Application of Saussurea in the synthesis of nano-selenium

Abstract

The present invention relates to the field of microorganisms, and more specifically to the application of snow lotus fungus in synthesizing nano-selenium. The application is to inoculate snow lotus fungus into milk, activate it continuously for 4-5 generations, inoculate the activated snow lotus fungus into milk containing sodium selenite, and perform selenium-enriched cultivation under constant temperature conditions to obtain snow lotus fungus containing nano-selenium. The present invention uses snow lotus fungus to convert toxic inorganic selenium into red elemental selenium of nanometer size (average particle size of 72.9nm) with biological activity, which is more conducive to the absorption of selenium by animals and humans.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to the application of Saussurea in synthesizing nano-selenium. Background Art

[0002] Selenium (Se) is located in the VI main group of the periodic table. It is an essential trace element for the human body. It cannot be synthesized in the human body and must be taken in from the outside. Selenium has a variety of prebiotic functions. The physiological functions of the trace element selenium include anti-oxidation, anti-aging, prevention of cardiovascular and cerebrovascular diseases, anti-tumor, detoxification, liver protection, and improvement of reproductive function. The lack of selenium in the human body can cause dysfunction of important organs and lead to the occurrence of more than 40 serious diseases such as Keshan disease and Kashin-Beck disease. A large number of pharmacological and clinical medical studies have further proved that selenium is irreplaceable in many physiological functions in the human body. The form of selenium determines its biological effects. Common forms of selenium are inorganic selenium, organic selenium and elemental selenium. The most common form of selenium in the inorganic state is inorganic selenium salts, including selenate (SeO 4 2- ), selenite (SeO 3 2- ) and selenide (Se 2- ), etc. Inorganic selenium mainly exists in the external environment such as soil and minerals; organic selenium includes selenoamino acids, selenoenzymes, selenopolysaccharides, etc. Among them, selenocystine (SeCys) and selenomethionine (SeMet) are the main organic selenium and are also selenides that can be used by humans; inorganic selenium itself is toxic, and organic selenium has lower toxicity, higher absorption and utilization rate, fewer side effects, and is easy to manage than inorganic selenium, so it has a broader research prospect. And elemental selenium (Se 0 ) is a method of reducing inorganic selenium to zero-valent selenium through chemical or microbial transformation. This elemental selenium is insoluble in water and has low toxicity. It exists in three forms: black, gray, and red. However, only red nano-selenium (about 100nm-500nm in size) is active and can be used. Nano-selenium synthesized by reducing inorganic selenium through biological transformation has low toxicity and high efficiency, and its biological effect is far superior to other forms of selenium.

[0003] Snow lotus fungus, also known as "Tibetan Linggu", "natural snow lotus fungus", "Tibetan Ganoderma lucidum" or "Tibetan snow lotus", is a milky white granular substance with irregular shape, mostly rice-like and sticky. It is fermented and cultured in milk, and its individual number will continue to increase. It is a symbiont of lactic acid bacteria, acetic acid bacteria, yeast and some other miscellaneous bacteria. Studies have shown that snow lotus fungus fermentation liquid has multiple medical and health care benefits. Long-term drinking of snow lotus fungus fermentation liquid can treat a variety of diseases, including regulating intestinal flora, anti-oxidation, immune regulation, lowering cholesterol, lowering blood pressure, lowering blood sugar, anti-fatigue, delaying aging and improving the body's immunity. Because snow lotus fungus produces alcohol, lactic acid and acetic acid during the fermentation process, its dairy products have a unique flavor of ordinary fermented milk, as well as a unique mellow and sour taste, and are deeply loved by consumers.

[0004] The traditional method of selenium supplementation is to use inorganic sodium selenite to make oral preparations, but sodium selenite has strong toxic side effects, while organic selenium compounds after inorganic selenium is biotransformed have low toxicity and higher absorption rate. There is no precedent for combining functional selenium and snow lotus fungus to ferment them to obtain small-sized nano-selenium.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide application of Saussurea schrenkiana in synthesizing nano-selenium.

[0007] Furthermore, the application is to inoculate snow lotus fungus into milk, activate it continuously for 4-5 generations, inoculate the activated snow lotus fungus into milk containing sodium selenite, and perform selenium-enriched culture under constant temperature conditions to obtain snow lotus fungus containing nano-selenium.

[0008] Furthermore, the milk is sterilized fresh milk.

[0009] Furthermore, the snow lotus bacteria is rinsed 3-5 times with sterile water before being inoculated into the sterilized fresh milk.

[0010] Furthermore, the washed snow lotus bacteria were inoculated into sterilized fresh milk at a mass volume ratio of 4-5 g / 100 mL.

[0011] Furthermore, the activation culture conditions are culturing at 27-29° C. for 23-25 ​​hours, filtering out the snow lotus bacteria, and continuously activating for 3-5 generations under the same conditions.

[0012] Furthermore, a sterilized sodium selenite solution is added to sterilized fresh milk to make the final concentration of the inorganic salt 48-52 μg / mL, and then the activated snow lotus fungus is inoculated into the sterilized fresh milk containing sodium selenite at a mass volume ratio of 1.0-4.0%.

[0013] Furthermore, the conditions for selenium-enriched culture are 27-29°C, 45-50h.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) The method of the present invention is the first to enrich the snow lotus fungus with selenium, and the total selenium-enriched content of the fungus is relatively high, exceeding the experimental expectations.

[0016] (2) The selenium-enriched snow lotus fungus prepared in the present invention can convert inorganic selenium into organic selenium while enriching it, and the organic selenium content is relatively high.

[0017] (3) Most of the existing technologies provide conditions for selenium enrichment by microorganisms, while the present invention analyzes the distribution of selenium on the basis of optimizing the selenium enrichment conditions.

[0018] (4) The nano-selenium particles generated by the method of the present invention are small in size (average particle size is 72.9 nm), which is more conducive to the absorption of selenium by animals and humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A flow chart of the method provided in Example 1 of the present invention;

[0020] Figure 2 This is the standard curve of selenium content;

[0021] Figure 3 The effect of the interaction between selenium concentration and culture time on selenium enrichment;

[0022] Figure 4 The effect of the interaction between selenium concentration and culture temperature on selenium enrichment;

[0023] Figure 5 The effect of the interaction between culture time and culture temperature on selenium enrichment;

[0024] Figure 6 The effect of the interaction between selenium concentration and culture time on the selenium enrichment rate;

[0025] Figure 7 The effect of the interaction between selenium concentration and culture temperature on the selenium enrichment rate;

[0026] Figure 8 The effect of the interaction between culture time and culture temperature on selenium enrichment rate;

[0027] Fig. 9 Flow chart for the fractional protein extraction of Saussurea ovata;

[0028] Fig.10 This is a scanning electron microscope image of snow lotus fungus;

[0029] Fig.11 This is a scanning electron microscope image of selenium-enriched snow lotus bacteria;

[0030] Fig.12 This is the SEM-EDX spectrum of Snow Lotus Bacteria;

[0031] Fig.13 This is the SEM-EDX spectrum of selenium-enriched snow lotus bacteria;

[0032] Fig.14 This is the particle size distribution diagram of snow lotus fungus nano-selenium;

[0033] Fig.15 This is the Fourier transform near infrared spectrum of snow lotus fungus;

[0034] Fig.16 This is the Raman spectrum of snow lotus fungus. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0036] Example 1: Optimization of preparation of selenium-enriched snow lotus fungus

[0037] The preparation method of selenium-enriched snow lotus bacteria provided in this embodiment is as follows Figure 1 As shown, the details are as follows:

[0038] 1. Activation of strains: After repeatedly washing the laboratory preserved snow lotus fungus (published in the paper "Technology Optimization of Snow Lotus Fermentation of Cowpea and Analysis of Volatile Flavor Components") with sterile water, inoculate it into sterilized fresh milk (fresh milk purchased from a local supermarket, protein 3.24g / 100g, fat 3.14g / 100g, lactose 4.83g / 100g, non-milk solids 8.85g / 100g) at a ratio of 5% (W / V), cultivate it at 28°C for 24h, filter out Tibetan mushrooms, add them into sterilized fresh milk again and cultivate them for 24h. Activate it continuously for five generations, and take the snow lotus fungus with relatively good growth for use.

[0039] 2. Selenium-enriched culture: Prepare 200 μg / mL sodium selenite solution, sterilize at 121°C for 20 minutes, and use it as the mother solution. Under aseptic conditions, add the corresponding volume of sodium selenite solution to sterilized fresh milk, with the final concentration of inorganic selenium being 10-70 μg / mL. After shaking well, inoculate 1.0-4.0% activated snow lotus bacteria respectively, place in a 19-37°C constant temperature incubator for selenium-enriched culture, and culture for 18-54 hours.

[0040] 3. Centrifugal separation: take the selenium-enriched snow lotus fungus after the cultivation is completed, centrifuge at 4000rpm for 10 minutes, discard the culture solution, collect the bacteria and wash them with deionized water several times, freeze-dry them in a vacuum freeze dryer for 24-48 hours, the cold trap temperature is ≤-62°, the vacuum degree is ≤10Pa, and freeze-dry to make selenium-enriched snow lotus fungus powder.

[0041] 4. Determination: Weigh a certain amount of freeze-dried selenium-enriched snow lotus powder and use hydride atomic fluorescence spectrometry to determine the selenium-enriched content of the bacteria, including microwave digestion and ICP-MS testing.

[0042] The detection methods are as follows:

[0043] (1) Microwave digestion: Weigh a certain amount of freeze-dried selenium-enriched snow lotus powder into a microwave digestion tube, add 5 mL of concentrated nitric acid, and pre-digest at 120°C for 40 minutes. After the digestion tank cools, add 1 mL of nitric acid and 1 mL of hydrogen peroxide. Set the program according to the microwave digestion steps shown in Table 1 below to digest the sample. After the digestion program is completed, remove the acid, reduce, and adjust the volume to be tested.

[0044] Table 1 Microwave digestion procedure

[0045]

[0046] (2) ICP-MS test: The digestion solution after the selenium content is fixed to a suitable concentration, and 10% hydrochloric acid is used as the carrier, 2% potassium borohydride and 0.5% sodium hydroxide solution are used as reducing agents, and the selenium concentration is detected by ICP-MS instrument. At the same time, a selenium standard curve solution is prepared, and 20% hydrochloric acid is used as the diluent to prepare the standard solution concentration. The fluorescence intensity of 0, 2, 4, 6, 8, 12, 16, and 20 μg / L selenium standards is measured. The standard curve is drawn with selenium concentration as the horizontal axis and fluorescence intensity as the vertical axis. The sample and blank determination methods are the same as above, and the fluorescence intensity of the sample and blank is substituted into the standard curve ( Figure 2 ), calculate the selenium content of the sample.

[0047] 5. Selection of optimization conditions: Selenium addition concentration (ug / mL), culture time (h) and culture temperature (℃) were selected as three factors to optimize the best selenium-enriched conditions, and response surface optimization was performed using the selenium-enriched amount and selenium-enriched rate of snow lotus fungus as indicators. The response surface test design and results are shown in Table 2.

[0048] Table 2 Response surface experimental design and results

[0049]

[0050] The response surface fitting results are shown in Tables 3, 4 and Figure 3 , 4, 5, 6, 7, and 8 show that the regression model is extremely significant (p < 0.01), the lack of fit term is not significant (p > 0.05), and the linear correlation coefficient of the regression model is R 2 is 0.9922, correction coefficient R adj 2 is 0.9822, both of which are relatively high, indicating that the model has a good fitting effect with the actual predicted value, the experimental design is reliable, and it can be used to optimize the process conditions of selenium enrichment of snow lotus fungus. Therefore, the model can predict the selenium content of selenium-enriched snow lotus fungus from the selenium addition concentration, culture time and culture temperature. The significance test of the regression coefficient shows that the selenium-enriched amount of A and A 2 , B 2 , C 2 The p value of A2, B2, and C2 is less than 0.01, indicating that it has a very significant effect on the selenium content of selenium-enriched snow lotus fungus; the p values ​​of B, C, AB, and BC are less than 0.05, indicating that they have a significant effect on the selenium content of selenium-enriched snow lotus fungus. The order of influence of each factor on the selenium-enriched effect is A>C>B, that is, selenium addition concentration>culture temperature>culture time. The p values ​​of A2, B2, and C2 of the selenium-enriched rate are less than 0.01, indicating that they have a very significant effect on the selenium-enriched rate of selenium-enriched snow lotus fungus; the p values ​​of B, C, AB, AC, and BC are less than 0.05, indicating that they have a significant effect on the selenium-enriched rate of selenium-enriched snow lotus fungus. The order of influence of each factor on the selenium-enriched effect is C>B>A, that is, culture temperature>culture time>selenium addition concentration.

[0051] Table 3 Significance test and variance analysis of the regression coefficient of selenium-rich regression model

[0052]

[0053] Note: P<0.05, *significant difference; P<0.01, **extremely significant difference

[0054] Table 4 Significance test and variance analysis of the regression coefficient of the selenium-enriched rate regression model

[0055]

[0056]

[0057] Note: P<0.05, *significant difference; P<0.01, **extremely significant difference

[0058] Example 2: Scanning electron microscopy analysis of selenium-enriched snow lotus bacteria

[0059] Scanning electron microscope analysis: After the selenium-rich bacterial sludge was resuspended in phosphate buffer and vortex-washed, a small amount of bacterial sludge was taken and applied to a small silicon wafer. After the bacterial sludge was naturally dried for a period of time, the silicon wafer was added to a 2.5% glutaraldehyde solution and left to stand overnight at 4°C. The glutaraldehyde was removed by centrifugation, and the phosphate buffer was used for suspension and washing 2 to 3 times, 10 minutes each time, and then eluted with 30%, 50%, 70%, 90%, and 100% ethanol gradient, 10 minutes each time. Supercritical CO 2 After drying, the sample was placed on a panel, sprayed with gold, and observed using a scanning electron microscope. At the same time, elemental analysis of specific areas was performed using SEM-EDX. Fig.10 and Fig.11 As shown in Figure 2, a large amount of selenium particles appeared around and on the surface of the selenium-enriched snow lotus fungus. The SEM-EDX spectrum is shown in Figure 2. Fig.12 and Fig.13 As shown, the results showed that specific characteristic peaks of zero-valent selenium element appeared near 1.3keV, 11.24keV and 12.50keV, that is, the snow lotus fungus can convert toxic inorganic selenium into nano-elemental selenium.

[0060] Example 3: Particle size analysis of snow lotus fungus nano-selenium

[0061] Particle size analysis: The freeze-dried powder of snow lotus fungus prepared under optimized conditions was suspended in sterile pure water and ultrasonically treated to make the nanoparticles dispersed evenly. The suspension was uniformly red and a nano-selenium solution was prepared. The solution was then diluted according to the instrument test requirements and the particle size distribution of the nanoparticles was measured using a ZEN3600 nano-laser particle size analyzer. This was repeated three times to detect the average size distribution. Fig.14 As shown, the average particle size of nano-selenium in aqueous solution appears as a narrow and symmetrical signal near 72.9 nm, that is, the average particle size of selenium nanoparticles is 72.9 nm.

[0062] Example 4: Fourier transform near infrared spectroscopy analysis of selenium-enriched snow lotus fungus

[0063] Infrared spectrum analysis: Weigh 200 mg of potassium bromide powder and 2 mg of freeze-dried selenium-enriched snow lotus powder, mix them evenly and grind them together. Use a tablet press at 8×10 7 The mixed powder was prepared into a transparent sheet under a pressure of 1.5 Pa, and the spectrum was detected by Fourier transform infrared spectrometer (FTIR). At the same time, potassium bromide was used as a blank sample group for baseline correction. The test wavelength range was set to 4000-400 cm -1 . Infrared spectra such as Fig.15 As shown, the selenium absorbed and transformed by snow lotus fungus will interact with other biological molecules such as proteins and carbohydrates and be coated by these macromolecules.

[0064] Example 5: Laser Micro-Raman Spectroscopy Analysis of Selenium-Enriched Snow Lotus Bacteria

[0065] Weigh a small amount of freeze-dried selenium-rich snow lotus powder, sieve it through 200 mesh, place it on a silicon wafer, and fix the sample stage for detection. The spectrum is recorded by a Raman microspectrometer equipped with a 785nm diode laser as the excitation source, and the integration time is 100 seconds. At the same time, the silicon peak deviation is corrected, and the first-order peak of the silicon peak of the standard sample is at 520.5±0.2cm -1 Raman spectroscopy Fig.16 As shown, the snow lotus fungus absorbed and transformed inorganic selenium, and precipitated nano-selenium particles.

[0066] Example 6: Determination of organic selenium content in selenium-enriched snow lotus fungus

[0067] Weigh a certain mass of freeze-dried selenium-enriched snow lotus fungus powder and place it in a volumetric flask, add 6 mol / L hydrochloric acid, ultrasonicate for 10 to 20 minutes to fully dissolve the fungus, then place it in a boiling water bath for 10 minutes and quickly cool it, retain the precipitate after centrifugation, and take the supernatant. The above-mentioned snow lotus fungus cell precipitate is repeatedly extracted with hydrochloric acid twice, the three supernatants are combined, and diluted to the scale. Subsequently, the content of inorganic selenium in the supernatant sample is determined by the method for determining the total selenium content, and the content of organic selenium is obtained by subtraction.

[0068] Example 7: Determination of selenium content in extracts from selenium-enriched snow lotus fungus

[0069] (1) Determination of polysaccharide selenium content: Weigh 0.2g of freeze-dried selenium-enriched snow lotus powder, add 8mL of water, stir and extract in a boiling water bath for 2h, cool and centrifuge at 6000rpm for 10min, collect the supernatant, and continue to add water to the precipitate for extraction, repeat twice, and ultrasonic assisted extraction can be used during the extraction process. Combine the supernatants of 3 times, add an equal volume of Sevage reagent (V (chloroform) : V (n-butanol) = 5:1) to the supernatant, use the Sevage method to remove the protein, collect the aqueous phase, add an equal volume of Sevage, repeat 7 times, measure the volume of the aqueous phase, add 3 to 4 times the volume of 95% ethanol, place at 4℃ for 12h, collect the precipitate by centrifugation, wash the precipitate with ethanol several times, and then wash with acetone. After the precipitate is freeze-dried, it is stored at -20℃ for use. The method for determining the polysaccharide selenium content is the same as the method for determining the total selenium content.

[0070] (2) Determination of protein selenium content: Weigh 0.2g of freeze-dried selenium-enriched snow lotus powder, add 10mL of 0.25mol / L NaOH solution, extract on a magnetic stirrer for 2h, centrifuge at 6000rpm for 10min, collect the supernatant, and continue to wash the precipitate with the solution twice. Then mix the supernatants and saturate them with ammonium sulfate at 80% at 4°C, place them at 4°C for 12h, collect the precipitate by centrifugation, and then dissolve them in 10.0ml of 50mM Tris-HCl (pH=8.0) solution, dialyze three times with 1.0L of 50mM Tris-HCl (pH=8.0) at 4°C to remove any other small molecules, put them into a 3500D dialysis bag and dialyze them. Finally, freeze-dry the dialysate and store it at -20°C for use. The method for determining protein selenium content is the same as the method for determining total selenium content.

[0071] (3) Determination of selenium content in graded protein: Weigh 0.2g of freeze-dried selenium-rich snow lotus powder, add different solutions and extract on a magnetic stirrer for 2h, centrifuge at 6000rpm for 10min, collect the supernatant, and continue to wash the precipitate with the solution twice. Then mix the supernatants, acidify the supernatant with 0.1mol / L hydrochloric acid solution, then slowly add ammonium sulfate to 80% saturation, place at 4℃ for 12h, collect the precipitate by centrifugation, and then dissolve it in a small amount of corresponding solution, put it into a 3500D dialysis bag and dialyze it. Finally, freeze-dry the dialysate and store it at -20℃ for use. The protein component extraction process adopts a continuous extraction method. The flow chart is as follows Fig. 9 .

[0072] (4) Determination of nucleic acid selenium content: Weigh 0.2 g of freeze-dried selenium-enriched snow lotus powder, add 6 mL of 12% NaCl solution, stir and extract in a boiling water bath for 4 h, cool and centrifuge at 4000 rpm for 10 min, collect the supernatant, and continue to extract the precipitate with NaCl, repeat twice, combine the supernatants of 3 times, add an equal volume of Sevage reagent (V (chloroform) : V (n-butanol) = 5: 1) to the supernatant, and use the Sevage method to remove the protein. Finally, adjust the pH value of the supernatant to 2.5 with 6 mol / L HCl, place at 4°C for 12 h, collect the precipitate by centrifugation, freeze-dry the precipitate and store at -20°C for use. The method for determining the nucleic acid selenium content is the same as the method for determining the total selenium content.

[0073] Results: The distribution of organic selenium in selenium-enriched snow lotus fungus is shown in Table 5, and the graded protein selenium content is shown in Table 6. The results show that the selenium in selenium-enriched snow lotus fungus mainly exists in the form of organic selenium, and the content of extracted organic selenium (including polysaccharides, nucleic acids, and proteins) is 50.74% of the total selenium, among which the content of protein organic selenium is 622.13±14.45μg / g, accounting for 25.86% of the total selenium; the content of polysaccharide organic selenium is 323.92±2.07μg / g, accounting for 13.46% of the total selenium; the content of nucleic acid organic selenium is 274.74±9.66μg / g, accounting for 11.42% of the total selenium, indicating that protein selenium is the main occurrence form of selenium in selenium-enriched snow lotus fungus, and its combined selenium content reaches more than 25% of the total selenium content of selenium-enriched snow lotus fungus. According to the amount of combined selenium, the distribution of organic selenium and total selenium in protein, polysaccharide and nucleic acid is protein selenium>polysaccharide selenium>nucleic acid selenium.

[0074] The amount of selenium bound by water-soluble protein, salt-soluble protein, alcohol-soluble protein and alkali-soluble protein in selenium-rich snow lotus fungus accounted for 24.10%, 13.45%, 1.36% and 16.26% of the total protein selenium, respectively. The order of bound selenium from large to small is: water-soluble protein > alkali-soluble protein > salt-soluble protein > alcohol-soluble protein. Water-soluble protein and alkali-soluble protein are the main proteins that bind selenium in snow lotus fungus, and the total bound selenium accounts for 6.23% and 4.21% of the total selenium.

[0075] This indicates that snow lotus fungus can absorb more than 70% of inorganic selenium from the culture medium and convert it into organic selenium; compared with other selenium-rich microorganisms, the present invention can convert selenium elements while enriching selenium to form organic selenium under the synergistic effect of the preparation method and snow lotus fungus, and this effect exceeds experimental expectations.

[0076] Table 5 Distribution of organic selenium in selenium-enriched snow lotus fungus

[0077]

[0078] Table 6 Protein selenium content in selenium-enriched snow lotus fungus

[0079]

[0080] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0081] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0082] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The application of snow lotus fungus in synthesizing nano elemental selenium and organic selenium is characterized in that: The method comprises the steps of: washing snow lotus fungus with sterile water for 3-5 times, inoculating the snow lotus fungus into sterilized fresh milk at a mass volume ratio of 4-5 g / 100 mL, continuously activating the snow lotus fungus for 4-5 generations, adding a sterilized sodium selenite solution into the sterilized fresh milk to make the final concentration of the sodium selenite be 48-52 μg / mL, and then inoculating the activated snow lotus fungus into sterilized fresh milk containing sodium selenite at a mass volume ratio of 1.0%-4.0%, and performing selenium-enriched culture under constant temperature to obtain snow lotus fungus containing nano-elemental selenium and organic selenium, wherein the selenium-enriched culture conditions are 27-29° C. for 45-50 hours.

2. The use according to claim 1, characterized in that: The activation culture conditions are culturing at 27-29° C. for 23-25 ​​hours, filtering out the snow lotus fungus, and continuously activating for 3-4 generations under the same conditions.

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