Cultivation method of ganoderma lucidum spore powder with high triterpenoid polysaccharide content

Abstract

The invention relates to the technical field of artificial cultivation of medicinal fungi, and discloses a cultivation method of ganoderma lucidum spore powder with high triterpenoid polysaccharide content, which comprises the following steps: S1, inoculating a ganoderma lucidum strain in a seed solution culture medium, and culturing to obtain a seed solution; s2, inoculating the seed solution to a fermentation culture medium, and culturing to obtain a fermentation solution; s3, carrying out oscillation-standing circulation culture on the fermentation liquid, and separating the bacterial liquid to obtain mycelia; s4, inoculating the mycelium into a cultivar culture medium for culturing to obtain a ganoderma lucidum cultivar; s5, transplanting ganoderma lucidum cultivated species on the substitute culture medium, and culturing to obtain ganoderma lucidum; s6, the ganoderma lucidum is sleeved with a net cover, and ganoderma lucidum spore powder is collected. The content of triterpenes and polysaccharides in the ganoderma lucidum spore powder can be increased.

Description

Technical Field

[0001] The invention relates to the technical field of artificial cultivation of medicinal fungi, in particular to a method for cultivating ganoderma lucidum spore powder with high triterpene polysaccharide content. Background Art

[0002] As a traditional Chinese medicinal material with rich medicinal value, Ganoderma lucidum has always been widely used in various health care fields such as enhancing immunity, anti-tumor, and anti-aging. Among them, Ganoderma lucidum spore powder is considered to be the essence of Ganoderma lucidum because it contains a variety of bioactive ingredients, especially triterpenoids and polysaccharides. Triterpenoids are believed to have significant effects on anti-cancer, anti-inflammatory and antioxidant effects, while polysaccharides are mainly believed to have immunomodulatory, antiviral, and anti-tumor effects. Therefore, increasing the content of triterpenoids and polysaccharides in Ganoderma lucidum spore powder is of great significance to improving its efficacy. Summary of the invention

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

[0004] The present invention solves the above technical problems by the following technical means:

[0005] The present invention provides a method for cultivating ganoderma lucidum spore powder with high triterpene polysaccharide content, comprising the following steps:

[0006] S1. Inoculate Ganoderma lucidum strains in a seed liquid culture medium to obtain a seed liquid;

[0007] S2. inoculating the seed liquid into the fermentation medium and culturing to obtain a fermentation liquid;

[0008] S3. The fermentation liquid is subjected to oscillation-stationary cycle culture, and the bacterial liquid is separated to obtain mycelium;

[0009] S4. The mycelium is inoculated into a culture medium to obtain a Ganoderma lucidum culture medium;

[0010] S5. Transplanting the cultivated species of Ganoderma lucidum on the substitute medium to obtain Ganoderma lucidum;

[0011] S6. Put a net on the Ganoderma lucidum to collect the Ganoderma lucidum spore powder.

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

[0013] Further preferably, the culture conditions of step S1 are a temperature of 25-27° C. and a shaking incubator at 140-160 rpm.

[0014] The seed liquid culture medium provides comprehensive nutritional support for Ganoderma lucidum strains by scientifically matching ingredients such as glucose, yeast powder, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and sodium taurocholate. These ingredients, as carbon sources, nitrogen sources, phosphorus sources, magnesium sources and bioactive additives, not only support the rapid growth and biomass accumulation of Ganoderma lucidum mycelium, but also promote its metabolic activity, especially the synthesis of secondary metabolites. It significantly improves the growth rate of Ganoderma lucidum mycelium, enhances the secretion of extracellular secondary metabolites (such as triterpenes and polysaccharides), and improves the tolerance and activity of the bacteria to various stress conditions. High-quality seed liquid preparation ensures the efficiency and stability of the fermentation process, and also provides high-quality strain resources for subsequent cultivation and substitute culture, which helps to achieve the goal of high triterpenoid and polysaccharide content in the final product, greatly optimizing the entire production process.

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

[0016] Further preferably, the culture conditions of step S2 are constant temperature and light-proof culture at a temperature of 19-23° C. and a humidity of 55-65%.

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

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

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

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

[0021] A3. The complex is dissolved in PBS to form a solution with a mass concentration of 0.5%-1.5%, and the lactoferrin solution is slowly added under stirring conditions, stirred and mixed at 3-5° C. for 11-13 hours, washed, and 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] Modified chitosan is prepared through chemical modification and composite technology, including the use of cyclodextrin and ascorbic acid for initial modification to improve its solubility and stability, and to generate a stable antioxidant structure; then combined with silver ions to form chitosan-silver complexes, using the antibacterial properties of silver ions to enhance its application effect in complex biological environments; finally, lactoferrin is introduced to further enhance the antibacterial ability and microbial affinity of the complex through hydrogen bonding and electrostatic effects. This series of steps not only improves the functionality of chitosan, but also particularly enhances its antibacterial activity and its ability to support the growth of Ganoderma lucidum mycelium.

[0024] Preferably, the shaking-stationary cycle culture in step S3 is shaking for 2-3 days, stationary for 7-8 days, shaking for 0.5-1 day, and stationary for 2-4 hours.

[0025] Oscillation-static cycle culture is a fermentation method that combines dynamic and static operations, aiming to provide the best environment for the growth of Ganoderma lucidum mycelium. The oscillation stage increases the solubility of oxygen and the uniform distribution of nutrients, promoting rapid growth in the early stage; while in the static stage, a stable low-disturbance environment is provided, which is conducive to the directional extension of the mycelium and the production of extracellular polymers such as polysaccharides and triterpenoids. The optimized cycle mode includes long-term oscillation in the early stage to activate growth, longer static in the middle stage to promote the accumulation of metabolites, short-term oscillation in the later stage to improve oxygen supply, and finally short static to ensure the operating conditions for bacterial liquid separation, thereby fully supporting the healthy growth and efficient metabolism of Ganoderma lucidum mycelium.

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

[0027] Further preferably, the culture conditions of step S4 are constant temperature culture at a temperature of 19-23° C. and a humidity of 55-65%; the culture is kept away from light for 9-11 days in the early stage, and the light intensity is 1000-1500 lx in the later stage.

[0028] Corn stalks and sawdust are used to provide carbon sources and cellulose, rapeseed meal is used to provide nitrogen sources, and functional additives such as humic acid and oligofructose are used to improve the matrix structure and promote microbial metabolic activity. At the same time, trace elements such as zinc sulfate, magnesium sulfate and sodium selenide are added to support metabolic enzyme activation and enhance antioxidant capacity. In addition, an appropriate amount of glucose is used as a rapidly available carbon source to ensure initial energy supply, and the introduction of honey fungus fermentation liquid not only helps decompose complex organic matter, but also enhances the matrix 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 residue, 30-40 parts of licorice 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 of the astragalus residue and licorice residue is: select fresh residues, mix them evenly, compost them, ferment them and dry them, crush them and pass them through a 40-60 mesh sieve.

[0031] Further preferably, the culture conditions of step S5 are culture at a temperature of 28-30° C. and a humidity of 60-75%; the culture is kept away from light for 9-11 days in the early stage, and the light intensity is 1000-1500 lx in the later stage.

[0032] After fermentation, the residues of Astragalus and Licorice can provide rich polysaccharides, flavonoids and trace active ingredients, which support the growth of Ganoderma lucidum mycelium and promote the accumulation of metabolites. Gypsum is used to adjust the pH value and improve air permeability, while providing calcium ions to support the formation of fruiting body structure. Auxiliary ingredients such as yeast powder and potassium dihydrogen phosphate further strengthen the metabolic process, promote the synthesis of triterpenes and polysaccharides, and improve stress resistance and metabolic efficiency. By optimizing pretreatment methods such as composting fermentation, crushing and screening, the stability and appropriate physical and chemical properties of the culture medium are ensured, providing an ideal growth environment for Ganoderma lucidum. The growth rate and quality of Ganoderma lucidum mycelium are significantly enhanced, the culture cycle is shortened, and the accumulation of effective ingredients such as polysaccharides and triterpenes is greatly increased. The physical and chemical properties of the culture medium have been optimized, becoming more loose and breathable, which is conducive to the healthy growth of mycelium. By recycling the residues, not only the production cost is reduced and the efficient use of resources is achieved, but also the environmental protection concept of green production is met, reducing environmental pollution.

[0033] Preferably, the S5 step also 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 the sodium acetate solution and the salicylic acid solution are both 1-4 mM, and the concentration of the methyl jasmonate solution is 0.2-0.3 mM.

[0035] The composite inducer is a mixture of sodium acetate, salicylic acid and methyl jasmonate in a ratio of 1:1:1. Each component acts on the growth and metabolism of Ganoderma lucidum through different physiological mechanisms. Sodium acetate regulates the intracellular acid-base balance and enhances antioxidant activity, promoting mycelial growth and accumulation of effective ingredients; salicylic acid, as a plant hormone, activates cell signal transduction pathways, enhances disease resistance and stress tolerance, and stimulates the production of bioactive substances; methyl jasmonate improves resistance to adversity and secondary metabolism by regulating plant hormone levels. The three work synergistically to enhance the metabolic activity, stress resistance and product accumulation ability of Ganoderma lucidum. Spraying application ensures that the inducing components can be quickly absorbed by the mycelium and take effect, optimizing the growth environment of Ganoderma lucidum.

[0036] Beneficial effects of the present invention:

[0037] (1) Based on the biological characteristics of Ganoderma lucidum, the present invention optimizes key process steps such as seed liquid culture, fermentation culture and substitute cultivation to regulate the secondary metabolic pathways of Ganoderma lucidum mycelium and promote the efficient synthesis of extracellular polymers (triterpenes and polysaccharides). Oscillation-static cycle culture is used to strengthen oxygen supply and metabolite secretion. At the same time, specific inducers and medicinal residues are introduced as culture medium components to provide rich nutrients and trace elements for the growth and secondary metabolism of Ganoderma lucidum, and ultimately increase the active ingredient content of Ganoderma lucidum spore powder. The content of triterpenes and polysaccharides in Ganoderma lucidum spore powder is significantly increased, enhancing the medicinal value and market competitiveness of the product. At the same time, the resource recycling strategy is used to introduce waste such as medicinal residues to reduce production costs.

[0038] (2) The modified chitosan significantly improved its water solubility and biological activity, ensuring that it was evenly dispersed in the culture medium and remained active for a long time. The addition of silver ions greatly enhanced the broad-spectrum antibacterial properties and protected the mycelium of Ganoderma lucidum 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 a variety of biological culture medium environments, providing strong technical support for the large-scale production of Ganoderma lucidum and other fungi.

[0039] (3) The oscillation-static cycle culture method of the present invention significantly improves the growth efficiency of Ganoderma lucidum mycelium and the synthesis of extracellular polymers (such as polysaccharides and triterpenes), and reduces the mechanical damage of mycelium 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, and increases the biological activity and effective ingredient content of the fermentation broth. Ultimately, this method simplifies the operating process, improves production efficiency, and provides ideal production conditions for subsequent bacterial broth separation, mycelium collection, and its transfer to substitute culture. DETAILED DESCRIPTION

[0040] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention:

[0041] Example 1

[0042] This embodiment is a method 1 for cultivating Ganoderma lucidum spore powder with high triterpene polysaccharide content.

[0043] The following steps are involved:

[0044] S1. Inoculate Ganoderma lucidum in a seed liquid medium, and culture at 25°C and 140 r / min shaking for 10 days to obtain a seed liquid;

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

[0046] S2. The seed solution was inoculated into the fermentation medium, and the fermentation liquid was obtained by constant temperature and dark culture at a temperature of 19°C and a humidity of 55% for 6 days;

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

[0048] The preparation steps of modified chitosan include:

[0049] A1. 120 g of chitosan was dissolved in 1000 mL of 0.5% acetic acid solution, 600 g of cyclodextrin was added, and then 40 g of ascorbic acid was added. The reaction was stirred at pH 9 and 55 ° C for 7 hours, the product was washed and dried to obtain 104 g of preliminary modified chitosan;

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

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

[0052] S3. The fermentation broth was subjected to oscillation - static cycle culture, and the oscillation - static cycle culture was specifically oscillated for 2 days, static for 7 days, then oscillated for 0.5 days, static for 2 hours, and the bacterial liquid was separated to obtain mycelium;

[0053] S4. The mycelium was inoculated into the culture medium to obtain the Ganoderma lucidum culture medium, and the culture was kept at a constant temperature of 19°C and a humidity of 55% for 50 days; the culture was kept away from light for 91 days, and the light intensity was 1000 lx in the later stage;

[0054] The cultivation medium 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 honey fungus fermentation liquid, 5 kg glucose, 1 kg fructooligosaccharide, 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 cultivated species comprises the following steps: mixing various raw materials according to a proportion, adding water to a water content of 50%, turning the pile for the first time when the temperature in the fermentation pile reaches 50°C, turning the pile once a day thereafter, and adding an appropriate amount of water, and ending the fermentation when the temperature of the fermentation pile drops below 35°C and the water content drops below 30%, indicating that the fermentation is mature; adjusting the water content to 60%, and sterilizing thoroughly.

[0056] S5. Ganoderma lucidum cultivars were transplanted on substitute medium to obtain Ganoderma lucidum; the culture conditions were 28°C and 60% humidity for 30 days; the culture was kept away from light for 9 days, and the light intensity was 1000 lx in the later stage.

[0057] A composite inducer was sprayed every 5 days. The composite inducer included sodium acetate, salicylic acid and methyl jasmonate, and the mixing ratio was 1:1:1. The concentrations of sodium acetate solution and salicylic acid solution were both 1mM, and the concentration of methyl jasmonate solution was 0.2mM.

[0058] The substitute culture medium comprises the following raw materials in parts by weight: 30 kg of astragalus residue, 30 kg of liquorice residue, 20 kg of wheat bran, 1 kg of gypsum, 6 kg of yeast powder, 0.3 kg of potassium dihydrogen phosphate, and the water content of the culture medium is 60%.

[0059] The pretreatment method of astragalus root residue and licorice root residue is as follows: fresh residue is selected, piled, fermented and dried, crushed and passed through a 40-mesh sieve.

[0060] S6. Put a net on the Ganoderma lucidum to collect the Ganoderma lucidum spore powder.

[0061] Example 2

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

[0063] The following steps are involved:

[0064] S1. Inoculate Ganoderma lucidum in a seed liquid culture medium, and culture at a temperature of 26°C and a shaking incubator at 150 rpm for 9 days to obtain a seed liquid;

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

[0066] S2. The seed solution was inoculated into the fermentation medium, and the fermentation liquid was obtained by constant temperature and dark culture at a temperature of 21°C and a humidity of 60% for 5 days;

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

[0068] The preparation steps of modified chitosan include:

[0069] A1. 120 g of chitosan was dissolved in 1000 mL of 1% acetic acid solution, 600 g of cyclodextrin was added, and 40 g of ascorbic acid was added. The mixture was stirred at pH 9 and 60 ° C for 6 hours, and the product was washed and dried to obtain 114 g of preliminary modified chitosan.

[0070] A2.114 g of preliminarily modified chitosan was dissolved in distilled water to prepare a solution with a mass concentration of 1%. 300 mL of a 25% AgNO3 solution was slowly added under stirring to adjust the pH of the solution to 6.5. The temperature was controlled at 25°C and stirred for 2 hours. The mixture was washed and freeze-dried to form a composite.

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

[0072] S3. The fermentation broth was subjected to oscillation - static cycle culture, and the oscillation - static cycle culture was specifically oscillated for 2.5 days, static for 7.5 days, then oscillated for 0.75 days, static for 3 hours, and the bacterial liquid was separated to obtain mycelium;

[0073] S4. The mycelium was inoculated into the culture medium to obtain the Ganoderma lucidum culture medium, and the culture was kept at a constant temperature of 21°C and a humidity of 60% for 42.5 days; the culture was kept away from light for 10 days in the early stage, and the light intensity was 1250lx in the later stage;

[0074] The cultivation medium includes 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 honey fungus fermentation liquid, 9 kg glucose, 2 kg fructooligosaccharide, 2 kg gypsum, 0.35 kg zinc sulfate, 0.75 kg magnesium sulfate, 0.015 kg sodium selenide, and the water content of the culture medium is 62.5%.

[0075] The preparation method of the culture medium for cultivated species comprises the following steps: mixing various raw materials according to a proportion, adding water to a water content of 60%, turning the pile for the first time when the temperature in the fermentation pile reaches 50°C, turning the pile once a day thereafter, and adding an appropriate amount of water, and ending the fermentation when the temperature of the fermentation pile drops below 35°C and the water content drops below 30%, indicating that the fermentation is mature; adjusting the water content to 62.5%, and sterilizing thoroughly.

[0076] S5. Ganoderma lucidum cultivars were transplanted on substitute medium to obtain Ganoderma lucidum; the culture conditions were 29°C and 67.5% humidity for 25 days; the culture was kept away from light for the first 10 days, and the light intensity was 1250lx in the later period.

[0077] A composite inducer was sprayed every 5 days. The composite inducer included sodium acetate, salicylic acid and methyl jasmonate, and the mixing ratio was 1:1:1. The concentrations of sodium acetate solution and salicylic acid solution were both 2.5 mM, and the concentration of methyl jasmonate solution was 0.25 mM.

[0078] The substitute culture medium includes the following raw materials in parts by weight: 35 kg of astragalus residue, 35 kg of liquorice residue, 22 kg of wheat bran, 2 kg of gypsum, 7 kg of yeast powder, and 0.45 kg of potassium dihydrogen phosphate, and the water content of the culture medium is 62.5%.

[0079] The pretreatment method of astragalus root residue and licorice root residue is as follows: fresh residue is selected, piled, fermented and dried, crushed and passed through a 50-mesh sieve.

[0080] S6. Put a net on the Ganoderma lucidum to collect the Ganoderma lucidum spore powder.

[0081] Example 3

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

[0083] The following steps are involved:

[0084] S1. Inoculate Ganoderma lucidum in a seed liquid culture medium, and culture at a temperature of 27°C and a shaking incubator at 160 rpm for 10 days to obtain a seed liquid;

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

[0086] S2. The seed solution was inoculated into the fermentation medium, and the fermentation liquid was obtained by constant temperature and dark culture at a temperature of 23°C and a humidity of 65% for 4 days;

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

[0088] The preparation steps of modified chitosan include:

[0089] A1. 120 g of chitosan was dissolved in 1000 mL of 1.5% acetic acid solution, 600 g of cyclodextrin was added, and 40 g of ascorbic acid was added. The mixture was stirred at pH 10 and 65 ° C for 5 hours, and the product was washed and dried to obtain 116 g of preliminary modified chitosan.

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

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

[0092] S3. The fermentation broth was subjected to oscillation-stationary cycle culture, and the oscillation-stationary cycle culture was specifically oscillated for 3 days, stationary for 8 days, then oscillated for 1 day, stationary for 4 hours, and the bacterial liquid was separated to obtain mycelium;

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

[0094] The cultivation medium 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 honey fungus fermentation liquid, 10 kg glucose, 3 kg fructooligosaccharide, 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 cultivated species comprises the following steps: mixing various raw materials according to a proportion, adding water to a water content of 0%, turning the pile for the first time when the temperature in the fermentation pile reaches 50°C, turning the pile twice a day thereafter, and adding an appropriate amount of water, and ending the fermentation when the temperature of the fermentation pile drops below 35°C and the water content drops below 30%, indicating that the fermentation is mature; adjusting the water content to 65%, and sterilizing thoroughly.

[0096] S5. Ganoderma lucidum cultivars were transplanted on substitute medium to obtain Ganoderma lucidum; the culture conditions were 30°C and 75% humidity for 20 days; the culture was kept away from light for the first 11 days, and the light intensity was 1500lx in the later period.

[0097] A composite inducer was sprayed every 5 days. The composite inducer included sodium acetate, salicylic acid and methyl jasmonate, and the mixing ratio was 1:1:1. The concentrations of sodium acetate solution and salicylic acid solution were both 4mM, and the concentration of methyl jasmonate solution was 0.3mM.

[0098] The substitute culture medium comprises the following raw materials in parts by weight: 40 kg of astragalus residue, 40 kg of liquorice residue, 24 kg of wheat bran, 3 kg of gypsum, 8 kg of yeast powder, and 0.6 kg of potassium dihydrogen phosphate, and the water content of the culture medium is 65%.

[0099] The pretreatment method of astragalus root residue and licorice root residue is as follows: fresh residue is selected, piled, fermented and dried, crushed and passed through a 60-mesh sieve.

[0100] S6. Put a net on the Ganoderma lucidum to 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 by ordinary chitosan.

[0103] Comparative Example 2

[0104] The difference from Example 2 is that the oligofructose in the fermentation medium is replaced by glucose.

[0105] Comparative Example 3

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

[0107] Comparative Example 4

[0108] The difference from Example 2 is: no composite derivant is sprayed

[0109] The ganoderma lucidum fruiting bodies of Examples 1-3 and Comparative Examples 1-3 were tested, and the results are shown in the following table. The testing methods refer to "Zhang Zhijun et al., Study on the Detection of Ganoderma Lucidum Polysaccharide Content by Phenol-sulfuric Acid Method, Food Industry Science and Technology, 2006(2):193-195"; "Li Baoming et al., Study on the Determination Method of Ganoderma Lucidum Total Triterpenic Acid Content, Chinese Journal of Traditional Chinese Medicine, 2006(12):1234-1236".

[0110]

[0111] When comparing Example 2 with Comparative Examples 1-4 provided previously, it can be clearly observed that Example 2 has a significant improvement in the content of triterpenes and polysaccharides in the 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, thereby standing out from similar products.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A method for cultivating Ganoderma lucidum spore powder with high triterpene polysaccharide content, characterized in that: The following steps are involved: S1. Inoculate Ganoderma lucidum strains in a seed liquid culture medium to obtain a seed liquid; S2. inoculating the seed liquid into the fermentation medium and culturing to obtain a fermentation liquid; S3. The fermentation liquid is subjected to oscillation-stationary cycle culture, and the bacterial liquid is separated to obtain mycelium; S4. The mycelium is inoculated into a culture medium to obtain a Ganoderma lucidum culture medium; S5. Transplanting the cultivated species of Ganoderma lucidum on the substitute medium to obtain Ganoderma lucidum; S6. Put a net on the Ganoderma lucidum to collect the Ganoderma lucidum spore powder.

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

3. The cultivation method according to claim 1, characterized in that The fermentation medium of step S2 comprises the following components: 10-25 g / L of oligofructose, 5-20 g / L of modified chitosan, 5-10 g / L of peptone, 0.5-8 g / L of yeast powder, 0.1-1.0 g / L of potassium dihydrogen phosphate, 1-2 g / L of magnesium sulfate heptahydrate, 0.05-0.1 g / L of vitamin B1, and 0.01-0.05 g / L of heme peptide.

4. The cultivation method according to claim 3, characterized in that: The preparation steps of the modified chitosan include: A1. Dissolve chitosan in acetic acid solution with a mass concentration of 0.5%-1.5%, add cyclodextrin, and then add ascorbic acid, stir and react at pH 9-10 and 55-65°C for 5-7 hours, wash the product, and dry it to obtain a preliminary modified chitosan; A2. The preliminarily modified chitosan was dissolved in distilled water to prepare a solution with a mass concentration of 0.5%-1.5%, and a 20%-30% AgNO3 solution was slowly added under stirring to adjust the solution pH to 6.5-7.0, and the temperature was controlled at 22-28°C and stirred for 1-3 hours, washed, and freeze-dried to form a complex; A3. The complex is dissolved in PBS to form a solution with a mass concentration of 0.5%-1.5%, and the lactoferrin solution is slowly added under stirring conditions, stirred and mixed at 3-5° C. for 11-13 hours, washed, and freeze-dried to form the modified chitosan.

5. The cultivation method according to claim 4, characterized in that: The mass ratio of cyclodextrin to chitosan in step A1 is 5:

1.

6. The cultivation method according to claim 5, characterized in that: The shaking-stationary cycle culture in step S3 specifically comprises shaking for 2-3 days, stationary for 7-8 days, shaking for 0.5-1 day, and stationary for 2-4 hours.

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

8. The cultivation method according to claim 1, characterized in that: The substitute culture medium in step S5 comprises the following raw materials in parts by weight: 30-40 parts of astragalus residue, 30-40 parts of liquorice 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%.

9. The cultivation method according to claim 8, characterized in that: The pretreatment method of the astragalus root residue and the licorice root residue is as follows: fresh residues are selected, mixed evenly, piled for fermentation and dried, crushed, and passed through a 40-60 mesh sieve.

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

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