阅读说明：本技术 一种利用虎奶菇生物合成纳米硒的方法 (Method for biosynthesizing nano-selenium by using Pleurotus tuber-regium ) 是由 陈磊 曹芳 郭宝婕 林演 刘京京 葛梦蝶 张薄博 张志强 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种利用虎奶菇生物合成纳米硒的方法,属于生物技术领域。本发明是虎奶菇菌丝体发酵到一定时间后,在发酵培养基中加入不同浓度的Se(IV),继续进行发酵,最终在菌体和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 Pleurotus tuber-regium, belonging to the field of biotechnology. After the Pleurotus tuber-regium mycelium is fermented for a certain time, Se (IV) with different concentrations is added into a fermentation medium for continuous fermentation, and finally nano selenium particles are synthesized in the mycelium and 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 tetravalent inorganic selenium salt is converted to produce nano-selenium (brick red zero-valent elemental selenium) in the fermentation process of Pleurotus tuber-regium.

2. The method of claim 1, wherein the nano-selenium in the fermentation product is collected; the fermentation product comprises Pleurotus tuber-regium mycelium, Pleurotus tuber regium lysate, fermentation liquor or precipitate obtained by centrifuging the fermentation liquor.

3. The method as claimed in claim 1 or 2, wherein the fermentation system is supplemented with Pleurotus tuber-regium after inoculation and fermentation to 4-6 daysAdding Na with the concentration of 0.1-20mM₂SeO₃Continuing fermenting the solution until the total time is 7-9 d.

4. The method according to claim 3, characterized in that it comprises in particular the steps of:

(1) activating the pleurotus tuber-regium strains by using a PDA culture medium;

(2) transferring the Pleurotus tuber-regium hyphae obtained by activation in the step (1) into a seed culture medium, and culturing at 26-32 ℃ for 3-5 days;

(3) transferring the seed liquid cultured in the step (2) into a fermentation medium containing tetravalent inorganic selenium salt in an amount of 1-15% by volume, and fermenting at the temperature of 26-32 ℃ and 200rpm for 7-9 days.

5. The method according to claim 1 or 4, wherein the fermentation product is collected and the nano-selenium is isolated and/or purified therefrom.

6. The method as claimed in claim 5, wherein the fermentation broth is sieved by a 30-mesh sieve, the Pleurotus tuber-regium thallus is collected, the Pleurotus tuber-regium thallus is washed for 3-5 times by deionized water, thallus cells are crushed, and thallus lysate is collected; 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.

7. The method as claimed in claim 5, wherein the fermentation broth is sieved with a 30-mesh sieve, the Pleurotus tuber-regium thallus is collected, washed with deionized water for 3-5 times, freeze-dried, ground and sieved with a 80-mesh sieve to obtain the nano-selenium dry powder.

8. The method as claimed in claim 5, wherein the fermentation broth is sieved by a 30-mesh sieve, the Pleurotus tuber-regium thallus is removed, the fermentation broth is filtered by quantitative filter paper to remove solid matters, and the fermentation broth is ultrafiltered by an ultrafiltration membrane with the molecular weight of 1 ten thousand, then the reflux liquid is collected, and the nano-selenium dry powder is obtained after freeze drying.

9. The nano selenium powder prepared by the method of any one of claims 1 to 8.

10. Pleurotus tuber-regium fruiting body, stipe, sclerotium or mycelium obtained by fermentation according to any one of claims 1-8.

Technical Field

The invention relates to a method for biosynthesizing nano-selenium by utilizing Pleurotus tuber-regium, belonging to the technical field of biological nano-technology

Background

Selenium (Selenium, Se) is a trace element necessary for human and animal to maintain normal physiological activity, is an important component of in vivo antioxidant enzyme, namely glutathione peroxidase (GSH-PX), and has multiple functions of enhancing immunity, resisting cancer, resisting oxidation, etc. Selenium deficiency can lead to a variety of diseases such as keshan disease, cardiovascular disease, hypertension, cirrhosis, and the like. The selenium distribution in China is extremely unbalanced, and the development of safe and efficient selenium supplement approaches is a problem to be solved urgently in China.

Selenium in nature has various forms including negative divalent, zero-valent, positive tetravalent, positive hexavalent and the like. Selenium has different forms, and has great difference in biological activity and toxicity. The nano selenium is different from gray elemental selenium (zero valence), inorganic selenium (negative divalent, positive tetravalent, positive hexavalent) and organic selenium, is brick red elemental selenium with a nano size, has far lower toxicity than inorganic selenium and organic selenium, and has higher biological activity than organic selenium (such as selenomethionine). Therefore, the nano-selenium is a safer and more effective approach for supplementing selenium.

In recent years, researchers have discovered that various microorganisms including algae, bacteria, fungi, actinomycetes, etc., have the ability to convert inorganic selenium into brick-red nano-selenium. The biotransformation method has the characteristics of environmental protection and energy conservation compared with the chemical/physical preparation method, so the biotransformation method is widely concerned. The large edible and medicinal fungi have more unique advantages in the aspect of nano selenium biosynthesis due to unique physiological activity and metabolic process, but related research is still in the initial stage.

Disclosure of Invention

The invention aims to provide a biosynthetic nano-selenium and a purification method, which are used for obtaining the pleurotus tuber-regium mycelium rich in nano-selenium through liquid state fermentation of pleurotus tuber-regium mycelium.

The first purpose of the invention is to provide a method for producing nano-selenium, which utilizes Pleurotus tuber-regium (Pleurotus tuber-regium) fermentation 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 Pleurotus tuber-regium mycelium, fermentation broth, precipitate after fermentation broth centrifugation, and Pleurotus tuber regium lysate.

In one embodiment, the method comprises adding Na with a concentration of 0.1-20mM to the fermentation broth after inoculation up to 4-6d₂SeO₃Continuing fermenting the solution until the total time is 7-9 d.

In one embodiment, the fermentation product is collected and the nano-selenium is isolated and/or purified therefrom.

In one embodiment, the method specifically comprises the steps of:

(1) activating the pleurotus tuber-regium strains by adopting a PDA culture medium;

(2) transferring the Pleurotus tuber-regium hyphae obtained by activation in the step (1) into a seed culture medium, and culturing at 26-32 ℃ for 3-5 days;

(3) the seed liquid cultured in the step (2) is treated according to the proportion of 10⁵Transferring the spore/mL fermentation liquid into a fermentation culture medium, and fermenting at 26-32 ℃ and 200rpm for 7-9 days.

In one embodiment, the seed medium comprises glucose 20-40g/L, yeast extract 2-8g/L, potassium dihydrogen phosphate (KH)₂PO₄)0.2-2g/L, magnesium sulfate heptahydrate (MgSO4 & 7H)₂O)0.1-2g/L。

In one embodiment, the fermentation medium comprises 20-40g/L glucose, 2-8g/L yeast extract powder, and potassium dihydrogen phosphate (KH)₂PO₄)0.2-2g/L, magnesium sulfate heptahydrate (MgSO4 & 7H)₂O)0.1-2g/L。

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

(1) 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, boiling 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 Pleurotus tuber regium strain on PDA flat plate, and culturing at 30 deg.C for 7 d;

(2) cutting PDA solid culture medium of Pleurotus tuber-regium mycelium into about 1cm in super clean bench²Selecting 5-15 pieces, adding seed culture solution, and culturing in a culture medium: glucose 20-40g/L, yeast extract powder 2-8g/L, potassium dihydrogen phosphate (KH)₂PO₄)0.2-2g/L, magnesium sulfate heptahydrate (MgSO4 & 7H)₂O)0.1-2 g/L. After inoculation, shaking the mixture for 2-5d at 26-32 ℃ at the rotating speed of 160-200 rpm;

(3) inoculating the seed culture solution into a sterilized liquid fermentation culture medium for liquid fermentation, wherein the inoculation amount of the liquid fermentation is 1-15%, the fermentation temperature is 26-32 ℃, the rotation speed of a shaking table is 150-. Adding Na with concentration of 0.1-20mM when fermenting to 4-6 days after inoculation₂SeO₃Continuing fermenting the solution until the total time is 7-9 d;

(4) and after the fermentation is finished, centrifuging, filtering, washing the thallus/precipitate fermentation liquor, freeze-drying and crushing to obtain the nano-selenium dry powder.

In one embodiment, the specific steps of step (4) are: sieving the fermentation liquor with a 30-mesh sieve, collecting thalli, washing for 3-5 times with deionized water, crushing thalli cells, and collecting 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 specific steps of step (4) are: and (3) sieving the fermentation liquor by a sieve of 30 meshes, collecting thalli, washing for 3-5 times by deionized water, freeze-drying the thalli, grinding the thalli, and sieving by a sieve of 80 meshes to obtain the nano-selenium dry powder.

In one embodiment, the specific steps of step (4) are: and (3) sieving the fermentation liquor by a 30-mesh sieve, removing thalli, filtering the fermentation liquor 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.

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 a pleurotus tuber-regium fruiting body, a stipe, a sclerotium or a mycelium obtained by the fermentation of the method.

Has the advantages that: the invention utilizes Pleurotus tuber-regium mycelium to biologically synthesize nano-selenium, the yield reaches 2.60 percent (namely 2.60g/100g dry weight of thallus), and the conversion rate reaches 81.45 percent. The method adopts a microbial 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 selenium enrichment of Pleurotus tuber-regium mycelia (actually brick red) under different inorganic selenium concentrations.

FIG. 2 shows the different Na content of Pleurotus tuber-regium mycelium₂SeO₃The yield and the conversion rate of the nano-selenium under the concentration.

FIG. 3 is a Transmission Electron Microscope (TEM) section view of the Pleurotus tuber-regium mycelium for intracellular synthesis of nano-selenium.

FIG. 4 shows the effect of Se (IV) addition time on the growth of cells.

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 nano selenium in the thallus/dry weight (g) multiplied by 100 percent of the thallus after fermentation

The conversion rate of nano selenium is equal to the total mole number (mol) of nano selenium in the thallus/mole number (mol) of added 4-valent selenium₂SeO₃Mole number) × 100% of