Cordyceps sinensis fermentation composition

Abstract

A cordyceps sinensis fermentation composition, containing erythritol and mannitol. The content of the erythritol is 1 wt% or more, and the content of the mannitol is 4 wt% or more. The fermentation composition has an excellent antioxidant effect and a synergistic technical effect.

Description

A Cordyceps sinensis fermentation composition

[0001] The present invention claims priority to a Chinese patent application filed with the Patent Office of China on June 15, 2023, with application number 202310716796X and invention name “A Cordyceps Sinensis Fermentation Composition”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The invention relates to a cordyceps sinensis fermentation composition, belonging to the technical 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 is naturally present in small amounts in fruits and vegetables such as pears, grapes, and mushrooms. It is also present in small amounts in fermented foods such as wine, beer, soy sauce, and sake. It is also present 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 refreshing 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 a unique metabolic pathway are among the metabolic characteristics of erythritol. With an energy coefficient of 0.88 kJ / g, erythritol has the lowest energy content among currently used polyol sweeteners. When erythritol is ingested, approximately 80% to 90% is absorbed by the small intestine through passive diffusion, while the remaining 10% enters the large intestine for fermentation as a carbon source. Furthermore, as the dosage of erythritol increases, the proportion of erythritol entering the large intestine also increases. Of this, erythritol absorbed through the small intestine is widely distributed throughout tissues. Approximately 5% to 10% of the erythritol is oxidized in the blood to erythrose, which is then rapidly further oxidized to erythrose esters and ultimately excreted in the urine. The remaining unoxidized erythritol cannot be broken down by the body's enzymes and must be filtered from the blood by the kidneys and excreted in the 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 to other sugar alcohols, the maximum non-laxative dose (MNLD) of erythritol is 0.66-1.0+ g / kg bw, significantly higher than other sugar alcohols. Studies on human intestinal tolerance have shown that healthy volunteers aged 18-24 years who ingest 20-35 g of erythritol in liquid form tolerate it well and experience no adverse symptoms. However, when erythritol intake reaches 50 g, diarrhea and nausea may be observed. Furthermore, clinical studies have shown that children aged 4-6 years who consume beverages containing 15 g of erythritol do not experience diarrhea and / or severe intestinal symptoms. Furthermore, it is important to note that diarrhea caused by excessive sugar alcohol intake is not a disease, but rather a simple osmotic reaction to slowly absorbed carbohydrates in the intestinal lumen. This means that sugar alcohols that are not absorbed in the small intestine may cause bloating, 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 inhibits its growth and adhesion in the mouth, thereby exerting a beneficial effect on the oral cavity. In a three-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] Currently, erythritol is mainly produced industrially through microbial fermentation. There are many microorganisms in nature that can synthesize erythritol, such as osmophilic yeasts such as Moniliella pollinis, Trichosporon, Trichosporon, and Yarrowia lipolytica, and bacteria such as Oenococcus oeni, Leuconostoc oeni, and Lactobacillus. The synthesis pathways of erythritol in yeast and bacteria are different. In yeast, erythritol is synthesized through the pentose phosphate pathway. The erythrose-4-phosphate produced by this pathway is dephosphorylated to form erythrose, which is then converted to erythritol under the action of erythrose reductase. In bacteria, erythritol can be produced through the regeneration of reduced coenzyme II (NADPH) in the phosphoketolase pathway.

[0008] Mannitol, also known as D-mannitol, hexanehexasol, and nicotol, is an isomer of sorbitol. Mannitol is present in both natural Cordyceps sinensis and fermented Cordyceps sinensis powder, but in varying amounts. As a typical hexavalent sugar alcohol, mannitol absorbs heat upon dissolution. After digestion and absorption by the human body, mannitol first reacts with NAD+ to generate acetyl-CoA and fructose. Through the action of fructokinase, two fructose phosphates are generated, which enter the triose cycle and complete mannitol's physiological metabolic process. Mannitol's metabolic process is unique, as it does not require the participation of insulin, and therefore does not cause significant fluctuations in blood sugar levels. Therefore, mannitol can be considered 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 consumed 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 for 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 disproportionation to prevent irreversible damage to penumbra tissues, thereby reducing neurological damage and can be used as a neuroprotective agent 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 cerebral vascular resistance, improve microcirculation, and increase perfusion of organ tissues.

[0012] Ergothioneine (EGT or ERG) is a natural, rare chiral amino acid antioxidant first discovered in the fungus Claviceps purpurea in 1909. It is a water-soluble, naturally occurring thiol compound that has been recognized as an antioxidant in the body and a protective factor that helps cells resist oxidative stress. It has multiple functions such as anti-oxidation, scavenging free radicals, chelating metal ions, preventing ultraviolet radiation damage, regulating intracellular redox reactions, participating in intracellular energy regulation, and acting as a cell physiological protector. It is an important active substance in the body.

[0013] Ergothioneine is distributed in certain tissues and organs of mammals, is mainly present in the semen of red blood cell and some animals, the content of ergothioneine in humans and mammalian tissue is 1-2mM (equivalent to 229.3-458.6mg / L) (Melville, 1959; Hartman, 1990 or The Natural Antioxidant Ergothioneine, Lipid Oxidation, 2013), this shows that ergothioneine may be used as a nontoxic antioxidant in vivo, and cell lacks ergothioneine and is easily subject to the influence of oxidative stress, thereby causes mitochondrial DNA damage, protein oxidation and lipid peroxidation to increase. But, up to now, also no experimental studies have shown that there is animal to synthesize this substance, and there is certainly the approach of absorbing and retaining ergothioneine in animal cells. Ergothioneine, with its unique biology, pharmacological properties and the advantages such as safety, stability, shows good application prospects in the fields such as cosmetics, functional foods, pharmacy and biomedicine, is subject to the extensive concern of scholars at home and abroad.

[0014] Ergothioneine is primarily found in mushrooms, but Melville DB and Eieh S have also found it in cereals. Subsequent research by Melville DB and Genghof DS has shown that ergothioneine is a common component of many microbial cells and can be synthesized in several fungi, but not in bacteria. Another reason for incorporating mushrooms into the human diet is that they are a rich source of antioxidants including ergothioneine. For example, Shin-Yu Chen et al. (Contents of lovastatin, g-aminobutyric acid and ergothioneine in mushroom fruiting bodies and mycelia, Food Science and Technology, 2012(47):274-278) reported that among mushroom fruiting bodies, Pleurotus citrinopileatus, P. ostreatus (Korea), P. ostreatus (Taiwan, China) and Pleurotus salteostramineus had the highest ergothioneine content (2850.7, 1829.4, 1458.4 and 1245.0 mg / kg, respectively), while among mycelia, Pleurotus eryngii had the highest ergothioneine content (1514.6 mg / kg). In order to further enhance the content of ergothioneine 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 content of ergothioneine in the enhanced mushroom fruiting body in mycelial culture by adding methionine. The difference between ergothioneine mushrooms and mushrooms was as high as 92.3 times. Adding 2mM methionine (Met) to mycelial culture medium can increase the ergothioneine content in the tested mushroom species, indicating that methionine is a beneficial additive for the production of ergothioneine in mushrooms.

[0015] Cordyceps militaris, also known as northern Chinese caterpillar fungus, has a scientific name of Cordyceps militairs (Vuill) Fr and an English name of Pupa insect grass. Its asexual form is Verticillium, and Liang Zongqi (1990) considered it to be a Cehalosporium. Patent document WO2014 / 055035 discloses a Cordyceps militaris strain cbs132098 and biologically active fruiting bodies, mycelial biomass, and extracts thereof. The ergothioneine content in the mycelial biomass of the Cordyceps militaris strain is 130.65 mg / kg (0.013%), corresponding to the fourth grade classification of Chen et al. (Chang et al., 2012). Bai-Xiong et al. (Enhancement of ergothioneine production by discovering and regulating its metabolic pathway in Cordyceps militaris. Bai-Xiong Chen et al, Microb Cell Fact. 2022 Aug 23; 21(1): 169) constructed a new ergothioneine synthesis pathway and successfully increased the component yield of the engineered strain by further introducing the pathway into the Cordyceps militaris genome, with the highest ergothioneine content reaching 2.5 g / kg dry weight.

[0016] Cordyceps sinensis (Borkeler) sacc. and Cordyceps militaris are two different species. Cordyceps sinensis is highly host-specific, parasitizing only the larvae of the bat moth (Hepialus armorieanus Oberthur). Its scientific name is Cordyceps sinensis (Berk) Sacc., and its English name is Chinese caterpillar fungus. Its asexual form has been confirmed by scholars and experts to be Hirsutella sinensis.Hui-Chen Lo et al. (A Systematic Review of the Mysterious Caterpillar Fungus Ophiocordyceps sinensis in DongChongXiaCao (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 Ophiocordyceps sinensis contain detectable ergothioneine, which is considered a secondary metabolite of fungal growth and not a small molecule organic compound necessary for the normal operation of its growth and development. Although Shin-Yu Chen et al. reported that the mycelia of Ophiocordyceps sinensis contain 142.0 ± 38.5 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 is only 35.70 ± 1.28 K, and the content in its fruiting bodies is 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 is 52.18 ± 2.75 μg / g. Therefore, there has been no wild Ophiocordyceps sinensis or natural Ophiocordyceps sinensis that stably contains a high level of ergothioneine, nor has there been a mycelium of wild Ophiocordyceps sinensis or natural Ophiocordyceps 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 Traditional Chinese Medicine, 2018). Natural Cordyceps sinensis sources are scarce and expensive. Using Cordyceps sinensis hyphae as a substitute for natural Cordyceps sinensis is a technological development trend. 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 sinensis, Mortierella spp., Cephalosporium sinensis, Mortierella sinensis, Toxicodendron sinensis, and Chrysosporium sinensis. A metabolomic study of Cordyceps sinensis at different growth stages (Zhang Zhou, Anhui Agricultural University, 2018) showed that there were significant differences in metabolites between different parts of Cordyceps sinensis, and significant differences also existed 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.

[0018] Summary of the Invention

[0019] The inventors unexpectedly discovered that, through process optimization, the provided Cordyceps sinensis fermentation composition contains erythritol at a level of 1% or more by weight, preferably 1%-6% by weight, more preferably 2-5% by weight, and even more preferably 2.5-4%. Furthermore, those skilled in the art generally believe that erythritol is merely a sweetener. However, the present inventors unexpectedly discovered through research that the combination of erythritol and adenosine exhibits a synergistic effect in scavenging hydroxyl radicals, thereby enhancing the overall efficacy of the Cordyceps sinensis fermentation composition. In particular, when erythritol is present within the range defined by the present invention, the combination with adenosine exhibits an excellent synergistic effect in scavenging hydroxyl radicals.

[0020] On the one hand, the present invention obtains Cordyceps sinensis fungi capable of producing erythritol by screening Cordyceps sinensis fungi, and then obtains a fermentation composition containing erythritol by fermentation. On the other hand, the present invention uses a low-temperature liquid fermentation technology to ensure that erythritol is present in a sufficient and stable amount in Cordyceps sinensis mycelium or powder. On the other hand, the present invention uses a low-temperature fermentation process to achieve the reasonable presence of effective ingredients such as erythritol and adenosine, thereby not only making the fermented Cordyceps sinensis composition more stable, but also providing a new utilization direction for the fermented Cordyceps sinensis powder.

[0021] Specifically, the present invention, through screening Cordyceps sinensis strains, identified Hirsutella sinensis from natural Cordyceps sinensis as having the ability to produce erythritol. Furthermore, the inventors obtained stable fermented mycelium or powder containing a high level of erythritol through fermentation of Hirsutella sinensis. This invention, through liquid fermentation, for the first time produces erythritol at levels similar to those found in natural Cordyceps sinensis, further enhancing the potential of Cordyceps sinensis fermentation compositions.

[0022] The Chinese Hirsutella (Hirsutella sinensis) strain is an asexual generation of the ergot fungus Cordyceps sinesis (Berk.) Sacc., which is isolated from fresh Cordyceps sinensis 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 from a commercial platform or obtained by independent screening and isolation, as long as it can be identified as Chinese Hirsutella (Hirsutella sinensis) according to identification principles such as microbial molecular genetics. Commercial channels include but are not limited to obtaining Chinese Hirsutella (Hirsutella sinensis) from the China Center for Type Culture Collection (CCTCC) or the China General Microorganism Culture Collection Center (CGMCC), for example, those obtained from the China Center for Type Culture Collection (CCTCC) include but are not limited to Chinese Hirsutella (Hirsutella sinensis) CCTCC No: M 2011278, for example, those obtained from the China General Microorganism Culture Collection Center (CGMCC) include but are not limited to Chinese Hirsutella (Hirsutella sinensis) CGMCC No. 3.14240 and Hirsutella sinensis CGMCC3.14243, for example, standard strains of Cordyceps sinensis (Ophiocordyceps sinensis or Cordyceps sinensis) purchased from the China Industrial 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 Biological 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. .

[0023] The growth cycle of Hirsutella sinensis is longer than that of most fungi, generally growing in an environment of 14-20°C, making it a mesophilic fungus. When cultured on solid media, the colonies can reach up to 1.5 cm in diameter. Their texture resembles hide glue, and they are flesh-colored to milky white. Initially, aerial hyphae are present on the surface, but over time, the surface becomes smooth and grooved, and the number of aerial hyphae gradually decreases. When cultured in liquid media, the mycelium is filamentous or grayish-globular, varying in size. During incubation, brown secretions are released into the medium. The mycelial pellets are firm, elastic, and hollow. It is known that the mycelium obtained from liquid fermentation of Hirsutella sinensis, when dried and then fermented with Cordyceps sinensis powder, can produce a variety of active substances, including polysaccharides, amino acids, nucleosides, sterols, mannitol, and trace metals. Signature components include adenosine, mannitol, polysaccharides, and amino acids. 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.

[0024] 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.

[0025] The slant culture of the present invention is to grow Hirsutella sinensis on the inclined surface of a solid culture medium, inoculate and spread Hirsutella sinensis on the slant, and culture under low temperature conditions, for example, at 8-18° C. for 20-60 days, preferably at 10-16° C. for 30-50 days, wherein the main raw materials of the slant culture medium are one or more of 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), and KH2PO4 (potassium dihydrogen phosphate or its hydrate); for example, the slant culture medium can be (the following % is weight-to-volume ratio g / 100 mL):

[0026] 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;

[0027] or

[0028] 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;

[0029] or

[0030] Silkworm pupa powder 1.8%, corn flour 1.5%, glucose 2.0%, MgSO4 0.01%, KH2PO4 0.02%, oatmeal 1.0-3.0g / 60mL, agar powder 0.65%, and the rest is water;

[0031] or

[0032] 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 portions;

[0033] or

[0034] Peptone 0.5-2.0%, broad bean flour 2.0-5.0%, corn flour 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;

[0035] or

[0036] 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%, solvent is water;

[0037] or

[0038] 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;

[0039] Those skilled in the art will understand that the specific slant culture medium listed above is only illustrative 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 Hirsutella sinensis fermentation, or slant culture media for Hirsutella sinensis fermentation known in the prior art.

[0040] Shake flask among the present invention / plant bottle culture bacterial classification is inoculated into the plant bottle from slant, shaking culture, or test tube bacterial classification is accessed in the triangular flask culture fluid of killing (killing) sterilization cooling, be placed in shaking culture on shaking table, for example, seeding amount is 2~6 slant-planes, preferably 2~3 slant-planes, cultivation temperature 10~18 ℃, preferred cultivation temperature 12~16 ℃, incubation time 8~16 days, preferred incubation time 9~14 days.When large-scale production, the mycelium of shake flask culture can be accessed seed tank as bacterial classification.In shake flask liquid culture, require mycelial growth fast, yield height, the bacterium ball formed is little, if bacterium ball is larger, the mycelium body at bacterium ball center will present the starvation state of anoxia and nutrition, the generation of unfavorable mycelial proliferation and secondary metabolites, affects fermentation quality and mycelial 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 (wheat bran), 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):

[0041] 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;

[0042] or

[0043] 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;

[0044] or

[0045] 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;

[0046] or

[0047] 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;

[0048] or

[0049] Peptone 5.0%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5.0%, solvent is water;

[0050] or

[0051] Fish peptone 0.5%, peptone 0.5%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5%, the rest is water;

[0052] or

[0053] 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;

[0054] Those skilled in the art will understand that the specific shake flask / seed flask culture medium listed above is only illustrative 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 media for Hirsutella sinensis fermentation, or shake flask / seed flask culture media for Hirsutella sinensis fermentation known in the prior art.

[0055] 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 of aerobic or anaerobic culture of pure microbial cells in a liquid submerged culture medium. Submerged fermentation is generally carried out in large fermentation tanks and can be automated and mechanized. The fermentation tank submerged culture process flow of Chinese Hirsutella spp. can be: first-level seed tank → second-level seed tank → fermentation tank; it can also be: seed tank → fermentation tank; continuous fermentation can also be used. For example, the culture temperature is 10-18°C, preferably the culture temperature is 12-16°C, the culture time is 6-20 days, preferably the culture time is 7-15 days, and preferably the culture time is 10-15 days. The main raw materials of the fermentation medium include one or more of the following: silkworm pupa powder, glucose, corn flour (slurry), yeast extract (extract paste or powder), NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate), polypeptide powder series (such as polypeptide powder 101, polypeptide powder 102, polypeptide powder 203, polypeptide powder 305, polypeptide powder 308, polypeptide powder 607, polypeptide powder 702, polypeptide powder 705) (the polypeptide powder series is purchased from Chengdu Wenfeng Biotechnology Co., Ltd.).

[0056] The fermentation medium of the present invention includes an organic nitrogen source for novel microbial fermentation, such as polypeptide powder "1" series. The polypeptide powder "1" series uses rice protein, corn protein and its hydrolyzate as main raw materials. The process of using multiple hydrolysis methods for staged hydrolysis is adopted, and the powdered products are separated, refined, concentrated and dried. The polypeptide powder "1" series includes polypeptide powder 101 and polypeptide powder 102. The polypeptide powder "2" series, such as polypeptide powder 203, uses corn protein as main raw material, adopts a process of multiple hydrolysis methods for staged hydrolysis (this process makes the product have different molecular weights, and the peptones and peptides are reasonably distributed, which is beneficial to the sustainable use of microorganisms), and the powdered products are separated, refined, concentrated and spray-dried.

[0057] The fermentation medium described herein includes a series of peptones (non-animal-derived) using a novel organic nitrogen source for microbial fermentation, such as polypeptide powder 305, polypeptide powder 308, and polypeptide powder 607. These peptones are primarily made from corn protein and yeast. They utilize a multi-step hydrolysis process (this yields products with varying molecular weights and a balanced distribution of peptones and peptides, facilitating sustainable microbial utilization) followed by separation, refining, concentration, and spray drying to produce a powdered product.

[0058] The fermentation medium of the present invention includes a series of peptones (non-animal derived) using novel organic nitrogen sources for microbial fermentation, such as polypeptide powder 702 and polypeptide powder 705. The polypeptide powder "7" series uses rice protein and corn protein as the main raw materials. The powdered product is prepared by separation, refining, concentration, and spray drying using a process of staged hydrolysis using modern biological methods and multiple hydrolysis methods (this process results in products with different molecular weights and a reasonable distribution of peptones and peptides, which is beneficial for the sustainable utilization of microorganisms).

[0059] The fermentation medium of the present invention may be (the following % are weight to volume ratio):

[0060] 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;

[0061] Preferably,

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

[0063] or

[0064] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 203 1%, and the rest is water;

[0065] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 305 1%, and the rest is water;

[0066] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 702 1%, and the rest is water;

[0067] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 705 1%, and the rest is water;

[0068] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 607 1%, and the rest is water.

[0069] The Cordyceps sinensis fermentation composition of the present invention refers to mycelium, wet pellets, dried mycelium powder, or dried mycelium powder of a fermentation filtrate obtained through slant culture, seed / shake flask culture, or fermentation culture of Hirsutella sinensis, preferably dried mycelium powder or dried mycelium powder of a fermentation filtrate. The fermentation filtrate includes a fermentation filtrate obtained through solvent separation and chromatography column separation (including membrane separation), or its effective fraction, effective ingredients, or various metabolites.

[0070] By optimizing the fermentation process, the present invention obtains a Cordyceps sinensis fermentation composition, in which the amounts of mycelium and metabolites are significantly improved. Among them, 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), 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 1% by weight or more, preferably 1-6% by weight, further preferably 1.5-5% by weight, 1.5-4% by weight, further preferably 2-5% by weight, and even further preferably 2.5-4% by weight, in a Cordyceps sinensis fermentation composition not only provides 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), more preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), more preferably 2-5% (weight percentage), and even more preferably 2.5-4% (weight percentage) is sufficient and stable. At the same time, the adenosine content is particularly preferably more than 0.02% (weight percentage), more than 0.03% (weight percentage), preferably 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), and preferably 0.03-0.08% (weight percentage).

[0072] Furthermore, previous studies have shown that adenosine can be converted from adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate. However, 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'-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 presence 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 more excellent technical effects. 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 significant. The highest adenosine obtained by solid fermentation 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 Pharmaceutical Journal, Vol. 56, No. 14, July 2021) recorded that the liquid fermentation culture conditions of Chinese Hirsutella spp. were optimized, and the dry weight of the optimized Chinese Hirsutella spp. 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). However, some studies have shown that the growth rate of the 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 the bacteria decreases, indicating that higher carbon and nitrogen source concentrations are not conducive to bacterial growth (CN103430777A).

[0073] The present invention unexpectedly discovered that the higher the adenosine content, 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), and preferably 0.03-0.08% (weight percentage), based on an erythritol content of 1% or more.

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

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

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

[0077] More specifically, the present invention preferably contains 0.02% to 1.00% adenosine (by weight) based on an erythritol content of 1.5 to 5% (by weight); or

[0078] More specifically, the present invention preferably contains 0.02% to 1.00% adenosine (by weight) based on an erythritol content of 1.5 to 4% (by weight); or

[0079] More specifically, the present invention preferably has an erythritol content of 2-5% and an adenosine content of 0.02%-1.00% (weight percentage); or

[0080] More specifically, the present invention preferably has an erythritol content of 2-5% and an adenosine content of 0.03%-0.80% or more (by weight); or

[0081] More specifically, the present invention preferably has an erythritol content of 2.5-4% or more and an adenosine content of 0.02%-1.00% (weight percentage).

[0082] More specifically, the present invention preferably has an erythritol content of 2.5-4% or more and an adenosine content of 0.03%-0.80% (weight percentage).

[0083] Those skilled in the art will understand that the above enumeration is only illustrative of the present invention and is not to be construed as limiting the present invention. Due to the study of the erythritol and adenosine content, the fermentation composition obtained within the above range has a very excellent antioxidant effect, especially a synergistic effect of hydroxyl radical scavenging rate.

[0084] Therefore, the present invention also provides a Cordyceps sinensis fermentation composition, which further includes one or more of mannitol, polysaccharides, and amino acids.

[0085] 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), and preferably more than 7% (weight percentage).

[0086] More specifically, the present invention preferably contains more than 4% mannitol (by weight) on the basis of more than 1% erythritol; or

[0087] More specifically, the present invention preferably contains more than 5% mannitol (by weight) on the basis of 1% erythritol; or

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

[0089] More specifically, the present invention preferably contains 1% erythritol and more than 7% mannitol (by weight); or

[0090] More specifically, the present invention preferably has an erythritol content of 1 to 6% and a mannitol content of 4% or more (by weight); or

[0091] More specifically, the present invention preferably has an erythritol content of 1 to 6% and a mannitol content of 5% or more (by weight); or

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

[0093] More specifically, the present invention preferably has an erythritol content of 1-6% and a mannitol content of 7% or more (by weight); or

[0094] More specifically, the present invention preferably contains more than 4% mannitol (by weight) on the basis of 1.5-5% erythritol; or

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

[0096] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 1.5-5% erythritol; or

[0097] More specifically, the present invention preferably contains more than 7% mannitol (by weight) on the basis of 1.5-5% erythritol; or

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

[0099] More specifically, the present invention preferably has an erythritol content of 2-4% and a mannitol content of more than 5% (by weight); or

[0100] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 2-4% erythritol; or

[0101] More specifically, the present invention preferably has an erythritol content of 2-4% and a mannitol content of more than 7% (by weight); or

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

[0103] More specifically, the present invention preferably contains more than 5% mannitol (by weight) on the basis of 2.5-4% erythritol; or

[0104] More specifically, the present invention preferably contains more than 6% mannitol (by weight) on the basis of 2.5-4% erythritol; or

[0105] More specifically, in the present invention, based on the erythritol content of 2.5-4%, the mannitol content is preferably above 7% (weight percentage).

[0106] 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), and preferably 2-10% (weight percentage).

[0107] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0108] Those skilled in the art will understand that the above enumeration is only for 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 polysaccharide, the fermentation composition obtained within the above range has very excellent technical effects.

[0109] 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).

[0110] Furthermore, 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 even more preferably 2.5-4% (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).

[0111] Furthermore, 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 even further preferably 2.5-4% (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).

[0112] Furthermore, 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 even more preferably 2.5-4% (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).

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

[0114] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of 1% or more (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of 0.03% or more (weight percentage), and a mannitol content of 5% or more (weight percentage); or,

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

[0116] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of 1% or more (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of 0.02% or more (weight percentage), a mannitol content of 5% or more (weight percentage), and a total amino acid content of 20% or more (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), more preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), more preferably 2-5% (weight percentage), and even more 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,

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

[0119] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an erythritol content of 1% or more (weight percentage), preferably 1-6% (weight percentage), further preferably 1.5-5% (weight percentage), 1.5-4% (weight percentage), further preferably 2-5% (weight percentage), and further preferably 2.5-4% (weight percentage), preferably an adenosine content of 0.02%-1.0% (weight percentage), a mannitol content of 5% or more (weight percentage), and a total amino acid content of 20% or more (weight percentage); or,

[0120] 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).

[0121] The Cordyceps sinensis fermentation composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, and capsules, or a single preparation can be obtained by mixing the components and encapsulating the mixture or forming the mixture into tablet form. Various dosage forms can also be prepared using acceptable carriers. With respect to oral administration compositions (e.g., tablets and capsules), the term "acceptable carrier" includes such carriers as common excipients, such as binders such as syrup, gum arabic, gelatin, sorbitol, gum tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose, and starch; fillers and carriers such as corn starch, gelatin, lactose, sucrose, and microcrystalline cellulose; and lubricants such as magnesium stearate, sodium stearate and other stearates, glyceryl stearate, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silicon dioxide. Flavoring agents such as peppermint, wintergreen oil, and cherry flavoring may also be used. Colorants may be added to distinguish the dosage form. 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.

[0122] 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 research on the erythritol and adenosine content, the fermentation composition obtained within the above range has a very excellent antioxidant effect, making the Cordyceps sinensis fermentation composition of the present invention show a better technical effect than the existing technology as a whole.

[0123] The pharmaceutical composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, and capsules, or a single preparation can be obtained by mixing the components and encapsulating the mixture or forming the mixture into tablets. Various dosage forms can also be prepared using acceptable carriers.

[0124] The pharmaceutical composition of the present invention has excellent antioxidant effect and synergistic technical effect.

[0125] The present invention further on the one hand is through the screening of Cordyceps sinensis, to the research on Cordyceps sinensis fermentation and beneficial component combination thereof, simultaneously obtains the Cordyceps sinensis that can produce thioneine, obtains the fermentation composition containing thioneine by fermentation, that is, realizes that thioneine in Cordyceps sinensis mycelium or bacterium powder is able to be stably present in sufficient amount 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, and provides the new utilization direction of fermented Cordyceps sinensis bacterium powder.

[0126] Specifically, the present invention is by the screening of Cordyceps sinensis strains on the one hand, and the inventor screens out Chinese Hirsutella (Hirsutella sinensis) from the bacterium of natural Cordyceps sinensis and has the function of producing erythritol, and there is the function of producing thioneine, on the other hand, the inventor obtains the fermentation mycelium or the bacterium powder that can stably exist, that contain higher content thioneine by Chinese Hirsutella fermentation.The present invention obtains the fermentation composition with natural Cordyceps sinensis content level thioneine or higher than natural Cordyceps sinensis content level thioneine first by liquid fermentation culture, plays the function that Cordyceps sinensis fermentation composition is more extensive.

[0127] The Chinese Hirsutella sinensis strain is an asexual generation of the ergot fungus Cordyceps sinesis (Berk.) Sacc., which is isolated from fresh Cordyceps sinensis on the Qinghai-Tibet Plateau. The Chinese Hirsutella sinensis described in the present invention can be any Chinese Hirsutella sinensis strain, for example, it can be purchased from a commercial platform or obtained by independent screening and isolation, as long as it can be identified as Chinese Hirsutella sinensis according to identification principles such as microbial molecular genetics. Commercial channels include but are not limited to obtaining Chinese Hirsutella sinensis from the China Center for Type Culture Collection (CCTCC) or the China General Microorganism Culture Collection Center (CGMCC), for example, those obtained from the 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 the China General Microorganism Culture Collection Center (CGMCC) include but are not limited to Chinese Hirsutella sinensis CGMCC No. 3.14240 and Hirsutella sinensis CGMCC3.14243, for example, standard strains of Cordyceps sinensis (Ophiocordyceps sinensis or Cordyceps sinensis) purchased from the China Industrial 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 Biological 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. .

[0128] The growth cycle of Hirsutella sinensis is longer than that of most fungi, generally growing in an environment of 14-20°C, making it a mesophilic fungus. When cultured on solid media, the colonies can reach up to 1.5 cm in diameter. Their texture resembles hide glue, and they are flesh-colored to milky white. Initially, aerial hyphae are present on the surface, but over time, the surface becomes smooth and grooved, and the number of aerial hyphae gradually decreases. When cultured in liquid media, the mycelium appears filamentous or grayish, spherical, and varies in size. As the culture progresses, brown secretions are released into the medium. The mycelial pellets are firm, elastic, and hollow. It is known that the dried mycelium obtained from liquid fermentation of Hirsutella sinensis can be fermented with Cordyceps sinensis powder to produce a variety of active substances, including polysaccharides, amino acids, nucleosides, sterols, mannitol, and trace metals. Signature components include adenosine, mannitol, polysaccharides, and amino acids. 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.

[0129] 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.

[0130] The slant culture of the present invention is to grow Hirsutella sinensis on the inclined surface of a solid culture medium, inoculate and spread Hirsutella sinensis on the slant, and culture under low temperature conditions, for example, at 8-18° C. for 20-60 days, preferably at 10-16° C. for 30-50 days, wherein the main raw materials of the slant culture medium are one or more of 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), and KH2PO4 (potassium dihydrogen phosphate or its hydrate); for example, the slant culture medium can be (the following % is weight-to-volume ratio g / 100 mL):

[0131] 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;

[0132] or

[0133] 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;

[0134] or

[0135] Silkworm pupa powder 1.8%, corn flour 1.5%, glucose 2.0%, MgSO4 0.01%, KH2PO4 0.02%, oatmeal 1.0-3.0g / 60mL, agar powder 0.65%, and the rest is water;

[0136] or

[0137] 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 portions;

[0138] or

[0139] Peptone 0.5-2.0%, broad bean flour 2.0-5.0%, corn flour 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;

[0140] or

[0141] 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%, solvent is water;

[0142] or

[0143] 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;

[0144] Those skilled in the art will understand that the specific slant culture medium listed above is only illustrative 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 Hirsutella sinensis fermentation, or slant culture media for Hirsutella sinensis fermentation known in the prior art.

[0145] Shake flask among the present invention / plant bottle culture bacterial classification is inoculated into the plant bottle from slant, shaking culture, or test tube bacterial classification is accessed in the triangular flask culture fluid of killing (killing) sterilization cooling, be placed in shaking culture on shaking table, for example, seeding amount is 2~6 slant-planes, preferably 2~3 slant-planes, cultivation temperature 10~18 ℃, preferred cultivation temperature 12~16 ℃, incubation time 8~16 days, preferred incubation time 9~14 days.When large-scale production, the mycelium of shake flask culture can be accessed seed tank as bacterial classification.In shake flask liquid culture, require mycelial growth fast, yield height, the bacterium ball formed is little, if bacterium ball is larger, the mycelium body at bacterium ball center will present the starvation state of anoxia and nutrition, the generation of unfavorable mycelial proliferation and secondary metabolites, affects fermentation quality and mycelial 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 (wheat bran), 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):

[0146] 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;

[0147] or

[0148] 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;

[0149] or

[0150] 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;

[0151] or

[0152] 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;

[0153] or

[0154] Peptone 5.0%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5.0%, solvent is water;

[0155] or

[0156] Fish peptone 0.5%, peptone 0.5%, glucose 2.0%, KH2PO4 0.1%, MgSO4 0.05%, wheat bran 5%, and the rest is water;

[0157] or

[0158] 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;

[0159] Those skilled in the art will understand that the specific shake flask / seed flask culture medium listed above is only illustrative 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 media for Hirsutella sinensis fermentation, or shake flask / seed flask culture media for Hirsutella sinensis fermentation known in the prior art.

[0160] 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 of aerobic or anaerobic culture of pure microbial cells in a liquid submerged culture medium. Submerged fermentation is generally carried out in large fermentation tanks and can be automated and mechanized. The fermentation tank submerged culture process flow of Chinese Hirsutella spp. can be: first-level seed tank → second-level seed tank → fermentation tank; it can also be: seed tank → fermentation tank; continuous fermentation can also be used. For example, the culture temperature is 10-18°C, preferably the culture temperature is 12-16°C, the culture time is 6-20 days, preferably the culture time is 7-15 days, and preferably the culture time is 10-15 days. The main raw materials of the fermentation medium include one or more of the following: silkworm pupa powder, glucose, corn flour (slurry), yeast extract (extract paste or powder), NaCl, MgCl2 (magnesium chloride or its hydrate), MgSO4 (magnesium sulfate or its hydrate), KCl, CaCl2 (calcium chloride or its hydrate), KH2PO4 (potassium dihydrogen phosphate or its hydrate), polypeptide powder series (such as polypeptide powder 101, polypeptide powder 102, polypeptide powder 203, polypeptide powder 305, polypeptide powder 308, polypeptide powder 607, polypeptide powder 702, polypeptide powder 705) (the polypeptide powder series is purchased from Chengdu Wenfeng Biotechnology Co., Ltd.).

[0161] The fermentation medium of the present invention includes an organic nitrogen source for novel microbial fermentation, such as polypeptide powder "1" series. The polypeptide powder "1" series uses rice protein, corn protein and its hydrolyzate as main raw materials. The process of using multiple hydrolysis methods for staged hydrolysis is adopted, and the powdered products are separated, refined, concentrated and dried. The polypeptide powder "1" series includes polypeptide powder 101 and polypeptide powder 102. The polypeptide powder "2" series, such as polypeptide powder 203, uses corn protein as main raw material, adopts a process of multiple hydrolysis methods for staged hydrolysis (this process makes the product have different molecular weights, and the peptones and peptides are reasonably distributed, which is beneficial to the sustainable use of microorganisms), and the powdered products are separated, refined, concentrated and spray-dried.

[0162] The fermentation medium described herein includes a series of peptones (non-animal-derived) using a novel organic nitrogen source for microbial fermentation, such as polypeptide powder 305, polypeptide powder 308, and polypeptide powder 607. These peptones are primarily made from corn protein and yeast. They utilize a multi-step hydrolysis process (this yields products with varying molecular weights and a balanced distribution of peptones and peptides, facilitating sustainable microbial utilization) followed by separation, refining, concentration, and spray drying to produce a powdered product.

[0163] The fermentation medium of the present invention includes a series of peptones (non-animal derived) using novel organic nitrogen sources for microbial fermentation, such as polypeptide powder 702 and polypeptide powder 705. The polypeptide powder "7" series uses rice protein and corn protein as the main raw materials. The powdered product is prepared by separation, refining, concentration, and spray drying using a process of staged hydrolysis using modern biological methods and multiple hydrolysis methods (this process results in products with different molecular weights and a reasonable distribution of peptones and peptides, which is beneficial for the sustainable utilization of microorganisms).

[0164] The fermentation medium of the present invention may be (the following % are weight to volume ratio):

[0165] 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;

[0166] Preferably,

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

[0168] or

[0169] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 203 1%, and the rest is water;

[0170] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 305 1%, and the rest is water;

[0171] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 702 1%, and the rest is water;

[0172] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 705 1%, and the rest is water;

[0173] Glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, polypeptide powder 607 1%, and the rest is water.

[0174] The Cordyceps sinensis fermentation composition of the present invention refers to mycelium, wet pellets, dried mycelium powder, or dried mycelium powder of a fermentation filtrate obtained through slant culture, seed / shake flask culture, or fermentation culture of Hirsutella sinensis, preferably dried mycelium powder or dried mycelium powder of a fermentation filtrate. The fermentation filtrate includes a fermentation filtrate obtained through solvent separation and chromatography column separation (including membrane separation), or its effective fraction, effective ingredients, or various metabolites.

[0175] The present invention optimizes the fermentation process, and the obtained 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);

[0176] 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.

[0177] 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 even more preferably 2.5-4% (weight percentage);

[0178] Further, the Cordyceps sinensis fermentation composition, its mannitol content is 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.

[0179] 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.

[0180] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0181] When the components contained in the Cordyceps sinensis fermentation composition provided by the present invention are within the above preferred ranges, the composition has excellent antioxidant and whitening effects as a whole.

[0182] Therefore, one aspect of the present invention is to achieve sufficient and stable presence of ergothioneine in Cordyceps sinensis mycelium or powder by low-temperature fermentation technology, at least 0.004% (weight percentage).

[0183] Ergothioneine (EGT) is a white, odorless, colorless crystalline powder in its solid state. In this solid state, only the thione form exists. Ergothioneine is readily soluble in water and exists as tautomers (thiol and thione forms) in solution. However, ergothioneine is very thermally stable, with a decomposition temperature 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 good effects 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. They 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.

[0184] The present invention unexpectedly finds that, in the Cordyceps sinensis fermentation composition, more than 0.004% (percentage by weight), preferably more than 0.006% (percentage by weight), preferably more than 0.008% (percentage by weight), preferably the presence of more than 0.01% (percentage by weight) ergothioneine is not only the first Cordyceps sinensis fermentation product with ergothioneine, and greatly enhances the effect of adenosine. The present invention shows that ergothioneine in the Cordyceps sinensis fermentation composition helps to enhance the antioxidant effect of adenosine by research experiments, so that ergothioneine and adenosine produce a synergistic effect, and by the optimization of the adenosine content range, the inventor unexpectedly finds that the Chinese Hirsutella spp. fermentation product or the fermentation composition as a whole show more excellent technical effects.

[0185] 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 present in sufficient quantity, 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% (percentage by weight), preferably 0.03-0.40% (percentage by weight), preferably 0.03-0.20% (percentage by weight), preferably 0.03-0.10% (percentage by weight), preferably 0.03-0.08% (percentage by weight); the ratio of ergothioneine to adenosine content is 1:1-1:20, preferably 1:1-1:15, preferably 1:1-1:10, preferably 1:1-1:8, preferably 1:1-1:7, preferably 1:1-1:6, preferably 1:1-1:5, preferably 1:1-1:4, preferably 1:1-1:3, preferably 1:1-1:2.

[0186] 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), guanosine, uridine and adenosine may be transformed during the extraction process. 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 adenosine conversion pathway during the water extraction of Cordyceps sinensis, World Chinese Medicine, 2016(11)5:758-762) found that under room temperature water extraction of Cordyceps sinensis, adenosine monophosphate was converted to adenosine, and adenosine was converted to inosine. They also found that under room temperature water extraction, adenosine triphosphate and adenosine diphosphate were also converted to adenosine. Among them, ATP is highly soluble in water and very stable in solutions with pH values ​​between 6.8 and 7.4, but it will be rapidly hydrolyzed at extreme pH values. Adenosine has activity in cardiovascular diseases. For example, Feng Yanhong et al. (Effects of adenosine on isolated rat heart function and myocardial cell apoptosis, Shandong Medicine, 2013, Vol. 53, No. 46) found that SOD is the most important antioxidant enzyme that can effectively eliminate 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.

[0187] The present invention unexpectedly finds that, in the Cordyceps sinensis fermentation composition, 0.004% or more (percentage by weight), preferably 0.006% or more (percentage by weight), preferably 0.008% or more (percentage by weight), preferably the presence of 0.01% or more (percentage by weight) ergothioneine 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 hydroxyl radical scavenging rate synergistic effect. That is, the present invention ensures that the ergothioneine of the obtained Cordyceps sinensis fermentation composition is more than 0.004% (weight percentage), preferably more than 0.006% (weight percentage), preferably more than 0.008% (weight percentage), preferably more than 0.01% (weight percentage) is present in sufficient and stable amount, and particularly preferably 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); 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.

[0188] 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 In Vitro Antioxidant Effect of 5′-acid adenosine (5′-AMP), Food and Machinery, Vol. 24, No. 1, January 2008) showed that AMP has a strong activity in scavenging hydroxyl radicals. Therefore, on the basis of ensuring that the stable presence 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 significant. The highest adenosine obtained by solid fermentation 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 Pharmaceutical Journal, Vol. 56, No. 14, July 2021) recorded that the liquid fermentation culture conditions of Chinese Hirsutella spp. were optimized, and the dry weight of the optimized Chinese Hirsutella spp. 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). However, some studies have shown that the growth rate of the 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 the bacteria decreases, indicating that higher carbon and nitrogen source concentrations are not conducive to bacterial growth (CN103430777A).

[0189] 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 0.03-0.4 ... 0.03-0.20% (percentage by weight), preferably 0.03-0.10% (percentage by weight), preferably 0.03-0.08% (percentage by weight); The ratio of thioneine 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 thioneine is within the preferred range, the Cordyceps sinensis fermentation composition as a whole can be made to have excellent antioxidant effect, well meets application requirements.

[0190] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:20; or,

[0191] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:15; or,

[0192] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1: 1-1: 10; or,

[0193] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:8; or,

[0194] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:7; or,

[0195] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:6; or,

[0196] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:5; or,

[0197] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:4; or,

[0198] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:3; or,

[0199] More specifically, the present invention is based on thioneine content 0.01% or more, preferably adenosine content 0.02% or more (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:2; or,

[0200] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:20; or,

[0201] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:15; or,

[0202] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:10; or,

[0203] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:8; or,

[0204] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:7; or,

[0205] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:6; or,

[0206] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:5; or,

[0207] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:4; or,

[0208] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:3; or,

[0209] More specifically, the present invention is based on thioneine content of more than 0.01%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:2; or,

[0210] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:20; or,

[0211] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:15; or,

[0212] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:10; or,

[0213] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:8; or,

[0214] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:7; or,

[0215] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:6; or,

[0216] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:5; or,

[0217] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:4; or,

[0218] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:3; or,

[0219] More specifically, the present invention is based on thioneine content 0.02% or more, preferably adenosine content 0.02% or more (percentage by weight), the ratio of thioneine to adenosine content is 1:1-1:2; or,

[0220] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:20; or,

[0221] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:15; or,

[0222] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:10; or,

[0223] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:8; or,

[0224] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:7; or,

[0225] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:6; or,

[0226] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:5; or,

[0227] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:4; or,

[0228] More specifically, the present invention is based on thioneine content of more than 0.02%, preferably adenosine content 0.03%-0.08% (percentage by weight), and the ratio of thioneine to adenosine content is 1:1-1:3; or,

[0229] More specifically, the present invention is based on an ergothioneine content of more than 0.02%, preferably an adenosine content of 0.03%-0.08% (percentage by weight), and the ratio of ergothioneine to adenosine content is 1:1-1:2.

[0230] Those skilled in the art will appreciate that the above-mentioned enumeration is only an explanation of the present invention, rather than being interpreted as limiting the application. Due to the research on thioneine and adenosine content, the fermented composition obtained in the described scope has very excellent antioxidant effect, especially the synergistic technology effect of hydroxyl radical scavenging rate.

[0231] On the other hand, the present invention is in conjunction with the content of thioneine, the conversion behavior of adenosine presence, on the basis of thioneine, erythritol, adenosine content, further study the yield of mycelium dry weight etc., that is, the inventors ensure thioneine, erythritol, adenosine content on the basis of, further by the optimization of fermentation process, obtained the higher cordyceps sinensis fermentation composition of mycelium yield, mycelium fermentation yield is more than 7g / L, preferably 7g / L-15g / L, 7g / L-20g / L, 8g / L-15g / L, 8g / L-20g / L, mycelium dry weight water content is less than 8%, preferably water content is less than 7%, preferably water content is less than 6%.

[0232] 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 content of 1:1-1:20, and a mycelium dry weight water content of less than 7%; or,

[0233] 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% (percentage by weight), a ratio of ergothioneine to adenosine content of 1:1-1:20, and a mycelial fermentation yield of more than 7g / L; or,

[0234] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more, preferably an adenosine content of 0.02%-0.2% (weight percentage), a ratio of ergothioneine to adenosine content of 1:1-1:20, and a mycelium dry weight water content of 7% or less; or,

[0235] 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%-0.2% (weight percentage), a ratio of ergothioneine to adenosine content of 1:1-1:20, and a mycelium fermentation yield of more than 7g / L.

[0236] Further, by the inventor's in-depth research, at thioneine 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 existence, the present invention unexpectedly finds that, in Cordyceps sinensis fermentation composition, the existence of more than 0.004% thioneine, is not only the first Cordyceps sinensis fermented product with thioneine, and greatly enhances the effect of other beneficial ingredients, the present invention shows that in Cordyceps sinensis fermentation composition, thioneine helps to enhance the effects such as adenosine by research experiment, and produces synergistic effect, especially in terms of antioxidant effect, makes Cordyceps sinensis fermentation composition as a whole show more excellent technique effect.

[0237] Therefore, another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the ergothioneine content is above 0.004% (weight percentage), preferably above 0.006% (weight percentage), preferably above 0.008% (weight percentage), preferably above 0.01% (weight percentage), and the fermentation composition further comprises one or more of adenosine, mannitol, amino acids, polysaccharides, and erythritol.

[0238] 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 even more preferably 2.5-4% (weight percentage).

[0239] Furthermore, the Cordyceps sinensis fermentation composition has a mannitol content of more than 4% (weight percentage), preferably more than 5%, preferably more than 6%, and preferably more than 7%.

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

[0242] 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), an adenosine content of 0.02% or more (by weight), and a mannitol content of 5% or more (by weight); 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% (by weight) or more, and a mannitol content of 5% or more (by weight); or

[0244] 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% (by weight) or more, and a mannitol content of 6% or more (by weight); or

[0245] 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).

[0246] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the ergothioneine content is 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage), and preferably a polysaccharide content of 2% or more (weight percentage); or,

[0247] Furthermore, the Cordyceps sinensis fermentation composition has a polysaccharide content of more than 2% (weight percentage), preferably more than 3% (weight percentage), and preferably 2-10% (weight percentage).

[0248] Furthermore, the Cordyceps sinensis fermentation composition of the present invention preferably has a polysaccharide content of 2% or more (by weight) and a total amino acid content of 20% or more, based on an ergothioneine content of 0.01% or more;

[0249] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more, and preferably has a polysaccharide content of 2%-10% (weight percentage) and a total amino acid content of 25-50%.

[0250] Those skilled in the art will appreciate that the above-mentioned enumeration 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 fermented composition obtained in the described scope has very excellent technical effect.

[0251] 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 erythritol content is above 1% (weight percentage), preferably 1-6% (weight percentage), and further preferably 2-5% (weight percentage). When the ergothioneine and erythritol contained in the Cordyceps sinensis fermentation composition provided by the present invention are within the preferred limited range of the present invention, the combination of the two exhibits a synergistic effect in scavenging hydroxyl radicals, thereby enhancing the overall antioxidant properties of the Cordyceps sinensis fermentation composition.

[0252] Another aspect of the present invention is to provide 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).

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

[0254] Furthermore, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01% (weight percentage), an adenosine content of more than 0.02% (weight percentage), a mannitol content of more than 4% (weight percentage), and a total amino acid content of more than 20% (weight percentage), preferably 25-50% (weight percentage).

[0255] Furthermore, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of more than 0.01% (weight percentage), an adenosine content of more than 0.02% (weight percentage), a mannitol content of more than 4% (weight percentage), a polysaccharide content of more than 2% (weight percentage), and a total amino acid content of more than 20% (weight percentage), preferably 25-50% (weight percentage).

[0256] 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% or more (by weight), a ratio of ergothioneine to adenosine content of 1:1-1:20, and a mannitol content of 5% or more (by weight); or,

[0257] More specifically, the Cordyceps sinensis fermentation composition of the present invention has an ergothioneine content of 0.01% or more, an adenosine content of 0.03% or more (by weight), a ratio of ergothioneine to adenosine content of 1:1-1:20, and a mannitol content of 5% or more (by weight); or

[0258] 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% or more (by weight), a ratio of ergothioneine to adenosine of 1:1-1:20, a mannitol content of 5% or more (by weight), and a polysaccharide content of 2% or more (by weight); or,

[0259] 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% or more (by weight), a ratio of ergothioneine to adenosine content of 1:1-1:20, a mannitol content of 5% or more (by weight), and a total amino acid content of 20% or more (by weight); 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 content of 1:1-1:20, and a mannitol content of 5% or more (weight percentage); or,

[0261] 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 content 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,

[0262] 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 content of 1:1-1:20, a mannitol content of 5% or more (weight percentage), and a total amino acid content of 20% or more (weight percentage); or,

[0263] 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).

[0264] 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%.

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

[0266] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol and polysaccharide, wherein the fermentation composition also comprises ergothioneine, and the content of ergothioneine (percentage by weight) is above 0.01%.

[0267] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine, mannitol, polysaccharides and amino acids, wherein the fermentation composition also comprises ergothioneine, and the content of ergothioneine (percentage by weight) is above 0.01%.

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

[0269] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes ergothioneine, and the content of ergothioneine (weight percentage) is more than 0.01%, and the ratio of ergothioneine to adenosine content is 1:1-1:20.

[0270] Further, the Cordyceps sinensis fermentation composition includes adenosine, wherein the fermentation composition also includes thioneine, and the content of thioneine (weight percentage) is more than 0.01%, the adenosine content is 0.02-1.0% (weight percentage), and the ratio of thioneine to adenosine content is 1:1-1:20.

[0271] Further, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition also comprises ergothioneine, and the content of ergothioneine (percentage by weight) is above 0.01%, the content of adenosine is 0.02-1.0% (percentage by weight), and the content of mannitol is above 4% (percentage by weight).

[0272] Further, described a kind of Cordyceps sinensis fermentation composition, including adenosine and mannitol, wherein, described fermentation composition also includes thioneine, and the content of thioneine (weight percentage) is more than 0.01%, adenosine content 0.02-1.0% (weight percentage), the ratio of thioneine to adenosine content is 1:1-1:20, and mannitol content is more than 4% (weight percentage).

[0273] Therefore, another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, wherein the content of ergothioneine is 0.004% or more (weight percentage), preferably 0.006% or more (weight percentage), preferably 0.008% or more (weight percentage), preferably 0.01% or more (weight percentage), the adenosine content is 0.02% or more (weight percentage), and the erythritol content is 1.0% or more (weight percentage).

[0274] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably contains 0.02% or more (by weight) of adenosine, on the basis of 0.01% or more of ergothioneine, the ratio of ergothioneine to adenosine is 1:1-1:20, and 1.0% or more (by weight) of erythritol; or

[0275] More specifically, the Cordyceps sinensis fermentation composition of the present invention preferably has an ergothioneine content of 0.01% or more, an adenosine content of 0.02%-1.0% (weight percentage), and an erythritol content of 1.0% or more (weight percentage).

[0276] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, comprising adenosine, wherein the fermentation composition further comprises ergothioneine and erythritol, and the content of ergothioneine (percentage by weight) is above 0.01%.

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

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

[0279] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, mannitol, polysaccharides and amino acids, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%.

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

[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%, and the ratio of ergothioneine to adenosine content is 1:1-1:20.

[0282] 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 adenosine content is 0.02-1.0% (weight percentage), and the ratio of ergothioneine to adenosine content is 1:1-1:20.

[0283] Furthermore, the Cordyceps sinensis fermentation composition comprises adenosine and mannitol, wherein the fermentation composition also comprises ergothioneine and erythritol, and the ergothioneine content (weight percentage) is above 0.01%, the adenosine content is 0.02-1.0% (weight percentage), and the mannitol content is above 4% (weight percentage).

[0284] Further, 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 more than 0.01%, the adenosine content is 0.02-1.0% (weight percentage), the ratio of ergothioneine to adenosine content is 1:1-1:20, and the mannitol content is more than 4% (weight percentage).

[0285] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, comprising adenosine, wherein the fermentation composition further comprises ergothioneine and erythritol, and the erythritol content is above 1% (weight percentage).

[0286] 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).

[0287] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine, mannitol and polysaccharide, wherein the fermentation composition also includes ergothioneine and erythritol, and the erythritol is more than 1% (weight percentage).

[0288] 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 content is above 1% (weight percentage).

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

[0290] 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).

[0291] 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 erythritol is above 1% (weight percentage), and the adenosine content is 0.02-1.0% (weight percentage).

[0292] Furthermore, the Cordyceps sinensis fermentation composition includes adenosine and mannitol, wherein the fermentation composition also includes ergothioneine and erythritol, and the content of ergothioneine (weight percentage) is above 0.01%, the erythritol is above 1% (weight percentage), the adenosine content is 0.02-1.0% (weight percentage), and the mannitol content is above 4% (weight percentage).

[0293] Another aspect of the present invention is to provide a Cordyceps sinensis fermentation composition, which comprises erythritol and mannitol, and the mannitol content 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 mannitol content of the Cordyceps sinensis fermentation composition is above 4% (weight percentage), preferably above 5%, preferably above 6%, and preferably above 7%.

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

[0295] 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), and preferably 0.03-0.08% (weight percentage).

[0296] 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), and preferably 2-10% (weight percentage).

[0297] Furthermore, the content of total amino acids is above 20% (weight percentage), preferably 25-50% (weight percentage).

[0298] The Cordyceps sinensis fermentation composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, and capsules, or a single preparation can be obtained by mixing the components and encapsulating the mixture or forming the mixture into tablet form. Various dosage forms can also be prepared using acceptable carriers. With respect to oral administration compositions (e.g., tablets and capsules), the term "acceptable carrier" includes such carriers as common excipients, such as binders such as syrup, gum arabic, gelatin, sorbitol, gum tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose, and starch; fillers and carriers such as corn starch, gelatin, lactose, sucrose, and microcrystalline cellulose; and lubricants such as magnesium stearate, sodium stearate and other stearates, glyceryl stearate, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silicon dioxide. Flavoring agents such as peppermint, wintergreen oil, and cherry flavoring may also be used. Colorants may be added to distinguish the dosage form. 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.

[0299] In summary, the cordyceps sinensis fermentation composition of the present invention is not only the first cordyceps sinensis fermentation product with thioneine, and, due to the research on thioneine 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 on the whole.

[0300] 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.

[0301] Further, the ratio of thioneine 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.

[0302] 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.

[0303] The pharmaceutical composition of the present invention can be directly prepared into various dosage forms, including powders, granules, tablets, pills, and capsules, or a single preparation can be obtained by mixing the components and encapsulating the mixture or forming the mixture into tablets. Various dosage forms can also be prepared using acceptable carriers.

[0304] The pharmaceutical composition of the present invention has excellent antioxidant effect and synergistic technical effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0305] Fig. 1 is thioneine standard substance HPLC collection of illustrative plates;

[0306] Fig. 2 is B-5 sample thioneine HPLC collection of illustrative plates;

[0307] Fig. 3 is B-7 sample thioneine HPLC collection of illustrative plates;

[0308] FIG4 is a HPLC profile of adenosine from sample CN101096641A of Example 4;

[0309] FIG5 is a HPLC profile of adenosine from Example 4B-2;

[0310] FIG6 is a HPLC spectrum of erythritol of sample B-2;

[0311] FIG7 is a standard curve diagram of polysaccharides. DETAILED DESCRIPTION

[0312] 1. Ergothioneine Detection Method

[0313] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0314] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; 0.2% heptafluorobutyric acid as mobile phase A, and methanol as mobile phase B; the detection wavelength was 255 nm. The theoretical plate number, calculated based on the ergothioneine peak, should be no less than 3000.

[0315] Preparation of reference solution: An appropriate amount of ergothioneine reference solution was accurately weighed and added with 0.5% phosphoric acid solution to prepare a solution containing μg per 1 mL. Gradient elution was performed according to the following table:

[0316] Preparation of the test solution: Take 0.5 g of fermented Cordyceps sinensis powder, accurately weigh it, place it in a stoppered conical flask, add 20 mL of ether, seal it tightly, soak it for 30 minutes, filter it, discard the ether, evaporate the ether from the residue, place it together with the filter paper in a stoppered conical flask, accurately add 50 mL of 0.5% phosphoric acid solution, seal it tightly, weigh it, and treat it with ultrasound for 30 minutes at a power of 250 W and a frequency of 33 kHz. Let it cool, shake it evenly, centrifuge it, and take the supernatant.

[0317] Determination method: Accurately aspirate 10 μL 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, accurately weigh it, 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, and treat it ultrasonically (power 250 W, frequency 40 kHz) for 60 min. Let it cool, weigh it again, make up the lost weight with deionized water, shake it well, filter it, and take the filtrate to obtain the product.

[0323] Detection method

[0324] Detector: RID differential detector;

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

[0326] Mobile phase: deionized water;

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

[0328] Flow 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 it 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, immediately shake well, let it stand for 15 minutes, immediately cool it in an ice water bath for 15 minutes, take it out and let it stand at room temperature for 10 minutes, use the corresponding reagent as the blank, measure the absorbance at 625 nm, and draw a standard curve with absorbance as the ordinate, as shown in Figure 7.

[0337] Preparation of test solution

[0338] Take about 2g of fermented Cordyceps sinensis powder, accurately weigh it, put it in a round-bottom flask, add 60mL of water, let it stand for 30min, heat under 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 under reflux for 2h, transfer it to a centrifuge tube while hot, centrifuge again, combine the supernatant, evaporate to dryness in a water bath, dissolve the residue in 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] Where:

[0343] W-polysaccharide content, %;

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

[0345] m-sample mass, mg;

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

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

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

[0349] Detection principle

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

[0351] Solution preparation

[0352] 1. Weigh 0.2 g of bacterial powder, add 2 mL of purified water, mix well, let stand at room temperature for 4 h, centrifuge at 10,000 rpm for 10 min, and take the supernatant for use.

[0353] 2. Other samples are reference solutions.

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

[0355] Add sample

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

[0357] Calculation: Test sample OH clearance rate (%) = [(A3-A3')-(A2-A0)] / (A1-A2)×100%

[0358] 5. The detection methods for adenosine, mannitol, total amino acid content and moisture shall be in accordance with the detection methods under the national drug standard for fermented Cordyceps sinensis powder.

[0359] 6. Detection method of tyrosinase inhibition rate

[0360] 1. Preparation of solution

[0361] (1) Preparation of PBS buffer: Weigh 3.4022 g of potassium dihydrogen phosphate and 5.7055 g of potassium dihydrogen phosphate trihydrate, dissolve them in appropriate amount of distilled water, and dilute to volume in a 1000 mL volumetric flask. Shake well to obtain a PBS buffer with a pH of 6.81 and a concentration of 0.05 mol·L-1. Store in a refrigerator at 4°C away from light.

[0362] (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, adjust the volume, shake well, and sonicate to obtain a 2 mmol·L-1 L-DOPA solution, which is used immediately after preparation.

[0363] (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 100mL volumetric flask, dilute to volume with PBS buffer, shake well to obtain 250U·mL-1 TYR solution, then dilute the mother liquor to 125U·mL-1 and store in a refrigerator at -20℃ for later use.

[0364] 2. Sample preparation: 250mg / ml erythritol stock solution: weigh 2.5g erythritol and add it to 10ml purified water; 250mg / ml mannitol stock solution: weigh 7.5g mannitol and add it to 30ml purified water, heat to dissolve, and dilute to the required concentration according to each reaction system before adding to the detection system.

[0365] 3. Determination of TYR inhibitory activity of samples

[0366] The reaction system, as shown in Table 4.2, uses 2 mmol·L⁻¹ L-DOPA as the substrate. PBS buffer, sample solution, and substrate are added sequentially to wells numbered 1, 2, 3, and 4 in 96-well plates, with each well repeated three times. The multi-functional microplate reader is set to 37°C. The plate is linearly shaken for 10 minutes, then removed from the plate. Finally, the TYR solution is added and quickly replaced. The detection wavelength is 475 nm. The total reaction volume is 240 μL. The absorbance is measured every 30 seconds for a total of 30 minutes. The inhibition rate is calculated based on the absorbance over the 30-minute period. The inhibition rate is calculated using the following formula. If the result is a negative number, the absolute value of the negative number is the activation rate.

[0367] A1: absorbance of the reaction solution with TYR added but no sample measured at 475 nm;

[0368] A2: absorbance of the reaction solution without sample and TYR at 475 nm;

[0369] A3: Absorbance of the reaction solution with sample and TYR measured at 475 nm;

[0370] A4: The absorbance of the reaction solution with sample added but not TYR measured at 475nm.

[0371] Example 1 Different mycelia ergothioneine (EGT) and erythritol content

[0372] The inventor's research shows that no ergothioneine is 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:

[0373] Although theoretically, the nutrient content and medicinal effects of the mycelium of 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. Not only are there differences with natural Cordyceps sinensis, but there are also large differences between the fermentation products of different Cordyceps sinensis. On the basis of studying natural Cordyceps sinensis, the inventors collected a number of 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, as well as Hirsutella sinensis CGMCC 3.14240 and Hirsutella sinensis CGMCC3.14243 purchased from China General Microorganism Culture Collection Center (CGMCC), and Paecilomyces hepiali 3.15540, Paecilomyces hepiali CGMCC3.7845, and Paecilomyces hepiali purchased from China General Microorganism Culture Collection Center (CGMCC). hepiali) CGMCC3.7908, further with reference to prior art, on the basis of having eliminated the possibility interference of exogenous thioneine (such as culture medium does not detect thioneine with silkworm pupa powder), studied the fermentation filtrate and mycelium dry powder in the existing Cordyceps sinensis fermentation process, not only all lower than the amount of thioneine that can be detected by natural Cordyceps sinensis, and most of all do not detect, but the inventor unexpectedly finds that Chinese Hirsutosporium fermentation mycelium can obtain trace thioneine.

[0374] Note: It means that according to the peak area, the content is only 3‰-10‰ of natural Cordyceps sinensis.

[0375] 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 in the fermentation filtrate and mycelium during the fermentation process.

[0376] Further, except the obtainable bacterial strain of above-mentioned preservation center, inventor is to commercially available many strains Hirsutella sinensis (Hirsutella sinensis), for example, the Cordyceps sinensis (Hirsutella spp. Hirsutella spp., Hirsutella sinensis) Ophiocordyceps sinensis standard strain CICC 14092, CICC 14095, CICC 14097, CICC 14099 purchased from China Industrial Microorganism Culture Collection Administration Center (CICC), and the Cordyceps sinensis (Hirsutella spp. Hirsutella spp., Hirsutella sinensis) Ophiocordyceps sinensis article number purchased from Tai Si Tuo biology is TS326189, TS326199 and Hirsutella sinensis (Hirsutella sinensis) article number is TS361737, the bacterial strain of TS392986, ferment with reference to prior art, all do not obtain thioneine at obtained fermentation filtrate and mycelium.

[0377] Similarly, the inventor is to commercially available multiple strains Paecilomyces hepiali (Paecilomyces hepiali), and such as deposit number is CGMCC 3.15421, CGMCC 3.14870, CGMCC 3.12108, CGMCC 3.10157, CCTCC NF 20081231, CCTCC NF 20081239, CCTCC NF 20081244, CCTCC NF 20081250 and ferments with reference to prior art, and thioneine is not all obtained at resulting fermentation filtrate and mycelium.

[0378] As shown in the table above, the inventors performed 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.

[0379] Furthermore, in addition to the strains available from the above-mentioned collection centers, the inventors fermented several commercially available strains of Hirsutella sinensis, for example, standard strains of Ophiocordyceps sinensis (Ophiocordyceps sinensis) CICC 14092, CICC 14095, CICC 14097, and CICC 14099 purchased from the China Industrial Culture Collection Center (CICC), and Ophiocordyceps sinensis (Ophiocordyceps sinensis) with product numbers TS326189 and TS326199 purchased from Testo Biotechnology, as well as strains of Hirsutella sinensis with product numbers TS361737 and TS392986, according to the prior art. However, no stable and meaningful content of erythritol was obtained in the resulting fermentation filtrate and mycelium.

[0380] Similarly, the inventors fermented several commercially available strains of Paecilomyces hepiali, such as those with accession numbers CGMCC 3.15421, CGMCC 3.14870, CGMCC 3.12108, CGMCC 3.10157, CCTCC NF 20081231, CCTCC NF 20081239, CCTCC NF 20081244, and CCTCC NF 20081250, according to the prior art. However, no stable and meaningful content of erythritol was obtained in the resulting fermentation filtrate and mycelium.

[0381] Example 2 has a fermented product that stably contains more than 0.004% ergothioneine (EGT)

[0382] Hirsutella sinensis was obtained from China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC No: M 2011278. Hirsutella sinensis was inoculated on a slant and cultured on the slant, in shake flasks / seed flasks, and fermented as follows:

[0383] 1. Slant culture

[0384] 1.1 Slant solid culture medium formula: silkworm pupa powder 1.8%, corn starch 1.5%, glucose 2.0%, MgSO4 0.01%, KH2PO4 0.02%, oatmeal 1.0-3.0 g / 60 mL, agar powder 0.65%, pH 6.8-7.2, prepared in water, sterilized at (118-122) °C for (30 ± 1) min.

[0385] 1.2 Under sterile conditions, use an inoculation shovel to dig out a 2×2 cm block of fungus, evenly spread the mycelium on a blank solid culture medium, place it in a biochemical incubator, and culture it at 15°C for 30 days.

[0386] 1.3 Store the cultured slant in a refrigerator at 0-8°C. The mycelium will grow slowly during this period. When the slant is fully grown, remove it and set aside.

[0387] 2 shake flask culture

[0388] 2.1 Shake flask culture 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;

[0389] 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.

[0390] 2.3 Culture medium preparation: Weigh the corresponding materials and dissolve them in the liquefied filtrate, adjust the volume, and divide the volume into shake flasks at a filling volume of 1500mL / 5000mL. Sterilize at (118-122)℃ for (30±1)min.

[0391] 2.4 Digging: Cut the slope into approximately 1.5 × 2.0 cm blocks. Use a sterile rake to rake the cut blocks into the prepared digging / seed bottle culture medium. Incubate at (13-16) ° C, (120-140) rpm, and shake for 9-14 days.

[0392] 3. Fermentation culture:

[0393] 3.1 Basic fermentation medium formula: glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01%, pH 7.0.

[0394] 3.2 Culture medium preparation: Shake flask volume 1000mL / 5000mL, (118-122)℃, sterilize for (30±1)min.

[0395] 3.3 Fermentation: inoculation amount 10%, temperature (13-16) °C, (120-140) rpm, shake culture for 7 days and then release the bottle.

[0396] 3.4 Take an appropriate volume of fermentation broth, vacuum filter to obtain wet bacterial slices, and dry to obtain bacterial powder samples.

[0397] Different components were added to the above-mentioned basic fermentation medium (glucose 2.8%, yeast extract 1%, KH2PO4 0.02%, MgSO4 0.01% (% are weight to volume ratios)) to obtain mycelial experimental numbered samples A-1 to A-8 and B-1 to B-8. The results of ergothioneine and erythritol detection are shown in the following table:

[0398] Furthermore, in addition to the strains disclosed in the above patents, the inventors have also conducted experiments on several commercially available strains of Hirsutella sinensis, such as Hirsutella sinensis CGMCC 3.14240 and Hirsutella sinensis CGMCC3.14243 purchased from the China General Microorganism Culture Collection Center (CGMCC), and standard strains of Ophiocordyceps sinensis CICC 14092, CICC 14095, CICC 14097, and CICC 14099 purchased from the China Industrial Microorganism Culture Collection Center (CICC), and Ophiocordyceps sinensis with item numbers TS326189, TS326199, and Hirsutella sinensis purchased from Testo Biological. The strains of Hirsutella sinensis with the deposit number TS361737 and TS392986 were fermented according to the fermentation method of Hirsutella sinensis with the deposit number CCTCC No: M 2011278 in Example 2. 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 powder samples obtained by fermentation of different Hirsutella sinensis were not significantly different from the contents of the components in the powder prepared with the deposit number CCTCC No: M 2011278 under the same conditions.

[0399] Example 3 Study on the effect of effective amount of ergothioneine or erythritol on the efficacy of Chinese Hirsutella spp. fermentation powder

[0400] 1. Results of the synergistic experiment on the scavenging rate of ergothioneine and adenosine hydroxyl radicals

[0401] 2. Experimental results on the synergistic effect of ergothioneine and erythritol on hydroxyl radical scavenging rate

[0402] 3. Results of synergistic experiment on erythritol and adenosine hydroxyl radical scavenging rate

[0403] The theoretical values ​​stated in the table are the simple superposition of the hydroxyl radical scavenging rates of the two components;

[0404] 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.

[0405] 4. Synergistic effect of erythritol and mannitol on tyrosinase inhibition rate

[0406] 5. Experimental results on the synergistic effect of erythritol and mannitol on hydroxyl radical scavenging rate

[0407] Example 4 Experimental results of hydroxyl radical scavenging rate of fermentation bacteria powder obtained by the present invention and fermentation bacteria powder of prior art

[0408] Experimental samples:

[0409] The preparation of mycelial samples B-2, B-5, and B-7 was the same as before;

[0410] Example 5 Sample preparation of CN108384726A:

[0411] 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.

[0412] Example 1 Sample preparation of CN101096641A:

[0413] The bacterial strain is inoculated onto a slant surface using a culture medium consisting of 1.5% glucose, 1.0% corn flour, 0.5% dextrin, 1.5% bran, 0.5% yeast powder, 1.5% silkworm pupa powder, 1.0% peptone, 0.01% magnesium sulfate, 0.02% potassium dihydrogen phosphate, 0.8% agar powder, and the remainder water, and cultured at 12°C for 25 days. The bacterial strain is then inoculated into a fermentation medium consisting of 1.0% glucose, 1.0% molasses, 0.5% silkworm pupa powder, 1.0% soybean cake powder, 0.5% yeast extract, 0.01% magnesium sulfate, 0.02% potassium dihydrogen phosphate, and the remainder water, and cultured on a shaker at 14°C for 8 days. The fermentation medium is then inoculated into a primary fermentation tank and cultured at 14°C for 8 days. The volume is then expanded by 10 times the volume of the culture medium, and fermented in stages until the volume reaches a 30-ton tank. After removal from the tank, solid-liquid separation is performed, and the solid is dried and pulverized.

[0414] Those skilled in the art will understand that the specific embodiments listed above are merely 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 within the scope of the present invention. Industrial Applicability

[0415] The cordyceps sinensis fermentation composition provided by the present invention is not only the first cordyceps sinensis fermentation product containing ergothioneine, but also, due to research on the content of ergothioneine 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 more excellent technical effect than the existing technology as a whole, and has strong industrial applicability.

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%, preferably 1.5-5wt%, more preferably 1.5-4wt%, and further preferably 2.5-4wt%.

3. The fermentation composition according to claim 1 or 2, characterized in that The content of mannitol is 5 wt % or more, preferably 6 wt % or more, and more preferably 7 wt % or more.

4. The fermentation composition according to any one of claims 1 to 3, characterized in that The fermented composition contains adenosine, and the content of the adenosine is greater than 0.02 wt %, preferably 0.03 wt % to 1.0 wt %.

5. The fermentation composition according to any one of claims 1 to 4, characterized in that The fermented composition contains ergothioneine, and the content of the ergothioneine is greater than 0.004 wt %, preferably greater than 0.01 wt %.

6. The fermentation composition according to any one of claims 1 to 5, characterized in that The fermented composition contains amino acids and / or polysaccharides.

7. The fermentation composition according to claim 6, characterized in that The content of the polysaccharide is 2 wt% or more, preferably 3 wt% or more, more preferably 4 wt% or more, and further preferably 2-10 wt%.

8. The fermentation composition according to claim 6 or 7, characterized in that The total content of the amino acids is above 20 wt %, preferably 25-50 wt %.

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.

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