A method for cultivating ganoderma spore powder with high triterpenoid polysaccharide content

Abstract

The present application relates to the technical field of artificial cultivation of medicinal fungi, and discloses a cultivation method of ganoderma spore powder with high triterpene polysaccharide content, which comprises the following steps: S1. inoculating ganoderma spores into a seed liquid culture medium to obtain a seed liquid through culture; S2. inoculating the seed liquid into a fermentation culture medium to obtain a fermentation liquid through culture; S3. carrying out oscillation-rest cycle culture on the fermentation liquid, and separating the mycelium from the fermentation liquid; S4. inoculating the mycelium into a cultivation seed culture medium to obtain a ganoderma cultivation seed through culture; S5. transplanting the ganoderma cultivation seed onto a substitute material culture medium to obtain ganoderma through culture; and S6. covering the ganoderma with a net cover, and collecting ganoderma spore powder. The present application can improve the content of triterpenes and polysaccharides in the ganoderma spore powder.

Description

Technical Field

[0001] This invention relates to the field of artificial cultivation technology of medicinal fungi, and in particular to a method for cultivating Ganoderma lucidum spore powder with high triterpenoid polysaccharide content. Background Technology

[0002] Reishi mushroom, a traditional Chinese medicine with rich medicinal value, has long been widely used in various health and wellness fields, including enhancing immunity, anti-tumor activity, and anti-aging. Among these, reishi spore powder is considered the essence of reishi due to its rich content of various bioactive components, especially triterpenoids and polysaccharides. Triterpenoids are believed to have significant anti-cancer, anti-inflammatory, and antioxidant effects, while polysaccharides are primarily considered to possess immunomodulatory, antiviral, and anti-tumor effects. Therefore, increasing the content of triterpenoids and polysaccharides in reishi spore powder is of great significance for improving its medicinal efficacy. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for cultivating Ganoderma lucidum spore powder with high triterpenoid polysaccharide content, which can increase the content of triterpenoids and polysaccharides in Ganoderma lucidum spore powder.

[0004] The present invention solves the above-mentioned technical problems through the following technical means:

[0005] This invention provides a method for cultivating Ganoderma lucidum spore powder with high triterpenoid polysaccharide content, comprising the following steps:

[0006] S1. Inoculate Ganoderma lucidum strains into seed culture medium and culture to obtain seed culture;

[0007] S2. Inoculate the seed culture into the fermentation medium and culture to obtain the fermentation broth;

[0008] S3. The fermentation broth is subjected to a shaking-static cycle culture, and the mycelium is obtained by separating the bacterial solution.

[0009] S4. Inoculate the mycelium into the culture medium and culture it to obtain the Ganoderma lucidum culture medium;

[0010] S5. Transplant Ganoderma lucidum cultivars onto a substrate culture medium to cultivate Ganoderma lucidum;

[0011] S6. Cover the Ganoderma lucidum with a net and collect the Ganoderma lucidum spore powder.

[0012] Preferably, the seed culture medium in step S1 comprises the following components: glucose 18-22 g / L, yeast powder 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate heptahydrate 1-2 g / L, and sodium taurocholate 2-3 g / L.

[0013] More preferably, the culture conditions in step S1 are a temperature of 25-27℃ and a shaking incubator at 140-160 r / min.

[0014] The seed culture medium, scientifically formulated with glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and sodium taurocholate, provides comprehensive nutritional support for Ganoderma lucidum mycelium. These components act as carbon, nitrogen, phosphorus, and magnesium sources, as well as bioactive additives, supporting not only the rapid growth and biomass accumulation of Ganoderma lucidum mycelium but also promoting its metabolic activity, particularly the synthesis of secondary metabolites. This significantly enhances the growth rate of Ganoderma lucidum mycelium, strengthens the secretion of extracellular secondary metabolites (such as triterpenes and polysaccharides), and improves the mycelium's tolerance and activity to various stress conditions. High-quality seed culture preparation ensures efficient and stable fermentation, while also providing excellent mycelial resources for subsequent cultivation and substrate culture, thus contributing to achieving the goal of high triterpenoid and polysaccharide content in the final product and greatly optimizing the entire production process.

[0015] Preferably, the fermentation medium in step S2 comprises the following components: fructooligosaccharides 10-25 g / L, modified chitosan 5-20 g / L, peptone 5-10 g / L, yeast extract 0.5-8 g / L, potassium dihydrogen phosphate 0.1-1.0 g / L, magnesium sulfate heptahydrate 1-2 g / L, vitamin B1 0.05-0.1 g / L, and heme peptide 0.01-0.05 g / L.

[0016] More preferably, the culture conditions in step S2 are constant temperature and light protection culture at a temperature of 19-23℃ and a humidity of 55-65%.

[0017] The fermentation medium, through precise proportioning of functional carbon sources (such as fructooligosaccharides and modified chitosan), nitrogen sources (peptone and yeast extract), inorganic salts (potassium dihydrogen phosphate and magnesium sulfate heptahydrate), vitamins (vitamin B1), and bioactive factors (heme peptides), provides an optimized growth environment for Ganoderma lucidum mycelium. These components work synergistically, supporting not only basic physiological activities such as energy metabolism, protein synthesis, and cell division in the mycelium, but also enhancing the synthesis efficiency of extracellular secondary metabolites (such as triterpenes and polysaccharides) through specific metabolic regulation mechanisms. The special components in the medium, modified chitosan and heme peptides, effectively enhance the mycelium's tolerance to potentially toxic elements and improve its antioxidant capacity, ensuring the stability and high efficiency of the fermentation process.

[0018] Preferably, the preparation steps of the modified chitosan include:

[0019] A1. Dissolve chitosan in an acetic acid solution with a mass concentration of 0.5%-1.5%, add cyclodextrin, then add ascorbic acid, stir and react for 5-7 hours at pH 9-10 and 55-65℃, wash the product, and dry to obtain preliminarily modified chitosan.

[0020] A2. Preliminary modified chitosan was dissolved in distilled water to prepare a solution with a mass concentration of 0.5%-1.5%. Under stirring, AgNO3 solution with a mass concentration of 20%-30% was slowly added to adjust the pH of the solution to 6.5-7.0. The temperature was controlled at 22-28℃ and the reaction was stirred for 1-3 hours. The mixture was then washed and freeze-dried to form a complex.

[0021] A3. The complex was dissolved in PBS to form a solution with a mass concentration of 0.5%-1.5%. Lactoferrin solution was slowly added under stirring conditions, and the mixture was stirred and mixed at 3-5°C for 11-13 hours. After washing, the mixture was freeze-dried to form the modified chitosan.

[0022] Preferably, in step A1, the mass ratio of cyclodextrin to chitosan is 5:1, and the mass ratio of ascorbic acid to chitosan is 1:3.

[0023] The preparation of modified chitosan involves chemical modification and compounding techniques. This includes initial modification with cyclodextrin and ascorbic acid to improve its solubility and stability, and to generate a stable antioxidant structure. Subsequently, it is combined with silver ions to form a chitosan-silver complex, leveraging the antibacterial properties of silver ions to enhance its application in complex biological environments. Finally, lactoferrin is introduced to further strengthen the complex's antibacterial ability and microbial affinity through hydrogen bonding and electrostatic interactions. This series of steps not only improves the functionality of chitosan but also significantly enhances its antibacterial activity and its support for the growth of Ganoderma lucidum mycelia.

[0024] Preferably, the oscillation-static cycle culture in step S3 specifically involves oscillating for 2-3 days, static for 7-8 days, then oscillating for 0.5-1 day, and static for 2-4 hours.

[0025] The oscillation-static cycle culture is a fermentation method combining dynamic and static operations, designed to provide an optimal environment for Ganoderma lucidum mycelial growth. The oscillation phase increases oxygen solubility and the uniform distribution of nutrients, promoting rapid initial growth; while the static phase provides a stable, low-disturbance environment, conducive to the directional extension of mycelia and the formation of extracellular polymers such as polysaccharides and triterpenoids. The optimized cycle includes a long initial oscillation to activate growth, a longer static phase to promote metabolite accumulation, a short initial oscillation to improve oxygen supply, and a final short static phase to ensure mycelial separation, thus comprehensively supporting the healthy growth and efficient metabolism of Ganoderma lucidum mycelia.

[0026] Preferably, the culture medium for cultivation in step S4 comprises the following raw materials in parts by weight: 40-60 parts corn stalks, 20-30 parts sawdust, 5-15 parts rapeseed meal, 5-8 parts humic acid, 8-10 parts Armillaria mellea fermentation broth, 5-10 parts glucose, 1-3 parts fructooligosaccharides, 1-3 parts gypsum, 0.2-0.5 parts zinc sulfate, 0.5-1 part magnesium sulfate, and 0.01-0.02 parts sodium selenide, with a water content of 60-65%.

[0027] More preferably, the culture conditions in step S4 are constant temperature culture at 19-23℃ and humidity at 55-65%; light-protected culture for 9-11 days in the early stage, and light intensity of 1000-1500 lx in the later stage.

[0028] By using corn stalks and sawdust to provide carbon and cellulose, and rapeseed meal to supply nitrogen, functional additives such as humic acid and fructooligosaccharides are used to improve the substrate structure and promote microbial metabolic activity. Simultaneously, trace elements such as zinc sulfate, magnesium sulfate, and sodium selenide are added to support metabolic enzyme activation and enhance antioxidant capacity. Furthermore, an appropriate amount of glucose serves as a readily available carbon source to ensure initial energy supply, while the introduction of Armillaria mellea fermentation broth not only helps decompose complex organic matter but also enhances the substrate's microecological environment.

[0029] Preferably, the substitute culture medium in step S5 comprises the following raw materials in parts by weight: 30-40 parts of Astragalus membranaceus residue, 30-40 parts of Glycyrrhiza uralensis residue, 20-24 parts of wheat bran, 1-3 parts of gypsum, 6-8 parts of yeast powder, 0.3-0.6 parts of potassium dihydrogen phosphate, and the water content of the culture medium is 60-65%.

[0030] Preferably, the pretreatment method for the astragalus residue and licorice residue is as follows: select fresh residue, mix them evenly, pile them up for fermentation and dry them, crush them, and pass them through a 40-60 mesh sieve.

[0031] More preferably, the culture conditions in step S5 are: a temperature of 28-30℃ and a humidity of 60-75%; 9-11 days of light-protected culture in the early stage, and a light intensity of 1000-1500 lx in the later stage.

[0032] Fermented astragalus and licorice residues provide abundant polysaccharides, flavonoids, and trace active ingredients, supporting Ganoderma lucidum mycelial growth and promoting the accumulation of metabolites. Gypsum is used to adjust pH and improve aeration, while providing calcium ions to support fruiting body structure formation. Auxiliary components such as yeast powder and potassium dihydrogen phosphate further enhance the metabolic process, promoting the synthesis of triterpenes and polysaccharides, and improving stress resistance and metabolic efficiency. Optimized pretreatment methods, including composting fermentation and crushing and sieving, ensured the stability and suitable physicochemical properties of the culture medium, providing an ideal growth environment for Ganoderma lucidum. This significantly enhanced the growth rate and quality of Ganoderma lucidum mycelia, shortened the cultivation cycle, and greatly increased the accumulation of effective components such as polysaccharides and triterpenes. The physicochemical properties of the culture medium were optimized, becoming more loose and breathable, which is conducive to healthy mycelial growth. Recycling the residues not only reduces production costs and achieves efficient resource utilization but also aligns with the environmental protection concept of green production, reducing environmental pollution.

[0033] Preferably, step S5 further includes spraying a composite inducer, which includes sodium acetate, salicylic acid and methyl jasmonate, in a mixing ratio of 1:1:1.

[0034] More preferably, the concentrations of sodium acetate solution and salicylic acid solution are both 1-4 mM, and the concentration of methyl jasmonate solution is 0.2-0.3 mM.

[0035] The compound inducer is composed of sodium acetate, salicylic acid, and methyl jasmonic acid in a 1:1:1 ratio. Each component works synergistically to promote the growth and metabolism of Ganoderma lucidum through different physiological mechanisms. Sodium acetate regulates intracellular acid-base balance and enhances antioxidant activity, promoting mycelial growth and accumulation of active ingredients. Salicylic acid, as a plant hormone, activates cell signaling pathways, enhancing disease resistance and stress tolerance, and stimulating the production of bioactive substances. Methyl jasmonic acid, by regulating plant hormone levels, enhances stress resistance and secondary metabolism. The synergistic effect of these three components enhances the metabolic activity, stress resistance, and product accumulation capacity of Ganoderma lucidum. Spraying ensures that the inducing components are rapidly absorbed by the mycelium and exert their effects, optimizing the growth environment of Ganoderma lucidum.

[0036] The beneficial effects of this invention are:

[0037] (1) Based on the biological characteristics of Ganoderma lucidum, this invention optimizes key process steps such as seed culture, fermentation culture, and substrate cultivation to regulate the secondary metabolic pathways of Ganoderma lucidum mycelium and promote the efficient synthesis of extracellular polymers (triterpenes and polysaccharides). A shaking-static cyclic culture is employed to enhance oxygen supply and metabolic product secretion. Simultaneously, specific inducers and medicinal residues are introduced as culture medium components, providing abundant nutrients and trace elements for the growth and secondary metabolism of Ganoderma lucidum, ultimately increasing the content of active ingredients in Ganoderma lucidum spore powder. This significantly increases the content of triterpenes and polysaccharides in Ganoderma lucidum spore powder, enhancing the product's medicinal value and market competitiveness. Furthermore, the use of a resource recycling strategy to introduce waste materials such as medicinal residues reduces production costs.

[0038] (2) The modified chitosan significantly improved water solubility and bioactivity, ensuring its uniform dispersion in the culture medium and long-term maintenance of activity. The addition of silver ions greatly enhanced its broad-spectrum antibacterial properties, protecting Ganoderma lucidum mycelia from contamination, while the synergistic effect of lactoferrin promoted mycelial growth and the accumulation of metabolites (such as polysaccharides and triterpenes). Ultimately, this modified chitosan is not only stable but also adaptable to various biological culture medium environments, providing strong technical support for the large-scale production of Ganoderma lucidum and other fungi.

[0039] (3) The oscillation-static cyclic culture method of this invention significantly improves the growth efficiency of Ganoderma lucidum mycelia and the synthesis of extracellular polymers (such as polysaccharides and triterpenes), and reduces mechanical damage to mycelia by balancing the relationship between oxygen supply and mechanical stress. In addition, this method not only accelerates the initial growth rate, but also promotes the accumulation of metabolites during steady-state growth, thereby increasing the bioactivity and effective ingredient content of the fermentation broth. Finally, this method simplifies the operation process, improves production efficiency, and provides ideal production conditions for subsequent mycelial separation, mycelial collection, and substrate culture transfer. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] This embodiment is a method for cultivating Ganoderma lucidum spore powder with high triterpenoid polysaccharide content.

[0043] Includes the following steps:

[0044] S1. Inoculate Ganoderma lucidum strain into seed culture medium and culture in a shaker at 25℃ and 140r / min for 10 days to obtain seed culture;

[0045] The seed culture medium contains the following components: glucose 18 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 1 g / L, and sodium taurocholate 2 g / L.

[0046] S2. Inoculate the seed culture into the fermentation medium and culture it at a constant temperature of 19℃ and a humidity of 55% for 6 days in the dark to obtain the fermentation broth;

[0047] The fermentation medium contains the following components: fructooligosaccharides 10 g / L, modified chitosan 5 g / L, peptone 5 g / L, yeast extract 0.5 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate heptahydrate 1 g / L, vitamin B1 0.05 g / L, and heme peptide 0.01 g / L.

[0048] The preparation steps of modified chitosan include:

[0049] A1. Dissolve 120g of chitosan in 1000mL of 0.5% acetic acid solution, add 600g of cyclodextrin, and then add 40g of ascorbic acid. Stir and react for 7 hours at pH 9 and 55℃. Wash the product and dry it to obtain 104g of pre-modified chitosan.

[0050] A2.104g of pre-modified chitosan was dissolved in distilled water to prepare a 0.5% solution. 300mL of 20% AgNO3 solution was slowly added under stirring to adjust the pH of the solution to 6.5. The temperature was controlled at 22℃ and the reaction was stirred for 1 hour. The mixture was washed and freeze-dried to form a complex.

[0051] A3. The complex was dissolved in PBS to form a 0.5% solution. 500 mL of lactoferrin solution was slowly added under stirring. The mixture was stirred and mixed at 3°C ​​for 13 hours. After washing, the mixture was freeze-dried to form the modified chitosan.

[0052] S3. The fermentation broth is subjected to a shaking-static cycle culture, specifically shaking for 2 days, static for 7 days, shaking for another 0.5 days, and static for 2 hours. Mycelia are obtained from the bacterial culture.

[0053] S4. The mycelium was inoculated into the culture medium to obtain the Ganoderma lucidum culture. It was cultured at a constant temperature of 19℃ and 55% humidity for 50 days. The first 91 days were cultured in the dark, and the light intensity was 1000 lx in the later stage.

[0054] The culture medium for cultivation includes the following raw materials in parts by weight: 40 kg corn stalks, 20 kg sawdust, 5 kg rapeseed meal, 5 kg humic acid, 8 kg Armillaria mellea fermentation broth, 5 kg glucose, 1 kg fructooligosaccharides, 1 kg gypsum, 0.2 kg zinc sulfate, 0.5 kg magnesium sulfate, 0.01 kg sodium selenide, and the water content of the culture medium is 60%.

[0055] The preparation method of the culture medium for the cultivar includes the following steps: mix the raw materials according to the ratio, add water to the moisture content of 50%, turn the pile for the first time when the temperature inside the fermentation pile reaches 50℃, turn the pile once a day thereafter, and add an appropriate amount of water. When the temperature inside the fermentation pile drops to below 35℃ and the moisture content drops to below 30%, it indicates that the fermentation is mature and the fermentation is ended; adjust the moisture content to 60% and sterilize thoroughly.

[0056] S5. Transplant Ganoderma lucidum cultivars onto a substrate culture medium to cultivate Ganoderma lucidum; the cultivation conditions are 28℃ and 60% humidity for 30 days; the initial 9 days are in the dark, and the light intensity is 1000 lx in the later stage.

[0057] A compound induction agent, consisting of sodium acetate, salicylic acid, and methyl jasmonate, is sprayed every 5 days in a 1:1:1 ratio. The sodium acetate and salicylic acid solutions are both 1 mM, and the methyl jasmonate solution is 0.2 mM.

[0058] The substrate culture medium consists of the following raw materials in parts by weight: 30 kg of Astragalus membranaceus residue, 30 kg of Glycyrrhiza uralensis residue, 20 kg of wheat bran, 1 kg of gypsum, 6 kg of yeast powder, and 0.3 kg of potassium dihydrogen phosphate. The water content of the culture medium is 60%.

[0059] The pretreatment method for Astragalus membranaceus residue and Licorice residue is as follows: select fresh residue, pile it up for fermentation and dry it, crush it and pass it through a 40-mesh sieve.

[0060] S6. Cover the Ganoderma lucidum with a net and collect the Ganoderma lucidum spore powder.

[0061] Example 2

[0062] This embodiment is a second method for cultivating Ganoderma lucidum spore powder with high triterpenoid polysaccharide content.

[0063] Includes the following steps:

[0064] S1. Inoculate Ganoderma lucidum strain into seed culture medium and culture in a shaker at 26℃ and 150r / min for 9 days to obtain seed culture;

[0065] The seed culture medium contains the following components: glucose 20 g / L, yeast extract 4 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, and sodium taurocholate 2.5 g / L.

[0066] S2. Inoculate the seed culture into the fermentation medium and culture it at a constant temperature of 21℃ and a humidity of 60% for 5 days in the dark to obtain the fermentation broth;

[0067] The fermentation medium contains the following components: fructooligosaccharides 17.5 g / L, modified chitosan 12.5 g / L, peptone 7.5 g / L, yeast extract 4.25 g / L, potassium dihydrogen phosphate 0.55 g / L, magnesium sulfate heptahydrate 1.5 g / L, vitamin B1 0.075 g / L, and heme peptide 0.03 g / L.

[0068] The preparation steps of modified chitosan include:

[0069] A1. Dissolve 120g of chitosan in 1000mL of 1% acetic acid solution, add 600g of cyclodextrin, and then add 40g of ascorbic acid. Stir the reaction for 6 hours at pH 9 and 60℃. Wash the product and dry it to obtain 114g of pre-modified chitosan.

[0070] A2.114g of pre-modified chitosan was dissolved in distilled water to prepare a 1% (w / w) solution. 300mL of 25% (w / w) AgNO3 solution was slowly added under stirring to adjust the pH of the solution to 6.5. The temperature was controlled at 25℃ and the reaction was stirred for 2 hours. The mixture was then washed and freeze-dried to form a complex.

[0071] A3. The complex was dissolved in PBS to form a 1% (w / w) solution. 500 mL of lactoferrin solution was slowly added under stirring. The mixture was stirred and mixed at 4 °C for 12 hours. After washing, the mixture was freeze-dried to form the modified chitosan.

[0072] S3. The fermentation broth was subjected to a shaking-static cycle culture, specifically shaking for 2.5 days, static for 7.5 days, shaking for another 0.75 days, and static for 3 hours. Mycelia were obtained from the bacterial culture.

[0073] S4. The mycelium was inoculated into the culture medium to obtain Ganoderma lucidum culture. The culture was kept at a constant temperature of 21℃ and 60% humidity for 42.5 days. The culture was carried out in the dark for the first 10 days and the light intensity was 1250 lx in the later stage.

[0074] The culture medium for cultivation comprises the following raw materials in parts by weight: 50 kg corn stalks, 25 kg sawdust, 10 kg rapeseed meal, 6.5 kg humic acid, 9 kg Armillaria mellea fermentation broth, 9 kg glucose, 2 kg fructooligosaccharides, 2 kg gypsum, 0.35 kg zinc sulfate, 0.75 kg magnesium sulfate, and 0.015 kg sodium selenide. The water content of the culture medium is 62.5%.

[0075] The preparation method of the culture medium for the cultivar includes the following steps: mix the raw materials according to the ratio, add water to the moisture content to 60%, turn the pile for the first time when the temperature inside the fermentation pile reaches 50℃, turn the pile once a day thereafter, and add an appropriate amount of water. When the temperature inside the fermentation pile drops to below 35℃ and the moisture content drops to below 30%, it indicates that the fermentation is mature and the fermentation is ended; adjust the moisture content to 62.5% and sterilize thoroughly.

[0076] S5. Ganoderma lucidum cultivars were transplanted onto a substrate culture medium to obtain Ganoderma lucidum. The culture conditions were 29℃ and 67.5% for 25 days. The culture was carried out in the dark for the first 10 days and the light intensity was 1250 lx in the later stage.

[0077] A compound induction agent, consisting of sodium acetate, salicylic acid, and methyl jasmonate, is sprayed every 5 days in a 1:1:1 ratio. The concentrations of the sodium acetate and salicylic acid solutions are both 2.5 mM, and the concentration of the methyl jasmonate solution is 0.25 mM.

[0078] The substrate culture medium consists of the following raw materials in parts by weight: 35 kg of Astragalus membranaceus residue, 35 kg of Glycyrrhiza uralensis residue, 22 kg of wheat bran, 2 kg of gypsum, 7 kg of yeast powder, and 0.45 kg of potassium dihydrogen phosphate. The water content of the culture medium is 62.5%.

[0079] The pretreatment method for Astragalus membranaceus residue and Licorice residue is as follows: select fresh residue, pile it up for fermentation and dry it, crush it and pass it through a 50-mesh sieve.

[0080] S6. Cover the Ganoderma lucidum with a net and collect the Ganoderma lucidum spore powder.

[0081] Example 3

[0082] This embodiment is a third method for cultivating Ganoderma lucidum spore powder with high triterpenoid polysaccharide content.

[0083] Includes the following steps:

[0084] S1. Inoculate Ganoderma lucidum strain into seed culture medium and culture in a shaker at 27℃ and 160r / min for 10 days to obtain seed culture;

[0085] The seed culture medium contains the following components: glucose 22 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 2 g / L, and sodium taurocholate 3 g / L.

[0086] S2. Inoculate the seed culture into the fermentation medium and culture it at a constant temperature of 23℃ and a humidity of 65% for 4 days in the dark to obtain the fermentation broth;

[0087] The fermentation medium contains the following components: fructooligosaccharides 25 g / L, modified chitosan 20 g / L, peptone 10 g / L, yeast extract 8 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 2 g / L, vitamin B1 0.1 g / L, and heme peptide 0.05 g / L.

[0088] The preparation steps of modified chitosan include:

[0089] A1. Dissolve 120g of chitosan in 1000mL of 1.5% acetic acid solution, add 600g of cyclodextrin, and then add 40g of ascorbic acid. Stir and react for 5 hours at pH 10 and 65℃. Wash the product and dry it to obtain 116g of pre-modified chitosan.

[0090] A2.116g of pre-modified chitosan was dissolved in distilled water to prepare a 1.5% solution. 300mL of 30% AgNO3 solution was slowly added under stirring to adjust the pH of the solution to 7.0. The temperature was controlled at 28℃ and the reaction was stirred for 3 hours. The mixture was washed and freeze-dried to form a complex.

[0091] A3. The complex was dissolved in PBS to form a 1.5% (w / w) solution. 500 mL of lactoferrin solution was slowly added under stirring. The mixture was stirred and mixed at 5 °C for 11 hours. After washing, the mixture was freeze-dried to form the modified chitosan.

[0092] S3. The fermentation broth is subjected to a shaking-static cycle culture, specifically shaking for 3 days, static for 8 days, shaking for 1 day, and static for 4 hours. Mycelia are obtained from the bacterial culture.

[0093] S4. The mycelium was inoculated into the culture medium to obtain the Ganoderma lucidum culture. The culture was kept at a constant temperature of 23℃ and 65% humidity for 35 days. The culture was kept in the dark for the first 11 days and the light intensity was 1500 lx in the later stage.

[0094] The culture medium for cultivation includes the following raw materials in parts by weight: 60 kg corn stalks, 30 kg sawdust, 15 kg rapeseed meal, 8 kg humic acid, 10 kg Armillaria mellea fermentation broth, 10 kg glucose, 3 kg fructooligosaccharides, 3 kg gypsum, 0.5 kg zinc sulfate, 1 kg magnesium sulfate, 0.02 kg sodium selenide, and the water content of the culture medium is 65%.

[0095] The preparation method of the culture medium for the cultivar includes the following steps: mix the raw materials according to the ratio, add water to the moisture content to 0%, turn the pile for the first time when the temperature inside the fermentation pile reaches 50℃, and then turn the pile twice a day and add an appropriate amount of water. When the temperature inside the fermentation pile drops to below 35℃ and the moisture content drops to below 30%, it indicates that the fermentation is mature and the fermentation is ended; adjust the moisture content to 65% and sterilize thoroughly.

[0096] S5. Transplant Ganoderma lucidum cultivars onto a substrate culture medium to cultivate Ganoderma lucidum; the cultivation conditions are 30℃ and 75% humidity for 20 days; the initial 11 days are in the dark, and the light intensity is 1500 lx in the later stage.

[0097] A compound induction agent, consisting of sodium acetate, salicylic acid, and methyl jasmonate, is sprayed every 5 days in a 1:1:1 ratio. The sodium acetate and salicylic acid solutions are both 4 mM, and the methyl jasmonate solution is 0.3 mM.

[0098] The substrate culture medium consists of the following raw materials in parts by weight: 40 kg of Astragalus membranaceus residue, 40 kg of Glycyrrhiza uralensis residue, 24 kg of wheat bran, 3 kg of gypsum, 8 kg of yeast powder, and 0.6 kg of potassium dihydrogen phosphate. The water content of the culture medium is 65%.

[0099] The pretreatment method for Astragalus membranaceus residue and licorice residue is as follows: select fresh residue, pile it up for fermentation and dry it, crush it and pass it through a 60-mesh sieve.

[0100] S6. Cover the Ganoderma lucidum with a net and collect the Ganoderma lucidum spore powder.

[0101] Comparative Example 1

[0102] The difference from Example 2 is that the modified chitosan in the fermentation medium is replaced with ordinary chitosan.

[0103] Comparative Example 2

[0104] The difference from Example 2 is that the fructooligosaccharides in the fermentation medium are replaced with glucose.

[0105] Comparative Example 3

[0106] The difference from Example 2 is that the culture medium for the cultivar and the substitute culture medium include the following components by weight: 80 parts sawdust, 18 parts wheat bran, 1 part sucrose, and 1 part gypsum powder.

[0107] Comparative Example 4

[0108] The difference from Example 2 is that no compound inducing agent is sprayed.

[0109] The fruiting bodies of Ganoderma lucidum in Examples 1-3 and Comparative Examples 1-3 were tested respectively, and the results are shown in the table below. The detection methods refer to "Zhang Zhijun et al., Study on the detection of polysaccharide content of Ganoderma lucidum by phenol sulfuric acid method, Food Industry Technology, 2006(2):193-195"; "Li Baoming et al., Study on the determination method of total triterpene acid content of Ganoderma lucidum, Chinese Journal of Traditional Chinese Medicine, 2006(12):1234-1236".

[0110]

[0111] When comparing Example 2 with the previously provided Comparative Examples 1-4, it can be clearly observed that Example 2 significantly increased the content of triterpenes and polysaccharides in Ganoderma lucidum spore powder. This improvement not only enhances the medicinal value of the product but also greatly improves its competitiveness in the market, while satisfying consumers' pursuit of high-quality health products, thus making it stand out among similar products.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for cultivating Ganoderma lucidum spore powder with high triterpenoid polysaccharide content, characterized in that, Includes the following steps: S1. Inoculate Ganoderma lucidum strains into seed culture medium and culture to obtain seed culture; S2. Inoculate the seed culture into the fermentation medium and culture to obtain the fermentation broth; S3. The fermentation broth is subjected to a shaking-static cycle culture, and the mycelium is obtained by separating the bacterial solution. S4. Inoculate the mycelium into the culture medium and culture it to obtain the Ganoderma lucidum culture medium; S5. Transplant Ganoderma lucidum cultivars onto a substrate culture medium to cultivate Ganoderma lucidum; S6. Cover the Ganoderma lucidum with a net and collect the Ganoderma lucidum spore powder; The fermentation medium in step S2 comprises the following components: fructooligosaccharides 10-25 g / L, modified chitosan 5-20 g / L, peptone 5-10 g / L, yeast extract 0.5-8 g / L, potassium dihydrogen phosphate 0.1-1.0 g / L, magnesium sulfate heptahydrate 1-2 g / L, vitamin B1 0.05-0.1 g / L, and heme peptide 0.01-0.05 g / L; The preparation steps of the modified chitosan include: A1. Dissolve chitosan in an acetic acid solution with a mass concentration of 0.5%-1.5%, add cyclodextrin, then add ascorbic acid, stir and react for 5-7 hours at pH 9-10 and 55-65℃, wash the product, and dry to obtain preliminarily modified chitosan. A2. Preliminary modified chitosan was dissolved in distilled water to prepare a solution with a mass concentration of 0.5%-1.5%. Under stirring, AgNO3 solution with a mass concentration of 20%-30% was slowly added to adjust the pH of the solution to 6.5-7.

0. The temperature was controlled at 22-28℃ and the reaction was stirred for 1-3 hours. The mixture was then washed and freeze-dried to form a complex. A3. The complex was dissolved in PBS to form a solution with a mass concentration of 0.5%-1.5%. Lactoferrin solution was slowly added under stirring conditions, and the mixture was stirred and mixed at 3-5°C for 11-13 hours. After washing, the mixture was freeze-dried to form the modified chitosan.

2. The cultivation method according to claim 1, characterized in that, The seed culture medium in step S1 comprises the following components: glucose 18-22 g / L, yeast powder 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate heptahydrate 1-2 g / L, and sodium taurocholate 2-3 g / L.

3. The cultivation method according to claim 1, characterized in that, In step A1, the mass ratio of cyclodextrin to chitosan is 5:

1.

4. The cultivation method according to claim 1, characterized in that, The specific steps of step S3, the oscillation-static cyclic culture, are as follows: oscillate for 2-3 days, static for 7-8 days, then oscillate for 0.5-1 day, and static for 2-4 hours.

5. The cultivation method according to claim 1, characterized in that, The culture medium for cultivation in step S4 comprises the following raw materials in parts by weight: 40-60 parts corn stalks, 20-30 parts sawdust, 5-15 parts rapeseed meal, 5-8 parts humic acid, 8-10 parts Armillaria mellea fermentation broth, 5-10 parts glucose, 1-3 parts fructooligosaccharides, 1-3 parts gypsum, 0.2-0.5 parts zinc sulfate, 0.5-1 part magnesium sulfate, and 0.01-0.02 parts sodium selenide. The water content of the culture medium is 60-65%.

6. The cultivation method according to claim 1, characterized in that, The substrate culture medium in step S5 comprises the following raw materials in parts by weight: 30-40 parts of Astragalus membranaceus residue, 30-40 parts of Glycyrrhiza uralensis residue, 20-24 parts of wheat bran, 1-3 parts of gypsum, 6-8 parts of yeast powder, 0.3-0.6 parts of potassium dihydrogen phosphate, and the water content of the culture medium is 60-65%.

7. The cultivation method according to claim 6, characterized in that, The pretreatment method for the astragalus and licorice residues is as follows: select fresh residues, mix them evenly, pile them up for fermentation and dry them, crush them, and pass them through a 40-60 mesh sieve.

8. The cultivation method according to claim 1, characterized in that, Step S5 also includes spraying a composite inducer, which consists of sodium acetate, salicylic acid and methyl jasmonate, in a mixing ratio of 1:1:1.

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