Soil sphaerisporus ch23 and method and application of synthesizing nano selenium thereof

Abstract

The application discloses soil sporosarcina CH23 and a method and application of synthesizing nano selenium by using the soil sporosarcina. Sporosarcina soli The nano selenium synthesis bacteria are soil sporosarcina (Sporosarcina siccitae) CH23, which are preserved in a Guangdong Microbial Culture Collection Center (GDMCC) and have a preservation number of GDMCC No. 67015. The soil sporosarcina CH23 can efficiently convert high-toxicity selenite into non-toxic nano selenium. The required culture condition is simple, low in cost and easy to operate. The produced nano selenium particles have the advantages of non-toxicity, high biological activity, stable property and almost no other selenium byproducts, and have the effects of antibiosis, antioxidation and anticancer in the medical field. In addition, the nano selenium can be used as a nano selenium fertilizer for plant selenium nutrition supplement, and has the advantages of higher efficiency, safety and environmental protection compared with traditional selenite selenium fertilizer.

Description

Technical Field

[0001] This invention belongs to the field of nano-selenium biosynthesis technology, specifically relating to a soil-sporogenous spore-forming Micrococcus CH23 and its method for synthesizing nano-selenium and its application. Background Technology

[0002] Nano-selenium possesses high bioavailability and strong antioxidant properties, with key functions including enhancing immunity, anti-aging, regulating metabolism, and assisting in anti-tumor activity. Currently, the main preparation method for nano-selenium is the chemical reduction method, which converts selenite into nano-selenium by adding reducing agents such as ascorbic acid. This method is energy-intensive, polluting, and the resulting nano-selenium particles are unstable and prone to aggregation, ultimately leading to a significant reduction in their biological activity. Therefore, there is an urgent need for an economical and environmentally friendly method to replace the traditional chemical reduction method for synthesizing stable, highly bioactive nano-selenium. Many microorganisms have been found to convert highly toxic selenite into non-toxic nano-selenium through detoxification mechanisms, resulting in nano-selenium with advantages such as small particle size, stable properties, and high biological activity. However, the conversion rates of selenite to nano-selenium by currently reported and applied microorganisms are generally low, easily producing highly toxic hydrogen selenide or organoselenium byproducts, hindering the promotion of nano-selenium biosynthesis technology in practical production applications. Therefore, obtaining highly efficient nano-selenium synthesizing microorganisms is of great significance for advancing the development of the nano-selenium biosynthesis industry. Summary of the Invention

[0003] The purpose of this invention is to provide a nano-selenium synthesizing bacterium, a method for synthesizing nano-selenium, and its application.

[0004] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0005] A nano-selenium synthesizing bacterium, named *Soil Sporocystis occulta* (… Sporosarcina soli CH23 was deposited on September 23, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No: 67015.

[0006] The second objective of this invention is to provide soil-sporogenous sarcodactylus ( Sporosarcina soli Application of CH23 in the synthesis of nano-selenium.

[0007] A third objective of this invention is to provide a method for microbial synthesis of selenium nanoparticles, comprising the following steps:

[0008] Using the above-mentioned soil-sporogenous spore-forming ... Sporosarcina soli CH23 is used to reduce selenium-containing salts to synthesize nano-selenium.

[0009] Preferably, the specific steps are as follows:

[0010] (1) Activation of strains: Soil sporogenous spore-forming ...

[0011] (2) Seed culture: Select a single colony of soil-sporogenous spore-forming occulta CH23, inoculate it into a liquid culture medium and carry out constant temperature shaking culture to obtain seed culture solution;

[0012] (3) Fermentation culture: The seed culture of soil-borne spore-forming ...

[0013] Preferably, the fermentation product rich in nano-selenium synthesized by *Sterilococcus solani* CH23 is centrifuged for the first time, the supernatant is discarded, and the precipitate is obtained. The precipitate is washed three times with 1.0-1.5% NaCl solution, and then resuspended in TE buffer. Lysozyme is added to destroy the cell wall of the bacteria. The bacteria are then sonicated and centrifuged a second time. The precipitate is discarded, and the supernatant containing nano-selenium is collected. The supernatant is centrifuged a third time, the precipitate is collected, and the precipitate is washed 2-4 times with deionized water to obtain pure nano-selenium particles.

[0014] Further preferred, in step (1), *Sphaerocera sporeans* CH23 is inoculated onto a solid activated culture medium at a volume ratio of 1%-5%. The solid culture medium consists of 10-25 g / L tryptone, 5-10 g / L sodium chloride, 2-5 g / L soybean papain hydrolysate, 1.5-4 g / L dipotassium hydrogen phosphate, 1.5-5 g / L glucose, 15-20 g / L agar powder, a pH range of 6.5-7.5, a culture temperature of 25-35℃, and a culture time of 24-48 h, until colonies with a diameter of 2-4 mm are formed.

[0015] Further preferred, in step (2), the activated soil-borne spore-forming Chlorophyllum spp. CH23 single colony is inoculated into a liquid culture medium. The liquid culture medium consists of 10-25 g / L tryptone, 5-10 g / L sodium chloride, 2-5 g / L soybean papain hydrolysate, 1.5-4 g / L dipotassium hydrogen phosphate, 1.5-5 g / L glucose, pH range 6.5-7.5, and culture conditions of 25-35°C, rotation speed 120-200 r / min, and shaking culture for 20-36 h.

[0016] Further preferred, in step (3), the seed culture solution is inoculated into a fermentation medium containing sodium selenite, with an inoculation amount of 1-5%. The liquid fermentation medium consists of 5-15 g / L tryptone, 2-10 g / L yeast extract, 5-10 g / L sodium chloride, and 1-20 mM sodium selenite. The culture conditions are 25-35°C, 120-200 r / min, and shaking culture for 24-72 h.

[0017] Further preferred, the centrifugation conditions are as follows: first centrifugation speed 10000-15000*g, time 10-20min; second centrifugation speed 3000-8000*g, time 5-15min; third centrifugation speed 10000-20000*g, time 20-30min.

[0018] Further preferably, the final concentration of the lysozyme is 10-15 mg / mL, and the reaction conditions are incubation at 37°C for 1-1.5 h.

[0019] Further preferred, the conditions for ultrasonic disruption of bacterial cells are: ultrasonication on ice, ultrasonic power of 100-180W, ultrasonic operation for 5 seconds followed by a 5-second pause, and a total operation time of 20-30 minutes.

[0020] The nano-selenium biosynthetic bacteria proposed in this invention can efficiently convert highly toxic selenite into non-toxic nano-selenium. The required cultivation conditions are simple, low-cost, and easy to operate. The recovered nano-selenium particles have advantages such as being non-toxic, high-purity, highly bioactive, and stable. In the pharmaceutical field, they exhibit antibacterial, antioxidant, and anticancer effects. They can also be used as nano-selenium fertilizer for plant selenium nutrient supplementation, offering greater efficiency, safety, and environmental friendliness compared to traditional selenite selenium fertilizers.

[0021] The beneficial effects of this invention are as follows:

[0022] The nano-selenium synthesizing bacterium *Dystrophus spp.* CH23 screened in this invention can efficiently convert highly toxic selenite into non-toxic nano-selenium. The required cultivation conditions are simple, low-cost, and easy to operate. The recovered nano-selenium particles have advantages such as being non-toxic, high-purity, highly bioactive, and stable. In the pharmaceutical field, they exhibit antibacterial, antioxidant, and anticancer effects. They can also be used as nano-selenium fertilizer for plant selenium nutrient supplementation, offering greater efficiency, safety, and environmental friendliness compared to traditional selenite fertilizers.

[0023] Soil-borne spore-forming ... Sporosarcina soli CH23 was deposited on September 23, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No. 67015. Attached Figure Description

[0024] Figure 1 The soil-sporogenous *Dystrophus* isolated in Example 1 of this invention (… Sporosarcina soli The CH23 pure strain was tested without the addition of selenite ( Figure 1 (left) and added 5mM selenite ( Figure 1 The growth on solid culture medium (right);

[0025] Figure 2 This is an example of the growth of *Soil Sporozoa occulta* CH23 in a liquid fermentation medium containing 5 mM sodium selenite in Example 2 of the present invention.

[0026] Figure 3 The curves showing the changes in the concentrations of selenite and nano-selenium of *Sphaerocera heterophylla* CH23 in a liquid fermentation medium containing 5 mM sodium selenite over time are shown in Example 2 of this invention.

[0027] Figure 4 This is a SEM image of the soil-sporogenous ostracod CH23 strain grown in the control group liquid culture medium without sodium selenite in Example 3 of the present invention.

[0028] Figure 5 This is a SEM image of the soil-sporogenous ostracod CH23 strain grown in the experimental group liquid fermentation medium containing 5 mM sodium selenite in Example 3 of the present invention.

[0029] Figure 6 This is a TEM image of the soil-sporogenous ostracod CH23 strain grown in the control group liquid culture medium without sodium selenite in Example 3 of the present invention;

[0030] Figure 7 This is a TEM image of the soil-sporogenous ostracod CH23 strain grown in the experimental group liquid fermentation medium containing 5 mM selenite in Example 3 of the present invention.

[0031] Figure 8 This shows the growth of the soil-sporogenous spore-forming Micrococcus CH23 strain in Example 4 of the present invention in different concentrations of selenite culture medium. Detailed Implementation

[0032] To further illustrate the features and effects of the present invention, the present invention will now be described in more detail with reference to the embodiments and accompanying drawings.

[0033] Example 1

[0034] The isolation and purification of nano-selenium synthesizing bacteria includes the following steps:

[0035] 1. Activated sludge was collected from Mingzhu Water Treatment Plant in Conghua District, Guangzhou City, Guangdong Province. 1g of activated sludge was added to 50mL of 0.01M sterile PBS and mixed thoroughly to prepare an activated sludge suspension.

[0036] 2. Enrichment of nano-selenium biosynthetic bacteria: 1 mL of activated sludge suspension was added to 50 mL of liquid enrichment medium containing 5 mM sodium selenite for cultivation. The enrichment medium consisted of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 20 mM sodium selenite, with water as the solvent. The preparation method involved mixing all components thoroughly and sterilizing before use. The cultivation conditions were 30°C, 150 rpm, and constant temperature shaking for 72 h to obtain the enriched solution of nano-selenium biosynthetic bacteria.

[0037] 3. Take 1 mL of enrichment solution and serially dilute it 10⁻¹⁰ times with 0.01 M sterile PBS. 1 -10 6 Take 0.1 mL of each dilution of the enrichment solution and spread it evenly on a solid culture medium containing 5 mM sodium selenite. The solid culture medium formula is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar powder, and 5 mM sodium selenite, with water as the solvent. The preparation method is to mix all components evenly and sterilize them before use. Incubate at 30°C for 48 h.

[0038] 4. After red colonies have grown on the plate, select a single red colony and streak it onto a fresh solid medium containing 5 mM sodium selenite for isolation and purification. Incubate at 30°C for 48 hours. Repeat this step 2-3 times until a pure strain is obtained.

[0039] 5. The pure strain was inoculated into liquid seed culture medium for cultivation. The liquid seed culture medium formulation was: 17 g / L tryptone, 5 g / L sodium chloride, 3 g / L soybean papain hydrolysate, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, pH 7.0, and water as the solvent. The preparation method was to mix all components thoroughly and sterilize before use. The cultivation conditions were: 30°C, 150 rpm, and shaking for 24 h. The obtained strain suspension was then cryopreserved using 15% sterile glycerol.

[0040] 6. After the pure strain was sent to Qingke Biotechnology Co., Ltd. for identification (its 16S rRNA sequence is shown in SEQ ID NO.1), it was named *Sphaerospora spp.* Sporosarcina soli CH23. Deposited on September 23, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, accession number: GDMCC No. 67015.

[0041] Example 2

[0042] Nano-selenium synthesizing bacteria, soil-sporidioidomycetes ( Sporosarcina soli The optimal culture time for the biosynthesis of selenium nanoparticles using CH23 includes the following steps:

[0043] 1. Inoculum activation: *Soil-borne spore-forming *Dystrophus* (…) Sporosarcina soli CH23 was inoculated onto a solid activation medium (the solvent was water, and the preparation method was to mix all the components evenly and sterilize it before use) containing 17 g / L tryptone, 5 g / L sodium chloride, 3 g / L soybean papain hydrolysate, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, 17 g / L agar powder, and pH 7.0. The medium was incubated at 30 °C for 24 h until colonies with a diameter of about 3 mm were formed.

[0044] 2. Seed culture: Single colonies of the soil-sporogenous spore-forming *Dystrophus spp.* CH23 were inoculated into a liquid culture medium containing 17 g / L tryptone, 5 g / L sodium chloride, 3 g / L soybean papain hydrolysate, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and pH 7.0. The culture was carried out under constant temperature and shaking conditions of 30°C, 150 r / min, and constant temperature and shaking for 24 h to obtain the seed culture solution.

[0045] 3. Fermentation culture: The seed culture was inoculated at a volume ratio of 1% into a liquid fermentation medium containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 5 mM sodium selenite (the solvent was water, which was prepared by mixing all components evenly and sterilizing before use). The culture temperature was 30°C, the rotation speed was 150 r / min, and the culture time was 0 h, 24 h, 30 h, 36 h, 48 h, and 72 h.

[0046] 4. Isolation and purification of nano-selenium: After fermentation, the bacterial suspension of the experimental group was centrifuged at 12000*g for 15 min. The supernatant was discarded, and the precipitate was obtained. The precipitate was washed three times with 1.0% NaCl solution, and then resuspended in TE buffer. Lysozyme was added to a final concentration of 10 mg / mL, and the bacterial cell walls were disrupted by incubation at 37°C for 1 h. Subsequently, the bacterial cells were sonicated on ice at a power of 150 W with a 5-second interval between sonication and a 5-second pause, for a total running time of 25 min. The sonicated sample was centrifuged at 5000*g for 5 min. The precipitate was discarded, and the supernatant containing nano-selenium was collected. The supernatant was centrifuged at 15000*g for 20 min, and the precipitate was collected. The precipitate was washed three times with deionized water to obtain pure nano-selenium particles.

[0047] 5. After the culture is completed, the remaining selenite and the content of biosynthesized nano-selenium in the solution are tested;

[0048] Experimental results: such as Figure 3 As shown, in a liquid fermentation medium with a sodium selenite concentration of approximately 5 mM, the selenite consumption rate of strain *Synthia spp.* CH23 reached 89% after 48 hours of constant temperature shaking culture, and 99% of the consumed selenite was converted into nano-selenium. The resulting nano-selenium had high purity and contained almost no other selenium byproducts.

[0049] Example 3

[0050] Characteristics of the biosynthesis of nano-selenium by the soil-sporogenous spore-forming Micrococcus CH23

[0051] 1. The seed culture solution of Example 2 was added at a volume ratio of 1% to the experimental group liquid fermentation medium containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 5 mM sodium selenite. The culture conditions were 30°C, 150 r / min and cultured for 36 h. A control group was set up with a liquid medium containing 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride and cultured under the same conditions.

[0052] 2. Scanning electron microscopy observation: After the culture was completed, the samples underwent ethanol gradient dehydration pretreatment, and then images were taken using a Czech Tescan MIRA LMS scanning electron microscope. Results for the blank control group are shown below. Figure 4 As shown, the results of the experimental group are as follows: Figure 5 As shown;

[0053] 3. Transmission electron microscopy observation: Glutaraldehyde was added to 0.01M PBS to prepare a 2.5% glutaraldehyde fixative. After incubation, the samples were fixed overnight at 4°C using the 2.5% glutaraldehyde fixative. The treated samples were fixed on copper grids and imaged using a Hitachi H-7650 transmission electron microscope. Results for the blank control group are shown below. Figure 6 As shown, the results of the experimental group are as follows: Figure 7 As shown.

[0054] Example 4

[0055] The tolerance test of soil-borne spore-forming Micrococcus chrysogenum CH23 to selenite includes the following steps:

[0056] Liquid culture media containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 0.01-100 mM sodium selenite were prepared. The seed culture of *Scolopendra subtilis* CH23 from Example 2 was added to liquid culture media with final concentrations of 0 mM, 0.1 mM, 1 mM, 5 mM, 20 mM, and 100 mM sodium selenite at an inoculation rate of 1% by volume. The culture temperature was 30°C, the rotation speed was 150 r / min, and the culture time was 48 h.

[0057] Experimental results: such as Figure 8 As shown, soil-sporogenous spore-forming spore-forming bacteria CH23 can grow well in 1-20 mM sodium selenite medium and synthesize a large amount of red nano-selenium.

Claims

1. A soil-sporophoric octopus ( Sporosarcina soli CH23, accession number: GDMCC No. 67015.

2. The soil-borne Sarcina sp. (S. albidifaciens) CH23 GDMCC No. 67015 for use in synthetic nanoselenium. Sporosarcina soli ) CH23 GDMCC No. 67015 for use in synthetic nanoselenium.

3. A method for preparing nano-selenium by using the nano-selenium synthesis bacteria, characterized in that, Includes the following steps: Sporosarcina ginsengisoli CH23 of claim 1 for reducing selenite to nano-selenium. Sporosarcina soli ) CH23 4. The method of claim 3, wherein, The specific steps are as follows: (1) Activation of microbial strains: The soil-sporogenous octopus ( Sporosarcina soli CH23 was inoculated into a solid activated medium and incubated at a constant temperature until the colonies covered the surface of the solid medium. (2) Seed culture: picking S. agalactiae (CH23) single colony, inoculating into liquid medium for constant temperature shaking culture to obtain seed culture solution; Sporosarcina soli ) (3) Fermentation culture: Soil-borne spore-forming Micrococcus occulta ( Sporosarcina soli CH23 seed culture solution was inoculated into a liquid fermentation medium containing sodium selenite and cultured under constant temperature and shaking to obtain a fermentation product rich in nano-selenium.

5. The method of claim 4, wherein, Soil-borne spore-forming Micrococcus ( Sporosarcina soli The fermentation product rich in nano-selenium synthesized by CH23 GDMCC No. 67015 was centrifuged for the first time, the supernatant was discarded, and the precipitate was obtained. The precipitate was washed three times with 1.0-1.5% NaCl solution, and then resuspended in TE buffer. Lysozyme was added to destroy the cell wall of the bacteria, and then the bacteria were sonicated. The mixture was centrifuged a second time, the precipitate was discarded, and the supernatant containing nano-selenium was collected. The supernatant was centrifuged a third time, the precipitate was collected, and the precipitate was washed 2-4 times with deionized water to obtain pure nano-selenium particles.

6. The method of claim 4, wherein, In step (1), the soil-sporidiogenic octopus ( Sporosarcina soli CH23 GDMCC No. 67015 was inoculated onto a solid activated medium at a volume ratio of 1%-5%. The solid medium consisted of 10-25 g / L tryptone, 5-10 g / L sodium chloride, 2-5 g / L soybean papain hydrolysate, 1.5-4 g / L dipotassium hydrogen phosphate, 1.5-5 g / L glucose, and 15-20 g / L agar powder. The pH range was 6.5-7.

5. The incubation temperature was 25-35℃, and the incubation time was 24-48 h, until colonies with a diameter of 2-4 mm were formed.

7. The method of claim 4, wherein, The step (2) involves activating the soil-borne spore-forming *Dystrophus* ( Sporosarcina soli A single colony of CH23 GDMCC No. 67015 was inoculated into liquid culture medium containing 10-25 g / L tryptone, 5-10 g / L sodium chloride, 2-5 g / L soybean papain hydrolysate, 1.5-4 g / L dipotassium hydrogen phosphate, and 1.5-5 g / L glucose, with a pH range of 6.5-7.

5. The culture conditions were 25-35°C, 120-200 r / min, and shaking culture for 20-36 h.

8. The method of claim 4, wherein, In step (3), the seed culture solution is inoculated into a fermentation medium containing sodium selenite at a volume ratio of 1-5%. The liquid fermentation medium consists of 5-15 g / L tryptone, 2-10 g / L yeast extract, 5-10 g / L sodium chloride, and 1-20 mM sodium selenite. The culture conditions are 25-35°C, 120-200 r / min, and shaking culture for 24-72 h.

9. The method of claim 5, wherein, The centrifugation conditions are as follows: first centrifugation speed 10000-15000*g, time 10-20min; second centrifugation speed 3000-8000*g, time 5-15min; third centrifugation speed 10000-20000*g, time 20-30min.

10. The method of claim 5, wherein, The final concentration of the lysozyme is 10-15 mg / mL, and the incubation conditions are 37°C for 1-1.5 h. The conditions for ultrasonic disruption of the bacterial cells are: ultrasonication on ice, ultrasonic power of 100-180 W, ultrasonication for 5 seconds followed by a 5-second pause, and a total running time of 20-30 min.

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