阅读说明：本技术 一种利用金针菇生物合成纳米硒的方法 (Method for biosynthesizing nano-selenium by utilizing needle mushrooms ) 是由 陈磊 李佳铭 吴凡 朱雨婕 代安然 杨崇婧 于 2019-10-10 设计创作，主要内容包括：本发明公开了一种利用金针菇生物合成纳米硒的方法,属于生物技术领域。本发明是利用金针菇子实体或液态发酵到一定时间的菌丝体,在培养基中加入不同浓度的Se(IV),继续进行培养,最终在菌体和/或胞外生物合成纳米硒颗粒。本发明的方法条件温和,工艺简单,环境友好,易于实现,具有潜在的工业化应用前景。(The invention discloses a method for biosynthesizing nano-selenium by utilizing needle mushrooms, belonging to the technical field of biology. The invention utilizes the needle mushroom fruiting body or mycelium fermented in liquid state to a certain time, adds Se (IV) with different concentrations into a culture medium, continues culturing, and finally synthesizes nano selenium particles in the thallus and/or extracellular organisms. The method has the advantages of mild conditions, simple process, environmental friendliness, easy realization and potential industrial application prospect.)

1. A method for producing nano-selenium is characterized in that Flammulina velutipes (Flammulina velutipes) fermentation and/or sporocarp immersion culture are utilized in an environment containing tetravalent inorganic selenium salt to produce nano-selenium.

2. The method of claim 1, wherein the nano-selenium in the fermentation product is collected; the fermentation product includes but is not limited to flammulina velutipes mycelium, fruiting body (containing stipe), fermentation liquor, precipitate after the fermentation liquor is centrifuged, and flammulina velutipes mycelium lysate.

3. The method according to claim 1 or 2, wherein the final concentration of selenium in the fermentation broth is 0.1-20mM by adding tetravalent inorganic selenium salt when fermenting to 6-9 days after inoculation, and continuing the fermentation for a total time of 9-12 days.

4. The process according to claim 1 or 2, characterized in that the solution of tetravalent inorganic selenium salt is Na₂SeO₃And (3) solution.

5. A method according to any one of claims 1 to 4, comprising the steps of:

(1) activating the flammulina velutipes strains by adopting a PDA culture medium;

(2) transferring the flammulina velutipes mycelium obtained by the activation in the step (1) into a seed culture medium, and culturing for 5-8 days at 20-28 ℃;

(3) the seed liquid cultured in the step (2) is treated according to the proportion of 10⁵Transferring the spores/mL fermentation liquor into a fermentation culture medium, and fermenting at 22-30 ℃ and 130-200 rpm for 6-9 days.

(4) Adding a tetravalent inorganic selenium salt Se (IV) solution into the fermentation liquor obtained in the step (3), continuing fermentation until the total fermentation time reaches 9-12d, and collecting nano selenium or a product containing the nano selenium obtained by fermentation according to any one of a, b and c:

a: directly collecting a fermentation product;

b: sterile filtering the flammulina velutipes fermentation liquor fermented in the step (3), collecting mycelia, washing the mycelia with sterile water for 3-5 times, transferring the mycelia to a solution containing tetravalent inorganic selenium salt, and continuously culturing for 2-4 days by using a shaking table to obtain selenium-rich flammulina velutipes mycelia;

c: and (3) performing static culture on the flammulina velutipes mycelium obtained in the step (4) at the temperature of 22-30 ℃ for 15-25 d to obtain flammulina velutipes sporocarp, immersing the flammulina velutipes sporocarp in tetravalent inorganic selenium salt solution, and performing static culture for 1-4d to obtain the selenium-rich flammulina velutipes sporocarp.

6. The method of claim 5, wherein the nanoselenium is also isolated from the fermentation product after the end of the fermentation and/or cultivation.

7. The method as claimed in claim 6, wherein the fermentation/culture product is sieved by a sieve of 30-50 meshes, thalli are collected, washed for 3-5 times, and cells are crushed to obtain thalli lysate; centrifuging the thallus lysate for 10-30min at 8000rpm of 5000-; and (3) resuspending the precipitate, dialyzing by a dialysis bag with the molecular weight cutoff of 3000-4000 Da, and freeze-drying to obtain the nano-selenium dry powder.

8. The method as claimed in claim 6, wherein the fermentation product after fermentation and/or culture is sieved by a 30-mesh sieve, the thalli are collected and washed for 3-5 times, and the thalli are freeze-dried, ground and sieved to obtain thalli powder containing nano-selenium.

9. The method of claim 6, wherein the fermentation product after fermentation and/or culture is sieved by a 30-mesh sieve, after thalli are removed, the fermentation liquor is filtered by quantitative filter paper to remove solid substances, and is ultrafiltered by an ultrafiltration membrane, reflux liquid is collected, and the nanometer selenium dry powder is obtained after freeze drying.

10. A product prepared by the method of any one of claims 1 to 9.

Technical Field

The invention relates to a method for biosynthesizing nano-selenium by utilizing flammulina velutipes, belonging to the technical field of biological nano-technology.

Background

Selenium, as a micronutrient essential to human body, is toxic, has a narrow safety range (i.e. between effective and toxic amounts), and is easily poisoned by excessive amounts. The nano-selenium prepared by the nano-technology, although being zero-valent selenium, can be absorbed and utilized by human bodies, can also play the biological and health-care functions of selenium, such as oxidation resistance, immunoregulation and the like, and is more important to be lower in toxicity than other selenium compounds such as inorganic selenium (sodium selenite), organic selenium (selenoprotein) and the like. The main basis for the approval of nano-selenium as a health product is also its safety.

The edible fungi have good biological enrichment and transformation effects, and have great potential in the fields of biosynthesis of metal or nonmetal nano materials for green manufacturing, environmental protection, functional food, medical materials and the like. Edible and medicinal fungus species such as shiitake mushroom, ganoderma lucidum and the like have also been used for enrichment and transformation of selenium, but organic selenium forms such as selenoprotein and the like are mostly reported. At present, the metabolic mechanism of the biosynthesis nanometer material is not clear, and the problems of poor particle uniformity, poor stability and the like often occur in the biosynthesis process of the nanometer material, so that the large-scale industrial production of the edible fungus-derived nanometer material cannot be realized, and the actual industrial application is not obtained. The flammulina velutipes, as a conventional cultivation and daily edible fungus, shows remarkable research potential and application prospect in the aspect of biotransformation and synthesis of nano materials.

Disclosure of Invention

The invention aims to provide a method for biologically synthesizing nano-selenium, which obtains needle mushroom mycelia and needle mushroom sporophores rich in nano-selenium by means of liquid fermentation of needle mushroom (Flammulinavelipes) mycelia and immersion culture of the needle mushroom sporophores.

The first purpose of the invention is to provide a method for producing nano-selenium, which utilizes Flammulina velutipes (Flammulina velutipes) fermentation and/or sporocarp immersion culture in an environment containing tetravalent inorganic selenium salt to produce nano-selenium.

In one embodiment, the method collects the nano-selenium in the fermentation product; the fermentation product includes but is not limited to flammulina velutipes mycelium, fruiting body (containing stipe), fermentation liquor, precipitate after the fermentation liquor is centrifuged, and flammulina velutipes mycelium lysate.

In one embodiment, the method comprises adding 0.1-20mM quaternary inorganic selenium salt solution to the fermentation solution after inoculation to 6-9 days, respectively, and continuing the fermentation for a total time of 9-12 days.

In one embodiment, the solution of tetravalent inorganic selenium salt is Na₂SeO₃And (3) solution.

In one embodiment, the Na₂SeO₃The concentration of the solution is 0.1-10 mM.

In one embodiment, the method comprises the steps of:

(1) activating the flammulina velutipes strains by adopting a PDA culture medium;

(2) transferring the flammulina velutipes mycelium obtained by the activation in the step (1) into a seed culture medium, and culturing for 5-8 days at 20-28 ℃;

(3) the seed liquid cultured in the step (2) is treated according to the proportion of 10⁵Transferring the spores/mL fermentation liquor into a fermentation culture medium, and fermenting at 22-30 ℃ and 130-200 rpm for 6-9 days.

(4) Adding a tetravalent inorganic selenium salt Se (IV) solution into the fermentation liquor obtained in the step (3), continuing fermentation until the total fermentation time reaches 9-12d, and collecting nano selenium or a product containing the nano selenium obtained by fermentation according to any one of a, b and c:

a: directly collecting a fermentation product;

b: sterile filtering the flammulina velutipes fermentation liquor fermented in the step (3), collecting mycelia, washing the mycelia with sterile water for 3-5 times, transferring the mycelia to a solution containing tetravalent inorganic selenium salt, and continuously culturing for 2-4d by a shaking table to obtain selenium-rich flammulina velutipes mycelia;

c: and (3) performing static culture on the flammulina velutipes mycelium obtained in the step (4) at the temperature of 22-30 ℃ for 15-25 d to obtain flammulina velutipes sporocarp, immersing the flammulina velutipes sporocarp in tetravalent inorganic selenium salt solution, and performing static culture for 1-4d to obtain the selenium-rich flammulina velutipes sporocarp.

In one embodiment, the nano-selenium in the fermentation product after the end of the fermentation and/or cultivation is also separated by: sieving the fermentation/culture product obtained in the step (4) by a 30-mesh sieve, collecting thalli, washing the thalli for 3-5 times by deionized water, and destroying the cell structure of the thalli by a tissue homogenizer to obtain a thalli lysate; centrifuging the thallus lysate for 10-20min at 8000rpm of 5000-; and (3) resuspending the precipitate in 20mL of deionized water, dialyzing by a dialysis bag with the molecular weight cutoff of 3000Da, and freeze-drying to obtain the nano-selenium dry powder.

In one embodiment, the nano-selenium in the fermentation product after the end of the fermentation and/or cultivation is also separated by: and (3) sieving the fermentation product obtained in the step (4) by a 30-mesh sieve, collecting thalli, washing the thalli for 3-5 times by deionized water, freezing and drying the thalli, grinding the thalli, and sieving the powder by a 80-mesh sieve to obtain thalli powder containing nano selenium.

In one embodiment, the nano-selenium in the fermentation product after the end of the fermentation and/or cultivation is also separated by: and (3) after the fermentation product collected in the step (4) is sieved by a 30-mesh sieve to remove thalli, filtering the fermentation liquor and/or the culture solution by quantitative filter paper to remove solid matters, ultrafiltering by an ultrafiltration membrane with the molecular weight of 1 ten thousand, collecting the reflux liquid, and freeze-drying to obtain the nano-selenium dry powder.

In one embodiment, the method comprises the following specific steps:

(1) strain activation

Activating and culturing the strain by using a PDA culture medium, wherein the PDA culture medium comprises the following components in parts by weight: dicing 200g peeled potato, adding 1L deionized water, decocting at 100 deg.C for 30-40min, filtering to obtain filtrate, adding 20g glucose and 20g agar, heating to dissolve, adding water to total volume of 1L, autoclaving at 121 deg.C for 20min, cooling, pouring into flat plate, solidifying, inoculating needle mushroom strain on PDA flat plate, and culturing at 25 deg.C for 7 d.

(2) Preparation of seed liquid

Preparing a needle mushroom seed solution, wherein a culture medium comprises: glucose 20g/L, yeast 20g/L, potassium dihydrogen phosphate (KH)₂PO₄)1g/L, magnesium sulfate heptahydrate (MgSO)₄·7H₂O)0.5g/L，VB₁0.5g/L, adding 200mL of culture medium into 500mL shake flask, autoclaving at 121 deg.C for 20min, and inoculating 5-15 pieces of 1 × 1cm²Slicing PDA plate of Flammulina velutipes, and culturing at 25 deg.C under 160rpm shaking table for 7 days.

(3) Shake flask fermentation

Preparing needle mushroom fermentation liquid (the formula is consistent with the seed liquid), wherein 190mL of needle mushroom fermentation liquid is contained in 500mL of shake flask, inoculating 10mL of seed liquid into each flask after sterilization and cooling, and culturing in a shaking table at 160rpm, wherein the fermentation temperature is 25 ℃. In the later stage of logarithmic growth in the fermentation process, at 6 th to 9 th days, adding a tetravalent inorganic selenium salt Se (IV) solution to ensure that the total fermentation time is 9 to 12 days, namely continuing to ferment for 3 days after adding Se (IV).

The second purpose of the invention is to provide the nano selenium powder prepared by applying any one of the methods.

The invention also claims the needle mushroom fruiting body, stipe or mycelium obtained by the method.

Has the advantages that: the method utilizes needle mushrooms (F.velutipes) to biologically synthesize nano-selenium, separates and purifies the biological nano-selenium, the yield reaches 5.06 percent (namely 5.06g/100g of dry weight of thalli), and the conversion rate can reach 99.58 percent at most. The method adopts an edible fungus fermentation process, has the characteristics of mild conditions, environmental friendliness, simplicity and convenience in operation, safety, high efficiency and the like, and the prepared nano selenium has potential industrial application prospects in the fields of selenium-rich functional foods, selenium-rich materials and medical products.

Drawings

FIG. 1 shows the effect of synthesizing nano-selenium from needle mushroom mycelia under different selenium concentrations.

FIG. 2 shows the presence of Flammulina velutipes mycelia in different Na₂SeO₃The yield and the conversion rate of the nano-selenium under the concentration.

FIG. 3 shows the biological synthesis effect of needle mushroom fruiting body on nano-selenium: a, before selenium enrichment; b, after enriching selenium.

Detailed Description

The method for calculating the yield and the nano-selenium conversion rate comprises the following steps:

the yield is the total mass (g) of the nano selenium in the thallus/fermentation, and the dry weight (g) of the thallus is multiplied by 100 percent;

the conversion rate of nano selenium is equal to the total mole number (mol) of nano selenium in the thallus/mole number (mol) of selenium with valence of 4 and Na₂SeO₃Mole) × 100%.