Selenium-rich pleurotus eryngii mycelium and fermentation method thereof

Abstract

The application discloses a kind of selenium-rich agaricus blazei murill mycelia and fermentation method thereof, comprising the following steps: agaricus blazei murill seed is inoculated to PDA culture medium slope, constant temperature culture is placed in incubator, after mycelium fills slope, wait for use;Potato is peeled and cut into small block and put into pot, 1000mL distilled water is added and boiled, potato filtrate is obtained after filtration, glucose, potassium dihydrogen phosphate and magnesium sulfate are added, and naphthalene acetic acid NAA and / or indole butyric acid IBA are added after heating and dissolving, and distilled water is supplemented to the total volume of solution reaches 1000m after, sealed and tied, sterilization is carried out at 121 DEG C for 15-30min, and seed liquid culture medium is obtained, agaricus blazei murill seed liquid is inoculated to seed liquid culture medium with 8%-14% (v / v) inoculation amount and is cultured to carry out liquid fermentation, after fermentation liquid cultured is filtered or centrifuged, mycelium is collected, after washing, selenium-rich agaricus blazei murill mycelia are obtained by drying.The application can shorten the cultivation time, and meanwhile improve the selenium content of agaricus blazei murill mycelium.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fungus cultivation, in particular to a selenium-rich agaricus blazei murrill mycelium and a fermentation method thereof. BACKGROUND

[0002] Agaricus blazei murrill is a new rare edible fungus with both medicinal and edible properties, which is rich in polysaccharides, proteins, mineral elements, vitamins and other nutrients, and has various pharmacological effects such as antioxidant and antitumor. Selenium is an essential trace element that cannot be synthesized by the human body and can only be obtained from food (drugs), drinking water and other external sources. Supplementing selenium is crucial for human health. There are three main forms of selenium: inorganic selenium, organic selenium and nano selenium. Compared with inorganic selenium, organic selenium has higher physiological activity, lower toxicity and higher utilization rate in the body.

[0003] Edible fungi have strong selenium enrichment ability. They can create a local high-concentration selenium environment, allowing the fungus to absorb selenium elements from the environment during growth. Selenium elements enter the cells, and the metabolism of intracellular substances can combine them with macromolecular active substances (such as polysaccharides and proteins) to convert them into organic forms of selenium such as polysaccharide selenium and protein selenium, thereby increasing the selenium content of edible fungi. The most commonly used method for selenium-enriched cultivation of edible fungi is cultivation technology, which involves adding inorganic selenium to the culture medium or using other selenium-containing raw materials (selenium-rich areas) to cultivate selenium-enriched edible fungi on a large scale. The optimal amount of selenium is determined based on selenium enrichment rate, recovery rate, yield and economic cost, but this method has the disadvantages of low selenium absorption rate and long production cycle. SUMMARY

[0004] The present application aims to provide a selenium-rich agaricus blazei murrill mycelium and a fermentation method thereof, which can shorten the cultivation time and increase the selenium content of agaricus blazei murrill mycelium.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] A fermentation method for selenium-rich agaricus blazei murrill mycelium, comprising the following steps:

[0007] Step 1: Activation of agaricus blazei murrill strain

[0008] Inoculate agaricus blazei murrill strain into PDA culture medium slant and incubate in a 25-30℃ incubator. When the mycelium covers the slant, it is ready for use.

[0009] Step 2: Preparation of seed liquid culture medium

[0010] A fermentation medium composition is selected, which includes 200-240 g of potatoes, 20-24 g of glucose, 0.8-1.2 g of potassium dihydrogen phosphate, and 0.1-0.5 g of magnesium sulfate, and 0.01-0.025 g of naphthalene acetic acid (NAA) and / or 0.005-0.02 g of indole butyric acid (IBA). The potatoes are peeled and cut into small pieces, which are placed in a pot, 1000 mL of distilled water is added, and boiled. After filtration, the potato filtrate is obtained. After the glucose, potassium dihydrogen phosphate, and magnesium sulfate, and the naphthalene acetic acid (NAA) and / or indole butyric acid (IBA) are added, heating is performed for dissolution, and distilled water is added to make up the total volume of the solution to 1000 mL. After sealing and bundling, sterilization is performed at 121°C for 15-30 min to obtain a seed liquid medium.

[0011] Step three: preparation of a Pleurotus eryngii seed liquid

[0012] The activated Pleurotus eryngii mycelial blocks are cut into 1 cm 2 small pieces, which are inoculated into 500 mL conical flasks containing 200 mL of the seed liquid medium under sterile conditions, 10 blocks are placed in each flask. First, they are placed in a constant temperature incubator at 25-30°C for 2 d, and then they are placed on a shaking bed for 4-6 d of shaking culture to obtain a Pleurotus eryngii seed liquid.

[0013] Step four: gradient selenium addition culture

[0014] The Pleurotus eryngii seed liquid is inoculated into the seed liquid medium at an inoculation amount 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 130-160 r / min, the entire fermentation time lasts for 7 d, and sodium selenite is added in batches according to a gradient during the fermentation process until the final concentration reaches 5-30 μg / mL on the 7th day. After the culture of the fermentation liquid is filtered or centrifuged, the mycelium is collected, washed, and dried to obtain selenium-enriched Pleurotus eryngii mycelium.

[0015] Further, the preparation method of the PDA medium in step one includes the following steps: the potatoes are peeled and cut into small pieces, which are placed in a pot, 1000 mL of distilled water is added, and boiled. After filtration, 200-240 g of the potato filtrate is taken, 20-24 g of glucose and agar are added, heating is performed, and a glass rod is continuously stirred to dissolve them. Distilled water is added to make up the total volume to 1000 mL. The medium is divided into triangular flasks and sealed and bundled. Sterilization is performed at 121°C for 15-30 min to obtain the PDA medium.

[0016] The specific way of adding sodium selenite in batches according to a gradient in step four is as follows: on the 2nd day after the start of fermentation, the concentration of sodium selenite in the medium is first increased to 1 μg / mL. Then, the concentration of sodium selenite in the medium is gradually increased in a linear gradient selenium addition manner every day until the final concentration reaches 5-30 μg / mL on the 7th day.

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

[0018] The selenium-rich agaricus blazei murrill mycelium cultivated 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 application has the following beneficial effects:

[0020] The present application selects a suitable seed liquid culture medium, in which glucose is used as a high-quality carbon source to quickly provide sufficient energy for the metabolic activity of the mycelium, potassium dihydrogen phosphate and magnesium sulfate are used to supplement essential mineral elements to promote the growth of agaricus blazei murrill mycelium and the absorption and conversion of selenium. In addition, naphthalene acetic acid NAA and / or indole butyric acid IBA are added to the seed liquid culture medium. 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 selenium by agaricus blazei murrill mycelium, thereby increasing the selenium content in the mycelium. When NAA and IBA act synergistically, they can regulate the permeability of the cell membrane of the mycelium, thereby improving the nutrient absorption capacity of agaricus blazei murrill mycelium in the culture medium. 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 culture process.

[0021] The present application also gradually adds sodium selenite to the culture medium in a gradient manner, which can ensure the reasonable absorption of selenium and avoid the inhibitory effect of excessive selenium on the growth of the mycelium. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be described in detail below with specific examples.

[0023] Example 1: A fermentation method of selenium-rich agaricus blazei murrill mycelium, comprising the following steps:

[0024] Step one: activation of agaricus blazei murrill spores

[0025] The agaricus blazei murrill spores are inoculated into a PDA culture medium slope and placed in a 25℃ incubator for constant temperature culture. After the mycelium grows on the slope, it is ready for use.

[0026] The preparation method of the PDA culture medium comprises the following steps: peeling potatoes and cutting them into small pieces, adding 1000 mL of distilled water, boiling and filtering, taking 200 g of the potato filtrate, adding 22 g of glucose and 22 g of agar, heating and continuously stirring with a glass rod to dissolve them, and making up the water to 1000 mL, the culture medium is divided into triangular flasks and sealed and bundled, sterilized at 121 ℃ for 20 min, and the PDA culture medium is obtained;

[0027] Step two: preparation of seed liquid culture medium

[0028] The fermentation medium composition is selected, and the fermentation medium composition comprises 200 g of potatoes, 20 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.022 g of naphthalene acetic acid NAA and 0.01 g of indole butyric acid IBA, the potatoes are peeled and cut into small pieces and put into a pot, 1000 mL of distilled water is added to boil, and the potato filtrate is obtained after filtering, the glucose, potassium dihydrogen phosphate and magnesium sulfate are added after heating and dissolving, and the distilled water is made up to 1000 mL of the total volume of the solution, and is divided into triangular flasks and sealed and bundled, sterilized at 121 ℃ for 30 min, and the seed liquid culture medium is obtained;

[0029] Step three: preparation of Pleurotus eryngii seed liquid

[0030] The activated Pleurotus eryngii mycelial block is cut into 1 cm 2 small pieces, inoculated into a 500 mL conical flask containing 200 mL of seed liquid culture medium under sterile conditions, 10 pieces are put into each flask, first placed in a 25 ℃ incubator for constant temperature culture for 2 d, and then placed on a shaking bed for shaking culture for 4 d, and the Pleurotus eryngii seed liquid is obtained;

[0031] Step four: gradient selenium addition culture

[0032] The Pleurotus eryngii seed liquid is inoculated into the seed liquid culture medium for culture, liquid fermentation is carried out, the inoculation amount is controlled at 10%, the fermentation environment temperature is maintained at 25 ℃, the rotation speed is set to 150 r / min, the whole fermentation time lasts for 7 d, and in the fermentation process, the sodium selenite concentration in the culture medium is first increased to 1 μg / mL on the 2nd day after the start of fermentation, and then the sodium selenite selenium concentration in the culture medium is gradually increased according to the linear gradient selenium addition mode every day, until the final concentration of 15 μg / mL is reached on the 7th day; the fermented liquid is filtered or centrifuged at a rotation speed of 10000 rpm, the mycelium is collected, washed and dried to obtain the selenium-rich Pleurotus eryngii mycelium.

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

[0034] Step one: activation of Pleurotus eryngii spores

[0035] PDA medium slope inoculated with Pleurotus eryngii spores, placed in a 30℃ incubator for constant temperature culture, and the mycelium was used after the slope was full;

[0036] The preparation method of the PDA culture medium includes the following steps: peeling and cutting potatoes into small pieces, adding 1000mL distilled water, boiling and filtering, taking 240g of potato filtrate, adding 20g of glucose and 20g of agar, heating and continuously stirring with a glass rod to dissolve, and making up the water to 1000mL, the culture medium is divided into triangular flasks and sealed and bundled, sterilized at 121℃ for 30min, and the PDA culture medium is obtained.

[0037] Step two: preparation of seed liquid medium

[0038] The fermentation medium composition is selected, and 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 naphthalene acetic acid NAA, and 0.015g of indole butyric acid IBA. The potatoes are peeled and cut into small pieces, added into a pot with 1000mL distilled water, boiled, filtered to obtain potato filtrate, and then heated and dissolved after adding glucose, potassium dihydrogen phosphate and magnesium sulfate. The distilled water is made up to 1000mL, and the solution is divided into triangular flasks and sealed and bundled. Sterilization is performed at 121℃ for 20min to obtain the seed liquid medium.

[0039] Step three: preparation of Pleurotus eryngii seed liquid

[0040] The activated Pleurotus eryngii mycelium block is cut into 1cm 2 small pieces, inoculated into a 500mL conical flask containing 200mL of seed liquid medium under sterile conditions, 10 pieces per flask, first placed in a 28℃ incubator for constant temperature culture for 2d, and then placed on a shaker for shaking culture for 5d to obtain the Pleurotus eryngii seed liquid.

[0041] Step four: gradient selenium addition culture

[0042] The Pleurotus eryngii seed liquid is inoculated into the seed liquid medium for culture, liquid fermentation is carried out, the inoculation amount is controlled at 8%, the fermentation environment temperature is maintained at 30℃, the rotation speed is set at 130r / min, the whole fermentation time lasts for 7d, and during the fermentation process, the sodium selenite concentration in the culture medium is first increased to 1μg / mL on the 2nd day after the start of fermentation, and then gradually increased according to the linear gradient selenium addition mode every day, until the final concentration of 5μg / mL is reached on the 7th day; the cultured fermentation broth is filtered or centrifuged at a rotation speed of 8000rpm, the mycelium is collected, washed and dried to obtain the selenium-enriched Pleurotus eryngii mycelium.

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

[0044] Step one: activation of pleurotus eryngii sp.

[0045] Pleurotus eryngii sp. is inoculated on PDA medium slant and placed in a 28℃ incubator for constant temperature culture. After the mycelium grows on the slant, it is ready for use.

[0046] The preparation method of PDA culture medium includes the following steps: peel the potatoes and cut them into small pieces, add 1000mL distilled water, boil and filter, take 210g potato filtrate, add 23g glucose and 23g agar, heat and continuously stir with a glass rod to dissolve, and make up the water to 1000mL, divide the culture medium into triangular flasks and seal and bundle, sterilize at 121℃ for 24min, and get PDA culture medium.

[0047] Step two: preparation of seed liquid culture medium

[0048] Select the fermentation medium composition, which includes 220g potatoes, 24g glucose, 0.9g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.01g naphthalene acetic acid NAA and 0.005g indole butyric acid IBA. Peel the potatoes and cut them into small pieces, add 1000mL distilled water to boil, filter to get potato filtrate, add glucose, potassium dihydrogen phosphate and magnesium sulfate, heat to dissolve, and make up the distilled water to 1000mL total volume, divide into triangular flasks and seal and bundle, sterilize at 121℃ for 15min, and get seed liquid culture medium.

[0049] Step three: preparation of pleurotus eryngii seed liquid

[0050] Cut the activated pleurotus eryngii mycelium into 1cm 2 small pieces, inoculate in 500mL conical flask containing 200mL seed liquid culture medium under sterile conditions, put 10 pieces in each flask, first place in a 26℃ incubator for constant temperature culture for 2d, then place on a shaker for shaking culture for 6d, and get pleurotus eryngii seed liquid.

[0051] Step four: gradient selenium culture

[0052] The Pleurotus eryngii seed liquid is inoculated into the seed liquid culture medium for liquid fermentation, and the culture conditions are as follows: the inoculation amount is controlled to be 14%, the fermentation environment temperature is maintained at 24 DEG C, the rotating speed is set to be 160 r / min, the whole fermentation time lasts for 7 days, and in the fermentation process, the sodium selenite concentration in the culture medium is first increased to 1 ug / mL at the second day after the fermentation starts, then the sodium selenite concentration in the culture medium is gradually increased according to a linear gradient every day, until the final concentration of 20 ug / mL is reached at the seventh day; the cultured fermentation liquid is filtered or treated by centrifugation at a rotating speed of 9000 rpm, then the mycelium is collected, washed and dried to obtain the selenium-rich Pleurotus eryngii mycelium.

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

[0054] Step one: activation of Pleurotus eryngii strain

[0055] The Pleurotus eryngii strain is inoculated into the PDA culture medium slope, and placed in a 26 DEG C incubator for constant temperature culture, and after the mycelium grows on the slope, it is used;

[0056] The preparation method of the PDA culture medium comprises the following steps: the potato is peeled and cut into small pieces and put into a pot, 1000 mL of distilled water is added, boiled, filtered, 220 g of potato filtrate is taken, 24 g of glucose and 24 g of agar are added, heated and continuously stirred with a glass rod to dissolve, and the water is made up to 1000 mL, the culture medium is divided into triangular flasks and sealed, sterilized at 121 DEG C for 18 min, and the PDA culture medium is obtained;

[0057] Step two: preparation of seed liquid culture medium

[0058] The fermentation medium composition is selected, and the fermentation medium composition comprises 230 g of potato, 20 g of glucose, 1.2 g of potassium dihydrogen phosphate, 0.4 g of magnesium sulfate, and 0.023 g of naphthalene acetic acid NAA. The potato is peeled and cut into small pieces and put into a pot, 1000 mL of distilled water is added, boiled, filtered to obtain potato filtrate, and then glucose, potassium dihydrogen phosphate and magnesium sulfate are added and dissolved by heating. The distilled water is made up to 1000 mL, and the solution is divided into triangular flasks and sealed, sterilized at 121 DEG C for 18 min, and the seed liquid culture medium is obtained;

[0059] Step three: preparation of Pleurotus eryngii seed liquid

[0060] The activated Pleurotus eryngii mycelium block is cut into 1 cm 2 small pieces, inoculated into a 500 mL conical flask containing 200 mL of seed liquid culture medium under sterile conditions, 10 pieces are put into each flask, first placed in a 27 DEG C incubator for constant temperature culture for 2 days, and then placed on a shaking bed for vibration culture for 5 days to obtain the Pleurotus eryngii seed liquid;

[0061] Step four: Gradient selenium culture

[0062] The Pleurotus eryngii seed liquid is inoculated into the seed liquid culture medium for liquid fermentation. The inoculation amount is controlled at 12%, the fermentation environment temperature is maintained at 28°C, the rotation speed is set at 140 r / min, the whole fermentation time lasts for 7 days, and during the fermentation process, the sodium selenite concentration in the culture medium is first increased to 1 μg / mL at the second day after the fermentation starts, and then the sodium selenite selenium concentration in the culture medium is gradually increased according to the linear gradient selenium addition mode every day until the final concentration of 30 μg / mL is reached at the seventh day. After the fermentation liquid is filtered or centrifuged at a rotation speed of 8500 rpm, the mycelium is collected, washed and dried to obtain the selenium-rich Pleurotus eryngii mycelium.

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

[0064] Step one: Activation of Pleurotus eryngii strain

[0065] The Pleurotus eryngii strain is inoculated into the PDA culture medium slope and placed in a 29°C incubator for constant temperature culture. After the mycelium grows on the slope, it is used.

[0066] The preparation method of the PDA culture medium comprises the following steps: the peeled potatoes are cut into small pieces and put into a pot, 1000 mL of distilled water is added, boiled, filtered, 230 g of potato filtrate is taken, 21 g of glucose and 21 g of agar are added, heated and continuously stirred with a glass rod to dissolve them, and the water is made up to 1000 mL, the culture medium is divided into triangular flasks and sealed, and sterilized at 121°C for 15 min to obtain the PDA culture medium.

[0067] Step two: Preparation of seed liquid culture medium

[0068] The fermentation medium composition is selected, which comprises 200 g of potatoes, 20 g of glucose, 1.1 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate, 0.0175 g of naphthalene acetic acid NAA and 0.0125 g of indole butyric acid IBA. The peeled potatoes are cut into small pieces and put into a pot, 1000 mL of distilled water is added and boiled, and then filtered to obtain the potato filtrate. After the glucose, potassium dihydrogen phosphate and magnesium sulfate are added, they are heated and dissolved, and the distilled water is made up to 1000 mL to make the total volume of the solution reach 1000 mL. It is divided into triangular flasks and sealed, and sterilized at 121°C for 25 min to obtain the seed liquid culture medium.

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

[0070] The activated Pleurotus eryngii mycelial block is cut into 1 cm 2Small pieces, sterile inoculation into a 500 mL conical flask containing 200 mL seed liquid medium, 10 pieces per flask, first placed in a 30℃ incubator for constant temperature culture for 2d, then placed on a shaker for shaking culture for 4d, to obtain P. eryngii seed liquid;

[0071] Step four: gradient selenium culture

[0072] P. eryngii seed liquid was inoculated into seed liquid medium for culture, liquid fermentation was carried out, the culture conditions were as follows: the inoculation amount was controlled at 13%, the fermentation environment temperature was maintained at 26℃, the rotation speed was set at 150r / min, the whole fermentation time lasted for 7d, and during the fermentation process, the sodium selenite concentration in the culture medium was first increased to 1μg / mL at the 2nd day after the start of fermentation, and then gradually increased according to the linear gradient selenium addition mode every day, until the final concentration of sodium selenite selenium in the culture medium reached 15μg / mL at the 7th day; after the culture of the fermentation broth, the mycelium was collected by filtration or centrifugation at a rotation speed of 10000rpm, and then washed and dried to obtain selenium-rich P. eryngii mycelium.

[0073] Example 6: A fermentation method of selenium-rich P. eryngii mycelium:

[0074] The preparation method of the present embodiment is only different from that of Example 1 in that in the present embodiment,

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

[0076] Example 7: A fermentation method of selenium-rich P. eryngii mycelium:

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

[0078] Example 8: A fermentation method of selenium-rich P. eryngii mycelium:

[0079] The preparation method of the present embodiment is only different from that of Example 1 in that in the present embodiment, the fermentation medium composition includes 200g of potato, 20g of glucose, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.025g of naphthalene acetic acid NAA and 0.02g of indole butyric acid IBA.

[0080] Example 9: A fermentation method of selenium-rich P. eryngii mycelium:

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

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

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

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

[0085] The preparation method of the present embodiment is only different from that of Example 1 in that in the present embodiment, the temperature of the fermentation environment is maintained at 24℃.

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

[0087] The preparation method of the present embodiment is only different from that of Example 1 in that in the present embodiment, the temperature of the fermentation environment is maintained at 30℃.

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

[0089] The preparation method of the present embodiment is only different from that of Example 1 in that in the present embodiment, on the 2nd day after the start of fermentation, the concentration of sodium selenite in the culture medium is first increased to 1 μg / mL, and then the concentration of sodium selenite in the culture medium is gradually increased every day according to a linear gradient selenium addition mode, until the final concentration of 5 μg / mL is reached on the 7th day.

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

[0091] The preparation method of the present embodiment is only different from that of Example 1 in that in the present embodiment, on the 2nd day after the start of fermentation, the concentration of sodium selenite in the culture medium is first increased to 1 μg / mL, and then the concentration of sodium selenite in the culture medium is gradually increased every day according to a linear gradient selenium addition mode, until the final concentration of 30 μg / mL is reached on the 7th day.

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

[0093] Step one: Activation of Pleurotus eryngii spores

[0094] The Pleurotus eryngii spores are inoculated into the PDA culture medium slope and incubated in a 25℃ incubator, and after the mycelium covers the slope, it is ready for use;

[0095] Step two: Preparation of seed liquid culture medium

[0096] The fermentation medium composition is selected as follows: 200 g of potatoes, 20 g of glucose, 1 g of potassium dihydrogen phosphate, and 0.5 g of magnesium sulfate. The potatoes are peeled and cut into small pieces, which are placed in a pot, 1000 mL of distilled water is added, and the mixture is boiled. After filtration, a potato filtrate is obtained. After the glucose, potassium dihydrogen phosphate, and magnesium sulfate are added, the mixture is heated and dissolved, and distilled water is added to make up the total volume of the solution to 1000 mL. The solution is divided into three-necked flasks, sealed, sterilized at 121°C for 30 min, and a seed liquid medium is obtained.

[0097] Step three: preparation of a seed liquid of Pleurotus eryngii

[0098] The activated Pleurotus eryngii mycelial blocks are cut into small pieces of 1 cm2, which are inoculated into 500 mL conical flasks containing 200 mL of the seed liquid medium under sterile conditions, 10 pieces are placed in each flask. The flasks are first placed in a 25°C incubator for constant temperature culture for 2 d, and then placed on a shaker for vibration culture for 6 d, and a seed liquid of Pleurotus eryngii is obtained.

[0099] Step four: one-time selenium addition culture

[0100] The seed liquid of Pleurotus eryngii is inoculated into the seed liquid medium for culture, and liquid fermentation is performed. The culture conditions are as follows: the inoculation amount is controlled at 10%, the fermentation temperature is maintained at 25°C, the rotation speed is set at 150 r / min, the whole fermentation time lasts for 7 d, and sodium selenite with a concentration of 15 μg / mL is added during the fermentation process. After the fermentation liquid is filtered or centrifuged at a rotation speed of 10000 rpm, the mycelium is collected, washed, and dried to obtain selenium-enriched Pleurotus eryngii mycelium.

[0101] Experimental part:

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

[0103] Table 1

[0104]

[0105] As can be seen from the comparison of the data of the comparative example 1 and the examples of the present application in Table 1, the mycelium of the selenium-enriched Pleurotus eryngii cultured by the fermentation method of the present application has improved mycelium biomass, total selenium content and organic selenium content. This shows that the addition of naphthalene acetic acid NAA and / or indole butyric acid IBA in the culture medium and the gradient selenium addition culture method can promote the growth rate of the mycelium and improve the selenium-enriched rate of the mycelium. 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] As can be seen from the data of Example 1 and Examples 6-8 in Table 1, the contents of each component in the mycelium are higher when naphthalene acetic acid NAA and indole butyric acid IBA are added in the culture medium than when only one of them is added. This shows that the synergistic effect of naphthalene acetic acid NAA and indole butyric acid IBA can improve the nutrient absorption capacity of the mycelium of Pleurotus eryngii in the culture medium, provide sufficient nutrients for the rapid growth of the mycelium, and thus increase the biomass and selenium content of the mycelium. When the contents of the components in the culture medium are 200 g of potatoes, 20 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.022 g of naphthalene acetic acid NAA and 0.010 g of indole butyric acid IBA, the mycelium biomass, total selenium content and organic selenium content are the highest.

[0107] As can be seen from the data of Example 1 and Examples 9 and 10 in Table 1, under the condition of selecting the same culture medium, the total selenium content and organic selenium content of the mycelium are the highest after the seed liquid of Pleurotus eryngii is inoculated into the seed liquid culture medium at an inoculation amount of 10% (v / v) for liquid fermentation. This shows that under this inoculation amount, the number of mycelium is larger, which means that the biomass is larger. These biomass can better absorb and transform selenium, so that the selenium enrichment effect is the best.

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

[0109] As can be seen from the data of Example 1 and Examples 11, 12 in Table 1, when the culture temperature is 25℃, the mycelium biomass, total selenium content and organic selenium content are the highest under the condition of selecting the same medium and inoculation amount, indicating that the activity of many enzymes in the Pleurotus eryngii mycelium is higher at 25℃, which is beneficial to the absorption and transformation of selenium by microorganisms.

[0110] When the temperature is too low, the low temperature will reduce the activity of enzymes, thereby slowing down the growth rate of mycelium. Under low temperature conditions, the mycelium expansion and metabolism process will become slow; due to the slow growth rate, the obtained mycelium biomass will be reduced, which will affect the absorption and transformation ability of mycelium to selenium, resulting in the reduction of 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 metabolic products, and further affect the growth and normal physiological function of mycelium. The mycelium will change in morphology under high temperature, such as thickening of mycelium and reduction of branching, which will affect its normal physiological function. High temperature will also cause the evaporation speed of the culture solution to increase, which will cause the change of the concentration of the culture solution, thereby affecting the growth environment of the mycelium.

[0112] As can be seen from the data of Example 1 and Examples 13, 14 in Table 1, under the same environment, when the final concentration of gradient selenium is 15μg / mL, the organic selenium content of mycelium is the highest, and under this concentration, microorganisms can convert inorganic selenium into organic selenium which is more easily absorbed and utilized. When the final concentration of gradient selenium is 30μg / mL, the total selenium content of mycelium is the highest, but high concentration of sodium selenite will destroy the normal metabolism and physiological function of mycelium, resulting in the inhibition of mycelium growth, and also weakening its transformation from inorganic selenium to organic selenium, resulting in a sharp decrease in organic selenium content. At the same time, when the concentration of sodium selenite is too low, the mycelium can absorb too little selenium, thereby resulting in the reduction of selenium enrichment.

Claims

1. A method for the fermentation of selenium-enriched Pleurotus eryngii mycelium, characterized by, The method comprises the following steps: Step 1: Activation of Pleurotus eryngii strain The Pleurotus eryngii strain is inoculated on a PDA medium slope and incubated in a 25-30℃ incubator, and the mycelium is used after the slope is fully covered with mycelium; Step 2: Preparation of seed liquid medium A fermentation medium composition is selected, which comprises 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 naphthalene acetic acid (NAA) and / or 0.005-0.02g of indole butyric acid (IBA). The potatoes are peeled and cut into small pieces, added into a pot with 1000mL of distilled water, boiled, filtered to obtain a potato filtrate, and then added with glucose, potassium dihydrogen phosphate, and magnesium sulfate, and naphthalene acetic acid (NAA) and / or indole butyric acid (IBA) to dissolve after heating, and then distilled water is added to make up the total volume of the solution to 1000mL, sealed and bundled, sterilized at 121℃ for 15-30min, and a seed liquid medium is obtained. Step 3: Preparation of Pleurotus eryngii seed liquid The activated Pleurotus eryngii mycelium block was cut into 1 cm 2 Small pieces were inoculated under sterile conditions into 500 mL conical flasks containing 200 mL of seed liquid medium, 10 pieces per flask, first placed in a constant temperature incubator at 25-30°C for 2 d, and then placed on a shaker for 4-6 d of shaking culture to obtain Pleurotus eryngii seed liquid; Step 4: Gradient selenium addition culture The Pleurotus eryngii seed liquid is inoculated into the seed liquid medium at an inoculation amount of 8%-14%(v / v) for liquid fermentation, and the fermentation conditions are as follows: the temperature of the fermentation environment is maintained at 24-30℃, the rotation speed is set to 130-160r / min, the whole fermentation time lasts for 7d, and sodium selenite is added in batches according to a gradient during the fermentation process until the final concentration reaches 5-30μg / mL on the 7th day; the fermented liquid is filtered or centrifuged, the mycelium is collected, washed and dried to obtain selenium-enriched Pleurotus eryngii mycelium. The specific method of adding sodium selenite in batches according to a gradient in step 4 is as follows: on the 2nd day after the start of fermentation, the concentration of sodium selenite in the culture medium is first increased to 1μg / mL, and then the concentration of sodium selenite in the culture medium is gradually increased according to a linear gradient selenium addition mode every day until the final concentration reaches 5-30μg / mL on the 7th day.

2. The method according to claim 1, wherein the method is characterized by, The preparation method of the PDA medium in step 1 comprises the following steps: the potatoes are peeled and cut into small pieces, added into a pot with 1000mL of distilled water, boiled, filtered, and then 200-240g of the potato filtrate is taken, 20-24g of glucose and agar are added, heated and continuously stirred with a glass rod to dissolve, and distilled water is added to make up the total volume to 1000mL, the medium is divided into triangular flasks and sealed and bundled, sterilized at 121℃ for 15-30min, and a PDA medium is obtained.

3. The method according to claim 1, wherein the method is characterized by, The rotation speed of the centrifugal treatment in step 4 is 8000-10000rpm.

4. The selenium-enriched Pleurotus eryngii mycelium according to any one of claims 1 to 3, characterized in that, The total selenium content of the Pleurotus eryngii mycelium is 288.3-351.22μg / g, and the organic selenium content is 267.93-335.67μg / g.

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