Hericium coralloides with antioxidant activity, its cultivation method and application

Abstract

The present invention provides a Hericium coralloides with antioxidant activity, its cultivation method and application, relating to the technical field of microbial fermentation. In the present invention, Schisandra chinensis is first used as a substrate for two-way fermentation with Hericium coralloides to obtain Hericium coralloides rich in γ-aminobutyric acid and its related products, providing a reference for developing Hericium coralloides into a functional food base material and related antioxidant foods.

Description

Technical Field

[0001] This application relates to the technical field of microbial fermentation, and particularly relates to a Hericium coralloides with antioxidant activity, its cultivation method and application. Background Art

[0002] Hericium coralloides, also known as Yuran, belongs to Basidiomycetes, Agaricomycetes, Russulales, Hericiaceae, Hericium, and is a famous rare and precious edible and medicinal mushroom in China. Hericium coralloides benefits the five internal organs, nourishes, aids digestion, and is mainly used to treat neurasthenia, gastric ulcer and other diseases. It is an excellent medicinal fungus with extremely high nutritional value and health care value. The main active ingredient of Hericium erinaceus is polysaccharide. In addition to polysaccharide, it also contains bioactive substances such as oligosaccharide, sterols, erinacines and erinakones, which have multiple effects such as improving immunity, anti-tumor, hypoglycemic, anti-mutation and anti-aging, and have been widely used in industries such as medicine.

[0003] γ-aminobutyric acid (GABA) is a non-protein amino acid widely present in animals, plants and microorganisms. Its molecular formula is C4H9NO2 and it was first discovered in potato tubers by Steward et al. in 1949. As an important endogenous inhibitory neurotransmitter in the mammalian brain, GABA plays a crucial role in maintaining the balance between excitation and inhibition of the neuron network. In addition, GABA also has physiological activities such as regulating blood pressure, anti-aging, improving liver and kidney functions, regulating the executive ability of bipolar disorder and assisting the body to maintain immune homeostasis in animals. Due to the important physiological functions of GABA for the human body, it is often added to foods, nutritional health products and drugs. The metabolism of GABA is balanced in healthy adults, but both aging and increased stress of the body may affect the normal accumulation of GABA, leading to symptoms such as anxiety, fatigue and restlessness, and it is necessary to supplement and intake from food.

[0004] However, there is no precedent for producing γ-aminobutyric acid using Hericium coralloides at present. Summary of the Invention

[0005] In view of the above problems, in this application, Schisandra chinensis is used as an inducing factor, and Hericium coralloides is used as a fermenting fungus for liquid fermentation. It is found that the content of γ-aminobutyric acid increases significantly during the fermentation process, which provides a reference and a new way for developing Hericium coralloides into a functional food base material and related antioxidant foods.

[0006] On the one hand, the present application provides a cultivation method of Hericium coralloides with antioxidant activity, and the method includes: inoculating Hericium coralloides in a fermentation medium for fermentation culture, and the fermentation medium includes Schisandra chinensis.

[0007] Preferably, the Hericium coralloides contains antioxidant active substances, and the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins, and / or γ-aminobutyric acid; preferably, γ-aminobutyric acid.

[0008] The antioxidant activity of Hericium coralloides obtained by fermentation using this method is improved, especially the content of γ-aminobutyric acid is increased.

[0009] Furthermore, the fermentation medium includes a carbon source, and the carbon source is selected from one or more of glucose, maltose, corn flour, sucrose, lactose, and mannitol; preferably, lactose; and / or, the fermentation medium further includes a nitrogen source, and the nitrogen source is selected from one or more of peptone, yeast extract powder, ammonium nitrate, beef extract, and yeast extract; preferably, peptone; and / or, the fermentation medium further includes potato, magnesium sulfate, dipotassium hydrogen phosphate, and VB1; preferably, the carbon source is 1.6%-2.4%, the nitrogen source is 0.1%-0.9%, the potato is 10%-50%, the magnesium sulfate is 0.1%-0.5%, the dipotassium hydrogen phosphate is 0.1%-0.5%, VB1 is 0.001%-0.01%, and Schisandra chinensis is 0.1%-0.3%; more preferably, the fermentation medium includes a carbon source of 1.9%, a nitrogen source of 0.3%, a potato of 20%, magnesium sulfate of 0.15%, dipotassium hydrogen phosphate of 0.3%, VB1 of 0.001%, and Schisandra chinensis of 0.24%, and the balance is water.

[0010] In a preferred embodiment, the fermentation medium includes: 200 g of potato (juice after boiling), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and 1000 mL of water.

[0011] Preferably, the potato is potato juice, and the preparation method of the potato juice includes: peeling the potato, cutting it into pieces, adding water, boiling for 30 min, filtering with gauze, making up the water to 100 mL, and sterilizing.

[0012] Furthermore, the conditions for the fermentation culture include: the inoculation amount is 0.1%-3%, the fermentation temperature is 18°C-32°C, the liquid loading amount is 24%-60%, the rotation speed is 100-180 rpm, and the pH is 4.0-7.0; preferably, the inoculation amount is 1%, the fermentation temperature is 25°C, the liquid loading amount is 32%, the rotation speed is 140 rpm, and the pH is 6.0.

[0013] Preferably, the fermentation time is 1-10 days, and more preferably, the fermentation time is 7 days.

[0014] Among them, the inoculation amount refers to the ratio of the volume of the transferred seed liquid to the volume of the culture medium after inoculation, and the loading amount of the liquid refers to the ratio of the volume of the culture medium filled in the Erlenmeyer flask to the total volume of the Erlenmeyer flask.

[0015] Furthermore, the Hericium coralloides is Hericium coralloides RT25, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC NO.: 41304.

[0016] In a preferred embodiment, a method for culturing Hericium coralloides with antioxidant activity comprises the following steps:

[0017] Step 1: Activate the Hericium coralloides RT25 strain to obtain a seed liquid;

[0018] Step 2: Inoculate the seed liquid into a fermentation medium for fermentation culture. The fermentation medium includes: lactose 1.6%-2.4%, peptone 0.1%-0.9%, potato 10%-50%, magnesium sulfate 0.1%-0.5%, dipotassium hydrogen phosphate 0.1%-0.5%, VB1 0.001%-0.01%, Schisandra chinensis 0.1%-0.3%. The conditions for fermentation culture include: the inoculation amount is 0.1%-3%, the fermentation temperature is 18°C-32°C, the loading amount of the liquid is 24%-60%, the rotation speed is 100-180 rpm, the pH is 4.0-7.0, and the fermentation time is 1-10 days.

[0019] Among them, the method commonly used in the art can be adopted to activate the Hericium coralloides strain to obtain a seed liquid.

[0020] In a preferred embodiment, the activation method of the Hericium coralloides strain includes: inoculating the Hericium coralloides mycelium block into a seed liquid medium, culturing it at 25°C and 140 r / min with shaking for 7 days.

[0021] Seed liquid medium: potato 20%, glucose 2%, peptone 0.3%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.15%, VB1 0.001%, and the balance is water.

[0022] On the other hand, the present application also provides the application of Schisandra chinensis or the method in improving the antioxidant activity of Hericium coralloides and / or improving the strain quality of Hericium coralloides.

[0023] Preferably, the Hericium coralloides is Hericium coralloides RT25 and / or Hericium coralloides SY26; more preferably, the Hericium coralloides is Hericium coralloides RT25.

[0024] Schisandra chinensis was first used as a substrate for fermenting Hericium coralloides in this application, and it was found that the Hericium coralloides obtained by fermentation using this method could improve the quality of Hericium coralloides strains during the fermentation process. The strain quality specifically includes mycelial biomass, spheroid density, etc., and enables the spheroid diameter to reach an appropriate spheroid density, and can promote Hericium coralloides to produce polysaccharides, polyphenols, flavonoids, γ-aminobutyric acid and other metabolites with antioxidant activity.

[0025] In this application, the fermentation formula was adjusted and optimized to be 200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and 1000 mL of water. The fermentation conditions were an inoculation amount of 1%, a fermentation time of 7 d, a shaker speed of 140 r / min, a pH of 6.0, a liquid loading volume of 80 mL / 250 mL in the shake flask, and a temperature of 25°C. The average value of the fermentation biomass obtained by the optimized process was 12.258 g, which was 98.24% of the predicted value (12.4780 g).

[0026] And on the basis of optimizing the Schisandra chinensis fermentation formula in this application, by comparing the active ingredients (γ-aminobutyric acid, polysaccharides, flavonoids, polyphenols, soluble proteins, triterpenoids) and antioxidant activities (total antioxidant capacity, hydroxyl radical (·OH) scavenging rate, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging rate, and superoxide anion (O 2－ ·) scavenging rate) in the fermented mycelium and fruiting body of Hericium coralloides, it was found that the content of γ-aminobutyric acid increased significantly, proving that Schisandra chinensis as an inducer is helpful for the synthesis of the antioxidant active substance γ-aminobutyric acid in Hericium coralloides, and there was no previous example of using Hericium coralloides to prepare γ-aminobutyric acid.

[0027] Therefore, the Hericium coralloides and its related products obtained by the above method, such as thalli, mycelia, fruiting bodies, fermentation broth, etc., all contain rich antioxidant active substances and have a certain antioxidant capacity.

[0028] On the other hand, this application also provides a strain of Hericium coralloides, and the Hericium coralloides is Hericium coralloides RT25, which is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC NO.: 41304.

[0029] A strain of Hericium coralloides for preparing antioxidant active substances, and the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenoids, soluble proteins, and / or γ-aminobutyric acid; preferably, γ-aminobutyric acid.

[0030] Preferably, the Hericium coralloides includes Hericium coralloides and its related products, such as:

[0031] A1) Hericium coralloides;

[0032] A2) Hericium coralloides inoculant;

[0033] A3) Suspension of dead Hericium coralloides;

[0034] A4) Metabolites of Hericium coralloides;

[0035] A5) Extracts of Hericium coralloides;

[0036] A6) Fruit bodies of Hericium coralloides;

[0037] A7) Mycelia of Hericium coralloides;

[0038] A8) Spores of Hericium coralloides.

[0039] On the other hand, the present application also provides Hericium coralloides containing antioxidant active substances prepared by the described method, and the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; preferably, γ-aminobutyric acid.

[0040] On the other hand, the present application also provides the use of Schisandra chinensis or the described method or the Hericium coralloides in the preparation of glutamate decarboxylase and / or in the preparation of antioxidant active substances; preferably, the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid.

[0041] On the other hand, the present application also provides the use of Schisandra chinensis as an inducer for the growth and / or antioxidant active substances of Hericium coralloides.

[0042] On the other hand, the present application also provides an inducer for the growth and / or antioxidant active substances of Hericium coralloides, and the inducer includes Schisandra chinensis.

[0043] Preferably, the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid.

[0044] It is first discovered in the present application that Schisandra chinensis can be used as an inducer for the growth and antioxidant active substances of Hericium coralloides, especially can induce the increase of the content of γ-aminobutyric acid, and it is by increasing the expression of the activity of glutamate decarboxylase, thereby improving the ability of Hericium coralloides to autonomously enrich γ-aminobutyric acid.

[0045] On the other hand, the present application also provides Hericium coralloides cultured by the described method or an antioxidant product of the Hericium coralloides.

[0046] The antioxidant product contains antioxidant active substances, which include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins, and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid.

[0047] The product may include excipients, which may be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

[0048] The product of the present application can be prepared by a general method, and one or more diluents or carriers can be added, such as pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0049] On the other hand, the present application also provides the use of Hericium coralloides cultured by the method described above, or the Hericium coralloides described above, or the antioxidant product in antioxidant.

[0050] The present application also provides the use of Hericium coralloides cultured by the method described above, or the Hericium coralloides described above, or the antioxidant product in antioxidant for non-disease treatment and diagnosis purposes.

[0051] The present invention has the following beneficial effects:

[0052] 1. In the present application, Schisandra chinensis is first used as a substrate for two-way fermentation with Hericium coralloides to obtain Hericium coralloides rich in antioxidant active ingredients and its related products.

[0053] 2. In the present application, by adjusting and optimizing the fermentation formula and fermentation conditions, the optimal fermentation process of Hericium coralloides is obtained. The obtained Hericium coralloides strain has the advantages of large mycelial ball density, good dispersion, good uniformity of mycelial ball diameter, thick mycelia, many clamp connections, dense and white mycelia, fast primordium formation, large quantity, large fruiting bodies, coral-like shape, soft spines, and fleshy texture.

[0054] 3. Hericium coralloides and its fermentation broth and other related products prepared by fermentation using the method of the present application contain rich antioxidant active ingredients, especially rich in γ-aminobutyric acid, providing a new biological source and green biological factory for γ-aminobutyric acid. Description of the Drawings

[0055] The accompanying drawings described herein are used to provide a further understanding of the present application and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings:

[0056] Figure 1 It is the gel electrophoresis diagram of PCR products;

[0057] Figure 2 It is the phylogenetic reverse tree diagram;

[0058] Figure 3 It is the schematic diagram of the fermentation substrate screening experiment results;

[0059] Figure 4 It is the statistical chart of the influence results of different fermentation substrates on the quality of the strain;

[0060] Figure 5 It is the schematic diagram of the influence results of different carbon sources on the liquid bacterial balls;

[0061] Figure 6 It is the statistical chart of the influence results of different carbon sources on the quality of the strain;

[0062] Figure 7 It is the schematic diagram of the influence results of different nitrogen sources on the liquid bacterial balls;

[0063] Figure 8 It is the statistical chart of the influence results of different nitrogen sources on the quality of the strain;

[0064] Figure 9 It is the statistical chart of the influence results of different peptone concentrations on the mycelial biomass;

[0065] Figure 10 It is the statistical chart of the influence results of different Schisandra chinensis concentrations on the mycelial biomass;

[0066] Figure 11 It is the statistical chart of the influence results of different lactose concentrations on the fermentation of Hericium coralloides;

[0067] Figure 12 It is the response surface diagram and contour map of the pairwise interaction of each factor;

[0068] Figure 13 It is the statistical chart of the influence results of different inoculation amounts on the quality of the strain;

[0069] Figure 14 It is the statistical chart of the influence results of different pH values on the quality of the strain;

[0070] Figure 15 It is the statistical chart of the influence results of different liquid volumes on the quality of the strain;

[0071] Figure 16 It is the statistical chart of the influence results of different fermentation temperatures on the quality of the strain;

[0072] Figure 17 Statistical chart of the influence of different shaker speeds on the quality of the strain;

[0073] Figure 18 Comparison chart of the growth of the strain in the experimental group and the control group;

[0074] Figure 19 Pie chart of the active ingredients of Hericium coralloides mycelium and fruiting body.

[0075] Biological material preservation:

[0076] A strain of Hericium coralloides RT25 was deposited on May 20, 2024 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO.: 41304, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. Detailed implementation manners

[0077] In order to more clearly illustrate the overall concept of the present application, the following will be described in detail by way of examples in conjunction with the accompanying drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0078] For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0079] Unless otherwise specified, in the following embodiments, for the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained by commercial purchase.

[0080] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture and related fields in the technical field.

[0081] Test strains:

[0082] Hericium coralloides RT25 was deposited on May 20, 2024 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO.: 41304, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0083] The control strain Hericium coralloides SY26 was purchased from Shenyang Shanhainongren Food Trading Co., Ltd.

[0084] The culture media involved in the following examples include:

[0085] Seed liquid culture medium: 200 g of potato (boiled juice), 20 g of glucose, 3 g of peptone, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, natural pH, 1000 mL of distilled water. Sterilized at 121 °C for 30 min.

[0086] Method for activating Hericium coralloides strain: Use a 9 mm hole punch to punch holes in the petri dish, and inoculate 3 pieces of fungal blocks into each bottle of seed liquid culture medium. After inoculation, place it in a shaker at 25 °C and 140 r / min for culturing for 7 days to obtain the seed liquid.

[0087] Basic fermentation culture medium (1 L): 20 g of glucose, 3 g of peptone, 200 g of potato (boiled juice), 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance is water.

[0088] Bagged material cultivation culture medium: 80% broad-leaved wood chips, 20% wheat bran, 1% each of gypsum and brown sugar, adjusted to a water content of 65%, natural pH.

[0089] Bagged material cultivation method: Use the bagged material cultivation culture medium to incubate in the dark for about 37 days until the cultivation bags are fully colonized, then move them out of the incubation room and carry out fruiting in a four-temperature zone constant temperature and humidity incubator. Fruiting occurs at the bag mouth, the temperature is controlled at 18 °C - 20 °C, ventilate twice a day, and harvest when the length of the mushroom spines of the Hericium coralloides fruiting bodies is more than 0.5 cm and the spores have not been ejected in large quantities.

[0090] Cultivation method for Hericium coralloides mycelium, primordium and fruiting body:

[0091] Step 1: After activating Hericium coralloides, carry out fermentation culture to obtain liquid spawn (or mycelium);

[0092] Step 2: The mycelium obtained by fermentation is rinsed with sterile water, filtered through gauze, dried at a constant temperature of 55 °C to constant weight, pulverized and passed through an 80-mesh sieve to obtain a mycelium powder sample for standby; inoculate the liquid spawn into the bagged material cultivation culture medium and culture it in a four-temperature zone constant temperature and humidity incubator, collect the primordium and fruiting body, dry at a constant temperature of 55 °C to constant weight, pulverize and pass through an 80-mesh sieve to obtain a primordium sample and a fruiting body sample for standby.

[0093] Among them, coix seed, fritillaria thunbergii, poria cocos, licorice, wheat bran, black bean, mulberry leaf, astragalus membranaceus, schizandra chinensis, schizandra chinensis, and notoginseng were all purchased from Chaoyang People's Kangtai Pharmacy (North Store); magnesium sulfate, potassium dihydrogen phosphate, anhydrous sodium sulfite, PDA potato agar medium, etc. were all purchased from Beijing Aoboxing Biotechnology Co., Ltd.; phenol, salicin, Folin-Ciocalteu phenol reagent, ethanol (analytical pure) were all purchased from Tianjin Kemiou Chemical Reagent Co., Ltd.; acid buffer salt solution (PBS, pH 7.8) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; hydroxyl radical scavenging ability kit, DPPH radical scavenging rate kit, superoxide anion scavenging ability kit were all purchased from Suzhou Grees Biotechnology Co., Ltd.

[0094] The instruments involved in the following examples include:

[0095] BHC-1300IIA / B2 biological safety cabinet (Shanghai Lishen Scientific Instruments Co., Ltd.); vertical constant temperature shaker (Shanghai Shiping Experimental Equipment Co., Ltd.); KQ-50DA numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); 723N visible spectrophotometer (Shanghai Yidian Analytical Instruments Co., Ltd.).

[0096] The detection methods involved in the following examples include:

[0097] The determination of the quality of the bacterial strain includes the determination of the diameter of the bacterial ball, the determination of the mycelial biomass, and the determination of the density of the bacterial ball.

[0098] Determination of the diameter of the bacterial ball: Randomly select 10 bacterial balls in a long row according to the proportion of the number of bacterial ball sizes contained in each culture solution, measure the total length, repeat 3 times, and calculate the average value.

[0099] Determination of the mycelial biomass: Determined by the difference method. Filter the cultured liquid bacterial strain, collect all the bacterial balls in a 250 mL shaking flask (the filter paper carrying the bacteria is dried to a constant weight before weighing), rinse 3 times with clear water, place in a constant temperature drying oven at 60 °C and dry to a constant weight, and calculate the average value.

[0100] Determination of the density of the bacterial ball: After shaking well from each repeated shaking flask of the liquid medium, place it in a petri dish, count the number of bacterial balls, repeat 3 times, and calculate the average value.

[0101] Determination of the content of active ingredients:

[0102] The determination of the polysaccharide content adopts the phenol-sulfuric acid method, and the polysaccharide standard curve equation: y = 6.1373x + 0.0284, R 2= 0.9947. Add 0.01 mL of the fermented sample solution to 1.99 mL of pure water, and the determination method is carried out according to the polysaccharide determination method optimized by Xie Cunyi et al. Calculate the polysaccharide content in the fermented sample solution according to the standard curve equation and gradient dilution (Reference: Xie Cunyi, Li Jianmei, Guo Lingling, et al. Comparison and Optimization of the Determination Method of Exopolysaccharide Content in Sanghuang Fermentation Broth [J]. Edible Fungi, 2022, 44(6): 69-73.).

[0103] The flavonoid content was determined by the aluminum nitrate-sodium nitrite colorimetric method. The standard curve equation of flavonoids: y = 2.6125x + 0.0466, R 2 = 0.9995. Take 3 mL of the fermented sample solution in a 25-mL graduated tube, add 2.0 mL of 70% ethanol and shake well; add 1.0 mL of 5% sodium nitrite solution and shake well, let stand for 6 min, add 1.0 mL of 10% aluminum nitrate solution and shake well, let stand for 6 min; add 10 mL of 4% sodium hydroxide solution, after standing for 10 min, dilute to 25 mL with 70% ethanol, and measure the absorbance value at 510 nm. Calculate the flavonoid content in the fermented sample solution according to the standard curve equation and gradient dilution.

[0104] The determination method of polyphenol content was improved according to the reference method. The spectrophotometric method was used. With the gallic acid concentration as the abscissa and the absorbance value D 750 as the ordinate, the standard curve equation was obtained: y = 4.379x - 0.0059, R 2 = 0.9962. Mix 80 μL of the fermented sample solution with 120 μL of water, add 1 mL of Folin-Ciocalteu reagent and react for 5 min, then add 0.8 mL of 7.5% Na2CO3, and then add 3 mL of pure water and let stand at 25 °C for 30 min. Measure the absorbance value of the fermented sample solution at 765 nm. Calculate the polyphenol content in the fermented sample solution according to the standard curve equation and gradient dilution (Reference: Li Jianmei, Zhu Wanqin, Chai Linshan, et al. Optimization of the Extraction Process of Polyphenols from Cordyceps militaris and Its Antioxidant Activity [J]. Food Research and Development, 2023, 44(8): 111-117.).

[0105] The content of triterpene compounds was detected by the vanillin-glacial acetic acid-perchloric acid method: sample treatment, fermentation broth was centrifuged at 8000×g for 5 min, the supernatant was discarded, an appropriate amount of isopropanol was added to the mycelium precipitate, heated to reflux extraction for 2 h, the filtrate was collected, the solvent was removed by vacuum distillation, and then dried to constant weight at 60°C; about 0.5 g of the fruiting body sample was weighed and put into a mortar, 2.5 mL of pH 7.0 phosphate buffer was added and ground into a homogenate, and isopropanol was refluxed for extraction for 2 h, the filtrate was collected, the solvent was removed by vacuum evaporation, and then dried to constant weight at 60°C. 10.4 mg of the standard betulin was weighed, dissolved in chloroform, and diluted to a 25 mL volumetric flask. 0, 0.25 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 4.0 mL of the mother solution were respectively taken in a 10 mL volumetric flask, diluted to a constant volume with chloroform, and 0.2 mL was taken in a test tube. After evaporation, 0.3 mL, 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid were added. After shaking, the mixture was placed in a 60°C water bath for 45 min, then cooled to room temperature in an ice-water bath, and 5 mL of anhydrous acetic acid was added. After fully shaking, the absorbance was measured at 548 nm to obtain the standard curve equation y=0.0022x+0.0687, R 2 =0.9991. 0.2mL of the extracted sample solution was placed in a test tube. The determination method was the same as the standard curve. The triterpene content was calculated based on the absorbance value and the standard curve equation.

[0106] Determination of γ-aminobutyric acid in samples: add 0.5mL borate buffer (0.2mol / L, pH10.0), 1mL 6% phenol, 1mL NaClO solution (active chlorine is 8%-12%) to 0.5mL of sample extract supernatant, mix well and put in boiling water bath for 10min, take out and immediately put in ice bath for 20min, after the solution turns blue-green, add 2.0mL of 60% ethanol solution and it can be used for determination (using the reagent solution without GABA standard solution as reference). Use 1cm colorimetric dish, scan at wavelength 645nm, obtain absorbance value, and calculate γ-aminobutyric acid concentration using standard curve equation.

[0107] Determination of γ-aminobutyric acid: Reference: Shi Xiaofeng, Luan Guangzhong, Cao Wanxin, Colorimetric determination of γ-aminobutyric acid in traditional fermented bean products, Chinese Condiments, 2008 (3), 76-81. Standard curve determination: Accurately weigh 2500.000 mg of γ-aminobutyric acid in a 500 mL volumetric flask and dissolve it in ultrapure water to make a 5.00 mg / mL standard solution. When using, accurately prepare 0, 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00 mg·mL -1 The GABA standard solution series was obtained to obtain the standard curve equation y = 0.9901x + 0.1337, R 2 =0.9996, where x is the concentration of γ-aminobutyric acid (mg / mL) and y is the absorbance value.

[0108] Determination of soluble protein content: Weigh about 0.5 g of the sample and put it into a mortar. Add 2.5 mL of phosphate buffer solution with pH 7.0 and grind it into a homogenate. Then transfer it to a centrifuge tube and centrifuge at 8000 r / min for 5 min. Discard the precipitate to obtain the extraction solution to be measured. Preparation of the standard curve: Take 6 clean test tubes labeled 1, 2, 3, 4, 5, and 6. The concentration of the stock solution is 0.5 mg / mL, and the volumes are 0, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, and 0.1 mL respectively. Then add 5.0 mL of Coomassie Brilliant Blue G-250 reagent, shake well, and let it stand for 2 min. Then measure the absorbance at 600 nm. Record the optical density value OD of each tube, and use the standard protein content (mg / mL) as the abscissa and the absorbance as the ordinate. Use Excel software to draw the standard curve (y = 5.8086x + 0.0322, R 2 = 0.9905). Then take 0.1 mL of the sample extraction solution and add it to the test tubes respectively. Add 0.1 mL of phosphate buffer solution with pH 7.0 to the blank control tube. Add 5.0 mL of Coomassie Brilliant Blue G-250 reagent and 0.9 mL of distilled water to each tube, shake well. After 2 min, measure the optical density value OD at 600 nm wavelength, and calculate the content of soluble protein through the regression equation.

[0109] Determination of free radical scavenging rate:

[0110] The antioxidant activity of the fermentation broth is expressed by the free radical scavenging rate. Refer to the method in the article by Xu Bin et al. to determine the DPPH free radical scavenging rate, superoxide anion scavenging rate, and hydroxyl free radical scavenging rate. In this experiment, the sample solution for free radical scavenging rate determination is the original solution and does not need to be diluted (Reference: Xu Bin, Zhou Yongkang, Li Huixing, et al. Analysis of the dose-effect relationship between antioxidant activity and active components of Cordyceps militaris mycelium [J]. Science and Technology of Food Industry, 2021, 42(2): 250-255.).

[0111] Refer to the kit instruction manual to test the DPPH free radical scavenging rate of the test sample solution. Calculate the DPPH free radical scavenging rate of the test sample solution according to formula (1).

[0112] DPPH free radical scavenging rate = [1 - (D 测定 - D 对照 ) ÷ D 空白 × 100% (1)

[0113] In the formula, D 测定 is the absorbance value of the sample and DPPH mixture; D 对照 is the absorbance value of the sample and absolute ethanol mixture; D 空白 is the absorbance value of DPPH and absolute ethanol mixture.

[0114] Determine the superoxide anion scavenging rate of the test sample with reference to the kit instructions, and calculate the superoxide anion scavenging rate of the test sample according to formula (2):

[0115] Superoxide anion scavenging rate = [1 - (D 测定 - D 对照 ) ÷ D 空白 × 100% (2)

[0116] In the formula, D 测定 is the absorbance value of the measurement tube; D 对照 is the absorbance value of the control tube; D 空白 is the absorbance value of the blank tube

[0117] Determine the hydroxyl radical scavenging rate of the test sample with reference to the kit instructions, and calculate the hydroxyl radical scavenging rate of the test sample according to formula (3):

[0118] Hydroxyl radical scavenging rate = [D 空白－ (D 测定 - D 对照 )] ÷ D 空白 × 100% (3)

[0119] In the formula, D 测定 is the absorbance value of the measurement tube; D 对照 is the absorbance value of the control tube; D 空白 is the absorbance value of the blank tube.

[0120] Use the T-AOC assay kit to perform the total antioxidant capacity assay and analysis, and calculate the total antioxidant capacity of the test sample according to formula (4):

[0121] Total antioxidant capacity / (μmol Trolox / g) = 0.3 × (△A + 0.029) ÷ W × D (4)

[0122] In the formula: △A = Ablank - (Ameasurement - Acontrol), where Ameasurement is the absorbance of the sample and the ABTS working solution, Acontrol is the absorbance of the sample and the PBS mixture, and Ablank is the absorbance of the ABTS working solution and the PBS mixture; W is the sample mass in g; D is the dilution factor of the sample.

[0123] All results are expressed as mean ± standard deviation. Use Excel software for data statistics and analysis, with P < 0.05 indicating significant difference and P < 0.01 indicating extremely significant difference. Use Design-Expert 11.0 software for model regression analysis and response surface interaction term analysis.

[0124] In addition, the "water" described in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, pure water, etc.

[0125] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0126] Example 1 Strain Identification and Strain Preservation

[0127] In this example, a new strain of Hericium coralloides was obtained through screening and named RT25. Molecular identification and phylogenetic studies were carried out on the above Hericium coralloides strain RT25, including genomic DNA extraction, PCR product sequencing, and phylogenetic analysis of the Hericium coralloides strain RT25. After purification, unidirectional sequencing was performed, and the rDNA ITS sequence of the tested strain was used to establish a phylogenetic tree in the GenBank nucleic acid sequence database. Among them, the electrophoresis pattern of the PCR product is as Figure 1 shown, and the phylogenetic tree is as Figure 2 shown.

[0128] The test strain Hericium coralloides strain RT25 was submitted to Shanghai Sangon Biotech Co., Ltd. for strain identification. Finally, the strain was identified as Hericium (genus Hericium), and it is Hericium coralloides. Currently, the strain Hericium coralloides RT25 has been deposited in the China General Microbiological Culture Collection Center on May 20, 2024, with the deposit number CGMCC NO.: 41304.

[0129] Example 2 Fermentation Substrate Screening Experiment

[0130] Different fermentation substrates have different effects on the quality of the Hericium coralloides RT25 strain. In this example, the fermentation substrates for Hericium coralloides RT25 were screened. The specific method includes:

[0131] The fermentation substrates Coix chinensis Tod., Fritillaria thunbergii, Wolfiporia cocos, Glycyrrhiza uralensis, Wheat bran, Glycine max, Folium Mori, Astragalus mongholicus Bunge, Schisandra chinensis, and Panax notoginseng were crushed by a pulverizer and sieved through a 60-mesh sample sieve for standby. According to 0.2 g·100 mL -1They were respectively added to the basic fermentation medium with natural pH, sterilized at 121 °C for 30 min to obtain the substrate medium. The blank control medium was the basic fermentation medium.

[0132] The above substrate medium and basic fermentation medium were used for the fermentation culture of Hericium coralloides RT25. The culture process was as follows: Step 1, Hericium coralloides RT25 was activated to obtain the seed liquid; Step 2, the seed liquid was inoculated into the above substrate medium containing different fermentation substrates at an inoculation amount of 1%, and fermentation culture was carried out. Liquid culture was carried out at 25 °C and 140 r / min with natural pH. After 7 days, the quality of the strains (hyphal biomass, pellet diameter, pellet density) of each formula was measured. The results were as Figure 3 , shown in Table 1. Based on the content of Table 1, Figure 4 .

[0133] Table 1 Effects of different fermentation substrates on the quality of strains

[0134]

[0135] As Figure 3 , shown in Table 1 and Figure 4 shown, Schisandra chinensis can significantly promote the growth of the strain, while Fritillaria thunbergii, Coix lacryma-jobi, and Panax notoginseng have significant inhibitory effects. Among them, when Schisandra chinensis is used as the fermentation substrate, the hyphal biomass is the highest, significantly higher than that of other substrates. The pellet diameter is not large (the pellet diameter does not block the nozzle during inoculation and can ensure sufficient hyphal amount), the pellet density is significantly higher than that of other substrates, and the pellet density is moderate, which is suitable for production as a liquid strain. Therefore, Schisandra chinensis was selected as the fermentation substrate for subsequent fermentation.

[0136] Example 3 Carbon source test

[0137] In this example, a single-factor experiment was designed to screen the carbon source. Liquid media were prepared according to different carbon sources, and liquid culture was carried out at an inoculation amount of 1%, 25 °C, and 140 r / min with natural pH. After 7 days, the quality of Hericium coralloides RT25 strains (hyphal biomass, pellet diameter, pellet density) of each formula was measured. The remaining culture methods were the same as those in Example 2. The test results were as Figure 5 and shown in Table 2. From Table 2, Figure 6 .

[0138] Among them, the formula of the liquid medium (1 L) was: carbon source 20 g, peptone 3 g, Schisandra chinensis 2 g, potato 200 g (boiled juice), dipotassium hydrogen phosphate 3 g, magnesium sulfate 1.5 g, VB1 0.01 g, and the balance was water.

[0139] The types of carbon sources included glucose, maltose, corn flour, sucrose, lactose, and mannitol.

[0140] The blank control medium was the basic fermentation medium.

[0141] Table 2 Influence of Different Carbon Sources on the Quality of Strains

[0142]

[0143] The screening results of different carbon sources are shown in Figure 5 and Table 2, and Figure 6 , in which when lactose is used as the carbon source, the mycelial biomass is the highest, reaching 8.208 g / L, significantly higher than several other carbon sources. Followed by maltose; when lactose is used as the carbon source, although the diameter of the mycelial pellets is not the smallest, it will not block the nozzle during inoculation, and at the same time, it can ensure a sufficient amount of mycelium. The density of the mycelial pellets is 205 per 100 mL, significantly higher than that of maltose and sucrose. The density of the mycelial pellets is moderate and suitable for production as liquid strains. Therefore, lactose can be used as the optimal carbon source for the production of liquid strains of Hericium coralloides.

[0144] Example 4 Nitrogen Source Test

[0145] A single-factor test was designed in the same way as in Example 3 to screen the nitrogen source. Liquid media were prepared according to different nitrogen sources, and liquid culture was carried out at an inoculation amount of 1%, 25 °C, and 140 r / min, with the pH natural. After 7 days, the quality of Hericium coralloides RT25 strains (mycelial biomass, mycelial pellet diameter, mycelial pellet density) of each formula was measured. The remaining culture methods were the same as in Example 2. The test results are as shown in Figure 7 and Table 3. Graphs were obtained from Table 3 as shown in Figure 8 .

[0146] Among them, the formula of the liquid medium (1 L) is: 3 g of nitrogen source, 20 g of glucose, 200 g of potato (boiled juice), 2 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance is water.

[0147] The types of nitrogen sources include peptone, yeast extract powder, ammonium nitrate, tryptone, beef extract, and yeast extract.

[0148] The blank control medium is the basic fermentation medium.

[0149] Table 3 Influence of Different Nitrogen Sources on the Quality of Strains

[0150]

[0151] The screening results of the nitrogen source for the liquid strains of Hericium coralloides are shown in Figure 7 , Table 3, Figure 8, when peptone is used as the nitrogen source, the mycelial biomass is 7.144 g / L, which is significantly higher than other nitrogen sources. The diameter of the mycelial pellets is 1.127 mm. This mycelial pellet diameter does not block the nozzle during inoculation and can ensure a sufficient amount of mycelium at the same time. The density of the mycelial pellets is 579 per 100 mL, which is suitable as the nitrogen source for liquid spawn; followed by yeast extract powder, with a mycelial biomass of 5.941 g / L. When yeast extract powder is used as the nitrogen source for liquid spawn, the density of the mycelial pellets is too low and the diameter of the mycelial pellets is too large. Therefore, peptone is selected as the best nitrogen source for the liquid medium of Hericium coralloides, while when ammonium nitrate inorganic nitrogen is used as the nitrogen source, both the mycelial biomass and the density of the mycelial pellets are low, which is not suitable as the nitrogen source for liquid spawn.

[0152] Example 5 Experiment on the Optimal Addition Amount of the Fermentation Medium

[0153] In this example, a single-factor experiment on the addition amount was carried out for the optimal medium components (peptone, potato, lactose, magnesium sulfate, dipotassium hydrogen phosphate, VB1, Schisandra chinensis) obtained in Examples 2-4. Hericium coralloides RT25 was cultured with media of different concentrations. Specifically, as shown in Table 4, liquid culture was carried out at an inoculation amount of 1%, 25 °C, and 140 r / min, with the pH natural. After 7 days, the quality of the spawn (mycelial biomass, mycelial pellet diameter, mycelial pellet density) and the content of γ-aminobutyric acid in the mycelium of each formulation were measured. The remaining culture methods were the same as those in Example 2. The test results are shown in Table 4. Plotting from Table 4 gives Figures 9 - 11 .

[0154] Among them, the formula of the fermentation medium (1 L) for the single-factor experiment on the addition amount of peptone is: peptone 1-9 g, potato 200 g (boiled juice), glucose 20 g, Schisandra chinensis 2 g, magnesium sulfate 1.5 g, dipotassium hydrogen phosphate 3 g, VB1 0.01 g, and water 1 L.

[0155] The formula of the fermentation medium (1 L) for the single-factor experiment on the addition amount of lactose is: lactose 16-24 g, potato 200 g (boiled juice), dipotassium hydrogen phosphate 3 g, magnesium sulfate 1.5 g, peptone 3 g, Schisandra chinensis 2 g, VB1 0.01 g, and the balance is water.

[0156] The formula of the fermentation medium (1 L) for the single-factor experiment on the addition amount of Schisandra chinensis is: Schisandra chinensis 1-3 g, peptone 3 g, potato 200 g (boiled juice), lactose 20 g, magnesium sulfate 1.5 g, dipotassium hydrogen phosphate 3 g, VB1 0.01 g, and the balance is water.

[0157] Table 4 Single-Factor Test on Fermentation Nutrient Components

[0158]

[0159]

[0160] From Table 4 andFigures 9 - 11 The results show that the formula of the optimal carbon-nitrogen ratio fermentation medium (1 L) is 3 g of peptone, 18 g of lactose, 2.5 g of Schisandra chinensis, 200 g of potato (boiled juice), 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance is water.

[0161] Moreover, in this example, it was first discovered that the addition of Schisandra chinensis could cause changes in the content of γ-aminobutyric acid in the strain.

[0162] Optimization of the fermentation formula in Example 6

[0163] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system, which has good water solubility and thermal stability. It has been confirmed that GABA, as a small molecular weight non-protein amino acid, has food safety and can be used in the production of foods such as beverages. Currently, research shows that the intake of a certain amount of GABA has physiological effects such as improving the sleep quality of the body and lowering blood pressure. Through comprehensive comparison, the active ingredient γ-aminobutyric acid has a significant correlation with antioxidant activity. In Example 5, it was preliminarily proved that γ-aminobutyric acid is the antioxidant active substance of Hericium coralloides. In this example, a single-factor experiment and response surface method optimization were further carried out on the RT25 fermentation medium of Hericium coralloides using the dual-index parameters of fermentation biomass and γ-aminobutyric acid content, in order to provide a reference for the development of Hericium coralloides fermentation products and improve their bioavailability.

[0164] According to the single-factor experiment results in Examples 2-5, fermentation experiments were carried out according to the response surface factor level design in Table 5. The weighted comprehensive score was used as the response value (Y). Combining with the single-factor experiment results, the maximum fermentation biomass of 12.772 was counted as 50 points, and its corresponding score was (N1 / 12.772)×50. The highest score of 28.778 for the γ-aminobutyric acid content was counted as 50 points, and its corresponding score was (N2 / 28.778)×50. The comprehensive score = (N1 / 12.772)×50+(N2 / 28.778)×50, where N1 is the fermentation biomass and N2 is the γ-aminobutyric acid content. The measurement results and comprehensive score results are shown in Table 6.

[0165] The results of the variance analysis of the regression model are shown in Table 7. After calculation, the regression equation between the comprehensive score (Y1) and lactose (X1), peptone (X2), and Schisandra chinensis (X3) is:

[0166] Y1 = 89.64 - 7.9X1 + 1.39X2 - 0.131X3 - 0.21X1X2 + 6.53X1X3 + 0.8065X2X3 - 16.05X1 2 - 20.57X2 2 - 16.58X

[0167] 3 2 (5)

[0168] Coefficient of determination R of the model 2 = 0.9128, adjusted coefficient of determination R 2 adj = 0.8007, indicating that 80.07% of the variation in the comprehensive score can be predicted by this model. The specific results are shown in Table 7. As can be seen from Table 7, the model F = 8.14, P < 0.01, indicating that the model is significant. The lack-of-fit term F = 1.63, P = 0.3174 > 0.05, indicating that the lack-of-fit test is not significant, the equation has a good fit, and the difference between the model and the experimental values is small. According to the F values of each item in Table 7: A 2 , B 2 , C 2 etc. have a highly significant effect on the comprehensive score, and A reaches a significant level. From the magnitude of the F values, it can also be judged that the order of influence on the comprehensive score is: lactose > peptone > Schisandra chinensis.

[0169] Table 5 Response surface factor levels

[0170]

[0171] Table 6 Response surface experimental design results

[0172]

[0173] Table 7 ANOVA of the regression model

[0174]

[0175] Note: * indicates that the correlation reaches a significant level (P < 0.05); ** indicates that the correlation reaches a highly significant level (P < 0.01)

[0176] The 3D response surface illustrates the interaction between the dependent variable and two test variables when other factors are fixed at zero level. Using Design-Expert 11.0 software to make an interaction surface plot for equation (5), we get Figure 12 , after fixing the value of one of the factors A, B, C in the regression model, the pairwise interactions of each factor all form a convex surface opening downward; within the value range of A, B, C, the comprehensive score Y shows a trend of first increasing and then decreasing with the increase of concentration, and there is a maximum point; from the contour plot, it can be seen that the response surface slope of the interaction between lactose and Schisandra chinensis (AC) is steep, and it has a significant effect on the comprehensive score Y; the interactions between lactose concentration and peptone concentration (AB) and between peptone concentration and Schisandra chinensis concentration (BC) have no significant effect on Y, and the interaction effect is AC > BC > AB, which is consistent with the ANOVA results.

[0177] Taking the first-order partial derivative of Equation (5) and after calculation, we get:

[0178]

[0179]

[0180] After calculation, when the response value is at its maximum, the optimal values of ABC are 19.054 g / L, 3.068 g / L, and 2.392 g / L respectively. Under these conditions, the maximum value of the comprehensive score is 90.644. The optimized fermentation process after adjustment is as follows: 200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, 1000 mL of water, an inoculation amount of 1%, a fermentation time of 7 d, a shaker speed of 140 r / min, a pH of 6.0 (natural), a liquid loading volume of 80 mL / 250 mL in the shaker flask, and a temperature of 25°C. Using the optimized process and repeating the experiment three times, the average value of the fermentation biomass obtained is 12.258 g / L, and the average value of the γ-amino acid content is 28.087 mg / g, indicating that this model is feasible and can provide a reference for the fermentation process of Hericium coralloides.

[0181] Example 7 Environmental Condition Test

[0182] Using the best fermentation medium formula obtained in Example 6 above, a single-factor experiment with 5 factors and 5 levels of inoculation amount, fermentation temperature, liquid loading volume, rotation speed, and pH was designed, as shown in Table 8 below. The remaining culture methods were the same as those in Example 2, and each group of experiments was repeated 3 times. And the quality of the strain was measured, and the measurement results were prepared according to Table 8 Figures 13 - 17 .

[0183] Table 8 Single-Factor Experiment on Fermentation Environmental Conditions of Hericium coralloides RT25

[0184]

[0185] As shown in Table 8 and Figures 13 - 17 For the experimental results of environmental conditions, combining the mycelial biomass, mycelial ball density, and average mycelial ball diameter, the appropriate inoculation amount was selected as 1%, the fermentation pH was 6, the liquid loading volume in the shaker flask was 80 mL / 250 mL, the fermentation temperature was 25°C, and the shaker speed was 140 r / min.

[0186] Example 8 Verification of Fermentation Formula

[0187] Fermentation and fruiting experiments were carried out using the optimized liquid fermentation medium obtained in Example 6 and the optimal fermentation conditions obtained in Example 7. The fermentation group of Hericium coralloides without Schisandra chinensis was used as the control group, and the fermentation medium added with Schisandra chinensis was used as the experimental group. The fruiting bodies of Hericium coralloides obtained by inoculating the fermentation liquid strain without Schisandra chinensis were used as the control group, and the fruiting bodies of Hericium coralloides inoculated with the fermentation liquid strain of the optimized medium of Schisandra chinensis were used as the experimental group. Taking the strain RT25 as an example, the quality of the strain was tested experimentally, and the mycelium was collected and cultivated in bagged materials to obtain primordia and fruiting bodies. The fermentation mycelium state of the strain, the growth rate of the fruiting mycelium, the number of days for the fruiting mycelium to grow full, the number of days for primordium formation, the fresh weight of the fruiting body, and the dry weight of the fruiting body were recorded. The results are shown in Table 9, Figure 18 as shown below.

[0188] Control group: The strain RT25 was inoculated into the control group medium (200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance was water, totaling 1 L), the inoculation amount was 1%, the fermentation time was 7 d, the shaker speed was 140 r / min, pH 6.0 (natural), the liquid loading amount in the shake flask was 80 mL / 250 mL, and the temperature was 25 °C for cultivation.

[0189] Experimental group: The strain RT25 was inoculated into the experimental group medium (200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance was water, totaling 1 L), the inoculation amount was 1%, the fermentation time was 7 d, the shaker speed was 140 r / min, pH 6.0 (natural), the liquid loading amount in the shake flask was 80 mL / 250 mL, and the temperature was 25 °C for cultivation.

[0190] From Table 9, Figure 18 it can be seen that the mycelial balls in the fermentation broth of the experimental group are more uniform and have a higher density than those in the control group. The growth rate of the mycelium, the size of the fruiting body, and the weight of the fruiting body in the experimental group are significantly improved compared with those in the control group, indicating that Schisandra chinensis has a promoting effect on the growth of Hericium coralloides. This fermentation formula has strong feasibility and broad application prospects.

[0191] Table 9 Comparison table between the control group and the experimental group

[0192]

[0193] Example 9 Mining of antioxidant active components

[0194] In this example, the effects of Schisandra chinensis and the strain RT25 on the antioxidant active components in the mycelium and fruiting body and their changes were further explored, and the strain SY26 was used as a control.

[0195] Strain RT25 experimental group: Inoculate strain RT25 into the experimental group medium (200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance is water, totaling 1 L), with an inoculation amount of 1%, a fermentation time of 7 d, a shaker rotation speed of 140 r / min, a natural pH of 6.0, a liquid loading volume of 80 mL / 250 mL in the shaking flask, and culture at 25 °C.

[0196] Strain RT25 control group: Inoculate strain RT25 into the control group medium (200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance is water, totaling 1 L), with an inoculation amount of 1%, a fermentation time of 7 d, a shaker rotation speed of 140 r / min, a natural pH of 6.0, a liquid loading volume of 80 mL / 250 mL in the shaking flask, and culture at 25 °C.

[0197] Strain SY26 experimental group: Inoculate strain SY26 into the experimental group medium (200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance is water, totaling 1 L), with an inoculation amount of 1%, a fermentation time of 7 d, a shaker rotation speed of 140 r / min, a natural pH of 6.0, a liquid loading volume of 80 mL / 250 mL in the shaking flask, and culture at 25 °C.

[0198] Strain SY26 control group: Inoculate strain SY26 into the control group medium (200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance is water, totaling 1 L), with an inoculation amount of 1%, a fermentation time of 7 d, a shaker rotation speed of 140 r / min, a natural pH of 6.0, a liquid loading volume of 80 mL / 250 mL in the shaking flask, and culture at 25 °C.

[0199] After obtaining the fermented mycelia by the above fermentation methods respectively, inoculate the fermented mycelia of the strain RT25 control group and the experimental group into the bagged cultivation medium to obtain the fruiting body control group and the experimental group of strain RT25; inoculate the mycelia of the RT26 control group and the experimental group into the bagged cultivation medium to obtain the fruiting body control group and the experimental group of strain RT26, and then measure the content of antioxidant active ingredients in them respectively.

[0200] The comparison results of the active ingredients and free radical scavenging rates in the mycelia and fruiting bodies of the test strain RT25 and the control strain SY26 are shown in Table 10. In this example, it is proved that after adding Schisandra chinensis as a fermentation factor, the active ingredients and free radical scavenging rates in the sample solution of the test group have increased, indicating that adding Schisandra chinensis as an inducing factor has a certain synergistic effect on the content of active substances and antioxidant activity in the mycelia and fruiting bodies of Hericium coralloides.

[0201] Pearson correlation analysis was used to determine the correlation between antioxidant active substances and antioxidant activity (References: Ge Xinhui, Meng Jikun, et al., Analysis of the phenolic substances composition and antioxidant activity of wild edible fungi in Wutai Mountain, Edible Fungi of China. 2022, 41(04)) (Table 11), and the percentage of each active substance in the total antioxidant active substances content was calculated ( Figure 19 ). It can be seen from the correlation analysis in Table 11 that the antioxidant ability of the mycelia and fruiting bodies of Hericium coralloides is the result of the synergistic action of multiple active ingredients. Among them, active ingredients such as γ-aminobutyric acid, polysaccharides, flavonoids, and polyphenols have different correlations with antioxidant ability: γ-aminobutyric acid has a very significant positive correlation with the DPPH free radical scavenging rate (P = 0.888) and a very significant positive correlation with the hydroxyl free radical scavenging rate (P = 0.945); polysaccharides have a very significant positive correlation with the hydroxyl free radical scavenging rate (P = 0.864) and a significant positive correlation with the DPPH free radical scavenging rate (P = 0.775); flavonoids have a very significant positive correlation with the hydroxyl free radical scavenging rate (P = 0.825) and a significant positive correlation with the DPPH free radical scavenging rate (P = 0.68); polyphenols have a significant positive correlation with the DPPH free radical scavenging rate and the hydroxyl free radical scavenging rate (P = 0.737, P = 0.812), and a positive correlation with the superoxide anion (P = 0.705).

[0202] Table 10 Active Ingredients and Antioxidant Activity of Hericium coralloides

[0203]

[0204] Table 11 Correlation Analysis

[0205]

[0206] Note: ***, **, * represent the significance levels of 1%, 5%, and 10% respectively

[0207] From Figure 19Analyze the increase in γ-aminobutyric acid. After the RT25 mycelium was induced to ferment by adding Schisandra chinensis, the content of γ-aminobutyric acid increased by 21% among the active ingredients. The increase in the proportion of γ-aminobutyric acid in the active ingredients of the mycelium of the control strain SY26 was 7%. The percentage increase in γ-aminobutyric acid in the fruiting body of RT25 was 7%, two percentage points more than that of the fruiting body of the control strain SY26. Therefore, the test strain RT25 can utilize the Schisandra chinensis fermentation medium better than the control strain SY26, promoting the biosynthesis of its antioxidant active substance γ-aminobutyric acid. That is, the Schisandra chinensis induction factor helps the synthesis of the antioxidant active substance γ-aminobutyric acid in Hericium coralloides, and its effect on the strain RT25 is particularly significant.

[0208] Example 10 Determination of the enzyme activity of glutamate decarboxylase (GAD) in Hericium coralloides

[0209] In this example, the reason for the rich γ-aminobutyric acid in Hericium coralloides was further explored, and the enzyme activity of glutamate decarboxylase (GAD) in it was determined using the mycelium as an example.

[0210] The specific method is as follows:

[0211] Step 1: Use a 250 mL Erlenmeyer flask for seed culture. The loading volume of the seed liquid medium is 100 mL. After inoculating Hericium coralloides RT25 or SY26, culture at 25 °C and 140 rpm for 7 days until the medium is covered with mycelial pellets the size of fish roe and the culture solution becomes clear, then the Hericium coralloides seed liquid is obtained.

[0212] Step 2: Inoculate the seed liquids of the strains RT25 and SY26 into the experimental group medium and the control group medium respectively, and ferment and culture until the medium is covered with mycelial pellets the size of rice grains (the specific grouping and culture process are as follows), then the Hericium coralloides mycelium is obtained. Filter the Hericium coralloides mycelium with a wire mesh, rinse it 3 times with physiological saline, and squeeze out the water to obtain the Hericium coralloides bacterial body.

[0213] The fermentation media and fermentation conditions of the experimental group of strain RT25, the control group of strain RT25, the experimental group of strain SY26, and the control group of strain SY26 are the same as those in Example 9.

[0214] Crude enzyme solution extraction: Collect 3 g of Hericium coralloides mycelium in Step 2, add 30 mL of Mac Ilvaine buffer (pH 4.8, containing 1 mmol / L mercaptoethanol and 0.1 mmol / L pyridoxal phosphate), break the cells by ultrasonic wave in an ice bath, the breaking condition is power 800 W, 15 min (working for 5 s, interval for 15 s), add 0.05 mL of Tween 80 and a little quartz sand, grind in an ice bath for 5 min, centrifuge (8000 r / min) to collect the supernatant, fully dialyze it in Mac Ilvaine buffer (pH 4.8, containing 1 mmol / L mercaptoethanol and 0.1 mmol / L pyridoxal phosphate), and then centrifuge to collect the supernatant as the crude enzyme solution of glutamate decarboxylase (GAD).

[0215] The enzyme reaction experiment includes the following steps:

[0216] Take 2 mL of GAD crude enzyme solution, 2 mL of glutamate solution (60 mmol / L, pH 4.8) and 2 mL of Mac Ilvaine buffer (pH 4.8, containing 0.4 mmol / L pyridoxal phosphate), mix them evenly and react at 40 °C for 20 h, take it out and place it in an ice bath to terminate the reaction. Use the GAD crude enzyme solution with inactivated enzyme activity (boiling water bath for 10 min) to carry out the same operation as the blank.

[0217] Use the phenol and sodium hypochlorite colorimetric method to determine GABA produced by the catalysis of glutamate decarboxylase (GAD). Finally, a blue substance is formed. This substance has obvious light absorption at 645 nm. The depth of its color is proportional to the content of GABA, and then the activity of GAD is obtained. The results are shown in Table 12.

[0218] Standard curve: y = 2.0976x - 0.0059, where x is the molar mass of the standard product (μmol), and y is the absorbance value ΔA. Calculated according to the fresh weight of the sample, unit definition: Producing 1 nmol of GABA per gram of tissue per minute is defined as one enzyme activity unit.

[0219] GAD (nmol / min / g fresh weight) = [(ΔA + 0.0059) ÷ 2.0976] × (V2 ÷ V3) ÷ (W × V1 ÷ V) ÷ T × 103 = 397.3 × (ΔA + 0.0059) ÷ W

[0220] V - - volume of the extraction solution, 1 mL; V1 - - volume of the sample added to the reaction system, 0.1 mL; V2 - - total volume of the reaction system: 0.5 mL; V3 - - volume of the supernatant in the color development stage, 0.2 mL; T - - reaction time, 30 min; W - - sample mass, g; molecular weight of standard product GABA - - 103.12.

[0221] Table 12 Glutamate decarboxylase activity in Hericium coralloides

[0222]

[0223] As can be seen from the results in Table 12, in this application, Schisandra chinensis is used as an inducing factor to ferment Hericium coralloides, and the expression of glutamate decarboxylase activity can be increased to varying degrees in mycelium, primordium, and fruiting body, thereby improving the ability of Hericium coralloides to autonomously enrich γ-aminobutyric acid.

[0224] Conclusion:

[0225] In recent years, the production range of Hericium coralloides has gradually spread southward from the Northeast region. With the proposal of the "going global" development strategy, Hericium coralloides has been successfully introduced to Zambia and integrated with supporting industrialization technologies for the tropical plateau. The main active components of Hericium coralloides include polysaccharides, sterols, hericenones, etc. Cheng Yanfen et al. found that Hericium coralloides polysaccharides can effectively regulate the blood lipid levels of hypercholesterolemic rats; Meng Junlong et al.'s research results showed that Hericium coralloides is an edible mushroom with high protein content, rich minerals, and complete essential amino acids, and its mycelial polysaccharides have the effect of enhancing the immune function of mice; Tang Peng et al. found that Hericium coralloides polysaccharides have a regulatory effect on the metabolism of rat liver. Currently, there is no report on the correlation analysis of the active components and antioxidant activity of the two-way liquid fermentation of Hericium coralloides-Schisandra chinensis.

[0226] In this study, for the first time, Schisandra chinensis was used as the fermentation substrate of Hericium coralloides for two-way fermentation, a liquid fermentation system of Hericium coralloides-Schisandra chinensis was constructed, the principle of antioxidant synergism of Hericium coralloides fermenting Schisandra chinensis was clarified, the characteristic two-way liquid fermentation technology of Hericium coralloides-Schisandra chinensis was broken through, and the fermentation formula was adjusted and optimized to be 200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and the balance was water, totaling 1000 mL. The fermentation conditions were an inoculation amount of 1%, a fermentation time of 7 d, a shaker speed of 140 r / min, a pH of 6.0 (natural), a liquid loading amount of 80 mL / 250 mL in the shake flask, and a temperature of 25°C. Using the optimized process, the average value of the fermentation biomass was 12.258 g, which was 98.24% of the predicted value (12.4780 g), and the average content of γ-amino acids was 28.087 mg / g; in terms of active components and antioxidant capacity, the antioxidant active component γ-aminobutyric acid of Hericium coralloides was excavated. Using the liquid fermentation system of Hericium coralloides-Schisandra chinensis, the test strain RT25 could utilize the Schisandra chinensis fermentation medium better than the control strain SY26. The Schisandra chinensis inducing factor contributed to the synthesis of the antioxidant active substance γ-aminobutyric acid of Hericium coralloides, and it had a particularly significant effect on the strain RT25.

[0227] In this study, the Hericium coralloides-Schisandra chinensis fermentation system achieved a simultaneous increase in the content of bioactive substances and antioxidant activity. The fermentation has "bidirectionality", and the antioxidant bioactive substance γ-aminobutyric acid in Hericium coralloides was discovered. Under the induction of the Hericium coralloides-Schisandra chinensis fermentation system, the increase in γ-aminobutyric acid in the mycelium and fruiting body of Hericium coralloides strain RT25 was significant. At the mycelium stage, based on the Hericium coralloides-Schisandra chinensis fermentation system, highly bioactive mycelium was obtained, increasing the yield of Hericium coralloides, which can also be used as a carrier for functional oral liquids. At the fruiting body stage, the Hericium coralloides-Schisandra chinensis fermentation system was inoculated to obtain fruiting bodies rich in antioxidant bioactive substances, thus providing a new biological material for biosynthetic preparations with high antioxidant activity and promoting growth and sleep. Through comprehensive analysis, the Hericium coralloides-Schisandra chinensis fermentation system has broad application prospects.

[0228] The above are only examples of this application and are not intended to limit this application. For those skilled in the art, various changes and modifications can be made to this application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of this application shall be included within the scope of the claims of this application.

Claims

1. A method for cultivating Hericium erinaceus with antioxidant activity, characterized in that: The method comprises: inoculating coral hericium into a fermentation medium for fermentation culture, wherein the coral hericium is coral hericium ( Hericium coralloides )RT25, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with a deposit number of CGMCC NO.: 41304; the fermentation medium includes 1.6%-2.4% carbon source, 0.1%-0.9% nitrogen source, 10%-50% potato, 0.1%-0.5% magnesium sulfate, 0.1%-0.5% dipotassium hydrogen phosphate, 0.001%-0.01% VB1, and 0.1%-0.3% Schisandra chinensis, the carbon source is lactose, and the nitrogen source is peptone.

2. The method according to claim 1, characterized in that The fermentation medium comprises 1.9% of carbon source, 0.3% of nitrogen source, 20% of potato, 0.15% of magnesium sulfate, 0.3% of dipotassium hydrogen phosphate, 0.001% of VB1, 0.24% of Schisandra chinensis, and the balance is water.

3. The method according to claim 1, characterized in that The fermentation culture conditions include: inoculation amount of 0.1%-3%, fermentation temperature of 18°C-32°C, liquid volume of 24%-60%, rotation speed of 100-180 rpm, and pH of 4.0-7.

0.

4. The method according to claim 3, characterized in that The fermentation culture conditions include: inoculation amount of 1%, fermentation temperature of 25°C, liquid volume of 32%, rotation speed of 140 rpm, and pH of 6.

0.

5. Application of the method according to any one of claims 1 to 4 in improving the antioxidant activity of Hericium erinaceus and / or improving the strain quality of Hericium erinaceus, characterized in that: The fermentation medium comprises 1.9% of carbon source, 0.3% of nitrogen source, 20% of potato, 0.15% of magnesium sulfate, 0.3% of dipotassium hydrogen phosphate, 0.001% of VB1, 0.24% of Schisandra chinensis, and the balance is water.

6. A coral hericium strain, characterized in that: The coral hericium is coral hericium RT25, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC NO.: 41304.

7. A coral hericium containing antioxidant active substances prepared by the method according to any one of claims 1 to 4, characterized in that: The antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or gamma-aminobutyric acid.

8. Use of the method according to any one of claims 1 to 4 or the coral hericium according to claim 6 or 7 in the preparation of glutamate decarboxylase and / or antioxidant active substances.

9. The use according to claim 8, characterized in that: The antioxidant active substances include polysaccharides, flavonoids, soluble proteins and / or gamma-aminobutyric acid.

10. The use according to claim 9, characterized in that: The antioxidant active substance is gamma-aminobutyric acid.

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