A Cordyceps sinensis fermentation composition

Abstract

The present invention provides a Cordyceps sinensis fermentation composition, which contains erythritol and mannitol, wherein the content of the erythritol is more than 1 wt %, and the content of the mannitol is more than 4 wt %. The fermentation composition of the present invention has excellent antioxidant effect and synergistic technical effect.

Description

Technical Field

[0001] The invention relates to a cordyceps sinensis fermentation composition and belongs to the field of microbial fermentation. Background Art

[0002] Erythritol, also known as erythritol, is a four-carbon polyol with the chemical name 1,2,3,4-butanetetrol and the molecular formula C4H 10 O4, with a relative molecular mass of 122.12. As a natural sweetener, erythritol is widely present in nature. It exists naturally in small amounts in fruits and vegetables such as pears, grapes, and mushrooms, and in fermented foods such as wine, beer, soy sauce, and sake. It also exists in small amounts in animal serum and eyeballs.

[0003] Erythritol has a small molecular weight and strong hydrophilicity. It does not contain reducing aldehyde groups and is highly stable to heat and acid. It is easily soluble in water, with a solubility of 37% at 25°C. It dissolves in water and absorbs heat, and produces a cool feeling when consumed in solid form. It has good crystallinity and low hygroscopicity, and does not absorb moisture in an environment of 20°C and 90% relative humidity. It has a sweetness of 60% to 80% of sucrose, and its sweetness is pure and refreshing without any unpleasant bitterness.

[0004] Low energy and special metabolic pathways are one of the metabolic characteristics of erythritol. The energy coefficient of erythritol is 0.88 kJ / g, which is the lowest energy among the polyol sweeteners currently used. When the human body ingests erythritol, about 80% to 90% of erythritol is absorbed by the small intestine through passive diffusion, and the remaining 10% enters the large intestine as a carbon source for fermentation. And as the dose of erythritol intake increases, the ratio of erythritol entering the large intestine will also increase. Among them, erythritol absorbed by the human body through the small intestine is widely distributed in the tissues, and about 5% to 10% of erythritol is oxidized into erythrose in the blood, and then rapidly further oxidized to form erythrose esters, and finally excreted from the urine. The remaining unoxidized erythritol cannot be decomposed by the enzyme system in the body, so it can only be filtered from the blood through the kidneys and excreted from the body through urine. Erythritol that enters the large intestine will be fermented by intestinal flora and converted into short-chain fatty acids (SCFAs). After being absorbed into the intestine, SCFAs can improve diet-induced metabolic disorders through their anti-inflammatory effects and have a protective effect on obesity and inflammatory bowel disease.

[0005] Good intestinal tolerance is another characteristic of erythritol. Compared with other sugar alcohols, the maximum non-laxative dose of erythritol is 0.66~1.0+ g / kgbw, which is significantly higher than other sugar alcohols. Studies on human intestinal tolerance show that when healthy volunteers aged 18 to 24 years old ingest 20 to 35 g of erythritol in liquid form, the intestinal tolerance is good and there are no adverse symptoms; when the intake of erythritol reaches 50 g, diarrhea and nausea can be observed. At the same time, clinical studies have shown that children aged 4 to 6 years old did not observe diarrhea and / or severe intestinal symptoms after consuming beverages containing 15 g of erythritol. In addition, it should be noted that diarrhea caused by excessive intake of sugar alcohols is not a disease, but a simple osmotic reaction to slowly absorbed carbohydrates in the intestinal cavity, that is, sugar alcohols that are not absorbed in the small intestine may cause abdominal distension, diarrhea and flatulence due to osmotic effects. Some people will experience this symptom after consuming it once. With continued consumption, most people will develop a certain degree of tolerance to sugar alcohols, and the related gastrointestinal symptoms will weaken or disappear. In addition, erythritol also has positive significance in oral protection. Studies have shown that erythritol is not only not used by Streptococcus mutans in the mouth, but also can inhibit its growth and adhesion in the mouth, thereby playing a beneficial role in the oral cavity. In a 3-year clinical trial, the levels of acetic acid and propionic acid in dental plaque and the counts of Streptococcus mutans in plaques of subjects treated with erythritol were significantly lower than those of subjects treated with xylitol or sorbitol.

[0006] At present, erythritol is mainly produced industrially by microbial fermentation. There are many microorganisms in nature that can synthesize erythritol, such as osmophilic yeasts such as Moniliella pollinis, Trichosporon, Trichosporon, and Yarrowia lipolytica, and bacteria such as Oenococcus oeni, Leuconostoc oeni, and Lactobacillus. The synthesis pathways of erythritol in yeast and bacteria are different. In yeast, erythritol is synthesized through the pentose phosphate pathway. The erythrose-4-phosphate produced by this pathway is dephosphorylated to form erythrose, and then erythritol is formed under the action of erythrose reductase; in bacteria, erythritol can be produced through the regeneration link of reduced coenzyme II (NADPH) in the phosphoacetolase pathway.

[0007] Mannitol, also known as D-mannitol, hexadecene, pyrifos, and sorbitol are isomers. Mannitol exists in both natural Cordyceps sinensis and fermented Cordyceps sinensis powder, but the content is different. As a typical hexavalent sugar alcohol, mannitol dissolves and absorbs heat. After being digested and absorbed by the human body, mannitol first reacts with NAD+ to generate acetyl coenzyme A and fructose. Through the action of fructokinase, two fructose phosphates are generated, which enter the linchuan triose cycle and complete the physiological metabolism of mannitol. It can be seen that the metabolic process of mannitol is relatively special. The whole process does not require the participation of insulin, so naturally it will not cause a large fluctuation in blood sugar concentration. Therefore, mannitol can be regarded as a low-calorie food additive.

[0008] Mannitol is also a low-calorie, low-sugar, refreshing sweetener with a sweetness equivalent to 60% of sucrose and 76% of xylitol. Due to its unique metabolic process, it can also be eaten by diabetics and obese patients. In chewing gum, it can be used as a sweetener and flavoring agent. As a sugar-free sweetener and additive, it is used in the production and processing of various foods, the storage of fruit flavors and the preparation of dried fruits.

[0009] Mannitol can be used in foods and daily chemicals to prevent or improve tooth decay, because mannitol, as a sweetener, does not provide nutrition to microorganisms in the mouth, and can inhibit the growth and reproduction of streptococci.

[0010] Mannitol is also a powerful free radical scavenger that can quickly clear some hydroxyl radicals in free radical chain reactions. When the two interact, they can form low-toxic mannitol free radicals, which are detoxified through dismutation to prevent irreversible damage to penumbra tissues, thereby reducing neurological damage and can be used as a neuroprotectant in clinical practice. It also has an inhibitory effect on the generation of oxygen free radicals. It can effectively clear free radicals produced by hypoxia in brain tissue, myocardial cells and limb muscle tissues, and can reduce blood concentration, reduce red blood cell aggregation and cerebrovascular resistance, improve microcirculation, and increase perfusion of organ tissues.

[0011] Ergothioneine (EGT or ERG) is a natural rare chiral amino acid antioxidant first discovered in the fungus Claviceps purpurea in 1909. It is a water-soluble, naturally occurring thiol compound. It has been recognized as an antioxidant in the body and a protective factor that helps cells resist oxidative stress. It has multiple functions such as anti-oxidation, scavenging free radicals, chelating metal ions, preventing ultraviolet radiation damage, regulating intracellular redox reactions, participating in intracellular energy regulation, and cell physiological protector. It is an important active substance in the body.

[0012] Ergothioneine is distributed in certain tissues and organs of mammals, mainly in red blood cells and semen of certain animals. The content of ergothioneine in human and mammalian tissues is 1-2 mM (equivalent to 229.3-458.6 mg / L) (Melville, 1959; Hartman, 1990 or The Natural Antioxidant Ergothioneine, LipidOxidation, 2013), which indicates that ergothioneine may act as a non-toxic antioxidant in the body. Cells lacking ergothioneine are easily affected by oxidative stress, leading to mitochondrial DNA damage, protein oxidation and increased lipid peroxidation. However, so far, no experimental studies have shown that animals can synthesize this substance, and there must be ways to absorb and retain ergothioneine in animal cells. Ergothioneine has shown good application prospects in the fields of cosmetics, functional foods, pharmaceuticals and biomedicine with its unique biological and pharmacological properties, safety, stability and other advantages, and has attracted extensive attention from scholars at home and abroad.

[0013] Ergothioneine is mainly found in mushrooms. Melville.DB and Eieh.S found that ergothioneine is also found in cereals. Subsequent studies by Melville.DB and Genghof.DS showed that ergothioneine is a common component of many microbial cells and can be synthesized in several fungi, but not in bacteria. Since mushrooms are a rich source of antioxidants including ergothioneine, this is another reason to include mushrooms in the human diet. For example, Shin-Yu Chen et al. (Contents of lovastatin, g-aminobutyric acid and ergothioneine in mushroom fruiting bodies and mycelia, Food Science and Technology, 2012(47):274-278) reported that among mushroom fruiting bodies, Pleurotus citrinopileatus, P. ostreatus (Korea), P. ostreatus (Taiwan, China) and Pleurotus salteostramineus had the highest ergothioneine content (2850.7, 1829.4, 1458.4 and 1245.0 mg / kg, respectively), while among mycelia, Pleurotus eryngii had the highest ergothioneine content (1514.6 mg / kg). In order to further enhance the ergothioneine content in mushrooms, Wi Yong Lee et al. (Ergothioneine Contents in Fruiting Bodies and Their Enhancement in Mycelial Cultures by the Addition of Methionine, Mycobiology, 2009 (37) 1: 43-7) reported the enhancement of ergothioneine content in mushroom fruiting bodies by adding methionine in mycelial culture. The difference between ergothioneine and mushrooms was as high as 92.3 times. Adding 2 mM methionine (Met) to the mycelial culture medium increased the ergothioneine content in the tested mushroom species, indicating that methionine is a beneficial additive for ergothioneine production in mushrooms.

[0014] Cordyceps militairs is also known as northern winter cordyceps. Its scientific name is Cordyceps militairs (Vuill) Fr, and its English name is Pupa insect grass. Its asexual form is Verticillium, and Liang Zongqi (1990) believed it to be a Cehalosporium. Patent document WO2014 / 055035 discloses a Cordyceps militairs strain cbs 132098 and biologically active fruiting bodies, mycelial biomass and extracts thereof. The ergothioneine in the mycelial biomass of Cordyceps militairs is 130.65 mg / kg (0.013%), corresponding to the fourth grade classification of Chen et al. (Chang et al., 2012). Bai-Xiong et al. (Enhancement of ergothioneine production by discovering and regulating its metabolic pathway in Cordyceps militaris. Bai-Xiong Chen et al, Microb Cell Fact. 2022 Aug 23;21(1):169) constructed a new ergothioneine synthesis pathway and successfully increased the component yield of the engineered strain by further introducing the pathway into the Cordyceps militaris genome, with the highest ergothioneine content reaching 2.5 g / kg dry weight.

[0015] Cordyceps sinensis (Borkeler) sacc. and Cordyceps militaris are two different species. Cordyceps sinensis is very specific in its host and can only parasitize the larvae of the bat moth (Hepialus armorieanus Oberthur). The scientific name of Cordyceps sinensis is Cordyceps sinensis (Berk) Sacc., and the English name is Chinese caterpillar fungus. The asexual form is named by scholars and experts as Chinese hairy spore ( Hirsutella sinensis). Hui-Chen Lo et al. (A Systematic Review of the Mysterious Caterpillar Fungus Ophiocordyceps sinensis in DongChongXiaCao (冬虫夏草 Dōng Chóng Xià Cǎo) and Related Bioactive Ingredients, Journal of Traditional and Complementary Medicine 2013(3)1:16 - 32) believe that the fruiting bodies of wild Ophiocordyceps sinensis contain detectable ergothioneine, and this ergothioneine is considered a secondary metabolite of fungal growth and not a small molecule organic compound necessary for the normal operation of its growth and development. Although Shin-Yu Chen et al. reported that the mycelia of Ophiocordyceps sinensis contain 142.0 ± 38.5IJK ergothioneine, Shin-Yi Lin et al. (Comparative Study of Contents of Several Bioactive Components in Fruiting Bodies and Mycelia of Culinary-Medicinal Mushrooms, International Journal of Medicinal Mushrooms, 2013(15)3: 315–323) reported that the content of ergothioneine in the mycelia of Ophiocordyceps sinensis is only 35.70 ± 1.28K, and the content in its fruiting bodies is even lower, only 12.20 ± 1.51E. Nachshol Cohen et al. (Chemical Composition and Nutritional and Medicinal Value of Fruit Bodies and Submerged Cultured Mycelia of Culinary-Medicinal Higher Basidiomycetes Mushrooms, International Journal of Medicinal Mushrooms, 2014(16)3: 273–291) reported that the content of ergothioneine in the mycelia of Ophiocordyceps sinensis is 52.18 ± 2.75ug / g, so there is no stable wild Cordyceps sinensis or natural Cordyceps sinensis containing higher ergothioneine, nor is there a stable wild Cordyceps sinensis or natural Cordyceps sinensis mycelium containing higher ergothioneine. Moreover, Cordyceps sinensis belongs to the Ascomycetes, Hypocreales, Ophiocordycipitaceae, Ophiocondyceps, Ophiocondyceps sinensis (Technical Specifications for Pollution-free Cultivation and Production of Chinese Medicinal Materials, 2018). Natural Cordyceps sinensis sources are scarce and expensive. Using Cordyceps sinensis hyphae as a substitute for natural Cordyceps sinensis is a trend in technological development. For decades, researchers have isolated more than 30 species of fungi from Cordyceps sinensis collected from various production areas, including Paecilomyces spp., Paecilomyces chinensis, Mortierella spp., Cephalosporium sinensis, Chinese Hirsutella, Chinese Toxoplasma, Chinese Chrysosporium, etc. In the metabolomycete study of Cordyceps sinensis at different growth stages (Zhang Zhou, Anhui Agricultural University, 2018), it was shown that there were significant differences in metabolites between different parts of Cordyceps sinensis, and there were also significant differences in the same part at different developmental stages. This difference was more obvious in the early stages of development. .

[0016] Therefore, the fermentation of Cordyceps sinensis and the combination of its beneficial ingredients remain technical issues that need to be solved. Summary of the invention

[0017] The inventor unexpectedly discovered that, through process optimization, the provided Cordyceps sinensis fermentation composition contains more than 1% (weight percentage) of erythritol, preferably 1%-6% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4%. Moreover, those skilled in the art generally believe that erythritol is only used as a sweetener, but the present invention unexpectedly discovered through research that the combination of erythritol and adenosine jointly exhibits a hydroxyl radical scavenging rate synergistic effect, thereby enhancing the overall effect of the Cordyceps sinensis fermentation composition, especially when erythritol is within the range defined by the present invention, the combination with adenosine can exhibit an excellent hydroxyl radical scavenging rate synergistic effect.

[0018] On the one hand, the present invention obtains Cordyceps sinensis fungi that can produce erythritol by screening Cordyceps sinensis fungi, and then obtains a fermentation composition containing erythritol by fermentation. On the other hand, the present invention realizes that erythritol exists in sufficient and stable amounts in Cordyceps sinensis mycelium or powder through low-temperature liquid fermentation technology. On the other hand, the present invention realizes the reasonable existence of effective ingredients such as erythritol and adenosine through a low-temperature fermentation process, which not only makes the fermented Cordyceps sinensis composition more stable, but also provides a new utilization direction of the fermented Cordyceps sinensis powder.

[0019] Specifically, the present invention screened Cordyceps sinensis strains and screened out Chinese Hirsutella sinensis ( Hirsutella sinensis ) has the function of producing erythritol. On the other hand, the inventor obtains a stable fermented mycelium or powder containing a high content of erythritol through fermentation of Chinese Hirsutella spp. The present invention obtains erythritol with a natural Cordyceps sinensis content level for the first time through liquid fermentation culture, and exerts a wider range of functions of the Cordyceps sinensis fermentation composition.

[0020] Chinese Hirsutella Hirsutella sinensis ) strain is an asexual generation of Cordyceps sinesis (Berk.) Sacc., a ergot fungus isolated from fresh Cordyceps sinesis from the Qinghai-Tibet Plateau. The Chinese Hirsutum spp. described in the present invention can be any Chinese Hirsutum spp. strain, for example, it can be purchased from a commercial platform or obtained through independent screening and separation, as long as it can be identified as Chinese Hirsutum spp. according to identification principles such as microbial molecular genetics. Hirsutella sinensis ), the commercial channels include but are not limited to obtaining Chinese Hirsutella spp. ( Hirsutella sinensis ), such as those obtained from the China Center for Type Culture Collection (CCTCC), including but not limited to Chinese Hirsutella sinensis ( Hirsutella sinensis )CCTCC No: M 2011278, such as those obtained from the China General Microbial Culture Collection Center (CGMCC), including but not limited to Chinese Hirsutella sinensis ( Hirsutella sinensis ) CGMCC 3.14240 and Hirsutella sinensis ( Hirsutella sinensis ) CGMCC3.14243, such as Cordyceps sinensis (Hairysporium batriaceum, Hairysporum sinensis) purchased from China Industrial Microbiological Culture Collection (CICC) Ophiocordyceps sinensis or Cordyceps sinensis (Hairy spores of the bat moth, Hairy spores of China) Cordyceps sinensisStandard strains include but are not limited to strain numbers: CICC 14016, CICC 14017, CICC 14088, CICC 14089, CICC 14090, CICC 14094, CICC 14096, CICC 14091, CICC 14092, CICC 14093, CICC 14095, CICC 14097, CICC 14098, CICC 14099, CICC 14100, CICC 14101, CICC 14102, CICC 14103, CICC 14104, CICC 14105, CICC 14106, CICC 14107, CICC 14108, CICC 14109, CICC 14110, CICC 14111, CICC 14112, CICC 14113, CICC 14114, CICC14115, CICC 14117, CICC 14118, CICC 14119, CICC 14120, CICC 50002, such as Cordyceps sinensis (Hairysporum batriae, Hairysporum sinensis) purchased from Testol Biotech Ophiocordyceps sinensis or Cordyceps sinensis (Hairy spores of the bat moth, Hairy spores of China) Cordyceps sinensis or Hirsutella sinensis ( Hirsutella sinensis ) including but not limited to article numbers TS324838, TS325049, TS325054, TS326189, TS326195, TS326196, TS326197, TS326198, TS326199, TS326200, TS326201, TS326202, TS326203, TS326204, TS326205, TS326206, TS326207, TS32 6210, TS326212, TS326213, TS326214, TS326408, TS326410, TS326412, TS342791, TS342792, TS3427 93. TS342794, TS342795, TS342796, TS342797, TS342798, TS342800, TS342809, TS361737, TS392986.

[0021] The growth cycle of Chinese hairy spore is longer than that of most fungi. It generally grows in an environment of 14-20°C and is a medium-low temperature fungus. When cultured on solid culture medium, the diameter can reach 1.5 cm. The texture of the colony is like leather glue, flesh-colored and milky white. There are aerial hyphae on the surface in the early stage of growth. As time goes by, the surface of the colony will appear smooth and grooved, and the aerial hyphae will gradually decrease. When cultured in liquid culture medium, the mycelium is filamentous or gray spherical, of varying sizes. As the culture progresses, brown will be secreted into the culture medium. The texture of the mycelium ball is tough and not loose, elastic, and hollow inside. It is known that the mycelium obtained by liquid fermentation culture of Chinese hairy spore is dried and fermented with Cordyceps sinensis powder, which can obtain a variety of active substances, including polysaccharides, amino acids, nucleosides, sterols, mannitol and trace metals. Among them, the characteristic components are adenosine, mannitol, polysaccharides, amino acids, etc. Fermented Cordyceps sinensis powder has the effects of enhancing immune function, anti-fibrosis, protecting kidney function, anti-tumor, and anti-inflammatory, and has a protective effect on organs such as the kidneys, lungs, and liver.

[0022] The fermentation in the present invention refers to the process of subjecting the existing Chinese Cordyceps sinensis asexual generation strain, namely, Chinese Cordyceps sinensis, to slant culture, seed bottle / shake bottle culture, and fermentation culture.

[0023] The slant culture of the present invention is to make the Chinese hairy spore grow on the inclined surface of the solid culture medium, inoculate and spread the Chinese hairy spore on the slant, and culture it under low temperature conditions, such as 8-18°C for 20-60 days, preferably 10-16°C for 30-50 days, wherein the main raw materials of the slant culture medium are glucose, potato juice (powder), corn flour, agar (powder), yeast extract (powder), wheat bran (skin), silkworm pupa powder, peptone, NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate); for example, the slant culture medium can be (the following % is weight-to-volume ratio g / 100ml):

[0024] Peptone 0.05-2.0%, yeast extract 0.05-2.0%, glucose 1.0-5.0%, agar 1.0-3.0%, wheat bran 2.0-10%, KH2PO4 0.02-0.2%, MgSO4 0.01-0.1%, and the rest is water;

[0025] or

[0026] Silkworm pupa powder 1.0-2.0%, corn flour 1.0-2.0%, glucose 2.0-5.0%, MgSO4 0.01-0.1%, KH2PO4 0.01-0.1%, oatmeal 1.0-3.0g / 60ml, agar powder 0.5-1%, and the rest is water;

[0027] or

[0028] Silkworm pupa powder 1.8%, corn flour 1.5%, glucose 2.0%, MgSO4 0.01%, KH2PO4 0.02%, oatmeal 1.0-3.0g / 60ml, agar powder 0.65%, and the rest is water;

[0029] or

[0030] Peptone 0.1%, yeast extract 0.1%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, agar 1.0-2.0%, wheat bran 5.0%, add water and boil for 30 minutes, then dispense;

[0031] or

[0032] Peptone 0.5-2.0%, broad bean powder 2.0-5.0%, corn powder 1.0-4.0%, glucose 2.0-5.0%, agar 0.5-2.0%, bran 0.5-4%, KH2PO4 0.01-1.0%, MgSO4 0.01-1.0%, and the rest is water;

[0033] or

[0034] Glucose 2.0%, corn flour 1.0%, potato juice 0.5%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm pupa powder 2.0%, peptone 1.0%, MgSO4 0.05%, KH2PO4 0.05%, agar powder 1.0%, the solvent is water;

[0035] or

[0036] Silkworm pupa powder 2.0-3.0%, corn flour 1.5%, bran 1.0-5.0%, glucose 2.0%, agar 1.8%, KH2PO4 0.02%, MgSO4 0.01%, and the rest is water;

[0037] Those skilled in the art will understand that the specific slant culture medium listed above is only an illustration of the present invention and is not to be construed as limiting the present invention. At the same time, those skilled in the art will understand that it may include other commonly used slant culture media for Chinese Hirsutella spp. fermentation, or a slant culture media for Chinese Hirsutella spp. fermentation known in the prior art.

[0038] The shake flask / seed bottle culture strain in the present invention is inoculated from the inclined surface to the seed bottle, and then shake culture is performed, or the test tube strain is accessed into the sterilized (sterilized) cooling triangular flask culture solution, and then placed on a shaking table for shaking culture, for example, the seed amount is 2 to 6 inclined surfaces, preferably 2 to 3 inclined surfaces, the culture temperature is 10 to 18 ° C, the preferred culture temperature is 12 to 16 ° C, the culture time is 8 to 16 days, and the preferred culture time is 9 to 14 days. In large-scale production, the mycelium of the shake flask culture can be accessed into the seed tank as the strain. In the shake flask liquid culture, it is required that the mycelium grows fast and the yield is high, and the formed bacterium ball is small. If the bacterium ball is larger, the mycelium in the center of the bacterium ball will present a starvation state of hypoxia and nutrition, which is unfavorable for the production of mycelium proliferation and secondary metabolites, and affects the fermentation quality and mycelium yield. The main raw materials of the seed flask / shake flask culture medium are one or more of peptone, fish peptone, beef extract, silkworm pupa powder, glucose, corn flour, dextrin, yeast extract (powder), wheat bran (skin), NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate); for example, the shake flask / seed flask culture medium can be (the following % is weight to volume ratio g / 100ml):

[0039] Silkworm pupa powder 2.0-3.0%, corn flour 2.0-3.0%, bran 2.0-3.0%, fish peptone 0.1-0.5%, peptone 0.1-1%, KH2PO4 0.01%-0.1%, glucose 2.0-5.0%, MgSO4 0.01%-0.1%, and the rest is water;

[0040] or

[0041] Silkworm pupa powder 2.0-3.0%, corn flour 1.5%, bran 1.0-5.0%, glucose 2.0%, KH2PO4 0.02%, MgSO4 0.01%, and the rest is water;

[0042] or

[0043] Silkworm pupa powder 2.0%, corn flour 2.5%, bran 2.5%, fish peptone 0.25%, peptone 0.75%, KH2PO4 0.02%, glucose 3.0%, MgSO4 0.01%, and the rest is water;

[0044] or

[0045] Glucose 2.0%, corn flour 1.0%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm pupa powder 2.0%, peptone 1.0%, MgSO4 0.05%, KH2PO4 0.05%, solvent is water;

[0046] or

[0047] Peptone 5.0%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5.0%, solvent is water;

[0048] or

[0049] Fish peptone 0.5%, peptone 0.5%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5%, the rest is water;

[0050] or

[0051] Glucose 2.0-4.5%, beef extract 2.0-4.5%, potato flour 1.0-2.5%, peptone 1.0-2.5%, KH2PO4 0.02-0.25%, MgSO4 0.01-0.05%, inulin 0.2-1.5%, vitamin B1 0.05-0.15%, zinc acetate 0.01-0.06%, and the rest is water;

[0052] Those skilled in the art will understand that the specific shake flask / seed flask culture medium listed above is only an illustration of the present invention and is not to be construed as limiting the present invention. At the same time, those skilled in the art will understand that it may include other commonly used shake flask / seed flask culture medium for Chinese Hirsutella spp. fermentation, or shake flask / seed flask culture medium for Chinese Hirsutella spp. fermentation known in the prior art.

[0053] Fermentation culture in the present invention refers to a culture medium for microorganisms to accumulate a large amount of metabolites. The fermentation described in the present invention includes the use of a fermentation medium. Submerged fermentation refers to a method in which pure strains of microbial cells are aerobically or anaerobically cultured in a liquid submerged culture medium. Submerged fermentation is generally carried out in a large fermentation tank and can be automated and mechanized. The fermentation tank submerged culture process of Chinese Hirsutella serrata can be: a primary seed tank → a secondary seed tank → a fermentation tank; it can also be: a seed tank → a fermentation tank; a continuous fermentation method can also be used. For example, the culture temperature is 10 ~ 18 ° C, preferably the culture temperature is 12 ~ 16 ° C, the culture time is 6 ~ 20 days, preferably the culture time is 7 ~ 15 days, and preferably the culture time is 10 ~ 15 days. The main raw materials of the fermentation medium include one or more of the following: silkworm pupa powder, glucose, corn flour (slurry), yeast extract (extract paste or powder), NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate), polypeptide powder series (such as polypeptide powder 101, polypeptide powder 102, polypeptide powder 203, polypeptide powder 305, polypeptide powder 308, polypeptide powder 607, polypeptide powder 702, polypeptide powder 705) (the polypeptide powder series is purchased from Chengdu Wenfeng Biotechnology Co., Ltd.).

[0054] The fermentation medium of the present invention includes an organic nitrogen source for fermentation with a novel microorganism, such as polypeptide powder "1" series, which is mainly made of rice protein, corn protein and its hydrolyzate. The process of segmented hydrolysis using multiple hydrolysis methods is used to separate, refine, concentrate and dry the powdered products. The polypeptide powder "1" series includes polypeptide powder 101 and polypeptide powder 102. The polypeptide powder "2" series, such as polypeptide powder 203, is a powdered product made of corn protein as the main raw material, using a process of segmented hydrolysis using multiple hydrolysis methods (this process enables the product to have different molecular weights, and the peptones and peptides are reasonably distributed, which is conducive to the continuous use of microorganisms), and is separated, refined, concentrated and spray-dried.

[0055] The fermentation medium of the present invention includes a peptone series (non-animal origin) using a novel organic nitrogen source for microbial fermentation, such as polypeptide powder 305, polypeptide powder 308, and polypeptide powder 607. The peptone series uses corn protein and yeast as the main raw materials. The powdered product is made by separation, refining, concentration, and spray drying using a multi-hydrolysis process (this process enables the product to have different molecular weights, reasonable distribution of peptones and peptides, and is conducive to the sustainable use of microorganisms).

[0056] The fermentation medium of the present invention includes a series of peptones (non-animal origin) using a novel organic nitrogen source for microbial fermentation, such as polypeptide powder 702 and polypeptide powder 705. The polypeptide powder "7" series uses rice protein and corn protein as the main raw materials. The powder product is made by separation, refining, concentration and spray drying using a modern biological method and a multi-hydrolysis method for segmented hydrolysis (this process enables the product to have different molecular weights, reasonable distribution of peptones and peptides, and is conducive to the sustainable use of microorganisms).

[0057] The fermentation medium of the present invention can be (the following % is weight to volume ratio):

[0058] Glucose 1-5%, yeast extract 0.5-5%, KH2PO4 0.01-0.1%, MgSO4 0.01-0.1%, the above polypeptide powder 0.5-5%, and the rest is water;

[0059] Preferably,

[0060] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, the above polypeptide powder 1%, and the rest is water;

[0061] or

[0062] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 203 1%, and the rest is water;

[0063] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 305 1%, and the rest is water;

[0064] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 702 1%, and the rest is water;

[0065] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 705 1%, and the rest is water;

[0066] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 607 1%, and the rest is water.

[0067] The Cordyceps sinensis fermentation composition of the present invention refers to mycelium, wet fungus sheet, dry mycelium powder, or dry mycelium powder of mycelium and fermentation filtrate obtained by slant culture, seed bottle / shake bottle culture, and fermentation culture of Hirsutella sinensis, preferably dry mycelium powder or dry mycelium powder of mycelium and fermentation filtrate. The fermentation filtrate includes the fermentation filtrate obtained by solvent separation and chromatography column separation (including membrane separation), or its effective parts, effective ingredients, or various metabolites.

[0068] The invention optimizes the fermentation process to obtain a Cordyceps sinensis fermentation composition, wherein the amount of mycelium and metabolites are significantly increased, wherein the content of erythritol is above 1% (weight percentage), preferably 1-6% (weight percentage), and further preferably 1.5-5% (weight percentage).

[0069] 1.5-4% (weight percentage), 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage).

[0070] The present invention unexpectedly discovered that the presence of erythritol in an amount of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage) in a Cordyceps sinensis fermentation composition is not only the first Cordyceps sinensis fermentation product with a stable erythritol content, but also enhances the effect of adenosine. The inventors unexpectedly discovered that the combination of erythritol and adenosine exhibits a significant synergistic effect in scavenging hydroxyl radicals. That is, the present invention ensures that the erythritol in the obtained Cordyceps sinensis fermentation composition is sufficient and stable at 1% or more (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage). At the same time, it is particularly preferred that the adenosine content is 0.02% or more (weight percentage), 0.03% or more (weight percentage), preferably 0.03% or more (weight percentage), preferably 0.03-1.00% (weight percentage), preferably 0.03-0.80% (weight percentage), preferably 0.03-0.60% (weight percentage), preferably 0.03-0.40% (weight percentage), preferably 0.03-0.20% (weight percentage), preferably 0.03-0.10% (weight percentage), and preferably 0.03-0.08% (weight percentage).

[0071] Furthermore, as previous studies have shown that adenosine may be converted from adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, nucleoside substances represented by adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate also have important physiological activities, such as Qian Jia et al. (Study on the in vitro antioxidant effect of 5′-acid adenosine (5′-AMP), Food and Machinery, Vol. 24, No. 1, January 2008) showed that AMP has a strong activity in scavenging hydroxyl free radicals. Therefore, on the basis of ensuring the stable existence of erythritol and adenosine with synergistic technical effects, the present invention controls the conversion amount of adenosine to a certain extent, so that the Cordyceps sinensis fermentation composition as a whole shows a more excellent technical effect. Previous studies such as Chen Jiaming et al. (Effects of different carbon sources and nitrogen sources on the solid fermentation process of Chinese Hirsutella serrata, Chinese Edible Fungi 2017, 37 (1): 55-60) showed that different carbon sources and nitrogen sources had a certain influence on the fermentation process of Chinese Hirsutella serrata ( Hirsutella sinensis ) The influence of solid fermentation process. The highest adenosine obtained by solid fermentation method was only 63.76 μg·g in formula G7. -1, and Zhang Ping et al. (Research Progress on Quality Evaluation and Control of Cordyceps Fermentation Products, Chinese Journal of Pharmacy, July 2021, Vol. 56, No. 14) recorded that the liquid fermentation culture conditions of Chinese Hirsutum were optimized, and the dry weight of the optimized Chinese Hirsutum mycelium increased by 36.2%, and the adenosine content increased by 46.8%. Amino nitrogen is the most important limiting matrix for bacterial growth during the fermentation process. Therefore, obtaining amino nitrogen during the liquefaction process is very important for increasing Cordyceps yield and effective utilization of raw materials (CN103444434A), but some studies have shown that the growth rate of bacteria is fastest when the concentration of carbon and nitrogen sources is low. As the concentration of carbon and nitrogen sources increases, the growth rate of bacteria decreases, indicating that higher concentrations of carbon and nitrogen sources are not conducive to bacterial growth (CN103430777A).

[0072] The present invention unexpectedly discovered that the higher the content of adenosine is, the better. The Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02% or more (weight percentage), 0.03% or more (weight percentage), preferably 0.03-1.00% (weight percentage), preferably 0.03-0.80% (weight percentage), preferably 0.03-0.60% (weight percentage), preferably 0.03-0.40% (weight percentage), preferably 0.03-0.20% (weight percentage), preferably 0.03-0.10% (weight percentage), preferably 0.03-0.08% (weight percentage), based on an erythritol content of more than 1%.

[0073] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 1% or more erythritol; or,

[0074] More specifically, the present invention preferably contains 0.02%-1.00% adenosine (weight percentage) on the basis of 1-6% erythritol content; or,

[0075] More specifically, the present invention preferably contains 0.03% to 0.80% adenosine (by weight) on the basis of 1% to 6% erythritol; or,

[0076] More specifically, the present invention preferably contains 0.02%-1.00% (weight percentage) of adenosine on the basis of 1.5-5% (weight percentage) of erythritol; or,

[0077] More specifically, the present invention preferably has an adenosine content of 0.02%-1.00% (weight percentage) based on an erythritol content of 1.5-4% (weight percentage); or, more specifically, the present invention preferably has an adenosine content of 0.02%-1.00% (weight percentage) based on an erythritol content of 2-5%; or,

[0078] More specifically, the present invention preferably contains 0.03% to 0.80% adenosine (by weight) on the basis of 2% to 5% erythritol; or,

[0079] More specifically, the present invention preferably has an adenosine content of 0.02%-1.00% (weight percentage) based on an erythritol content of 2.5-4% or more.

[0080] More specifically, the present invention preferably has an adenosine content of 0.03%-0.80% (weight percentage) based on an erythritol content of 2.5-4% or more.

[0081] Those skilled in the art will understand that the above list is only an illustration of the present invention and is not to be construed as limiting the present application. Due to the study of the content of erythritol and adenosine, the fermentation composition obtained within the above range has a very excellent antioxidant effect, especially the synergistic technical effect of hydroxyl radical scavenging rate.

[0082] Therefore, the present invention also provides a Cordyceps sinensis fermentation composition, wherein the fermentation composition further comprises one or more of mannitol, polysaccharides, and amino acids.

[0083] Furthermore, the Cordyceps sinensis fermentation composition has a mannitol content of more than 4% (weight percentage), preferably more than 5% (weight percentage), preferably more than 6% (weight percentage), preferably more than 7% (weight percentage).

[0084] More specifically, in the present invention, based on the erythritol content of more than 1%, the mannitol content is preferably more than 4% (by weight); or,

[0085] More specifically, the present invention preferably contains more than 5% mannitol (by weight) on the basis of 1% erythritol; or,

[0086] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 1% erythritol; or,

[0087] More specifically, the present invention preferably contains 1% erythritol and more than 7% mannitol (by weight); or,

[0088] More specifically, the present invention preferably contains more than 4% mannitol (by weight) on the basis of 1-6% erythritol; or,

[0089] More specifically, the present invention preferably contains more than 5% mannitol (by weight) on the basis of 1-6% erythritol; or,

[0090] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 1-6% erythritol; or,

[0091] More specifically, the present invention preferably contains more than 7% mannitol (by weight) on the basis of 1-6% erythritol; or,

[0092] More specifically, the present invention preferably contains more than 4% mannitol (by weight) on the basis of 1.5-5% erythritol; or,

[0093] More specifically, the present invention preferably contains more than 5% mannitol (by weight) on the basis of 1.5-5% erythritol; or,

[0094] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 1.5-5% erythritol; or,

[0095] More specifically, the present invention preferably contains more than 7% mannitol (by weight) on the basis of 1.5-5% erythritol; or,

[0096] More specifically, the present invention preferably contains more than 4% mannitol (by weight) on the basis of 2-4% erythritol; or,

[0097] More specifically, the present invention preferably contains more than 5% mannitol (by weight) on the basis of 2-4% erythritol; or,

[0098] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 2-4% erythritol; or,

[0099] More specifically, the present invention preferably contains more than 7% mannitol (by weight) on the basis of 2-4% erythritol; or,

[0100] More specifically, the present invention preferably contains more than 4% mannitol (by weight) on the basis of 2.5-4% erythritol; or,

[0101] More specifically, the present invention preferably contains more than 5% mannitol (by weight) on the basis of 2.5-4% erythritol; or,

[0102] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 2.5-4% erythritol; or,

[0103] More specifically, in the present invention, based on the erythritol content of 2.5-4%, the mannitol content is preferably above 7% (weight percentage).

[0104] Furthermore, the Cordyceps sinensis fermentation composition has a polysaccharide content of more than 2% (weight percentage), preferably more than 3% (weight percentage), preferably more than 4% (weight percentage), preferably 2-10% (weight percentage).

[0105] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0106] Those skilled in the art will understand that the above list is only an illustration of the present invention and is not to be construed as limiting the present invention. Due to the research on erythritol, adenosine content, mannitol, and polysaccharides, the fermentation composition obtained within the above range has very excellent technical effects.

[0107] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the content of erythritol is above 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), the content of adenosine is above 0.02% (weight percentage), and the content of mannitol is above 4% (weight percentage).

[0108] Furthermore, in the Cordyceps sinensis fermentation composition of the present invention, the content of erythritol is above 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), the content of adenosine is above 0.02% (weight percentage), the content of mannitol is above 4% (weight percentage), and the content of polysaccharides is above 2% (weight percentage).

[0109] Furthermore, in the Cordyceps sinensis fermentation composition of the present invention, the content of erythritol is above 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), the content of adenosine is above 0.02% (weight percentage), the content of mannitol is above 4% (weight percentage), and the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0110] Furthermore, in the Cordyceps sinensis fermentation composition of the present invention, the content of erythritol is above 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), the content of adenosine is above 0.02% (weight percentage), the content of mannitol is above 4% (weight percentage), the content of polysaccharides is above 2% (weight percentage), and the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0111] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of more than 0.02% (weight percentage), and a mannitol content of more than 5% (weight percentage); or,

[0112] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of more than 0.03% (weight percentage), and a mannitol content of more than 5% (weight percentage); or,

[0113] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of more than 0.02% (weight percentage), a mannitol content of more than 5% (weight percentage), and a polysaccharide content of more than 2% (weight percentage); or,

[0114] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of more than 0.02% (weight percentage), a mannitol content of more than 5% (weight percentage), and a total amino acid content of more than 20% (weight percentage); or,

[0115] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of 0.02%-1.0% (weight percentage), and a mannitol content of more than 5% (weight percentage); or,

[0116] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of 0.02%-1.0% (weight percentage), a mannitol content of more than 5% (weight percentage), and a polysaccharide content of more than 2% (weight percentage); or,

[0117] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of 0.02%-1.0% (weight percentage), a mannitol content of more than 5% (weight percentage), and a total amino acid content of more than 20% (weight percentage); or,

[0118] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of 0.02%-1.0% (weight percentage), a mannitol content of more than 5% (weight percentage), a polysaccharide content of more than 2% (weight percentage), and a total amino acid content of more than 20% (weight percentage).

[0119] The Cordyceps sinensis fermentation composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, capsules, or the parts are mixed and the mixture is encapsulated or the mixture is made into tablet form to obtain a single preparation. It can also be prepared into various dosage forms with acceptable carriers. For oral administration compositions (such as tablets and capsules), the term "acceptable carrier" includes such carriers, such as common excipients, such as adhesives, such as syrup, gum arabic, gelatin, sorbitol, tragacanth gum, polyvinyl pyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose and starch; fillers and carriers such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose; lubricants such as magnesium stearate, sodium stearate and other stearates, glycerol stearate, stearic acid, silicone fluid, talc, wax, oil and colloidal silicon dioxide. Flavoring agents such as mint, wintergreen oil, cherry seasoning, etc. can also be used. Colorants may need to be added to distinguish the dosage forms. Tablets may also be coated by methods well known in the art. Tablets may be compressed or molded, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine a free-flowing form of the active ingredient, such as a powder or granules, optionally mixed with one or more binders, lubricants, inert diluents, preservatives, surface active agents or dispersants.

[0120] In summary, the Cordyceps sinensis fermentation composition of the present invention is not only the first Cordyceps sinensis fermentation product containing a stable content of erythritol, but also, due to the study on the content of erythritol and adenosine, the fermentation composition obtained within the said range has a very excellent antioxidant effect, so that the Cordyceps sinensis fermentation composition of the present invention exhibits a better technical effect than the prior art as a whole.

[0121] The pharmaceutical composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, capsules, or mixed with the parts and the mixture is filled into capsules or the mixture is made into tablets to obtain a single preparation. It can also be prepared into various dosage forms using acceptable carriers.

[0122] The pharmaceutical composition of the present invention has excellent antioxidant effect and synergistic technical effect.

[0123] The present invention also screens cordyceps sinensis, studies the fermentation of cordyceps sinensis and the combination of its beneficial components, obtains cordyceps sinensis that can produce thioneine, obtains a fermentation composition containing thioneine by fermentation, that is, realizes that thioneine in cordyceps sinensis mycelium or powder is present in sufficient amount and stably by low-temperature liquid fermentation technology, realizes the reasonable presence of effective ingredients such as thioneine and adenosine by low-temperature fermentation process, not only makes the composition of fermented cordyceps sinensis more stable, but also provides a new utilization direction of fermented cordyceps sinensis powder.

[0124] Specifically, the present invention screened Cordyceps sinensis strains and screened out Chinese Hirsutella sinensis ( Hirsutella sinensis ) has the function of producing erythritol and ergothioneine. On the other hand, the inventor obtains a fermented mycelium or powder containing a high content of ergothioneine that can exist stably through fermentation of Chinese Hirsutella spp. The present invention obtains a fermentation composition having a natural Cordyceps sinensis content of ergothioneine or ergothioneine content higher than the natural Cordyceps sinensis content for the first time through liquid fermentation culture, and exerts a wider range of functions of the Cordyceps sinensis fermentation composition.

[0125] Chinese Hirsutella Hirsutella sinensis ) strain is an asexual generation of Cordyceps sinesis (Berk.) Sacc., a ergot fungus isolated from fresh Cordyceps sinesis from the Qinghai-Tibet Plateau. The Chinese Hirsutum spp. described in the present invention can be any Chinese Hirsutum spp. strain, for example, it can be purchased from a commercial platform or obtained through independent screening and separation, as long as it can be identified as Chinese Hirsutum spp. according to identification principles such as microbial molecular genetics. Hirsutella sinensis ), the commercial channels include but are not limited to obtaining Chinese Hirsutella spp. ( Hirsutella sinensis ), such as those obtained from the China Center for Type Culture Collection (CCTCC), including but not limited to Chinese Hirsutella sinensis ( Hirsutella sinensis )CCTCC No: M 2011278, such as those obtained from the China General Microbial Culture Collection Center (CGMCC), including but not limited to Chinese Hirsutella sinensis ( Hirsutella sinensis ) CGMCC 3.14240 and Hirsutella sinensis ( Hirsutella sinensis ) CGMCC3.14243, such as Cordyceps sinensis (Hairysporium batriaceum, Hairysporum sinensis) purchased from China Industrial Microbiological Culture Collection (CICC) Ophiocordyceps sinensisor Cordyceps sinensis (Hairy spores of the bat moth, Hairy spores of China) Cordyceps sinensis Standard strains include but are not limited to strain numbers: CICC 14016, CICC 14017, CICC 14088, CICC 14089, CICC 14090, CICC 14094, CICC 14096, CICC 14091, CICC 14092, CICC 14093, CICC 14095, CICC 14097, CICC 14098, CICC 14099, CICC 14100, CICC 14101, CICC 14102, CICC 14103, CICC 14104, CICC 14105, CICC 14106, CICC 14107, CICC 14108, CICC 14109, CICC 14110, CICC 14111, CICC 14112, CICC 14113, CICC 14114, CICC14115, CICC 14117, CICC 14118, CICC 14119, CICC 14120, CICC 50002, such as Cordyceps sinensis (Hairysporum batriae, Hairysporum sinensis) purchased from Testol Biotech Ophiocordyceps sinensis or Cordyceps sinensis (Hairy spores of the bat moth, Hairy spores of China) Cordyceps sinensis or Hirsutella sinensis ( Hirsutella sinensis ) including but not limited to article numbers TS324838, TS325049, TS325054, TS326189, TS326195, TS326196, TS326197, TS326198, TS326199, TS326200, TS326201, TS326202, TS326203, TS326204, TS326205, TS326206, TS326207, TS32 6210, TS326212, TS326213, TS326214, TS326408, TS326410, TS326412, TS342791, TS342792, TS3427 93. TS342794, TS342795, TS342796, TS342797, TS342798, TS342800, TS342809, TS361737, TS392986.

[0126] The growth cycle of Chinese hairy spore is longer than that of most fungi. It generally grows in an environment of 14-20°C and is a medium-low temperature fungus. When cultured on solid culture medium, the diameter can reach 1.5 cm. The texture of the colony is like leather glue, flesh-colored and milky white. There are aerial hyphae on the surface in the early stage of growth. As time goes by, the surface of the colony will appear smooth and grooved, and the aerial hyphae will gradually decrease. When cultured in liquid culture medium, the mycelium is filamentous or gray spherical, of varying sizes. As the culture progresses, brown will be secreted into the culture medium. The texture of the mycelium ball is tough and not loose, elastic, and hollow inside. It is known that the mycelium obtained by liquid fermentation culture of Chinese hairy spore is dried and fermented with Cordyceps sinensis powder, which can obtain a variety of active substances, including polysaccharides, amino acids, nucleosides, sterols, mannitol and trace metals. Among them, the characteristic components are adenosine, mannitol, polysaccharides, amino acids, etc. Fermented Cordyceps sinensis powder has the effects of enhancing immune function, anti-fibrosis, protecting kidney function, anti-tumor, and anti-inflammatory, and has a protective effect on organs such as the kidneys, lungs, and liver.

[0127] The fermentation in the present invention refers to the process of subjecting the existing Chinese Cordyceps sinensis asexual generation strain, namely, Chinese Cordyceps sinensis, to slant culture, seed bottle / shake bottle culture, and fermentation culture.

[0128] The slant culture of the present invention is to make the Chinese hairy spore grow on the inclined surface of the solid culture medium, inoculate and spread the Chinese hairy spore on the slant, and culture it under low temperature conditions, such as 8-18°C for 20-60 days, preferably 10-16°C for 30-50 days, wherein the main raw materials of the slant culture medium are glucose, potato juice (powder), corn flour, agar (powder), yeast extract (powder), wheat bran (skin), silkworm pupa powder, peptone, NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate); for example, the slant culture medium can be (the following % is weight-to-volume ratio g / 100ml):

[0129] Peptone 0.05-2.0%, yeast extract 0.05-2.0%, glucose 1.0-5.0%, agar 1.0-3.0%, wheat bran 2.0-10%, KH2PO4 0.02-0.2%, MgSO4 0.01-0.1%, and the rest is water;

[0130] or

[0131] Silkworm pupa powder 1.0-2.0%, corn flour 1.0-2.0%, glucose 2.0-5.0%, MgSO4 0.01-0.1%, KH2PO4 0.01-0.1%, oatmeal 1.0-3.0g / 60ml, agar powder 0.5-1%, and the rest is water;

[0132] or

[0133] Silkworm pupa powder 1.8%, corn flour 1.5%, glucose 2.0%, MgSO4 0.01%, KH2PO4 0.02%, oatmeal 1.0-3.0g / 60ml, agar powder 0.65%, and the rest is water;

[0134] or

[0135] Peptone 0.1%, yeast extract 0.1%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, agar 1.0-2.0%, wheat bran 5.0%, add water and boil for 30 minutes, then dispense;

[0136] or

[0137] Peptone 0.5-2.0%, broad bean powder 2.0-5.0%, corn powder 1.0-4.0%, glucose 2.0-5.0%, agar 0.5-2.0%, bran 0.5-4%, KH2PO4 0.01-1.0%, MgSO4 0.01-1.0%, and the rest is water;

[0138] or

[0139] Glucose 2.0%, corn flour 1.0%, potato juice 0.5%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm pupa powder 2.0%, peptone 1.0%, MgSO4 0.05%, KH2PO4 0.05%, agar powder 1.0%, the solvent is water;

[0140] or

[0141] Silkworm pupa powder 2.0-3.0%, corn flour 1.5%, bran 1.0-5.0%, glucose 2.0%, agar 1.8%, KH2PO4 0.02%, MgSO4 0.01%, and the rest is water;

[0142] Those skilled in the art will understand that the specific slant culture medium listed above is only an illustration of the present invention and is not to be construed as limiting the present invention. At the same time, those skilled in the art will understand that it may include other commonly used slant culture media for Chinese Hirsutella spp. fermentation, or a slant culture media for Chinese Hirsutella spp. fermentation known in the prior art.

[0143] The shake flask / seed bottle culture strain in the present invention is inoculated from the inclined surface to the seed bottle, and then shake culture is performed, or the test tube strain is accessed into the sterilized (sterilized) cooling triangular flask culture solution, and then placed on a shaking table for shaking culture, for example, the seed amount is 2 to 6 inclined surfaces, preferably 2 to 3 inclined surfaces, the culture temperature is 10 to 18 ° C, the preferred culture temperature is 12 to 16 ° C, the culture time is 8 to 16 days, and the preferred culture time is 9 to 14 days. In large-scale production, the mycelium of the shake flask culture can be accessed into the seed tank as the strain. In the shake flask liquid culture, it is required that the mycelium grows fast and the yield is high, and the formed bacterium ball is small. If the bacterium ball is larger, the mycelium in the center of the bacterium ball will present a starvation state of hypoxia and nutrition, which is unfavorable for the production of mycelium proliferation and secondary metabolites, and affects the fermentation quality and mycelium yield. The main raw materials of the seed flask / shake flask culture medium are one or more of peptone, fish peptone, beef extract, silkworm pupa powder, glucose, corn flour, dextrin, yeast extract (powder), wheat bran (skin), NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate); for example, the shake flask / seed flask culture medium can be (the following % is weight to volume ratio g / 100ml):

[0144] Silkworm pupa powder 2.0-3.0%, corn flour 2.0-3.0%, bran 2.0-3.0%, fish peptone 0.1-0.5%, peptone 0.1-1%, KH2PO4 0.01%-0.1%, glucose 2.0-5.0%, MgSO4 0.01%-0.1%, and the rest is water;

[0145] or

[0146] Silkworm pupa powder 2.0-3.0%, corn flour 1.5%, bran 1.0-5.0%, glucose 2.0%, KH2PO4 0.02%, MgSO4 0.01%, and the rest is water;

[0147] or

[0148] Silkworm pupa powder 2.0%, corn flour 2.5%, bran 2.5%, fish peptone 0.25%, peptone 0.75%, KH2PO4 0.02%, glucose 3.0%, MgSO4 0.01%, and the rest is water;

[0149] or

[0150] Glucose 2.0%, corn flour 1.0%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm pupa powder 2.0%, peptone 1.0%, MgSO4 0.05%, KH2PO4 0.05%, solvent is water;

[0151] or

[0152] Peptone 5.0%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5.0%, solvent is water;

[0153] or

[0154] Fish peptone 0.5%, peptone 0.5%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5%, the rest is water;

[0155] or

[0156] Glucose 2.0-4.5%, beef extract 2.0-4.5%, potato flour 1.0-2.5%, peptone 1.0-2.5%, KH2PO4 0.02-0.25%, MgSO4 0.01-0.05%, inulin 0.2-1.5%, vitamin B1 0.05-0.15%, zinc acetate 0.01-0.06%, and the rest is water;

[0157] Those skilled in the art will understand that the specific shake flask / seed flask culture medium listed above is only an illustration of the present invention and is not to be construed as limiting the present invention. At the same time, those skilled in the art will understand that it may include other commonly used shake flask / seed flask culture medium for Chinese Hirsutella spp. fermentation, or shake flask / seed flask culture medium for Chinese Hirsutella spp. fermentation known in the prior art.

[0158] Fermentation culture in the present invention refers to a culture medium for microorganisms to accumulate a large amount of metabolites. The fermentation described in the present invention includes the use of a fermentation medium. Submerged fermentation refers to a method in which pure strains of microbial cells are aerobically or anaerobically cultured in a liquid submerged culture medium. Submerged fermentation is generally carried out in a large fermentation tank and can be automated and mechanized. The fermentation tank submerged culture process of Chinese Hirsutella serrata can be: a primary seed tank → a secondary seed tank → a fermentation tank; it can also be: a seed tank → a fermentation tank; a continuous fermentation method can also be used. For example, the culture temperature is 10 ~ 18 ° C, preferably the culture temperature is 12 ~ 16 ° C, the culture time is 6 ~ 20 days, preferably the culture time is 7 ~ 15 days, and preferably the culture time is 10 ~ 15 days. The main raw materials of the fermentation medium include one or more of the following: silkworm pupa powder, glucose, corn flour (slurry), yeast extract (extract paste or powder), NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate), polypeptide powder series (such as polypeptide powder 101, polypeptide powder 102, polypeptide powder 203, polypeptide powder 305, polypeptide powder 308, polypeptide powder 607, polypeptide powder 702, polypeptide powder 705) (the polypeptide powder series is purchased from Chengdu Wenfeng Biotechnology Co., Ltd.).

[0159] The fermentation medium of the present invention includes an organic nitrogen source for fermentation with a novel microorganism, such as polypeptide powder "1" series, which is mainly made of rice protein, corn protein and its hydrolyzate. The process of segmented hydrolysis using multiple hydrolysis methods is used to separate, refine, concentrate and dry the powdered products. The polypeptide powder "1" series includes polypeptide powder 101 and polypeptide powder 102. The polypeptide powder "2" series, such as polypeptide powder 203, is a powdered product made of corn protein as the main raw material, using a process of segmented hydrolysis using multiple hydrolysis methods (this process enables the product to have different molecular weights, and the peptones and peptides are reasonably distributed, which is conducive to the continuous use of microorganisms), and is separated, refined, concentrated and spray-dried.

[0160] The fermentation medium of the present invention includes a peptone series (non-animal origin) using a novel organic nitrogen source for microbial fermentation, such as polypeptide powder 305, polypeptide powder 308, and polypeptide powder 607. The peptone series uses corn protein and yeast as the main raw materials. The powdered product is made by separation, refining, concentration, and spray drying using a multi-hydrolysis process (this process enables the product to have different molecular weights, reasonable distribution of peptones and peptides, and is conducive to the sustainable use of microorganisms).

[0161] The fermentation medium of the present invention includes a series of peptones (non-animal origin) using a novel organic nitrogen source for microbial fermentation, such as polypeptide powder 702 and polypeptide powder 705. The polypeptide powder "7" series uses rice protein and corn protein as the main raw materials. The powder product is made by separation, refining, concentration and spray drying using a modern biological method and a multi-hydrolysis method for segmented hydrolysis (this process enables the product to have different molecular weights, reasonable distribution of peptones and peptides, and is conducive to the sustainable use of microorganisms).

[0162] The fermentation medium of the present invention can be (the following % is weight to volume ratio):

[0163] Glucose 1-5%, yeast extract 0.5-5%, KH2PO4 0.01-0.1%, MgSO4 0.01-0.1%, the above polypeptide powder 0.5-5%, and the rest is water;

[0164] Preferably,

[0165] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, the above polypeptide powder 1%, and the rest is water;

[0166] or

[0167] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 203 1%, and the rest is water;

[0168] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 305 1%, and the rest is water;

[0169] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 702 1%, and the rest is water;

[0170] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 705 1%, and the rest is water;

[0171] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 607 1%, and the rest is water.

[0172] The Cordyceps sinensis fermentation composition of the present invention refers to mycelium, wet fungus sheet, dry mycelium powder, or dry mycelium powder of mycelium and fermentation filtrate obtained by slant culture, seed bottle / shake bottle culture, and fermentation culture of Hirsutella sinensis, preferably dry mycelium powder or dry mycelium powder of mycelium and fermentation filtrate. The fermentation filtrate includes the fermentation filtrate obtained by solvent separation and chromatography column separation (including membrane separation), or its effective parts, effective ingredients, or various metabolites.

[0173] The invention optimizes the fermentation process to obtain a Cordyceps sinensis fermentation composition, wherein the amount of mycelium and metabolites are significantly improved, wherein the content of ergothioneine is 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage), preferably 0.01-0.10% (weight percentage), preferably 0.01-0.08% (weight percentage), or preferably 0.02% or more (weight percentage), preferably 0.02-0.10% (weight percentage). Ratio), preferably 0.02-0.08% (weight percentage); the content of adenosine is above 0.02% (weight percentage), above 0.03% (weight percentage), preferably 0.03-1.00% (weight percentage), preferably 0.03-0.80% (weight percentage), preferably 0.03-0.60% (weight percentage), preferably 0.03-0.40% (weight percentage), preferably 0.03-0.20% (weight percentage), preferably 0.03-0.10% (weight percentage), preferably 0.03-0.08% (weight percentage);

[0174] The ratio of ergothioneine to adenosine content is 1:1-1:20, preferably 1:1-1:15, preferably 1:1-1:10, preferably 1:1-1:8, preferably 1:1-1:7, preferably 1:1-1:6, preferably 1:1-1:5, preferably 1:1-1:4, preferably 1:1-1:3, preferably 1:1-1:2.

[0175] Furthermore, the Cordyceps sinensis fermentation composition has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage);

[0176] Furthermore, the Cordyceps sinensis fermentation composition has a mannitol content of more than 4% (weight percentage), preferably more than 5% (weight percentage), preferably more than 6% (weight percentage), preferably more than 7% (weight percentage); the ratio of ergothioneine to mannitol content is 1:1-1:1200, preferably 1:1-1:1100, preferably 1:1-1:1000, preferably 1:1-1:900, preferably 1:1-1:800, preferably 1:1-1:700, preferably 1:1-1:600, preferably 1:1-1:500, preferably 1:1-1:400, preferably 1:1-1:300.

[0177] Furthermore, the Cordyceps sinensis fermentation composition has a polysaccharide content of more than 2% (weight percentage), preferably more than 3% (weight percentage), preferably more than 4% (weight percentage), preferably 2-10% (weight percentage); the ratio of ergothioneine to polysaccharide content is 1:1-1:500, preferably 1:1-1:400, preferably 1:1-1:300, preferably 1:1-1:200, preferably 1:1-1:100, preferably 1:1-1:50.

[0178] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0179] When the components contained in the Cordyceps sinensis fermentation composition provided by the present invention are within the above preferred range, the composition has excellent antioxidant and whitening effects as a whole.

[0180] Therefore, one aspect of the present invention is to achieve sufficient and stable presence of ergothioneine in Cordyceps sinensis mycelium or powder by low-temperature fermentation technology, at least 0.004% (weight percentage).

[0181] Ergothioneine (EGT) is a white, odorless, colorless crystalline powder in the solid state. In this solid state, only the thione form exists. Ergothioneine is easily soluble in water and has tautomers (thione and thione forms) in the solution. However, ergothioneine has a very high thermal stability, and its decomposition temperature is as high as 262-265°C. Ergothioneine cannot be synthesized by the animal body itself and can only be obtained from food. It is a rare amino acid. Mammals and humans mainly obtain it through diet. Ergothioneine can be highly absorbed by the human body after ingestion. Ergothioneine has strong antioxidant properties and plays a unique role in the body's cell synthesis process. It has a good effect in preventing diabetes, kidney disease, tissue fibrosis, eye diseases and cancer, resisting UV damage, anti-inflammation, anti-oxidation, promoting neuronal differentiation, and enhancing memory and learning ability. The European Food Safety Authority and the U.S. Food and Drug Administration have both affirmed the important position of ergothioneine in new functional foods, and recognized the safety of producing ergothioneine through fermentation, and even believe that ergothioneine can be added to the diet of pregnant women and maternal and infant food. The antioxidant activity of ergothioneine is related to at least four molecular activities: direct scavenging of reactive oxygen species; chelation of various divalent metal cations; activation of antioxidant enzymes such as glutathione peroxidase and manganese superoxide dismutase, and inhibition of superoxidative kinases such as cytochrome c reductase (NADPH); and influence on the oxidation of various heme proteins such as heme and myoglobin.

[0182] The present invention unexpectedly discovered that the presence of 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage) of ergothioneine in the Cordyceps sinensis fermentation composition is not only the first Cordyceps sinensis fermentation product with ergothioneine, but also greatly enhances the effect of adenosine. The present invention shows through research experiments that ergothioneine in the Cordyceps sinensis fermentation composition helps to enhance the antioxidant effect of adenosine, so that ergothioneine and adenosine produce a synergistic effect, and through the optimization of the adenosine content range, the inventors also unexpectedly discovered that the Chinese Hirsutella spp. fermentation product or fermentation composition exhibits more excellent technical effects as a whole.

[0183] Therefore, on the other hand, the present invention ensures that the ergothioneine in the fermented Cordyceps sinensis composition obtained is more than 0.004% (weight percentage), preferably more than 0.006% (weight percentage), preferably more than 0.008% (weight percentage), preferably more than 0.01% (weight percentage) is present in sufficient amount and stably, and it is particularly preferred that the adenosine content is more than 0.02% (weight percentage), more than 0.03% (weight percentage), preferably 0.03-1.00% (weight percentage), preferably 0.03-0.80% (weight percentage), preferably 0.03-0.60% (weight percentage). (weight percentage), preferably 0.03-0.40% (weight percentage), preferably 0.03-0.20% (weight percentage), preferably 0.03-0.10% (weight percentage), preferably 0.03-0.08% (weight percentage); the ratio of ergothioneine to adenosine content is 1:1-1:20, preferably 1:1-1:15, preferably 1:1-1:10, preferably 1:1-1:8, preferably 1:1-1:7, preferably 1:1-1:6, preferably 1:1-1:5, preferably 1:1-1:4, preferably 1:1-1:3, preferably 1:1-1:2.

[0184] Adenosine is slightly soluble in cold water, soluble in water at room temperature, easily soluble in hot water, and insoluble in alcohol. Studies have shown that, such as Li Heyu et al. (Determination of five nucleoside components in cordyceps products and quality analysis of Cordyceps militaris, Chinese Herbal Medicine, 2018(49)22:5410-5417), there may be transformations in the extraction process of guanosine, uridine and adenosine. For example, adenosine triphosphate, adenosine diphosphate and adenosine monophosphate can be converted into adenosine during ultrasonic extraction using water or mixed reagents with high water content. For example, Qian Zhengming et al. (Study on the transformation pathway of adenosine during water extraction of Cordyceps sinensis, World Traditional Chinese Medicine, 2016(11)5:758-762) found that under room temperature water extraction of Cordyceps sinensis, adenosine monophosphate was converted into adenosine, and adenosine was converted into inosine. It was also found that under room temperature water extraction, adenosine triphosphate and adenosine diphosphate were also converted into adenosine. Among them, ATP is highly soluble in water and very stable in solutions between pH 6.8 and 7.4, but it will be rapidly hydrolyzed at extreme pH values. Adenosine is active in cardiovascular diseases. For example, Feng Yanhong et al. (Effects of adenosine on isolated cardiac function and myocardial cell apoptosis in rats, Shandong Medicine, Vol. 53, No. 46, 2013) showed that SOD is the most important antioxidant enzyme, which can effectively remove excess free radicals in the body. The SOD activity index in myocardial tissue of the adenosine group was better than that of the ischemia-reperfusion group.

[0185] The present invention unexpectedly discovered that the presence of 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage) of ergothioneine in the Cordyceps sinensis fermentation composition is not only the first Cordyceps sinensis fermentation product with ergothioneine, but also enhances the effect of adenosine. The inventors unexpectedly discovered that the combination of ergothioneine and adenosine exhibits a significant synergistic effect in scavenging hydroxyl radicals. That is, the present invention ensures that the ergothioneine in the obtained Cordyceps sinensis fermentation composition is sufficiently and stably present at a content of 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage), and at the same time, the adenosine content is particularly preferably 0.02% or more (weight percentage), 0.03% or more (weight percentage), preferably 0.03-1.00% (weight percentage), preferably 0.03-0.80% (weight percentage), preferably 0. 03-0.60% (weight percentage), preferably 0.03-0.40% (weight percentage), preferably 0.03-0.20% (weight percentage), preferably 0.03-0.10% (weight percentage), preferably 0.03-0.08% (weight percentage); the ratio of ergothioneine to adenosine content is 1:1-1:20, preferably 1:1-1:15, preferably 1:1-1:10, preferably 1:1-1:8, preferably 1:1-1:7, preferably 1:1-1:6, preferably 1:1-1:5, preferably 1:1-1:4, preferably 1:1-1:3, preferably 1:1-1:2.

[0186] Furthermore, as previous studies have shown that adenosine may be converted from adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, nucleoside substances represented by adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate also have important physiological activities, such as Qian Jia et al. (Study on the in vitro antioxidant effect of 5′-acid adenosine (5′-AMP), Food and Machinery, Vol. 24, No. 1, January 2008) showed that AMP has a strong activity in scavenging hydroxyl free radicals. Therefore, on the basis of ensuring the stable existence of ergothioneine and adenosine with synergistic technical effects, the present invention controls the conversion amount of adenosine to a certain extent, so that the Cordyceps sinensis fermentation composition as a whole shows a more excellent technical effect. Previous studies such as Chen Jiaming et al. (Effects of different carbon sources and nitrogen sources on the solid fermentation process of Chinese hairy spores, Chinese Edible Fungi 2017, 37 (1): 55-60) showed that different carbon sources and nitrogen sources had a synergistic effect on the solid fermentation process of Chinese hairy spores ( Hirsutella sinensis ) The influence of solid fermentation process. The highest adenosine obtained by solid fermentation method was only 63.76 μg·g in formula G7. -1, and Zhang Ping et al. (Research Progress on Quality Evaluation and Control of Cordyceps Fermentation Products, Chinese Journal of Pharmacy, July 2021, Vol. 56, No. 14) recorded that the liquid fermentation culture conditions of Chinese Hirsutum were optimized, and the dry weight of the optimized Chinese Hirsutum mycelium increased by 36.2%, and the adenosine content increased by 46.8%. Amino nitrogen is the most important limiting matrix for bacterial growth during the fermentation process. Therefore, obtaining amino nitrogen during the liquefaction process is very important for increasing Cordyceps yield and effective utilization of raw materials (CN103444434A), but some studies have shown that the growth rate of bacteria is fastest when the concentration of carbon and nitrogen sources is low. As the concentration of carbon and nitrogen sources increases, the growth rate of bacteria decreases, indicating that higher concentrations of carbon and nitrogen sources are not conducive to bacterial growth (CN103430777A).

[0187] The present invention unexpectedly found that the content of adenosine is not as high as possible. The Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02% or more (weight percentage), 0.03% or more (weight percentage), preferably 0.03-1.00% (weight percentage), preferably 0.03-0.80% (weight percentage), preferably 0.03-0.60% (weight percentage), preferably 0.03-0.40% (weight percentage), preferably 0.03-0.50% (weight percentage), preferably 0.03-0.60% (weight percentage), preferably 0.03-0.70% (weight percentage), preferably 0.03-0.8 ... 0.03-0.20% (weight percentage), preferably 0.03-0.10% (weight percentage), preferably 0.03-0.08% (weight percentage); the ratio of ergothioneine to adenosine is 1: 1-1: 20, preferably 1: 1-1: 15, preferably 1: 1-1: 10, preferably 1: 1-1: 8, preferably 1: 1-1: 7, preferably 1: 1-1: 6, preferably 1: 1-1: 5, preferably 1: 1-1: 4, preferably 1: 1-1: 3, preferably 1: 1-1: 2. In the present invention, when the content of adenosine and ergothioneine is within the above preferred range, the Cordyceps sinensis fermentation composition as a whole can have excellent antioxidant effect, which well meets the application requirements.

[0188] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:20; or,

[0189] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:15; or,

[0190] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:10; or,

[0191] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:8; or,

[0192] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:7; or,

[0193] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:6; or,

[0194] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:5; or,

[0195] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:4; or,

[0196] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:3; or,

[0197] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:2; or,

[0198] More specifically, the present invention preferably has an ergothioneine content of 0.01% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:20; or,

[0199] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine of 1:1-1:15; or,

[0200] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine of 1:1-1:10; or,

[0201] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine of 1:1-1:8; or,

[0202] More specifically, the present invention preferably has an ergothioneine content of 0.01% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:7; or,

[0203] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine of 1:1-1:6; or,

[0204] More specifically, the present invention preferably has an ergothioneine content of 0.01% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:5; or,

[0205] More specifically, the present invention preferably has an ergothioneine content of 0.01% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:4; or,

[0206] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine of 1:1-1:3; or,

[0207] More specifically, the present invention preferably has an ergothioneine content of 0.01% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:2; or,

[0208] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.02% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:20; or,

[0209] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.02% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:15; or,

[0210] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.02% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:10; or,

[0211] More specifically, the present invention preferably contains more than 0.02% adenosine (by weight) on the basis of more than 0.02% ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:8; or,

[0212] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.02% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:7; or,

[0213] More specifically, the present invention preferably contains more than 0.02% adenosine (by weight) on the basis of more than 0.02% ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:6; or,

[0214] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.02% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:5; or,

[0215] More specifically, the present invention preferably contains more than 0.02% adenosine (by weight) on the basis of more than 0.02% ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:4; or,

[0216] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.02% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:3; or,

[0217] More specifically, the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.02% or more ergothioneine, and the ratio of ergothioneine to adenosine is 1:1-1:2; or,

[0218] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine of 1:1-1:20; or,

[0219] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine of 1:1-1:15; or,

[0220] More specifically, the present invention preferably has an ergothioneine content of 0.02% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:10; or,

[0221] More specifically, the present invention preferably has an ergothioneine content of 0.02% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:8; or,

[0222] More specifically, the present invention preferably has an ergothioneine content of 0.02% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:7; or,

[0223] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine of 1:1-1:6; or,

[0224] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine of 1:1-1:5; or,

[0225] More specifically, the present invention preferably has an ergothioneine content of 0.02% or more and an adenosine content of 0.03%-0.08% (percentage by weight), and a ratio of ergothioneine to adenosine of 1:1-1:4; or,

[0226] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (weight percentage) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine of 1:1-1:3; or,

[0227] More specifically, the present invention preferably has an ergothioneine content of 0.02% or more and an adenosine content of 0.03%-0.08% (weight percentage), and the ratio of ergothioneine to adenosine is 1:1-1:2.

[0228] Those skilled in the art will appreciate that the above-mentioned is only an explanation of the present invention, and is not interpreted as limiting the application. Due to the research on ergothioneine and adenosine content, the fermented composition obtained in the described scope has a very excellent antioxidant effect, especially the synergistic technical effect of hydroxyl radical scavenging rate.

[0229] On the other hand, the present invention combines the content of ergothioneine, the conversion behavior of adenosine, on the basis of ergothioneine, erythritol, adenosine content, further studies the yield of mycelium dry weight, that is, the inventors ensure ergothioneine, erythritol, adenosine content on the basis of further through the optimization of fermentation process, obtain mycelium yield higher Cordyceps sinensis fermentation composition, mycelium fermentation yield is more than 7g / L, preferably 7g / L-15g / L, 7g / L-20g / L, 8g / L-15g / L, 8g / L-20g / L, mycelium dry weight water content of 8% or less, preferably water content of 7% or less, preferably water content of 6% or less.

[0230] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02% or more (by weight) on the basis of an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, and a mycelium dry weight water content of 7% or less; or,

[0231] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02% or more (by weight) on the basis of an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, and a mycelium fermentation yield of 7 g / L or more; or,

[0232] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-0.2% (weight percentage) on the basis of an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, and a mycelium dry weight water content of 7% or less; or,

[0233] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-0.2% (weight percentage) based on an ergothioneine content of more than 0.01%, a ratio of ergothioneine to adenosine of 1:1-1:20, and a mycelium fermentation yield of more than 7 g / L.

[0234] Further, through in-depth research by the inventors, on the basis of the presence of ergothioneine at a content of more than 0.004% (weight percentage), preferably more than 0.006% (weight percentage), preferably more than 0.008% (weight percentage), preferably more than 0.01%, the present invention unexpectedly found that the presence of more than 0.004% ergothioneine in the Cordyceps sinensis fermentation composition is not only the first Cordyceps sinensis fermentation product with ergothioneine, but also greatly enhances the effects of other beneficial ingredients. The present invention shows through research experiments that ergothioneine in the Cordyceps sinensis fermentation composition helps to enhance the effects of adenosine and the like, and produces a synergistic effect, especially in terms of antioxidant effect, so that the Cordyceps sinensis fermentation composition as a whole shows a more excellent technical effect.

[0235] Therefore, another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the ergothioneine content is above 0.004% (weight percentage), preferably above 0.006% (weight percentage), preferably above 0.008% (weight percentage), preferably above 0.01% (weight percentage), and the fermentation composition further comprises one or more of adenosine, mannitol, amino acids, polysaccharides, and erythritol.

[0236] Furthermore, the Cordyceps sinensis fermentation composition has an erythritol content of more than 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage).

[0237] Furthermore, the Cordyceps sinensis fermentation composition has a mannitol content of more than 4% (weight percentage), preferably more than 5%, preferably more than 6%, preferably more than 7%.

[0238] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more and an erythritol content of 1% or more (percentage by weight); or

[0239] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more, an erythritol content of 1% or more (weight percentage), and an adenosine content of 0.02% or more (weight percentage); or,

[0240] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more, an erythritol content of 1% or more (weight percentage), an adenosine content of 0.02% or more (weight percentage), and a mannitol content of 5% or more (weight percentage); or,

[0241] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more, an adenosine content of 0.02% (weight percentage) or more, and a mannitol content of 5% or more (weight percentage); or,

[0242] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more, an adenosine content of 0.02% (weight percentage) or more, and a mannitol content of 6% or more (weight percentage); or,

[0243] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01%, an adenosine content of more than 0.02% (weight percentage), and a mannitol content of more than 7% (weight percentage).

[0244] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the ergothioneine content is 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage), and the polysaccharide content is preferably 2% or more (weight percentage); or,

[0245] Furthermore, the Cordyceps sinensis fermentation composition has a polysaccharide content of more than 2% (weight percentage), preferably more than 3% (weight percentage), preferably 2-10% (weight percentage).

[0246] Furthermore, the Cordyceps sinensis fermentation composition of the present invention preferably has a polysaccharide content of 2% or more (by weight) and a total amino acid content of 20% or more, based on an ergothioneine content of 0.01% or more;

[0247] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has a polysaccharide content of 2%-10% (weight percentage) and a total amino acid content of 25-50% based on an ergothioneine content of more than 0.01%.

[0248] Those skilled in the art will appreciate that the above-mentioned is only an explanation of the present invention, and is not interpreted as limiting the present invention. Due to the research on ergothioneine, adenosine content, mannitol, polysaccharide, erythritol, the fermentation composition obtained within the scope has very excellent technical effect.

[0249] On the other hand, the present invention also provides a Cordyceps sinensis fermentation composition, wherein the ergothioneine content is above 0.004% (weight percentage), preferably above 0.006% (weight percentage), preferably above 0.008% (weight percentage), preferably above 0.01% (weight percentage), and the erythritol content is above 1% (weight percentage), preferably 1-6% (weight percentage), and further preferably 2-5% (weight percentage). When the ergothioneine and erythritol contained in the Cordyceps sinensis fermentation composition provided by the present invention are within the preferred limited range of the present invention, the combination of the two exhibits a synergistic effect in scavenging hydroxyl radicals, thereby enhancing the overall antioxidant property of the Cordyceps sinensis fermentation composition.

[0250] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the ergothioneine content is above 0.004% (weight percentage), preferably above 0.006% (weight percentage), preferably above 0.008% (weight percentage), preferably above 0.01% (weight percentage), the adenosine content is above 0.02% (weight percentage), and the mannitol content is above 4% (weight percentage).

[0251] Furthermore, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01% (weight percentage), an adenosine content of more than 0.02% (weight percentage), a mannitol content of more than 4% (weight percentage), and a polysaccharide content of more than 2% (weight percentage).

[0252] Furthermore, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01% (weight percentage), an adenosine content of more than 0.02% (weight percentage), a mannitol content of more than 4% (weight percentage), and a total amino acid content of more than 20% (weight percentage), preferably 25-50% (weight percentage).

[0253] Furthermore, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01% (weight percentage), an adenosine content of more than 0.02% (weight percentage), a mannitol content of more than 4% (weight percentage), a polysaccharide content of more than 2% (weight percentage), and a total amino acid content of more than 20% (weight percentage), preferably 25-50% (weight percentage).

[0254] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02% or more (by weight) based on an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, and a mannitol content of 5% or more (by weight); or,

[0255] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.03% or more (by weight) based on an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, and a mannitol content of 5% or more (by weight); or,

[0256] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02% or more (by weight) based on an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, a mannitol content of 5% or more (by weight), and a polysaccharide content of 2% or more (by weight); or,

[0257] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02% or more (weight percentage) on the basis of an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, a mannitol content of 5% or more (weight percentage), and a total amino acid content of 20% or more (weight percentage); or,

[0258] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-1.0% (weight percentage) based on an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, and a mannitol content of 5% or more (weight percentage); or,

[0259] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-1.0% (weight percentage) based on an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, a mannitol content of 5% or more (weight percentage), and a polysaccharide content of 2% or more (weight percentage); or,

[0260] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-1.0% (weight percentage) on the basis of an ergothioneine content of 0.01% or more, a ratio of ergothioneine to adenosine of 1:1-1:20, a mannitol content of 5% or more (weight percentage), and a total amino acid content of 20% or more (weight percentage); or,

[0261] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-1.0% (weight percentage) on the basis of an ergothioneine content of more than 0.01%, a ratio of ergothioneine to adenosine of 1:1-1:20, a mannitol content of more than 5% (weight percentage), a polysaccharide content of more than 2% (weight percentage), and a total amino acid content of more than 20% (weight percentage).

[0262] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, comprising adenosine, wherein the fermentation composition further comprises ergothioneine, and the content (weight percentage) of ergothioneine is above 0.01%.

[0263] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition further comprises ergothioneine, and the content (weight percentage) of ergothioneine is above 0.01%.

[0264] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol and polysaccharide, wherein the fermentation composition further comprises ergothioneine, and the content (weight percentage) of ergothioneine is above 0.01%.

[0265] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol, polysaccharides and amino acids, wherein the fermentation composition also comprises ergothioneine, and the content (weight percentage) of ergothioneine is above 0.01%.

[0266] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine, and the content of ergothioneine (weight percentage) is above 0.01%, and the content of adenosine is 0.02-1.0% (weight percentage).

[0267] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine, and the content (weight percentage) of ergothioneine is above 0.01%, and the ratio of ergothioneine to adenosine is 1:1-1:20.

[0268] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine, and the content of ergothioneine (weight percentage) is above 0.01%, the content of adenosine is 0.02-1.0% (weight percentage), and the ratio of ergothioneine to adenosine is 1:1-1:20.

[0269] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition also comprises ergothioneine, and the content of ergothioneine (weight percentage) is above 0.01%, the content of adenosine is 0.02-1.0% (weight percentage), and the content of mannitol is above 4% (weight percentage).

[0270] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine and mannitol, wherein the fermentation composition also includes ergothioneine, and the content of ergothioneine (weight percentage) is above 0.01%, the content of adenosine is 0.02-1.0% (weight percentage), the ratio of ergothioneine to adenosine is 1:1-1:20, and the content of mannitol is above 4% (weight percentage).

[0271] Therefore, another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the content of ergothioneine is 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage), the content of adenosine is 0.02% or more (weight percentage), and the content of erythritol is 1.0% or more (weight percentage).

[0272] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably contains 0.02% or more adenosine (by weight) on the basis of 0.01% or more ergothioneine, the ratio of ergothioneine to adenosine is 1:1-1:20, and 1.0% or more erythritol (by weight); or,

[0273] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-1.0% (weight percentage) and an erythritol content of 1.0% or more (weight percentage) based on an ergothioneine content of 0.01% or more.

[0274] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, comprising adenosine, wherein the fermentation composition further comprises ergothioneine and erythritol, and the content (weight percentage) of ergothioneine is above 0.01%.

[0275] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition further comprises ergothioneine and erythritol, and the content (weight percentage) of ergothioneine is above 0.01%.

[0276] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol and polysaccharide, wherein the fermentation composition further comprises ergothioneine and erythritol, and the content (weight percentage) of ergothioneine is above 0.01%.

[0277] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, mannitol, polysaccharides and amino acids, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%.

[0278] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%, and the content of adenosine is 0.02-1.0% (weight percentage).

[0279] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine and erythritol, and the content (weight percentage) of ergothioneine is above 0.01%, and the ratio of ergothioneine to adenosine is 1:1-1:20.

[0280] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%, the content of adenosine is 0.02-1.0% (weight percentage), and the ratio of ergothioneine to adenosine is 1:1-1:20.

[0281] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine and mannitol, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%, the content of adenosine is 0.02-1.0% (weight percentage), and the content of mannitol is above 4% (weight percentage).

[0282] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine and mannitol, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%, the content of adenosine is 0.02-1.0% (weight percentage), the ratio of ergothioneine to adenosine is 1:1-1:20, and the content of mannitol is above 4% (weight percentage).

[0283] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, including adenosine, wherein the fermentation composition also includes ergothioneine and erythritol, and the erythritol is more than 1% (weight percentage).

[0284] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine and mannitol, wherein the fermentation composition also includes ergothioneine and erythritol, and the erythritol is more than 1% (weight percentage).

[0285] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, mannitol and polysaccharide, wherein the fermentation composition also includes ergothioneine and erythritol, and the erythritol is more than 1% (weight percentage).

[0286] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, mannitol, polysaccharides and amino acids, wherein the fermentation composition also includes ergothioneine and erythritol, and the erythritol is more than 1% (weight percentage).

[0287] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine and erythritol, and the erythritol content is above 1% (weight percentage), and the adenosine content is 0.02-1.0% (weight percentage).

[0288] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine and erythritol, and the content (weight percentage) of ergothioneine is above 0.01%, and the content of erythritol is above 1% (weight percentage).

[0289] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%, the content of erythritol is above 1% (weight percentage), and the content of adenosine is 0.02-1.0% (weight percentage).

[0290] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine and mannitol, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%, the content of erythritol is above 1% (weight percentage), the content of adenosine is 0.02-1.0% (weight percentage), and the content of mannitol is above 4% (weight percentage).

[0291] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, which comprises erythritol and mannitol, and the content of mannitol is above 1% (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage). The Cordyceps sinensis fermentation composition has a mannitol content of above 4% (weight percentage), preferably above 5%, preferably above 6%, and preferably above 7%.

[0292] Furthermore, the Cordyceps sinensis fermentation composition has a polysaccharide content of more than 2% (weight percentage), preferably more than 3% (weight percentage), preferably 2-10% (weight percentage).

[0293] Furthermore, the Cordyceps sinensis fermentation composition of the present invention has an adenosine content of more than 0.02% (weight percentage), more than 0.03% (weight percentage), preferably 0.03-1.00% (weight percentage), preferably 0.03-0.80% (weight percentage), preferably 0.03-0.60% (weight percentage), preferably 0.03-0.40% (weight percentage), preferably 0.03-0.20% (weight percentage), preferably 0.03-0.10% (weight percentage), preferably 0.03-0.08% (weight percentage).

[0294] Furthermore, the Cordyceps sinensis fermentation composition has a polysaccharide content of more than 2% (weight percentage), preferably more than 3% (weight percentage), preferably more than 4% (weight percentage), preferably 2-10% (weight percentage).

[0295] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0296] The Cordyceps sinensis fermentation composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, capsules, or the parts are mixed and the mixture is encapsulated or the mixture is made into tablet form to obtain a single preparation. It can also be prepared into various dosage forms with acceptable carriers. For oral administration compositions (such as tablets and capsules), the term "acceptable carrier" includes such carriers, such as common excipients, such as adhesives, such as syrup, gum arabic, gelatin, sorbitol, tragacanth gum, polyvinyl pyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose and starch; fillers and carriers such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose; lubricants such as magnesium stearate, sodium stearate and other stearates, glycerol stearate, stearic acid, silicone fluid, talc, wax, oil and colloidal silicon dioxide. Flavoring agents such as mint, wintergreen oil, cherry seasoning, etc. can also be used. Colorants may need to be added to distinguish the dosage forms. Tablets may also be coated by methods well known in the art. Tablets may be compressed or molded, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine a free-flowing form of the active ingredient, such as a powder or granules, optionally mixed with one or more binders, lubricants, inert diluents, preservatives, surface active agents or dispersants.

[0297] In summary, the Cordyceps sinensis fermentation composition of the present invention is not only the first Cordyceps sinensis fermentation product with ergothioneine, but also, due to the research on ergothioneine and adenosine content, the fermentation composition obtained within the scope has a very excellent antioxidant effect, so that the Cordyceps sinensis fermentation composition of the present invention shows a more excellent technical effect than the prior art as a whole.

[0298] Therefore, the present invention also provides a pharmaceutical composition, comprising a combination of ergothioneine and adenosine, wherein the combination ratio of ergothioneine to adenosine is 1:1-1:20.

[0299] Further, the ratio of ergothioneine to adenosine content is preferably 1:1-1:15, preferably 1:1-1:10, preferably 1:1-1:8, preferably 1:1-1:7, preferably 1:1-1:6, preferably 1:1-1:5, preferably 1:1-1:4, preferably 1:1-1:3, preferably 1:1-1:2.

[0300] The present invention also provides a pharmaceutical composition, comprising a combination of ergothioneine and erythritol, wherein the combination ratio of ergothioneine to erythritol is 1:1-1:600, preferably 1:1-1:500, preferably 1:1-1:400, preferably 1:1-1:300, preferably 1:1-1:200, preferably 1:1-1:100.

[0301] The pharmaceutical composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, capsules, or mixed with the parts and the mixture is filled into capsules or the mixture is made into tablets to obtain a single preparation. It can also be prepared into various dosage forms using acceptable carriers.

[0302] The pharmaceutical composition of the present invention has excellent antioxidant effect and synergistic technical effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0303] Figure 1 HPLC spectrum of ergothioneine standard

[0304] Figure 2 HPLC spectrum of ergothioneine of sample B-5

[0305] Figure 3 HPLC spectrum of ergothioneine of B-7 sample

[0306] Figure 4 Example 4: Adenosine HPLC spectrum of CN101096641A sample

[0307] Figure 5 HPLC spectrum of adenosine of sample B-2 in Example 4

[0308] Figure 6 HPLC spectrum of erythritol of sample B-2

[0309] Figure 7 The standard curve of polysaccharide DETAILED DESCRIPTION

[0310] 1. Ergothioneine Detection Method

[0311] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0312] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; 0.2% heptafluorobutyric acid was used as mobile phase A, and methanol was used as mobile phase B; the detection wavelength was 255 nm. The theoretical plate number calculated based on the ergothioneine peak should be no less than 3000.

[0313] Preparation of reference solution: An appropriate amount of ergothioneine reference substance is accurately weighed and added with 0.5% phosphoric acid solution to make a solution containing μg per 1 ml. Gradient elution is performed according to the following table:

[0314]

[0315] Preparation of the test solution: Take 0.5 g of fermented Cordyceps sinensis powder, weigh it accurately, put it in a stoppered conical flask, add 20 ml of ether, stopper it, soak it for 30 minutes, filter it, discard the ether, evaporate the ether from the residue, put it in a stoppered conical flask together with the filter paper, accurately add 50 ml of 0.5% phosphoric acid solution, stopper it, weigh it, and treat it with ultrasound for 30 minutes at a power of 250 W and a frequency of 33 kHz. Cool it, shake it well, centrifuge it, and take the supernatant.

[0316] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0317] 2. Detection method of erythritol

[0318] Preparation of reference solution

[0319] Take an appropriate amount of erythritol reference substance, weigh it accurately, and add deionized water to make a solution containing 1000 μg per 1 ml.

[0320] Preparation of test solution

[0321] Take 0.5 g of fermented Cordyceps sinensis powder, accurately weigh it, put it into a stoppered conical flask, accurately add 10 ml of deionized water, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 60 min, cool it, weigh it again, make up the lost weight with deionized water, shake it well, filter it, and take the filtrate to obtain it.

[0322] Detection Methods

[0323] Detector: RID differential detector;

[0324] Chromatographic column: Model Agilent Hi-Plex Ca, 300×7.7mm, 8um;

[0325] Mobile phase: deionized water;

[0326] Column flow rate: 0.6 ml / min; column temperature: 80°C; injection volume: 10 ul; detection time: 50 min;

[0327] Circulation cell temperature: 35°C.

[0328] 3. Anthrone-sulfuric acid detection method for polysaccharides

[0329] Solution preparation

[0330] Anthrone sulfate solution: Accurately weigh 0.1 g of anthrone, add 100 mL of sulfuric acid solution to dissolve it, shake well, and place in a brown bottle.

[0331] Preparation of standard curve

[0332] Preparation of reference solution

[0333] Take an appropriate amount of anhydrous glucose reference substance, accurately weigh it, and add water to make a solution containing 0.1 mg per 1 mL.

[0334] Standard curve drawing

[0335] Accurately measure 0, 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of the reference solution and place them in 20 mL stoppered test tubes, respectively. Add water to 2.0 mL each, quickly and accurately add 6 mL of anthrone sulfate solution, shake well immediately, let stand for 15 min, immediately cool in an ice water bath for 15 min, take out and let stand at room temperature for 10 min, use the corresponding reagent as blank, measure the absorbance at 625 nm, and draw a standard curve with absorbance as the ordinate, see Figure 7 .

[0336] Preparation of test solution

[0337] Take about 2 g of fermented Cordyceps sinensis powder, weigh it accurately, put it in a round-bottom flask, add 60 mL of water, let it stand for 30 min, heat to reflux for 3 h, transfer it to a centrifuge tube while it is hot, wash the filter and filter residue with a small amount of hot water, centrifuge at 5000rpm for 10 min, take the supernatant, transfer the precipitate to a round-bottom flask with a small amount of hot water, add 60 mL of water, heat to reflux for 2 h, transfer it to a centrifuge tube while it is hot, centrifuge again, combine the supernatant, evaporate it in a water bath, dissolve the residue with 5 mL of water, slowly add 75 mL of ethanol while stirring, shake well, let it stand at 4°C for 12 h, centrifuge, discard the supernatant, dissolve the precipitate with hot water and transfer it to 50 mL, let it cool, add water to the scale, shake well, take an appropriate amount of solution, centrifuge, accurately measure 1 mL of supernatant, put it in a 25 mL volumetric flask, add water to the scale, shake well, and obtain it.

[0338] Determination

[0339] Accurately measure 2 mL of the test solution and place it in a 10 mL stoppered test tube. Follow the method under the standard curve preparation, starting from "rapidly and accurately add 6 mL of anthrone sulfate solution", and operate in the same way to measure the absorbance. Read the anhydrous glucose content in the test solution from the standard curve and calculate it.

[0340] Result calculation

[0341]

[0342] Where:

[0343] W -Polysaccharide content, %;

[0344] c- Polysaccharide concentration of the sample from the standard curve, mg / mL;

[0345] m - Sample mass, mg;

[0346] 8 / 2 and 25 / 2 indicate dilution multiples;

[0347] 50-The volume of the precipitate obtained after water extraction and alcohol precipitation is dissolved in hot water to make up the volume.

[0348] 4. Hydroxyl free radical scavenging rate detection (Fenton colorimetric method)

[0349] Detection principle

[0350] H2O2 / Fe 2+ Hydroxyl radicals are generated by Fenton reaction and Fe 2+ Oxidized to Fe 3+ , resulting in the red phenanthroline-Fe 2+ Oxidized to colorless o-dihydrophenanthrene-Fe 3+ , making o-phenanthroline-Fe 2+ The maximum absorption peak at 536 nm disappears, and the change in absorbance at 530-540 nm can be measured by a spectrophotometer. Based on this, the change in the content of hydroxyl free radicals can be calculated, and the hydroxyl free radical scavenging rate or scavenging capacity of the sample can be calculated.

[0351] Solution preparation

[0352] 1. Weigh 0.2 g bacterial powder, add 2 ml purified water, mix well, let stand at room temperature for 4 h, centrifuge at 10000 rpm for 10 min, and take the supernatant for later use.

[0353] 2. Other samples are reference solutions.

[0354] The spectrophotometer was turned on and preheated for 30 min, the wavelength was adjusted to 536 nm, and the purified water was used to zero the instrument.

[0355] Add sample

[0356] Set up blank tubes, undamaged tubes, damaged tubes, control tubes, and assay tubes according to the table below. Add the solutions in sequence, mix well, and place in a 37°C water bath for 1 h.

[0357]

[0358] Calculation: OH removal rate of the sample to be tested (%) = [(A3-A3')-(A2-A0)] / (A1-A2)×100%

[0359] 5. The detection methods for adenosine, mannitol, total amino acid content and moisture shall be in accordance with the detection methods for fermented Cordyceps sinensis powder in the national drug standards.

[0360] 6. Detection method of tyrosinase inhibition rate

[0361] 1. Solution preparation

[0362] (1) Preparation of PBS buffer: Weigh 3.4022 g of potassium dihydrogen phosphate and 5.7055 g of potassium dihydrogen phosphate trihydrate respectively, dissolve them in appropriate amount of distilled water, make up to volume in a 1000 mL volumetric flask, shake well to obtain a PBS buffer with pH = 6.81 and 0.05 mol·L-1, and store it in a refrigerator at 4°C away from light.

[0363] (2) Preparation of L-DOPA solution: Accurately weigh 0.0395 g of L-DOPA, dissolve it in the prepared PBS buffer, transfer it to a 100 mL volumetric flask, make up to volume, shake well, and sonicate to obtain a 2 mmol·L-1 L-DOPA solution, which is ready for use.

[0364] (3) Preparation of TYR (tyrosine kinase) solution: Dissolve 25 KU of TYR in a small amount of PBS buffer, repeat washing several times, transfer the washing solution to a 100 mL volumetric flask, make up to volume with PBS buffer, shake well to obtain 250 U·mL-1 TYR solution, then dilute the mother liquor to 125 U·mL-1 and store in a refrigerator at -20℃ for later use.

[0365] 2. Sample preparation:

[0366] 250mg / ml erythritol mother solution: weigh 2.5g erythritol and add to 10ml purified water; 250mg / ml mannitol mother solution: weigh 7.5g mannitol and add to 30ml purified water, heat to dissolve, and dilute to the required concentration according to each reaction system and then add to the detection system.

[0367] 3. Determination of the inhibitory activity of samples on TYR

[0368] The reaction system is shown in the table below. With 2 mmol·L-1 L-DOPA as the substrate, PBS buffer, sample solution and substrate are added to 96-well plates numbered 1, 2, 3, and 4 in sequence, and each well is repeated three times. The temperature of the multifunctional microplate reader is set to 37°C, and the plate is shaken linearly for 10 min before being taken out. Finally, TYR solution is added and quickly put back. The detection wavelength is 475 nm. The total volume of the reaction system is 240 μL. The absorbance is measured every 30 s for a total of 30 min. The inhibition rate is calculated based on the absorbance of 30 min. The inhibition rate calculation formula is as follows. When the calculation result is a negative number, the absolute value of the negative number is the activation rate.

[0369]

[0370] (4.1)

[0371] A1: absorbance of the reaction solution without adding sample but adding TYR at 475 nm;

[0372] A2: absorbance of the reaction solution without sample and TYR at 475 nm;

[0373] A3: absorbance of the reaction solution with sample and TYR measured at 475 nm;

[0374] A4: The absorbance of the reaction solution with sample added but without TYR measured at 475 nm.

[0375] Example 1 Ergothioneine (EGT) and Erythritol Contents in Different Mycelia

[0376] The inventor's research shows that ergothioneine is not detected in the culture soil, larvae, and silkworm pupae of natural Cordyceps sinensis, and only 0.008% of ergothioneine is detected in some fresh insect bodies and dry insect bodies. Therefore, it is difficult to detect ergothioneine from the mycelium of natural Cordyceps sinensis:

[0377]

[0378] Although in theory, the content of nutrients and medicinal effects of fungi isolated from natural Cordyceps sinensis in the mycelium are similar to those of natural Cordyceps sinensis, the components of Cordyceps sinensis fermentation products are still different from those of natural Cordyceps sinensis, especially the metabolites and secondary metabolites in the fermentation process, which are not only different from those of natural Cordyceps sinensis, but also have great differences between the fermentation products of different Cordyceps sinensis. Based on the research of natural Cordyceps sinensis, the inventors collected several strains of Chinese Hirsutella sibiricum ( Hirsutella sinensis ) Such as China Center for Type Culture Collection (CCTCC) Chinese Hirsutella Hirsutella sinensis ), with a collection number of CCTCC No: M 2011278, and Hirsutella sinensis purchased from China General Microbiological Culture Collection Center (CGMCC) Hirsutella sinensis ) CGMCC 3.14240 and Hirsutella sinensis ( Hirsutella sinensis )CGMCC3.14243 was purchased from China General Microbiological Culture Collection Center (CGMCC) Paecilomyces hepiali ) 3.15540, Paecilomyces pyralis ( Paecilomyces hepiali )CGMCC3.7845, Paecilomyces pyralis ( Paecilomyces hepiali)CGMCC3.7908, further referring to the prior art, on the basis of excluding the possible interference of exogenous ergothioneine (for example, ergothioneine was not detected in the culture medium using silkworm pupa powder), the fermentation filtrate and mycelium dry powder in the existing Cordyceps sinensis fermentation process were studied. Not only were the amounts of ergothioneine both lower than those detectable in natural Cordyceps sinensis, but most of them were not detected. However, the inventor unexpectedly discovered that trace amounts of ergothioneine could be obtained by fermenting mycelium of Hirsutella sibiricum.

[0379]

[0380] Note: ∓ refers to the peak area, the content is only 3‰-10‰ of natural Cordyceps sinensis.

[0381] As shown in the table above, the inventors performed fermentation with reference to the method disclosed in the prior art, and no significant ergothioneine was obtained from the fermentation filtrate and mycelium during the fermentation process.

[0382] Furthermore, in addition to the strains available from the above-mentioned collection centers, the inventors also tested several commercially available strains of Hirsutella sinensis ( Hirsutella sinensis ), for example, Cordyceps sinensis (Hairysporium batriaceum, Hairysporium sinensis) purchased from the China Industrial Culture Collection Center (CICC) Ophiocordyceps sinensis Standard strains CICC 14092, CICC 14095, CICC 14097, CICC 14099, and Cordyceps sinensis (Hairysporium batriaceum, Hairysporium sinensis) purchased from Testol Biotechnology Ophiocordyceps sinensis The product numbers are TS326189, TS326199 and Chinese Hirsutella sinensis ( Hirsutella sinensis ) The strains with item numbers TS361737 and TS392986 were fermented according to the prior art, and no ergothioneine was obtained in the obtained fermentation filtrate and mycelium.

[0383] Similarly, the inventors tested several commercially available strains of Paecilomyces pyralis ( Paecilomyces hepiali ), for example, deposit numbers are CGMCC 3.15421, CGMCC 3.14870, CGMCC 3.12108, CGMCC 3.10157, CCTCC NF20081231, CCTCC NF 20081239, CCTCC NF 20081244, and CCTCC NF 20081250, which are fermented with reference to prior art, and thioneine is not obtained in the resulting fermentation filtrate and mycelium.

[0384]

[0385] As shown in the table above, the inventors carried out fermentation according to the method disclosed in the prior art, but failed to obtain a stable and meaningful amount of erythritol in the fermentation filtrate and mycelium during the fermentation process.

[0386] Furthermore, in addition to the strains available from the above-mentioned collection centers, the inventors also tested several commercially available strains of Hirsutella sinensis ( Hirsutella sinensis ), for example, Cordyceps sinensis (Hairysporium batriaceum, Hairysporium sinensis) purchased from the China Industrial Culture Collection Center (CICC) Ophiocordyceps sinensis Standard strains CICC 14092, CICC 14095, CICC 14097, CICC 14099, and Cordyceps sinensis (Hairysporium batriaceum, Hairysporium sinensis) purchased from Testol Biotechnology Ophiocordyceps sinensis The product numbers are TS326189, TS326199 and Chinese Hirsutella sinensis ( Hirsutella sinensis ) The strains with item numbers TS361737 and TS392986 were fermented according to the prior art, but no stable and meaningful content of erythritol was obtained in the fermentation filtrate and mycelium.

[0387] Similarly, the inventors tested several commercially available strains of Paecilomyces pyralis ( Paecilomyces hepiali ), for example, the deposit numbers are CGMCC 3.15421, CGMCC 3.14870, CGMCC 3.12108, CGMCC 3.10157, CCTCC NF20081231, CCTCC NF 20081239, CCTCC NF 20081244, CCTCC NF 20081250. Fermentation is performed according to the prior art, and no stable and meaningful content of erythritol is obtained in the obtained fermentation filtrate and mycelium.

[0388] Example 2 Fermented product with stable content of ergothioneine (EGT) above 0.004%

[0389] Hirsutella sinensis ( Hirsutella sinensis ), the deposit number is CCTCC No: M 2011278, the Chinese Hirsutella species is inoculated on a slant, and slant culture, shake flask / seed bottle culture, and fermentation culture are performed as follows:

[0390] 1 Slant culture

[0391] 1.1 Formula of slant solid culture medium: 1.8% silkworm pupa powder, 1.5% corn flour, 2.0% glucose, 0.01% MgSO4, 0.02% KH2PO4, 1.0-3.0g / 60ml oatmeal, 0.65% agar powder, pH 6.8-7.2, prepared with water, sterilized at (118-122)℃ for (30±1)min.

[0392] 1.2 Under sterile conditions, use an inoculation shovel to dig out a 2×2 cm block of fungi, spread the mycelium evenly on a blank solid culture medium, place it in a biochemical incubator, and culture it at 15°C for 30 days.

[0393] 1.3 Store the cultured slant in a refrigerator at 0-8℃. The mycelium will grow slowly during this period. When the slant is fully grown, take it out and set aside.

[0394] 2 Shake flask culture

[0395] 2.1 Shake flask medium formula: silkworm pupa powder 2.0%, corn flour 2.5%, bran 2.5%, fish peptone 0.25%, peptone 0.75%, KH2PO4 0.02%, glucose 3.0%, MgSO4 0.01%, pH 6.8-7.2;

[0396] 2.2 Liquefaction: Weigh silkworm pupa powder, corn flour and bran according to the volume of liquefied material, add water and liquefy at (118-122)℃ for 30 minutes, take out the liquefied material and let it stand until the upper and lower layers are separated, then filter it with a filter screen with 6 layers of gauze to obtain the filtrate.

[0397] 2.3 Preparation of culture medium: Weigh the corresponding materials and dissolve them in the liquefied filtrate, make up to volume, and divide them into shake bottles with a filling volume of 1500ml / 5000ml. Sterilize at (118-122)℃ for (30±1)min.

[0398] 2.4 Digging: Cut the slope into approximately 1.5×2.0 cm blocks. Use a sterile rake to rake the cut blocks into the prepared digging / seed bottle culture medium. Culture at (13-16)°C, (120-140) rpm, and shake for 9-14 days.

[0399] 3. Fermentation culture:

[0400] 3.1 Basic fermentation medium formula: glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, pH 7.0.

[0401] 3.2 Culture medium preparation: shake flask volume 1000ml / 5000ml, (118-122)℃, sterilize for (30±1)min.

[0402] 3.3 Fermentation: inoculation amount 10%, temperature (13-16)℃, (120-140)rpm, shake culture for 7 days before bottling.

[0403] 3.4 Take an appropriate volume of fermentation liquid, vacuum filter to obtain wet bacterial slices, and dry to obtain bacterial powder samples.

[0404] Different ingredients were added to the above basic fermentation medium (glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01% (% are weight to volume ratio)) to obtain mycelium experimental samples numbered A-1 to A-8 and B-1 to B-8. The results of testing ergothioneine and erythritol are shown in the following table:

[0405]

[0406] Furthermore, in addition to the strains disclosed in the above patents, the inventors have also conducted studies on several commercially available strains of Hirsutella sinensis ( Hirsutella sinensis ), such as the Hirsutella sinensis purchased from the China General Microbiological Culture Collection Center (CGMCC) Hirsutella sinensis ) CGMCC 3.14240 and Hirsutella sinensis ( Hirsutella sinensis ) CGMCC3.14243, and Cordyceps sinensis (Hairysporium batriaceum, Hairysporium sinensis) purchased from China Industrial Microbiological Culture Collection (CICC) Ophiocordyceps sinensis Standard strains CICC 14092, CICC 14095, CICC14097, CICC 14099, and Cordyceps sinensis (Hairysporium batriaceum, Hairysporium sinensis) purchased from Testol Biotechnology Ophiocordyceps sinensis The product numbers are TS326189, TS326199 and Chinese Hirsutella sinensis ( Hirsutella sinensis ) The strains with article numbers TS361737 and TS392986 were prepared according to Example 2 Chinese Hirsutella spp. ( Hirsutella sinensis ) The fermentation method with the deposit number of CCTCC No: M 2011278 was repeated under the conditions of different culture media shown in the above table to perform fermentation, and different Chinese Hirsutella spp. ( Hirsutella sinensis ) The EGT content, adenosine content (%), and mannitol and erythritol content (%) in the bacterial powder samples fermented by the same conditions were not significantly different from those in the bacterial powder prepared with the preservation number CCTCC No: M2011278.

[0407] Example 3 Study on the effect of effective amount of ergothioneine or erythritol on the efficacy of Chinese Hirsutella serrata fermentation powder

[0408] 1. Results of synergistic experiment on scavenging rate of ergothioneine and adenosine hydroxyl radical

[0409]

[0410] 2. Experimental results on the synergistic effect of ergothioneine and erythritol on hydroxyl radical scavenging rate

[0411]

[0412] 3. Results of synergistic experiment on erythritol and adenosine hydroxyl radical scavenging rate

[0413]

[0414]

[0415] The theoretical values ​​stated in the table are the simple superposition of the hydroxyl radical scavenging rates of the two components;

[0416] From the above data, it can be seen that the combination of erythritol and adenosine produces a significant synergistic effect, and the hydroxyl free radical scavenging effect of the two components is at least 4.76 times higher than the theoretical value.

[0417] 4. Synergistic effect of erythritol and mannitol on tyrosinase inhibition rate

[0418]

[0419]

[0420]

[0421] 5. Experimental results on the synergistic effect of erythritol and mannitol on hydroxyl free radical scavenging rate

[0422]

[0423]

[0424]

[0425]

[0426]

[0427] Example 4 Experimental results of hydroxyl radical scavenging rate of fermented bacteria powder obtained by the present invention and fermented bacteria powder in the prior art

[0428] Experimental samples:

[0429] The preparation of mycelial samples B-2, B-5, and B-7 was the same as before;

[0430] Example 5 Sample preparation of CN108384726A:

[0431] 2.0% sorbitol, 0.1% sea cucumber extract and 0.15% angelica extract were added to the basic culture medium described in Example 1, and the mycelium was fermented using the culture medium.

[0432] Example 1 Sample preparation of CN101096641A:

[0433] The strains were inoculated on the slant, the culture medium formula was glucose 1.5%, corn flour 1.0%, dextrin 0.5%, bran 1.5%, yeast powder 0.5%, silkworm pupa powder 1.5%, peptone 1.0%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, agar powder 0.8%, and the rest was water, and cultured at 12°C for 25 days; then the strains were inoculated in the fermentation medium, the culture medium formula was glucose 1.0%, molasses 1.0%, silkworm pupa powder 0.5%, soybean cake powder 1.0%, yeast paste 0.5%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, and the rest was water, placed on a shaker, and cultured at 14°C for 8 days; then inoculated in the primary fermentation tank, cultured at 14°C for 8 days, and then expanded by 10 times the amount of the culture medium, fermented step by step, until a 30-ton tank. After leaving the tank, solid-liquid separation was carried out, and the solid was dried and crushed.

[0434]

[0435] Those skilled in the art will understand that the specific embodiments listed above are only illustrative of the present invention and are not to be construed as limiting the present invention. At the same time, those skilled in the art will understand that, in accordance with the spirit of the present invention, they can implement different preparation examples to achieve the same technical effects, which are all included in the scope of the present invention.

Claims

1. A Cordyceps sinensis fermentation composition, characterized in that: The fermentation composition contains erythritol, mannitol and adenosine, wherein the content of erythritol is 1-2.5 wt %, the content of mannitol is more than 4 wt %, and the content of adenosine is more than 0.02 wt %; The fermented composition is prepared from Hirsutella chinensis ( Hirsutella sinensis ) is obtained by fermentation.

2. The fermentation composition according to claim 1, characterized in that The content of mannitol is above 5 wt %.

3. The fermentation composition according to claim 2, characterized in that The content of mannitol is above 6 wt %.

4. The fermentation composition according to claim 1, characterized in that The fermented composition contains ergothioneine, and the content of the ergothioneine is greater than 0.004 wt%.

5. The fermentation composition according to claim 1, characterized in that The fermented composition contains amino acids and / or polysaccharides.

6. Use of the fermentation composition according to any one of claims 1 to 5 in the preparation of medicines or health foods.

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