Organic cultivation method of selenium-rich ganoderma lucidum spore powder

Abstract

The present application relates to a kind of organic cultivation methods of selenium-rich ganoderma spore powder, it is related to ganoderma cultivation technical field;Wherein the organic cultivation method of selenium-rich ganoderma spore powder includes base material preparation, inoculation and bacteria cultivation, cultivation management, collection step;The present application is realized by high-efficiency conversion of inorganic selenium to organic selenium, improves the proportion of organic selenium in ganoderma spore powder, also further improves total selenium content, and then guarantees the high quality of ganoderma spore powder, finally realizes the large-scale, standardization organic cultivation of selenium-rich ganoderma spore powder.

Description

Technical Field

[0001] This invention relates to the field of Ganoderma lucidum cultivation technology, and in particular to an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder. Background Technology

[0002] Selenium is an essential trace element for the human body, which can improve human immunity and anti-aging. Ganoderma lucidum has the natural ability to enrich and convert selenium, and can be used as an ideal selenium carrier. Selenium-enriched Ganoderma lucidum spore powder, as the essence of Ganoderma lucidum, has the dual health benefits of Ganoderma lucidum and selenium, and has become a research hotspot in recent years.

[0003] In the cultivation of selenium-enriched Ganoderma lucidum, the form in which selenium exists directly determines its biological activity, safety, and human absorption rate. Organic selenium, such as selenomethionine and selenocysteine, has significant advantages over inorganic selenium, including higher absorption rates, lower toxicity, and stronger physiological activity. Therefore, the key to cultivating selenium-enriched Ganoderma lucidum spore powder lies not only in increasing the total selenium content in the spore powder, but more importantly, in increasing the proportion of organic selenium within the total selenium. Only by efficiently converting exogenous selenium into easily absorbed organic selenium and enriching it in the spore powder can the synergistic health benefits of Ganoderma lucidum and selenium be truly realized, thereby enhancing the product's market competitiveness and application value.

[0004] Currently, Chinese invention patent CN117678472A, published on March 12, 2024, proposes a method for cultivating selenium-enriched Ganoderma lucidum spore powder. The method includes selection, breeding, and cultivation steps. In the breeding step, the preparation of the selenium-enriched growth solution includes the following steps: preparing a solution of selenite, adding vermiculite powder, sonicating for 5-10 minutes, then adding glucose, vitamins, and sodium chloride, and stirring for 5-10 minutes to obtain the selenium-enriched growth solution. This technical solution, through selenium-tolerant and selenium-enriched cultivation of Ganoderma lucidum strains, yields Ganoderma lucidum strains with high selenium absorption efficiency and utilization rate, thereby reducing the amount of selenium used and lowering costs.

[0005] However, the inventors discovered that in the above-mentioned selenium-enriched method, the selenium content is increased by adding selenium-enriched growth solution and using vermiculite powder and sodium chloride. However, when Ganoderma lucidum mycelium comes into contact with selenium-enriched growth solution during the growth process, although it can absorb some inorganic selenium, it cannot perform preliminary conversion. As a result, the unconverted inorganic selenium remains in the mycelium in its original form, ultimately leading to a low proportion of organic selenium in the spore powder. Summary of the Invention

[0006] In order to increase the proportion of organic selenium in Ganoderma lucidum spore powder, this invention provides an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0007] Firstly, this application provides an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder, employing the following technical solution: An organic cultivation method for selenium-enriched Ganoderma lucidum spore powder includes the following steps: Preparation of substrate: The substrate comprises, by mass parts: 72-83 parts of virgin sawdust, 17-22 parts of wheat bran, 5-8 parts of corn flour, 0.8-1.2 parts of lime, and 63-65 parts of water; Mix the above substrate, stir evenly, pack into bags, and then autoclave to obtain culture medium bags; Inoculation and cultivation: Cool the culture medium to 26-28℃, inoculate with Ganoderma lucidum liquid spawn to obtain spawn bags, transfer the spawn bags to the cultivation workshop, and cultivate at 23-25℃ for 65-75 days to obtain Ganoderma lucidum spawn bags; Cultivation and management: First, place the seedlings on shelves and then manage their growth under the conditions of 26-30℃, humidity 88-92%, carbon dioxide concentration 500-800ppm, and light intensity 400-900Lux. Collection: Collect spores after 38–45 days of management; The raw wood chips are first pretreated by adding a selenium-enriching agent, including sodium selenite, to the raw wood chips and pre-piling for 24 to 48 hours; the amount of selenium-enriching agent added is 0.006 to 0.008 wt% of the total mass of the base material.

[0008] By adopting the above technical solution, firstly, the raw sawdust provides the carbon source for Ganoderma lucidum growth, while wheat bran and corn flour supplement sufficient nitrogen source and trace elements. Lime adjusts the pH of the substrate to a neutral to slightly alkaline condition for Ganoderma lucidum growth, providing a foundation for Ganoderma lucidum mycelial metabolism and selenium absorption and conversion.

[0009] Secondly, the pretreatment of raw sawdust allows selenium to penetrate evenly into the sawdust fibers, increasing the contact area with Ganoderma lucidum mycelium and reducing selenium loss. On the other hand, during the pre-piling process, the sawdust's own microorganisms and enzymes will carry out preliminary transformation of sodium selenite, converting some inorganic selenium into simple organic selenium precursors that are easily absorbed by the mycelium, such as simple selenized compounds.

[0010] Specifically, native sawdust naturally contains a variety of microorganisms, mainly including Bacillus, yeast, and actinomycetes. Under the temperature and humidity conditions of pre-piling, these microorganisms will multiply in large numbers and activate their own metabolic systems. The microorganisms' own selenium metabolism pathways will first reduce inorganic sodium selenite to elemental selenium through the catalytic action of reductase, making it easier for microorganisms to adsorb and further transform it. Subsequently, small molecules such as amino acids and polypeptides produced by microbial metabolism will combine with elemental selenium under the action of transaminase to form simple organic selenium precursors. These compounds mainly include primary derivatives of selenomethionine and selenocysteine, which are more likely to penetrate the cell membrane of Ganoderma lucidum hyphae and be actively absorbed by the hyphae cells.

[0011] Finally, inoculation and cultivation management provide a metabolic environment for the conversion of organic selenium, which allows the absorbed inorganic selenium to be further converted into the organic selenium of Ganoderma lucidum itself, such as selenoamino acids and selenoproteins, and then transported to the spore powder through mycelium, ultimately achieving a significant increase in the proportion of organic selenium in the spore powder.

[0012] Optionally, a compound microbial agent is evenly sprayed onto the raw sawdust after the addition of selenium-enriching agent, followed by pre-piling treatment; the amount of the compound microbial agent added accounts for 0.36 to 0.60 wt% of the raw sawdust before pre-treatment; the compound microbial agent includes Bacillus subtilis agent and Trichoderma harzianum agent, wherein the mass ratio of Bacillus subtilis agent to Trichoderma harzianum agent is 5 to 8:1.

[0013] By employing the above technical solutions, Bacillus subtilis secretes hydrolytic enzymes such as proteases and cellulases to degrade recalcitrant substances such as cellulose and lignin in sawdust, converting them into small molecule nutrients such as glucose and amino acids that can be directly absorbed and utilized by Ganoderma lucidum mycelia. At the same time, its metabolites regulate the microenvironment of the substrate, promote the dissolution and activation of selenium in the selenium-enriching agent, and make selenium more easily recognized and absorbed by the subsequently inoculated Ganoderma lucidum mycelia. The strong antagonistic effect of Trichoderma harzianum agent inhibits the growth and reproduction of harmful bacteria such as molds and bacteria during the pre-piling process, preventing harmful bacteria from competing for selenium and nutrients, and ensuring the dominant growth position of Ganoderma lucidum mycelia.

[0014] Optionally, during the pre-stacking process, 3 to 5 ventilating ducts extending to the bottom are inserted into the stack to ensure that the oxygen concentration inside the stack is ≥8%.

[0015] By adopting the above technical solution, during the pre-stacking process, a permeable conduit extending to the bottom is inserted into the stack to construct an internal gas circulation channel, maintaining the oxygen concentration inside the stack at ≥8%. This meets the aerobic requirements of Bacillus subtilis and other bacteria, enabling them to maintain a highly efficient metabolic state, continuously secrete hydrolytic enzymes to degrade substrate nutrients, and activate selenium, providing sufficient raw materials for Ganoderma lucidum mycelium to absorb selenium and convert it into organic selenium. At the same time, sufficient oxygen inhibits the growth of anaerobic bacteria, ensuring the stability of the pre-stacking process and preventing substrate deterioration. In addition, sufficient oxygen can also promote the aerobic metabolism of the virgin sawdust itself, accelerating the penetration and initial conversion of selenium in the sawdust.

[0016] Optionally, the selenium-enriching agent further includes selenized carrageenan and L-glutamine, wherein the mass ratio of selenized carrageenan, L-glutamine, and sodium selenite is 1:0.4-0.8:3-5.

[0017] By adopting the above technical solution, selenized carrageenan can be directly absorbed and utilized by Ganoderma lucidum mycelium, directly increasing the basic content of organic selenium in spore powder. At the same time, after being absorbed by mycelium, it will regulate cell membrane permeability, activate the activity of selenium transport proteins on the cell membrane, increase the selenium transport channels, and thus improve the transmembrane absorption efficiency of sodium selenite. Meanwhile, the simple organic selenium precursor produced by the preliminary conversion of sodium selenite can provide a substrate for the further metabolism of selenized carrageenan, accelerating its conversion into selenoproteins that are more easily accumulated in spore powder, thus achieving a two-way promotion of the absorption and conversion of organic and inorganic selenium.

[0018] L-glutamine activates the activity of enzymes such as selenocysteine ​​synthase and methyltransferase, accelerating the conversion of inorganic selenium and promoting the metabolism of selenized carrageenan in mycelia, converting it into a more biologically active and easily accumulated organic selenium form. At the same time, the increased absorption of selenized carrageenan and sodium selenite promotes the synthesis and utilization of L-glutamine, enhancing its precursor-mediated effect.

[0019] Optionally, the high-pressure sterilization method is: high-pressure sterilization at 121°C for 2.5 to 3.5 hours.

[0020] By adopting the above technical solutions, the high-pressure sterilization environment is used to thoroughly kill all kinds of miscellaneous bacteria and spores in the substrate, eliminating competition from miscellaneous bacteria; at the same time, excessive destruction of nutrients is avoided, and nutrients in the substrate that are beneficial to mycelial growth and selenium metabolism are retained, ensuring the robust growth of Ganoderma lucidum mycelium, maintaining good selenium absorption and conversion capacity, and thus improving the stability of the proportion of organic selenium.

[0021] Optionally, in the inoculation and culture steps, based on a culture cycle of 65-75 days, a culture inducer is added on the 10th-16th day of culture, and the amount of the culture inducer added accounts for 0.02-0.04 wt% of the total mass of the substrate. The method for preparing the culture inducer is as follows: dry powder of Cordyceps cephalosporin mycelium is selected and culture inducers are prepared by acid hydrolysis.

[0022] By adopting the above technical solution and using Cordyceps cephalosporin mycelium, the small molecule peptides produced after its degradation have high compatibility with the metabolic system of Ganoderma lucidum mycelium. This can quickly trigger the enhancement of the activity of selenocysteine ​​synthase and selenoprotein synthase in Ganoderma lucidum cells, further activating the selenium metabolic pathway of Ganoderma lucidum mycelium and promoting the conversion of inorganic selenium to organic selenium. Moreover, the degradation products are non-toxic and harmless small molecule peptides and nutrients, which will not inhibit the growth of Ganoderma lucidum mycelium or cause harmful residues in the spore powder, thus ensuring safety.

[0023] Days 10 to 16 represent the initial to mid-stage of rapid mycelial growth in Ganoderma lucidum. During this period, the mycelial mass increases rapidly, cell membrane permeability increases, and the metabolic system is fully activated. Enzymes related to selenium metabolism are in an inducible and easily activated state. After adding a culture inducer, the active ingredients in the inducer, such as small molecule peptides and polysaccharides, quickly penetrate the mycelial cell membrane and act as signaling molecules to trigger the activation of selenium metabolic pathways within Ganoderma lucidum cells. This promotes the expression and enhanced activity of key enzymes such as selenocysteine ​​synthase and selenoprotein synthase, accelerates the conversion of inorganic selenium to organic selenium, and simultaneously promotes the transport of organic selenium to the mycelial tip, which is beneficial for the accumulation of organic selenium after spore formation.

[0024] In addition, after the addition of the inducing agent, the active ingredients continue to work for 10 to 15 days, covering the rapid growth period of Ganoderma lucidum mycelium, ensuring that the induction effect covers the entire key process of organic selenium conversion, and continuously increasing the induction effect.

[0025] Optionally, the step of preparing culture inducers by acid hydrolysis is as follows: Raw material pretreatment: Add sterile deionized water to the dried powder of Cordyceps cephalosporin mycelium, adjust the pH to 5.5-6.0, add neutral protease, cellulase and pectinase, and carry out enzymatic hydrolysis to obtain enzymatic hydrolysate; Membrane separation and purification: The enzymatic hydrolysate is coarsely filtered through a filter screen to remove mycelial residue, and then sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 5000 Da, 3000 Da, and 1000 Da to collect small molecule peptides of 1000–3000 Da. Impurity removal and concentration: Disperse the small molecule peptides with sterile deionized water, let stand for 8 hours, centrifuge to remove trace amounts of precipitated impurities, and then evaporate and concentrate to 1 / 5 of the original volume to obtain a concentrated solution. Drying and sterilization: The concentrate is freeze-dried to obtain crude inducing agent; after pulverizing, it is passed through a 100-mesh sieve and then sterilized by gamma ray irradiation.

[0026] By employing the above technical solution, the raw material pretreatment involves enzymatic hydrolysis to degrade macromolecules in Cordyceps cephalosporin mycelium, releasing small molecule components with inducible activity. Membrane separation and purification utilize ultrafiltration membranes with different molecular weight cutoffs to collect the desired small peptides, remove macromolecular impurities, and improve the purity of the inducible factor. Impurity removal and concentration further remove trace precipitates, concentrate active ingredients, and enhance the induction effect. The drying and sterilization steps involve freeze-drying to preserve the inducible factor's activity and gamma-ray irradiation sterilization to prevent contamination by other microorganisms, ensuring the inducible factor's safety and effectiveness. This process yields highly active and pure inducible factors.

[0027] Optionally, the step of preparing culture inducers by acid hydrolysis further includes an enzyme inactivation step, wherein the enzyme inactivation step is performed after the raw material pretreatment step and before the membrane separation and purification step; the enzyme inactivation step includes: Keep warm at 50-60℃ for 25-35 minutes, then cool down to 20-25℃ within 10-20 minutes.

[0028] By adopting the above technical solution, an enzyme inactivation step is used to inactivate residual enzymes, destroy the spatial structure of enzymes, and thus lose their catalytic activity; rapid cooling avoids the continuous impact of high temperature on the active ingredients of the culture inducer, prevents small molecule peptides from denaturing due to high temperature, and maximizes the preservation of the activity of the inducer, thereby ensuring that the active ingredients of the prepared culture inducer are not destroyed, ensuring the stability of the induction effect, and thus steadily improving the organic selenium conversion efficiency of Ganoderma lucidum mycelium.

[0029] Optionally, the step of preparing culture inducers by acid hydrolysis further includes a centrifugal purification step, which is performed after the membrane separation purification step and before impurity removal and concentration; the centrifugal purification step includes: Centrifuge at 8000–10000 r / min for 15–20 min, and collect the supernatant after centrifugation.

[0030] By adopting the above technical solution, centrifugal force is used to completely separate the trace precipitates and suspended particles remaining after membrane separation. The supernatant is then used to obtain a small molecule peptide solution with higher purity, thereby improving the purity and activity of the culture inducer and ensuring that it can more efficiently activate the selenium metabolism of Ganoderma lucidum mycelium, and further promote the improvement of organic selenium conversion efficiency.

[0031] Optionally, in the drying and sterilization step, the crude culture inducer obtained after freeze-drying is mixed with β-cyclodextrin to obtain a mixture, and sterile deionized water is added at 5 to 7 times the total mass of the mixture. The mixture is stirred at 30 to 35°C and 150 to 200 r / min for 30 to 40 min to form an inclusion complex suspension. The inclusion complex suspension is freeze-dried and then pulverized through a 100-mesh sieve. The mass ratio of the crude culture inducer to β-cyclodextrin is 1:0.8 to 1.2.

[0032] By employing the above technical solution, β-cyclodextrin is used to encapsulate the active ingredient, a small molecule signal peptide, within a cavity of the elicitor, isolating it from external factors such as oxygen and moisture, preventing oxidative denaturation, and improving the stability and shelf life of the elicitor. When the inclusion complex is added to the substrate and enters the microenvironment of Ganoderma lucidum mycelium growth, the hydrophilic outer layer of β-cyclodextrin binds with the moisture in the substrate, causing its cavity structure to open slightly, and the small molecule peptide encapsulated inside is slowly released. At the same time, enzymes and small molecules produced during the metabolism of Ganoderma lucidum mycelium further trigger the inclusion balance of β-cyclodextrin to shift towards the release direction, allowing the signal molecules to be continuously and slowly released around the mycelium.

[0033] In summary, this application includes at least one of the following beneficial technical effects: 1. By pre-treating the raw sawdust, the contact area with Ganoderma lucidum mycelium is expanded, reducing the loss of selenium. At the same time, during the pre-piling process, the microorganisms and enzymes in the sawdust itself will carry out preliminary transformation of sodium selenite, converting some inorganic selenium into simple organic selenium precursors that are easily absorbed by the mycelium, ultimately achieving a significant increase in the proportion of organic selenium in the spore powder.

[0034] 2. By spraying Bacillus subtilis and Trichoderma harzianum into the raw sawdust, the dissolution and activation of selenium in the selenium-enriching agent are promoted, making it easier for the selenium to be recognized and absorbed by the Ganoderma lucidum mycelium that is subsequently inoculated. The strong antagonistic effect of Trichoderma harzianum is used to prevent other bacteria from competing for selenium and nutrients. At the same time, during the pre-piling treatment, aeration tubes extending to the bottom of the pile are inserted into the pile to ensure the dominant growth position of the Ganoderma lucidum mycelium.

[0035] 3. By using selenium-enriched carrageenan, cell membrane permeability is regulated after direct absorption by mycelia, and the transmembrane absorption efficiency of sodium selenite is improved; L-glutamine accelerates the conversion of inorganic selenium and promotes the metabolism of selenium-enriched carrageenan in mycelia, converting it into a more bioactive and easily accumulated organic selenium form.

[0036] 4. By adding cultivation inducers, the selenium metabolism pathway of Ganoderma lucidum mycelium is activated, increasing selenium absorption and promoting the conversion of inorganic selenium to organic selenium. Moreover, the degradation products are non-toxic and harmless small molecule peptides and nutrients, which will not inhibit the growth of Ganoderma lucidum mycelium or cause harmful residues in spore powder, ensuring safety. Attached Figure Description

[0037] Figure 1 This is a comparison chart of the total selenium and organic selenium content of Ganoderma lucidum spore powder collected in Examples 3, 9, and Comparative Examples 1-3. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments.

[0039] Unless otherwise specified, the experimental methods used in the embodiments of this application are conventional methods, and the materials used are commercially available unless otherwise specified.

[0040] Example 1: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0041] 1. An organic cultivation method for selenium-enriched Ganoderma lucidum spore powder, comprising the following steps: Preparation of substrate: The substrate comprises, by mass, 72 parts of virgin sawdust, 22 parts of wheat bran, 5 parts of corn flour, 0.8 parts of lime, and 63 parts of water. Mix the above substrate, stir evenly, pack into polypropylene bags, each bag with a wet weight of 1.4 ± 0.03 kg, and sterilize in an autoclave at 121°C for 3 hours to obtain the culture medium package. Inoculation and cultivation: After sterilization, the culture medium pack is naturally cooled to 27℃. Ganoderma lucidum liquid inoculum is used for inoculation, with an inoculation amount of 5wt% of the wet weight of the culture medium pack. The resulting spawn pack is then transferred to the cultivation workshop, where the temperature is maintained at 24±1℃ and the humidity at 60±5%. After 70 days of cultivation, the spawn pack reaches physiological maturity and is obtained. Cultivation and Management: The cultivated Ganoderma lucidum bags are transported to a digital greenhouse, placed on shelves with an 8cm spacing, and then managed for growth under the conditions of 28℃, 90% humidity, 650ppm carbon dioxide concentration and 650Lux light intensity. Collection: After 42 days of management during the Ganoderma lucidum growth period, the spore powder was collected. In this embodiment, a clean tent-type Ganoderma lucidum spore powder collection system was used to collect the spore powder. The raw wood chips are first pretreated by adding a selenium-enriching agent, sodium selenite, to the raw wood chips. The raw wood chips after adding the selenium-enriching agent are stirred evenly and then pre-piled. The pre-piling environment is 22.5±2.5℃ and 60% humidity. The pile is then formed into a pile 1.2m high and 1.5m wide and left to stand naturally for 36 hours, turning it over once every 12 hours. The amount of selenium-enriching agent added accounts for 0.007wt% of the total mass of the base material.

[0042] The components include: ① raw sawdust, which can be one or a combination of oak sawdust, birch sawdust, linden sawdust, and poplar sawdust; oak sawdust is used in this embodiment; ② wheat bran, which is organic wheat bran with a moisture content ≤10wt% and a crude protein content ≥18wt%; ③ corn flour with a moisture content ≤10wt% and a starch content ≥70wt%; ④ lime, which is quicklime calcium oxide; and ⑤ Ganoderma lucidum liquid spawn, which is Ganoderma lucidum and Ganoderma sinense liquid spawn with a spawn concentration of 1.2×10⁻⁶. 8 CFU / mL.

[0043] 2. Testing: The collected Ganoderma lucidum spore powder was tested for the following indicators: (1) Total selenium content (mg / kg): This reflects the overall enrichment capacity of Ganoderma lucidum spore powder for exogenous selenium. It is a basic indicator for judging selenium absorption efficiency and directly reflects the total amount of selenium transported from the substrate to the fruiting body / spores. The detection method is based on GB5009.93-2017 Method I and is performed using a fluorescence spectrometer. (2) Organic selenium content (mg / kg): reflects the ability of Ganoderma lucidum mycelium to convert inorganic selenium into organic forms of selenium such as selenoproteins, selenium polysaccharides, and selenoamino acids; the test is performed in accordance with Appendix A.2 of GB1903.23-2016. (3) Organic selenium conversion rate (%): reflects the conversion efficiency of inorganic selenium to safe and highly bioavailable organic selenium. The calculation formula is: Organic selenium conversion rate = (organic selenium content / total selenium content) × 100%.

[0044] Example 2: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0045] An organic cultivation method for selenium-enriched Ganoderma lucidum spore powder, wherein in the base material preparation step, the base material comprises, by mass parts: 83 parts of virgin sawdust, 17 parts of wheat bran, 8 parts of corn flour, 1.2 parts of lime, and 65 parts of water; the above base materials are mixed, stirred evenly, bagged, and then sterilized under high pressure to obtain a culture medium package; Everything else is the same as in Example 1.

[0046] Example 3: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0047] An organic cultivation method for selenium-enriched Ganoderma lucidum spore powder, wherein in the base material preparation step, the base material comprises, by mass parts: 77.5 parts of virgin sawdust, 19.5 parts of wheat bran, 6.5 parts of corn flour, 1.0 part of lime, and 64 parts of water; the above base materials are mixed, stirred evenly, bagged, and then sterilized under high pressure to obtain a culture medium package; Everything else is the same as in Example 1.

[0048] Example 4: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0049] In an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder, a compound microbial agent is uniformly sprayed onto native sawdust after the addition of a selenium-enriching agent, followed by pre-piling treatment; the amount of the compound microbial agent added accounts for 0.48 wt% of the native sawdust before pre-treatment; the compound microbial agent includes Bacillus subtilis agent and Trichoderma harzianum agent, wherein the mass ratio of Bacillus subtilis agent to Trichoderma harzianum agent is 6.5:1; In this embodiment, the viable count of Bacillus subtilis inoculant is ≥2.0 × 10⁻⁶. 9 CFU / mL; viable count of Trichoderma harzianum fungicide ≥1.5×10⁻⁶ 9 CFU / mL; Everything else is the same as in Example 3.

[0050] Example 5: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0051] In this embodiment, during the pre-stacking process, four ventilating conduits extending to the bottom are inserted into the stack. The initial oxygen concentration inside the stack is 12%. During the pre-stacking period, the oxygen concentration inside the stack is checked every 12 hours. If it is lower than 8%, the stack is turned over once more. The ventilating conduits can be either stainless steel or PVC. In this embodiment, a PVC conduit with a diameter of 1.5 cm is selected, and ventilating holes with a diameter of 0.3 cm are evenly distributed on the surface of the conduit. Everything else is the same as in Example 4.

[0052] Example 6: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0053] In this embodiment, the selenium-enriching agent further includes selenized carrageenan and L-glutamine, wherein the mass ratio of selenized carrageenan, L-glutamine, and sodium selenite is 1:0.6:4; wherein the purity of selenized carrageenan is ≥98% and the selenium content is ≥150mg / kg; and the purity of L-glutamine is ≥99%. Everything else is the same as in Example 5.

[0054] Example 7: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0055] An organic cultivation method for selenium-enriched Ganoderma lucidum spore powder, wherein in the inoculation and culturing steps, based on a 70-day cultivation cycle, a cultivation inducer is added on the 13th day of cultivation, and the amount of the cultivation inducer added accounts for 0.03 wt% of the total mass of the substrate; The method for preparing the culture inducer is as follows: using dried Cordyceps cephalosporin mycelium powder with a water content ≤8wt%, the culture inducer is prepared by acid hydrolysis. The steps for preparing culture inducers by acid hydrolysis are as follows: 1) Raw material pretreatment: The dried Cordyceps cephalosporin mycelium powder was pulverized to 90±10 mesh, and 9 times its weight of sterile deionized water was added. The mixture was stirred evenly to obtain a mycelium suspension. Citric acid was added to the mycelium suspension to adjust the pH of the system to 5.8. The mixture was stirred for 10 min to ensure uniform dispersion of the citric acid. The mixture was placed in a constant temperature water bath and the temperature was controlled at 30℃. The stirring speed was 120 r / min, and the mixture was acid-hydrolyzed for 3.5 h. The citric acid used in this embodiment was food grade. 2) Purification: Add sodium bicarbonate to the above system while stirring, adjust the pH of the system to 7.0, let it stand for 2 hours, and then filter it coarsely with a 300-mesh sterile filter to remove precipitates and undissolved impurities; 3) Concentration: Place it in a low-temperature rotary evaporator, concentrate at 35℃ and 0.03MPa, and evaporate and concentrate to 1 / 5 of the original volume; 4) Drying and sterilization: The concentrated acid hydrolysate was placed in a vacuum freeze dryer and freeze-dried at -40℃ and 0.02MPa for 14 hours. After pulverization, it was passed through a 100-mesh sieve and then sterilized using a Co... 60 Sterilization was carried out by irradiation with a dose of 2 kGy using gamma rays to obtain culture inducers.

[0056] Everything else is the same as in Example 6.

[0057] Example 8: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0058] In this embodiment, the step of preparing culture inducers by acid hydrolysis is as follows: Raw material pretreatment: The dried Cordyceps cephalosporin mycelium powder was pulverized to 90±10 mesh, and sterile deionized water was added in 9 times its weight. The mixture was stirred evenly to obtain a mycelium suspension. Citric acid was added to the mycelium suspension to adjust the pH of the system to 5.8. Then, 0.4 wt% of neutral protease, 0.3 wt% of cellulase, and 0.15 wt% of pectinase were added to the dried Cordyceps cephalosporin mycelium powder. The mixture was enzymatically hydrolyzed at 45℃ and 180 r / min for 2.5 h to obtain the enzymatic hydrolysate. Enzyme inactivation: Incubate at 55℃ for 30 min, then cool to 22℃ within 15 min; Membrane separation and purification: The enzymatic hydrolysate is coarsely filtered through a 300-mesh filter to remove mycelial residue, and then sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 5000 Da, 3000 Da, and 1000 Da. Small peptides of 1000–3000 Da are collected, while large molecular impurities greater than 5000 Da and small molecules less than 1000 Da are discarded. In this embodiment, a polysulfone ultrafiltration membrane is used. Centrifugal purification: Centrifuge at 9000 r / min for 18 min, and collect the supernatant after centrifugation; Impurity removal and concentration: The small molecule peptides were dispersed in sterile deionized water. The concentration of the dispersed small molecule peptide solution was 6.5 mg / mL. The solution was allowed to stand for 8 hours at 2-8℃, and then centrifuged at 5000 r / min for 10 min to remove trace amounts of precipitated impurities. The solution was then placed in a low-temperature rotary evaporator and concentrated at 35℃ and 0.03 MPa to evaporate and concentrate to 1 / 5 of the original volume to obtain the concentrated solution. Drying and sterilization: The concentrated solution was placed in a vacuum freeze dryer and freeze-dried at -40℃ and 0.02MPa for 14 hours to obtain crude inducing agents. The crude inducing agents were then pulverized and passed through a 100-mesh sieve, and then sterilized using a Co... 60 Sterilization was carried out by irradiation with a dose of 2 kGy using gamma rays to obtain culture inducers; Everything else is the same as in Example 7.

[0059] Example 9: This example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0060] In the drying and sterilization steps of preparing culture inducers by acid hydrolysis, the crude culture inducer obtained after freeze-drying was mixed with β-cyclodextrin to obtain a mixture. Sterile deionized water, six times the total mass of the mixture, was added, and the mixture was stirred at 32°C and 180 r / min for 35 min to form an inclusion complex suspension. The inclusion complex suspension was freeze-dried using the same parameters as in Example 8. It was then pulverized through a 100-mesh sieve, and subsequent steps were the same as in Example 8. The mass ratio of the crude culture inducer to β-cyclodextrin was 1:1. Everything else is the same as in Example 8.

[0061] Comparative Example 1: This comparative example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0062] In this comparative example, no selenium enrichment agent was added to the virgin wood chips during pretreatment. Everything else is the same as in Example 3.

[0063] The following indicators were tested on the Ganoderma lucidum spore powder collected in Example 3 and Comparative Example 1, respectively, as shown in Table 1.

[0064] Serial Number Testing items unit standard Comparative Example 1 Example 3 1 Cell wall breakage rate % ≥95 99.2 99.5 2 Moisture % ≤9.0 6.50 6.23 3 Total Ash % ≤3.0 1.40 1.10 4 Lead (Pb) mg / kg ≤2.0 Not detected (<0.04) Not detected (<0.04) 5 Total mercury (Hg) mg / kg ≤0.1 Not detected (<0.01) Not detected (<0.01) 6 Cadmium (Cd) mg / kg ≤0.5 0.074 0.056 7 Nickel (Ni) mg / kg ≤1.0 0.746 0.512 8 Chromium (Cr) mg / kg ≤2.0 0.98 0.61 9 Polysaccharides (calculated as anhydrous glucose) % ≥0.9 1.7 1.7 10 Total triterpenes g / 100g － 8.32 8.07 11 Peroxide value (based on Ganoderma lucidum spore oil) g / 100g ≤0.20 0.078 0.049 The above test results show that: (1) Basic quality indicators: ① Cell wall breakage rate: an indicator to measure the cell wall breakage effect of Ganoderma lucidum spore powder. Both Comparative Example 1 and Example 3 far exceeded the standard; ② Moisture: an indicator to reflect the storage stability of spore powder. Both measured values ​​met the standard. Example 3 was better than Comparative Example 1, indicating that Example 3 is more conducive to long-term storage; ③ Total ash: an indicator to reflect the purity of spore powder. Both met the standard. Example 3 was lower than Comparative Example 1, indicating higher purity.

[0065] (2) Heavy metal indicators (lead, total mercury, cadmium, nickel, chromium): These indicators reflect safety and all meet safety standards. Furthermore, the cadmium, nickel, and chromium content in Example 3 is lower than that in Comparative Example 1, indicating that the cultivation scheme in Example 3 is more conducive to reducing heavy metal residues in spore powder and is safer.

[0066] (3) Nutritional activity indicators: ① Polysaccharides: These are the core active ingredients of Ganoderma lucidum spore powder. Both Comparative Example 1 and Example 3 meet the standards and there is no difference, indicating that selenium-enriched pretreatment does not affect the polysaccharide content; ② Total triterpenes: One of the main active ingredients of Ganoderma lucidum. Comparative Example 1 is slightly higher than Example 3. Both are within a reasonable range and the difference is not significant.

[0067] (4) Oxidation index: ① Peroxide value: reflects the degree of oxidation of Ganoderma lucidum spore oil. Both Comparative Example 1 and Example 3 are much lower than the standard. Example 3 has an even lower degree of oxidation, indicating that the spore powder is more stable and easier to store.

[0068] Comparative Example 2: This comparative example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0069] In this comparative example, during the pretreatment of virgin wood chips, sodium selenite, a selenium-enriching agent, was added to the virgin wood chips at an amount of 0.0035 wt% of the total mass of the base material. Everything else is the same as in Example 3.

[0070] Comparative Example 3: This comparative example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0071] In this comparative example, during the pretreatment of virgin wood chips, sodium selenite, a selenium-enriching agent, was added to the virgin wood chips at an amount of 0.0105 wt% of the total mass of the base material. Everything else is the same as in Example 3.

[0072] Comparative Example 4: This comparative example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0073] In this comparative example, the autoclaving method was: autoclaving at 121℃ for 1 hour; Everything else is the same as in Example 3.

[0074] Comparative Example 5: This comparative example discloses an organic cultivation method for selenium-enriched Ganoderma lucidum spore powder.

[0075] In this comparative cultivation and management process, after the seedlings are placed on shelves and packaged, the spacing is maintained at 8cm. Then, the seedlings are managed under the conditions of 32°C, 85% humidity, 900ppm carbon dioxide concentration, and 300Lux light intensity. Other steps are the same as in Example 3.

[0076] The Ganoderma lucidum spore powders collected in Examples 1-9 and Comparative Examples 1-5 were tested, and the results are shown in Table 2. A comparison chart of the total selenium and organic selenium detection data for Examples 3, 9, and Comparative Examples 1-3 is also included. Figure 1 : Table 2. Detection results of Examples 1-9 and Comparative Examples 1-5 Experimental Groups Total selenium content (mg / kg) Organic selenium content (mg / kg) Organic selenium conversion rate (%) Example 1 148.00 147.40 99.59 Example 2 158.00 157.23 99.51 Example 3 154.00 153.54 99.70 Example 4 224.00 223.40 99.73 Example 5 274.00 273.60 99.86 Example 6 344.00 343.70 99.91 Example 7 364.30 364.12 99.95 Example 8 384.00 383.90 99.97 Example 9 398.11 398.02 99.98 Comparative Example 1 4.30 4.30 100.00 Comparative Example 2 94.70 94.38 99.66 Comparative Example 3 156.00 154.53 99.06 Comparative Example 4 149.00 138.60 93.02 Comparative Example 5 132.00 121.80 92.27 Comparing Examples 1-3, it can be seen that Example 1 has the highest proportion of wheat bran. The organic nutrients in wheat bran provide sufficient carbon and nitrogen sources for Ganoderma lucidum mycelium, promoting the metabolic activity of mycelium and thus improving the conversion efficiency of selenium, which is better than Example 2. Example 2 has the highest proportion of virgin sawdust. As the main carrier of selenium enrichment agent, its total selenium content is the highest among the three examples, but the reduced proportion of wheat bran leads to insufficient organic nutrients, resulting in the lowest conversion rate among the three examples. Example 3 is relatively balanced overall, with the highest organic selenium conversion rate. Overall, all three examples can meet the cultivation requirements of selenium-enriched Ganoderma lucidum spore powder.

[0077] By comparing Example 4 with Example 3, it can be seen that Example 4 added a compound microbial agent composed of Bacillus subtilis and Trichoderma harzianum, which improved the microenvironment of the substrate, inhibited the growth of miscellaneous bacteria, promoted the growth and reproduction of Ganoderma lucidum mycelium, enhanced the mycelium's adsorption and absorption capacity for selenium, and thus improved the metabolic efficiency of mycelium in converting inorganic selenium into organic selenium. Therefore, the total selenium and organic selenium content were significantly increased, and the conversion rate was also improved.

[0078] By comparing Example 5 with Example 4, it can be seen that in Example 5, during the pre-stack treatment, the insertion of a ventilated conduit into the stack effectively improved the oxygen supply inside the stack, maintaining the oxygen concentration inside the stack within a suitable range. Oxygen is a necessary condition for the aerobic respiration and metabolic activities of Bacillus subtilis and Trichoderma harzianum agents. Sufficient oxygen can significantly enhance the activity of the agents, thus the total selenium, organic selenium content and conversion rate are steadily improved.

[0079] By comparing Example 6 with Example 5, it can be seen that the selenium-enriching agent used in Example 6 adds selenized carrageenan and L-glutamine to sodium selenite. Selenized carrageenan can improve the solubility of selenium in the base material, reduce selenium precipitation loss, and make the mycelium more easily absorbed. L-glutamine, as an amino acid, promotes the metabolic synthesis of Ganoderma lucidum mycelium, enhances the bioconversion capacity of mycelium for selenium, and thus continuously increases the content of total selenium and organic selenium, and further improves the conversion rate.

[0080] By comparing Example 7 with Example 6, it can be seen that Example 7 added a cultivation inducer. The cultivation inducer was prepared by acid hydrolysis of dried Cordyceps cephalosporin mycelium powder. Its active ingredients can induce the expression of Ganoderma lucidum mycelium-related metabolic enzymes, further enhancing the mycelium's ability to enrich and transform selenium.

[0081] By comparing Example 8 with Example 7, it can be seen that Example 8 optimized the preparation process of the inducing agent, and used enzymatic hydrolysis and membrane separation to replace the traditional acid hydrolysis method, which can obtain small molecule peptide inducing substances with higher purity and stronger activity, further enhance the induction effect, promote the absorption and transformation of selenium by mycelium, and all indicators have increased to a certain extent.

[0082] By comparing Example 9 with Example 8, it can be seen that the addition of β-cyclodextrin in Example 9 to include the culture inducer improves the stability of the inducer and reduces its activity loss during the culture process, thereby maintaining the high conversion efficiency of the mycelium. The total selenium content and conversion rate are slightly increased, but the long-term effect is more sustained.

[0083] By comparing Comparative Example 1 and Example 3, it can be seen that no selenium enrichment agent was added during the pretreatment of raw sawdust in Comparative Example 1. The base material only contains trace amounts of selenium from the raw material itself. The Ganoderma lucidum mycelium does not have sufficient selenium source to absorb and enrich. Therefore, the total selenium and organic selenium content is significantly lower than that in Example 3, far below the level required for the actual application of selenium-enriched products. Moreover, because the total amount of selenium source is extremely low, it is only a theoretical conversion rate and has no practical significance for production and application, and cannot meet the product requirements of selenium-enriched Ganoderma lucidum spore powder.

[0084] By comparing Comparative Example 2 with Example 3, it can be seen that all indicators of Comparative Example 2 are lower than those of Example 3. The amount of selenium enrichment agent added in Comparative Example 2 is only 1 / 2 of that in Example 3. The selenium source supply in the base material is insufficient, and the total amount of inorganic selenium that Ganoderma lucidum mycelium can absorb is reduced, resulting in a significant decrease in both total selenium and organic selenium content. The conversion rate is only slightly lower than that of Example 3, which may be due to the reduction in selenium source and the slight difference in the proportion of trace losses during the conversion process.

[0085] By comparing Comparative Example 3 with Example 3, it can be seen that the amount of selenium-enriching agent added in Comparative Example 3 is 1.5 times that in Example 3, but the total selenium content only increases slightly. It is speculated that the selenium element is excessively supplied. However, under this condition, the absorption of the original sawdust and the subsequent absorption and utilization of Ganoderma lucidum mycelium have basically reached the maximum value under this condition. Therefore, the effect of increasing total selenium and organic selenium is limited.

[0086] By comparing Comparative Example 4 with Example 3, it can be seen that the difference between Comparative Example 4 and Example 3 is the high-pressure sterilization process. The sterilization time of Comparative Example 4 is shortened to 1 hour, which is much shorter than the 3 hours of Example 3. This results in incomplete sterilization of the substrate. The residual bacteria in the substrate will compete with the Ganoderma lucidum mycelium for nutrients, and at the same time inhibit the growth, reproduction and metabolic activity of the Ganoderma lucidum mycelium, weakening the mycelium's ability to absorb selenium and its conversion efficiency. Therefore, even though the total selenium content is close to that of Example 3, the organic selenium content and conversion rate still decrease.

[0087] By comparing Comparative Example 5 with Example 3, it can be seen that the environmental parameters of the cultivation management of Comparative Example 5 deviated from the suitable range. The temperature, humidity, carbon dioxide concentration and light intensity were all changed. The unsuitable environment will inhibit the activity of Ganoderma lucidum mycelium, slow down the absorption rate of selenium in the substrate by the mycelium, and reduce the metabolic efficiency of the mycelium in converting inorganic selenium into organic selenium, resulting in a decrease in both total selenium and organic selenium content, and a significant decrease in conversion rate.

[0088] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. An organic cultivation method for selenium-enriched Ganoderma lucidum spore powder, characterized in that, Includes the following steps: Preparation of substrate: The substrate comprises, by mass parts: 72-83 parts of virgin sawdust, 17-22 parts of wheat bran, 5-8 parts of corn flour, 0.8-1.2 parts of lime, and 63-65 parts of water; Mix the above substrate, stir evenly, pack into bags, and then autoclave to obtain culture medium bags; Inoculation and cultivation: Cool the culture medium to 26-28℃, inoculate with Ganoderma lucidum liquid spawn to obtain spawn bags, transfer the spawn bags to the cultivation workshop, and cultivate at 23-25℃ for 65-75 days to obtain Ganoderma lucidum spawn bags; Cultivation and management: First, place the seedlings on shelves and then manage their growth under the conditions of 26-30℃, humidity 88-92%, carbon dioxide concentration 500-800ppm, and light intensity 400-900Lux. Collection: Collect spores after 38–45 days of management; The raw wood chips are first pretreated by adding a selenium-enriching agent, including sodium selenite, to the raw wood chips and pre-piling for 24 to 48 hours; the amount of selenium-enriching agent added is 0.006 to 0.008 wt% of the total mass of the base material.

2. The organic cultivation method for selenium-enriched Ganoderma lucidum spore powder according to claim 1, characterized in that, A compound microbial agent is evenly sprayed onto the raw sawdust after the addition of selenium-enriching agent, followed by pre-piling treatment; the amount of the compound microbial agent added accounts for 0.36 to 0.60 wt% of the raw sawdust before pre-treatment; the compound microbial agent includes Bacillus subtilis agent and Trichoderma harzianum agent, wherein the mass ratio of Bacillus subtilis agent to Trichoderma harzianum agent is 5 to 8:

1.

3. The organic cultivation method for selenium-enriched Ganoderma lucidum spore powder according to claim 2, characterized in that, During the pre-stacking process, 3 to 5 ventilated conduits extending to the bottom are inserted into the stack to ensure that the oxygen concentration inside the stack is ≥8%.

4. The organic cultivation method for selenium-enriched Ganoderma lucidum spore powder according to claim 3, characterized in that, The selenium-enriching agent also includes selenized carrageenan and L-glutamine, wherein the mass ratio of selenized carrageenan, L-glutamine, and sodium selenite is 1:0.4-0.8:3-5.

5. The organic cultivation method for selenium-enriched Ganoderma lucidum spore powder according to claim 1, characterized in that, The high-pressure sterilization method is as follows: high-pressure sterilization at 121℃ for 2.5 to 3.5 hours.

6. The method for organic cultivation of selenium-enriched Ganoderma lucidum spore powder according to any one of claims 1-5, characterized in that, In the inoculation and culture steps, based on a culture cycle of 65-75 days, a culture inducer is added on the 10th to 16th day of culture, and the amount of the culture inducer added accounts for 0.02-0.04 wt% of the total mass of the substrate. The method for preparing the culture inducer is as follows: dry powder of Cordyceps cephalosporin mycelium is selected and culture inducers are prepared by acid hydrolysis.

7. The organic cultivation method for selenium-enriched Ganoderma lucidum spore powder according to claim 6, characterized in that, The steps for preparing culture inducers by acid hydrolysis are as follows: Raw material pretreatment: Add sterile deionized water to the dried powder of Cordyceps cephalosporin mycelium, adjust the pH to 5.5-6.0, add neutral protease, cellulase and pectinase, and carry out enzymatic hydrolysis to obtain enzymatic hydrolysate; Membrane separation and purification: The enzymatic hydrolysate is coarsely filtered through a filter screen to remove mycelial residue, and then sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 5000 Da, 3000 Da, and 1000 Da to collect small molecule peptides of 1000–3000 Da. Impurity removal and concentration: Disperse the small molecule peptides with sterile deionized water, let stand for 8 hours, centrifuge to remove trace amounts of precipitated impurities, and then evaporate and concentrate to 1 / 5 of the original volume to obtain a concentrated solution. Drying and sterilization: The concentrate is freeze-dried to obtain crude inducing agent; after pulverizing, it is passed through a 100-mesh sieve and then sterilized by gamma ray irradiation.

8. The organic cultivation method for selenium-enriched Ganoderma lucidum spore powder according to claim 7, characterized in that, The step of preparing culture inducers by acid hydrolysis further includes an enzyme inactivation step, which is performed after the raw material pretreatment step and before the membrane separation and purification step; the enzyme inactivation step includes: Keep warm at 50-60℃ for 25-35 minutes, then cool down to 20-25℃ within 10-20 minutes.

9. The method for organic cultivation of selenium-enriched Ganoderma lucidum spore powder according to claim 8, characterized in that, The step of preparing culture inducers by acid hydrolysis also includes a centrifugal purification step, which is set after the membrane separation purification step and before impurity removal and concentration. The centrifugal purification step includes: Centrifuge at 8000–10000 r / min for 15–20 min, and collect the supernatant after centrifugation.

10. The method for organic cultivation of selenium-enriched Ganoderma lucidum spore powder according to any one of claims 7-9, characterized in that, In the drying and sterilization steps, the crude culture inducer obtained after freeze-drying is mixed with β-cyclodextrin to obtain a mixture. Sterile deionized water of 5 to 7 times the total mass of the mixture is added, and the mixture is stirred at 30 to 35°C and 150 to 200 r / min for 30 to 40 min to form an inclusion complex suspension. The inclusion complex suspension is freeze-dried and then pulverized through a 100-mesh sieve. The mass ratio of the crude culture inducer to β-cyclodextrin is 1:0.8 to 1.2.

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