Cordyceps sinensis mycelium rich in ergothioneine as well as preparation process and application thereof

Abstract

The invention provides cordyceps sinensis mycelia rich in ergothioneine as well as a preparation process and application thereof. The content of ergothioneine in the cordyceps sinensis mycelia is not less than 350mg / kg by dry weight. The cordyceps sinensis mycelium is obtained by adding at least one of the following three amino acids in a mycelium fermentation culture process: Met, Cys and His. Tests prove that the cordyceps sinensis mycelium rich in ergothioneine has outstanding biological activity, has excellent series of physiological effects of cell repair, oxidation resistance, free radical removal, aging resistance, beauty maintaining, young keeping and the like, and has wide application prospects in the industries of cosmetics, food, biological medicine and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fungus culture and active ingredient extraction, and in particular to cordyceps sinensis mycelium rich in ergothioneine and a preparation process and application thereof. BACKGROUND

[0002] Ergothioneine (EGT) is a rare amino acid derived from histidine, which has stable chemical properties. Ergothioneine has physiological effects such as antioxidant, free radical scavenging, anti-aging, and skin beautifying, and is increasingly attracting attention, being continuously researched and developed, and showing great application value. Some scholars have found that natural ergothioneine mainly exists in fungi and cereal plants, and edible fungi are the richest biological source. Cordyceps sinensis (Berk.) Sacc., also known as Cordyceps, is a parasitic fungus. The Catalogue of Cosmetics Ingredients (2021 Edition) (No. 62 of the State Drug Administration in 2021) includes two raw material categories of Cordyceps sinensis, including Cordyceps sinensis (CORDYCEPS SINENSIS) extract and Cordyceps sinensis (CORDYCEPS SINENSIS) mycelium powder. The former is mainly extracted from Cordyceps sinensis medicinal materials. Due to its strict parasitic nature and special growth environment requirements, the wild resources of Cordyceps sinensis are very limited, so the extract is extremely expensive and its production and use are severely limited. The latter mycelium powder is obtained through liquid submerged fermentation, one of modern bioengineering technologies. The fermentation process of Cordyceps sinensis (anamorphic form of Hirsutella sinensis) mycelium has bottlenecks such as very slow growth, harsh temperature, low yield, long cycle, high energy consumption, and low efficiency, which hinder the development and application of Cordyceps sinensis mycelium. In addition, the EGT content in Cordyceps sinensis mycelium is not as high as that in other fungi, and the biological activities such as antioxidant activity are not outstanding enough, so Cordyceps sinensis has not shown potential value in the application field permitted by regulations. The ergothioneine content in common edible fungi is as follows: Hericium erinaceus 0.53-1.12 mg / g, Tricholoma matsutake 0.74-2.56 mg / g, Ganoderma lucidum 0.06-0.56 mg / g, and Boletus 0.52-7.27 mg / g (Yang Linlei, Shen Zhenhui, Luo Xiangying, et al. Research Progress of Edible Fungus Ergothioneine [J]. Bioengineering Bulletin, 2025, 41(02): 574-587. DOI:10.13345 / j.cjb.240495). Chinese patent CN 118766973 A discloses a Cordyceps sinensis fermentation composition containing ergothioneine, and the actual EGT content in different embodiments is in the range of 40 mg / kg-310 mg / kg, which is still at the level under conventional process conditions. SUMMARY

[0003] Therefore, the present application aims to further study the fermentation process of Cordyceps sinensis mycelium, so as to obtain Cordyceps sinensis mycelium rich in ergothioneine and explore its application value in skin function. Further, the present application provides Cordyceps sinensis mycelium rich in ergothioneine, a preparation process thereof and an application thereof.

[0004] To achieve the above technical purposes, the technical scheme adopted by the present application is as follows:

[0005] In a first aspect, the present application provides Cordyceps sinensis mycelium rich in ergothioneine, wherein the content of ergothioneine in the Cordyceps sinensis mycelium is not less than 350 mg / kg in dry weight.

[0006] Preferably, the Cordyceps sinensis mycelium is obtained by adding at least one of the following three amino acids: Met, Cys and His during the fermentation culture of mycelium. Further preferably, the amino acid is added after 5-7 days of fermentation culture of mycelium; more preferably, the addition amount of the amino acid is 0.1-2.0 g / L in final concentration.

[0007] More preferably, the amino acid is Met and / or His, and the addition amount is 0.75-1.5 g / L in final concentration; or the amino acid is Cys, and the addition amount is 0.1-0.5 g / L in final concentration.

[0008] In a second aspect, the present application provides a preparation method of the Cordyceps sinensis mycelium of the first aspect, comprising the following steps:

[0009] Step 1, activating culture and rejuvenation culture of Cordyceps sinensis;

[0010] Step 2, seed liquid culture of the rejuvenation cultured strain;

[0011] Step 3, fermentation culture of the Cordyceps sinensis seed liquid during mycelium culture, and adding at least one of the following three amino acids: Met, Cys and His during the mycelium culture and fermentation culture to obtain the Cordyceps sinensis mycelium.

[0012] In a third aspect, the present application provides another preparation method of the Cordyceps sinensis mycelium of the first aspect, comprising the following steps:

[0013] Step 1, activating culture and rejuvenation culture of Cordyceps sinensis;

[0014] Step 2, seed liquid culture of the rejuvenation cultured strain;

[0015] Step 3, mycelium basic culture of the Cordyceps sinensis seed liquid;

[0016] Step 4), the culture obtained by mycelium culture is subjected to fermentation culture, and at least one of the following three amino acids, Met, Cys and His, is added during the fermentation culture to obtain the Cordyceps sinensis mycelium.

[0017] Preferably, the preparation method further comprises: drying or freeze-drying the Cordyceps sinensis mycelium, and crushing to obtain Cordyceps sinensis mycelium powder.

[0018] Preferably, in the step 1 or step 1), the activation culture temperature is 16-20℃, and further preferably 18±1℃; the time is 1-5 days, and further preferably 3-4 days; and / or, the rejuvenation culture temperature is 16-20℃, and further preferably 18±1℃; the time is 10-30 days, and further preferably 16-24 days.

[0019] Preferably, in the step 2 or step 2), the seed liquid culture temperature is 16-20℃, and preferably 18±1℃, and the time is 16-24 days; or, in the step 3), the mycelium culture and fermentation culture temperature is 16-20℃, and preferably 18±1℃, and the time is 15-24 days; or, in the step 3), the mycelium culture temperature is 16-20℃, and preferably 18±1℃, and the time is 5-7 days; or, in the step 4), the fermentation culture temperature is 16-20℃, and preferably 18±1℃, and the time is 8-12 days.

[0020] Further preferably, in the seed liquid culture, the composition of the seed liquid culture medium is: glucose 20-30g / L, yeast extract 3-7g / L, peptone 1-3g / L, inorganic salt in proper amount, and water as solvent.

[0021] Preferably, in the culture system of the step 3 or the step 4), the addition amount of the amino acid is 0.1-2.0g / L in terms of final concentration; further preferably, the addition amount of Met and / or His is 0.5-1.5g / L in terms of final concentration, and / or the addition amount of Cys is 0.1-0.5g / L in terms of final concentration.

[0022] Further preferably, in the step 3 or the step 3) or the step 4), the composition of the basic culture medium is: glucose 40-50g / L, yeast extract 8-12g / L, peptone 3-7g / L, inorganic salt in proper amount, and water as solvent.

[0023] In a fourth aspect, the present application provides the use of the Cordyceps sinensis mycelium of the first aspect or the Cordyceps sinensis mycelium obtained by the preparation method of the second aspect or the third aspect in the preparation of a skin external composition.

[0024] In a fifth aspect, the present application provides a skin external use composition comprising the Cordyceps sinensis mycelium of the first aspect or the Cordyceps sinensis mycelium obtained by the preparation method of the second or third aspect.

[0025] In a sixth aspect, the present application provides at least one skin product selected from the group consisting of 1) to 7) below, comprising the skin external use composition of the fifth aspect:

[0026] 1) a skin product for repairing damaged cells;

[0027] 2) a skin product for eliminating inflammation;

[0028] 3) a skin product for acne removal;

[0029] 4) a skin product for bacteria removal;

[0030] 5) a skin product for oil control;

[0031] 6) a skin product for moisturizing;

[0032] 7) a skin product for anti-aging and wrinkle removal;

[0033] 8) a skin product for anti-oxidation.

[0034] The skin product can optionally further comprise, but is not limited to, various adjuvants such as surfactants, diluents, emulsifiers, thickening agents, dispersants, etc. The surfactants are, for example, cetyl stearyl olivate, sorbitan olivate, polysorbate-60, polysorbate-80, methyl glucose sesquistearate, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-butyl polyether-26, PEG-4 polyglyceryl-2 stearate, etc. The diluents are, for example, glycerol, dipropylene glycol, butylene glycol, etc. The emulsifiers are, for example, one or more of polyglyceryl-3 methyl glucose distearate, glyceryl stearate citrate, polyglyceryl-10 stearate, polyglyceryl-10 myristate, polyglyceryl-10 dioleate, polyglyceryl-10 laurate, polyglyceryl-10 isostearate, polyglyceryl-10 oleate, polyglyceryl-10 diisostearate, polyglyceryl-6 laurate, polyglyceryl-6 myristate, sucrose stearate, sucrose polystearate, etc. The thickening agents are, for example, one or more of carbomers, acrylates and their derivatives, xanthan gum, gum arabic, polyethylene glycol-14M, polyethylene glycol-90M, succinoglucan, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc. The dispersants are, for example, gelatin, pectin, starch, polyvinyl alcohol, polyacrylic acid, etc.

[0035] The skin product can also optionally contain other active ingredients such as one or more of tocopherol (vitamin E), retinol, retinol palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydrocurcumin, ellagic acid, ubiquinone, lactoprotein, polypeptide, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicyloyl phytosphingosine, concentrated birch sap, silymarin, silk fibroin protein, sodium tocopheryl phosphate, ribonucleic acid (RNA), dipeptide diaminobutyroyl hydrazide benzylamide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptide, palmitoyl tetrapeptide-7, VITIS VINIFERA (GRAPE) SEED EXTRACT, PTEROCARPUS MARSUPIUM (KINO) BARK EXTRACT, CAMELLIA SINENSIS (GREEN TEA) POLYPHENOLS, WINE EXTRACT, APPLE SEED EXTRACT, FAGUS SYLVATICA (BEECH) BUD EXTRACT, HYDROLYZED ADANSONIA DIGITATA (BAOBAB) EXTRACT, ARTEMIA EXTRACT, IRIS FLORENTINA (ORCHID) ROOT EXTRACT, HEPANTRINE, GINSENOSIDE, SALVIA MILTIORRHIZA (DANSHEN) EXTRACT, NICOTINAMIDE, URSOLIC ACID, SODIUM HYALURONATE, SODIUM ACETYLATED HYALURONATE, SODIUM HYDROLYZED HYALURONATE, LYCOPENE, COFFEA ARABICA (COFFEE) EXTRACT, DIPEPTIDE-2, LACTIC ACID, SUPER OXIDE DISMUTASE (SOD), OENOTHERA BIENNIS (EVENING PRIMROSE) OIL, CERAMIDE, DIPALMITOYL HYDROXYPROLINE, HYDROXYSTEARIC ACID, SALICYLIC ACID, ERGOTHIONE, LYSOLECTIN, CARNOSINE, DECARBOXYLASE CARNOSINE HCL, THIOCTIC ACID, ADENOSINE, GLYCOGEN, RESVERATROL, FERULIC ACID, SCHIZACHYRYUM MOUNTAIN FERMENT, LACTOBACILLUS FERMENT, etc.

[0036] The dry powder of cordyceps sinensis mycelium enriched with ergothioneine or its extract according to the present application can be mixed with other pharmaceutical ingredients or cosmetic ingredients to obtain a pharmaceutical composition or a cosmetic composition according to any method known in the skin product industry. The other pharmaceutical ingredients or cosmetic ingredients are the same as those commonly used in skin products as described above.

[0037] In addition, the skin product can be prepared into various dosage forms such as solution, suspension, paste, cream, emulsion, gel, powder or spray, etc. according to the need.

[0038] Compared with the prior art, the present application has the following beneficial effects:

[0039] Firstly, the ergothioneine-rich cordyceps sinensis mycelium preparation process of the present application, the cordyceps sinensis mycelium enters the logarithmic growth phase 4-5 days after inoculation in the fermentation culture process, and the fermentation period is shortened from the conventional 22-24 days to 15-17 days, that is, the whole fermentation period is shortened by more than 5 days compared with the conventional process, and the production efficiency is significantly improved, which creates favorable conditions for large-scale production. And the ergothioneine content of the cordyceps sinensis mycelium obtained is significantly higher than that obtained by the conventional process of the 5L fermenter and the process of directly adding Met for fermentation.

[0040] Secondly, the cordyceps sinensis mycelium of the present application is rich in ergothioneine, and the content is significantly higher than that obtained by the conventional process of the 5L fermenter and the process of directly adding Met or not adding Met for fermentation. Experimental results show that the cordyceps sinensis mycelium of the present application has outstanding biological activity, excellent cell repair, antioxidant, free radical scavenging, anti-aging, beauty and skin care, and other series of physiological effects, and has broad application prospects in the cosmetics, food and biological medicine industries. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 It is a preparation process schematic diagram of the ergothioneine-rich cordyceps sinensis mycelium in the scale-up process of the present application 6.

[0042] Figure 2 It is the cell scratch repair test results of the ergothioneine-rich cordyceps sinensis mycelium in the application example 1 of the present application, wherein A~C are respectively the test results of the comparative example 3, the example 6 and the positive control.

[0043] Figure 3 It is the antioxidant test results of the ergothioneine-rich cordyceps sinensis mycelium in the application example 1 of the present application.

[0044] Figure 4 It is the anti-wrinkle and firming test results of the ergothioneine-rich cordyceps sinensis mycelium in the application example 1 of the present application. DETAILED DESCRIPTION

[0045] In the example of the present application, the EGT in the obtained cordyceps sinensis mycelium is detected according to NY T 3872-2021 Determination of L-ergothioneine in Edible Fungi by Ultra Performance Liquid Chromatography.

[0046] In the example of the present application, the cordyceps sinensis strain used is Chinese hirsutella, and the CGMCC number is 3.14240. Before use, it is activated and rejuvenated first, then seed liquid culture is carried out, followed by mycelium basic culture and fermentation culture at the same time, and at least one of the following three kinds of amino acids is added during the culture process: methionine (Met), cysteine (Cys), histidine (His). The three kinds of amino acids can be added alone or in combination of two or even three, as described in the example.

[0047] In the embodiment of the present application, the fermentation tank preparation method of the Ophiocordyceps sinensis mycelium with industrial prospect comprises the following steps:

[0048] Step 1), the Ophiocordyceps sinensis strain is activated and rejuvenated. The low-temperature preserved Ophiocordyceps sinensis strain is cultured at 16-20℃ for 1-5 days, preferably at 18±1℃ for 3-4 days, so as to restore normal activity. Then it is transferred to PDA plate culture medium for rejuvenation culture for 10-30 days, preferably 16-24 days, and grows into a colony with a diameter of 1-2 cm.

[0049] Step 2), the rejuvenated Ophiocordyceps sinensis strain is seed liquid cultured. The specific process is that the plate colony is cut into small pieces and inoculated into a seed culture medium and shaken at 16-20℃ for 16-24 days. The seed culture medium is composed of glucose, yeast extract, peptone, inorganic salt, etc.

[0050] Step 3), the seed liquid is enlarged in volume for mycelium basic culture. The specific process is that the cultured seed liquid is inoculated into a fermentation tank containing a basic culture medium at a certain ratio, and pre-fermented for 5-7 days, so that the mycelium enters the logarithmic growth phase. This process is the mycelium culture stage.

[0051] Step 5) EGT synthesis of mycelium. A sterile amino acid solution is added to the fermentation tank mycelium culture by aseptic means, and the fermentation is continued for 8-12 days, and the EGT-rich Ophiocordyceps sinensis mycelium is collected;

[0052] Step 6), the Ophiocordyceps sinensis mycelium is dried.

[0053] In the above process, before the fermentation culture of the Ophiocordyceps sinensis mycelium for EGT synthesis, the mycelium basic culture is carried out. The purpose of the basic culture is to make the Ophiocordyceps sinensis strain enter the logarithmic growth phase. The basic culture can make the Ophiocordyceps sinensis mycelium quickly adapt to the nutritional environment, enter the logarithmic growth state, and obtain a certain biomass. In order to avoid the interference of exogenous amino acid to the early growth of mycelium, which leads to the influence of mycelium biomass. This process is the conventional process of Ophiocordyceps sinensis culture. Under this condition, the early growth of Ophiocordyceps sinensis mycelium is not affected by the exogenous addition of amino acid, and it grows stably to form a certain mycelium biomass, which is beneficial to the addition of Met in the later stage to promote the synthesis of EGT by Ophiocordyceps sinensis mycelium.

[0054] In the above process, before the mycelium basic culture of the Ophiocordyceps sinensis strain, the Ophiocordyceps sinensis strain is seed liquid cultured, and then the culture obtained by the seed liquid culture is basic cultured. The purpose of the seed liquid culture is to amplify the culture of the Ophiocordyceps sinensis strain, and the amplification culture is carried out in a larger volume, such as in a 5L fermentation tank. The amplification culture mode is not limited to the aforementioned mode.

[0055] In the above process, before seed liquid culture of the Ophiocordyceps sinensis, the Ophiocordyceps sinensis is activated and / or rejuvenated, and then the culture obtained by the activation and / or rejuvenation is subjected to seed liquid culture. The purpose of the activation and / or rejuvenation is to restore the vitality of the Ophiocordyceps sinensis.

[0056] Further, in the description of the present application, it should be noted that, in the examples, the specific conditions not indicated are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not indicated by the manufacturers are the conventional products that can be obtained by commercial purchase.

[0057] Unless otherwise defined, all the professional terms used herein have the same meanings as generally understood by the person skilled in the art. The professional terms used herein are only for the purpose of describing the specific examples, and are not intended to limit the protection scope of the present application.

[0058] In order to facilitate the understanding of the present application, the present application will be described in more detail from the small-scale preparation of the Ophiocordyceps sinensis mycelium rich in ergothioneine to the scale-up in combination with the accompanying drawings and examples, but the protection scope of the present application is not limited to the following specific examples.

[0059] Example 1

[0060] The present example provides a small-scale example of the preparation of the Ophiocordyceps sinensis mycelium rich in ergothioneine, and the specific process is as follows:

[0061] Step 1, activation and rejuvenation of the Ophiocordyceps sinensis strain: the low-temperature preserved Ophiocordyceps sinensis slant is cultured at 18℃ for 3 days to restore normal vitality; then the strain is transferred to PDA flat plate culture medium for rejuvenation culture for 20 days, and grows into a colony with a diameter of about 1.5 cm.

[0062] Step 2, seed liquid culture of the rejuvenated Ophiocordyceps sinensis strain: the flat plate colony is cut into small pieces and inoculated into a 250 mL triangular flask containing 75 mL of seed culture medium, and is subjected to 18℃ shaking culture for 20 days to prepare the Ophiocordyceps sinensis seed liquid. The seed culture medium formula is glucose 30 g / L, yeast extract 5 g / L, peptone 2 g / L, and inorganic salt in an appropriate amount, and the solvent is water.

[0063] Step 3, mycelium culture and fermentation culture of O. sinensis seed liquid: 200 mL of basic medium was put into 1 L flask, sterilized and cooled. The formula of the basic medium was glucose 50 g / L, yeast extract 10 g / L, peptone 5 g / L, and inorganic salts in proper amount, and the solvent was water. Then Met was dissolved in purified water to prepare a solution with a concentration of 48 mg / mL, filtered and sterilized by a 0.22 μm filter, and then added to the above basic medium and mixed uniformly. Two test groups were set, one with Met added at 0.75 g / L (final concentration, the same below), and the other with Met added at 1.5 g / L. The cultured O. sinensis seed liquid was inoculated into 1 L flask at a ratio of 10%, and cultured at 18°C for 24 days. The mycelium was spherical, grew well, and had a diameter of 2-3 mm and high density. Two parallel repeats were set for each test group. The obtained O. sinensis mycelium was dried, and the yield of mycelium was 7-9 g / L. HPLC detection showed that the ergothioneine content in the O. sinensis mycelium obtained in the test group with Met added at 0.75 g / L was 637 mg / kg on average, and the ergothioneine content in the O. sinensis mycelium obtained in the test group with Met added at 1.5 g / L was 545 mg / kg on average. A lower amount of Met added resulted in a better ergothioneine content.

[0064] Example 2

[0065] The difference between this example and Example 1 is that His was added to the basic medium, and the others were the same as in Example 1. At the end of the culture, the culture solution was black-brown, the mycelium was spherical, grew well, and had a diameter of 2-3 mm and high density. Two parallel repeats were set for each test group. The obtained O. sinensis mycelium was dried, and the yield of mycelium was 10-12 g / L. HPLC detection showed that the ergothioneine content in the O. sinensis mycelium obtained in the test group with His added at 0.75 g / L was 136 mg / kg on average, and the ergothioneine content in the O. sinensis mycelium obtained in the test group with His added at 1.5 g / L was 118 mg / kg on average. This shows that the effect of His addition is far less than that of Met.

[0066] Example 3

[0067] The difference between this example and Example 1 is that Cys was added to the basic medium, and the others were the same as in Example 1. At the end of the culture, the O. sinensis mycelium did not grow obviously compared with the initial inoculation, and only a small amount of original mycelial balls remained. This example did not obtain sufficient dry weight of O. sinensis mycelium. This shows that the addition of Cys in the basic medium at a concentration exceeding 0.75 g / L is not conducive to the growth of O. sinensis mycelium.

[0068] Cys at the amount of 0.75 and 1.5 g / L, the combination of Example 1 and Example 2 amino acid addition amount is low, ergothioneine content is high, further reduce the amount of Cys added to 0.25 g / L, mycelium culture and fermentation culture for 26 days, the other same as Example 1 operation. At the end of the culture, the culture solution is black-brown, the mycelium is spherical and the density is low. Each test set 2 parallel repeats. The obtained cordyceps mycelium is dried to obtain the mycelium yield of 5-6 g / L. The obtained cordyceps mycelium is detected by HPLC to obtain the average content of ergothioneine in the cordyceps mycelium of 576 mg / kg. Although the addition amount under this condition, Cys can also improve the content of ergothioneine in cordyceps mycelium, and the effect is close to Met, but the biomass is obviously low, which is not an economic and efficient way.

[0069] Example 4:

[0070] The difference between this example and Example 1 is that Met and His are added to the basic medium at the same time, and the addition amount of Met and His is 0.75 g / L. After adding, the mycelium culture and fermentation culture time is 26 days, and the other is the same as Example 1. Each test group is set 2 parallel repeats. The obtained cordyceps mycelium is dried to obtain the mycelium yield of 4-6 g / L. The average content of ergothioneine in the cordyceps mycelium detected by HPLC is 410 mg / kg. Similarly, the combination of Met and His can also improve the content of ergothioneine in cordyceps mycelium, but the effect is much worse than Met.

[0071] Example 5:

[0072] The difference between this example and Example 1 is that Met, His and Cys are added during fermentation culture, and the addition amount of Met, His and Cys is 0.75 g / L, 0.75 g / L and 0.25 g / L respectively. After adding, the mycelium culture and fermentation culture time is 26 h, and the other is the same as Example 1. Each test group is set 2 parallel repeats. The obtained cordyceps mycelium is dried to obtain the mycelium yield of 4-5 g / L. The average content of ergothioneine in the cordyceps mycelium detected by HPLC is 528 mg / kg, which is significantly lower than Met.

[0073] Comparative Example 1

[0074] This comparative example provides a conventional fermentation method of cordyceps mycelium, and the specific operation is as follows:

[0075] Step 1: Activate and rejuvenate the Cordyceps sinensis strain. Inoculate the low-temperature preserved Cordyceps sinensis slant at 18℃ for 3 days to restore its normal activity. Then, transfer the strain to PDA agar plates and rejuvenate for 20 days, until colonies approximately 1.5 cm in diameter are formed.

[0076] Step 2 involves seed culture of the revitalized Cordyceps sinensis. The specific process is as follows: The plate colonies are cut into small pieces and inoculated into a 250mL Erlenmeyer flask containing 75mL of seed culture medium. The flask is then incubated at 18°C ​​with shaking for 15-20 days to prepare the Cordyceps sinensis seed culture. The seed culture medium formula is the same as in Example 1.

[0077] Step 3: Next, 200 mL of basal culture medium will be added to a 1 L Erlenmeyer flask, sterilized, and then cooled. The basal culture medium formula is the same as in Example 1. The cultured Cordyceps sinensis seed solution will be inoculated into the 1 L Erlenmeyer flask at a ratio of 10%, and cultured at 18°C ​​with shaking for 20-30 days. At the end of the culture, the culture solution will be dark brown, the mycelium will be spherical, showing good growth, with a diameter of 2-3 mm and high density. The obtained Cordyceps sinensis mycelium will be dried, yielding a mycelium yield of 7-9 g / L. Multiple batches of this comparative example, analyzed by HPLC, showed that the ergothioneine content in the Cordyceps sinensis mycelium was 130-200 mg / kg.

[0078] Example 6

[0079] This embodiment provides an example of preparing Cordyceps sinensis mycelium rich in ergothioneine, the process of which is as follows: Figure 1 The diagram shows the scale-up process of the fermenter, and the specific steps are as follows:

[0080] Step 1: Activate and rejuvenate the Cordyceps sinensis strain: Inoculate the low-temperature preserved Cordyceps sinensis slant at 18℃ for 4 days to restore its normal activity; then transfer the strain to PDA plate medium and rejuvenate it for 20 days to grow into colonies with a diameter of about 1.5cm.

[0081] Step 2: Seed culture is then performed: The revitalized Cordyceps sinensis strain is cultured in seed culture. Specifically, the plate colonies are cut into small pieces and inoculated into a 250mL Erlenmeyer flask containing 75mL of seed culture medium. The flask is then incubated at 18°C ​​with shaking for 20 days to prepare the Cordyceps sinensis seed culture. The seed culture medium formula is the same as in Example 1.

[0082] Step 3, proceed with mycelial culture (i.e., basic culture): Increase the volume of the seed culture for basic mycelial culture. The basic culture medium formula is the same as in Example 1. Specifically, the cultured seed culture is inoculated into a glass fermenter containing basic culture medium at a ratio of 10%. After pre-fermentation for 5 days, the mycelium enters the logarithmic growth phase. This process is the mycelial culture stage.

[0083] Step 4, then mycelium EGT fermentation culture: Met was dissolved in purified water to prepare a solution with a concentration of 48 mg / mL, filtered with a 0.22 μm filter to remove bacteria; then added to the fermentation tank which had completed the mycelium basic culture, the amount of Met added was 0.75 g / L, continued to ferment for 10 days, and collected Cordyceps mycelium. Low temperature drying or freeze drying. Parallel to carry out 3 batches of scale-up experiments, the dry weight yield of mycelium in each liter of fermentation broth was 10.4 g / L, 10.5 g / L, and 11.1 g / L, respectively, and the EGT content was 355 mg / kg, 450 mg / kg, and 386 mg / kg, respectively.

[0084] Comparative Example 2

[0085] The difference between this comparative example and Example 6 is that there is no basic culture process, and the fermentation culture process is as follows: the basic culture medium is loaded into a glass fermentation tank and autoclaved at high temperature. Met is dissolved in purified water to prepare a solution with a concentration of 48 mg / mL, filtered to remove bacteria, and then added directly to the fermentation tank by sterile means, with an addition concentration of 0.75 g / L, and then the prepared Cordyceps seed solution is inoculated. In the early stage of culture, the mycelium grows slowly; after 12-14 days, the growth accelerates. On the 20th day, the mycelium is collected from the tank, washed, and dried or freeze-dried. The calculated dry weight yield of mycelium per liter of fermentation broth is 4.2 g / L, and the EGT content is 210 mg / kg.

[0086] Comparative Example 3

[0087] This comparative example provides a conventional fermentation method for Cordyceps mycelium, which is a fermentation tank scale-up process, and the specific operation is as follows:

[0088] Step 1, activation and rejuvenation of Cordyceps strain. The low-temperature preserved Cordyceps slant was cultured at 18°C for 5 days to restore normal activity. Then the strain was transferred to PDA plate culture medium and cultured for 20 days to grow into colonies with a diameter of about 1.5 cm.

[0089] Step 2, then seed liquid culture: the rejuvenated Cordyceps strain was cultured in seed liquid. The specific process is to cut the plate colonies into small pieces and inoculate into a 250 mL triangular flask containing 75 mL of seed culture medium and shake culture at 18°C for 22 days to prepare the seed liquid of Cordyceps strain. The seed culture medium formula is the same as that of Example 1.

[0090] Step 3, mycelium regular cultivation: the specific process is that the seed liquid is inoculated into the fermentation tank containing the basic medium (the formula is the same as that in Example 1) at a proportion of 10%, and the mycelium is cultured at 18°C for 24 days, and the mycelium is collected and dried at low temperature. In this process, the mycelium naturally synthesizes EGT using the nutritional components of the basic medium. Three batches of experiments are carried out in parallel, and the average yield of mycelium dry weight in each liter of fermentation liquid is 8.3 g / L. The average content of EGT in the mycelium is 160 mg / kg.

[0091] From the results of Examples 1-6 and Comparative Examples 1-3, it can be seen that using the method of the present embodiment, the cordyceps mycelium enters the logarithmic growth phase 4-5 days after inoculation in the fermentation culture process, and the fermentation period is shortened from the conventional 22-24 days to 15-17 days. The total fermentation period is shortened by more than 5 days compared with the conventional process, and the production efficiency is significantly improved, creating favorable conditions for large-scale production. And the ergothioneine content in the cordyceps mycelium obtained is significantly higher than that obtained by the conventional process of 5L fermentation tank and the process of directly adding Met for fermentation.

[0092] Application Example 1

[0093] The cordyceps mycelium with an ergothioneine content of 450 mg / kg obtained in Example 6 was subjected to efficacy evaluation test, as follows:

[0094] (1) Cell scratch test

[0095] A scratch test model was established using commercially available human immortalized keratinocytes HaCaT. HaCaT cells were taken from a T25 bottle of HaCaT cells cultured to 80-90% confluence and in good condition, the old culture medium was removed, washed once with 1xPBS, 1 mL of 0.25% trypsin was added to the culture bottle and placed in the incubator for 7-8 min, then 3 mL of DMEM complete medium (containing 10% FBS) was added to terminate digestion, and the cell suspension was prepared by repeated blowing and beating, centrifuged at 200xg for 5 min, resuspended and counted to adjust the cell density to 3x10 6 cells / mL; the scratch insert (source: Kujian Biological, item number: CR211-2) was inserted into the 24-well plate, and the cell suspension was inoculated into the insert hole at 100 μL per well (i.e. 3x10 5HaCaT cells were seeded in 6-well plates at a density of 1 x 105 cells per well and incubated at 37°C in a 5% CO2 incubator for 24 h. The inserts were removed and the wells were gently washed with PBS. 1 mL of the extract of O. sinensis mycelium diluted in DMEM base medium was added to each well. The concentration range was 0%-0.5%. The positive control group was added with 10% FBS (fetal bovine serum) in DMEM medium. Two duplicate wells were set up for each group. Photographs were taken under a 10x microscope and recorded as 0 h. After 24 h of continuous culture, photographs were taken under the same conditions. The area of the scratch region was measured using Image J software. The cell migration rate was calculated according to the formula "cell migration rate = [(0 h scratch area-24 h scratch area) / 0 h scratch area] x 100%". The results are shown in Table 1 and Figure 2 As shown in Table 1, the EGT-rich O. sinensis mycelium sample had a migration rate of 45.99% at a concentration of 0.1% in the cell scratch test, which was significantly higher than the migration rate of 22.63% of the conventional sample (i.e., O. sinensis mycelium fermented in a medium without Met) at the same concentration.

[0096] Table 1 Migration rate data of O. sinensis mycelium obtained by the conventional process and the process of the present application

[0097]

[0098] (2) Antioxidant experiment

[0099] Further, DPPH antioxidant experiment. DPPH is a relatively stable organic free radical and is currently widely used as an evaluation of in vitro antioxidant activity. When dissolved in ethanol, it presents a purple color with the strongest absorption at 517 nm. When a free radical scavenger is present, the single electron of DPPH is paired, causing the solution color to become lighter and the absorbance to decrease. The degree of decrease is quantitatively related to the degree of DPPH scavenging.

[0100] Specific operation: 12 mg of 1,1-diphenyl-2-trinitrophenylhydrazine (CAS No. 1898-66-4; source: Aldrich, purity 97%) was added to 100 mL of 95% ethanol and stirred until dissolved to prepare a 0.12 mg / mL DPPH working solution.

[0101] Take dried Cordyceps sinensis mycelium powder from Examples 6 and 3 at a material-to-liquid ratio of 1:20, add purified water, and sonicate (400W, 40kHz) for 60 minutes, repeatedly shaking and mixing during the process. Centrifuge at 4000rpm for 20 minutes, and use the supernatant as the stock solution of the test sample at 25mg / mL. Dilute it with ultrapure water to a series of concentration gradients of 1.0mg / mL, 2.0mg / mL, and 3.0mg / mL.

[0102] Weigh 0.05 g of L-ascorbic acid powder (CAS No. 50-81-7, source: Aladdin, purity 99%), dissolve in 10 mL of purified water to obtain a 5 mg / mL vitamin C solution. Dilute the vitamin C solution to a series of concentration gradients: 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL.

[0103] The reaction sample additions are shown in Table 2:

[0104] Table 2 Reaction Sampling Details

[0105] Reagent name (μL) Blank tube Sample tube Sample background Positive control Sample solution 0 100 100 0 Vc solution 0 0 0 100 Purified water 100 0 100 0 DPPH solution 100 100 0 100

[0106] The above sample loading method was applied to a 96-well plate (repeated three times). After standing at room temperature in the dark for 40 min, the absorbance was measured at a wavelength of 517 nm. The wells were labeled A (blank tube), S (sample tube), S0 (sample background), and A0 (positive control). The clearance rate R was calculated using the formula: R = [1 - (S - S0) / A0] × 100%.

[0107] like Figure 3 As shown, when the sample concentration was 1 mg / mL, the EGT-rich Cordyceps sinensis mycelium extract exhibited a DPPH scavenging rate of up to 33.10%, significantly higher than the scavenging rate of 13.95% for samples processed under conventional conditions. When the sample concentration was 2 mg / mL, the EGT-rich Cordyceps sinensis mycelium extract exhibited a DPPH scavenging rate of up to 55.67%, significantly higher than the scavenging rate of 28.61% for samples processed under conventional conditions. When the sample concentration was 3 mg / mL, the EGT-rich Cordyceps sinensis mycelium extract exhibited a DPPH scavenging rate of up to 70.74%, significantly higher than the scavenging rate of 42.20% for samples processed under conventional conditions. This indicates that the Cordyceps sinensis mycelium of the present invention possesses superior antioxidant activity.

[0108] (3) Anti-wrinkle and firming test

[0109] Human elastase has the ability to degrade collagen and elastin. Skin aging is closely related to the degradation activity of elastase. The anti-wrinkle and firming effects of a sample can be evaluated by testing the inhibition rate of elastase by Cordyceps sinensis mycelium extract. A higher inhibition rate indicates a stronger anti-wrinkle effect.

[0110] Specific operation: Take the dry Cordyceps sinensis mycelium powder of Example 6 and Comparative Example 3 in a centrifuge tube, add purified water at a material-liquid ratio of 1:20, vortex uniformly, and then place in a 60℃ water bath for 2h. Take out, centrifuge at 4000rpm for 10min, take the supernatant and pass through a 0.22μm filter membrane. Dilute the filtrate with purified water to gradient solutions of 0.13mg / mL, 0.25mg / mL and 0.5mg / mL.

[0111] Add buffer solution, different concentrations of sample dilutions and substrate AAAPNA (from Aladdin) in a 96-well plate, shake and mix uniformly, and then incubate at 25℃ for 20min. Then add elastase (from Shanghai Yuanye) solution, immediately shake and mix uniformly and detect OD 400nm , incubate for 10min, then detect OD 400nm again, and obtain the degree of influence of the sample on elastase activity.

[0112] Inhibition rate (%) = (1-V 样 / V0) x 100, wherein V 样 is the enzyme activity rate of the test group (with sample added); and V0 is the reaction rate of the negative control group (100% activity).

[0113] The results are shown in Table Figure 4 In the elastase inhibition test reaction system, the effect of the Cordyceps sinensis mycelium rich in EGT of the application is obviously better than that of the Cordyceps sinensis mycelium obtained under conventional process conditions, and when the sample concentration is 0.5mg / mL, the inhibition rate of the elastase activity of the Cordyceps sinensis mycelium extract sample rich in EGT reaches 50.6%, and the inhibition rate of the sample under conventional process conditions is 38.2%, and the difference in anti-aging between the two is the largest. This also shows that within a certain concentration range, increasing the dosage of the Cordyceps sinensis mycelium of the application can significantly improve the anti-wrinkle effect.

[0114] In summary, ergothioneine has physiological effects such as antioxidant, free radical scavenging, anti-aging, beauty and skin care, and in combination with the experimental results of the application, the Cordyceps sinensis mycelium rich in ergothioneine is expected to be developed into the following skin products and popularized and applied: 1) skin products for repairing damaged cells; 2) skin products for eliminating inflammation; 3) skin products for acne removal; 4) skin products for bacteria removal; 5) skin products for oil control; 6) skin products for moisturizing; 7) skin products for anti-aging and wrinkle removal; and 8) skin products for antioxidant.

[0115] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Cordyceps sinensis mycelium rich in ergothioneine, characterized in that, The ergothioneine content in the Cordyceps sinensis mycelium is not less than 350 mg / kg on a dry weight basis.

2. The Cordyceps sinensis mycelium according to claim 1, characterized in that, The Cordyceps sinensis mycelium is obtained by adding at least one of the following three amino acids during the mycelium fermentation culture process: Met, Cys, and His.

3. A method for preparing Cordyceps sinensis mycelium according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Activate and rejuvenate the Cordyceps sinensis fungus. Step 2: Culture the rejuvenated strain in seed culture. Step 3: The Cordyceps sinensis seed liquid is cultured as mycelium; at the same time, fermentation culture is carried out. During the mycelium culture and fermentation culture, at least one of the following three amino acids is added: Met, Cys, and His, to obtain the Cordyceps sinensis mycelium.

4. Another method for preparing Cordyceps sinensis mycelium according to claim 1 or 2, characterized in that, Includes the following steps: Step 1) Activate and rejuvenate the Cordyceps sinensis fungus; Step 2), the rejuvenated strain is cultured in seed culture; Step 3) Further culture the mycelium of the Cordyceps sinensis seed liquid; Step 4) The culture obtained from mycelial culture is fermented. During the fermentation process, at least one of the following three amino acids is added: Met, Cys, and His to obtain the Cordyceps sinensis mycelium.

5. The preparation method according to claim 3 or 4, characterized in that, Also includes: The Cordyceps sinensis mycelium is dried or freeze-dried, and then pulverized to obtain Cordyceps sinensis mycelium powder.

6. The preparation method according to any one of claims 3 to 5, characterized in that, In step 1 or step 1), the activation culture temperature is 16-20℃, preferably 18±1℃; the time is 1-5 days, preferably 3-4 days; and / or, the rejuvenation culture temperature is 16-20℃, preferably 18±1℃; the time is 10-30 days, preferably 16-24 days.

7. The preparation method according to claim 6, characterized in that, In step 2 or step 2), the seed culture temperature is 16–20℃, preferably 18±1℃, and the time is 16–24 days; or, in step 3, the mycelial culture and fermentation culture temperature is 16–20℃, preferably 18±1℃, and the time is 15–24 days; or, in step 3), the mycelial culture temperature is 16–20℃, preferably 18±1℃, and the time is 5–7 days; or, in step 4), the fermentation culture temperature is 16–20℃, preferably 18±1℃, and the time is 8–12 days.

8. The application of the Cordyceps sinensis mycelium according to claim 1 or 2, or the Cordyceps sinensis mycelium obtained by the preparation method according to any one of claims 3 to 7, in the preparation of topical skin compositions.

9. A topical skin composition, characterized in that, include: The Cordyceps mycelium as described in claim 1 or 2, or the Cordyceps mycelium obtained by the preparation method described in any one of claims 3 to 7.

10. At least one skin product according to 1) to 7) below, characterized in that, Including the topical skin composition of claim 9: 1) Skin products that repair damaged cells; 2) Skin products that reduce inflammation; 3) Acne-fighting skin products; 4) Antibacterial skin products; 5) Oil-controlling skin products; 6) Moisturizing skin products; 7) Anti-aging and wrinkle-reducing skin products; 8) Antioxidant skin products.

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