Cordyceps sinensis fermented composition

Abstract

The invention provides a cordyceps sinensis fermentation composition, the cordyceps sinensis fermentation composition contains erythritol and mannitol, the content of the erythritol is 1 wt% or above, the content of the mannitol is 4 wt% or above, and the fermentation composition has an excellent anti-oxidation effect and has a synergistic technical effect.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of June 14, 2024, application number 2024107650911, and invention name “A Cordyceps Sinensis Fermentation Composition”. Technical Field

[0002] The invention relates to a cordyceps sinensis fermentation composition and belongs to the field of microbial fermentation. Background Art

[0003] 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.

[0004] 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.

[0005] Low energy and special metabolic pathways are one of the metabolic characteristics of erythritol. The energy coefficient of erythritol is 0.88kJ / 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.

[0006] 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 35g of erythritol in liquid form, the intestinal tolerance is good and there are no adverse symptoms; when the intake of erythritol reaches 50g, 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 15g 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 exerting a beneficial effect on the oral cavity. In a 3-year clinical trial, the levels of acetic acid and propionic acid in the dental plaque and the counts of Streptococcus mutans in the plaque of subjects treated with erythritol were significantly lower than those of subjects treated with xylitol or sorbitol.

[0007] 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, and 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.

[0008] Mannitol, also known as D-mannitol, hexadecene, and sorbitol, is an isomer of mannitol. 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] Ergothioneine (EGT or ERG) is a natural rare chiral amino acid strong 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 antioxidant, free radical scavenging, 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.

[0013] Ergothioneine is distributed in some tissues and organs of mammals, mainly present in the semen of red blood cells and some animals, and the content of ergothioneine in humans and mammalian tissues is 1-2mM (equivalent to 229.3-458.6mg / L) (Melville, 1959; Hartman, 1990 or The Natural Antioxidant Ergothioneine, LipidOxidation, 2013), which shows that ergothioneine may be used as a non-toxic antioxidant in vivo, and cells lacking ergothioneine are easily affected by oxidative stress, thereby causing mitochondrial DNA damage, protein oxidation and lipid peroxidation to increase. However, up to now, no experimental studies have shown that there are animals that can synthesize the substance, and there is definitely a way to absorb and retain ergothioneine in animal cells. Ergothioneine, with its unique biology, pharmacological characteristics and safety, stability and other advantages, shows good application prospects in the fields of cosmetics, functional foods, pharmacy and biomedicine, and is widely concerned by scholars at home and abroad.

[0014] 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 (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 the ergothioneine content in mushroom fruiting bodies by adding methionine in mycelial culture. The difference between ergothioneine mushrooms 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 the production of ergothioneine in mushrooms.

[0015] Cordyceps militairs is also known as northern winter cordyceps. The scientific name of Cordyceps militairs is Cordyceps militairs (Vuill) Fr, and the English name is Pupa insect grass. The asexual form is Verticillium, and Liang Zongqi (1990) believed that it is 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 militaris is 130.65 mg / kg (0.013%), corresponding to the fourth level 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.

[0016] Cordyceps sinensis (Borkeler) sacc.) and Cordyceps militaris are two different species. Cordyceps sinensis has a strong host specificity and can only parasitize the larvae of bat moth (Hepialus armorieanus Oberthur). The scientific name of Cordyceps sinensis is Cordyceps sinensis (Berk) Sacc., and its English name is Chinese caterpillar fungus. The name of the asexual form has been confirmed 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 (Cordyceps sinensis) and Related Bioactive Ingredients, Journal of Traditional and Complementary Medicine 2013(3)1:16-32) believed that the fruiting bodies of wild Cordyceps sinensis contained detectable ergothioneine, and the ergothioneine among them was 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 Cordyceps sinensis contained 142.0±38.5 IJK 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 was only 35.70±1.28 K, and the content in its fruiting bodies was even lower, only 12.20±1.51 E. 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 was 52.18±2.75 μg / g. Therefore, there has been no wild Cordyceps sinensis or natural Cordyceps sinensis that stably contains a high level of ergothioneine, nor has there been a mycelium of wild Cordyceps sinensis or natural Cordyceps sinensis that stably contains a high level of ergothioneine.Moreover, Cordyceps sinensis belongs to the Ascomycetes, Hypocreales, Ophiocordycipitaceae, Ophiocondyceps family, 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. Over the past few decades, researchers have isolated more than 30 species of fungi from Cordyceps sinensis collected from various production areas, including Paecilomyces spp., Paecilomyces spp., Mortierella spp., Cephalosporium sinensis, Chinese Hairy Spore, Chinese Townectomyces spp., and Chinese Chrysosporium. 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 the 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.

[0017] 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

[0018] The inventor unexpectedly found that, through the optimization of the process, 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 found 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.

[0019] 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.

[0020] Specifically, the inventors screened out Hirsutella sinensis from natural Cordyceps sinensis strains to find that Hirsutella sinensis has the function of producing erythritol. On the other hand, the inventors obtained stable fermented mycelium or powder containing a high content of erythritol through Hirsutella sinensis fermentation. The inventors obtained erythritol with the content of natural Cordyceps sinensis for the first time through liquid fermentation culture, and exerted a wider range of functions of the Cordyceps sinensis fermentation composition.

[0021] The Chinese Hirsutella sinensis strain is an asexual generation of the ergot fungus, cordyceps sinesis (Berk.) Sacc., which is isolated from fresh Cordyceps sinesis on the Qinghai-Tibet Plateau. The Chinese Hirsutella described in the present invention can be any Chinese Hirsutella strain, for example, it can be purchased through a commercial platform, or it can be obtained by independent screening and separation, as long as it can be identified as Chinese Hirsutella sinensis according to identification principles such as microbial molecular genetics. The commercial channels include but are not limited to obtaining Chinese Hirsutella sinensis from China Center for Type Culture Collection (CCTCC) or China General Microbiological Culture Collection Administration Center (CGMCC), for example, those obtained from China Center for Type Culture Collection (CCTCC) include but are not limited to Chinese Hirsutella sinensis CCTCC No: M 2011278, for example, those obtained from China General Microbiological Culture Collection Administration Center (CGMCC) include but are not limited to Chinese Hirsutella sinensis CGMCC 3.14240 and Hirsutella sinensis CGMCC3.14243, for example, standard strains of Cordyceps sinensis (Ophiocordyceps sinensis or Cordyceps sinensis) purchased from China Industrial Microbiological Culture Collection Center (CICC) 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, CICC 14115, CICC 14117, CICC 14118, CICC 14119, CICC 14120, CICC 50002, such as Ophiocordyceps sinensis or Cordyceps sinensis (Hirsutella truncatula, Hirsutella sinensis) purchased from Testo Biotech sinensis) including but not limited to TS324838, TS325049, TS325054, TS326189, TS326195, TS326196, TS326197, TS326198, TS326199, TS326200, TS326201, TS326202, TS326203, TS326204, TS326205, TS326206, TS326207, TS326210, TS326212, TS326213, TS326214, TS326408, TS326410, TS326412, TS342791, TS342792, TS34 2793, TS342794, TS342795, TS342796, TS342797, TS342798, TS342800, TS342809, TS361737, TS392986. .

[0022] The growth cycle of Chinese Hirsutum is longer than that of most fungi. It generally grows in an environment of 14-20℃ and is a medium-low temperature fungus. When cultured on solid culture medium, the diameter can reach 1.5cm. 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 become 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 mycelium ball is tough and not loose, elastic, and hollow inside. It is known that the mycelium obtained by liquid fermentation culture of Chinese Hirsutum can be dried and fermented with Cordyceps sinensis powder to 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.

[0023] 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.

[0024] 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):

[0025] 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;

[0026] or

[0027] 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;

[0028] or

[0029] 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%, the rest is water;

[0030] or

[0031] 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 divide into packages;

[0032] or

[0033] 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;

[0034] or

[0035] 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;

[0036] or

[0037] 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;

[0038] 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.

[0039] The shake flask / seed bottle culture strain in the present invention is inoculated from the slant to the seed bottle, and then shake culture is performed, or the test tube strain is accessed into the sterilized (sterilized) cooled triangular flask culture solution, and placed on a shaker for shaking culture, for example, the seed amount is 2 to 6 slants, preferably 2 to 3 slants, the culture temperature is 10 to 18 ° C, preferably the 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 a strain. In the shake flask liquid culture, it is required that the mycelium grows fast, the yield is high, and the formed ball is small. If the ball is larger, the mycelium in the center of the ball will show a state of anoxia and nutrient starvation, which is unfavorable for the proliferation of mycelium and the production of 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):

[0040] 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;

[0041] or

[0042] 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;

[0043] or

[0044] 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;

[0045] or

[0046] 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;

[0047] or

[0048] Peptone 5.0%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5.0%, solvent is water;

[0049] or

[0050] Fish peptone 0.5%, peptone 0.5%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5%, the rest is water;

[0051] or

[0052] 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;

[0053] 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.

[0054] The 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 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 produced automatically and mechanized. The fermentation tank submerged culture process flow of Chinese Hirsutella spores can be: primary seed tank → secondary seed tank → fermentation tank; it can also be: seed tank → fermentation tank; continuous fermentation can also be used. For example, the culture temperature is 10 to 18°C, preferably the culture temperature is 12 to 16°C, the culture time is 6 to 20 days, preferably the culture time is 7 to 15 days, and preferably the culture time is 10 to 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.).

[0055] 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.

[0056] 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 main raw materials. The powder product is prepared 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).

[0057] 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 702 and polypeptide powder 705. The polypeptide powder "7" series uses rice protein and corn protein as main raw materials. The powder product is prepared by separation, refining, concentration and spray drying using a process of segmented hydrolysis by modern biological methods and multiple hydrolysis methods (the process enables the product to have different molecular weights, reasonable distribution of peptones and peptides, and is conducive to the continuous use of microorganisms).

[0058] The fermentation medium of the present invention may be (the following % is weight to volume ratio):

[0059] 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;

[0060] Preferably,

[0061] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, the above polypeptide powder 1%, and the rest is water;

[0062] or

[0063] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 203 1%, and the rest is water;

[0064] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 305 1%, the rest is water;

[0065] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 702 1%, the rest is water;

[0066] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 705 1%, the rest is water;

[0067] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 607 1%, and the rest is water.

[0068] The Cordyceps sinensis fermentation composition of the present invention refers to mycelium, wet bacterial slices, dry bacterial powder of mycelium, or dry bacterial powder of mycelium and fermentation filtrate obtained by slant culture, seed bottle / shake bottle culture, and fermentation culture of Hirsutella sinensis, preferably dry bacterial powder of mycelium and dry bacterial 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.

[0069] The invention optimizes the fermentation process to obtain a Cordyceps sinensis fermentation composition, wherein the amount of mycelium and metabolites of the Cordyceps sinensis fermentation composition 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).

[0070] 1.5-4% (weight percentage), 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage).

[0071] The present invention unexpectedly discovered that the presence of erythritol in an amount of more than 1% (by weight), preferably 1-6% (by weight), further preferably 1.5-5% (by weight), 1.5-4% (by weight), further preferably 2-5% (by weight), and further preferably 2.5-4% (by weight) in the 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 content of the obtained Cordyceps sinensis fermentation composition is 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) is sufficient and stable, and at the same time, it is particularly preferred that the adenosine content is more than 0.02% (weight percentage), more than 0.03% (weight percentage), preferably the adenosine content is 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).

[0072] Furthermore, previous studies have shown that adenosine may be converted from adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, but nucleoside substances represented by adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate also have important physiological activities. For example, Qian Jia et al. (Study on the Antioxidant Effect of 5′-acid adenosine (5′-AMP) in Vitro, Food and Machinery, Vol. 24, No. 1, January 2008) showed that AMP has a strong activity in scavenging hydroxyl radicals. Therefore, on the basis of ensuring the stable existence of erythritol and adenosine with a synergistic technical effect, the present invention controls the conversion amount of adenosine to a certain extent, so that the Cordyceps sinensis fermentation composition as a whole exhibits 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 Hirsutella sinensis, Chinese Edible Fungi, 2017, 37(1): 55-60) showed that the effects of different carbon sources and nitrogen sources on the solid fermentation process of Hirsutella sinensis were the highest adenosine obtained by solid fermentation method, which 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, Vol. 56, No. 14, July 2021) recorded that the liquid fermentation culture conditions of Chinese Hirsutella were optimized, and the dry weight of the optimized Chinese Hirsutella 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).

[0073] 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%.

[0074] More specifically, the present invention preferably has an adenosine content of 0.02% or more (by weight) on the basis of an erythritol content of 1% or more; or

[0075] More specifically, the present invention preferably has an adenosine content of 0.02%-1.00% (weight percentage) based on an erythritol content of 1-6%; or,

[0076] More specifically, the present invention preferably has an adenosine content of 0.03%-0.80% (by weight) based on an erythritol content of 1-6%; or,

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

[0078] More specifically, the present invention preferably has an adenosine content of 0.02%-1.00% (weight percent) based on an erythritol content of 1.5-4% (weight percent); or, more specifically, the present invention preferably has an adenosine content of 0.02%-1.00% (weight percent) based on an erythritol content of 2-5%; or,

[0079] More specifically, the present invention preferably has an adenosine content of 0.03% to 0.80% (by weight) on the basis of an erythritol content of 2 to 5%; or

[0080] 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.

[0081] More specifically, the present invention preferably has an adenosine content of 0.03%-0.80% (by weight) based on an erythritol content of 2.5-4% or more.

[0082] 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.

[0083] 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.

[0084] 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).

[0085] More specifically, the present invention preferably has a mannitol content of 4% or more (by weight) on the basis of an erythritol content of 1% or more; or

[0086] More specifically, the present invention preferably has a mannitol content of 5% or more (by weight) on the basis of 1% erythritol content; or,

[0087] More specifically, the present invention preferably has a mannitol content of 6% or more (by weight) on the basis of 1% erythritol content; or,

[0088] More specifically, the present invention preferably has a mannitol content of 7% or more (by weight) on the basis of 1% erythritol content; or,

[0089] More specifically, the present invention preferably has a mannitol content of 4% or more (by weight) on the basis of an erythritol content of 1 to 6%; or

[0090] More specifically, the present invention preferably has a mannitol content of more than 5% (by weight) on the basis of an erythritol content of 1 to 6%; or,

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

[0092] More specifically, the present invention preferably has a mannitol content of more than 7% (by weight) on the basis of an erythritol content of 1-6%; or,

[0093] More specifically, the present invention preferably has a mannitol content of more than 4% (by weight) on the basis of an erythritol content of 1.5-5%; or,

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

[0095] More specifically, the present invention preferably has a mannitol content of more than 6% (by weight) on the basis of an erythritol content of 1.5-5%; or,

[0096] More specifically, the present invention preferably has a mannitol content of more than 7% (by weight) on the basis of an erythritol content of 1.5-5%; or,

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

[0098] More specifically, the present invention preferably has a mannitol content of more than 5% (by weight) on the basis of an erythritol content of 2-4%; or,

[0099] More specifically, the present invention preferably has a mannitol content of more than 6% (by weight) on the basis of an erythritol content of 2-4%; or,

[0100] More specifically, the present invention preferably has a mannitol content of more than 7% (by weight) on the basis of an erythritol content of 2-4%; or,

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

[0102] More specifically, the present invention preferably has a mannitol content of more than 5% (by weight) on the basis of an erythritol content of 2.5-4%; or,

[0103] More specifically, the present invention preferably has a mannitol content of more than 6% (by weight) on the basis of an erythritol content of 2.5-4%; or,

[0104] More specifically, in the present invention, based on the erythritol content of 2.5-4%, the mannitol content is preferably above 7% (weight percentage).

[0105] 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).

[0106] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0107] 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.

[0108] 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).

[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 polysaccharides is above 2% (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), and the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0111] 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).

[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 0.02% (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 0.03% (weight percentage), and a mannitol content of more than 5% (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 polysaccharide content of more than 2% (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 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,

[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), and a mannitol content of more than 5% (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 polysaccharide content of more than 2% (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), and a total amino acid content of more than 20% (weight percentage); or,

[0119] 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).

[0120] 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.

[0121] 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.

[0122] 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.

[0123] The pharmaceutical composition of the present invention has excellent antioxidant effect and synergistic technical effect.

[0124] 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.

[0125] Specifically, the present invention is by the screening of Cordyceps sinensis strains on the one hand, the inventor screens out Chinese Hirsutella sinensis from the bacterium of natural Cordyceps sinensis and has the function of producing erythritol, and has the function of producing ergothioneine, on the other hand, the inventor obtains the fermentation mycelium or the bacterial powder that can stably exist, contain higher content ergothioneine by Chinese Hirsutella fermentation. The present invention obtains the fermentation composition with natural Cordyceps sinensis content level ergothioneine or higher than natural Cordyceps sinensis content level ergothioneine for the first time by liquid fermentation culture, and plays the wider function of Cordyceps sinensis fermentation composition.

[0126] The Chinese Hirsutella sinensis strain is an asexual generation of the ergot fungus, cordyceps sinesis (Berk.) Sacc., which is isolated from fresh Cordyceps sinesis on the Qinghai-Tibet Plateau. The Chinese Hirsutella described in the present invention can be any Chinese Hirsutella strain, for example, it can be purchased through a commercial platform, or it can be obtained by independent screening and separation, as long as it can be identified as Chinese Hirsutella sinensis according to identification principles such as microbial molecular genetics. The commercial channels include but are not limited to obtaining Chinese Hirsutella sinensis from China Center for Type Culture Collection (CCTCC) or China General Microbiological Culture Collection Administration Center (CGMCC), for example, those obtained from China Center for Type Culture Collection (CCTCC) include but are not limited to Chinese Hirsutella sinensis CCTCC No: M 2011278, for example, those obtained from China General Microbiological Culture Collection Administration Center (CGMCC) include but are not limited to Chinese Hirsutella sinensis CGMCC 3.14240 and Hirsutella sinensis CGMCC3.14243, for example, standard strains of Cordyceps sinensis (Ophiocordyceps sinensis or Cordyceps sinensis) purchased from China Industrial Microbiological Culture Collection Center (CICC) 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, CICC 14115, CICC 14117, CICC 14118, CICC 14119, CICC 14120, CICC 50002, such as Ophiocordyceps sinensis or Cordyceps sinensis (Hirsutella truncatula, Hirsutella sinensis) purchased from Testo Biotech sinensis) including but not limited to TS324838, TS325049, TS325054, TS326189, TS326195, TS326196, TS326197, TS326198, TS326199, TS326200, TS326201, TS326202, TS326203, TS326204, TS326205, TS326206, TS326207, TS326210, TS326212, TS326213, TS326214, TS326408, TS326410, TS326412, TS342791, TS342792, TS34 2793, TS342794, TS342795, TS342796, TS342797, TS342798, TS342800, TS342809, TS361737, TS392986. .

[0127] The growth cycle of Chinese Hirsutum is longer than that of most fungi. It generally grows in an environment of 14-20℃ and is a medium-low temperature fungus. When cultured on solid culture medium, the diameter can reach 1.5cm. 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 become 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 mycelium ball is tough and not loose, elastic, and hollow inside. It is known that the mycelium obtained by liquid fermentation culture of Chinese Hirsutum can be dried and fermented with Cordyceps sinensis powder to 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.

[0128] 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.

[0129] 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):

[0130] 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;

[0131] or

[0132] 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;

[0133] or

[0134] 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%, the rest is water;

[0135] or

[0136] 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 divide into packages;

[0137] or

[0138] 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;

[0139] or

[0140] 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;

[0141] or

[0142] 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;

[0143] 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.

[0144] The shake flask / seed bottle culture strain in the present invention is inoculated from the slant to the seed bottle, and then shake culture is performed, or the test tube strain is accessed into the sterilized (sterilized) cooled triangular flask culture solution, and placed on a shaker for shaking culture, for example, the seed amount is 2 to 6 slants, preferably 2 to 3 slants, the culture temperature is 10 to 18 ° C, preferably the 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 a strain. In the shake flask liquid culture, it is required that the mycelium grows fast, the yield is high, and the formed ball is small. If the ball is larger, the mycelium in the center of the ball will show a state of anoxia and nutrient starvation, which is unfavorable for the proliferation of mycelium and the production of 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):

[0145] 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;

[0146] or

[0147] 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;

[0148] or

[0149] 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;

[0150] or

[0151] 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;

[0152] or

[0153] Peptone 5.0%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5.0%, solvent is water;

[0154] or

[0155] Fish peptone 0.5%, peptone 0.5%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5%, the rest is water;

[0156] or

[0157] 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;

[0158] 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.

[0159] The 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 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 produced automatically and mechanized. The fermentation tank submerged culture process flow of Chinese Hirsutella spores can be: primary seed tank → secondary seed tank → fermentation tank; it can also be: seed tank → fermentation tank; continuous fermentation can also be used. For example, the culture temperature is 10 to 18°C, preferably the culture temperature is 12 to 16°C, the culture time is 6 to 20 days, preferably the culture time is 7 to 15 days, and preferably the culture time is 10 to 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.).

[0160] 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.

[0161] 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 main raw materials. The powder product is prepared 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).

[0162] 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 702 and polypeptide powder 705. The polypeptide powder "7" series uses rice protein and corn protein as main raw materials. The powder product is prepared by separation, refining, concentration and spray drying using a process of segmented hydrolysis by modern biological methods and multiple hydrolysis methods (the process enables the product to have different molecular weights, reasonable distribution of peptones and peptides, and is conducive to the continuous use of microorganisms).

[0163] The fermentation medium of the present invention may be (the following % is weight to volume ratio):

[0164] 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;

[0165] Preferably,

[0166] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, the above polypeptide powder 1%, and the rest is water;

[0167] or

[0168] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 203 1%, and the rest is water;

[0169] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 305 1%, the rest is water;

[0170] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 702 1%, the rest is water;

[0171] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 705 1%, the rest is water;

[0172] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 607 1%, and the rest is water.

[0173] The Cordyceps sinensis fermentation composition of the present invention refers to mycelium, wet bacterial slices, dry bacterial powder of mycelium, or dry bacterial powder of mycelium and fermentation filtrate obtained by slant culture, seed bottle / shake bottle culture, and fermentation culture of Hirsutella sinensis, preferably dry bacterial powder of mycelium and dry bacterial 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.

[0174] The present invention optimizes the fermentation process to obtain the Cordyceps sinensis fermentation composition, the amount of mycelium and metabolites are significantly improved, wherein the content of ergothioneine 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), preferably 0.01-0.10% (weight percentage), preferably 0.01-0.08% (weight percentage), or preferably more than 0.02% (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);

[0175] 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.

[0176] Further, 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);

[0177] Further, 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.

[0178] Further, 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.

[0179] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0180] 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.

[0181] 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% (percentage by weight).

[0182] 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 thiol 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.

[0183] The present invention unexpectedly finds that the presence of ergothioneine in the Cordyceps sinensis fermentation composition of more than 0.004% (percentage by weight), preferably more than 0.006% (percentage by weight), preferably more than 0.008% (percentage by weight), preferably more than 0.01% (percentage by weight) 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 inventor unexpectedly finds that the Chinese Hirsutella spore fermentation product or the fermentation composition as a whole shows a more excellent technical effect.

[0184] Therefore, on the other hand, the present invention ensures that the ergothioneine of 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 sufficient and stable, 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), 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.

[0185] 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 is 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.

[0186] The present invention unexpectedly finds that the presence of 0.004% or more (percentage by weight), preferably 0.006% or more (percentage by weight), preferably 0.008% or more (percentage by weight), preferably 0.01% or more (percentage by weight) 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 inventor unexpectedly finds that the combination of ergothioneine and adenosine shows an obvious synergistic effect of hydroxyl radical scavenging rate. That is, the present invention ensures that the ergothioneine content of the obtained Cordyceps sinensis fermentation composition is above 0.004% (weight percentage), preferably above 0.006% (weight percentage), preferably above 0.008% (weight percentage), preferably above 0.01% (weight percentage) in sufficient and stable amount, and particularly preferably the adenosine content is above 0.02% (weight percentage), above 0.03% (weight percentage), preferably the adenosine content is 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); 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.

[0187] Further, since previous studies have shown that adenosine may be converted from adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, but nucleoside substances represented by adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate also have important physiological activity, as shown by 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), AMP has a strong activity of scavenging hydroxyl radicals. Therefore, on the basis of ensuring that the stable existence of ergothioneine and adenosine has a synergistic technical effect, 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 Hirsutella sinensis, Chinese Edible Fungi, 2017, 37(1): 55-60) showed that the effects of different carbon sources and nitrogen sources on the solid fermentation process of Hirsutella sinensis were the highest adenosine obtained by solid fermentation method, which 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, Vol. 56, No. 14, July 2021) recorded that the liquid fermentation culture conditions of Chinese Hirsutella were optimized, and the dry weight of the optimized Chinese Hirsutella 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).

[0188] 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 0.03-0.60% (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 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. In the present invention, when the content of adenosine and ergothioneine is within the above-mentioned preferred range, the Cordyceps sinensis fermentation composition as a whole can have excellent antioxidant effect, which well meets the application requirements.

[0189] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:20; or,

[0190] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:15; or,

[0191] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:10; or,

[0192] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:8; or,

[0193] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:7; or,

[0194] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:6; or,

[0195] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:5; or,

[0196] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:4; or,

[0197] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:3; or,

[0198] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:2; or,

[0199] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of more than 0.01%, and a ratio of ergothioneine to adenosine content of 1:1-1:20; or,

[0200] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of more than 0.01%, and a ratio of ergothioneine to adenosine content of 1:1-1:15; or,

[0201] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of more than 0.01%, and a ratio of ergothioneine to adenosine content of 1:1-1:10; or,

[0202] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:8; or,

[0203] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:7; or,

[0204] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:6; or,

[0205] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:5; or,

[0206] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:4; or,

[0207] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:3; or,

[0208] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.01% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:2; or,

[0209] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and the ratio of ergothioneine to adenosine content is 1:1-1:20; or,

[0210] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and the ratio of ergothioneine to adenosine content is 1:1-1:15; or,

[0211] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and the ratio of ergothioneine to adenosine content is 1:1-1:10; or,

[0212] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:8; or,

[0213] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:7; or,

[0214] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:6; or,

[0215] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:5; or,

[0216] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:4; or,

[0217] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:3; or,

[0218] More specifically, the present invention preferably has an adenosine content of 0.02% or more (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and the ratio of ergothioneine to adenosine content is 1:1-1:2; or,

[0219] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of more than 0.02%, and a ratio of ergothioneine to adenosine content of 1:1-1:20; or,

[0220] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of more than 0.02%, and a ratio of ergothioneine to adenosine content of 1:1-1:15; or,

[0221] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:10; or,

[0222] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:8; or,

[0223] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:7; or,

[0224] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:6; or,

[0225] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:5; or,

[0226] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:4; or,

[0227] More specifically, the present invention preferably has an adenosine content of 0.03%-0.08% (percentage by weight) on the basis of an ergothioneine content of 0.02% or more, and a ratio of ergothioneine to adenosine content of 1:1-1:3; or,

[0228] 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 more than 0.02%, and a ratio of ergothioneine to adenosine is 1:1-1:2.

[0229] 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.

[0230] On the other hand, the present invention combines the content of ergothioneine and the conversion behavior of adenosine, and on the basis of ergothioneine, erythritol and adenosine contents, further studies the yield of mycelium dry weight, that is, the inventors further optimize the fermentation process by ensuring the content of ergothioneine, erythritol and adenosine, and obtain a Cordyceps sinensis fermentation composition with a higher mycelium yield, the 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, and the mycelium dry weight water content is less than 8%, preferably less than 7%, preferably less than 6%.

[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 dry weight water content of 7% or less; or,

[0232] 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 7g / L or more; or,

[0233] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an adenosine content of 0.02%-0.2% (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,

[0234] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01%, preferably an adenosine content of 0.02%-0.2% (weight percentage), a ratio of ergothioneine to adenosine of 1:1-1:20, and a mycelium fermentation yield of more than 7g / L.

[0235] Further, by the inventor's in-depth research, at ergothioneine more than 0.004% (percentage by weight), preferably more than 0.006% (percentage by weight), preferably more than 0.008% (percentage by weight), preferably on the basis of 0.01% or more content, the present invention unexpectedly found that the presence of more than 0.004% ergothioneine in Cordyceps sinensis fermentation composition is not only the first Cordyceps sinensis fermentation product with ergothioneine, and greatly enhances the effect of other beneficial ingredients, the present invention shows that ergothioneine in Cordyceps sinensis fermentation composition helps to enhance the effects such as adenosine by research experiments, and produces synergistic effect, especially in terms of antioxidant effect, makes Cordyceps sinensis fermentation composition as a whole show more excellent technical effect.

[0236] 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 also includes one or more of adenosine, mannitol, amino acids, polysaccharides, and erythritol.

[0237] 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).

[0238] Furthermore, the Cordyceps sinensis fermentation composition has a mannitol content of more than 4% (by weight), preferably more than 5%, preferably more than 6%, and preferably more than 7%.

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

[0241] 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,

[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 5% or more (weight percentage); or,

[0243] 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,

[0244] 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).

[0245] 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 preferably the polysaccharide content is above 2% (weight percentage); or,

[0246] 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).

[0247] Furthermore, the Cordyceps sinensis fermentation composition of the present invention preferably has a polysaccharide content of more than 2% (by weight) and a total amino acid content of more than 20% based on an ergothioneine content of more than 0.01%;

[0248] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01%, preferably a polysaccharide content of 2%-10% (weight percentage), and a total amino acid content of 25-50%.

[0249] 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.

[0250] On the other hand, the present invention also provides a Cordyceps sinensis fermentation composition, wherein the content of ergothioneine 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 content of erythritol 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 hydroxyl radical scavenging rate, thereby enhancing the overall antioxidant property of the Cordyceps sinensis fermentation composition.

[0251] On the other hand, the present invention also provides a Cordyceps sinensis fermentation composition, wherein the ergothioneine content 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), the adenosine content is more than 0.02% (weight percentage), and the mannitol content is more than 4% (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 polysaccharide content of more than 2% (weight percentage).

[0253] Further, 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).

[0254] Further, 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).

[0255] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01%, preferably an adenosine content of more than 0.02% (by weight), a ratio of ergothioneine to adenosine of 1:1-1:20, and a mannitol content of more than 5% (by weight); or,

[0256] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01%, preferably an adenosine content of more than 0.03% (by weight), a ratio of ergothioneine to adenosine of 1:1-1:20, and a mannitol content of more than 5% (by weight); or,

[0257] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01%, preferably an adenosine content of more than 0.02% (weight percentage), a ratio of ergothioneine to adenosine content of 1:1-1:20, a mannitol content of more than 5% (weight percentage), and a polysaccharide content of more than 2% (weight percentage); or,

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

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

[0260] 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%-1.0% (weight percentage), 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,

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

[0262] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01%, an adenosine content of preferably 0.02%-1.0% (weight percentage), a ratio of ergothioneine to adenosine content 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).

[0263] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, comprising adenosine, wherein the fermentation composition also comprises ergothioneine, and the content (weight percentage) of ergothioneine is above 0.01%.

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

[0265] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol and polysaccharide, 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 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%.

[0267] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, wherein the fermentation composition also comprises ergothioneine, and the content (weight percentage) of ergothioneine is above 0.01%, and the content of adenosine is 0.02-1.0% (weight percentage).

[0268] 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.

[0269] 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.

[0270] Further, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition also comprises ergothioneine, and the content (weight percentage) of ergothioneine 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).

[0271] Further, 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), the ratio of ergothioneine to adenosine content is 1:1-1:20, and the content of mannitol is above 4% (weight percentage).

[0272] Therefore, on the other hand, the present invention also provides a Cordyceps sinensis fermentation composition, the content of ergothioneine 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), the adenosine content is more than 0.02% (weight percentage), and the erythritol is more than 1.0% (weight percentage).

[0273] 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,

[0274] 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.

[0275] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, comprising adenosine, wherein the fermentation composition also comprises ergothioneine and erythritol, and the content (weight percentage) of ergothioneine is above 0.01%.

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

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

[0278] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol, polysaccharides and amino acids, wherein the fermentation composition also comprises ergothioneine and erythritol, and the content (weight percentage) of ergothioneine is above 0.01%.

[0279] 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).

[0280] 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.

[0281] 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.

[0282] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition also comprises ergothioneine and erythritol, and the content (weight percentage) of ergothioneine 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).

[0283] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition also comprises 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).

[0284] 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).

[0285] 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).

[0286] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol and polysaccharide, wherein the fermentation composition also comprises ergothioneine and erythritol, and the erythritol is above 1% (weight percentage).

[0287] 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).

[0288] 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).

[0289] 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).

[0290] 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).

[0291] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition also comprises ergothioneine and erythritol, and the content (weight percentage) of ergothioneine 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).

[0292] 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%.

[0293] 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).

[0294] Furthermore, the Cordyceps sinensis fermentation composition of the present invention 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). ;

[0295] 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).

[0296] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0297] 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 loaded into capsules or the mixture is made into tablets 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, 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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.

[0303] The pharmaceutical composition of the present invention has excellent antioxidant effect and synergistic technical effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0304] Figure 1 HPLC spectrum of ergothioneine standard

[0305] Figure 2 HPLC spectrum of ergothioneine of sample B-5

[0306] Figure 3 HPLC spectrum of ergothioneine of B-7 sample

[0307] Figure 4 HPLC profile of adenosine of sample CN101096641A in Example 4

[0308] Figure 5 HPLC profile of adenosine of Example 4B-2

[0309] Figure 6 HPLC spectrum of erythritol of sample B-2

[0310] Figure 7 The standard curve of polysaccharide DETAILED DESCRIPTION

[0311] 1. Ergothioneine Detection Method

[0312] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0313] 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 255nm. The theoretical plate number calculated based on the ergothioneine peak should be no less than 3000.

[0314] Preparation of reference solution: An appropriate amount of ergothioneine reference substance is accurately weighed and added with 0.5% phosphoric acid solution to prepare a solution containing μg per 1 ml. Gradient elution is performed according to the following table:

[0315] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 12.5 100 0 13 40 60 18 40 60 19 100 0 33 100 0

[0316] 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.

[0317] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0318] 2. Detection method of erythritol

[0319] Preparation of reference solution

[0320] 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.

[0321] Preparation of test solution

[0322] 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.

[0323] Detection Methods

[0324] Detector: RID differential detector;

[0325] Chromatographic column: Model Agilent Hi-Plex Ca, 300×7.7mm, 8um;

[0326] Mobile phase: deionized water;

[0327] Column flow rate: 0.6 ml / min; column temperature: 80 °C; injection volume: 10 ul; detection time: 50 min;

[0328] Circulation cell temperature: 35°C.

[0329] 3. Anthrone-sulfuric acid detection method for polysaccharides

[0330] Solution preparation

[0331] 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.

[0332] Preparation of standard curve

[0333] Preparation of reference solution

[0334] 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.

[0335] Standard curve drawing

[0336] 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 .

[0337] Preparation of test solution

[0338] Take about 2g of fermented Cordyceps sinensis powder, weigh it accurately, put it in a round-bottom flask, add 60mL of water, let it stand for 30min, heat to reflux for 3h, transfer it to a centrifuge tube while hot, wash the filter and filter residue with a small amount of hot water, centrifuge at 5000rpm for 10min, take the supernatant, transfer the precipitate to a round-bottom flask with a small amount of hot water, add 60mL of water, heat to reflux for 2h, transfer it to a centrifuge tube while hot, centrifuge again, combine the supernatant, evaporate it in a water bath, dissolve the residue with 5mL of water, slowly add 75mL of ethanol while stirring, shake well, let it stand at 4℃ for 12h, centrifuge, discard the supernatant, dissolve the precipitate with hot water and transfer it to 50mL, cool, add water to the scale, shake well, take an appropriate amount of solution, centrifuge, accurately measure 1mL of supernatant, put it in a 25mL volumetric flask, add water to the scale, shake well, and obtain.

[0339] Determination

[0340] 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.

[0341] Result calculation

[0342]

[0343] Where:

[0344] W-polysaccharide content, %;

[0345] c-polysaccharide concentration of the sample checked from the standard curve, mg / mL;

[0346] m-sample mass, mg;

[0347] 8 / 2 and 25 / 2 indicate dilution multiples;

[0348] 50-The volume of the precipitate obtained after water extraction and alcohol precipitation is dissolved in hot water to make up the volume.

[0349] 4. Hydroxyl free radical scavenging rate detection (Fenton colorimetric method)

[0350] Detection principle

[0351] 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 536nm disappears, and the change in absorbance at 530-540nm 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.

[0352] Solution preparation

[0353] 1. Weigh 0.2 g of bacterial powder, add 2 ml of purified water, mix well, let stand at room temperature for 4 hours, centrifuge at 10000 rpm for 10 minutes, and take the supernatant for later use.

[0354] 2. Other samples are reference solutions.

[0355] The spectrophotometer was turned on and preheated for 30 minutes, the wavelength was adjusted to 536 nm, and the purified water was used to zero the instrument.

[0356] Add sample

[0357] 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 hour.

[0358]

[0359] Calculation: Test sample OH removal rate (%) = [(A3-A3')-(A2-A0)] / (A1-A2)×100%

[0360] 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.

[0361] 6. Detection method of tyrosinase inhibition rate

[0362] 1. Solution preparation

[0363] (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.

[0364] (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 used immediately after preparation.

[0365] (3) Preparation of TYR (tyrosine kinase) solution: Dissolve 25KU 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 a 250U·mL-1 TYR solution, then dilute the mother liquor to 125U·mL-1 and store in a refrigerator at -20°C for later use.

[0366] 2. Sample preparation:

[0367] 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.

[0368] 3. Determination of the inhibitory activity of samples on TYR

[0369] The reaction system is shown in the table below. With 2mmol·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, the plate is linearly shaken for 10 minutes and then taken out, and finally the TYR solution is added and quickly put back. The detection wavelength is 475nm, the total volume of the reaction system is 240μL, and the absorbance is measured every 30s for a total of 30min. The inhibition rate is calculated based on the absorbance of 30min. 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.

[0370]

[0371]

[0372] A1: absorbance of the reaction solution without adding sample but adding TYR at 475nm;

[0373] A2: absorbance of the reaction solution without sample and TYR at 475 nm;

[0374] A3: absorbance of the reaction solution with sample and TYR measured at 475nm;

[0375] A4: The absorbance of the reaction solution with sample added but without TYR measured at 475nm.

[0376] Example 1 Ergothioneine (EGT) and erythritol content in different mycelia

[0377] 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:

[0378]

[0379] Although in theory the nutrient content and medicinal effects of the mycelium from fungi isolated from natural Cordyceps sinensis are similar to those of natural Cordyceps sinensis, there are still differences in the composition of the fermentation products of Cordyceps sinensis and that of natural Cordyceps sinensis, especially the metabolites and secondary metabolites produced during the fermentation process, which are not only different from those of natural Cordyceps sinensis, but also vary greatly between the fermentation products of different Cordyceps sinensis. On the basis of studying natural Cordyceps sinensis, the inventors collected several strains of Hirsutella sinensis from preservation centers, such as Hirsutella sinensis from China Center for Type Culture Collection (CCTCC), with a preservation number of CCTCC No: M 2011278, Hirsutella sinensis CGMCC 3.14240 and Hirsutella sinensis CGMCC3.14243 purchased from China General Microbiological Culture Collection Center (CGMCC), and Paecilomyces hepiali 3.15540, Paecilomyces hepiali CGMCC3.7845, and Paecilomyces hepiali CGMCC3.15543 purchased from China General Microbiological Culture Collection Center (CGMCC). hepiali) CGMCC3.7908, further with reference 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 with silkworm pupa powder), the fermentation filtrate and mycelium dry powder in the existing Cordyceps sinensis fermentation process were studied, not only were all lower than the amount of ergothioneine that can be detected in natural Cordyceps sinensis, but most of them were not detected, but the inventor unexpectedly found that the Chinese Hirsutosporium fermentation mycelium can obtain trace amounts of ergothioneine.

[0380]

[0381] Note: It means that according to the peak area, the content is only 3‰-10‰ of natural Cordyceps sinensis.

[0382] 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.

[0383] Further, except the bacterial strain available in above-mentioned preservation center, inventor is to commercially available multiple Chinese hairy spores (Hirsutella sinensis), for example, Cordyceps sinensis (bat moth hairy spore, Chinese hairy spore) Ophiocordyceps sinensis standard strain CICC 14092, CICC14095, CICC 14097, CICC 14099 purchased from China Industrial Microorganism Culture Collection Administration Center (CICC), and Cordyceps sinensis (bat moth hairy spore, Chinese hairy spore) Ophiocordyceps sinensis article number purchased from Taisituo biology is TS326189, TS326199 and Chinese hairy spore (Hirsutella sinensis) article number is TS361737, TS392986 bacterial strain, ferment with reference to prior art, ergothioneine is not obtained at obtained fermentation filtrate and mycelium.

[0384] Similarly, the inventor fermented commercially available multiple strains of Paecilomyces hepiali, such as CGMCC 3.15421, CGMCC 3.14870, CGMCC 3.12108, CGMCC 3.10157, CCTCC NF20081231, CCTCC NF 20081239, CCTCC NF 20081244, CCTCC NF 20081250 with reference to prior art, and ergothioneine was not obtained in the obtained fermentation filtrate and mycelium.

[0385]

[0386] 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.

[0387] Further, in addition to the strains available from the above-mentioned collection centers, the inventors fermented multiple commercially available strains of Hirsutella sinensis, for example, standard strains of Ophiocordyceps sinensis CICC 14092, CICC 14095, CICC 14097, and CICC 14099 purchased from the China Industrial Microbiological Culture Collection Center (CICC), and Ophiocordyceps sinensis with item numbers TS326189 and TS326199 purchased from Testol Biotechnology, as well as strains of Hirsutella sinensis with item numbers TS361737 and TS392986, with reference to the prior art, and failed to obtain a stable and meaningful content of erythritol in the obtained fermentation filtrate and mycelium.

[0388] Similarly, the inventors fermented several commercially available strains of Paecilomyces hepiali, such as those with the deposit numbers CGMCC 3.15421, CGMCC 3.14870, CGMCC 3.12108, CGMCC 3.10157, CCTCC NF20081231, CCTCC NF 20081239, CCTCC NF 20081244, and CCTCC NF 20081250, according to the prior art, but failed to obtain a stable and meaningful amount of erythritol in the obtained fermentation filtrate and mycelium.

[0389] Embodiment 2 has the fermentation product of stably containing 0.004% or more ergothioneine (EGT)

[0390] Hirsutella sinensis was obtained from China Center for Type Culture Collection (CCTCC), with the collection number of CCTCC No: M 2011278. Hirsutella sinensis was inoculated on a slant, and slant culture, shake flask / seed bottle culture, and fermentation culture were performed as follows:

[0391] 1 Slant culture

[0392] 1.1 Slant solid culture medium formula: 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%, pH 6.8-7.2, prepared with water, sterilized at (118-122)℃ for (30±1)min.

[0393] 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.

[0394] 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.

[0395] 2 Shake flask culture

[0396] 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;

[0397] 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.

[0398] 2.3 Preparation of culture medium: Weigh the corresponding materials and dissolve them with liquefied filtrate, make up to volume, and divide into shake bottles with a filling volume of 1500ml / 5000ml, (118-122)℃, and sterilize for (30±1)min.

[0399] 2.4 Digging blocks: Cut the slope into about 1.5×2.0 cm blocks, use a sterile rake to rake the cut blocks into the prepared digging block / seed bottle culture medium, (13-16)℃, (120-140)rpm, and shake culture for 9-14 days.

[0400] 3. Fermentation culture:

[0401] 3.1 Basic fermentation medium formula: glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, pH 7.0.

[0402] 3.2 Preparation of culture medium: shake flask liquid volume 1000ml / 5000ml, (118-122)℃, sterilize for (30±1)min.

[0403] 3.3 Fermentation: inoculation amount 10%, temperature (13-16) °C, (120-140) rpm, shake culture for 7 days and then release the bottle.

[0404] 3.4 Take an appropriate volume of fermentation liquid, vacuum filter to obtain wet bacterial slices, and dry to obtain bacterial powder samples.

[0405] Different ingredients were added to the above basic fermentation medium (glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01% (% are weight volume ratio)), and the mycelium experimental number samples A-1 to A-8 and B-1 to B-8 were obtained by fermentation. The results of detecting ergothioneine and erythritol are shown in the following table:

[0406]

[0407] Furthermore, in addition to the strains disclosed in the above patents, the inventors conducted a number of commercially available strains of Hirsutella sinensis, such as Hirsutella sinensis CGMCC 3.14240 and Hirsutella sinensis CGMCC3.14243 purchased from the China General Microbiological Culture Collection Center (CGMCC), and Cordyceps sinensis (Ophiocordyceps sinensis) standard strains CICC 14092, CICC 14095, CICC14097, CICC 14099 purchased from the China Industrial Microbiological Culture Collection Center (CICC), and Cordyceps sinensis (Ophiocordyceps sinensis) purchased from Testo Biotechnology. The strains of Hirsutella sinensis with the item numbers TS326189, TS326199 and Hirsutella sinensis with the item numbers TS361737 and TS392986 were fermented according to the fermentation method of Hirsutella sinensis with the deposit number CCTCC No: M 2011278 in Example 2, and the experiment was repeated under the conditions of different culture media shown in the table above. The EGT content, adenosine content %, and mannitol and erythritol content % in the bacterial powder samples obtained by fermentation of different Hirsutella sinensis were not significantly different from the contents of the components in the bacterial powder prepared with the deposit number CCTCC No: M2011278 under the same conditions.

[0408] Example 3 Study on the effect of effective amount of ergothioneine or erythritol on the efficacy of Chinese Hirsutella spp. fermentation powder 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] 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

[0427] Experimental samples:

[0428] The preparation of mycelial samples B-2, B-5, and B-7 was the same as before;

[0429] Example 5 Sample preparation of CN108384726A:

[0430] 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.

[0431] Example 1 Sample preparation of CN101096641A:

[0432] The strains are inoculated on the slant, the culture medium formula is 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%, the rest is water, and cultured at 12°C for 25 days; then the strains are inoculated in the fermentation medium, the culture medium formula is 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%, the rest is water, placed on a shaker, cultured at 14°C for 8 days; then inoculated in the first-level 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 is carried out, and the solid is dried and crushed.

[0433]

[0434] 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 fermented composition contains erythritol and mannitol, wherein the content of the erythritol is greater than 1 wt %, and the content of the mannitol is greater than 4 wt %.

2. The fermentation composition according to claim 1, characterized in that The content of erythritol is 1-6wt%.

3. The fermentation composition according to claim 2, characterized in that The content of erythritol is 1.5-4wt%.

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

5. The fermentation composition according to claim 4, characterized in that The content of mannitol is above 6wt%.

6. The fermentation composition according to claim 1, characterized in that The fermented composition contains adenosine, and the content of the adenosine is greater than 0.02 wt %.

7. 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%.

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

9. The fermentation composition according to any one of claims 1 to 8, characterized in that The fermented composition is prepared by fermenting Hirsutella sinensis.

10. Use of the fermentation composition according to any one of claims 1 to 9 in the preparation of medicines or health foods.

Images