CN111264299A

CN111264299A - Method for biosynthesizing organic selenium by utilizing hypsizygus marmoreus

Info

Publication number: CN111264299A
Application number: CN202010072347.2A
Authority: CN
Inventors: 郭岩彬; 胡婷; 赵桂慎; 丁惠; 惠改芳
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2020-06-12
Anticipated expiration: 2040-01-21
Also published as: CN111264299B

Abstract

The invention provides a method for biologically synthesizing organic selenium by utilizing hypsizygus marmoreus. The invention provides a hypsizygus marmoreus mutant strain which is obtained by ultraviolet mutagenesis and can efficiently convert inorganic selenium in a culture medium into organic selenium, and the preservation number is CCTCC M2019981. The seafood mushroom has short fruiting and growth time, controllable cultivation conditions, controllable selenium adding amount and stable growth state. According to the invention, sodium selenate, sodium selenite and nano-selenium are preferably used as addition forms, the biomass of the hypsizygus marmoreus fruiting bodies is not influenced under the condition that the concentration reaches 40mg/kg, the yield of the hypsizygus marmoreus fruiting bodies can be improved to a certain extent, and the selenium methyl selenocysteine can reach 39.4% of the total selenium content at most. In the cultivation process, the hypsizygus marmoreus hyphae grow fast, fungus bags are filled in a short time, the hypsizygus marmoreus is white and tender in color and high in yield, and a powerful means is provided for preparing organic selenium such as selenomethylselenocysteine and the like.

Description

Method for biosynthesizing organic selenium by utilizing hypsizygus marmoreus

Technical Field

The invention relates to the field of microbiology, in particular to a method for biosynthesizing organic selenium by utilizing hypsizygus marmoreus.

Background

Hypsizygus marmoreus belongs to the order Agaricales, Agaricaceae, Basidiomycotina, and is a large wood saprophytic fungus with delicious meat quality and good taste. The Hypsizygus marmoreus contains abundant amino acids, wherein the content of lysine and arginine is higher than that of common mushrooms, which is beneficial to improving intelligence of teenagers. The extract of the hypsizygus marmoreus has various physiological active components, wherein the fungus polysaccharide, purine and adenosine can enhance immunity, promote antibody formation, prevent aging, resist and prevent cancer, delay aging and the like. Meanwhile, the hypsizygus marmoreus is a low-calorie and low-fat health food.

Selenium is a necessary trace element for human bodies, and the human bodies can not spontaneously synthesize the selenium and can only ingest the selenium through food so as to ensure that the necessary ingestion of the selenium is achieved and the health of the human bodies is maintained. Selenium in natural environment mainly comprises two categories of inorganic selenium and organic selenium, and the organic selenium has higher biological activity than the inorganic selenium and is easier to be digested and absorbed by animals. Meanwhile, organic selenium (including selenomethylselenocysteine MeSeCys, selenomethionine SeMet, selenocysteine SeCys)₂Etc.) have the effect of preventing certain cancers. Mescys is also considered to be the most beneficial dietary source of selenium supplementation in humans, and several studies have shown that mescys appears to be more effective in chemoprevention in animal disease models. MeSeCys can be used clinically for treating selenium deficiency, as a nutrient supplement, and as a food additive for nutrition enrichment of foods. The currently reported MeSeCys is mainly synthesized chemically, see CN200710051362.3 and CN200910115585.0, etc., while MeSeCys obtained by biosynthesis is not found. A large number of researches find that MeSeCys is widely present in garlic, onion, broccoli and other plants, but the MeSeCys accounts for a small proportion of edible fungi. The invention provides a large-scale edible fungus seafood mushroom strain capable of efficiently converting inorganic selenium in a culture medium into MeSeCys, and conditions for synthesizing MeSeCys by seafood mushroom are optimized, so that sporocarps with high MeSeCys content are obtainedAnd (3) a body.

Disclosure of Invention

The invention aims to provide a method for biosynthesizing organic selenium (particularly MeSeCys) by utilizing hypsizygus marmoreus.

Another objective of the present invention is to provide a novel biosynthetic carrier of organic selenium (particularly MeSeCys), which utilizes the high nutritional quality large-scale fungus hypsizygus marmoreus as a selenium-rich carrier with high biological safety to obtain fruiting bodies with high organic selenium content.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a Hypsizygusmarmoreus HM1 (hypsizygusguard HM1), which is a mutant strain (HM1) capable of efficiently converting inorganic selenium in a medium into organic selenium, obtained by uv mutagenesis on the basis of Hypsizygusmarmoreus H023. Is preserved in China center for type culture Collection with the address: wuhan, Wuhan university, post code 430072, preservation number CCTCC M2019981, preservation date 2019, 11 months and 28 days.

In a second aspect, the present invention provides a food, pharmaceutical, nutraceutical or cosmetic product comprising hypsizygus marmoreus HM 1.

In a third aspect, the present invention provides a composition comprising hypsizygus marmoreus HM 1.

In a fourth aspect, the invention provides a culture medium for cultivating hypsizygus marmoreus, which is prepared from douglas fir sawdust, corn cobs, soybean hulls and wheat bran with uniform granularity in a weight ratio of 36-43: 28-32: 12-16: 12-16 (preferably, the weight ratio is 40: 30: 15: 15), adjusting the pH value to 8 with lime powder, uniformly mixing, bagging and sterilizing at high pressure, wherein the water content of the culture medium is 70-80%.

The sterilization conditions of the culture medium are as follows: 120-123 ℃ sterilization for 2-3h (preferably 121 ℃ sterilization for 2 h).

In a fifth aspect, the invention provides a method for biosynthesis of organic selenium by utilizing hypsizygus marmoreus, which comprises the steps of adding a selenium source into a culture medium, inoculating hypsizygus marmoreus HM1, and culturing at a temperature of 17 +/-1 ℃ and a relative humidity of 90 +/-5% in the presence of CO₂Culturing in dark at 1500 + -100 mg/L for 70-80 days (preferably 80 days), collecting fruiting body of Hypsizygus marmoreus, and separating organic selenium from fruiting body.

In the present invention, the selenium source is inorganic selenium, and preferably at least one of selenate (such as sodium selenate), selenite (such as sodium selenite) and nano-selenium. The preparation of nano-selenium can be found in ZL201410520106.4, ZL201610946282.3, ZL201610566900.1, ZL201510047365.4, ZL201610952997.X, ZL201610946230.6, but is not limited to the methods described in the above patents.

The concentration of the added selenium source in the culture medium is 0.5-40.0mg/kg, preferably 0.5mg/kg, 1.0mg/kg, 2.5mg/kg, 5.0mg/kg, 10.0mg/kg, 20.0mg/kg, 40.0 mg/kg.

The organic selenium includes but is not limited to selenomethylselenocysteine, selenomethionine, selenocysteine, and preferably selenomethylselenocysteine.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

according to the invention, various inorganic selenium is used as an additive selenium source of the hypsizygus marmoreus, and selenium-rich cultivation of the hypsizygus marmoreus is carried out by selecting a selenium concentration from low to high, so that selenium-containing hypsizygus marmoreus fruiting bodies which can be directly eaten in daily life and fruiting bodies with high organic selenium proportion which can be used as raw materials for extracting MeSeCys are obtained. The seafood mushroom has short fruiting and growth time, controllable cultivation conditions, controllable selenium adding amount and stable growth state. Preferably, the sodium selenate, sodium selenite and nano-selenium are used as the adding forms, the biomass of the hypsizygus marmoreus fruiting body is not influenced under the condition that the concentration reaches 40mg/kg, the yield of the hypsizygus marmoreus fruiting body can be improved to a certain extent, and the selenium methyl selenocysteine can reach 39.4 percent of the total selenium content at most. During the cultivation process, the hypsizygus marmoreus hyphae grow fast, the fungus bags are filled in a short time, and the hypsizygus marmoreus is white and tender in color and high in yield (figure 1). Provides a powerful means for preparing the organic selenium.

Drawings

FIG. 1 shows the shape of the hypsizygus marmoreus HM1 cultivated and grown according to the present invention.

FIG. 2 shows the resistance of HM023 strain to selenate and selenite in example 1 of the present invention.

FIG. 3 shows the colony morphology of the colonies of the Hypsizygus marmoreus of different strains on the PDA plate in example 1 of the present invention.

FIG. 4 shows the colonies grown by each of the Hypsizygus marmoreus strains on the PDA plate containing 5ml/L sodium selenite in example 1 of the present invention.

FIG. 5 shows the sodium selenate treated Hypsizygus marmoreus biomass in example 2 of the present invention. Wherein, different lower case letters a, b and c represent significant difference, and p is less than 0.05.

FIG. 6 shows the selenium form in the fruit body treated with sodium selenate in example 2 of the present invention.

FIG. 7 shows the sodium selenite treatment of Hypsizygus marmoreus biomass in example 3 of the present invention. Wherein, different lower case letters a, b and c represent significant difference, and p is less than 0.05.

FIG. 8 shows the selenium form in the fruit body treated with sodium selenite in example 3 of the present invention.

FIG. 9 chemical nano-selenium treatment of Hypsizygus marmoreus biomass in example 4 of the present invention. Wherein, different lower case letters a, b and c represent significant difference, and p is less than 0.05.

FIG. 10 shows the selenium form in the fruiting body treated with chemical nano-selenium in example 4 of the present invention.

FIG. 11 biological nano-selenium treatment of Hypsizygus marmoreus biomass in example 5 of the present invention. Wherein, different lower case letters a, b and c represent significant difference, and p is less than 0.05.

FIG. 12 shows the selenium form in the fruiting body treated with biological nano-selenium in example 5 of the present invention.

Detailed Description

The invention provides a method for biologically synthesizing organic selenium (particularly selenium methyl selenocysteine) by utilizing hypsizygus marmoreus, which utilizes douglas fir sawdust, corn cobs, soybean hulls and wheat bran with uniform granularity in a weight ratio of 40: 30: 15: 15, adding lime powder to adjust the pH to 8, and preparing the culture medium with the water content of 80%, setting the concentration gradient of selenate, selenite and chemical nano-selenium to be 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, filling 2.0kg of mixed materials into each culture bag, and sterilizing the mixed materials for 2 hours at the high temperature of 121 ℃ and under high pressure. Taking a bacterial cake with uniform size of 5mm from a test tube for preserving the bacterial strain, inoculating the bacterial cake into a 500mL conical shaking flask containing 150mL liquid PDA culture medium, and carrying out shaking culture at the temperature of 21-23 ℃ for 4 days at 180r/min for 170-. From overgrowth with bacteria5mL of strain is sucked by a sterile pipette in a primary strain shake flask of the silk ball and transferred to a 500mL shake flask containing 150mL PDA liquid culture medium for secondary strain culture, the secondary strain is subjected to shaking culture at the temperature of 21-23 ℃ for 3 days at 180r/min, and 10mL of secondary bacterial liquid is inoculated and inoculated into a sterilized cultivation bag. Cultivation conditions are as follows: the temperature is set to be 17 +/-1 ℃, the relative humidity is 90 +/-5 percent, and CO is added₂The amount of the extract is 1500 +/-100 mg/L, and the extract is cultured in the dark for 80 days. Harvesting hypsizygus marmoreus sporocarp after maturation, measuring the yield, performing freeze-drying treatment, crushing, and analyzing the form and content of organic selenium.

In the invention, the method for measuring the organic selenium comprises the following steps: the detection is carried out by adopting a high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) method, and the specific method can be seen in Hu T, Liang Y, ZHao G, Wu W, Li H and Guo Y.2019. Selenium biochemical and antibiotic activity in Cordycepsmilitaris applied with selected, or selected theory. biological Trace Element research, 187:553-561.Doi:10.1007/s 12011-018-1386-Y; and TingHu, Li Li, Gaifang Hui, Junjie Zhang, Huafen Li, Wenliang Wu, Xuehong Wei, YanbinGuo.2019.Selenium biodistribution and its effect on multimedia-element change in Australia auricular. food Chemistry 295: 206-213. DOI:10.1016/j. foodchem.2019.05.101. The method mainly comprises the following steps: freeze drying Hypsizygus marmoreus, pulverizing, collecting 0.2g, adding 2mg mL^-1Protease XIV is subjected to enzymolysis at 37 deg.C under 150rpm for 24 hr, then centrifuged at 8000g for 5min to remove cell debris, the supernatant is filtered with 0.22 μm filter, and separated with liquid chromatography column Hamilton RPR-X100 (Flexa; Pekinelmer, USA), and the mobile phase is 40mM (NH)₄)₂HPO₄At a flow rate of 1mL min^-1And detecting selenium in different forms by using Inductively Coupled Plasma Mass Spectrometry (ICPMS) as a detector. Se-methylselenocysteine, Se-methionine, Se-cysteine, selenate and selenite are used as standard samples. The method has high detection accuracy, and the selenium form of 1 mug/L can be effectively detected; the selenium recovery rate is up to more than 80%.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

EXAMPLE 1 Strain Breeding and selenium-enriched vector acquisition

Culturing Hypsizygus marmoreus strain H023 stored in edible fungi research laboratory of China agricultural university on PDA culture medium for 14 days, taking 5mm fungus cakes from the growth edge of the colony to respectively inoculate on plates with the contents of sodium selenate and sodium selenite of 1.0mg/kg, 5.0mg/kg and 20.0mg/kg, culturing for 10 days at 21 ℃, and measuring the diameter of the colony, wherein the result shows that the growth of the colony is obviously inhibited under the treatment of 5.0mg/kg of sodium selenite by H023; the H023 hypha growth was significantly inhibited under the treatment of 25.0mg/kg sodium selenate (FIG. 2).

Culturing Hypsizygus marmoreus strain H023 stored in edible fungi research laboratory of China agricultural university on PDA culture medium for 14 days, inoculating 5mm fungus cakes from the growth edge of the colony to 10 PDA plates respectively for culturing for 2 days, placing under an ultraviolet lamp tube of 30W for 25cm to irradiate for 3min, culturing in dark for 24H, then irradiating for 2 nd time, and repeating the irradiation for 3 times. After the mutagenized strain was cultured for 7 days, 5mm of cake was taken from the edge of the colony and inoculated on PDA medium at 21 ℃ for 14 days, and the colony diameter was measured. Among them, HM1, HM3, HM4 and HM7 showed no difference in growth from the control strain H023 (fig. 3).

The colony diameters of HM1, HM3, HM4, HM7 and H023 strains were inoculated to PDA plates containing 5mg/L of sodium selenate and sodium selenite, each treated with 10 plates, cultured at 21 ℃ for 10 days, and measured, and the results showed that the colony diameters of HM1 and HM3 strains were not significantly different from the growth of PDA plates containing no selenium and were significantly higher than the colony diameter of H023 on PDA plates containing 5mg/L of sodium selenate and sodium selenite (FIG. 4). The two strains HM1 and HM3 are respectively cultivated in 5mg/L cultivation medium of sodium selenate and sodium selenite (the weight ratio of the douglas fir sawdust to the corn cob to the soybean hull to the wheat bran is 40: 30: 15: 15, and the water content is 80%), and the result shows that the fresh weight of the hypsizygus marmoreus cultivated in the 5mg/L cultivation medium of sodium selenate and sodium selenite is respectively increased by 7.9% and 11.6%, but the yield of the HM3 strain is respectively reduced by 15% and 12%. HM1 strain is preferred strain, and is preserved in China center for type culture Collection with strain number of CCTCC M2019981.

Example 2 sodium selenate treatment selenium-rich cultivation

The weight ratio of the douglas fir sawdust, the corn cobs, the soybean hulls and the wheat bran with uniform granularity is 40: 30: 15: 15, adjusting the pH to 8 by using lime powder, setting the water content to be 80 percent, setting the selenate concentration gradient to be 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, filling 2.0kg of mixed materials into each culture bag, and sterilizing the pot for 2 hours at the high temperature of 121 ℃. After cooling to room temperature, the strain was inoculated. Taking a bacterial cake with uniform size of 5mm from a test tube for preserving the bacterial strain, inoculating the bacterial cake into a 500mL conical shaking flask containing 150mL liquid PDA culture medium, and carrying out shaking culture at the temperature of 21-23 ℃ for 4 days at 180r/min for 170-. 5mL of strains are sucked from a primary strain shake flask full of mycelial pellets by a sterile pipette and transferred to a 500mL shake flask containing 150mL of PDA liquid culture medium for secondary strain culture, the secondary strain is subjected to shaking culture at the temperature of 21-23 ℃ for 3 days at 180r/min at 170-. The culture conditions are as follows: the temperature is set to be 17 +/-1 ℃, the relative humidity is 90 +/-5 percent, and CO is added₂The amount of the extract is 1500 +/-100 mg/L, and the extract is cultured in the dark for 80 days. Collecting the hypsizygus marmoreus fruiting body, measuring the yield, freeze-drying, grinding, and measuring the selenium form in the fruiting body. The biomass of the fruit bodies of the hypsizygus marmoreus under the condition of different concentrations of sodium selenate treatment is 454.5 +/-11.52, 360.0 +/-38.0, 374.2 +/-40.2,366.8 +/-59.6, 374.3 +/-20.6, 367.6 +/-39.4 and 342.5 +/-32.3 g/rod (fresh weight), and the biomass of the hypsizygus marmoreus in a control group (without sodium selenate addition) is 379.6 +/-44.0 g/rod (figure 5). Under the condition of treatment with sodium selenate with different concentrations, the total selenium content in the hypsizygus marmoreus sporocarp is respectively 0.7 +/-0.1, 1.3 +/-0.1, 3.8 +/-0.2, 5.3 +/-0.9, 7.0 +/-0.6, 13.6 +/-2.8 and 29.7 +/-4.1 mg/kg, and the MeSeCys accounts for 15.9-23.9 percent of the total selenium. When the selenate concentration reaches 40mg/kg, the selenium form in the fruiting body is shown in FIG. 5. With the increase of the addition concentration of the sodium selenate, the selenium content in the hypsizygus marmoreus sporocarp is increased. The selenium form in the fruiting body of Hypsizygus marmoreus is mainly MeSeCys, SeMet and SeCys₂The three forms exist, and the hypsizygus marmoreus fruiting body has no inorganic selenium residue, and the proportion of organic selenium is 100% (figure 6). SeCys in the fruit body of Hypsizygus marmoreus treated with 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, 40.0mg/kg sodium selenate₂The unknown selenium form, the proportions of MeSeCys and SeMet are: 11.9%, 0.5%, 14.9% and 72.7%; 10.5%, 0.4%, 15.4% and 73.7%; 6.9%, 0.6%, 15.8% and 76.7%; 5.5%, 0.5%, 16.9% and 77.1%; 4.9%, 0.6%, 17.4% and 77.1%; 5.9%, 0.6%, 16.9% and 76.6%; 3.1%, 1.8%, 18.8% and 76.3%. Wherein the content of MeSeCys in the sodium selenate-treated fruiting body is 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, and the content of MeSeCys in the sodium selenate-treated fruiting body is 0.1mg/kg, 0.2mg/kg, 0.6mg/kg, 0.9mg/kg, 1.2mg/kg, 2.3mg/kg and 5.6mg/kg respectively.

Example 3 sodium selenite treatment selenium-rich cultivation

The weight ratio of the douglas fir sawdust, the corn cobs, the soybean hulls and the wheat bran with uniform granularity is 40: 30: 15: 15, adjusting the pH to 8 by using lime powder, wherein the water content is 80%, the selenite concentration gradient is set to be 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, each culture bag is filled with 2.0kg of mixed materials, and the autoclave is sterilized at the high temperature of 121 ℃ for 2 hours. After cooling to room temperature, the strain was inoculated. Taking a bacterial cake with uniform size of 5mm from a test tube for preserving the bacterial strain, inoculating the bacterial cake into a 500mL conical shaking flask containing 150mL liquid PDA culture medium, and carrying out shaking culture at the temperature of 21-23 ℃ for 4 days at 180r/min for 170-. 5mL of strains are sucked from a primary strain shake flask full of mycelial pellets by a sterile pipette and transferred to a 500mL shake flask containing 150mL of PDA liquid culture medium for secondary strain culture, the secondary strain is subjected to shaking culture at the temperature of 21-23 ℃ for 3 days at 180r/min at 170-. The culture conditions are as follows: the temperature is set to be 17 +/-1 ℃, the relative humidity is 90 +/-5 percent, and CO is added₂The amount of the extract is 1500 +/-100 mg/L, and the extract is cultured in the dark for 80 days. Collecting the hypsizygus marmoreus fruiting body, measuring the yield, freeze-drying, grinding, and measuring the selenium form in the fruiting body. The biomass of the hypsizygus marmoreus fruiting body under the sodium selenite treatment condition is 397.4 + -44.0, 434.3 + -64.9, 405.0 + -67.1, 405.4 + -65.7, 414.9 + -38.2, 432.9 + -44.5, 404.8 + -67.2, 397.4 + -81.0 g/rod (fresh weight), which is obviously higher than that of the control group (without adding sodium selenate) hypsizygus marmoreus biomass 379.6 + -44.0 g/rod (figure 7). Under the treatment conditions of sodium selenite with different concentrations, the total selenium content in the hypsizygus marmoreus sporocarp is respectively 0.4 plus or minus 0.1,0.8 plus or minus 0.2, 1.5 plus or minus 0.1, 1.9 plus or minus 0.2, 3.5 plus or minus 0.2, 6.4 plus or minus 0.3, 12.8 plus or minus 3.1mg/kg, and the proportion of MeSeCys in the total selenium is 33.7-43.9%. When the selenite concentration reaches 40mg/kg, the selenium form in the fruiting body is shown in FIG. 8. With the increase of the addition concentration of the sodium selenite, the selenium content in the hypsizygus marmoreus sporocarp is increased. The selenium form in the fruiting body of Hypsizygus marmoreus is mainly MeSeCys, SeMet, SeCys₂And small amounts of Se (IV) are present in four forms (FIG. 8). SeCys in the fruit body of Hypsizygus marmoreus treated with 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, 40.0mg/kg sodium selenite₂The unknown selenium form, the proportions of MeSeCys, Se (IV) and SeMet are respectively: 13.8%, 0.2%, 34.8%, 0 and 51.2%; 12.0%, 0.7%, 33.8%, 0.1% and 53.4%; 10.5%, 0.5%, 36.9%, 0.4% and 51.7%; 10.5%, 0.5%, 36.9, 0.4% and 51.7%; 7.9%, 0.7%, 35.0%, 0.9% and 55.5%; 3.8%, 1.4%, 36.9%, 2.3% and 55.6%; 2.8%, 1.7%, 39.4%, 4.4% and 51.7%. Wherein the content of MeSeCys in the sodium selenite-treated fruiting body is 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, respectively, 0.2mg/kg, 0.3mg/kg, 0.5mg/kg, 0.7mg/kg, 1.3mg/kg, 2.4mg/kg and 5.0 mg/kg.

Example 4 chemical nano-selenium treatment selenium-rich cultivation

The chemical nano-selenium used in this example can be prepared as described in ZL201410520106.4 example 1.

The weight ratio of the douglas fir sawdust, the corn cobs, the soybean hulls and the wheat bran with uniform granularity is 40: 30: 15: and 15, adjusting the pH to 8 by using lime powder, wherein the water content is 80%, the concentration gradient of the chemical nano selenium is set to be 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, each culture bag is filled with 2.0kg of mixed materials, and the high-temperature and high-pressure sterilization pot is sterilized at 121 ℃ for 2 hours. After cooling to room temperature, the strain was inoculated. Taking a bacterial cake with uniform size of 5mm from a test tube for preserving the bacterial strain, inoculating the bacterial cake into a 500mL conical shaking flask containing 150mL liquid PDA culture medium, and carrying out shaking culture at the temperature of 21-23 ℃ for 4 days at 180r/min for 170-. Sucking 5mL of strain from the first-stage strain shake flask full of mycelial pellets by using a sterile pipette, transferring the strain to a 500mL shake flask containing 150mL of PDA liquid culture medium, and performing second-stage strain cultureThe strain is subjected to shaking culture at the temperature of 21-23 ℃ for 3 days at 170-180r/min, and 10mL of secondary bacterial liquid is inoculated into a sterilized culture bag. The culture conditions are as follows: the temperature is set to be 17 +/-1 ℃, the relative humidity is 90 +/-5 percent, and CO is added₂The amount of the extract is 1500 +/-100 mg/L, and the extract is cultured in the dark for 80 days. Collecting the hypsizygus marmoreus fruiting body, measuring the yield, freeze-drying, grinding, and measuring the selenium form in the fruiting body. The biomass of the hypsizygus marmoreus fruiting body under the chemical nano-selenium treatment condition is 379.6 +/-44.0, 340.1 +/-54.9, 367.6 +/-53.7, 387.6 +/-27.0, 374.8 +/-43.7, 391.5 +/-40.5, 449.3 +/-18.0, 450.0 +/-4.2 g/rod (fresh weight), and the biomass of the hypsizygus marmoreus (without sodium selenate) in a control group is 379.6 +/-44.0 g/rod (figure 9). Under the condition of treatment with sodium selenate with different concentrations, the total selenium content in the hypsizygus marmoreus sporocarp is respectively 0.6 +/-0.1, 1.0 +/-0.1, 1.8 +/-0.1, 2.7 +/-0.2, 4.2 +/-0.4, 7.2 +/-0.7 and 15.0 +/-0.3 mg/kg, and the MeSeCys accounts for 18.9-30.2 percent of the total selenium. When the chemical nano-selenium concentration reaches 40mg/kg, the selenium form in the fruiting body is shown in fig. 10. With the increase of the adding concentration of the chemical nano-selenium, the selenium content in the hypsizygus marmoreus sporocarp is increased. The selenium form in the fruiting body of Hypsizygus marmoreus is mainly MeSeCys, SeMet, SeCys₂And small amounts of Se (IV) are present in four forms (FIG. 10). SeCys in the fruit body of the hypsizygus marmoreus under the treatment of 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg of chemical nano-selenium₂The unknown selenium form, the proportions of MeSeCys, Se (IV) and SeMet are respectively: 7.4%, 0, 22.5%, 0 and 70.1%; 7.1%, 0, 20.6%, 0 and 72.3%; 7.1%, 0, 20.6%, 0 and 72.3%; 5.5%, 0.4%, 21.9%, 0.1% and 72.1%; 4.8%, 0.3%, 22.7%, 0.4% and 71.8%; 2.5%, 0.3%, 23.8%, 1.5% and 71.9%; 2.2%, 0.4%, 22.3%, 3.4% and 71.7%. Wherein the content of MeSeCys in the fruiting body treated by 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg of chemical nano selenium is 0.1mg/kg, 0.2mg/kg, 0.4mg/kg, 0.6mg/kg, 1.0mg/kg, 1.7mg/kg and 3.3mg/kg respectively.

Example 5 biological Nano selenium treatment selenium-enriched cultivation

The preparation method of the biological nano selenium used in the embodiment can be seen in ZL201610946282.3 embodiment 1.

By utilizing the uniformity of the particle sizeThe weight ratio of the douglas fir sawdust to the corn cobs to the soybean hulls to the wheat bran is 40: 30: 15: 15, adjusting the pH to 8 by using lime powder, setting the water content to be 80 percent, setting the concentration gradient of the biological nano selenium to be 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, filling 2.0kg of mixed materials into each culture bag, and sterilizing the pot for 2 hours at the high temperature of 121 ℃. After cooling to room temperature, the strain was inoculated. Taking a bacterial cake with uniform size of 5mm from a test tube for preserving the bacterial strain, inoculating the bacterial cake into a 500mL conical shaking flask containing 150mL liquid PDA culture medium, and carrying out shaking culture at the temperature of 21-23 ℃ for 4 days at 180r/min for 170-. 5mL of strains are sucked from a primary strain shake flask full of mycelial pellets by a sterile pipette and transferred to a 500mL shake flask containing 150mL of PDA liquid culture medium for secondary strain culture, the secondary strain is subjected to shaking culture at the temperature of 21-23 ℃ for 3 days at 180r/min at 170-. The culture conditions are as follows: the temperature is set to be 17 +/-1 ℃, the relative humidity is 90 +/-5 percent, and CO is added₂The amount of the extract is 1500 +/-100 mg/L, and the extract is cultured in the dark for 80 days. Collecting the hypsizygus marmoreus fruiting body, measuring the yield, freeze-drying, grinding, and measuring the selenium form in the fruiting body. The biomass of the fruit bodies of the hypsizygus marmoreus under the biological nano-selenium treatment condition is 382.6 +/-41.0, 385.7 +/-42.4, 391.2 +/-24.8, 397.0 +/-20.6, 391.1 +/-16.7, 402.9 +/-26.2, 423.1 +/-22.1, 438.3 +/-26.1 g/rod (fresh weight), and the biomass of the hypsizygus marmoreus in a control group (without sodium selenate) is 376.6 +/-34.2 g/rod (figure 11). Under the condition of treatment with sodium selenate with different concentrations, the total selenium content in the hypsizygus marmoreus sporocarp is respectively 0.5 +/-0.1, 1.0 +/-0.2, 1.8 +/-0.1, 2.6 +/-0.2, 4.3 +/-0.4, 7.3 +/-0.5 and 15.5 +/-0.6 mg/kg, and the MeSeCys accounts for 18.8-32.1 percent of the total selenium. When the biological nano selenium concentration reaches 40mg/kg, the selenium form in the fruiting body is shown in figure 12. Along with the increase of the adding concentration of the biological nano selenium, the selenium content in the hypsizygus marmoreus sporocarp is increased. The selenium form in the fruiting body of Hypsizygus marmoreus is mainly MeSeCys, SeMet, SeCys₂And small amounts of Se (IV) are present in four forms (FIG. 12). SeCys in the fruit body of the hypsizygus marmoreus under the treatment of 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg biological nano selenium₂The proportions of MeSeCys, Se (IV) and SeMet are respectively as follows: 8.8%, 23.6%, 0 and 67.6%; 7.0%, 22.7%, 0 and 70.3%; 7.0%, 20.8%, 0 and 72.2%;4.4%, 22.9%, 0 and 72.7%; 2.7%, 24.0%, 0 and 73.3%; 2.8%, 24.0%, 0 and 73.3%; 2.5%, 24.8%, 0 and 71.5%. Wherein the content of MeSeCys in the biological nano selenium-treated fruiting body is 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 40.0mg/kg, and the content of MeSeCys in the biological nano selenium-treated fruiting body is 0.1mg/kg, 0.2mg/kg, 0.4mg/kg, 0.6mg/kg, 1.0mg/kg, 1.8mg/kg and 3.8mg/kg respectively.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. Hypsizygus marmoreus HM1(Hypsizygus marmoreus HM1), characterized by the preservation number of CCTCC M2019981.

2. A food, pharmaceutical, nutraceutical or cosmetic product comprising hypsizygus marmoreus HM1 of claim 1.

3. A composition comprising hypsizygus marmoreus HM1 of claim 1.

4. The cultivation medium of hypsizygus marmoreus HM1 as claimed in claim 1, which is prepared from douglas fir sawdust, corn cobs, soybean hulls and wheat bran with uniform particle size by weight ratio of 36-43: 28-32: 12-16: 12-16, adjusting the pH value to 8 by using lime powder, uniformly mixing, bagging, and carrying out autoclaving to obtain the culture medium, wherein the water content of the culture medium is 70-80%;

preferably, the weight ratio of douglas fir sawdust, corn cobs, soybean hulls and wheat bran is 40: 30: 15: 15.

5. the cultivation medium as claimed in claim 4, wherein the sterilization conditions are: sterilizing at the temperature of 120 ℃ and 123 ℃ for 2-3 h.

6. By using the sea fresh mushroom biologyA method for producing organic selenium, characterized in that selenium source is added into the culture medium of claim 4 or 5, and then the hypsizygus marmoreus HM1 of claim 1 is inoculated, at the temperature of 17 + -1 deg.C, relative humidity of 90 + -5%, CO₂Culturing in dark at 1500 + -100 mg/L for 70-80 days, collecting fruiting body of Hypsizygus marmoreus, and separating organic selenium from the fruiting body.

7. The method according to claim 6, wherein the selenium source is inorganic selenium, preferably at least one of selenate, selenite and nano-selenium.

8. The method of claim 7, wherein the selenium source is added at a concentration of 0.5-40.0mg/kg in the cultivation medium.

9. The method according to any one of claims 6 to 8, wherein the organic selenium comprises selenomethylselenocysteine, selenomethionine, selenocysteine.

10. The method of claim 9, wherein the organic selenium is selenomethylselenocysteine.