阅读说明：本技术 一种利用金针菇发酵产物合成纳米银的方法 (Method for synthesizing nano-silver by utilizing needle mushroom fermentation product ) 是由 陈磊 朱雨婕 代安然 杨崇婧 吴凡 李佳铭 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发酵产物合成纳米银的方法,属于生物纳米技术领域。本发明是将金针菇菌丝体进行液态发酵,发酵到一定阶段后,换为无营养成分发酵液,继续发酵,利用所得发酵产物,在一定温度条件下还原AgNO<Sub>3</Sub>生成稳定的纳米银溶液。本发明的方法工艺简单,易于操作,环境友好,具有潜在的工业化应用前景。(The invention discloses a method for synthesizing nano-silver by utilizing a needle mushroom fermentation product, belonging to the technical field of biological nano-technology. The invention carries out liquid fermentation on the flammulina velutipes mycelium, the flammulina velutipes mycelium is changed into nutrient-free fermentation liquor after being fermented for a certain period, the fermentation is continued, and AgNO is reduced by utilizing the obtained fermentation product under the condition of a certain temperature 3 And generating stable nano silver solution. The method has the advantages of simple process, easy operation, environmental protection and potential industrial application prospect.)

1. A method for synthesizing nano silver is characterized in that silver ions are converted into nano silver by using a product of liquid fermentation of Flammulina velutipes mycelium.

2. The method of claim 1, wherein the fermentation is a two-stage fermentation, the first stage comprises liquid fermentation in a fermentation medium, the second stage comprises transferring mycelium obtained from the liquid fermentation to a nutrient-free system for further fermentation, and the fermentation product is placed in an environment containing silver ions to synthesize the nano-silver.

3. The method of claim 2, wherein the non-nutrient system comprises sterile deionized water, PBS buffer, menadione solution, diethyl phthalate solution, or hydrogen peroxide solution, respectively.

4. The method of claim 3, wherein the PBS buffer is a solution with a pH of 6.0-10.0 and a concentration of 0.1-1M.

5. The method according to claim 3, wherein the menadione solution is at a concentration of 1-15 μ M; the concentration of the diethyl phthalate solution is 0.1-10 mM; the concentration of the hydrogen peroxide solution is 5-40 mM.

6. The method according to any one of claims 1 to 5, wherein the medium for fermentation comprises: 15-25 g/L glucose, 8-25 g/L yeast extract, 0.5-1.5 g/L potassium dihydrogen phosphate, 0.2-0.8 g/L magnesium sulfate heptahydrate, VB₁0.1～1.0g/L。

7. The method according to any one of claims 1 to 6, wherein the needle mushroom is inoculated into a fermentation medium and fermented at 22 to 28 ℃ and 130 to 180rpm for 5 to 8 days; and then, the hyphae are washed by sterile water and transferred to a non-nutrient fermentation system, the hyphae are continuously cultured for 2-5 days at 22-28 ℃ and 130-180 rpm, and a fermentation product is collected and used for synthesizing the nano silver.

8. The method of claim 7, wherein the synthesis of the nano silver is carried out at 50-120 ℃ for 5-60 min.

9. The method according to any one of claims 1 to 8, wherein the pH of the liquid environment for synthesizing the nano silver is controlled to be 6.0 to 14.0.

10. Use of the method of any one of claims 1 to 9 for the preparation of nanosilver containing products.

Technical Field

The invention relates to a method for synthesizing nano-silver by utilizing a needle mushroom fermentation product, belonging to the technical field of biological nano-technology.

Background

The nano silver is a metallic silver simple substance with the particle size of 1-100 nm, has unique properties of heat, light, electricity, catalysis and the like, has the characteristics of low toxicity, high activity and the like which are not possessed by common simple substance particles, inorganic particles, organic particles and the like, and greatly reduces the harm to human bodies while exerting the physiological functions of the nano silver, thereby avoiding the side effect on the human bodies caused by excessive ingestion. In addition, nanoparticles have significant advantages in combating bacterial and fungal infections: 1) the silver nanoparticles can play a bacteriostatic effect at a smaller concentration (3-7 mu g/mL); 2) the silver nanoparticles have broad-spectrum antibacterial action on bacteria and fungi, and the action of the silver nanoparticles is not obviously different (P is more than 0.05); 3) compared with two common antibiotics of gentamicin and neomycin, the silver nanoparticles have obvious advantages in the aspect of inhibiting bacteria and fungi.

At present, the synthesis mode of the nano material is mainly a chemical method and a physical method, and the two methods have some defects: the physical method has high requirements on instruments and equipment, high cost and high energy consumption; the chemical method requires more reducing agents, which are expensive, toxic and seriously polluting. The current situation seriously restricts the popularization and application of the nano material in the life and health fields. In recent years, the biological synthesis of nano materials is receiving attention, for example, the nano particles of gold or silver are synthesized by using plant leaching liquor or plant extracting agent, etc., and have unique biological activity, such as enhanced cell activity, stronger bacteriostatic ability, etc. The edible fungi have unique biological enrichment effect, and are also regarded as important biotransformation objects for the biosynthesis research of nano materials. The flammulina velutipes as a conventional cultivation and daily edible fungus for synthesizing the nano-silver has the advantages of wide material source, low price, mild and controllable reaction conditions, and is a clean, nontoxic, environment-friendly and sustainable development synthesis method.

Disclosure of Invention

The invention aims to provide a method for synthesizing nano-silver, which utilizes flammulina velutipes (Flammulinavelipes) liquid fermentation to convert silver ions into nano-silver.

In one embodiment, the method utilizes flammulina velutipes mycelium to carry out two-stage fermentation, the first stage carries out liquid fermentation in a fermentation medium, the second stage transfers the mycelium obtained by the liquid fermentation to a nutrient-free system for continuous fermentation for a certain time, and the fermentation product is placed in an environment containing silver ions to synthesize the nano-silver.

In one embodiment, the non-nutritive system comprises sterile deionized water, PBS buffer, menadione solution, diethyl phthalate solution, or hydrogen peroxide solution.

In one embodiment, the non-nutrient system comprises sterile deionized water, PBS buffer (0.1-1M) with pH of 6.0-10.0, and any one of menadione solution with the concentration of 1-15 μ M, diethyl phthalate solution with the concentration of 0.1-10mM, or hydrogen peroxide solution with the concentration of 5-40 mM.

In one embodiment, the fermentation medium comprises: 15-25 g/L glucose, 8-25 g/L yeast, and potassium dihydrogen phosphate (KH)₂PO₄) 0.5-1.5 g/L magnesium sulfate heptahydrate (MgSO)₄·7H₂O)0.2～0.8g/L，VB₁0.1～1.0g/L。

In one embodiment, the method comprises the steps of inoculating the needle mushrooms into a fermentation medium, and fermenting at the temperature of 22-28 ℃ and the rpm of 130-180 for 5-8 days; and then, the hyphae are washed by sterile water and transferred to a non-nutrient fermentation system, and are continuously cultured for 2-5 days at 22-28 ℃ and 130-180 rpm.

In one embodiment, the method comprises the steps of:

(1) preparing a seed solution: inoculating needle mushroom hyphae into a seed culture medium, wherein the seed culture medium contains: 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, wherein the inoculation is to cut the PDA plate full of needle mushroom hypha into 1 × 1cm²5-10 pieces of the culture medium are added into each bottle, and the mixture is cultured for 7 days in a shaking table at the temperature of 25 ℃ and the rpm of 160;

(2) and (3) shaking flask fermentation: inoculating 2.5-5% of the seed liquid cultured in the step (1) into a fermentation medium according to the volume, and fermenting for 5-8 d under the conditions of 25 ℃ and the rotating speed of a shaking table of 160 rpm;

(3) and (3) after-fermentation: sterile filtering needle mushroom fermentation liquor growing for 5-8 d, collecting mycelia, washing the mycelia for 3-5 times by using sterile water, and transferring the mycelia to a nutrient-free culture solution for continuous culture for 2-5 d;

(4) and (3) synthesizing nano silver: filtering the culture solution obtained in the step (3) in a suction manner, removing mycelia, collecting the fermentation liquor obtained in the step (3), and adding the fermentation liquor into 6mM AgNO according to the volume ratio of 1: 3-10: 3₃And (3) reacting in the solution for 0-60 min at the temperature of 50-100 ℃ respectively to synthesize the nano silver.

In one embodiment of the invention, the pH of the sterile deionized water fermentation liquor used for synthesizing the nano-silver in the step (4) is adjusted to 6.0-14.0 by acetic acid solution or sodium hydroxide solution before use.

The invention also claims the application of the method in preparing products containing nano silver.

Has the advantages that: according to the method, a stable nano-silver solution is efficiently synthesized by using a liquid fermentation product of flammulina velutipes (F.velutipes), the yield of nano-silver can reach 0.2-0.5 mM (different solution systems are different), the method adopts an edible fungus fermentation process combined with a heating reaction, the reaction condition is mild, the energy consumption is low, no pollution is caused, the operation is simple and convenient, and the prepared nano-silver solution has potential industrial application prospects in the fields of catalyst materials, anti-static materials, low-temperature superconducting materials, electronic slurry, biosensor materials, biomedicine and the like.

Drawings

FIG. 1 shows the effect of the concentration of the sterile water fermentation broth on the synthesis of AgNPs.

FIG. 2 shows the absorption wavelength of the nano-silver solution produced by the reaction.

FIG. 3 shows the ability of fermentation liquids with different concentrations to produce nano-silver at 100 ℃.

Detailed Description