Application of Trichosporon axylodis in the preparation of nano-selenium

Abstract

The present invention relates to the technical field of selenium reduction, and discloses the application of Trichosporon arfuei in the preparation of nano-selenium, the deposit number of Trichosporon arfuei is CCTCC M 2022759, and the particle size of the nano-selenium is 100nm-300nm. The Trichosporon arfuei in the present invention has good tolerance to selenite, can reduce selenite, and the prepared nano-selenium provides a new application direction for Trichosporon arfuei.

Description

Technical Field

[0001] The invention relates to the technical field of selenium reduction, and in particular to the application of Trichosporon axotii in the preparation of nano-selenium. Background Art

[0002] Selenium is an indispensable trace element in the life activities of humans and animals. It plays a very important role in antioxidant defense, maintaining redox homeostasis, regulating immune function, and promoting growth and development. Selenium deficiency can lead to Keshan disease, Kashin-Beck disease, thyroid disease and cardiovascular disease. However, selenium intake will produce toxic effects when it exceeds the recommended dietary intake, and the concentration range from beneficial to harmful is only one order of magnitude. Therefore, small changes in selenium concentration in the natural environment may lead to selenium pollution, which will cause serious harm to organisms and the ecological environment.

[0003] In the natural environment, selenium mainly exists in the form of selenide, elemental selenium, selenite and selenate, and its toxicity depends not only on its concentration but also on its valence state. Studies have shown that reducing highly toxic oxidized selenium to safe and non-toxic elemental selenium is not only of great significance in the remediation of environmental selenium pollution, but also the produced elemental selenium nanoparticles have multiple biological activities such as antioxidant, antibacterial, antiviral and anti-tumor, and have wide application value in food, medicine, optoelectronic industry and agricultural production.

[0004] Traditionally, chemical methods are usually used to reduce selenite to prepare nano-selenium, such as adding vitamin C reducing agent and bovine serum albumin stabilizer to selenite. However, this method is relatively costly, and the synthesized nano-selenium has low purity, uneven particle size distribution, and poor stability, which limits the application of nano-selenium. The microbial method refers to the synthesis of biological nano-selenium by microbial dissimilatory reduction. The conversion conditions are mild, no specific equipment is required, it is highly safe, environmentally friendly and economical; and the nano-selenium synthesized by microorganisms has a more stable structure, can withstand high temperature and high pressure, and the synthesized nano-selenium has a uniform particle size, stable size, and good dispersibility. At present, the application of Trichosporon aberrantis in the production of flavonoid compounds has been reported, but the application of Trichosporon aberrantis in the preparation of nano-selenium has not been disclosed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of Trichosporon axotii in the preparation of nano-selenium.

[0006] In order to achieve the above-mentioned purpose, the technical solution of the present invention is specifically as follows.

[0007] Application of Trichosporon affluvium in the preparation of nano-selenium, the preservation number of the Trichosporon affluvium is CCTCC M2022759, and the particle size of the nano-selenium is 100nm-300nm.

[0008] The Trichosporon axylodispermea of ​​the present invention has good tolerance to selenite, the highest tolerance concentration can reach 2M, and the reduction speed of selenite is fast, nano-selenium with a particle size of 100nm to 300nm can be obtained, and the nano-selenium yield can reach 31.5mg / g.

[0009] In another preferred embodiment, the preparation of nano-selenium refers to reducing selenite using the Trichosporon axotii to obtain nano-selenium.

[0010] In another preferred embodiment, the selenite is sodium selenite.

[0011] In another preferred embodiment, the specific process of preparing nano-selenium is as follows:

[0012] The Trichosporon alderyi was inoculated into a YPD medium, and a sodium selenite solution was added for culturing. The sodium selenite was reduced by the Trichosporon alderyi to obtain nano-selenium.

[0013] In another preferred embodiment, the concentration of the sodium selenite solution is 0.1 mM to 2 mM.

[0014] In another preferred embodiment, the inoculation amount of Trichosporon axotii is 10% of the YPD medium; the viable count is 1×10 8 cfu / mL~2×10 8 cfu / mL.

[0015] In another preferred embodiment, the sodium selenite solution is added 6 h to 12 h after inoculating Trichosporon axotii into the YPD medium.

[0016] In another preferred embodiment, the YPD medium comprises the following raw materials:

[0017] Glucose 20g / L, peptone 20g / L, yeast extract 10g / L;

[0018] The pH of the YPD medium is 7.2-7.4.

[0019] In another preferred embodiment, the culture conditions are as follows:

[0020] Temperature: 27.5℃~28.5℃.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The Trichosporon axylodispermeable strain of the present invention has good tolerance and fast reduction rate during the selenium reduction process. The nano-selenium content can reach 18.44 mg / g under the condition of 2 mM sodium selenite; compared with the highest concentration of 800 mM reported in bacteria at present, the maximum selenite tolerance concentration of the nano-selenium producing strain Trichosporon axylodispermeable strain of the present invention can reach 2 M, and the nano-selenium yield can reach 31.5 mg / g after optimizing the culture conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the tolerance of Trichosporon axylodis multiflorus to different concentrations of sodium selenite.

[0024] Figure 2 This is a graph showing the effects of different concentrations of sodium selenite on the growth of Trichosporon axolotl and the synthesis of nano-selenium.

[0025] Figure 3 This is a quantitative graph showing the apparent effects of different sodium selenite concentrations on the growth and nano-selenium synthesis of Trichosporon axolotl strains.

[0026] Figure 4 This graph shows the effect of different sodium selenite addition times on the growth of Trichosporon axolotl strains and the synthesis of nano-selenium.

[0027] Figure 5 These are scanning electron microscope and transmission electron microscope characterization images of the synthesis of nano-selenium by the Trichosporon axyloglucosporum strain in the embodiment of the present invention; wherein A is a scanning electron microscope photo of the strain cells, and B is a transmission electron microscope photo of the strain cells.

[0028] Figure 6 These are transmission electron micrographs of different elements in nano-selenium synthesized by the Trichosporon axolotl strain in the embodiments of the present invention, wherein A is a transmission electron micrograph of nano-selenium, B is a transmission electron micrograph of element C, C is a transmission electron micrograph of element Se, D is a transmission electron micrograph of element O, E is a transmission electron micrograph of element Na, and F is a transmission electron micrograph of element P.

[0029] Figure 7 This is a particle size analysis diagram of nano-selenium synthesized by the Trichosporon axolotl strain in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0032] In the natural environment, selenium mainly exists in the form of selenide, elemental selenium, selenite and selenate, and its toxicity depends not only on the concentration but also on its valence state. Studies have shown that highly toxic oxidized selenium can be reduced to safe and non-toxic elemental selenium. The physical method for synthesizing nano-selenium has the problems of high energy consumption, easy contamination of samples and uneven particle size. The chemical method for synthesizing nano-selenium has the disadvantages of high cost, low purity of nano-selenium, uneven particle size distribution and poor stability. The existing microbial synthesis method is not conducive to industrial production and the strain has low tolerance to selenite.

[0033] The embodiment of the present invention utilizes Trichosporon akadori to reduce selenite. Trichosporon akadori has good tolerance to selenite and has a high yield of synthesized nano-selenium, which provides a new application direction for Trichosporon akadori.

[0034] The Trichosporon aberrantis in the following embodiments has been disclosed in a Chinese patent with publication number CN116396873A and titled "Flavonoid Compound Production Strains and Applications Thereof". The deposit number of Trichosporon aberrantis is CCTCC M 2022759, the deposit date is May 30, 2022, and the deposit address is China Center for Type Culture Collection (CCTCC), No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Trichosporon aberrantis is hereinafter referred to as HZ10.

[0035] The selenites in the following examples are all sodium selenite.

[0036] YPD medium includes the following ingredients:

[0037] 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract; the pH of the YPD medium is 7.2-7.4.

[0038] Example 1

[0039] A method for preparing nano-selenium by Trichosporon axylodispersion comprises the following steps:

[0040] The Trichosporon axylodispermea was inoculated into the YPD medium, and a 0.1 mM sodium selenite solution was directly added thereto. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon axylodispermea to obtain nano-selenium.

[0041] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 1×10 8 cfu / mL.

[0042] Example 2

[0043] A method for preparing nano-selenium by Trichosporon axylodispersion comprises the following steps:

[0044] The Trichosporon axylodispermea was inoculated into the YPD medium, and a 0.5 mM sodium selenite solution was directly added thereto. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon axylodispermea to obtain nano-selenium.

[0045] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 1×10 8 cfu / mL.

[0046] Example 3

[0047] A method for preparing nano-selenium by Trichosporon axylodispersion comprises the following steps:

[0048] The Trichosporon axylodispermea was inoculated into the YPD medium, and a 1 mM sodium selenite solution was directly added thereto. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon axylodispermea to obtain nano-selenium.

[0049] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 2×10 8 cfu / mL.

[0050] Example 4

[0051] A method for preparing nano-selenium by Trichosporon axylodispersion comprises the following steps:

[0052] The Trichosporon axylodispermea was inoculated into the YPD medium, and a 2 mM sodium selenite solution was directly added thereto. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon axylodispermea to obtain nano-selenium.

[0053] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 2×10 8 cfu / mL.

[0054] Example 5

[0055] A method for preparing nano-selenium by Trichosporon axylodispermea, except that the time of adding the sodium selenite solution is different from that in Example 4, the remaining steps are the same as those in Example 4, and include the following steps:

[0056] The Trichosporon axylodispermea was inoculated into the YPD medium, and a sodium selenite solution with a concentration of 2 mM was added after 6 hours. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon axylodispermea to obtain nano-selenium.

[0057] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 2×10 8 cfu / mL.

[0058] Example 6

[0059] A method for preparing nano-selenium by Trichosporon axylodispermea, except that the time of adding the sodium selenite solution is different from that in Example 4, the remaining steps are the same as those in Example 4, and include the following steps:

[0060] The Trichosporon axylodispermea was inoculated into the YPD medium, and 2 mM sodium selenite solution was added after 12 hours. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon axylodispermea to obtain nano-selenium.

[0061] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 2×10 8 cfu / mL.

[0062] Example 7

[0063] A method for preparing nano-selenium by Trichosporon axylodispermea, except that the time of adding the sodium selenite solution is different from that in Example 4, the remaining steps are the same as those in Example 4, and include the following steps:

[0064] The Trichosporon axylodispermea was inoculated into the YPD medium, and 2 mM sodium selenite solution was added after 18 hours. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon axylodispermea to obtain nano-selenium.

[0065] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 2×10 8 cfu / mL.

[0066] Example 8

[0067] A method for preparing nano-selenium by Trichosporon axylodispermea, except that the time of adding the sodium selenite solution is different from that in Example 4, the remaining steps are the same as those in Example 4, and include the following steps:

[0068] The Trichosporon affluent was inoculated into the YPD medium, and 2 mM sodium selenite solution was added after 24 hours. The culture was carried out at 28° C. and 200 rpm for 5 days. The sodium selenite was reduced by the Trichosporon affluent to obtain nano-selenium.

[0069] The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of YPD medium; the viable count was 2×10 8 cfu / mL.

[0070] 1. Tolerance of Trichosporon axolotl to sodium selenite

[0071] The tolerance of HZ10 strain to different concentrations of sodium selenite was determined by 96-well plate. Figure 1 As shown. Figure 1It can be seen that the HZ10 strain can still grow at a sodium selenite concentration of 2M and has the ability to convert and synthesize nano-selenium, indicating that the maximum sodium selenite tolerance concentration of the HZ10 strain can reach 2M, which is much higher than the highest selenite tolerance concentration of 800mM reported so far.

[0072] 2. Synthesis of Nano-Selenium

[0073] The nano-selenium yields of Examples 1 to 4 were measured respectively, and the results are as follows: Figure 2 shown.

[0074] from Figure 2 It can be seen that the ability of HZ10 strain to synthesize nano-selenium increases with the increase of sodium selenite concentration, while the growth ability of strain decreases with the increase of sodium selenite concentration. This is mainly due to the toxic effect of sodium selenite. The higher the concentration, the greater the toxicity, thus inhibiting the growth of strains. As a substrate for reducing and synthesizing nano-selenium, the higher the concentration of selenite, the higher the yield of synthesized nano-selenium. Finally, the nano-selenium content of HZ10 strain under 2mM sodium selenite was measured to be 18.44mg / g.

[0075] The nano-selenium prepared in Examples 4 to 8 was measured, and the results are as follows: Figure 3 As shown. Figure 3 It can be seen that the later sodium selenite is added, the more conducive it is to the growth of HZ10 strain, which is mainly due to the toxic effect of high concentration sodium selenite; and when sodium selenite is added at 6h, the content of nano-selenium generated is the highest, reaching 31.5mg / g, indicating that adding sodium selenite in the early logarithmic phase is more conducive to the reduction and production of nano-selenium.

[0076] The structure of the nano-selenium prepared in Example 5 was characterized, and the results were as follows:

[0077] 1) Transmission electron microscopy and scanning electron microscopy

[0078] like Figure 4 As shown, from Figure 4 It can be seen that nano-selenium particles located in the cells of the HZ10 strain were observed under a transmission electron microscope, namely Figure 4 The area indicated by the arrow B in the figure was observed under a scanning electron microscope, where nano-selenium particles were located on the cell wall surface of the HZ10 strain and secreted outside the cell, namely Figure 4 The area indicated by the arrow A in the figure.

[0079] 2) Transmission electron microscopy characterization and energy spectrum analysis

[0080] The nano-selenium particles obtained in Example 5 were purified, and the specific purification method was as follows:

[0081] After the fermentation, the culture solution was poured into a 50ml centrifuge tube, centrifuged at 12000r / min for 15min, the supernatant was poured out, the bacterial precipitate was washed once with 30ml sterile water, centrifuged at 12000r / min for 15min to remove the supernatant, the precipitate was resuspended with 10ml sterile water, and the cell wall was broken by ultrasound at 350W power for 30min under ice bath conditions. After the cell wall was broken, it was washed once with Tris-HCl, SDS, and sterile water at pH 8.3, centrifuged at 12000r / min for 15min, and finally resuspended with 6ml sterile water, 3ml n-octanol was added, and the mixture was centrifuged at 12000r / min for 15min. The precipitate was the nano-selenium particles, which were washed once with chloroform, ethanol, and sterile water, centrifuged at 10000r / min for 15min, and the precipitate was dried with a freeze dryer to obtain purified nano-selenium particles.

[0082] The purified nano-selenium particles were characterized by transmission electron microscopy and energy spectrum analysis. Figure 5 and Figure 6 As shown, the results show that the HZ10 strain can synthesize selenium particles of different sizes and shapes, with a size of nanometers. Most of the nano-selenium is spherical, and a few are irregular ellipsoidal or rod-shaped. The results of energy dispersive X-ray spectroscopy analysis show that the metal element contained in the nano-selenium particles synthesized by the HZ10 strain is Se.

[0083] 3) Particle size analysis

[0084] The present invention uses a particle size analyzer to analyze the diameter of the nano-selenium particles synthesized by the HZ10 strain after 5 days of cultivation. The results show that the size of the nano-selenium particles finally obtained is between 100nm and 300nm, and the average particle size is 226.4nm. Figure 7 As shown. The particle size of nano-selenium is the main factor that determines its chemical properties and biological activity. The smaller the particle size, the better the in vitro antioxidant effect and the stronger the ability to scavenge free radicals.

[0085] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of Trichosporon axylodispermoides in the preparation of nano-selenium is characterized in that: The preservation number of the Trichosporon axolotl is CCTCC M 2022759, and the particle size of the nano-selenium is 100nm-300nm.

2. The use of Trichosporon axylodispermea according to claim 1 in the preparation of nano-selenium, characterized in that: The preparation of nano-selenium refers to reducing selenite by using the Trichosporon axylodispermosus to obtain nano-selenium.

3. The use of Trichosporon axylodispermea according to claim 2 in the preparation of nano-selenium, characterized in that: The selenite is sodium selenite.

4. The use of Trichosporon axylodispermoides according to claim 3 in the preparation of nano-selenium, characterized in that: The specific process of preparing nano-selenium is as follows: The Trichosporon alderyi was inoculated into a YPD medium, and a sodium selenite solution was added for culturing. The sodium selenite was reduced by the Trichosporon alderyi to obtain nano-selenium.

5. The use of Trichosporon axylodispermoides according to claim 4 in the preparation of nano-selenium, characterized in that: The concentration of the sodium selenite solution is 0.1 mM to 2 mM.

6. The use of Trichosporon axylodispermoides according to claim 4 in the preparation of nano-selenium, characterized in that: The inoculation amount of Trichosporon axylodispermoides was 10% of the mass of the YPD medium, and the viable count was 1×10 8 cfu / mL~2×10 8 cfu / mL.

7. The use of Trichosporon axylodispermoides according to claim 4 in the preparation of nano-selenium, characterized in that: The sodium selenite solution is added 6 to 12 hours after the yeast Trichosporon axotii is inoculated into the YPD medium.

8. The use of Trichosporon axylodispermoides according to claim 4 in the preparation of nano-selenium, characterized in that: The YPD medium includes the following raw materials: Glucose 20g / L, peptone 20g / L, yeast extract 10g / L; The pH of the YPD medium is 7.2-7.

4.

9. The use of Trichosporon affluentifolia in preparing nano-selenium according to claim 4, characterized in that: The culture conditions are as follows: Temperature: 27.5℃~28.5℃.

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