Selenium-rich pleurotus eryngii mycelium and fermentation method thereof

Abstract

The fermentation method comprises the following steps that a pleurotus eryngii strain is inoculated to a PDA culture medium slant, the PDA culture medium slant is placed in an incubator for constant-temperature culture, and the slant is full of the pleurotus eryngii strain for standby application; the method comprises the following steps: peeling potatoes, cutting into small blocks, putting into a pot, adding 1000mL of distilled water, boiling, filtering to obtain potato filtrate, adding glucose, monopotassium phosphate, magnesium sulfate, naphthylacetic acid (NAA) and / or indolebutyric acid (IBA), heating and dissolving, complementing the distilled water until the total volume of the solution reaches 1000m, sealing and bundling, sterilizing at 121 DEG C for 15-30 minutes to obtain a seed solution culture medium, and inoculating the seed solution culture medium into a culture medium for later use; the method comprises the following steps: inoculating a pleurotus eryngii seed solution into a seed solution culture medium according to the inoculum size of 8-14% (v / v), culturing for liquid fermentation, filtering or centrifuging the cultured fermentation liquor, collecting mycelia, and washing and drying to obtain the selenium-enriched pleurotus eryngii mycelia. The cultivation time can be shortened, and meanwhile the selenium content of the pleurotus eryngii mycelium is increased.

Description

Technical Field

[0001] The invention relates to the technical field of fungus cultivation, and in particular to selenium-enriched Pleurotus eryngii mycelium and a fermentation method thereof. Background Art

[0002] Pleurotus eryngii is a new variety of rare edible fungi that can be used as both medicine and food. It is not only rich in nutrients such as polysaccharides, proteins, mineral elements and vitamins, but also has multiple pharmacological effects such as antioxidant and anti-tumor. Selenium is an essential trace element that the human body cannot synthesize on its own and can only obtain from external sources such as food (drugs) and drinking water. Supplementing selenium is crucial to human health. There are three main forms of selenium: inorganic selenium, organic selenium, and nano-selenium. Compared with inorganic forms, organic selenium has higher physiological activity, lower toxicity, and higher utilization rate by the body.

[0003] Edible fungi have strong selenium enrichment ability. By creating a local high-concentration selenium environment, the fungi can absorb selenium in the environment during growth. The selenium enters the cells, and the material metabolism in the cells can bind it to macromolecular active substances (such as polysaccharides and proteins), and convert it into organic forms of selenium such as polysaccharide selenium and protein selenium, thereby increasing the selenium content of the edible fungi themselves. The current edible fungi selenium enrichment cultivation technology most commonly uses cultivation technology, that is, by adding inorganic selenium to the culture material or using other selenium-containing raw materials (selenium-rich areas) to cultivate selenium-enriched edible fungi on a large scale, and the optimal amount of selenium added is determined based on the selenium enrichment rate, recovery rate, yield and economic cost, but there are disadvantages such as low selenium absorption rate and long production cycle. Summary of the invention

[0004] The object of the present invention is to provide a selenium-enriched Pleurotus eryngii mycelium and a fermentation method thereof, which can shorten the cultivation time and improve the selenium content of the Pleurotus eryngii mycelium.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A fermentation method for selenium-enriched Pleurotus eryngii mycelium comprises the following steps:

[0007] Step 1: Activation of Pleurotus eryngii strains

[0008] Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 25-30℃ incubator for constant temperature culture, and wait for use after the mycelium has covered the slant;

[0009] Step 2: Prepare seed culture medium

[0010] Selecting a fermentation medium composition, the fermentation medium composition includes 200-240g of potatoes, 20-24g of glucose, 0.8-1.2g of potassium dihydrogen phosphate and 0.1-0.5g of magnesium sulfate, and 0.01-0.025g of naphthylacetic acid NAA and / or 0.005-0.02g of indolebutyric acid IBA, peeling the potatoes, cutting them into small pieces and putting them into a pot, adding 1000mL of distilled water and boiling, filtering to obtain a potato filtrate, adding glucose, potassium dihydrogen phosphate and magnesium sulfate, and naphthylacetic acid NAA and / or indolebutyric acid IBA, heating to dissolve, supplementing with distilled water until the total volume of the solution reaches 1000mL, sealing and bundling, and sterilizing at 121°C for 15-30min to obtain a seed liquid culture medium;

[0011] Step 3: Preparation of Pleurotus eryngii seed liquid

[0012] Cut the activated Pleurotus eryngii mycelium into 1 cm 2 Small pieces are inoculated into a 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, with 10 pieces placed in each flask, first placed in a 25-30℃ incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 4-6 days to obtain Pleurotus eryngii seed liquid;

[0013] Step 4: Gradient Selenium Culture

[0014] The Pleurotus eryngii seed liquid is inoculated into a seed liquid culture medium at an inoculation rate of 8%-14% (v / v) for liquid fermentation. The culture conditions are as follows: the temperature of the fermentation environment is maintained at 24-30°C, the rotation speed is set to 130-160r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, sodium selenite is added in batches according to a gradient until the final concentration reaches 5-30μg / mL on the 7th day; the cultured fermentation liquid is filtered or centrifuged, and the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained through washing and drying.

[0015] Furthermore, the preparation method of the PDA culture medium in step 1 includes the following steps: peeling potatoes, cutting them into small pieces and putting them into a pot, adding 1000 mL of distilled water, boiling and filtering, taking 200-240 g of potato filtrate, adding 20-24 g of glucose and agar respectively, heating and stirring continuously with a glass rod to dissolve them, and adding water to 1000 mL, dividing the culture medium into conical flasks and sealing and tying them, sterilizing at 121° C. for 15-30 min, and obtaining the PDA culture medium.

[0016] The specific method of adding sodium selenite in batches according to the gradient in step 4 is as follows: on the second day after the start of fermentation, the sodium selenite concentration in the culture medium is first increased to 1 μg / mL, and then the sodium selenite selenium concentration in the culture medium is gradually increased every day according to the linear gradient selenium addition method until the final concentration reaches 5-30 μg / mL on the 7th day.

[0017] Furthermore, the rotation speed of the centrifugal treatment in step 4 is 8000-10000 rpm.

[0018] The selenium-rich Pleurotus eryngii mycelium cultured by the fermentation method has a total selenium content of 288.3-351.22 μg / g and an organic selenium content of 267.93-335.67 μg / g.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention selects a suitable seed liquid culture medium, wherein glucose is used as a high-quality carbon source to quickly provide sufficient energy for the metabolic activities of the mycelium, and potassium dihydrogen phosphate and magnesium sulfate supplement essential mineral elements to promote the growth of Pleurotus eryngii mycelium and the absorption and conversion of selenium. In addition, naphthaleneacetic acid NAA and / or indolebutyric acid IBA are also added to the seed liquid culture medium, and naphthaleneacetic acid NAA can affect the relaxation of the cell wall of the mycelium, thereby promoting the extension and branching of the mycelium and accelerating the growth rate of the mycelium. The addition of IBA helps to improve the absorption and conversion efficiency of Pleurotus eryngii mycelium to selenium, thereby increasing the selenium content in the mycelium. When NAA and IBA act synergistically, the permeability of the mycelium cell membrane can be adjusted, and the nutrient absorption capacity of Pleurotus eryngii mycelium in the culture medium can be improved. At the same time, the addition of NAA and IBA can also optimize the internal structure of the mycelium, making it more compact and uniform, which helps to increase the biomass and selenium content of the mycelium, and is also beneficial to the subsequent fermentation and cultivation process.

[0021] The present invention also gradually adds sodium selenite to the culture medium in a gradient selenium addition manner, thereby ensuring the reasonable absorption of selenium and avoiding the inhibitory effect of excessive selenium on mycelium growth. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below in conjunction with specific embodiments.

[0023] Embodiment 1: A fermentation method of selenium-enriched Pleurotus eryngii mycelium, comprising the following steps:

[0024] Step 1: Activation of Pleurotus eryngii strains

[0025] Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 25°C incubator for constant temperature culture, and wait for use after the mycelium has covered the slant;

[0026] The preparation method of the PDA medium comprises the following steps: peeling potatoes, cutting them into small pieces and putting them into a pot, adding 1000 mL of distilled water, boiling and filtering, taking 200 g of potato filtrate, adding 22 g of glucose and 22 g of agar, heating and stirring with a glass rod to dissolve them, and adding water to 1000 mL, filling the medium into conical flasks and sealing and tying them, sterilizing at 121° C. for 20 minutes, and obtaining the PDA medium;

[0027] Step 2: Prepare seed culture medium

[0028] Selecting a fermentation medium composition, the fermentation medium composition includes 200g of potatoes, 20g of glucose, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.022g of naphthylacetic acid NAA and 0.01g of indolebutyric acid IBA, peeling the potatoes, cutting them into small pieces and putting them into a pot, adding 1000mL of distilled water and boiling, filtering to obtain a potato filtrate, adding glucose, potassium dihydrogen phosphate and magnesium sulfate and heating to dissolve, and supplementing with distilled water until the total volume of the solution reaches 1000mL, dividing them into Erlenmeyer flasks, sealing and bundling them, and sterilizing them at 121°C for 30min to obtain a seed liquid culture medium;

[0029] Step 3: Preparation of Pleurotus eryngii seed liquid

[0030] Cut the activated Pleurotus eryngii mycelium into 1 cm 2 Small pieces were inoculated into a 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, with 10 pieces placed in each flask, first placed in a 25℃ incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 4 days to obtain Pleurotus eryngii seed liquid;

[0031] Step 4: Gradient Selenium Culture

[0032] The seed liquid of Pleurotus eryngii is inoculated into a seed liquid culture medium for cultivation to carry out liquid fermentation. The culture conditions are as follows: the inoculation amount is controlled at 10%, the temperature of the fermentation environment is maintained at 25°C, the rotation speed is set at 150r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, on the second day after the start of the fermentation, the sodium selenite concentration in the culture medium reaches 1μg / mL for the first time, and then the sodium selenite selenium concentration in the culture medium is gradually increased according to a linear gradient selenium addition method every day until the final concentration reaches 15μg / mL on the seventh day; the cultured fermentation liquid is filtered or centrifuged at a speed of 10000rpm, the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained through washing and drying.

[0033] Embodiment 2: A fermentation method of selenium-enriched Pleurotus eryngii mycelium, comprising the following steps:

[0034] Step 1: Activation of Pleurotus eryngii strains

[0035] Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 30°C incubator for constant temperature culture, and wait for use after the mycelium has covered the slant;

[0036] The preparation method of the PDA medium comprises the following steps: peeling potatoes, cutting them into small pieces and putting them into a pot, adding 1000 mL of distilled water, boiling and filtering, taking 240 g of potato filtrate, adding 20 g of glucose and 20 g of agar, heating and stirring with a glass rod to dissolve them, and adding water to 1000 mL, dividing the culture medium into conical flasks and sealing and tying them, and sterilizing them at 121° C. for 30 minutes to obtain the PDA medium;

[0037] Step 2: Prepare seed culture medium

[0038] Selecting a fermentation medium composition, the fermentation medium composition includes 240g of potatoes, 22g of glucose, 0.8g of potassium dihydrogen phosphate, 0.3g of magnesium sulfate, 0.025g of naphthylacetic acid NAA and 0.015g of indolebutyric acid IBA, peeling the potatoes, cutting them into small pieces and putting them into a pot, adding 1000mL of distilled water and boiling, filtering to obtain a potato filtrate, adding glucose, potassium dihydrogen phosphate and magnesium sulfate and heating to dissolve, and supplementing with distilled water until the total volume of the solution reaches 1000mL, dividing them into Erlenmeyer flasks, sealing and bundling them, and sterilizing them at 121°C for 20min to obtain a seed liquid culture medium;

[0039] Step 3: Preparation of Pleurotus eryngii seed liquid

[0040] Cut the activated Pleurotus eryngii mycelium into 1 cm 2 Small pieces were inoculated into a 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, with 10 pieces placed in each flask, first placed in a 28℃ incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 5 days to obtain Pleurotus eryngii seed liquid;

[0041] Step 4: Gradient Selenium Culture

[0042] The seed liquid of Pleurotus eryngii is inoculated into a seed liquid culture medium for cultivation to carry out liquid fermentation. The culture conditions are as follows: the inoculation amount is controlled at 8%, the temperature of the fermentation environment is maintained at 30°C, the rotation speed is set at 130r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, on the second day after the start of the fermentation, the sodium selenite concentration in the culture medium is first made to reach 1μg / mL, and then the sodium selenite selenium concentration in the culture medium is gradually increased every day according to a linear gradient selenium addition method until a final concentration of 5μg / mL is reached on the seventh day; the cultured fermentation liquid is filtered or centrifuged at a speed of 8000rpm, and the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained after washing and drying.

[0043] Embodiment 3: A fermentation method of selenium-enriched Pleurotus eryngii mycelium, comprising the following steps:

[0044] Step 1: Activation of Pleurotus eryngii strains

[0045] Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 28°C incubator for constant temperature culture, and wait for use after the mycelium has grown all over the slant;

[0046] The preparation method of the PDA medium comprises the following steps: peeling potatoes, cutting them into small pieces and putting them into a pot, adding 1000 mL of distilled water, boiling and filtering, taking 210 g of potato filtrate, adding 23 g of glucose and 23 g of agar, heating and stirring with a glass rod to dissolve them, and adding water to 1000 mL, dividing the culture medium into conical flasks and sealing and tying them, sterilizing at 121° C. for 24 minutes, and obtaining the PDA medium;

[0047] Step 2: Prepare seed culture medium

[0048] Selecting a fermentation medium composition, the fermentation medium composition includes 220g of potatoes, 24g of glucose, 0.9g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate, 0.01g of naphthylacetic acid NAA and 0.005g of indolebutyric acid IBA, peeling the potatoes, cutting them into small pieces and putting them into a pot, adding 1000mL of distilled water and boiling, filtering to obtain a potato filtrate, adding glucose, potassium dihydrogen phosphate and magnesium sulfate and heating to dissolve, and supplementing with distilled water until the total volume of the solution reaches 1000mL, dividing them into Erlenmeyer flasks, sealing and bundling them, and sterilizing them at 121°C for 15min to obtain a seed liquid culture medium;

[0049] Step 3: Preparation of Pleurotus eryngii seed liquid

[0050] Cut the activated Pleurotus eryngii mycelium into 1 cm 2 Small pieces were inoculated into a 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, with 10 pieces placed in each flask, first placed in a 26℃ incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 6 days to obtain Pleurotus eryngii seed liquid;

[0051] Step 4: Gradient Selenium Culture

[0052] The seed liquid of Pleurotus eryngii is inoculated into a seed liquid culture medium for cultivation to carry out liquid fermentation. The culture conditions are as follows: the inoculation amount is controlled at 14%, the temperature of the fermentation environment is maintained at 24°C, the rotation speed is set at 160r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, on the second day after the start of the fermentation, the sodium selenite concentration in the culture medium is first made to reach 1μg / mL, and then the sodium selenite selenium concentration in the culture medium is gradually increased according to a linear gradient selenium addition method every day until a final concentration of 20μg / mL is reached on the seventh day; the cultured fermentation liquid is filtered or centrifuged at a speed of 9000rpm, and the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained through washing and drying.

[0053] Embodiment 4: A fermentation method of selenium-enriched Pleurotus eryngii mycelium, comprising the following steps:

[0054] Step 1: Activation of Pleurotus eryngii strains

[0055] Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 26°C incubator for constant temperature culture, and wait for use after the mycelium has grown all over the slant;

[0056] The preparation method of the PDA medium comprises the following steps: peeling potatoes, cutting them into small pieces and putting them into a pot, adding 1000 mL of distilled water, boiling and filtering, taking 220 g of potato filtrate, adding 24 g of glucose and 24 g of agar, heating and stirring with a glass rod to dissolve them, and adding water to 1000 mL, filling the medium into conical flasks and sealing and tying them, sterilizing at 121° C. for 18 minutes, and obtaining the PDA medium;

[0057] Step 2: Prepare seed culture medium

[0058] Selecting a fermentation medium composition, the fermentation medium composition includes 230g of potato, 20g of glucose, 1.2g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate, and 0.023g of naphthylacetic acid NAA; peeling the potato, cutting it into small pieces and putting it into a pot; adding 1000mL of distilled water and boiling it; filtering to obtain a potato filtrate; adding glucose, potassium dihydrogen phosphate and magnesium sulfate and heating to dissolve them; supplementing with distilled water until the total volume of the solution reaches 1000mL; dividing it into Erlenmeyer flasks, sealing and bundling them, and sterilizing them at 121°C for 18min to obtain a seed liquid culture medium;

[0059] Step 3: Preparation of Pleurotus eryngii seed liquid

[0060] Cut the activated Pleurotus eryngii mycelium into 1 cm 2 Small pieces were inoculated into a 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, with 10 pieces placed in each flask, first placed in a 27℃ incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 5 days to obtain Pleurotus eryngii seed liquid;

[0061] Step 4: Gradient Selenium Culture

[0062] The seed liquid of Pleurotus eryngii is inoculated into the seed liquid culture medium for cultivation to carry out liquid fermentation. The culture conditions are as follows: the inoculation amount is controlled at 12%, the temperature of the fermentation environment is maintained at 28°C, the rotation speed is set at 140r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, on the second day after the start of the fermentation, the sodium selenite concentration in the culture medium is first made to reach 1μg / mL, and then the sodium selenite selenium concentration in the culture medium is gradually increased according to the linear gradient selenium addition method every day until the final concentration reaches 30μg / mL on the seventh day; the cultured fermentation liquid is filtered or centrifuged at a speed of 8500rpm, the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained after washing and drying.

[0063] Embodiment 5: A fermentation method of selenium-enriched Pleurotus eryngii mycelium, comprising the following steps:

[0064] Step 1: Activation of Pleurotus eryngii strains

[0065] Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 29°C incubator for constant temperature culture, and wait for use after the mycelium has grown all over the slant;

[0066] The preparation method of the PDA medium comprises the following steps: peeling potatoes, cutting them into small pieces and putting them into a pot, adding 1000 mL of distilled water, boiling and filtering, taking 230 g of potato filtrate, adding 21 g of glucose and 21 g of agar, heating and stirring with a glass rod to dissolve them, and adding water to 1000 mL, filling the medium into conical flasks and sealing and tying them, sterilizing at 121° C. for 15 minutes, and obtaining the PDA medium;

[0067] Step 2: Prepare seed culture medium

[0068] Selecting a fermentation medium composition, the fermentation medium composition includes 200g of potatoes, 20g of glucose, 1.1g of potassium dihydrogen phosphate, 0.1g of magnesium sulfate, 0.0175g of naphthylacetic acid NAA and 0.0125g of indolebutyric acid IBA, peeling the potatoes, cutting them into small pieces and putting them into a pot, adding 1000mL of distilled water and boiling, filtering to obtain a potato filtrate, adding glucose, potassium dihydrogen phosphate and magnesium sulfate and heating to dissolve, supplementing with distilled water until the total volume of the solution reaches 1000mL, dividing them into Erlenmeyer flasks, sealing and bundling them, and sterilizing them at 121°C for 25min to obtain a seed liquid culture medium;

[0069] Step 3: Preparation of Pleurotus eryngii seed liquid

[0070] Cut the activated Pleurotus eryngii mycelium into 1 cm 2Small pieces were inoculated into a 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, with 10 pieces placed in each flask, first placed in a 30℃ incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 4 days to obtain Pleurotus eryngii seed liquid;

[0071] Step 4: Gradient Selenium Culture

[0072] The seed liquid of Pleurotus eryngii is inoculated into a seed liquid culture medium for cultivation to carry out liquid fermentation. The culture conditions are as follows: the inoculation amount is controlled at 13%, the temperature of the fermentation environment is maintained at 26°C, the rotation speed is set at 150r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, on the second day after the start of the fermentation, the sodium selenite concentration in the culture medium reaches 1μg / mL for the first time, and then the sodium selenite selenium concentration in the culture medium is gradually increased according to a linear gradient selenium addition method every day until the final concentration reaches 15μg / mL on the seventh day; the cultured fermentation liquid is filtered or centrifuged at a speed of 10000rpm, the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained through washing and drying.

[0073] Example 6: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0074] The preparation method of this embodiment is different from that of embodiment 1 only in that: in this embodiment,

[0075] The fermentation medium composition included 200 g potato, 20 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate and 0.01 g indolebutyric acid IBA.

[0076] Example 7: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0077] The preparation method of this embodiment is different from that of Embodiment 1 only in that: in this embodiment, the fermentation medium composition includes 200 g of potato, 20 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 0.022 g of naphthylacetic acid NAA.

[0078] Example 8: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0079] The preparation method of this embodiment is different from that of Embodiment 1 only in that: in this embodiment, the fermentation medium composition includes 200 g potato, 20 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.025 g naphthylacetic acid NAA and 0.02 g indolebutyric acid IBA.

[0080] Example 9: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0081] The preparation method of this embodiment is different from that of Embodiment 1 only in that: in this embodiment, Pleurotus eryngii seed liquid is inoculated into the seed liquid culture medium at an inoculation rate of 8% (v / v) for liquid fermentation.

[0082] Example 10: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0083] The preparation method of this embodiment is different from that of Embodiment 1 only in that: in this embodiment, Pleurotus eryngii seed liquid is inoculated into the seed liquid culture medium at an inoculation rate of 14% (v / v) for liquid fermentation.

[0084] Example 11: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0085] The preparation method of this embodiment is different from that of Embodiment 1 only in that: in this embodiment, the temperature of the fermentation environment is maintained at 24°C.

[0086] Example 12: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0087] The preparation method of this embodiment is different from that of Embodiment 1 only in that: in this embodiment, the temperature of the fermentation environment is maintained at 30°C.

[0088] Example 13: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0089] The preparation method of this embodiment is different from that of Example 1 only in that: in this embodiment, on the second day after the start of fermentation, the sodium selenite concentration in the culture medium is first made to reach 1 μg / mL, and then the sodium selenite selenium concentration in the culture medium is gradually increased every day according to a linear gradient selenium addition method until the final concentration reaches 5 μg / mL on the 7th day.

[0090] Example 14: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0091] The preparation method of this embodiment is different from that of Example 1 only in that: in this embodiment, on the second day after the start of fermentation, the sodium selenite concentration in the culture medium is first made to reach 1 μg / mL, and then the sodium selenite selenium concentration in the culture medium is gradually increased every day according to a linear gradient selenium addition method until the final concentration reaches 30 μg / mL on the 7th day.

[0092] Comparative Example 1: A fermentation method for selenium-enriched Pleurotus eryngii mycelium:

[0093] Step 1: Activation of Pleurotus eryngii strains

[0094] Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 25°C incubator for constant temperature culture, and wait for use after the mycelium has covered the slant;

[0095] Step 2: Prepare seed culture medium

[0096] Selecting a fermentation medium composition, the fermentation medium composition includes 200g of potatoes, 20g of glucose, 1g of potassium dihydrogen phosphate, and 0.5g of magnesium sulfate, peeling the potatoes, cutting them into small pieces and putting them into a pot, adding 1000mL of distilled water and boiling, filtering to obtain a potato filtrate, adding glucose, potassium dihydrogen phosphate and magnesium sulfate and heating to dissolve, and supplementing with distilled water until the total volume of the solution reaches 1000mL, dividing them into Erlenmeyer flasks, sealing and bundling them, and sterilizing them at 121°C for 30min to obtain a seed liquid culture medium;

[0097] Step 3: Preparation of Pleurotus eryngii seed liquid

[0098] The activated Pleurotus eryngii mycelium blocks were cut into 1 cm2 small pieces, inoculated into 500 mL conical flasks containing 200 mL seed liquid culture medium under sterile conditions, 10 pieces were placed in each bottle, and first placed in a 25°C incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 6 days to obtain Pleurotus eryngii seed liquid;

[0099] Step 4: One-time selenium culture

[0100] The Pleurotus eryngii seed liquid is inoculated into a seed liquid culture medium for liquid fermentation. The culture conditions are as follows: the inoculation amount is controlled at 10%, the temperature of the fermentation environment is maintained at 25°C, the rotation speed is set at 150r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, sodium selenite is added at a concentration of 15μg / mL; the cultured fermentation liquid is filtered or centrifuged at a speed of 10000rpm, the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained through washing and drying.

[0101] Experimental part:

[0102] The selenium-enriched Pleurotus eryngii mycelia cultured in Comparative Example 1, Example 1, Examples 6-8, and Examples 9-14 were selected to determine the mycelial biomass, and the total selenium content and organic selenium content were determined by inductively coupled plasma mass spectrometry (ICP-MS). The results are shown in Table 1.

[0103] Table 1

[0104]

[0105] In Table 1, it can be seen from the data comparison between Comparative Example 1 and the embodiments of the present invention that the mycelium biomass, total selenium content and organic selenium content of the selenium-enriched Pleurotus eryngii mycelium cultured by the fermentation method of the present invention are all improved. This shows that the addition of naphthylacetic acid NAA and (or) indolebutyric acid IBA to the culture medium and the gradient selenium addition culture method can promote the growth rate of mycelium and increase the selenium enrichment rate of mycelium; and the mycelium cultured in Example 1 has the highest organic selenium content. The total selenium content of Example 1 is 340.82 μg / g, and the organic selenium content is 335.67 μg / g. Compared with the traditional selenium-enriched method in Comparative Example 1, the total selenium content of Example 1 is increased by 121.27 μg / g, and the organic selenium content is increased by 128.08 μg / g.

[0106] In Table 1, it can be seen from the data of Example 1 and Examples 6-8 that the content of each in the mycelium is higher when both naphthylacetic acid NAA and indolebutyric acid IBA are added to the culture medium than when either one is added alone, indicating that the synergistic effect of naphthylacetic acid NAA and indolebutyric acid IBA can improve the nutrient absorption capacity of Pleurotus eryngii mycelium in the culture medium, provide sufficient nutrients for the rapid growth of mycelium, and thus increase the biomass and selenium content of mycelium. And when the contents of each component in the culture medium are: 200g potato, 20g glucose, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.022g naphthylacetic acid NAA and 0.010g indolebutyric acid IBA, the mycelium biomass, total selenium content and organic selenium are the highest.

[0107] In Table 1, it can be seen from the data of Examples 1 and 9 and 10 that, when the same culture medium is selected, after the Pleurotus eryngii seed liquid is inoculated into the seed liquid culture medium at an inoculation amount of 10% (v / v) for liquid fermentation, the total selenium content and organic selenium content of the mycelium are the highest. This indicates that at this inoculation amount, the number of mycelium is greater, which means that the biomass is greater, and the biomass can better absorb and transform selenium, thereby achieving the best selenium enrichment effect.

[0108] When the inoculation amount is lower than 8% (v / v), too low an inoculation amount means that the initial number of mycelium is small, which will cause the growth rate of the mycelium in the culture medium to slow down, thereby prolonging the time required to reach the maximum biomass. Due to the small number of mycelium, the overall biotransformation capacity is reduced, resulting in a decrease in the conversion efficiency of sodium selenite or other selenium sources, thereby affecting the final selenium enrichment. At the same time, when the inoculation amount is higher than 14% (v / v), the excessively high inoculation amount leads to intensified competition between mycelium and accelerated consumption of nutrients, which in turn reduces the selenium enrichment efficiency.

[0109] In Table 1, it can be seen from the data of Example 1 and Examples 11 and 12 that, when the same culture medium and inoculation amount are selected, the mycelium biomass, total selenium content and organic selenium content are the highest when the culture temperature is 25°C, indicating that many enzymes in the mycelium of Pleurotus eryngii have higher activities at 25°C, which is beneficial to the absorption and conversion of selenium by microorganisms.

[0110] When the temperature is too low, it will reduce the activity of enzymes, thereby slowing down the growth of mycelium. Under low temperature conditions, mycelium expansion and metabolic processes will become slow; due to the slowed growth rate, the final mycelium biomass will be reduced, which will in turn affect the mycelium's ability to absorb and convert selenium, resulting in a decrease in selenium enrichment efficiency.

[0111] When the temperature is higher than 30℃, high temperature will interfere with the normal metabolic pathway of mycelium, affect the synthesis and secretion of its metabolites, and further affect the growth and normal physiological function of mycelium. Mycelium will undergo morphological changes under high temperature, such as thickening of mycelium and reduction of branches, which will affect its normal physiological function. High temperature will also accelerate the evaporation rate of the culture solution, causing changes in the concentration of the culture solution, thus affecting the growth environment of the mycelium.

[0112] In Table 1, it can be seen from the data of Example 1 and Examples 13 and 14 that, under the same environment, when the final concentration of gradient selenium addition is 15 μg / mL, the organic selenium content of the mycelium is the highest, and at this concentration, the microorganism can convert inorganic selenium into organic selenium that is easier to absorb and utilize. When the final concentration of gradient selenium addition is 30 μg / mL, the total selenium content of the mycelium is the highest, but high concentrations of sodium selenite will destroy the normal metabolism and physiological functions of the mycelium, resulting in inhibition of mycelium growth, and also weakening its conversion from inorganic selenium to organic selenium, resulting in a sharp decrease in the organic selenium content. At the same time, when the concentration of sodium selenite is too low, the mycelium can absorb too little selenium, resulting in a reduction in selenium enrichment.

Claims

1. A fermentation method for selenium-enriched Pleurotus eryngii mycelium, characterized in that: The steps include: Step 1: Activation of Pleurotus eryngii strains Inoculate Pleurotus eryngii strains onto the slant of PDA culture medium, place in a 25-30℃ incubator for constant temperature culture, and wait for use after the mycelium has covered the slant; Step 2: Prepare seed culture medium Selecting a fermentation medium composition, the fermentation medium composition includes 200-240g of potatoes, 20-24g of glucose, 0.8-1.2g of potassium dihydrogen phosphate and 0.1-0.5g of magnesium sulfate, and 0.01-0.025g of naphthylacetic acid NAA and / or 0.005-0.02g of indolebutyric acid IBA, peeling the potatoes, cutting them into small pieces and putting them into a pot, adding 1000mL of distilled water and boiling, filtering to obtain a potato filtrate, adding glucose, potassium dihydrogen phosphate and magnesium sulfate, and naphthylacetic acid NAA and / or indolebutyric acid IBA, heating to dissolve, supplementing with distilled water until the total volume of the solution reaches 1000mL, sealing and bundling, and sterilizing at 121°C for 15-30min to obtain a seed liquid culture medium; Step 3: Preparation of Pleurotus eryngii seed liquid Cut the activated Pleurotus eryngii mycelium into 1 cm 2 Small pieces are inoculated into a 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, with 10 pieces placed in each flask, first placed in a 25-30℃ incubator for constant temperature culture for 2 days, and then placed on a shaker for shaking culture for 4-6 days to obtain Pleurotus eryngii seed liquid; Step 4: Gradient Selenium Culture The Pleurotus eryngii seed liquid is inoculated into a seed liquid culture medium at an inoculation rate of 8%-14% (v / v) for liquid fermentation. The culture conditions are as follows: the temperature of the fermentation environment is maintained at 24-30°C, the rotation speed is set to 130-160r / min, the entire fermentation time lasts for 7 days, and during the fermentation process, sodium selenite is added in batches according to a gradient until the final concentration reaches 5-30μg / mL on the 7th day; the cultured fermentation liquid is filtered or centrifuged, and the mycelium is collected, and the selenium-enriched Pleurotus eryngii mycelium is obtained through washing and drying.

2. The fermentation method of a selenium-enriched Pleurotus eryngii mycelium according to claim 1, characterized in that: Step 1 The preparation method of the PDA culture medium comprises the following steps: peeling potatoes, cutting them into small pieces and putting them into a pot, adding 1000 mL of distilled water, boiling and filtering, taking 200-240 g of potato filtrate, adding 20-24 g of glucose and agar respectively, heating and stirring with a glass rod to dissolve them, and adding water to 1000 mL, dividing the culture medium into conical flasks and sealing and tying them, sterilizing at 121° C. for 15-30 min, and obtaining the PDA culture medium.

3. The fermentation method of a selenium-enriched Pleurotus eryngii mycelium according to claim 1, characterized in that: The specific method of adding sodium selenite in batches according to the gradient in step 4 is as follows: on the second day after the start of fermentation, the sodium selenite concentration in the culture medium is first increased to 1 μg / mL, and then the sodium selenite selenium concentration in the culture medium is gradually increased every day according to the linear gradient selenium addition method until the final concentration reaches 5-30 μg / mL on the 7th day.

4. The fermentation method of a selenium-enriched Pleurotus eryngii mycelium according to claim 1, characterized in that: The rotation speed of the centrifugal treatment in step 4 is 8000-10000 rpm.

5. A selenium-enriched Pleurotus eryngii mycelium cultured by the fermentation method according to any one of claims 1 to 4, characterized in that: The total selenium content of the Pleurotus eryngii mycelium is 288.3-351.22 μg / g, and the organic selenium content is 267.93-335.67 μg / g.

Images