A production method and application of selenium-enriched Gastrodia elata

Abstract

This invention relates to the field of Gastrodia elata cultivation technology, specifically to a method for producing and applying selenium-enriched Gastrodia elata. By using selenium-enriched Armillaria mellea spawn for Gastrodia elata cultivation, this invention significantly increases the selenium content in commercial Gastrodia elata, significantly improves its yield, and enhances the effective components specified in the Chinese Pharmacopoeia of Gastrodia elata. While increasing the selenium content, it also improves the yield and quality of Gastrodia elata.

Description

Technical Field

[0001] This invention relates to the field of Gastrodia elata cultivation technology, specifically to a method for producing selenium-enriched Gastrodia elata and its application. Background Technology

[0002] Selenium (Se) is an essential trace element for the human body to maintain normal life activities. It can regulate redox balance, participate in thyroid hormone metabolism, maintain immune function, and play an important role in physiological processes such as antioxidation, anti-aging, and tumor suppression. The human body can only obtain selenium from the external environment, and consuming selenium-rich animal and plant products is the most effective way to supplement selenium. Therefore, developing selenium-enriched foods as a means of exogenous selenium supplementation is of great significance.

[0003] Gastrodia elata is a plant belonging to the genus Gastrodia in the Orchidaceae family. Its dried tuber is not only a traditional Chinese medicine, but also demonstrates significant application value in the fields of both medicinal and edible uses. Modern research has confirmed that Gastrodia elata is rich in various bioactive substances such as phenols, polysaccharides, and organic acids. These components endow it with multiple physiological functions, including antioxidation, neuroprotection, and immune regulation, enabling it to play an important role in both clinical treatment and daily health care, and its application value has been widely recognized.

[0004] Exogenous selenium supplementation for enhanced cultivation is a highly efficient method for plant selenium enrichment. This technology induces plants to absorb and accumulate selenium by adjusting the selenium content in the soil or substrate, thereby significantly increasing the selenium content of crops and achieving the production of "selenium-enriched agricultural products." Currently, there are reports on the use of selenium-enriched germination bacteria, selenium-enriched Armillaria mellea, and Gastrodia elata seeds for cultivation. The highest selenium content in Gastrodia elata is 99.328 μg / kg, but the selenium accumulation rate is low, limiting the application scope of selenium-enriched Gastrodia elata and restricting its development value as a highly efficient selenium supplement. Therefore, it is urgent to optimize the production methods of selenium-enriched Gastrodia elata to improve the selenium accumulation effect. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for producing selenium-enriched Gastrodia elata and its application.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for producing selenium-enriched Gastrodia elata, comprising cultivating Gastrodia elata using selenium-enriched Armillaria mellea spawn; wherein the amount of exogenous selenium added to the selenium-enriched Armillaria mellea spawn is 0.1~50 ppm.

[0007] Furthermore, the amount of exogenous selenium added to the selenium-enriched Armillaria mellea spawn is 0.1~40 ppm.

[0008] Furthermore, the amount of exogenous selenium added is 0.1~35 ppm, specifically 0.1 ppm, 2 ppm, 4 ppm, 8 ppm, 16 ppm, 32 ppm or 35 ppm, etc., but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0009] Furthermore, the exogenous selenium includes inorganic selenium or organic selenium. The inorganic selenium includes sodium selenite, and the organic selenium includes barley grass.

[0010] Furthermore, the cultivation of Gastrodia elata using selenium-enriched Armillaria mellea spawn includes: planting seed hemp (white-headed hemp) around the selenium-enriched Armillaria mellea spawn, with the tail of the seed hemp facing the selenium-enriched Armillaria mellea spawn and the bud facing outwards; covering it with a 5-10 cm thick layer of Gastrodia elata planting substrate; and covering the top layer with a 5-10 cm thick layer of mulch for moisture retention.

[0011] Furthermore, the cultivation of Gastrodia elata using selenium-enriched Armillaria mellea spawn includes: from November to May of the following year, field cultivation of Gastrodia elata using selenium-enriched Armillaria mellea spawn; selecting fresh spawn material about 15 cm in length and placing it vertically in a cultivation box, filling the gaps with Gastrodia elata planting soil or substrate, then placing the selenium-enriched Armillaria mellea spawn on the spawn material, filling the sides of the spawn with fresh spawn material (chestnut wood), filling the gaps with Gastrodia elata planting soil or substrate, planting seed hemp around the upper part of the spawn, with the tail of the seed hemp facing the spawn and the bud facing outward, then filling and completely covering it with Gastrodia elata planting soil or substrate to a thickness of 5-10 cm, and covering it with a 5-10 cm thick layer of covering material to retain moisture.

[0012] Furthermore, the fungal material is selected from either oak or chestnut wood.

[0013] Furthermore, the covering material is selected from one or more of straw, sawdust, leaves, and cogongrass.

[0014] Furthermore, the preparation method of the selenium-enriched Armillaria mellea spawn includes: weighing the base material, adding exogenous selenium, adding water to the above materials and stirring evenly, then adding lime, gypsum, KH2PO4 and MgSO4, stirring evenly, maintaining the water content at 50%~60%, obtaining a mixture, sealing the mixture in bags, sterilizing, and obtaining selenium-enriched base material spawn; inoculating the Armillaria mellea fermentation broth or Armillaria mellea solid inoculum (secondary inoculum) into the selenium-enriched base material spawn, sealing the bags after inoculation, and culturing in a culture room at 15℃-25℃ for 2~3 months to obtain the selenium-enriched Armillaria mellea spawn.

[0015] Furthermore, the carbon-nitrogen ratio of the base material is 25-50, and the base material is selected from two or more combinations of chestnut wood chips, cottonseed hulls, corn kernels, corn cobs, soybean meal, and wheat bran.

[0016] Furthermore, the amounts of lime, gypsum, KH2PO4 and MgSO4 added are 0.4%, 0.4%, 0.04% and 0.06% of the mass of the base material, respectively.

[0017] Furthermore, the bagging and sealing of the mixture includes: packing the mixture into a polyethylene plastic bag with a length of 25-45 cm and a diameter of 8-15 cm, with 1000-3500 g of filler per bag, and sealing it.

[0018] Furthermore, the Armillaria mellea fermentation broth contains more than 90% Armillaria mellea mycelial balls.

[0019] Furthermore, the inoculation volume of Armillaria mellea fermentation broth in a single selenium-enriched substrate stick is 30-60 mL.

[0020] Furthermore, the preparation of the Armillaria mellea fermentation broth includes: inoculating test tubes of Armillaria mellea into a liquid culture medium for fermentation culture.

[0021] Furthermore, the fermentation culture conditions are: 24℃, 140 rpm for 7-10 days.

[0022] Furthermore, the liquid culture medium formula for Armillaria mellea is as follows: 5 g / L of slag, 2 g / L of corn flour, 20 g / L of glucose, 2 g / L of peptone, 20 g / L of yeast extract, 0.6 g / L of potassium dihydrogen phosphate, and 0.6 g / L of magnesium sulfate, sterilized at high temperature for later use.

[0023] Furthermore, the Armillaria mellea is either Qiangmi 41 or A9.

[0024] A second aspect of the present invention provides the application of the above-described production method in the production of selenium-enriched Gastrodia elata.

[0025] The beneficial technical effects of one or more of the above technical solutions are as follows: This invention provides a method for producing selenium-enriched Gastrodia elata. By using selenium-enriched Armillaria mellea spawn for Gastrodia elata cultivation, the selenium content in commercial Gastrodia elata can be significantly increased, with the total selenium content reaching up to 32.41 mg / kg. This significantly increases the yield of Gastrodia elata and also significantly enhances the effective components specified in the Chinese Pharmacopoeia of Gastrodia elata. While increasing the selenium content of Gastrodia elata, the yield and quality of Gastrodia elata are also improved. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1The diagram shows the growth of Armillaria mellea after adding organic selenium (mint rice) and inorganic selenium (sodium selenite) to the solid culture medium. A is a schematic diagram of the growth of Armillaria mellea after adding organic selenium to the culture medium, and B is a schematic diagram of the growth of Armillaria mellea after adding different concentrations of inorganic selenium to the culture medium.

[0028] Figure 2 The diagram shows the total selenium content of the mycelial cords of Armillaria mellea after adding organic selenium (mineralized rice) and inorganic selenium (sodium selenite) to the solid culture medium of Armillaria mellea. A shows the result of the determination of the total selenium content of the mycelial cords of Armillaria mellea after adding organic selenium to the culture medium, and B shows the result of the determination of the total selenium content of the mycelial cords of Armillaria mellea after adding inorganic selenium to the culture medium.

[0029] Figure 3 The selenium content and speciation of selenium in the mycelium of *Armillaria mellea* were determined by adding inorganic selenium (sodium selenite) to the liquid culture medium. A represents the total selenium content, M1 indicates that the amount of sodium selenite added is 21 mg / 600 mL, M2 indicates that the amount of sodium selenite added is 42 mg / 600 mL, and M3 indicates that the amount of sodium selenite added is 84 mg / 600 mL; B is the chromatogram of different selenium speciation standards; C is the speciation of selenium in the mycelium of *Armillaria mellea*.

[0030] Figure 4 This is a schematic diagram of the fermentation broth of Armillaria mellea.

[0031] Figure 5 The growth of Armillaria mellea on mycelium sticks with different concentrations of exogenous selenium is shown. A is the blank control, BD is the treatment group with inorganic selenium (sodium selenite) added, with selenium concentrations of 2.5 ppm, 7.5 ppm and 15 ppm, respectively, and EG is the treatment group with organic selenium (Cephalotaxus fortunei) added, with selenium concentrations of 2.5 ppm, 7.5 ppm and 15 ppm, respectively.

[0032] Figure 6 This diagram illustrates the effect of using selenium-enriched (organic selenium, *Armillaria mellea*) mycelium sticks on the growth of *Gastrodia elata*. A represents the growth of *Gastrodia elata* cultivated with mycelium sticks without exogenous selenium treatment (blank); B represents the growth of *Gastrodia elata* cultivated with mycelium sticks containing 2.22 ppm exogenous organic selenium; C represents the growth of *Gastrodia elata* cultivated with mycelium sticks containing 6.66 ppm exogenous organic selenium; and D represents the growth of *Gastrodia elata* cultivated with mycelium sticks containing 13.33 ppm exogenous organic selenium.

[0033] Figure 7 This is a schematic diagram illustrating the effect of using selenium-enriched Armillaria mellea spawn on the yield of Gastrodia elata; in the bar chart, the same letter indicates no significant difference, and different letters indicate significant differences.

[0034] Figure 8This diagram illustrates the effect of using selenium-enriched (organic selenium, *Armillaria mellea*) mycelium sticks to cultivate *Gastrodia elata* on the total selenium content in *Gastrodia elata*. A shows the change in total selenium content in mother *Gastrodia elata* (seedlings) treated with different exogenous selenium concentrations; B shows the change in total selenium content in commercial *Gastrodia elata* treated with different exogenous selenium concentrations; and C shows the results of selenium determination in different forms in commercial *Gastrodia elata*. In the bar chart, the same letter indicates no significant difference, and different letters indicate significant differences.

[0035] Figure 9 This diagram illustrates the effect of using selenium-enriched (Eriocaulon buergerianum) Armillaria mellea spawn on the content of effective components in Gastrodia elata. A represents the results of gastrodin content determination, B represents the results of p-hydroxybenzyl alcohol determination, C represents the results of barisonoside E determination, D represents the results of barisonoside C determination, E represents the results of barisonoside B determination, and F represents the results of barisonoside A determination.

[0036] Figure 10 The effect of using common Armillaria mellea spawn to cultivate Gastrodia elata and applying exogenous inorganic selenium fertilizer on the selenium enrichment effect of Gastrodia elata; in the bar chart, the same letter indicates no significant difference, and different letters indicate significant differences. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise stated, the experimental methods disclosed in this invention employ conventional techniques in the art, and the reagents and raw materials used in the examples are commercially available.

[0038] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, or combinations thereof.

[0039] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0040] The Armillaria mellea used in the examples was Qiangmi 41, purchased from Hanzhong Lisen Technology Development Co., Ltd.; the broken rice grass was provided by Enshi Academy of Agricultural Sciences; sodium selenite was purchased from Sinopharm Group analytical grade reagent, catalog number TO069750G; and the seed hemp (white-headed hemp) was black and red hybrid Gastrodia elata.

[0041] Armillaria mellea solid culture medium formula (g / L): Take 20 g of chestnut sawdust and 50 g of wheat bran, add water and boil for 30 min. After boiling, filter with gauze, take the filtrate, add 4 g of corn flour, 20 g of glucose, 6 g of soybean meal, 2 g of peptone, 2 g of yeast extract, 0.6 g of potassium dihydrogen phosphate, 0.6 g of magnesium sulfate, and 7.5 g of agar. Add water to make up to 1 L, autoclave and set aside for later use.

[0042] Formula for Armillaria mellea liquid culture medium: Weigh out 5 g / L of mixed residue, 2 g / L of corn flour, 20 g / L of glucose, 2 g / L of peptone, 2 g / L of yeast extract, 0.6 g / L of potassium dihydrogen phosphate, and 0.6 g / L of magnesium sulfate. Sterilize at high temperature for later use.

[0043] Example 1: Cultivation of selenium-enriched Armillaria mellea Appropriate amounts of exogenous selenium can promote the growth of Armillaria mellea. The purpose of this embodiment is to confirm the enrichment effect of exogenous selenium by Armillaria mellea and to screen the amount of exogenous selenium to be added.

[0044] 1. Solid culture: 0.1~32 ppm organic selenium (mineralized rice) / 0.1~32 ppm inorganic selenium (sodium selenite) were added to the solid culture medium of Armillaria mellea to supplement the exogenous selenium agent. After inoculation with Armillaria mellea, the medium was placed in a constant temperature culture at 25℃. The growth of Armillaria mellea was observed and photographed on the day of inoculation, day 10, and day 15. Mycelial cords of Armillaria mellea that had grown for more than 15 days were collected for determination of total selenium content.

[0045] like Figure 1 As shown in A and B, compared to the control, the application of 0.1–32 ppm organic selenium (Citrus malachite) can promote the growth of Armillaria mellea mycelia in the early stage and promote the formation of mycelial cords in the later stage of growth. The application of 0.1–32 ppm inorganic selenium (sodium selenite) can promote the formation of mycelial cords in the later stage. Especially at 15 days of treatment, the cells turned red at concentrations of 16 ppm and 32 ppm, indicating that elemental selenium may be converted and accumulated.

[0046] Armillaria mellea can absorb exogenous selenium from the culture medium, and the total selenium content in Armillaria mellea increases with increasing exogenous selenium concentration, such as... Figure 2 As shown in A and B, when organic selenium is added, the peak total selenium content in Armillaria mellea can reach about 300 mg / kg, and when inorganic selenium is added, the peak total selenium content in Armillaria mellea can reach about 1500 mg / kg.

[0047] 2. Liquid fermentation: Inorganic selenium (sodium selenite) was added to the liquid culture medium to supplement exogenous selenium. The sodium selenite addition amounts were 21 mg / 600 mL, 42 mg / 600 mL, and 84 mg / 600 mL, respectively. After inoculation with Armillaria mellea, the mixture was placed on a shaker at 24 ℃ and 140 rpm for fermentation. The growth of Armillaria mellea was observed. After 10 days, the fermentation mycelial balls were collected by filtration, freeze-dried, and then the total selenium content and different selenium forms were determined.

[0048] like Figure 3 As shown in Figure A, when inorganic selenium (sodium selenite) was added to the liquid culture medium for the fermentation culture of Armillaria mellea, the total selenium content in the Armillaria mellea cells increased with the increase of sodium selenite addition. The results of speciation of selenium are shown in Figure A. Figure 3 As shown in B and C, various organic selenium forms exist in Armillaria mellea cells.

[0049] Example 2: Cultivation of Armillaria mellea in selenium-containing substrate Obtaining Armillaria mellea culture: Take a 1*1cm piece of crushed Armillaria mellea mycelial cord cultured on solid culture medium, inoculate it into 600 mL of liquid culture medium, and ferment at 24℃ and 140 rpm for 8 days. Then add 600 mL of the fermented culture to a fermenter containing 800 L of liquid culture medium and ferment at 24℃ for 8 days to obtain the Armillaria mellea fermentation culture. Figure 4 As shown, Armillaria mellea mycelial balls account for more than 90% of the fermentation broth.

[0050] 2. Preparation of selenium-containing base material bars: Weigh sawdust, corn kernels, wheat bran, and soybean meal according to the dosages in Table 1. The final carbon-nitrogen ratio of the culture medium should be between 25 and 50. Add exogenous selenium agents (inorganic selenium (sodium selenite) treatment group, with selenium concentrations of 2.5 ppm, 7.5 ppm, and 15 ppm respectively; organic selenium (chopped rice) treatment group, with selenium concentrations of 2.5 ppm, 7.5 ppm, and 15 ppm respectively). Add water to the above materials and stir evenly. Then add lime, gypsum, KH2PO4, and MgSO4, add water and stir evenly to make the moisture content of the materials 50% to 60%. Pack the materials into polyethylene plastic bags with a length of 42 cm and a diameter of 10 to 12 cm, with 3000 to 3500 g of filler per bag. Tie the bag tightly and autoclave at 121°C for 1 to 2 hours to obtain sterilized selenium-containing base material bars.

[0051] Table 1. Composition ratio of basic feed bar components from different selenium sources

[0052] 3. Cultivation of *Armillaria mellea* in selenium-enriched substrate: Inoculate each sterilized selenium-enriched substrate with 40-45 mL of fermented *Armillaria mellea* culture. After inoculation, seal the substrate in a bag and incubate at 15℃-25℃ for 2-3 months. Figure 5 As shown in A, B, C, D, E, F, and G, adding exogenous selenium to the substrate can promote the growth of Armillaria mellea. The growth of Armillaria mellea on substrates with added organic selenium is better than that on substrates with added inorganic selenium.

[0053] Example 3 Production of selenium-enriched Gastrodia elata 1. The preparation of Armillaria mellea bacterial solution is the same as in Example 2.

[0054] 2. Preparation of selenium-containing substrate sticks: Weigh sawdust, cottonseed hulls, corn kernels, corn cobs, soybean meal, and broken watercress according to the dosages in Table 2. While adding water, stir in a mixer, and add 0.75 kg of lime, 0.75 kg of gypsum, 0.08 kg of KH₂PO₄, and 0.11 kg of MgSO₄. Stir until homogeneous, add water to achieve a moisture content of 50%–60%, and pack into polyethylene plastic bags (42 cm long, 10–12 cm in diameter), using 3000–3500 g of filler per bag. Seal the bags tightly and autoclave at 121℃ for 1–2 hours to obtain sterilized selenium-containing substrate sticks. The selenium concentrations of the organic selenium (broken watercress) are 2.22 ppm, 6.66 ppm, and 13.33 ppm, respectively.

[0055] Table 2. Component ratios for mushroom substrates with different selenium contents

[0056] 3. Preparation of selenium-enriched Armillaria mellea spawn: In a sterile inoculation room, each sterilized selenium-containing substrate spawn is inoculated with 40-45 mL of fermented Armillaria mellea broth. After inoculation, the spawn is sealed in a bag and placed in an incubation room at 15℃-25℃ for 2-3 months to obtain the spawn.

[0057] 4. Cultivation of Gastrodia elata: From November to May of the following year, Gastrodia elata is cultivated in the field using selenium-enriched Armillaria mellea spawn. Select fresh spawn (chestnut wood) approximately 15 cm in length and place it vertically in the incubation box. Fill the gaps with Gastrodia elata planting soil or substrate. Then place the selenium-enriched Armillaria mellea spawn on the spawn, filling the sides of the spawn with fresh spawn (chestnut wood). Fill the gaps with Gastrodia elata planting soil or substrate. Plant seed hemp (white-headed hemp) around the top of the spawn, with the tail of the seed hemp facing the spawn and the buds facing outwards. Fill and completely cover with Gastrodia elata planting soil or substrate, ensuring a thickness of 5-10 cm. Cover with a 5-10 cm thick layer of mulch (pine needles) to retain moisture. Perform routine field management.

[0058] 5. Gastrodia elata harvesting and processing: Gastrodia elata samples were collected, and the total fresh weight of Gastrodia elata collected from a single mycelium was weighed. Grades 1-3 Gastrodia elata were collected and transported back to the laboratory. After washing, they were steamed thoroughly, sliced, and dried in a constant temperature drying oven at 55℃ until constant weight. The powder was then passed through a No. 4 sieve. Each treatment was repeated 3 times.

[0059] 6. Determination of Total Selenium Content and Specified Selenium in Gastrodia elata: The total selenium content in both seed and commercial Gastrodia elata was determined using inductively coupled plasma mass spectrometry (ICP-MS) according to GB 5009.268-2016 National Food Safety Standard for the Determination of Multiple Elements in Food. A standard curve was prepared, and the selenium content was determined using the instrument, with parallel blank correction. Specified selenium was determined using HPLC-ICP-MS.

[0060] 7. Determination of active ingredients in Gastrodia elata: The determination was performed according to the method for determining active ingredients under the Gastrodia elata section of the Chinese Pharmacopoeia. The preparation method of the standard is as follows: Accurately weigh appropriate amounts of each reference standard and prepare a mixed reference solution containing 397.80 μg / mL of gastrodin, 74.00 μg / mL of p-hydroxybenzyl alcohol, 1378.40 μg / mL of parisonoside E, 814.32 μg / mL of parisonoside B, 219.42 μg / mL of parisonoside C, and 1111.286 μg / mL of parisonoside A in 50% methanol. The mixed reference solution was then determined under chromatographic conditions.

[0061] Exogenous selenium can be transferred to Gastrodia elata through Armillaria mellea. Using selenium-enriched (organic selenium, crushed rice) Armillaria mellea substrate to cultivate Gastrodia elata can improve its yield and quality. For example... Figure 6 A, B, C, D and Figure 7 As shown, the yield of Gastrodia elata was higher than that of the control after the addition of exogenous selenium. Moreover, the yield of Gastrodia elata was significantly higher than that of the control when the concentration of selenium was 6.66 ppm and 13.33 ppm. With the increase of exogenous selenium concentration, the yield of Gastrodia elata could be increased by 1.67% to 29.51% compared with the control.

[0062] Treatment with different concentrations of selenium can increase the total selenium content in Gastrodia elata, such as Figure 8 As shown in A, the total selenium content in both mother hemp (seedlings) and commercial hemp significantly increased with increasing exogenous selenium concentration, with the highest total selenium content in mother hemp reaching 58.78 mg / kg. Figure 8 As shown in B, the total selenium content in commercial Gastrodia elata can reach up to 32.41 mg / kg. Different forms of selenium were determined in the collected Gastrodia elata samples, and the results were compared with reference standards. Figure 8 As shown in C, various organic selenium substances, such as selenocysteine, methylselenocysteine, and selenomethionine, were found in the Gastrodia elata sample.

[0063] like Figure 9As shown in Figures A, B, C, D, E, and F, except for a decrease in the content of balithinoside E in different treatment groups compared to the control, the contents of gastrodin, p-hydroxybenzyl alcohol, balithinoside C, balithinoside B, and balithinoside A in different treatment groups all showed an increasing trend compared to the control group. At an exogenous selenium application concentration of 2.22 ppm, gastrodin, p-hydroxybenzyl alcohol, balithinoside C, and balithinoside B showed their maximum values. Balithinoside A peaked at the 6.66 ppm treatment group.

[0064] Comparative Example 1: Effect of exogenous selenium fertilizer application on the selenium enrichment capacity of Gastrodia elata 1. The preparation of Armillaria mellea bacterial solution is the same as in Example 2.

[0065] 2. Preparation of mushroom substrate: Weigh out 125 kg of sawdust, 15 kg of cottonseed hulls, 21 kg of corn kernels, 21 kg of corn cobs, and 6.75 kg of soybean meal. Add water to the mixture while stirring, and add 0.75 kg of lime, 0.75 kg of gypsum, 0.08 kg of KH2PO4, and 0.11 kg of MgSO4. Stir until uniform, add water to make the moisture content 50%~60%, and pack the mixture into polyethylene plastic bags 42 cm long and 10~12 cm in diameter, with 3000~3500 g of filler per bag to obtain selenium-enriched substrate substrate.

[0066] 3. Preparation of Armillaria mellea spawn: The selenium-containing substrate spawn is autoclaved and cooled. In a sterile inoculation room, each spawn is inoculated with 40-45 mL of fermented Armillaria mellea broth. After inoculation, the spawn is sealed in a bag and placed in a culture room at 15℃-25℃ for 2-3 months to obtain the spawn.

[0067] 4. Cultivation of Gastrodia elata: Select fresh fungal material (chestnut wood) about 15 cm in length and place it vertically in the incubator. Fill the gaps with Gastrodia elata planting substrate, then place the Armillaria mellea spawn on the chestnut wood, placing fresh fungal material (chestnut wood) on both sides of the spawn. Fill the gaps with Gastrodia elata planting soil or substrate. Plant seed hemp (white-headed hemp) around the top of the spawn, with the tail of the seed hemp facing the spawn and the bud facing outward. After applying 2 kg of planting substrate with inorganic selenium added (sodium selenite added at 16 ppm and 24 ppm respectively) to the top of the spawn, fill and completely cover with Gastrodia elata planting soil or substrate to a thickness of 5-10 cm. Cover with another 5-10 cm thick layer of mulch (pine needles) to retain moisture. Perform routine field management.

[0068] 5. The harvesting and processing of Gastrodia elata, and the determination of the total selenium content in Gastrodia elata are the same as in Example 3.

[0069] The results are as follows Figure 10 As shown, when Gastrodia elata is cultivated with exogenous inorganic selenium (sodium selenite), selenium can be enriched in the commercial Gastrodia elata, but the selenium content is low, with a maximum of only 0.35 mg / kg.

[0070] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for producing selenium-enriched Gastrodia elata, characterized by, This includes cultivating Gastrodia elata using selenium-enriched Armillaria mellea spawn; the amount of exogenous selenium added to the selenium-enriched Armillaria mellea spawn is 0.1~32 ppm; the exogenous selenium is organic selenium, and the organic selenium is *Cephalotaxus fortunei*.

2. The production method according to claim 1, characterized by, Cultivating Gastrodia elata using selenium-enriched Armillaria mellea spawn sticks involves: planting Gastrodia elata seedlings around the selenium-enriched Armillaria mellea spawn sticks, with the tail of the seedlings facing the selenium-enriched Armillaria mellea spawn sticks and the buds facing outwards; covering them with Gastrodia elata planting soil or substrate for 5-10 cm; and finally covering the top layer with 5-10 cm of mulch material to retain moisture.

3. The production method according to claim 1, wherein The preparation method of the selenium-enriched Armillaria mellea spawn includes: weighing the base material, adding exogenous selenium, adding water to the base material and stirring evenly, then adding lime, gypsum, KH2PO4 and MgSO4, stirring evenly, maintaining the moisture content at 50%~60%, obtaining a mixture, sealing the mixture in bags, sterilizing, and obtaining selenium-enriched base material spawn; inoculating the Armillaria mellea fermentation broth or Armillaria mellea solid inoculum into the selenium-enriched base material spawn, sealing the bags after inoculation, and culturing in a culture room at 15℃-25℃ for 2~3 months to obtain the selenium-enriched Armillaria mellea spawn.

4. The production method as described in claim 3, characterized in that, The carbon-to-nitrogen ratio of the base material is 25-50.

5. The production method as described in claim 3, characterized in that, The amounts of lime, gypsum, KH2PO4 and MgSO4 added are 0.4%, 0.4%, 0.04% and 0.06% of the mass of the base material, respectively.

6. The production method as described in claim 3, characterized in that, The bagging and sealing of the mixture includes: packing the mixture into polyethylene plastic bags with a length of 25-45 cm and a diameter of 8-15 cm, with 1000-3500 g of filler per bag.

7. The production method as described in claim 3, characterized in that, The inoculation volume of Armillaria mellea fermentation broth in a single selenium-enriched substrate bar is 30-60 mL.

8. The production method as described in claim 3, characterized in that, The preparation of the Armillaria mellea fermentation broth includes: inoculating test tubes of Armillaria mellea into a liquid culture medium for fermentation culture.

9. The production method as described in claim 8, characterized in that, The fermentation conditions are: 140 rpm, 24℃ for 7-10 days.

10. The application of the production method according to any one of claims 1-9 in the production of selenium-enriched Gastrodia elata.

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