Phellinus igniarius-Chinese yam fermentation mycoplasm as well as preparation method and application thereof

Abstract

The invention provides phellinus igniarius-Chinese yam fermentation mycoplasm as well as a preparation method and application thereof, and relates to the technical field of microbial fermentation. Through a bidirectional fermentation technology, the Chinese yam is used as a fermentation culture medium, the black beans are used as a supplementary nitrogen source, and the phellinus igniarius strain is used as a fermentation strain for bidirectional fermentation. On one hand, by adopting the fermentation method, the growth speed of phellinus igniarius mycelia and the synthesis efficiency of active ingredients are remarkably improved, and the content of various active ingredients such as flavone, polyphenol and polysaccharide in a fermentation product is remarkably improved. On the other hand, the fermented mycoplasm obtained by the method is also rich in various active ingredients such as flavone, polyphenol, polysaccharide, alkaloid and the like, and has remarkable oxidation resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, and particularly relates to a Phellinus linteus-Dioscorea opposita fermentation fungus, a preparation method and application thereof. BACKGROUND

[0002] Phellinus linteus belongs to the family of Hymenochaetaceae and is a large fungus with important medicinal value. Phellinus linteus is rich in polysaccharides, flavonoids, polyphenols, triterpenoids, alkaloids and other bioactive components, and has significant antioxidant, antitumor, immunomodulatory, anti-inflammatory and other effects. Traditional Chinese medicine literature such as Materia Medica and Compendium of Materia Medica have recorded that Phellinus linteus has the effects of stopping bleeding, dispersing blood, and resisting tumors. Modern research shows that the active ingredients of Phellinus linteus have inhibitory effects on various cancers such as gastric cancer, liver cancer, lung cancer, breast cancer, etc., and the flavonoids, polyphenols and polysaccharides in the fruiting bodies and mycelium of Phellinus linteus have significant antioxidant and anti-aging effects, can enhance the activity of immune cells, and improve the body's immunity.

[0003] Dioscorea opposita is a plant with both medicinal and edible values, and is rich in protein, vitamins, amino acids and various trace elements, and also contains starch, saponins, choline and other nutrients, and has very high nutritional and medicinal value. Compendium of Materia Medica records that Dioscorea opposita has the effects of “benefiting kidney qi, invigorating spleen and stomach, stopping diarrhea, resolving phlegm and sputum, and moisturizing skin and hair”. Modern research shows that Dioscorea opposita not only can supplement the nutrients needed by the body, but also has the pharmacological effects of benefiting qi and nourishing yin, tonifying spleen and stomach, generating fluid and benefiting lung, and tonifying kidney and astringing essence, and is widely used in the fields of food, medicine and health care products.

[0004] Although Phellinus linteus and Dioscorea opposita each have significant medicinal value, the traditional single fermentation or extraction method often cannot fully exert the potential of the active ingredients. The existing fermentation technology of Phellinus linteus mostly uses a single culture medium, and the synthesis efficiency of active ingredients is low during the fermentation process, and the nutritional components of Dioscorea opposita cannot be fully utilized in the traditional processing process. Therefore, how to further improve the content of active ingredients and the bioavailability of Phellinus linteus and Dioscorea opposita through innovative fermentation technology has become an important research direction. SUMMARY

[0005] In the present application, Dioscorea opposita is used as a fermentation culture medium, black beans are used as a supplementary nitrogen source, and Phellinus linteus strain is used as a fermentation strain for bidirectional fermentation, aiming to further promote the growth and metabolism of Phellinus linteus strain, promote the production of active products, and obtain a fungus with high antioxidant capacity.

[0006] Two-way fermentation technology combines medicinal fungi with traditional Chinese medicinal materials to achieve a two-way enhancement of active ingredients, significantly improving the bioavailability and pharmacological activity of the fermentation products. In the two-way fermentation process described in this invention, *Sanghuang* mycelium secretes various enzymes (such as cellulase, amylase, and protease) to degrade the cell wall structure in the yam substrate, releasing active ingredients such as polysaccharides, flavonoids, and polyphenols from the yam. Simultaneously, the flavonoids and polyphenols synthesized by the *Sanghuang* mycelium during fermentation are also significantly enhanced. Experiments show that the antioxidant capacity of the *Sanghuang*-yam mycelium after two-way fermentation is significantly higher than that of single fermentation products, and it has a stronger scavenging ability against various free radicals (such as DPPH free radicals and hydroxyl free radicals). Furthermore, two-way fermentation technology is characterized by its simple fermentation process and high efficiency. Therefore, two-way fermentation technology provides a new approach for the deep processing of traditional Chinese medicinal materials such as yam. By using yam as a fermentation substrate, not only can its nutritional components be fully utilized, but its medicinal value can also be significantly enhanced. Fermented yam substrate has stronger antioxidant activity and pharmacological functions, and can be widely used in the fields of food, medicine, cosmetics and health products.

[0007] On the one hand, this application provides a method for preparing Phellinus linteus-yam fermentation mycelium, the method comprising: inoculating Phellinus linteus into a culture medium containing yam for fermentation.

[0008] Preferably, the yam is yam powder with a moisture content of 8%-12% and a mesh size of 20-100.

[0009] More preferably, the yam can be any value among 20 mesh, 30 mesh, 40 mesh, 50 mesh, 60 mesh, 70 mesh, 80 mesh, 90 mesh, and 100 mesh.

[0010] More preferably, the moisture content of the yam powder can be any value among 8%, 9%, 10%, 11%, and 12%.

[0011] Furthermore, the culture medium also includes a nitrogen source; preferably, the nitrogen source is selected from one or more of black beans, peanuts, fish meal, coffee grounds, soybean protein powder, and urea; more preferably, the nitrogen source is black beans; more preferably, the mass ratio of yam to nitrogen source in the culture medium is 5:(0.5-2); more preferably, the mass ratio of yam to nitrogen source in the culture medium is 5:1.

[0012] The mass ratio of yam to nitrogen source in the culture medium is any one of 5:0.5, 5:1, 5:1.5, or 5:2.

[0013] Preferably, the black beans are black bean powder with a mesh size of 20-100 and a moisture content of 8-12%.

[0014] More preferably, the black beans can be any one of 20 mesh, 30 mesh, 40 mesh, 50 mesh, 60 mesh, 70 mesh, 80 mesh, 90 mesh, 100 mesh.

[0015] More preferably, the water content of the black bean powder can be any one of 8%, 9%, 10%, 11%, 12%.

[0016] Further, the preparation method of the culture medium comprises: adding yam and nitrogen source into water according to a solid-liquid ratio of 1: (1-2.3); preferably, the solid-liquid ratio is 1:1.2.

[0017] Preferably, the solid-liquid ratio can be any one of 1:1.0, 1:1.2, 1:1.5, 1:1.8, 1:2.3.

[0018] More preferably, the preparation method of the culture medium further comprises a sterilization step.

[0019] In a preferred embodiment, the sterilization is sterilization at 121℃ for 40 min.

[0020] The skilled in the art can choose an existing sterilization method for sterilization as long as the purpose of sterilization in the culture medium can be achieved.

[0021] Further, the inoculated strain OD 600 value is 0.5-1.0, and the inoculation amount is 4mL / 100g-14mL / 100g; more preferably, the inoculation amount is 10mL / 100g.

[0022] Preferably, the inoculation amount can be any one of 4mL / 100g, 6mL / 100g, 8mL / 100g, 10mL / 100g, 12mL / 100g, 14mL / 100g.

[0023] Preferably, the method further comprises a step of activating the Phellinus igniarius strain.

[0024] The skilled in the art can activate the Phellinus igniarius by using a general method.

[0025] In a preferred embodiment, the Phellinus igniarius activation method comprises: inoculating the Phellinus igniarius into an activation culture medium, and culturing in the dark at 20℃-30℃ for 5-10d.

[0026] In a preferred embodiment, the Phellinus igniarius activation method comprises: inoculating the Phellinus igniarius into an activation culture medium, and culturing in the dark at 25℃ for 7d.

[0027] In a preferred embodiment, the Phellinus igniarius activation method comprises:

[0028] Step one, inoculate the Phellinus baumii to the activated culture medium, cultivate in dark at 25℃ for 7 days, and obtain the first activated seed;

[0029] Step two, inoculate the first activated seed to the seed liquid culture medium, inoculation amount is 3×3mm 2 mycelium block, cultivate at 25℃, 130r / min constant temperature vibration, cultivate until OD 600 value is 0.5-1.0.

[0030] More preferably, the activated culture medium is PDA (potato dextrose agar) medium.

[0031] More preferably, the activated culture medium includes: potato, glucose, yeast powder, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1, water.

[0032] More preferably, the activated culture medium includes: potato 10%-50%, glucose 1%-5%, yeast powder 0.1%-0.5%, potassium dihydrogen phosphate 0.1%-0.5%, magnesium sulfate 0.1%-0.5%, vitamin B1 1%-5%, and the rest is water.

[0033] More preferably, the activated culture medium includes: potato 20%, glucose 2%, yeast powder 0.3%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1%, vitamin B1 1%, and the rest is water.

[0034] In a preferred embodiment, the activated culture medium includes: potato 200g, glucose 20g, yeast powder 3g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, vitamin B1 10mg, water 1000mL.

[0035] More preferably, the seed liquid culture medium includes: potato, sucrose, yeast powder, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1, water.

[0036] More preferably, the activated culture medium includes: potato 10%-50%, sucrose 1%-5%, yeast powder 0.1%-0.5%, potassium dihydrogen phosphate 0.1%-0.5%, magnesium sulfate 0.1%-0.5%, vitamin B1 1%-5%, and the rest is water.

[0037] More preferably, the activated culture medium includes: potato 20%, sucrose 2%, yeast powder 0.3%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1%, vitamin B1 1%, and the rest is water.

[0038] In a preferred embodiment, the seed liquid culture medium includes: potato 200g, sucrose 20g, yeast powder 3g, potassium dihydrogen phosphate 2.0g, magnesium sulfate 1.0g, vitamin B1 10mg, water 1000mL.

[0039] More preferably, the method further comprises the step of propagating the Phellinus baumii strain.

[0040] In a preferred embodiment, the propagation method of the Phellinus baumii comprises inoculating the Phellinus baumii into the propagation medium, and culturing at 20-30℃ in the dark until the surface of the medium is covered.

[0041] In a preferred embodiment, the propagation method of the Phellinus baumii comprises inoculating the Phellinus baumii into the propagation medium, and culturing at 25℃ in the dark until the surface of the medium is covered.

[0042] More preferably, the propagation medium is PDA (potato dextrose agar) medium.

[0043] More preferably, the propagation medium comprises potato, glucose, yeast powder, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1, and water.

[0044] More preferably, the propagation medium comprises 10-50% of potato, 1-5% of glucose, 0.1-0.5% of yeast powder, 0.1-0.5% of potassium dihydrogen phosphate, 0.1-0.5% of magnesium sulfate, and 1-5% of vitamin B1, and the rest is water.

[0045] More preferably, the propagation medium comprises 20% of potato, 2% of glucose, 0.3% of yeast powder, 0.2% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, and 1% of vitamin B1, and the rest is water.

[0046] In a preferred embodiment, the propagation medium comprises 200g of potato, 20g of glucose, 3g of yeast powder, 2g of potassium dihydrogen phosphate, 1g of magnesium sulfate, 10mg of vitamin B1, and 1000mL of water.

[0047] The person skilled in the art can select the propagation method of the Phellinus baumii or decide whether to perform the propagation operation according to the actual situation, which is not limited here.

[0048] In a preferred embodiment, the activation method of the Phellinus baumii comprises:

[0049] Step one, inoculating the Phellinus baumii into the activation medium, and culturing at 25℃ in the dark for 7 days to obtain the first activated seed;

[0050] Step two, inoculating the first activated seed into the propagation medium, and culturing at 25℃ in the dark until the surface of the medium is covered to obtain the propagation strain;

[0051] Step three, inoculating the propagation strain into the seed liquid medium, and the inoculation amount is 3×3mm 2mycelial block, under the condition of constant temperature oscillation at 25℃, 130r / min, and the culture is carried out until OD 600 value is 0.5-1.0.

[0052] Further, the fermentation condition comprises: 15℃-35℃ fermentation for 5-40 days; preferably, 25℃ fermentation for 25 days.

[0053] The fermentation temperature can be any value among 15℃, 20℃, 25℃, 30℃ and 35℃.

[0054] The fermentation time can be any value among 5, 10, 15, 20, 25, 30, 35 and 40 days.

[0055] In a preferred embodiment, the preparation method of the Phellinus baumii-Dioscorea fermenting mycelium comprises the following steps:

[0056] Step one, mixing Dioscorea and black beans with the mass ratio of 5:1, adding water according to the proportion of 1:1.2, sterilizing, and obtaining the culture medium;

[0057] Step two, inoculating Phellinus baumii in the culture medium with the inoculation amount of 10mL / 100g, and fermenting under the condition of 25℃, 130r / min, and the constant temperature oscillation, and the culture is carried out until OD 600 value is 0.5-1.0, and obtaining the Phellinus baumii-Dioscorea fermenting mycelium.

[0058] Preferably, the method further comprises the steps of drying and / or crushing the Phellinus baumii-Dioscorea fermenting mycelium.

[0059] More preferably, the drying condition is ≥65℃.

[0060] In a preferred embodiment, the preparation method of the Phellinus baumii-Dioscorea fermenting mycelium comprises the following steps:

[0061] Step one, mixing Dioscorea and black beans with the mass ratio of 5:1, adding water according to the proportion of 1:1.2, sterilizing, and obtaining the culture medium;

[0062] Step two, inoculating Phellinus baumii in the culture medium with the inoculation amount of 10mL / 100g, and fermenting under the condition of 25℃, 130r / min, and the constant temperature oscillation, and the culture is carried out until OD 600 value is 0.5-1.0, and obtaining the Phellinus baumii-Dioscorea fermenting mycelium.

[0063] On the other hand, the application also provides the Phellinus baumii-Dioscorea fermenting mycelium prepared by the method.

[0064] Further, the fermented fungus of Phellinus baumii-Dioscorea opposita contains antioxidant active substances; preferably, the antioxidant active substances include one or more of polyphenols, flavonoids, polysaccharides, and alkaloids.

[0065] Preferably, the content of polyphenols in the fermented fungus of Phellinus baumii-Dioscorea opposita is greater than or equal to 1.6 mg / g, and more preferably greater than or equal to 3 mg / g.

[0066] Preferably, the content of flavonoids in the fermented fungus of Phellinus baumii-Dioscorea opposita is greater than or equal to 2.1 mg / g, and more preferably greater than or equal to 5 mg / g.

[0067] Preferably, the content of polysaccharides in the fermented fungus of Phellinus baumii-Dioscorea opposita is greater than or equal to 168 mg / g, and more preferably greater than or equal to 240 mg / g.

[0068] Preferably, the content of alkaloids in the fermented fungus of Phellinus baumii-Dioscorea opposita is greater than or equal to 2 mg / g, and more preferably 2.6 mg / g.

[0069] The present application first proves that the method of bidirectional fermentation of Phellinus baumii-Dioscorea opposita can improve the content of effective components in the fermented fungus. Compared with the Dioscorea opposita culture medium without fermentation, the flavonoids in the fermented fungus are increased by 228.6%, the polyphenols are increased by 92.8%, the polysaccharides are increased by 37.4%, and the alkaloids are increased by 451.2%, all of which have a significant growth effect.

[0070] In another aspect, the present application also provides a composition comprising the fermented fungus of Phellinus baumii-Dioscorea opposita.

[0071] The composition of the present application can also add adjuvants, which can be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integration agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filtration aids, release retardants, etc.

[0072] The composition of the present application can be prepared by general methods, in which one or more diluents or carriers can be added, such as pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0073] In another aspect, the present application also provides the use of the method or the fermented fungus of Phellinus baumii-Dioscorea opposita or the composition in antioxidant and / or preparation of antioxidant products.

[0074] Preferably, the antioxidant activity includes improving DHHP scavenging rate, hydroxyl radical scavenging rate, and / or total antioxidant activity.

[0075] More preferably, the DHHP removal rate is greater than or equal to 89%.

[0076] More preferably, the hydroxyl radical scavenging rate is greater than or equal to 79%.

[0077] More preferably, the total antioxidant activity is greater than or equal to 80%.

[0078] On the other hand, this application also provides the application of yam or the method described above in the cultivation of Phellinus linteus, the promotion of Phellinus linteus growth and / or the increase of the content of characteristic active ingredients of Phellinus linteus; preferably, the content of the characteristic active ingredients includes one or more of polyphenols, flavonoids, polysaccharides and alkaloids.

[0079] A method for cultivating Phellinus linteus, promoting its growth, and / or increasing the content of its characteristic active ingredients, the method comprising: inoculating Phellinus linteus into a culture medium containing Dioscorea opposita for fermentation.

[0080] In a preferred embodiment, the method includes the following steps:

[0081] Step 1: Mix yam and black beans in a mass ratio of 5:(0.5-2), add them to water in a material-to-liquid ratio of 1:(1-2.3), sterilize, and obtain the culture medium;

[0082] Step 2, bacterial OD 600 The value is 0.5-1.0. Inoculate Phellinus linteus into the culture medium with an inoculation amount of 4mL / 100g-14mL / 100g (or inoculate 3×3mm mycelial blocks) for fermentation. Fermentation conditions include: 15℃-35℃ fermentation in the dark for 5-40 days.

[0083] More preferably, the culture medium may further include one or more of the following: potato, glucose, sucrose, yeast powder, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1, agar, and water.

[0084] In a preferred embodiment, the culture medium comprises: 0.5% yam, 0.1% black bean, 10%-50% potato, 1%-5% sucrose or glucose, 0.1%-0.5% yeast powder, 0.1%-0.5% potassium dihydrogen phosphate, 0.1%-0.5% magnesium sulfate, 1%-5% vitamin B1, and the balance being water. Those skilled in the art can select the amount of agar added according to the actual situation.

[0085] Preferably, the average daily growth rate of the *Sanghuang* mycelium is greater than or equal to 4 mm / d, and the dry weight of the mycelium is greater than or equal to 0.5 g / 100 mL.

[0086] The present invention has the following beneficial effects:

[0087] The present application uses yam as the fermentation culture medium and black beans as the supplementary nitrogen source to carry out bidirectional fermentation by using Phellinus baumii as the fermentation strain. On the one hand, the fermentation method significantly improves the growth rate of Phellinus baumii mycelium and the synthesis efficiency of active ingredients, and the content of flavonoids, polyphenols, polysaccharides and other active ingredients in the fermentation product is significantly improved. On the other hand, the fermentation medium obtained by the method is also rich in flavonoids, polyphenols, polysaccharides, alkaloids and other active ingredients, and the medium has significant antioxidant capacity, and the DPPH free radical scavenging rate and hydroxyl radical scavenging rate reach 92.14% and 80.52% respectively, and the total antioxidant activity of the medium after the fermentation process is improved by 14.7%.

[0088] The prepared Phellinus baumii and Phellinus baumii-yam medium are rich in rare edible and medicinal fungus cell proteins and antioxidant, tumor inhibition and other functional ingredients, which are beneficial to people's health, can be used as new raw materials for health foods, medicines and cosmetics, and have a wide application prospect.

[0089] In the present application, the fermentation process is optimized, and a preparation method with simple process and easy industrial production is finally obtained, which is suitable for wide promotion. BRIEF DESCRIPTION OF DRAWINGS

[0090] The drawings described herein are used to provide further understanding of the present application, constitute a part of the present application, the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application. In the drawings:

[0091] Figure 1 It is a growth chart of Phellinus baumii mycelium on different Chinese herbal medicine culture media;

[0092] Figure 2 It is a chart showing the influence of different liquid-solid ratios on the content of active substances in the medium;

[0093] Figure 3 It is a chart showing the influence of different temperatures on the growth of Phellinus baumii mycelium, and the temperatures from left to right are 15℃, 20℃, 25℃, 30℃ and 35℃;

[0094] Figure 4 It is a chart showing the influence of different temperatures on the content of active substances in the medium;

[0095] Figure 5 It is a chart showing the influence of different inoculation amounts on the content of active substances in the medium;

[0096] Figure 6 It is a chart showing the influence of growth cycle on the growth of mycelium, and the charts from left to right are the growth charts of Phellinus baumii mycelium inoculated for 5d, 10d, 15d, 20d, 25d, 30d, 35d and 40d respectively.

[0097] Figure 7 Figure 1 is the appearance of the fermented substrate of Phellinus baumii-Dioscorea opposita;

[0098] Figure 8 Figure 2 is the electron microscope picture, the left is the unfermented substrate, and the right is the fermented substrate of Phellinus baumii-Dioscorea opposita. DETAILED DESCRIPTION

[0099] Technical terms:

[0100] Substrate: refers to the nutrient substance composed of bacteria and the culture medium fermented by bacteria.

[0101] In order to more clearly illustrate the overall concept of the present application, the following will be described in detail in the form of examples in conjunction with the accompanying drawings. In the following description, a large number of specific details are given in order to provide a more thorough understanding of the present application. However, it is obvious to those skilled in the art that the present application can be implemented without one or more of these details. In other examples, in order to avoid obscuring the present application, some technical features known in the art are not described.

[0102] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application. The test methods in the following examples, if not specified, are generally carried out under conventional conditions, or under the conditions recommended by the manufacturers.

[0103] It should be noted that the terms used herein are only for the purpose of describing the specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in the specification, there is a feature, step, operation, device, component and / or combination thereof.

[0104] When the embodiments give numerical ranges, it should be understood that, unless otherwise stated by the present application, each numerical range of two endpoints and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the examples, any method, device and material of the prior art similar or equivalent to the methods, devices and materials described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.

[0105] In the following examples, the reagents or instruments used are not specified by the manufacturer unless otherwise stated, and are all conventional products that can be obtained commercially.

[0106] In the following examples, the reagents or instruments used are not specified by the manufacturer unless otherwise stated, and are all conventional products that can be obtained commercially.

[0107] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application all employ conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields.

[0108] In addition, the "water" described in the present application includes deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and any feasible water that can be used in the field.

[0109] In the following examples, % means wt%, i.e., weight percentage, unless otherwise specified.

[0110] Example 1: Effect of different Chinese medicinal materials on the growth of Phellinus linteus mycelium

[0111] 1. Activation and culture of Phellinus linteus strain

[0112] (1) Preparation of PDA (potato dextrose agar) comprehensive slant medium: The formula of PDA comprehensive slant medium is potato 200 g, glucose 20 g, yeast powder 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 1 g, vitamin B1 10 mg, agar 20 g, and water 1000 mL. After preparing the medium according to the above formula, sterilize it at 121°C for 25 min, and then place it in a slant for standby use.

[0113] (2) Activation of strain: inoculate the Phellinus linteus strain onto the PDA comprehensive slant medium, and place it in a 25°C dark environment for 7 days. Select the strain with dense mycelium and fast growth rate as the Phellinus linteus strain.

[0114] (3) Preparation of plate medium: The formula of plate medium is potato 200 g, glucose 20 g, yeast powder 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 1 g, vitamin B1 10 mg, agar 20 g, and water 1000 mL. After preparing the medium according to the above formula, sterilize it at 121°C for 25 min, and then pour it into a sterile culture dish to prepare a plate medium for standby use.

[0115] (4) Strain propagation: inoculate the activated Phellinus linteus strain (fresh slant culture) in step (2) to the center of the plate medium, and place it in a 25°C inverted, dark environment for culture. Terminate the culture when the mycelium of Phellinus linteus covers the surface of the plate medium, and then store it for standby use.

[0116] 2. Preparation of different Chinese medicinal media

[0117] (1) Pre-treatment of Chinese medicinal materials: Different Chinese medicinal materials (yam, black bean, mulberry leaf, poria cocos, gynostemma pentaphyllum, astragalus membranaceus, American ginseng, licorice, schisandra chinensis, notoginseng, and fritillaria thunbergii) with a moisture content of 10% are pulverized with a pulverizer and passed through an 80-mesh sieve for later use.

[0118] (2) Preparation of culture medium for traditional Chinese medicine

[0119] The culture medium formula is as follows: 200g potato, 20g sucrose, 3g yeast powder, 2.0g potassium dihydrogen phosphate, 1.0g magnesium sulfate, 10mg vitamin B1, 5g Chinese herbal medicine powder, 20g agar, and 1000mL water;

[0120] After preparing the culture medium according to the above ratio, sterilize it in the culture medium at 121℃ for 25 minutes, and aseptically pour it into petri dishes to prepare plate culture medium for later use.

[0121] 3. Microbial culture

[0122] Using a sterile punch, take a small piece of the *Sanghuang* strain obtained in step 1 with a diameter of 8 mm, and aseptically inoculate it in the center of a solid plate culture medium (TCM culture medium) containing different Chinese herbal medicines. Incubate the medium upside down and in the dark at 25°C, observe the mycelial growth, and measure the mycelial growth rate.

[0123] In this embodiment, the growth of *Sanghuang* strain on different traditional Chinese medicine culture media is as follows: Figure 1 As shown in Table 1, the growth rate is represented by CK, which represents the growth of Sanghuang fungus in a culture medium without any added Chinese medicinal materials (the culture medium formula is: 200g potato, 20g sucrose, 3g yeast powder, 2.0g potassium dihydrogen phosphate, 1.0g magnesium sulfate, 10mg vitamin B1, 20g agar, and 1000mL water).

[0124] Table 1. Effects of different Chinese medicinal herbs on the growth of Phellinus linteus mycelium.

[0125]

[0126] From Table 1, Figure 1 It was found that *Sanghuang* could grow on culture media containing 11 kinds of medicinal and edible herbs with antioxidant and antitumor properties, but the growth rate and vigor of *Sanghuang* mycelium differed significantly on the media containing different herbs. Table 1 shows that, in terms of mycelial growth rate, the order from fastest to slowest is: Dioscorea opposita > Gynostemma pentaphyllum > Poria cocos > Black soybean > Mulberry leaf > CK > Astragalus membranaceus > Licorice > American ginseng > Schisandra chinensis > Panax notoginseng > Fritillaria thunbergii. Figure 1It is shown that the mycelium of P. igniarius is denser than that of the control group in the medium containing yam, black bean, mulberry leaf, fuling and huangqi, and the mycelium in the yam medium is the densest and most vigorous. In summary, yam has a significant growth-promoting effect on P. igniarius strains. Considering the growth rate and vigor, yam, black bean, mulberry leaf, fuling and huangqi are selected for further experiments.

[0127] Example 2 Effects of different Chinese herbal medicines on the biomass and active ingredient synthesis capacity of P. igniarius mycelium

[0128] 1. The activation steps of P. igniarius strains are the same as in Example 1.

[0129] 2. Preparation of P. igniarius strain medium with different Chinese herbal medicine additives

[0130] (1) Yam, black bean, mulberry leaf, fuling and huangqi with a water content of 10% were crushed with a crusher, sieved through an 80-mesh sieve, and reserved for use.

[0131] (2) Preparation of medium with different Chinese herbal medicine additives

[0132] The medium formula is: potato 200 g, sucrose 20 g, yeast powder 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 1.0 g, vitamin B1 10 mg, and 0.5% (5 g) of Chinese herbal medicine powder (yam, black bean, mulberry leaf, fuling and huangqi) added, respectively, water 1000 mL; 100 mL / 250 mL triangular bottles were used for sub-packaging, sterilized at 121°C for 25 min, and reserved for use.

[0133] 3. Inoculation and culture

[0134] The activated P. igniarius strains in step 1 were inoculated into the medium containing Chinese herbal medicines, 3×3 mm mycelial blocks were inoculated per bottle, and the culture was incubated at 25°C and 130 r / min under constant temperature and shaking for 10 days. After the culture was completed, the mycelium was collected by filtering with gauze, dried at 55°C, and reserved for use. In this example, the method for measuring the dry weight of the mycelium was to dry it to a constant weight at 55°C, and then measure the mass of the mycelium using an analytical balance; the method for detecting the content of polyphenols was in accordance with GB / T 44349-2024, the method for detecting the content of flavonoids was in accordance with GB / T 20574-2006, and the method for detecting the content of polysaccharides was the phenol-sulfuric acid method. The detection of the dry weight of P. igniarius mycelium and the content of active ingredients in this example is shown in Table 2. The control group (CK) was P. igniarius strains cultured in a medium without any Chinese herbal medicine additives (same as in Example 1) under the same conditions.

[0135] Table 2 Effects of different Chinese herbal medicines on the biomass and characteristic active ingredient content of P. igniarius mycelium

[0136]

[0137] As shown in Table 2, the five Chinese medicinal materials in Example 1 were added to the liquid culture medium for fermentation for 10 days, and the fermentation was terminated. The five Chinese medicinal materials had significant effects on mycelial growth and content of characteristic active ingredients. In terms of mycelial growth, the medium with Dioscorea opposita had the highest mycelial yield (0.523 g / 100 mL), which was significantly higher than that of the control (0.452 g / mL). In terms of characteristic active ingredients, in terms of polyphenol content, the medium with Dioscorea opposita had the highest polyphenol content (0.106 mg / mL), which was significantly higher than that of the control (0.094 mg / mL); in terms of flavonoid content, the medium with Dioscorea opposita, black beans, mulberry leaves, and Poria cocos all had higher flavonoid content than the control, and the medium with Dioscorea opposita had the highest flavonoid content (0.051 mg / mL); in terms of polysaccharide content, the medium with Poria cocos had the highest polysaccharide content (4.417 mg / mL), and the medium with Dioscorea opposita had the second highest polysaccharide content (3.765 mg / mL) and was significantly higher than that of the control. In summary, the medium with Dioscorea opposita had higher mycelial biomass and active substance content after fermentation. It can be seen that Dioscorea opposita can not only significantly promote the growth of Phellinus baumii, but also significantly improve the ability of Phellinus baumii to synthesize active ingredients such as flavonoids and polyphenols. Therefore, Dioscorea opposita is the best medium for fermentation.

[0138] Example 3: Effect of supplemental nitrogen source on mycelial biomass

[0139] 1. The activation step of the Phellinus baumii strain was the same as in Example 1.

[0140] 2. Preparation of the medium for the Phellinus baumii strain with a supplemental nitrogen source

[0141] (1) Black beans, peanuts, fish meal, coffee grounds, soybean protein powder, and urea with a water content of 10% were ground and sieved through an 80-mesh sieve for use;

[0142] (2) Preparation of media with different supplemental nitrogen sources

[0143] The medium formula was: potato 200 g, sucrose 20 g, yeast powder 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 1.0 g, vitamin B1 10 mg, Dioscorea opposita powder 5 g, and water 1000 mL. On this basis, 0.05%, 0.10%, 0.15%, and 0.20% of black beans, peanuts, fish meal, coffee grounds, soybean protein powder, and urea were added, respectively. The medium was divided into 100 mL / 250 mL triangular flasks, sterilized at 121°C for 25 min, and prepared for use.

[0144] The control group (CK) medium formula was: potato 200 g, sucrose 20 g, yeast powder 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 1.0 g, vitamin B1 10 mg, Dioscorea opposita powder 5 g, and water 1000 mL.

[0145] 3. Inoculation and culture

[0146] The activated Phellinus sp. strain in step 1 was inoculated into a triangular flask containing Chinese herbal medicine, 3x3mm mycelium blocks were inoculated in each flask, and the flask was incubated at 25℃ and 130r / min for 10 days. After the incubation, the mycelium was collected by filtering with gauze and dried at 55℃ for standby. The dry weight of the mycelium was measured by the same method as in Example 2, and the results are shown in Table 3.

[0147] Table 3 Influence of different concentrations and types of supplemental nitrogen sources on the biomass of Phellinus sp. mycelium

[0148]

[0149]

[0150] As shown in Table 3, black beans, peanuts, fish meal, coffee grounds, soybean protein, and urea were added as supplemental nitrogen sources in the medium with yam as the additive at different concentrations. Black beans, peanuts, and soybean protein significantly promoted the growth of mycelium, with black beans being the most significant. The optimal addition amount was 0.10%, and the mycelium biomass reached 0.659g / mL, which was 26% higher than the control. Therefore, the best supplemental nitrogen source was black beans, and the optimal addition amount was 0.10%. Moreover, the results showed that yam and black beans as medium components had a synergistic effect on the growth of Phellinus sp.

[0151] Example 4 Optimization of the solid-liquid ratio of Phellinus sp.-yam microbial fermentation medium

[0152] 1. Activation of Phellinus sp. strain and strain culture

[0153] (1) Preparation of PDA (potato dextrose agar) comprehensive slope medium: The formula of PDA comprehensive slope medium is potato 200g, glucose 20g, yeast powder 3g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, vitamin B1 10mg, agar 20g, and water 1000mL. After preparing the medium according to the above formula, sterilize it at 121℃ for 25min, and place it on a slope for standby.

[0154] (2) Strain activation: inoculate the Phellinus sp. strain into the PDA comprehensive slope medium, and incubate it at 25℃ in the dark for 7 days. Select the strain with dense mycelium and fast growth rate as the Phellinus sp. strain.

[0155] (3) Preparation of plate medium: The formula of plate medium is potato 200g, glucose 20g, yeast powder 3g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, vitamin B1 10mg, agar 20g, and water 1000mL. After preparing the medium according to the above formula, sterilize it at 121℃ for 25min, and pour it into a sterile petri dish to prepare a plate medium for standby.

[0156] (4) Strain propagation: the activated Phellinus baumii strain (fresh slant culture) in step (2) was inoculated into the center of the plate medium, and cultured at 25°C under inverted and light-avoiding conditions. The culture was terminated when the Phellinus baumii mycelium covered the surface of the plate medium, and was stored for later use.

[0157] (5) Seed liquid preparation: the Phellinus baumii strain propagated in step (4) was inoculated into liquid medium, 3x3mm of mycelium block was inoculated into each bottle, and the liquid medium was incubated at 25°C and 130r / min under constant temperature and shaking. The OD value of the liquid seed was 0.5-1.0, and the incubation was terminated after about 8-10 days. The preparation of the liquid medium: potato 200g, sucrose 20g, yeast powder 3g, potassium dihydrogen phosphate 2.0g, magnesium sulfate 1.0g, vitamin B1 10mg, water 1000mL; after the medium was prepared according to the above ratio, it was sterilized at 121°C for 25min. 2 600

[0158] 2. Yam fermentation substrate preparation

[0159] (1) Yam pretreatment: the yam with a water content of 10% was crushed into debris by a crusher, and was sieved through an 80-mesh sieve for later use; black bean pretreatment: the black bean with a water content of 10% was crushed into debris by a crusher, and was sieved through an 80-mesh sieve for later use;

[0160] (2) Yam fermentation substrate preparation: 20g of yam powder was divided into 50mL of Erlenmeyer flask as the culture container, and black bean powder was added as the nitrogen source supplement according to a ratio of 5:1.

[0161] Water was added according to a ratio of 1:1.0, 1:1.2, 1:1.5, 1:1.8, and 1:2.3 (g / mL), respectively, and was sterilized at 121°C for 40min to obtain the yam fermentation substrate, which was cooled for later use.

[0162] 3. Preparation of Phellinus baumii-yam fermentation substrate

[0163] The Phellinus baumii strain prepared in step 1 was inoculated into the yam fermentation substrate obtained in step 2, and the inoculation amount was 10.0mL / 100g. The fermentation was carried out at 25°C under light-avoiding conditions for 25 days to obtain the Phellinus baumii-yam fermentation substrate.

[0164] 4. Preparation of Phellinus baumii-yam fermentation substrate powder

[0165] The fermentation substrate was dried and crushed at ≥65°C to obtain the Phellinus baumii-yam fermentation substrate powder.

[0166] The control group (CK) was the yam fermentation substrate (ratio of solid to liquid (g / mL) was 1:1.5) without inoculation of the Phellinus baumii strain.

[0167] ​​The active substance content of the *Sanghuang*-yam mycelium obtained in this embodiment was determined using the same method as in Example 2, and the results are as follows: Figure 2 As shown.

[0168] Depend on Figure 2 It is evident that the mycelium of *Sanghuang* exhibits good growth within a material-to-liquid ratio (g / mL) of 1:1.0–1:2.3. As the material-to-liquid ratio increases, the content of active ingredients initially increases and then decreases. The highest content of flavonoids is observed at a ratio of 1:1.8, followed by polyphenols at 1:1.5, and polysaccharides at 1:1.2. Considering both mycelial growth and the content of the three active ingredients, a material-to-liquid ratio of 1:1.2–1:1.8 (g / mL) is considered suitable for mycelial fermentation. Since polysaccharides have a relatively high proportion of the three active substances, the optimal material-to-liquid ratio for *Sanghuang*-yam mycelial fermentation is 1:1.2, which will be used for further experiments.

[0169] Example 5: Optimization of the temperature for bidirectional fermentation culture of Phellinus linteus and Dioscorea opposita.

[0170] 1. The activation steps for the Sanghuang strain are the same as in Example 4.

[0171] 2. Preparation of yam fermentation substrate

[0172] (1) The pretreatment of yam and black beans is the same as in Example 4;

[0173] (2) Preparation of yam fermentation substrate: Take 20g of yam powder and divide it into 50mL Erlenmeyer flasks or other culture containers. Add black bean powder as a nitrogen source supplement at a ratio of 5:1. Then add water at a ratio of 1:1.2 (g / mL) and sterilize at 121℃ for 40min to obtain the fermentation substrate. Cool it for later use.

[0174] 3. Preparation of fermentation mycelium of Phellinus linteus and Dioscorea opposita

[0175] The *Sanghuang* strain prepared in step 1 was inoculated into the yam fermentation substrate obtained in step 2 at an inoculation amount of 10.0 mL / 100 g. Fermentation was carried out at 15℃, 20℃, 25℃, 30℃ and 35℃ for 25 days to obtain *Sanghuang*-yam fermentation mycelium.

[0176] The control group (CK) consisted of yam fermentation substrate that was not inoculated with Phellinus linteus, and the treatment temperature was 25℃.

[0177] 4. Preparation of Sanghuang-Yam Fermented Mycelium Powder: Same as in Example 4; Method for Determining Active Substance Content: Same as in Example 2; Results are as follows. Figure 4 Different strains of Phellinus linteus and Dioscorea opposita grow as follows Figure 3 As shown.

[0178] from Figure 3 , Figure 4It can be seen that temperature has a significant effect on the growth of P. baumii mycelium and the accumulation of active ingredients. The mycelium grows slowly at 15-20°C, and grows well at 25-35°C. In terms of the accumulation of active ingredients, the contents of flavonoids and polyphenols increase first and then tend to be stable at 25°C, and the content of polysaccharides shows no obvious trend at different temperatures. Overall, P. baumii strain can grow well at 25-35°C, and the contents of flavonoids, polyphenols and polysaccharides in the mycelium maintain at a high level. Considering the energy consumption factor, the contents of flavonoids, polyphenols and polysaccharides in the mycelium obtained at 25°C are optimal. Therefore, the optimal culture temperature of P. baumii-D. japonica mycelium is 25°C, and this parameter is used for the next experiment.

[0179] Example 6 Optimization of Inoculum Size for P. baumii-D. japonica Mycelium Two-way Fermentation Culture

[0180] 1. The activation step of P. baumii strain is the same as that in Example 4.

[0181] 2. Preparation of D. japonica fermentation substrate

[0182] (1) The pretreatment of D. japonica and black beans is the same as that in Example 4.

[0183] (2) Preparation of D. japonica fermentation substrate: 20 g of D. japonica powder was divided into 50 mL conical flasks as culture containers, and black bean powder was added as a nitrogen source supplement at a ratio of 5:1. Then, water was added at a ratio of 1:1.2 (g / mL), and the fermentation substrate was obtained by sterilization at 121°C for 40 min, and then cooled for standby.

[0184] 3. Preparation of P. baumii-D. japonica fermentation substrate

[0185] The P. baumii strain prepared in step 1 was inoculated into the D. japonica fermentation substrate obtained in step 2, and the inoculum size was 4, 6, 8, 10, 12 and 14 mL / 100 g, respectively. The P. baumii-D. japonica fermentation substrate was obtained by fermentation at 25°C in the dark for 25 days.

[0186] The control group (CK) was the D. japonica fermentation substrate without inoculation of P. baumii strain.

[0187] 4. Preparation of P. baumii-D. japonica fermentation substrate powder, the same as in Example 4, and the determination method of active substance content is the same as in Example 2, and the results are shown in Figure 5 .

[0188] From Figure 5It was found that different inoculum amounts had a certain impact on mycelial growth and vigor. In the early stages of growth, a larger inoculum amount helped the mycelium grow rapidly and cover the entire substrate surface. However, the inoculum amount had a relatively small impact on mycelial growth during the experimental culture period, with better mycelial growth occurring within the range of 4%-14%. The inoculum amount significantly affected the content of flavonoids, polyphenols, and polysaccharides in the mycelium. Flavonoids and polyphenols showed a trend of first increasing and then decreasing, while polysaccharide content first decreased, then increased, and then decreased again. When the inoculum amount reached 10.0 mL / 100g, the contents of flavonoids, polyphenols, and polysaccharides in the mycelium all reached their peak. Then, with further increases in inoculum amount, the contents of polyphenols, flavonoids, and cordycepin showed a slight decreasing trend. Therefore, the most suitable inoculum amount was selected as 10.0 mL / 100g, and this was used as the parameter for the next stage of the experiment.

[0189] Example 7: Optimization of the fermentation cycle for the bidirectional fermentation of Phellinus linteus and Dioscorea mycelium.

[0190] 1. The activation steps for the Sanghuang strain are the same as in Example 4.

[0191] 2. Preparation of yam fermentation substrate, same as in Example 6.

[0192] 3. Preparation of Phellinus linteus-Dioscorea mycelium

[0193] The fermentation substrate prepared in step 2 was inoculated with Phellinus linteus liquid inoculum: Phellinus linteus inoculum was inoculated into the yam fermentation substrate at an inoculation rate of 10.0 mL / 100 g, and fermented in the dark at 25 °C; with the uninoculated fermentation substrate as the control (0d), samples were taken at 5, 10, 15, 20, 25, 30, 35 and 40 days to obtain Phellinus linteus-yam fermentation inoculum.

[0194] The control group (CK) consisted of yam fermentation substrate that was not inoculated with Phellinus linteus.

[0195] 4. Preparation of Sanghuang-Yam fermented mycelium powder: Same as in Example 4. The method for determining the content of active substances was the same as in Example 2. The methods for determining total antioxidant capacity, DPPH scavenging capacity, and hydroxyl radical scavenging capacity were performed using kits from Suzhou Greens Biotechnology Co., Ltd. (catalog numbers G0142F, G0128F, and G0153F, respectively). Specific methods were described in the kit instructions. The results are shown in Table 4. The mycelial growth after inoculation was as follows: Figure 6 As shown.

[0196] Table 4. Active ingredients and antioxidant capacity at different growth stages

[0197]

[0198] Depend on Figure 6 It can be seen that the mycelium grew to about half of the substrate by day 5, and basically covered the substrate surface by day 10. After that, the aerial mycelium increased and the mycelium thickened. Yellow water was produced after day 30 of cultivation, indicating the appearance of brown spots.

[0199] From Table 4, it can be seen that, compared with the control, the flavonoid content increased with the increase of fermentation period, increased faster in the early stage, tended to slow down in the later stage, and was the highest (6.62 mg / g) at the 40th day. The polyphenol content first increased and then decreased, and was the highest (3.96 mg / g) at the 25th day. The polysaccharide content first increased and then decreased, and rapidly decreased (145.35 mg / g) after the 30th day, which was significantly lower than that of the control (179.41 mg / g). The total antioxidant capacity, DPPH clearance rate and hydroxyl radical clearance rate of the mycelium were all significantly increased compared with the control, among which the total antioxidant capacity was stable at more than 80% after the 20th day, the DPPH clearance rate was more than 80% after the 10th day, and the hydroxyl radical clearance rate was the highest (79.02%) at the 25th day. In combination with the mycelial growth, the content of active ingredients and the antioxidant activity, the optimal culture period of Phellinus baumii-sandwort substrate was 25 d, and was used as the optimization condition for the next experiment.

[0200] Example 8 Preparation of mycelium under the optimized condition and analysis of the effect of bidirectional fermentation

[0201] The bidirectional fermentation of Phellinus baumii-dosage was carried out under the optimized fermentation conditions of Examples 2-7, and the Phellinus baumii-dosage fermentation mycelium was obtained for the following experiments.

[0202] The preparation method of the mycelium under the optimized condition is as follows:

[0203] 1. Activation and culture of Phellinus baumii strain

[0204] (1) Preparation of PDA (potato dextrose agar) comprehensive slope medium: the formula of PDA comprehensive slope medium is potato 200 g, glucose 20 g, yeast powder 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 1 g, vitamin B1 10 mg, agar 20 g, and water 1000 mL; after the medium is prepared according to the above formula, sterilize it at 121℃ for 25 min, and place it into a slope for standby.

[0205] (2) Activation of strain: inoculate the Phellinus baumii strain on the PDA comprehensive slope medium, and place it in the dark at 25℃ for 7 d; select the strain with dense mycelium and fast growth speed as the Phellinus baumii strain.

[0206] (3) Preparation of plate medium: the formula of plate medium is potato 200 g, glucose 20 g, yeast powder 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 1 g, vitamin B1 10 mg, agar 20 g, and water 1000 mL; after the medium is prepared according to the above formula, sterilize it at 121℃ for 25 min, and pour it into a sterile culture dish to prepare the plate medium for standby.

[0207] (4) Strain propagation: the activated Phellinus baumii strain (fresh slant culture) in step (2) was inoculated into the center of the plate culture medium, and cultured at 25°C under inverted and light-avoiding conditions. The culture was terminated when the Phellinus baumii mycelium covered the surface of the plate culture medium, and the Phellinus baumii strain was preserved for later use.

[0208] (5) Seed liquid preparation: the Phellinus baumii strain propagated in step (4) was inoculated into liquid culture medium, 3x3mm mycelium pieces were inoculated into each bottle, and the liquid culture was incubated at 25°C and 130r / min. The OD value of the liquid seed was 0.5-1.0, and the culture was terminated after about 8-10 days. The liquid culture medium was prepared by mixing 200g potato, 20g sucrose, 3g yeast powder, 2.0g potassium dihydrogen phosphate, 1.0g magnesium sulfate, 10mg vitamin B1, and 1000mL water, and sterilized at 121°C for 25min. 2 600 (5) Seed liquid preparation: the Phellinus baumii strain propagated in step (4) was inoculated into liquid culture medium, 3x3mm mycelium pieces were inoculated into each bottle, and the liquid culture was incubated at 25°C and 130r / min. The OD value of the liquid seed was 0.5-1.0, and the culture was terminated after about 8-10 days. The liquid culture medium was prepared by mixing 200g potato, 20g sucrose, 3g yeast powder, 2.0g potassium dihydrogen phosphate, 1.0g magnesium sulfate, 10mg vitamin B1, and 1000mL water, and sterilized at 121°C for 25min.

[0209] 2. Yam fermentation substrate preparation

[0210] (1) Yam pretreatment: the yam with a water content of 10% was crushed into debris by a crusher, and the debris was sieved through an 80-mesh sieve for later use. Black bean pretreatment: the black bean with a water content of 10% was crushed into debris by a crusher, and the debris was sieved through an 80-mesh sieve for later use.

[0211] (2) Yam fermentation substrate preparation: 20g yam powder was separately placed in 50mL triangular flasks as culture containers, black bean powder was added as nitrogen source supplement at a ratio of 5:1, and then water was added at a solid-liquid ratio of 1:1.2 (g / mL). The yam fermentation substrate was obtained by sterilizing at 121°C for 40min, and then cooled for later use.

[0212] 3. Preparation of Phellinus baumii-yam fermentation substrate

[0213] The Phellinus baumii strain prepared in step 1 was inoculated into the yam fermentation substrate obtained in step 2, and the inoculation amount was 10.0mL / 100g. The Phellinus baumii-yam fermentation substrate was obtained by fermentation at 25°C for 25 days in the dark.

[0214] 4. Preparation of Phellinus baumii-yam fermentation substrate powder: the fermentation substrate was dried and crushed at ≥65°C to obtain the Phellinus baumii-yam fermentation substrate powder.

[0215] The yam fermentation substrate inoculated with the Phellinus baumii strain was used as the experimental group (EG), and the yam fermentation substrate without inoculation of the Phellinus baumii strain was used as the control group (CK). The medium containing only yam was used instead of the yam fermentation substrate, and the rest of the treatment methods were the same as those of the yam group. The medium containing only black bean was used instead of the yam fermentation substrate, and the rest of the treatment methods were the same as those of the black bean group.

[0216] The appearance and electron microscope images of the Phellinus baumii-yam fermentation substrate powder obtained in this example are shown in FIG. 1 and FIG. 2, respectively. Figure 7 ​, Figure 8 The method for determining the content of active substances is the same as in Example 2, the method for measuring the antioxidant activity is the same as in Example 7, and the method for determining alkaloids is as follows: 2.0 g of the sample is added to 50 mL of 0.1 mol / L hydrochloric acid solution, and reflux extraction is performed for 1 h. After cooling and filtration, the absorbance is measured at 265 nm. Berberine (content ≥98%) is used as a standard, and a standard curve equation y=8.42x+0.015 (R 2 =0.9986) is determined. The content of alkaloids is calculated according to the curve equation. The content of the main active ingredients and the measured antioxidant activity are shown in Table 5.

[0217] Table 5 Content of main active substances in Phellinus baumii and yam fermentation substrate and antioxidant activity

[0218]

[0219]

[0220] As shown in Table 5, under the optimized conditions, the content of active substances such as flavonoids, polyphenols, polysaccharides, and the total antioxidant capacity, DPPH scavenging capacity, and hydroxyl radical scavenging capacity of the Phellinus baumii-yam fermentation substrate are all better than those of the unfermented control group. Among them, the flavonoids are 228.6% higher, the polyphenols are 92.2.8% higher, and the polysaccharides are 37.4% higher. The analysis of the content of alkaloids in the fermentation substrate shows that the content of alkaloids in the fermentation substrate is significantly increased, reaching 2.62 mg / g, which is 451.2% higher than that of the unfermented substrate. In addition, the antioxidant capacity of the fermentation substrate is all more than 80%, the total antioxidant activity is increased by 14.7%, the DPPH scavenging capacity is increased by 298.9%, and the hydroxyl radical scavenging capacity is increased by 17.4%. In addition, the Phellinus baumii-yam fermentation substrate has higher content of active substances and antioxidant capacity than the yam substrate and the black bean nitrogen source. In particular, the antioxidant capacity is significantly improved. It can be seen that the fermentation of yam by Phellinus baumii is conducive to the synthesis of secondary metabolites by Phellinus baumii using yam as a nutrient source. On the other hand, the structure of yam is changed by the growth of Phellinus baumii mycelium, which promotes the release of active ingredients in the yam substrate and enhances the antioxidant activity of the fermentation substrate.

[0221] From the results of the electron microscope, Figure 7 It can be seen that the Phellinus baumii-yam fermentation substrate has a golden yellow color, and the morphology and structure of the fermentation substrate are significantly changed under the action of Phellinus baumii fermentation. The electron microscope results show that Figure 8)The inside of the substrate is filled with mycelium after fermentation treatment, the growth of the mycelium can penetrate the inside of the substrate, destroy the structure of the substrate, make the active substance more easily release, at the same time, the enzymes produced by the growth of the mycelium of the Phellinus baumii effectively decompose the substances in the substrate to convert into nutrient and active substance with small molecules, and the increase of the active substance content promotes the change of the pharmacological active substance of the fermented substrate, so as to show the potential in the antioxidant capacity and pharmacological function.

[0222] In conclusion, the application combines the characteristics of the raw dioscorea that is homology of food and medicine, provides nutrient substances for the growth and metabolism of the mycelium of the Phellinus baumii after physical crushing, the enzyme system in the Phellinus baumii can change the ingredient composition and spatial structure of the dioscorea, and significantly improve the content of the effective ingredients such as flavonoids, polyphenols and polysaccharides in the dioscorea and the antioxidant activity.

[0223] The above only describes the embodiments of the application and is not used to limit the application. The application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the scope of claims of the application.

Claims

1. A method for preparing fermented mycelium of Phellinus linteus and Dioscorea opposita, characterized in that, The method includes: inoculating Phellinus linteus into a culture medium containing Dioscorea opposita for fermentation.

2. The method according to claim 1, characterized in that, The culture medium further includes a nitrogen source; preferably, the nitrogen source is selected from one or more of black beans, peanuts, fish meal, coffee grounds, soybean protein powder, and urea; more preferably, the nitrogen source is black beans; more preferably, the mass ratio of yam to nitrogen source in the culture medium is 5:(0.5-2); more preferably, the mass ratio of yam to nitrogen source in the culture medium is 5:

1.

3. The method according to claim 2, characterized in that, The method for preparing the culture medium includes: adding yam and nitrogen source to water at a material-to-liquid ratio of 1:(1-2.3); preferably, the material-to-liquid ratio is 1:1.

2.

4. The method according to claim 1, characterized in that, The inoculated bacterial strain OD 600 The value is 0.5-1.0, and the inoculation amount is 4mL / 100g-14mL / 100g; more preferably, the inoculation amount is 10mL / 100g.

5. The method according to claim 1, characterized in that, The fermentation conditions include: fermentation at 15℃-35℃ for 5-40 days; preferably, fermentation at 25℃ for 25 days.

6. The *Sanghuang*-yam fermentation mycelium prepared by any one of the methods described in claims 1-5.

7. The *Sanghuang*-yam fermentation mycelium according to claim 6, characterized in that, The fermented mycelium of Phellinus linteus-yam contains antioxidant active substances; preferably, the antioxidant active substances include one or more of polyphenols, flavonoids, polysaccharides, and alkaloids.

8. A composition, characterized in that, The composition comprises the *Sanghuang*-yam fermentation mycelium as described in claim 6 or 7.

9. The use of the method as described in any one of claims 1-5, or the fermented mycelium of Phellinus linteus and Dioscorea opposita as described in claim 6 or 7, or the composition as described in claim 8, in antioxidant and / or preparation of antioxidant products.

10. The application of yam or the method described in any one of claims 1-5 in the cultivation of Phellinus linteus, the promotion of Phellinus linteus growth and / or the increase of the content of characteristic active ingredients of Phellinus linteus; preferably, the content of the characteristic active ingredients includes one or more of polyphenols, flavonoids, polysaccharides and alkaloids.

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