring the fermentation effect.

[0052] 4. When the present invention uses Pleurotus citrinopileatus mycelium to ferment and produce ergothioneine, hydroxytyrosol fermentation is carried out by supplementing fresh culture medium (modified M9 fermentation medium) in batches in the second fermentation stage, which improves the production efficiency, reduces the production energy consumption, and reduces environmental pollution. Especially when adding the modified M9 fermentation medium after high-temperature sterilization as the feeding liquid, fermentation failure is avoided.

[0053] 5. The present invention uses an enzymatic extraction method to extract the obtained ergothioneine-hydroxytyrosol mixed fermentation broth, which destroys components such as the cell wall and cytoplasmic matrix of the raw materials, enables ergothioneine and hydroxytyrosol to be fully released into the extraction solution, and improves the extraction rate.

[0054] 6. The present invention combines the extraction solution obtained by enzymatic extraction with D-101 macroporous resin for macroporous adsorption and elution with an ethanol aqueous solution of 50-70 wt%, enriches and purifies the natural active chemical components, significantly increases their content in the ergothioneine-hydroxytyrosol mixture, and is beneficial for later applications. Specific embodiments

[0055] The information of the raw materials for the preparation methods of the ergothioneine-hydroxytyrosol mixtures provided in the examples and comparative examples of this application is as follows:

[0056] Glucose; peptone; agar powder; yeast powder are all from Sinopharm Chemical Reagent Co., Ltd. (analytical pure); other raw materials are all commercially available products.

[0057] Example 1

[0058] Example 1 of the present invention provides a preparation method of an ergothioneine-hydroxytyrosol mixture, which includes the following steps:

[0059] (1) Deep liquid fermentation of Pleurotus citrinopileatus mycelium to produce an ergothioneine fermentation filtrate, including: (1.1) Activating the strain to obtain an activated strain; (1.2) Preparing a shake flask medium; (1.3) Inoculating the activated strain into the shake flask medium to culture a shake flask liquid strain; (1.4) Preparing a fermentation tank medium; (1.5) Inoculating the shake flask liquid strain into the fermentation tank medium for fermentation tank culture to obtain a fermentation broth; (1.6) Post-treating the fermentation broth to obtain an ergothioneine fermentation filtrate;

[0060] (2) Co-fermentation culture of hydroxytyrosol to obtain an ergothioneine-hydroxytyrosol mixed fermentation broth, including: (2.1) Activating the strain to obtain an activated strain; (2.2) Preparing a shake flask seed medium; (2.3) Inoculating the activated strain into the shake flask seed medium to culture a shake flask seed liquid; (2.4) Preparing a fermentation medium; (2.5) Inoculating the shake flask seed liquid into the fermentation tank medium added with the fermentation medium for fermentation culture to obtain an ergothioneine-hydroxytyrosol mixed fermentation broth;

[0061] (3) Extracting and purifying the ergothioneine-hydroxytyrosol mixed fermentation broth to prepare an ergothioneine-hydroxytyrosol mixture.

[0062] The steps of the strain activation include: preparing a strain activation medium, inoculating the Pleurotus citrinopileatus strain under sterile conditions, culturing for 10 days at 25°C under light-shielded conditions, and using the activated strain after passing the test.

[0063] The Pleurotus citrinopileatus Sing. strain is Changbaishan No. 1 from Jiangdu Tianda Edible Fungi Research Institute.

[0064] By weight, the formula of the strain activation medium includes: 200 parts of potato, 18 parts of glucose, 7 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.2 part of magnesium sulfate, 18 parts of agar powder, and 1000 parts of water.

[0065] The steps for preparing the shake flask medium include: preparing the shake flask medium according to the shake flask medium formula and performing autoclaving; by weight, the shake flask medium formula includes: 200 parts of potato, 17 parts of glucose, 6 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate, 0.1 part of agar powder, and 1000 parts of water.

[0066] The steps for autoclaving include: dispensing the prepared shake flask medium into Erlenmeyer flasks, sealing with double-layer polypropylene film; placing in an autoclave and sterilizing at 121 °C for 20 min.

[0067] The steps for culturing the shake flask liquid strain include: inoculating the activated strain obtained in step (1.1) into the shake flask medium prepared in step (1.2), and performing shaking culture at a rotation speed of 130 r / min and a temperature of 25 °C for 6 days to obtain the shake flask liquid strain.

[0068] The steps for preparing the fermenter medium include: adding the fermenter medium to the ergothioneine fermenter according to the formula to prepare the fermenter medium, and using it after high-temperature sterilization (the high-temperature sterilization conditions are: sterilizing at 121 °C for 20 min).

[0069] By weight, the formula of the fermenter medium includes: 10 parts of starch, 10 parts of wheat bran, 20 parts of glucose, 5 parts of peptone, 0.5 part of potassium dihydrogen phosphate, 0.2 part of magnesium sulfate, and 1000 parts of water.

[0070] The steps for fermenter culture include: inoculating the shake flask liquid strain cultured in step (1.3) into the ergothioneine fermenter in step (1.4) under aseptic conditions for aerobic fermentation culture to obtain the fermentation broth, and the temperature of the fermentation culture is 25 °C.

[0071] The steps for post-treatment include: filtering half of the total volume of the fermentation broth in the ergothioneine fermenter in step (1.5) to obtain the filtrate; adding activated carbon to the filtrate, and the mass of the added activated carbon is 2% of the volume of the filtrate, and stirring and adsorbing at 35 °C and 350 rpm for 50 min to obtain the mixture; filtering the mixture successively with 100-mesh, 200-mesh, and 400-mesh filter cloths to obtain the light yellow to colorless ergothioneine fermentation filtrate.

[0072] The steps for activating the strain include: preparing an activation medium (modified solid LB), inoculating the strain (TOP 10 from Beijing BioWin Biotechnology Co., Ltd.) by streaking under sterile conditions, culturing at 37°C in the dark for 48 h, and using the activated strain after passing the test.

[0073] By weight, the formula of the activation medium (modified solid LB) includes: 15 parts of glucose, 7 parts of peptone, 5 parts of yeast powder, 10 parts of sodium chloride, 18 parts of agar powder, and 1000 parts of water.

[0074] The steps for preparing the shake flask seed medium include: preparing the shake flask seed medium according to the shake flask seed medium formula, dispensing the prepared shake flask seed medium into Erlenmeyer flasks, and sealing with cotton plugs or double-layer polypropylene films; placing in an autoclave and sterilizing at 121°C for 20 min.

[0075] The shake flask seed medium includes a modified LB medium and a modified M9 fermentation medium.

[0076] By weight, the formula of the modified LB medium includes: 20 parts of glucose, 10 parts of peptone, 7 parts of yeast powder, 7 parts of sodium chloride, and 1000 parts of water.

[0077] By weight, the formula of the modified M9 fermentation medium includes: 3 parts of yeast powder, 20 parts of glucose, 3 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of sodium chloride, 10 parts of disodium hydrogen phosphate, 0.5 part of ammonium chloride, and 1000 parts of water.

[0078] The steps for culturing the shake flask seed liquid include: inoculating the activated strain (single colony) obtained in step (2.1) into the modified LB medium prepared in step (2.2), culturing with shaking at a rotation speed of 200 r / min and a temperature of 37°C for 12 h, and then collecting the cells; inoculating the cells into the modified M9 fermentation medium prepared in step (2.2), culturing with shaking at a rotation speed of 200 r / min and a temperature of 30°C for 48 h, and adding 1 mmol / L of IPTG (isopropyl-β-D-thiogalactoside) for induction when the cell concentration OD600 reaches 0.5 to obtain the shake flask seed liquid.

[0079] The steps for preparing the fermentation medium include: preparing the modified M9 fermentation medium and performing high-temperature sterilization (the formula of the modified M9 fermentation medium and the conditions for high-temperature sterilization are the same as those in step 2.2).

[0080] The steps for fermentation culture include: inoculating the shake flask seed liquid cultured in step (2.3) into the ergothioneine fermentation tank in step (1.5) supplemented with the fermentation medium prepared in step (2.4), and continuing the fermentation culture. During the fermentation culture process, the fermentation medium is supplemented in batches until the fermentation is completed.

[0081] The conditions for the fermentation culture include: the fermentation aeration ratio is 1.5; two-stage temperature-controlled fermentation is adopted, the fermentation temperature is 36°C for 0-14h, and the fermentation temperature is 30°C for 14-48h.

[0082] In the present invention, the peeled and boiled potato juice is used, specifically the water from the peeled and boiled potatoes.

[0083] The extraction and purification include the following steps: the ergothioneine-hydroxytyrosol mixed fermentation broth is concentrated under reduced pressure to 50% of the original volume, a mixed enzyme composition of dextranase, chitinase and cellulase is added, the pH is adjusted to 5.0, the enzymolysis temperature is 24°C, and the enzymolysis time is 24h to obtain a hydrolyzate; the hydrolyzate is heated to 60°C, the pH is adjusted to 8.5, protease hydrolysis is carried out, and the supernatant is taken after centrifugation; the supernatant is concentrated to 20% of the original volume, 4 times the volume of 95wt% ethanol aqueous solution is added under stirring, and it is left standing at 4°C for 24h, and the extract is obtained by centrifugation; the extract is adsorbed, eluted and concentrated under reduced pressure to obtain the ergothioneine-hydroxytyrosol mixture.

[0084] Based on the volume of the ergothioneine-hydroxytyrosol mixed fermentation broth after concentration under reduced pressure, the addition amount of dextranase is 0.9% (w / v), the addition amount of chitinase is 0.6% (w / v), and the addition amount of cellulase is 0.4% (w / v).

[0085] Based on the volume of the hydrolyzate, the addition amount of protease is 0.8% (w / v).

[0086] The adsorption step includes: mixing the extract with D-101 macroporous resin (Sinopharm Chemical Reagent Co., Ltd.) and shaking for adsorption for 24h.

[0087] The elution step includes: eluting with deionized water until neutral, and then eluting with 60wt% ethanol aqueous solution.

[0088] Example 2

[0089] Example 2 of the present invention provides a preparation method of an ergothioneine-hydroxytyrosol mixture, and the difference in its specific implementation from Example 1 is as follows:

[0090] By weight, the formula of the strain activation medium includes: 200 parts of potato, 20 parts of glucose, 6 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate, 18 parts of agar powder, and 1000 parts of water.

[0091] By weight, the formula of the shake flask medium includes: 200 parts of potato, 20 parts of glucose, 7 parts of peptone, 0.5 part of potassium dihydrogen phosphate, 0.2 part of magnesium sulfate, 0.1 part of agar powder, and 1000 parts of water.

[0092] By weight, the formula of the fermentation tank medium includes: 7 parts of starch, 7 parts of wheat bran, 20 parts of glucose, 6 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate, and 1000 parts of water.

[0093] By weight, the formula of the activation medium (modified solid LB) includes: 20 parts of glucose, 6 parts of peptone, 6 parts of yeast powder, 7 parts of sodium chloride, 18 parts of agar powder, and 1000 parts of water.

[0094] By weight, the formula of the modified LB medium includes: 20 parts of glucose, 8 parts of peptone, 8 parts of yeast powder, 10 parts of sodium chloride, and 1000 parts of water.

[0095] By weight, the formula of the modified M9 fermentation medium includes: 5 parts of yeast powder, 20 parts of glucose, 5 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of sodium chloride, 10 parts of disodium hydrogen phosphate, 0.5 part of ammonium chloride, and 1000 parts of water.

[0096] Based on the volume of the ergot thioneine-hydroxytyrosol mixed fermentation broth after vacuum concentration, the addition amount of dextranase is 0.8% (w / v), the addition amount of chitinase is 0.5% (w / v), and the addition amount of cellulase is 0.3% (w / v).

[0097] Comparative Example 1

[0098] Comparative Example 1 of the present invention provides a method for co-fermenting Pleurotus citrinopileatus and Tricholoma matsutake mycelia to produce ergot thioneine, comprising the following steps:

[0099] a. Inoculate tissue blocks of Pleurotus citrinopileatus and Tricholoma matsutake on a solid medium respectively and culture at 25°C for 5 days;

[0100] b. Inoculate Pleurotus citrinopileatus mycelia into a seed medium and culture for 3 days;

[0101] c. Inoculate Tricholoma matsutake mycelia into the Pleurotus citrinopileatus seed liquid obtained by culturing in step b and supplement with fresh seed medium, and continue to culture for 3 days;

[0102] d. Inoculate the seed liquid containing Pleurotus citrinopileatus and Tricholoma matsutake obtained by culturing in step c into a fermentation medium at an inoculation amount of 10% of the volume of the fermentation medium and add a precursor substance. After fermenting for 4 days, supplement the precursor substance again and continue to ferment for 4 days;

[0103] e. Treat the fermented fermentation broth to obtain a fermentation broth containing ergot thioneine.

[0104] Among them: the precursor substances include: histidine 75 mg / L, histidine betaine 20 mg / L, mercaptohistidine betaine 10 mg / L, betaine 1.5 g / L, cysteine 10 mg / L, γ-glutamylcysteine 10 mg / L, and the solvent is water.

[0105] By weight, the seed culture medium formula is: 20 parts of potato, 8 parts of corn flour, 8 parts of soybean cake powder, 0.5 part of magnesium sulfate, 1.5 parts of potassium dihydrogen phosphate, 0.002 part of vitamin B1, and 1000 parts of water.

[0106] Comparative Example 2

[0107] Comparative Example 2 of the present invention provides a preparation method of ergothioneine-hydroxytyrosol mixture, and its specific implementation manner is the same as that of Example 1, except that, by weight, the shake flask culture medium formula includes: 200 parts of potato, 17 parts of glucose, 2 parts of peptone, 1 part of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate, 0.1 part of agar powder, and 1000 parts of water.

[0108] Comparative Example 3

[0109] Comparative Example 3 of the present invention provides a preparation method of ergothioneine-hydroxytyrosol mixture, and its specific implementation manner is the same as that of Example 1, except that the steps of fermenter culture include: inoculating the shake flask liquid strain cultured in step (1.3) into the ergothioneine fermenter in step (1.4) under sterile conditions and performing aeration fermentation culture to obtain a fermentation broth, and the temperature of the fermentation culture is 30 °C.

[0110] Comparative Example 4

[0111] Comparative Example 4 of the present invention provides a preparation method of ergothioneine-hydroxytyrosol mixture, and its specific implementation manner is the same as that of Example 1, except that the extraction and purification include the following steps: concentrating the ergothioneine-hydroxytyrosol mixed fermentation broth under reduced pressure to 50% of the original volume, adding a mixed enzyme composition of dextranase, chitinase and cellulase, adjusting the pH to 5.0, the enzyme hydrolysis temperature to 45 °C, and the enzyme hydrolysis time to 24 h to obtain a hydrolyzate; heating the hydrolyzate to 60 °C, adjusting the pH to 8.5, adding protease for hydrolysis, and taking the supernatant after centrifugation; concentrating the supernatant to 20% of the original volume, adding 4 times the volume of 95 wt% ethanol aqueous solution under stirring, standing at 4 °C for 24 h, and centrifuging to obtain an extract; subjecting the extract to adsorption, elution, and concentration under reduced pressure to obtain the ergothioneine-hydroxytyrosol mixture.

[0112] Comparative Example 5

[0113] Comparative Example 5 of the present invention provides a method for preparing a mixture of ergothioneine and hydroxytyrosol. The specific implementation manner is the same as that of Example 1, except that the elution step includes: eluting with deionized water until neutral, and then eluting with an aqueous ethanol solution of 30 wt%.

[0114] Comparative Example 6

[0115] Comparative Example 6 of the present invention provides a method for preparing a mixture of ergothioneine and hydroxytyrosol. The specific implementation manner is the same as that of Example 1, except that the step of preparing the fermentation medium includes: preparing a modified M9 fermentation medium without high-temperature sterilization.

[0116] Performance test

[0117] Test the contents of ergothioneine and hydroxytyrosol in the mixtures of ergothioneine and hydroxytyrosol obtained in the test examples and comparative examples. The results are shown in Table 1.

[0118] Table 1

[0119]

[0120] It can be seen from the data in Table 1 that compared with Comparative Examples 1-6, the methods for preparing the mixtures of ergothioneine and hydroxytyrosol provided in Examples 1-2 can successfully obtain the mixtures of ergothioneine and hydroxytyrosol, while ensuring the yields of ergothioneine and hydroxytyrosol, and better meeting the actual application requirements.

Claims

1. A method for preparing a mixture of ergothioneine and hydroxytyrosol, characterized in that, It includes at least the following steps: (1) Deep liquid fermentation of Pleurotus citrinopileatus mycelium to produce ergothioneine fermentation filtrate, including: (1.1) Activating the strain to obtain an activated strain; (1.2) Preparing a shake flask medium; (1.3) Inoculating the activated strain into the shake flask medium to culture shake flask liquid strain; (1.4) Preparing a fermenter medium; (1.5) Inoculating the shake flask liquid strain into the fermenter medium for fermenter culture to obtain a fermentation broth; (1.6) Post-treating the fermentation broth to obtain ergothioneine fermentation filtrate; (2) Co-fermentation culture of hydroxytyrosol to obtain an ergothioneine-hydroxytyrosol mixed fermentation broth, including: (2.1) Activating the strain to obtain an activated strain; (2.2) Preparing a shake flask seed medium; (2.3) Inoculating the activated strain into the shake flask seed medium to culture shake flask seed liquid; (2.4) Preparing a fermentation medium; (2.5) Inoculating the shake flask seed liquid into the fermenter medium added with the fermentation medium for fermentation culture to obtain an ergothioneine-hydroxytyrosol mixed fermentation broth; (3) Extracting and purifying the ergothioneine-hydroxytyrosol mixed fermentation broth to prepare an ergothioneine-hydroxytyrosol mixture.

2. The preparation method of the ergothioneine-hydroxytyrosol mixture according to claim 1, characterized in that, The step of preparing the shake flask medium includes: preparing the shake flask medium according to the shake flask medium formula and performing high-pressure sterilization; by weight, the shake flask medium formula includes: 150 - 250 parts of potato, 15 - 20 parts of glucose, 5 - 7 parts of peptone, 0.5 - 1 part of potassium dihydrogen phosphate, 0.2 - 0.5 part of magnesium sulfate, 0.1 - 0.3 part of agar powder, and 1000 parts of water.

3. The preparation method of the ergothioneine-hydroxytyrosol mixture according to claim 1, characterized in that, The step of fermenter culture includes: inoculating the shake flask liquid strain cultured in step (1.3) into the ergothioneine fermenter in step (1.4) under sterile conditions for aerated fermentation culture to obtain a fermentation broth, and the temperature of the fermentation culture is 24 - 26 °C.

4. The preparation method of the ergothioneine-hydroxytyrosol mixture according to claim 1, characterized in that, The extraction and purification includes the following steps: concentrating the ergothioneine-hydroxytyrosol mixed fermentation broth under reduced pressure to 40 - 60% of the original volume, adding a mixed enzyme composition of dextranase, chitinase, and cellulase, adjusting the pH to 4.5 - 5.5, the enzyme hydrolysis temperature is 20 - 30 °C, and the enzyme hydrolysis time is 20 - 24 h to obtain a hydrolyzate; heating the hydrolyzate to 55 - 65 °C, adjusting the pH to 8 - 9, adding protease for hydrolysis, and taking the supernatant after centrifugation; concentrating the supernatant to 10 - 30% of the original volume, adding 3 - 5 times the volume of 90 - 98 wt% ethanol aqueous solution under stirring, standing at 0 - 5 °C for 20 - 24 h, and centrifuging to obtain an extract; subjecting the extract to adsorption, elution, and concentration under reduced pressure to obtain an ergothioneine-hydroxytyrosol mixture.

5. The preparation method of the ergothioneine-hydroxytyrosol mixture according to claim 4, characterized in that, The step of elution includes: eluting with deionized water until neutral, and then eluting with 50 - 70 wt% ethanol aqueous solution.

6. The preparation method of the ergothioneine-hydroxytyrosol mixture according to claim 3, characterized in that, The step of preparing the fermentation medium includes: preparing a modified M9 fermentation medium and performing high-temperature sterilization.

7. The preparation method of the ergothioneine-hydroxytyrosol mixture according to claim 1, characterized in that, The step of preparing the fermenter medium includes: adding the fermenter medium to the ergothioneine fermenter according to the formula to prepare the fermenter medium, and using it after high-temperature sterilization.

8. The preparation method of the ergothioneine-hydroxytyrosol mixture according to claim 7, characterized in that, By weight, the formula of the fermentor culture medium comprises: 5-10 parts of starch, 5-10 parts of wheat bran, 15-20 parts of glucose, 5-7 parts of peptone, 0.5-1 part of potassium dihydrogen phosphate, 0.2-0.5 part of magnesium sulfate, and 1000 parts of water.

9. An ergothioneine-hydroxytyrosol mixture prepared by the preparation method of the ergothioneine-hydroxytyrosol mixture according to any one of claims 1-8, characterized in that, The ergothioneine-hydroxytyrosol mixture comprises at least the following components: ergothioneine and hydroxytyrosol.

10. Use of the ergothioneine-hydroxytyrosol mixture according to claim 9, characterized in that, It is applied to the preparation of foods, cosmetics and pharmaceuticals.

Images