Selenium-rich ganoderma lucidum mycelium as well as culture method and application thereof

Abstract

The invention provides selenium-rich ganoderma lucidum mycelia and a culture method and application thereof, and belongs to the technical field of edible fungus culture. The method comprises the following steps: inoculating a lucid ganoderma strain to a solid culture medium, and culturing; inoculating the strain cultured in the step (1) to a liquid culture medium, and carrying out shake cultivation; and (3) inoculating the strain obtained by shake cultivation in the step (2) to a liquid culture medium, and performing enlarged cultivation to obtain the selenium-rich ganoderma lucidum mycelium. According to the culture method disclosed by the invention, the content of organic selenium in ganoderma lucidum mycelia, namely the content of organic selenium in ganoderma lucidum basswood cultivation seeds, can be greatly increased, so that the content of organic selenium in ganoderma lucidum sporocarp and ganoderma lucidum spore powder which can be harvested from ganoderma lucidum is increased. In the selenium-rich culture process, the selenium content has the effects of low-concentration promotion and high-concentration inhibition on the growth of the plants.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi culture technology, and particularly relates to a selenium-enriched Ganoderma lucidum mycelium, its culture method, and its application. Background Technology

[0002] Reishi mushroom, the dried fruiting body of Ganoderma lucidum or Ganoderma sinense, is a fungus belonging to the Polyporaceae family. It has the effects of tonifying qi and calming the mind, relieving cough and asthma. Its mycelium is rich in various bioactive components such as Ganoderma lucidum polysaccharides, triterpenoids, and amino acids. It has a variety of pharmacological effects such as enhancing immunity, anti-oxidation, and regulating metabolism. It is widely used in the research and development and production of pharmaceuticals, health foods and functional foods.

[0003] Selenium is an essential trace element for the human body, participating in the synthesis and activation of various enzymes such as glutathione peroxidase. It plays a vital role in maintaining immune function, preventing cardiovascular disease, and combating oxidative damage. However, the human body cannot synthesize selenium and must obtain it through dietary intake. Inorganic selenium has low bioavailability and poor safety, with the maximum effective dose and minimum toxic dose of inorganic selenium being similar. Organic selenium, on the other hand, has advantages such as high bioavailability and low toxicity, making it more suitable as a dietary source of selenium. By utilizing the biotransformation of Ganoderma lucidum mycelium, inorganic selenium in the culture medium is converted into organic selenium accumulated within the mycelium, thus preparing selenium-enriched Ganoderma lucidum mycelium. This process preserves the inherent biological activity of Ganoderma lucidum while also endowing it with selenium supplementation functions, achieving the dual value of being both a food and a medicine and fortifying trace elements.

[0004] Currently, the cultivation technology of selenium-enriched Ganoderma lucidum mycelium mainly focuses on two directions: liquid submerged fermentation and solid-state cultivation. Although existing technologies have made some progress, there are still many technical bottlenecks that need to be solved in practical applications, which limit the large-scale production and quality improvement of selenium-enriched Ganoderma lucidum mycelium: First, the rationality of selenium source addition is insufficient, with problems such as improper timing of addition or uncontrolled concentration. Selenium concentration exceeding the strain's tolerance threshold will significantly inhibit mycelial growth, reduce selenium conversion rate, and some high-selenium concentration cultures will also lead to mycelial cell damage and off-odors in the fermentation broth. Second, the optimization of culture medium formulation is insufficient. Existing formulations mostly use the carbon-nitrogen ratio and composition of ordinary Ganoderma lucidum mycelium culture without targeted adjustments for the selenium conversion process, resulting in low selenium conversion efficiency of mycelium and difficulty in increasing the proportion of organic selenium. Third, the control of culture conditions is complex. The selenium bioconversion process is affected by multiple factors such as temperature and culture time, resulting in large fluctuations in mycelial selenium content between batches and poor product quality stability.

[0005] Furthermore, existing selenium-enriched Ganoderma lucidum mycelium culture technologies still suffer from problems such as insufficient organic selenium enrichment and some products failing to meet national standards for selenium content. Therefore, there is an urgent need to develop a selenium-enriched Ganoderma lucidum mycelium culture method that features precise selenium source addition, scientific culture medium formulation, and controllable culture parameters, enabling efficient selenium enrichment of mycelium, stable biomass, and complete preservation of active ingredients. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a selenium-enriched Ganoderma lucidum mycelium, its cultivation method and application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for culturing selenium-enriched Ganoderma lucidum mycelium, comprising the following steps: (1) Inoculate the Ganoderma lucidum strain onto a solid culture medium and culture it; (2) Inoculate the bacterial strain obtained in step (1) onto a liquid culture medium and culture it in a shaker; (3) The strain obtained from the shaker culture in step (2) is inoculated onto a liquid culture medium and expanded to obtain selenium-enriched Ganoderma lucidum mycelium; The liquid culture medium comprises the following components: glucose 6-7 g / L, soybean meal 6-7 g / L, sucrose 15-25 g / L, yeast extract 0.3-0.4 g / L, potassium dihydrogen phosphate 0.1-0.3 g / L, vitamin B1 0.01-0.02 g / L, MgSO4·7H2O 0.01-0.02 g / L, corn flour 1-1.5 g / L, selenium powder 1-30 g / L, Ganoderma lucidum spore powder residue 1-3 g / L, and Ganoderma lucidum spore oil 0.04-0.08 g / L.

[0008] Preferably, the solid culture medium in step (1) is PDA culture medium, the culture temperature is 22~27℃, and the culture time is 4~6 days.

[0009] Preferably, the inoculation amount of the strain in step (2) is 2 to 4% of the total mass of the liquid culture medium.

[0010] Preferably, the shaking speed of the shaker culture in step (2) is 100~200 rpm, the shaking temperature is 25~30℃, and the shaking time is 4~6 days.

[0011] Preferably, the inoculation amount of the strain in step (3) is 3 to 7% of the total mass of the liquid culture medium.

[0012] Preferably, the rotation speed of the expansion culture in step (3) is 100~200 rpm, the temperature of the expansion culture is 25~30℃, and the expansion culture time is 6~8 days.

[0013] The present invention also provides selenium-enriched Ganoderma lucidum mycelium obtained by the aforementioned cultivation method.

[0014] The present invention also provides the application of the aforementioned selenium-enriched Ganoderma lucidum mycelium in the preparation of selenium-enriched Ganoderma lucidum products.

[0015] Preferably, the selenium-enriched Ganoderma lucidum product includes selenium-enriched Ganoderma lucidum fruiting body, selenium-enriched Ganoderma lucidum spore powder, and selenium-enriched Ganoderma lucidum spore oil.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The cultivation method of this invention can significantly increase the content of organic selenium in Ganoderma lucidum mycelium, that is, the content of organic selenium in the seeds used for Ganoderma lucidum cultivation on linden wood, thereby increasing the content of organic selenium in the harvestable fruiting bodies and spore powder of Ganoderma lucidum. During selenium-enriched cultivation, selenium content promotes growth at low concentrations and inhibits growth at high concentrations. Furthermore, the organic selenium enriched in Ganoderma lucidum products has a beneficial regulatory effect on human physiology, particularly playing a crucial role in the growth of mitochondria in somatic cells, the repair of organelles, and the immune regulation of human diseases. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the fermentation mycelium of Qifeng Ganoderma lucidum in liquid culture medium; Figure 2 It is a stock solution of selenium-containing bacterial powder; Figure 3 This is a schematic diagram of the mycelium fermentation in a shaker on liquid culture medium for Qifeng Ganoderma lucidum; Figure 4 This is a schematic diagram of the microbial activity test in a fermenter; Figure 5 This is a schematic diagram showing the mycelial fermentation before and after fermentation under different concentrations of selenium powder. Detailed Implementation

[0018] This invention provides a method for culturing selenium-enriched Ganoderma lucidum mycelium, comprising the following steps: (1) Inoculate the Ganoderma lucidum strain onto a solid culture medium and culture it; (2) Inoculate the bacterial strain obtained in step (1) onto a liquid culture medium and culture it in a shaker; (3) The strain obtained from the shaker culture in step (2) is inoculated onto a liquid culture medium and expanded to obtain selenium-enriched Ganoderma lucidum mycelium.

[0019] In this invention, Ganoderma lucidum strains are inoculated onto a solid culture medium and cultured. The solid culture medium is PDA medium, and the culture temperature is preferably 22-27°C, more preferably 23-26°C, and even more preferably 25°C; the culture time is preferably 4-6 days, more preferably 4.5-5.5 days, and even more preferably 5 days.

[0020] In this invention, the bacterial strain obtained in step (1) is inoculated onto a liquid culture medium and cultured on a shaker. The inoculation amount of the bacterial strain is preferably 2-4% of the total mass of the liquid culture medium, more preferably 2.5-3.5%, and even more preferably 3%. The liquid culture medium comprises the following components: glucose 6-7 g / L, soybean meal 6-7 g / L, sucrose 15-25 g / L, yeast extract 0.3-0.4 g / L, potassium dihydrogen phosphate 0.1-0.3 g / L, vitamin B1 0.01-0.02 g / L, and MgSO4·7H2O. The following ingredients are used: 0.01~0.02 g / L, corn flour 1~1.5 g / L, selenium powder 1~30 g / L, Ganoderma lucidum spore powder residue 1~3 g / L, and Ganoderma lucidum spore oil 0.04~0.08 g / L; the preferred amount of glucose is 6.2~6.8 g / L, more preferably 6.5 g / L; the preferred amount of soybean meal is 6.2~6.8 g / L, more preferably 6.5 g / L; the preferred amount of sucrose is 18~22 g / L. The preferred concentration of the first step is 20 g / L; the preferred concentration of the yeast extract is 0.32~0.38 g / L, more preferably 0.35 g / L; the preferred concentration of potassium dihydrogen phosphate is 0.15~0.25 g / L, more preferably 0.2%; the preferred concentration of vitamin B1 is 0.012~0.018 g / L, more preferably 0.015 g / L; the preferred concentration of MgSO4·7H2O is 0.012~0.018 g / L. / L, more preferably 0.015g / L; the amount of selenium powder added is preferably 5g / L; the amount of corn flour added is preferably 1~1.5g / L, more preferably 1.1~1.4g / L, and even more preferably 1.2g / L; the amount of Ganoderma lucidum spore powder residue added is preferably 1~3g / L, more preferably 1.5~2.5g / L, and even more preferably 2g / L; the Ganoderma lucidum spore powder residue is the waste residue left after extracting Ganoderma lucidum spore oil from Ganoderma lucidum spore powder, that is, Ganoderma lucidum spore powder residue. This residue is usually discarded. Adding Ganoderma lucidum spore powder residue to the culture medium is to reuse the water-soluble effective components remaining in the Ganoderma lucidum spore powder residue after extracting Ganoderma lucidum spore oil from Ganoderma lucidum spore powder. These water-soluble effective components are not lost in the Ganoderma lucidum spore powder oil extraction process and can be reused because only the fat-soluble effective components in Ganoderma lucidum spore powder are transferred to Ganoderma lucidum spore oil in the extraction process. The preferred dosage of the added Ganoderma lucidum spore oil is 0.04~0.08 g / L, more preferably 0.05~0.07 g / L, and even more preferably 0.06 g / L. Ganoderma lucidum spore powder residue and Ganoderma lucidum spore oil can accelerate the growth rate and quantity of Ganoderma lucidum mycelium. Compared with the culture medium without the addition of Ganoderma lucidum spore powder residue and Ganoderma lucidum spore oil, the Ganoderma lucidum mycelium with these two materials grows 3~4 days faster.The rotation speed of the shaker culture is preferably 100-200 rpm, more preferably 120-180 rpm, and even more preferably 150 rpm; the temperature of the shaker culture is preferably 25-30℃, more preferably 26-29℃, and even more preferably 28℃; the shaker culture time is preferably 4-6 days, more preferably 4.5-5.5 days, and even more preferably 5 days.

[0021] In this invention, the solid culture medium and the liquid culture medium are sterilized before use. The sterilization temperature is preferably 118~125℃, more preferably 120~123℃, and even more preferably 121℃. The sterilization time is preferably 15~25min, more preferably 18~22min, and even more preferably 20min.

[0022] In this invention, the bacterial strain obtained from the shaker culture in step (2) is inoculated onto a liquid culture medium for expansion culture. The culture medium is sterilized before use. The sterilization temperature is preferably 105-121℃, more preferably 110-120℃; the sterilization pressure is preferably 1-2 MPa, more preferably 1.5 MPa; the sterilization time is preferably 30-50 min, more preferably 35-45 min, and even more preferably 40 min; the inoculation amount of the bacterial strain is preferably 3-7% of the total mass of the culture medium, more preferably 4-6%, and even more preferably 5%; the expansion culture rotation speed is preferably 100-200 rpm, more preferably 120-180 rpm, and even more preferably 150 rpm; the expansion culture temperature is preferably 25-30℃, more preferably 26-29℃, and even more preferably 28℃; the expansion culture time is preferably 6-8 days, more preferably 6.5-7.5 days, and even more preferably 7 days.

[0023] The present invention also provides selenium-enriched Ganoderma lucidum mycelium obtained by the aforementioned cultivation method.

[0024] This invention also provides the application of the aforementioned selenium-enriched Ganoderma lucidum mycelium in the preparation of selenium-enriched Ganoderma lucidum products. In this invention, the selenium-enriched Ganoderma lucidum products include selenium-enriched Ganoderma lucidum fruiting bodies, selenium-enriched Ganoderma lucidum spore powder, and selenium-enriched Ganoderma lucidum spore oil.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] The cultivation method for selenium-enriched Ganoderma lucidum mycelium is as follows: I. Culture medium preparation 1. Prepare the culture medium.

[0028] 2. Dissolve.

[0029] 3. Fill the Erlenmeyer flasks (1L flasks, filled with 500-600 ml of culture medium).

[0030] 4. Bandage and sterilize (121℃, 20min).

[0031] II. Inoculation and Culture (Primary Fermentation) for Preparation of Basic Seeds

[0032] 1. Activation: Take the test tube mother culture of Qifeng Ganoderma (obtained from mature Ganoderma fruiting bodies by tissue culture from Longquan mushroom sticks purchased by Ouyuan Edible Fungus Professional Cooperative in Longquan City, Zhejiang Province) out of the refrigerator and place it at room temperature for 1 day.

[0033] 2. Inoculate with PDA solid medium: Pick a pea-sized piece of bacteria from the activated test tube mother culture and place it in a 90mm PDA agar dish. Incubate at 25℃ for 5 days. During this period, you can also perform another blind passage.

[0034] 3. Inoculation with liquid culture medium: Take the inoculum from the agar plate and inoculate it into an Erlenmeyer flask with liquid culture medium (6.5 g / L glucose, 6.5 g / L soybean meal, 20 g / L sucrose, 0.35 g / L yeast extract, 0.2 g / L potassium dihydrogen phosphate, 0.015 g / L vitamin B1, 0.015 g / L MgSO4·7H2O, 1.2 g / L corn flour, 5 g / L selenium powder (the selenium powder purchased from Jiangsu Duoyang Bioengineering Technology Co., Ltd. was ground and pulverized, then a suitable amount of water was added and quickly stirred into a homogeneous mixture. After standing, the supernatant was collected to obtain the selenium powder solution). Figure 2 As shown), 2 g / L of Ganoderma lucidum spore powder residue (the waste residue remaining after extracting Ganoderma lucidum spore oil from Ganoderma lucidum spore powder), and 0.06 g / L of Ganoderma lucidum spore oil (preparation method see CN106318616B-A Ganoderma lucidum spore oil / CN106381220B-A Ganoderma lucidum spore oil preparation process) are used, with the inoculation amount being 3% of the total mass of the liquid culture medium in the Erlenmeyer flask. Figure 1 and Figure 3 As shown.

[0035] 4. Shake incubation, 150 rpm, 28℃, 5 days.

[0036] III. Expanded culture (secondary fermentation) to prepare working seeds

[0037] 1. Prepare liquid culture medium in a culture tank (1500L).

[0038] 2. Culture medium sterilization: 120℃, 1.5mPa, 40min.

[0039] 3. Inoculation: Inoculate 5% of the total mass of the culture medium in the culture vessel.

[0040] 4. Incubation: 28℃, 150 rpm, 7 days. The viability of the microorganisms in the fermenter should be tested. Figure 4 As shown.

[0041] Experimental Example 1

[0042] Effects of selenium concentration on the growth of Ganoderma lucidum mycelium

[0043] The basic culture medium formula is as follows: glucose 6.5 g / L, sucrose 20 g / L, yeast extract 0.35 g / L, potassium dihydrogen phosphate 0.2 g / L, vitamin B1 0.015 g / L, MgSO4·7H2O 0.015 g / L, soybean meal 6.5 g / L, and corn flour 1.2 g / L.

[0044] The selenium powder was divided into five dosage groups: 5, 10, 15, 20, and 25 g / L, and was added according to the method in Example 1.

[0045] Add 0.2 cm of liquid culture medium to a 250 ml Erlenmeyer flask containing 100 ml of liquid culture medium. 2 PDA solid mycelium was cultured at 25℃ for 7 days, then centrifuged, filtered, and dried. Wet weight, dry weight, cell yield, and moisture content were measured. The mycelial fermentation status before and after fermentation is shown in the figure. Figure 5 As shown.

[0046] Cell yield = Dry weight of mycelium after culture (g) / Volume of fermentation broth (L) = g / L Moisture content (water content) = (Wet weight of mycelium after culture - Dry weight of mycelium after culture / Wet weight of mycelium after culture) 100.

[0047] Experimental results are shown in Table 1.

[0048] Table 1. Effects of different selenium concentrations on the growth of Ganoderma lucidum mycelium.

[0049] As shown in Table 1, under liquid culture conditions, the addition of selenium showed a dose-dependent inhibitory effect on mycelial biomass accumulation within the tested concentration range.

[0050] Biomass showed a significant decreasing trend. Both mycelial dry weight and yield decreased significantly and continuously with increasing selenium concentration. The highest dry weight (13.37 g) occurred at the lowest concentration of 5 g / L, while at the highest concentration of 25 g / L, the dry weight had dropped to about half of that (6.40 g). This indicates that under the experimental conditions, selenium mainly inhibited the growth of Ganoderma lucidum mycelium, and the higher the concentration, the stronger the inhibition.

[0051] Unlike the linear decrease in dry weight, the moisture content did not change monotonically with increasing selenium concentration, exhibiting non-linear fluctuations. It reached its lowest point (10.50%) at 15 g / L and peaked at 20 g / L (22.59%). This indicates that different concentrations of selenium affect the metabolic activity or cell wall permeability of mycelial cells. Selenium content promotes growth at low concentrations and inhibits growth at high concentrations.

[0052] The mycelial yield reached its highest point (53.48 g / L) at a selenium powder concentration of 5 g / L, and then continuously decreased, reaching its lowest point (25.60 g / L) at a selenium powder concentration of 25 g / L. The highest mycelial yield was observed at a selenium powder concentration of 5 g / L, confirming 5 g / L as the optimal concentration for Ganoderma lucidum mycelial growth.

[0053] Experiment Example 2

[0054] The Nanjing Municipal Institute of Product Quality Supervision and Inspection was commissioned to test the content of total selenium and organic selenium in the bacterial broth and mycelium before and after primary and secondary fermentation in Example 1.

[0055] Experimental results are shown in Table 2.

[0056] Table 2. Content of total selenium and organic selenium in bacterial culture and mycelium at different stages.

[0057] Table 2 shows that, taking total selenium as an example, the total selenium content in the bacterial broth before primary fermentation was 16.2 mg / kg, while after primary fermentation it was 0.329 mg / kg. This indicates a significant decrease in the total selenium content in the fermentation broth, suggesting that the total selenium in the fermentation broth (culture medium) was transferred from the fermentation broth to the Ganoderma lucidum mycelium during fermentation. After primary fermentation, the total selenium content in the mycelium was 51 mg / kg, while before secondary fermentation it was 5.3 mg / kg. This more directly demonstrates that as fermentation progresses, selenium gradually transfers from the fermentation broth (culture medium) to the mycelium, increasing the selenium content in the mycelium. The same principle applies to organic selenium.

[0058] Experimental Example 3

[0059] The selenium-enriched Ganoderma lucidum mycelium obtained in Example 1 was regenerated and inoculated onto sterilized linden wood. The linden wood was then transplanted to soil within a greenhouse for cultivation. Selenium-enriched Ganoderma lucidum fruiting bodies were grown in the greenhouse. These fruiting bodies were then processed into selenium-enriched Ganoderma lucidum spore powder according to the method described in CN106318616B - A Method for Extracting Ganoderma lucidum Spore Oil. Ganoderma lucidum spore oil was extracted from the spore powder. The organic selenium content and absorption attenuation conversion rate in the Ganoderma lucidum fruiting bodies and selenium-enriched Ganoderma lucidum spore powder were measured. The absorption attenuation conversion rate was calculated as: (Selenium content of harvested Ganoderma lucidum fruiting bodies (organic selenium)) / (Selenium content of harvested Ganoderma lucidum spore powder (organic selenium)).

[0060] Experimental results: The harvested Ganoderma lucidum fruiting bodies contained 68.5 mg / kg of mycelium selenium, and the broken-cell wall Ganoderma lucidum spore powder contained 6.883 mg / kg of mycelium selenium, with an absorption attenuation conversion rate of 10.05%.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing selenium-enriched Ganoderma lucidum mycelium, characterized in that, Includes the following steps: (1) Inoculate the Ganoderma lucidum strain onto a solid culture medium and culture it; (2) Inoculate the bacterial strain obtained in step (1) onto a liquid culture medium and culture it in a shaker; (3) The strain obtained from the shaker culture in step (2) is inoculated onto a liquid culture medium and expanded to obtain selenium-enriched Ganoderma lucidum mycelium; The liquid culture medium comprises the following components: glucose 6-7 g / L, soybean meal 6-7 g / L, sucrose 15-25 g / L, yeast extract 0.3-0.4 g / L, potassium dihydrogen phosphate 0.1-0.3 g / L, vitamin B1 0.01-0.02 g / L, MgSO4·7H2O 0.01-0.02 g / L, corn flour 1-1.5 g / L, selenium powder 1-30 g / L, Ganoderma lucidum spore powder residue 1-3 g / L, and Ganoderma lucidum spore oil 0.04-0.08 g / L.

2. The cultivation method according to claim 1, characterized in that, The solid culture medium in step (1) is PDA culture medium, the culture temperature is 22~27℃, and the culture time is 4~6 days.

3. The cultivation method according to claim 1, characterized in that, The inoculation amount of the strain in step (2) is 2-4% of the total mass of the liquid culture medium.

4. The cultivation method according to claim 1, characterized in that, In step (2), the shaking speed of the shaker culture is 100~200 rpm, the temperature of the shaker culture is 25~30℃, and the shaking time is 4~6 days.

5. The cultivation method according to claim 1, characterized in that, The inoculation amount of the strain in step (3) is 3-7% of the total mass of the liquid culture medium.

6. The cultivation method according to claim 1, characterized in that, The rotation speed of the expansion culture in step (3) is 100~200 rpm, the temperature of the expansion culture is 25~30℃, and the expansion culture time is 6~8 days.

7. Selenium-enriched Ganoderma lucidum mycelium cultured by the culture method according to any one of claims 1 to 6.

8. The application of the selenium-enriched Ganoderma lucidum mycelium as described in claim 7 in the preparation of selenium-enriched Ganoderma lucidum products.

9. The application according to claim 8, characterized in that, The selenium-enriched Ganoderma lucidum products include selenium-enriched Ganoderma lucidum fruiting bodies, selenium-enriched Ganoderma lucidum spore powder, and selenium-enriched Ganoderma lucidum spore oil.

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