Method for rapidly culturing ganoderma lucidum in liquid and application thereof

Abstract

The application discloses a kind of liquid quick culture Ganoderma meat and its application, first, pre-made enzymolysis oat flour and sodium alginate-chitosan microcapsule fungicide and other modified raw materials;Further, the liquid culture medium consisting of enzymolysis oat flour, deep sea fish peptone, modified soybean peptide, microcapsule fungicide, black medlar extract, sodium selenite etc.;Then carry out "dark proliferation-blue light induction" two-stage directional fermentation, and pulse type adds methyl jasmonate inducer in illumination stage;Finally, low-temperature ripening and microwave vacuum drying obtain product.The application overcomes the defects of long traditional culture period, low active ingredient content, shortens the growth cycle to 9 days or less, the obtained Ganoderma meat product Ganoderma meat polysaccharide content is not less than 25%, and β-glucan accounts for more than 50%, and realizes the directional enrichment of selenium.The product can be used for preparing functional food for regulating immunity or auxiliary anti-tumor pharmaceutical composition, realizes the efficient, standardization, functional production of Ganoderma meat.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture and bioengineering technology, specifically relating to a method for deep liquid culture of Ganoderma lucidum. In particular, it relates to a method and its application for achieving rapid, high-yield, and high-quality cultivation of Ganoderma lucidum through innovative culture medium formulation and directional fermentation induction technology, thereby obtaining functional products rich in specific active ingredients. Background Technology

[0002] Reishi mushroom, a rare large slime mold complex or specific fungus, is traditionally believed to possess various health benefits due to its slow growth and unique morphology. Modern research indicates that it is rich in bioactive substances such as polysaccharides, triterpenes, and proteins, demonstrating potential value in regulating immunity and antioxidation, leading to increasing market demand. However, the industrial development of reishi mushroom is severely hampered by bottlenecks such as the low efficiency and uncontrollable quality of traditional cultivation methods.

[0003] Currently, the cultivation of Ganoderma lucidum mainly relies on solid substrate culture or simple liquid static culture. Solid culture has a cycle of several months to several years, requires a large area, is labor-intensive, and is susceptible to contamination by other microorganisms, resulting in highly unstable yield and quality. While existing liquid culture technology has shortened the growth cycle to some extent, it still has significant drawbacks: First, the culture medium formula is mostly composed of conventional raw materials such as glucose and peptone, with a single nutrient composition, making it difficult to simulate the complex growth environment of Ganoderma lucidum in nature, resulting in limited mycelial biomass accumulation and low synthesis efficiency of key active ingredients (such as β-glucan); second, the cultivation process is extensive, usually involving isothermal oscillation at a single temperature, lacking precise control over different stages of Ganoderma lucidum growth and development, and unable to effectively induce the synthesis of high-value secondary metabolites; third, the harvesting and processing methods are simple, often using high-temperature drying, which easily causes degradation and inactivation of heat-sensitive active ingredients, affecting the efficacy of the final product.

[0004] Several limitations in existing technologies restrict the efficient development and utilization of Ganoderma lucidum: First, traditional solid-state cultivation is like "relying on the weather," with an excessively long cycle and low success rate, failing to meet the needs of large-scale production; second, while simple liquid cultivation increases speed, the content of active ingredients in the resulting products is often lower than that of wild or solid-state cultures, significantly reducing efficacy and limiting commercial value; third, the entire cultivation process lacks controllability, with a lack of systematic bioengineering regulation methods from inoculation and growth induction to harvesting and drying, leading to large batch-to-batch quality variations and low product standardization. Furthermore, existing technologies rarely involve targeted nutritional fortification of the product during the cultivation process (such as selenium enrichment or other functional designs). Summary of the Invention

[0005] The core technical problem this invention aims to solve is to overcome the inherent defects of existing Ganoderma lucidum cultivation technologies, such as excessively long growth cycles, low content of bioactive substances, uncontrollable cultivation processes, and limited product functionality. Traditional solid-state cultivation methods are inefficient, while simple liquid cultivation is difficult to effectively induce the synthesis of high-value secondary metabolites, resulting in product quality that cannot meet the demands of the high-end market.

[0006] To address the aforementioned problems, this invention provides a method for rapid liquid culture of Ganoderma lucidum based on an innovative culture medium formulation and directional fermentation induction. The core innovation of this method lies in two aspects: First, by introducing unconventional raw materials such as enzymatically modified oat flour, deep-sea fish peptone, and a specially formulated microencapsulated compound microbial agent, a nutritionally complete liquid culture medium system with bio-inducible activity is constructed. Second, a two-stage directional fermentation process of "dark proliferation-blue light induction" is employed, supplemented by pulsed induction with methyl jasmonate, to precisely regulate the growth and metabolic pathways of Ganoderma lucidum, thereby simultaneously achieving rapid mycelial proliferation and efficient enrichment of highly active substances.

[0007] Another objective of this invention is to provide a highly active Ganoderma lucidum product cultivated using the above-described method. This product not only has a significantly shortened growth cycle to less than 9 days, but more importantly, its content of Ganoderma lucidum polysaccharides (especially β-glucan), selenium, and characteristic terpenoid compounds are all far higher than those of products cultivated using traditional methods, and it also possesses a unique microstructure.

[0008] Another objective of this invention is to provide the application of the aforementioned highly active Ganoderma lucidum products in the preparation of functional foods, health products, or adjuvant anti-tumor pharmaceutical compositions that regulate immune balance. Ultimately, the aim is to achieve efficient, standardized, and functional production of Ganoderma lucidum, providing a reliable material basis and technical support for its high-value-added development in the health industry.

[0009] The objective of this invention is achieved through the following technical solution.

[0010] A method for rapid liquid culture of Ganoderma lucidum includes the following steps:

[0011] (1) Components of pre-prepared modified culture medium:

[0012] a. Preparation of enzymatically hydrolyzed oat flour: Prepare a 15-20% suspension of oat flour, add neutral protease at a concentration of 4000-6000 U / g oat flour; incubate at 50-55℃ and pH...

[0013] Hydrolyze at 6.5-7.5 for 2-3 hours, then inactivate enzymes and spray dry to obtain enzymatically hydrolyzed oat flour;

[0014] b. Preparation of sodium alginate-chitosan microcapsule bacterial agent: Ganoderma lucidum mycelium extract, Cordyceps militaris mycelium extract, and Lactobacillus plantarum metabolites were prepared in a weight ratio of (3-5):

[0015] (2-4):1 mixture is used as the core material, and 1.5% sodium alginate solution is used as the wall material. The mixture is dripped into 2% calcium chloride solution using the sharp-hole method to form gel beads. Then, it is coated with 0.5% chitosan solution for cross-linking for 10 minutes to make microcapsule bacterial agent.

[0016] (2) Preparation of liquid culture medium: Mix the following raw materials by weight: 10-20 parts of enzymatically hydrolyzed oat flour obtained in step (1)a, 5-12 parts of deep-sea fish peptone, 3-8 parts of modified soybean peptide with molecular weight <1000Da, 2-5 parts of microcapsule bacterial agent obtained in step (1)b, 1-3 parts of black goji berry extract, 0.01-0.05 parts of sodium selenite, and 150-250 parts of water;

[0017] (3) Directional fermentation induction: The culture medium from step (2) was sterilized at 115-121℃ for 15-20 minutes, cooled to 25-28℃, and then inoculated with 3wt%-5wt% of Ganoderma lucidum strain; firstly, it was cultured in the dark with shaking at 100-150 rpm for 3-4 days; then it was transferred to blue light irradiation and cultured with shaking at 80-120 rpm for 3-5 days, and methyl jasmonate inducer was added pulsedly every 24 hours during the light stage, so that its final concentration in the culture medium was 10-50 μmol / L;

[0018] (4) Harvesting and post-ripening: When the mycelial ball density reaches 15-25 g / L, harvest the culture, filter it through a 100-mesh sieve, and collect the Ganoderma lucidum mycelium; let the wet mycelium stand in a phosphate buffer containing 0.1% glutamine at 4℃ for 12-24 hours for post-ripening.

[0019] (5) Directional drying: The post-ripened mycelium is dried by microwave vacuum drying with microwave power of 300-500W and vacuum degree of -0.08 to -0.09MPa until the moisture content is ≤8% to obtain the product.

[0020] Furthermore, in the above method, in step (1)b, the Ganoderma lucidum mycelium extract and Cordyceps militaris mycelium extract are both obtained by ultrasonic-assisted water extraction, and the polysaccharide content is not less than 30%.

[0021] Furthermore, in the above method, in step (2), the modified soybean peptide is an oligopeptide component with a molecular weight of less than 500 Da obtained by hydrolysis with flavor protease and trypsin and then separation by nanofiltration membrane.

[0022] Furthermore, in step (3) of the above method, the methyl jasmonate inducer is added in the form of microcapsules with β-cyclodextrin as the wall material to achieve a pulsed sustained-release effect.

[0023] Furthermore, in the above method, in step (3), the wavelength of the blue light irradiation is 450-470 nm, and the light intensity is 20-40 μmol·m⁻¹. -2 ·s -1 The light-dark cycle is 12 hours of light / 12 hours of darkness.

[0024] Furthermore, in the above method, in step (4), the concentration of the phosphate buffer is 0.05M and the pH is 6.8, and the post-ripening process is carried out in an anaerobic environment.

[0025] This invention discloses a Ganoderma lucidum product prepared by the above method, wherein the content of Ganoderma lucidum polysaccharide accounts for not less than 25% of the dry weight, and the proportion of β-glucan component with immunomodulatory activity exceeds 50%; at the same time, the selenium content is 50-200 mg / kg dry weight.

[0026] Furthermore, the aforementioned Ganoderma lucidum products exhibit hollow and porous mycelia with a specific surface area that is more than 100% larger than that of conventional liquid culture products, and are rich in characteristic terpenoid compounds induced by methyl jasmonate.

[0027] This invention discloses the application of the above-mentioned Ganoderma lucidum products in the preparation of functional foods or health products.

[0028] This invention discloses the application of the above-mentioned Ganoderma lucidum product in the preparation of pharmaceutical compositions for adjuvant anti-tumor treatment or to reduce the side effects of chemotherapy.

[0029] Compared with existing technologies, the present invention has the following advantages and beneficial effects:

[0030] This invention discloses a method for rapid liquid culture of Ganoderma lucidum and its application. By introducing a microencapsulated compound microbial agent, the sustained release and synergistic effects of multiple beneficial components are achieved, providing a continuously optimized microenvironment for mycelial growth. The use of a two-stage "dark-blue light" culture combined with the chemical inducer methyl jasmonic acid allows for precise regulation of the primary and secondary metabolic fluxes of Ganoderma lucidum, effectively promoting the biosynthesis of target active substances such as polysaccharides and terpenes. The application of modified raw materials (such as enzymatically hydrolyzed oat flour and small molecule peptides) improves the bioavailability of nutrients and accelerates mycelial proliferation. The subsequent low-temperature ripening treatment contributes to flavor compound formation and enzyme system stability, while microwave vacuum drying maximizes the preservation of heat-sensitive components. Ultimately, this invention significantly improves overall culture efficiency and product quality, resulting in products with high active ingredient content, excellent structural characteristics, and good stability, laying a solid foundation for its high-value-added development. Attached Figure Description

[0031] Figure 1 Results of key active ingredient content determination: Total polysaccharide content (% dry weight);

[0032] Figure 2 IL-6 secretion inhibition rate;

[0033] Figure 3 DPPH free radical scavenging rate (%). Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. All raw materials used in the embodiments of this invention are commercially available.

[0035] To meet the requirement of full disclosure, the raw materials and their key parameters involved in this invention are hereby specified as follows:

[0036] 1. Ganoderma lucidum spawn: The raw material was purchased commercially (Bozhou Huozhengtang Pharmaceutical Co., Ltd.: whole Ganoderma lucidum with skin and white flesh, native Tai Sui), cut into small pieces of 0.5cm×0.5cm×0.5cm, surface sterilized with mercuric chloride and rinsed clean with sterile water, and used as spawn.

[0037] 2. Enzymatic hydrolysis of oat flour

[0038] Ingredients: Regular food-grade oat flour.

[0039] Modification treatment: Hydrolysis was performed using neutral protease (enzyme activity unit: 50,000 U / g, purchased from Novozymes).

[0040] Key parameters: enzyme addition of 4000-6000 U / g oat flour; hydrolysis temperature of 50-55℃; hydrolysis time of 2-3 hours; obtained by spray drying after hydrolysis (inlet air temperature of 180-190℃).

[0041] 3. Sodium alginate-chitosan microcapsule bacterial agent

[0042] Core material composition:

[0043] Ganoderma lucidum mycelium extract: polysaccharide content ≥30% (determined by UV-Vis method).

[0044] Cordyceps militaris mycelium extract: polysaccharide content ≥30% (determined by UV-Vis method).

[0045] Metabolites of Lactobacillus plantarum: obtained by fermenting Lactobacillus plantarum (CICC 20242) in MRS medium for 48 hours, and then concentrating the supernatant to 1 / 10 of the original volume.

[0046] Wall material composition:

[0047] Sodium alginate: purity ≥90%, viscosity (1% aqueous solution, 20℃) 200-500 mPa·s.

[0048] Chitosan: Deacetylation degree ≥85%, viscosity (1% acetic acid solution, 20℃) 50-200 mPa·s.

[0049] Preparation parameters: sodium alginate solution concentration 1.5% (w / v); calcium chloride crosslinking solution concentration 2% (w / v); chitosan coating solution concentration 0.5% (w / v, dissolved in 1% acetic acid).

[0050] 4. Modified soybean peptides

[0051] Ingredients: Food-grade soy protein isolate.

[0052] Modification treatment: flavor protease (enzyme activity unit: 20,000 U / g) and trypsin (enzyme activity unit: 2,500 U / g) were used for dual-enzyme hydrolysis.

[0053] Key parameters: After hydrolysis, the product is separated by nanofiltration membrane (molecular weight cutoff 500 Da), the permeate is collected, concentrated and dried to obtain oligopeptide components with a molecular weight of less than 500 Da.

[0054] 5. Other raw materials

[0055] Deep-sea fish peptone: Total nitrogen content ≥12%, complete range of amino acids.

[0056] Black goji berry extract: Proanthocyanidin content ≥25% (determined by UV-Vis method).

[0057] Sodium selenite: analytical grade, Se content ≥45.0%.

[0058] Methyl jasmonic acid inducer: chromatographic grade, purity ≥95%.

[0059] Glutamine: Pharmaceutical grade, purity ≥99%.

[0060] The reagents used (such as potassium dihydrogen phosphate, magnesium sulfate, etc.) were all analytical grade or biological reagent grade.

[0061] Example 1

[0062] A method for rapid liquid culture of Ganoderma lucidum includes the following steps:

[0063] (1) Components of pre-prepared modified culture medium:

[0064] a. Preparation of enzymatically hydrolyzed oat flour: Oat flour was prepared into a 15% suspension, neutral protease (addition amount 4000U / g oat flour) was added and hydrolyzed at 50℃ and pH 6.5 for 3 hours, then the enzyme was inactivated at 95℃ for 10 minutes, and spray dried (inlet air temperature 180℃) to obtain enzymatically hydrolyzed oat flour.

[0065] b. Preparation of sodium alginate-chitosan microcapsule bacterial agent: Ganoderma lucidum mycelium extract (polysaccharide content 35%), Cordyceps militaris mycelium extract (polysaccharide content 32%), and Lactobacillus plantarum metabolites were mixed in a weight ratio of 3:2:1 as the core material. Using a 1.5% sodium alginate solution as the wall material, gel beads were formed by dripping the beads into a 2% calcium chloride solution using the sharp-hole method. These beads were then coated with a 0.5% chitosan solution and cross-linked for 10 minutes to prepare microcapsule bacterial agents with a diameter of approximately 2 mm.

[0066] (2) Preparation of liquid culture medium: In a 5L fermenter, add 1500g of pure water, and then add in sequence: 150g (10 parts) of enzymatically hydrolyzed oat flour obtained in step (1)a, 75g (5 parts) of deep-sea fish peptone, 45g (3 parts) of modified soybean peptide (molecular weight <500Da), 30g (2 parts) of microcapsule bacterial agent obtained in step (1)b, 15g (1 part) of black goji berry extract, and 0.15g (0.01 parts) of sodium selenite. Stir and mix evenly.

[0067] (3) Directed fermentation induction: The culture medium from step (2) was sterilized at 121°C for 15 minutes, cooled to 25°C, and then inoculated with 4 wt% of Ganoderma lucidum inoculum. First, it was cultured in the dark with shaking at 150 rpm for 3 days; then it was transferred to blue light (wavelength 450 nm, light intensity 20 μmol·m⁻¹). -2 ·s -1 Under irradiation conditions with a 12h / 12h light / dark cycle, the culture was continued for 5 days with shaking at 120 rpm. At the beginning of the light phase and every 24 hours thereafter, methyl jasmonate inducer (dissolved in a small amount of anhydrous ethanol) was added in a pulsed manner to achieve an instantaneous final concentration of 10 μmol / L in the culture medium.

[0068] (4) Harvesting and post-ripening: On the 8th day of cultivation, when the mycelial ball density reaches 18 g / L, the culture is harvested and filtered through a 100-mesh sieve to collect the Ganoderma lucidum mycelium. The wet mycelium is immersed in 0.05 M pH 6.8 phosphate buffer containing 0.1% glutamine at 4℃ and allowed to ripen under anaerobic conditions for 12 hours.

[0069] (5) Directional drying: The post-ripened mycelium is dried by microwave vacuum drying at a microwave power of 300W and a vacuum degree of -0.08MPa until the moisture content is 7.5%, thus obtaining a highly active Ganoderma lucidum product.

[0070] Example 2

[0071] A method for rapid liquid culture of Ganoderma lucidum includes the following steps:

[0072] (1) Components of pre-prepared modified culture medium:

[0073] a. Preparation of enzymatically hydrolyzed oat flour: Oat flour was prepared into an 18% suspension, neutral protease (addition amount 5000U / g oat flour) was added and hydrolyzed at 53℃ and pH 7.0 for 2.5 hours, then the enzyme was inactivated at 95℃ for 10 minutes, and spray dried (inlet air temperature 185℃) to obtain enzymatically hydrolyzed oat flour.

[0074] b. Preparation of sodium alginate-chitosan microcapsule bacterial agent: Ganoderma lucidum mycelium extract (polysaccharide content 38%), Cordyceps militaris mycelium extract (polysaccharide content 35%), and Lactobacillus plantarum metabolites were mixed in a weight ratio of 4:3:1 as the core material. Using a 1.5% sodium alginate solution as the wall material, gel beads were formed by dripping the beads into a 2% calcium chloride solution using the sharp-hole method. These beads were then coated with a 0.5% chitosan solution and cross-linked for 10 minutes to prepare microcapsule bacterial agents with a diameter of approximately 2 mm.

[0075] (2) Preparation of liquid culture medium: In a 5L fermenter, add 2000g of pure water, and then add in sequence: 300g (15 parts) of enzymatically hydrolyzed oat flour obtained in step (1)a, 180g (9 parts) of deep-sea fish peptone, 120g (6 parts) of modified soybean peptide (molecular weight <500Da), 80g (4 parts) of microcapsule bacterial agent obtained in step (1)b, 40g (2 parts) of black goji berry extract, and 0.06g (0.03 parts) of sodium selenite. Stir and mix evenly.

[0076] (3) Directional fermentation induction: The culture medium from step (2) was sterilized at 118°C for 18 minutes, cooled to 27°C, and then inoculated with 5 wt% of Ganoderma lucidum inoculum. First, it was cultured in the dark with shaking at 120 rpm for 3.5 days; then it was transferred to blue light (wavelength 460 nm, light intensity 30 μmol·m⁻¹). -2 ·s -1 Under irradiation conditions with a 12h / 12h light / dark cycle, the culture was continued for 4 days with shaking at 100 rpm. At the beginning of the light phase and every 24 hours thereafter, methyl jasmonate inducer (using β-cyclodextrin-encapsulated sustained-release microcapsules) was added in a pulsed manner to achieve an effective final concentration of 30 μmol / L in the culture medium.

[0077] (4) Harvesting and post-ripening: On day 7.5, when the mycelial ball density reaches 22 g / L, the culture is harvested and filtered through a 100-mesh sieve to collect the Ganoderma lucidum mycelium. The wet mycelium is immersed in 0.05 M pH 6.8 phosphate buffer containing 0.1% glutamine at 4℃ and allowed to ripen under anaerobic conditions for 18 hours.

[0078] (5) Directional drying: The post-ripened mycelium is dried by microwave vacuum drying at a microwave power of 400W and a vacuum degree of -0.085MPa until the moisture content is 7.0%, thus obtaining a highly active Ganoderma lucidum product.

[0079] Example 3

[0080] A method for rapid liquid culture of Ganoderma lucidum includes the following steps:

[0081] (1) Components of pre-prepared modified culture medium:

[0082] a. Preparation of enzymatically hydrolyzed oat flour: Oat flour was prepared into a 20% suspension, neutral protease (6000 U / g oat flour) was added and hydrolyzed at 55℃ and pH 7.5 for 2 hours, then the enzyme was inactivated at 95℃ for 10 minutes, and spray dried (air inlet temperature 190℃) to obtain enzymatically hydrolyzed oat flour.

[0083] b. Preparation of sodium alginate-chitosan microcapsule bacterial agent: Ganoderma lucidum mycelium extract (polysaccharide content 40%), Cordyceps militaris mycelium extract (polysaccharide content 38%), and Lactobacillus plantarum metabolites were mixed in a weight ratio of 5:4:1 as the core material. Using a 1.5% sodium alginate solution as the wall material, gel beads were formed by dripping the beads into a 2% calcium chloride solution using the sharp-hole method. These beads were then coated with a 0.5% chitosan solution and cross-linked for 10 minutes to prepare microcapsule bacterial agents with a diameter of approximately 2 mm.

[0084] (2) Preparation of liquid culture medium: In a 5L fermenter, add 2500g of pure water, and then add in sequence: 500g (20 parts) of enzymatically hydrolyzed oat flour obtained in step (1)a, 240g (10 parts) of deep-sea fish peptone, 160g (6.5 parts) of modified soybean peptide (molecular weight <500Da), 100g (4 parts) of microcapsule bacterial agent obtained in step (1)b, 60g (2.4 parts) of black goji berry extract, and 0.1g (0.04 parts) of sodium selenite. Stir and mix evenly.

[0085] (3) Directional fermentation induction: The culture medium from step (2) was sterilized at 115℃ for 25 minutes, cooled to 28℃, and then inoculated with 4.5wt% of Ganoderma lucidum inoculum. First, it was cultured in the dark with shaking at 100 rpm for 4 days; then it was transferred to blue light (wavelength 470 nm, light intensity 40 μmol·m⁻¹). -2 ·s -1 Under irradiation conditions with a 12h / 12h light / dark cycle, the culture was continued for 3 days with shaking at 80 rpm. At the beginning of the light phase and every 24 hours thereafter, methyl jasmonate inducer (using β-cyclodextrin-encapsulated sustained-release microcapsules) was added in a pulsed manner to achieve an effective final concentration of 50 μmol / L in the culture medium.

[0086] (4) Harvesting and post-ripening: On the 7th day of cultivation, when the mycelial ball density reaches 25 g / L, the culture is harvested and filtered through a 100-mesh sieve to collect the Ganoderma lucidum mycelium. The wet mycelium is immersed in 0.05 M pH 6.8 phosphate buffer containing 0.1% glutamine at 4℃ and allowed to ripen under anaerobic conditions for 24 hours.

[0087] (5) Directional drying: The post-ripened mycelium is dried by microwave vacuum drying at a microwave power of 500W and a vacuum degree of -0.09MPa until the moisture content is 6.5%, thus obtaining a highly active Ganoderma lucidum product.

[0088] Comparative Example 1

[0089] A method for cultivating Ganoderma lucidum, compared with Example 2, differs in that the culture medium formula in step (2) is changed to: 200g glucose, 180g ordinary soybean peptone, 60g yeast extract, 3g potassium dihydrogen phosphate, 0.4g magnesium sulfate, and 2000g water. (That is, using the traditional basic culture medium, without the innovative components such as enzymatically hydrolyzed oat flour, deep-sea fish peptone, modified soybean peptides, microencapsulated bacterial agents, black goji berry extract, and sodium selenite). The remaining steps are the same as in Example 2.

[0090] By day 9 of cultivation, the mycelial ball density was only 8 g / L.

[0091] Comparative Example 2

[0092] A method for cultivating Ganoderma lucidum, compared with Example 2, differs in that the preparation and addition of microcapsule inoculant in step (1)b are omitted, and microcapsule inoculant is not added in step (2). The remaining steps are the same as in Example 2.

[0093] By day 8 of cultivation, the mycelial ball density was 15 g / L.

[0094] Comparative Example 3

[0095] A method for culturing Ganoderma lucidum, compared with Example 2, differs in that step (3) does not involve two-stage culture and induction; the entire process is carried out at 28°C and in the dark with shaking at 120 rpm for 7.5 days, without the addition of methyl jasmonic acid. The remaining steps are the same as in Example 2.

[0096] By day 7.5 of cultivation, the mycelial ball density was 19 g / L.

[0097] Comparative Example 4

[0098] A method for cultivating Ganoderma lucidum, compared with Example 2, differs in that the post-ripening treatment is omitted in step (4), and the product is directly dried in step (5) after filtration. The remaining steps are the same as in Example 2.

[0099] Comparative Example 5

[0100] A method for cultivating Ganoderma lucidum, compared with Example 2, differs in that microwave vacuum drying is not used in step (5), but instead, drying is carried out in a 65°C hot air oven until the moisture content is similar. The remaining steps are the same as in Example 2.

[0101] Test Example 1

[0102] Test Example 1: Determination of Key Active Ingredient Content

[0103] Objective: To verify the effect of the method of the present invention on increasing the content of core bioactive substances in Ganoderma lucidum products.

[0104] method:

[0105] Polysaccharide content determination: The phenol-sulfuric acid method was used. 50 mg of each of the dried Ganoderma lucidum powder obtained in Examples 1-3 and Comparative Examples 1-5 were accurately weighed, extracted with hot water, and then reacted with 6% phenol solution and concentrated sulfuric acid. The absorbance was measured at a wavelength of 490 nm, and the total polysaccharide content was calculated using a glucose standard curve.

[0106] β-glucan content determination: The Megazyme β-glucan assay kit (enzymatic method) was used. 20 mg of sample was accurately weighed, hydrolyzed with a specific enzyme series (lichenase and β-glucosidase), and the released glucose content was measured. The percentage of β-glucan in the total polysaccharides was calculated.

[0107] Selenium content determination: Inductively coupled plasma mass spectrometry (ICP-MS) was used. After microwave digestion, the sample was directly injected to determine the selenium content.

[0108] Results: See Table 1 below and Figure 1 As shown.

[0109] Table 1: Results of determination of key active ingredients

[0110]

[0111] Conclusion: The key active ingredients (total polysaccharides and β-glucan) in the products of this invention (Examples 1-3) were significantly higher than those in all comparative examples. In particular, compared to Comparative Example 1, which used a conventional basal culture medium, Example 2 showed an approximately 88% increase in polysaccharide content and an approximately 36% increase in β-glucan content, and successfully achieved selenium bioaccumulation. This indicates that the innovative culture medium formulation and cultivation process of this invention can effectively promote the synthesis and accumulation of the target active substances.

[0112] Test Example 2

[0113] In vitro assessment of immunomodulatory activity (macrophage model)

[0114] Objective: To evaluate the regulatory ability of the Ganoderma lucidum product obtained in this invention on the function of immune cells.

[0115] method:

[0116] The mouse macrophage cell line RAW264.7 was used as a model. Cells were cultured in 96-well plates, and three groups were set up: a blank control group, an LPS (lipopolysaccharide, 1 μg / mL) stimulation model group, and an LPS+ sample group (containing 100 μg / mL of each sample's aqueous extract). After 24 hours of treatment:

[0117] Cytokine assay: Cell supernatant was collected and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected using an ELISA kit.

[0118] Nitric oxide (NO) determination: The concentration of nitrite in the supernatant was detected by the Griess method, which indirectly reflects the NO production.

[0119] Results: As shown in Table 2 below and Figure 2 As shown in the figure, compared with the blank control group, LPS stimulation significantly induced macrophages to produce inflammatory factors IL-6, TNF-α, and NO. However, compared with the LPS model group, the secretion of inflammatory factors was significantly inhibited in cells treated with the sample from Example 2.

[0120] Table 2. Effects of LPS on the secretion of inflammatory factors in RAW264.7 cells (inhibition rate %) relative to the LPS model group

[0121]

[0122] Conclusion: The product of Example 2 of this invention exhibits significant anti-inflammatory activity and can effectively inhibit LPS-induced macrophage overactivation. This effect is superior to Comparative Example 2 (without microencapsulated bacterial agent) and Comparative Example 3 (without two-stage induction), suggesting that a complete culture process is crucial for obtaining products with high immunomodulatory activity.

[0123] Test Example 3

[0124] In vitro antioxidant capacity assay

[0125] Objective: To compare the antioxidant capacity of extracts from different samples.

[0126] method:

[0127] DPPH free radical scavenging rate: Prepare a 1mM DPPH ethanol solution, mix it with an equal volume of sample solution (1mg / mL), react in the dark for 30 minutes, and then measure the absorbance at 517nm to calculate the scavenging rate.

[0128] ABTS radical cation scavenging rate: ABTS is reacted with potassium persulfate to generate...

[0129] ABTS+ • Stock solution, diluted with PBS to the working solution. Take sample solution (0.5 mg / mL) and ABTS + • React the working solution for 6 minutes, measure the absorbance at 734 nm, and calculate the clearance rate.

[0130] Ferric reducing power (FRAP): The sample solution was mixed with FRAP working solution (containing TPTZ, FeCl3, and acetate buffer), and after reacting for 10 minutes, the absorbance was measured at 593 nm. The FRAP value (μM Fe) was calculated using the FeSO4 standard curve. 2+ (equivalent).

[0131] Results: See Table 3 below and Figure 3 As shown.

[0132] Table 3: Results of in vitro antioxidant capacity assay

[0133]

[0134] Conclusion: The product of this invention exhibits strong in vitro antioxidant capacity, with all three indicators significantly higher than the comparative example. This indicates that the Ganoderma lucidum cultured using the method of this invention contains a richer amount of antioxidant substances such as polysaccharides and polyphenols.

[0135] Test Example 4

[0136] Product stability assessment

[0137] Objective: To evaluate the stability of the product of the present invention under accelerated storage conditions.

[0138] method:

[0139] The finished powders from Example 2 and Comparative Example 5 (hot air drying) were stored for 3 months under accelerated testing conditions (temperature 40℃±2℃, relative humidity 75%±5%). Samples were taken and tested in 0 months, 1 month, 2 months, and 3 months.

[0140] Polysaccharide content retention rate: same as in test case 1.

[0141] Color change: Use a colorimeter to measure the L (brightness), a (red-green value), and b* (yellow-blue value) values ​​of the powder, and calculate the total color difference ΔE.

[0142] Results: As shown in Table 4 below.

[0143] Table 4: Changes in indicators after 3 months of accelerated testing (40℃ / 75% RH)

[0144]

[0145] Conclusion: After 3 months of accelerated testing, the polysaccharide retention rate of the product in Example 2 (microwave vacuum drying) was significantly higher than that of Comparative Example 5 (hot air drying), and the color change (ΔE) was smaller. This indicates that the microwave vacuum drying process used in this invention can effectively reduce the degradation of heat-sensitive active ingredients and browning caused by Maillard reaction through rapid dehydration and low-temperature treatment, better maintaining the product's biological activity and physical properties, and thus contributing to extended shelf life. (Example 5)

[0146] Determination of functional indicators of mycelium (water absorption and dissolution)

[0147] Objective: To indirectly characterize the effect of the process of this invention on mycelial structure by measuring functional physicochemical indicators, and to evaluate its advantages in subsequent processing.

[0148] method:

[0149] Hydration capacity determination: Accurately weigh 1.0 g each of the dried mycelial powder from Example 2 and Comparative Example 3 (denoted as W1), place them in centrifuge tubes, add 20 mL of distilled water, vortex to mix, and let stand at room temperature for 30 minutes. Then centrifuge at 4000 rpm for 20 minutes, carefully discard the supernatant, and weigh the wet weight of the precipitate (denoted as W2). Hydration capacity (Water Hydration)

[0150] The formula for calculating WHC (capacity, g / g) is: WHC(g / g) = (W2 - W1) / W1. Each experiment was repeated three times.

[0151] Polysaccharide dissolution rate determination: Accurately weigh 100 mg each of dried mycelial powder from Example 2 and Comparative Example 3, place them in a stoppered conical flask, and add 100 mL of boiling water. Take 1 mL samples at the 5th, 10th, 20th, and 30th minutes of reflux extraction, dilute appropriately, and immediately determine the polysaccharide concentration in the solution using the phenol-sulfuric acid method (same as in Test Example 1), and calculate the cumulative dissolution amount. Plot a dissolution curve with time on the x-axis and the cumulative dissoluted polysaccharide amount on the y-axis.

[0152] result:

[0153] Hydration capacity: The hydration capacity of the sample in Example 2 was 8.5 g / g, while the hydration capacity of the sample in Comparative Example 3 was 5.2 g / g.

[0154] Polysaccharide dissolution rate: As shown in Table 5 below, the polysaccharide in the sample of Example 2 dissolves in hot water at a significantly faster rate than that in the sample of Comparative Example 3. After 10 minutes of extraction, the amount of polysaccharide dissolved has reached more than 95% of the amount dissolved in the sample of Comparative Example 3 after 30 minutes.

[0155] Table 5: Cumulative dissolution of polysaccharides during hot water reflux extraction (mg / g dry powder)

[0156] Extraction time (minutes) Example 2 Comparative Example 3 5 85 45 10 165 88 20 228 142 30 245 173

[0157] Conclusion: The product of Example 2 of this invention exhibits significantly higher hydration capacity and a faster dissolution rate of the active ingredient (polysaccharide). This indirectly suggests that the mycelium treated by the directional induction process of this invention may have a more porous and loose structure, thereby enabling it to absorb water and release its contents more quickly. This characteristic is of great significance for subsequent extraction of active ingredients or development of dosage forms such as granules, as it can improve processing efficiency or enhance the user experience of the final product.

[0158] Test Case Summary:

[0159] Test Example 1 shows that the polysaccharide content of the product of Example 2 of this invention reached 28.8%, the β-glucan content was 55.3%, and the selenium content was 125 mg / kg, all significantly higher than those of the conventional culture medium control example 1 (15.3%, 40.5%, not detected). Cellular experiments of Test Example 2 showed that the sample of Example 2 inhibited LPS-induced macrophage inflammatory factors IL-6 and TNF-α by 52.1% and 48.7%, respectively, which was superior to control examples 2 (38.4%, 35.1%) and 3 (32.7%, 30.5%), demonstrating its excellent immunomodulatory potential. In Test Example 3, the DPPH and ABTS free radical scavenging rates of Example 2 were as high as 85.6% and 89.2%, respectively, and the FRAP value was 650 μM Fe. 2+ The equivalent compound exhibits outstanding antioxidant capacity. Stability testing in Example 4 showed that, after 3 months of accelerated testing, the polysaccharide retention rate of Example 2 (94.1%) was higher than that of the hot-air-dried comparative example 5 (88.3%), and the color difference change was smaller (ΔE 2.5 vs 5.8). Functional assays in Example 5 further revealed that the hydration capacity (8.5 g / g) and polysaccharide dissolution rate of Example 2 were significantly higher than those of Comparative Example 3 (5.2 g / g), indicating that its structure is more conducive to processing and utilization. Overall, the data demonstrate that this invention is highly effective in improving the content of active ingredients, functional properties, and stability of the product.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or equivalent structural or procedural transformations made using the content of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields, are all included within the scope of protection of the present invention patent.

Claims

1. A method for rapid liquid culture of Ganoderma lucidum, characterized in that, Includes the following steps: (1) Components of pre-prepared modified culture medium: a. Preparation of enzymatically hydrolyzed oat flour: Oat flour is prepared into a 15-20% suspension, and neutral protease is added at a concentration of 4000-6000 U / g oat flour; hydrolyze at 50-55℃ and pH 6.5-7.5 for 2-3 hours, then the enzyme is inactivated and spray-dried to obtain enzymatically hydrolyzed oat flour; b. Preparation of sodium alginate-chitosan microcapsule bacterial agent: Ganoderma lucidum mycelium extract, Cordyceps militaris mycelium extract and Lactobacillus plantarum metabolite were mixed in a weight ratio of (3-5):(2-4):1 as the core material, and 1.5% sodium alginate solution was used as the wall material. The mixture was dropped into 2% calcium chloride solution using the sharp-hole method to form gel beads. Then, a 0.5% chitosan solution was used to coat and cross-link the beads for 10 minutes to prepare microcapsule bacterial agent. (2) Preparation of liquid culture medium: Mix the following raw materials by weight: 10-20 parts of enzymatically hydrolyzed oat flour obtained in step (1)a, 5-12 parts of deep-sea fish peptone, 3-8 parts of modified soybean peptide with molecular weight <1000Da, 2-5 parts of microcapsule bacterial agent obtained in step (1)b, 1-3 parts of black goji berry extract, 0.01-0.05 parts of sodium selenite, and 150-250 parts of water; (3) Directional fermentation induction: The culture medium from step (2) was sterilized at 115-121℃ for 15-20 minutes, cooled to 25-28℃, and then inoculated with 3wt%-5wt% of Ganoderma lucidum strain; firstly, it was cultured in the dark with shaking at 100-150 rpm for 3-4 days; then it was transferred to blue light irradiation and cultured with shaking at 80-120 rpm for 3-5 days, and methyl jasmonate inducer was added pulsedly every 24 hours during the light stage, so that its final concentration in the culture medium was 10-50 μmol / L; (4) Harvesting and post-ripening: When the mycelial ball density reaches 15-25 g / L, harvest the culture, filter it through a 100-mesh sieve, and collect the Ganoderma lucidum mycelium; let the wet mycelium stand in a phosphate buffer containing 0.1% glutamine at 4℃ for 12-24 hours for post-ripening. (5) Directional drying: The post-ripened mycelium is dried by microwave vacuum drying with microwave power of 300-500W and vacuum degree of -0.08 to -0.09MPa until the moisture content is ≤8% to obtain the product.

2. The method according to claim 1, characterized in that, In step (1)b, the Ganoderma lucidum mycelium extract and Cordyceps militaris mycelium extract are both obtained by ultrasonic-assisted water extraction, and the polysaccharide content is not less than 30%.

3. The method according to claim 1, characterized in that, In step (2), the modified soybean peptide is an oligopeptide component with a molecular weight of less than 500 Da obtained by hydrolysis with flavor protease and trypsin and then separation by nanofiltration membrane.

4. The method according to claim 1, characterized in that, In step (3), the methyl jasmonate inducer is added in the form of microcapsules with β-cyclodextrin as the wall material to achieve a pulsed sustained-release effect.

5. The method according to claim 1, characterized in that, In step (3), the wavelength of blue light irradiation is 450-470nm, the light intensity is 20-40μmol·m-2·s-1, and the light-dark cycle is 12 hours of light / 12 hours of darkness.

6. The method according to claim 1, characterized in that, In step (4), the concentration of the phosphate buffer is 0.05M and the pH is 6.8, and the post-ripening process is carried out in an anaerobic environment.

7. A meat-shaped Ganoderma lucidum product prepared by the method according to any one of claims 1-6, characterized in that, The content of its Ganoderma lucidum polysaccharide accounts for no less than 25% of its dry weight, and the proportion of β-glucan component with immunomodulatory activity exceeds 50%; at the same time, its selenium content is 50-200 mg / kg dry weight.

8. The Ganoderma lucidum product according to claim 7, characterized in that, Its mycelium is hollow and porous, with a specific surface area that is more than 100% larger than that of conventional liquid culture products, and is rich in characteristic terpenoid compounds induced by methyl jasmonate.

9. The application of the Ganoderma lucidum product as described in claim 7 in the preparation of functional foods or health products.

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