Soil sporosarcina CH23, method for synthesizing nano-selenium by soil sporosarcina CH23 and application of soil sporosarcina CH23

Abstract

The invention discloses soil sporosarcina CH23 as well as a method for synthesizing nano-selenium by using the soil sporosarcina CH23 and application of the soil sporosarcina CH23. The nano-selenium synthetic bacterium is sporosarcina soli CH23, is preserved in the Guangdong Microbial Culture Collection Center (GDMCC), and has the preservation number of GDMCC No.67015. The invention further discloses a preparation method of the nano-selenium synthetic bacterium. The soil sporosarcina CH23 can be used for efficiently converting high-toxicity selenite into non-toxic nano-selenium. The required culture conditions are simple, the cost is low, and the operation is easy. The produced nano-selenium particles have the advantages of being non-toxic, high in biological activity, stable in property, almost free of other selenium by-products and the like, have the effects of resisting bacteria, resisting oxidation, resisting cancers and the like in the field of medicines, can also be used as a nano-selenium fertilizer for plant selenium nutrition supplement, and have the advantages of being more efficient, safer and more environmentally friendly compared with a traditional selenite selenium fertilizer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanometer selenium biosynthesis, and particularly relates to a soil sporosarcina CH23 and a method and application thereof for synthesizing nanometer selenium. BACKGROUND

[0002] Nanometer selenium has high bioavailability and strong antioxidant properties, and its main effects include enhancing immunity, anti-aging, regulating metabolism, and assisting in anti-tumor. At present, the main preparation method of nanometer selenium is chemical reduction method, which converts selenite into nanometer selenium by adding reducing agents such as ascorbic acid. This method is high in energy consumption and heavy in pollution, and the generated nanometer selenium particles are unstable and prone to agglomeration, which ultimately leads to a significant decrease in the bioactivity of nanometer selenium. Therefore, an economical and environmentally friendly method is urgently needed to replace the traditional chemical reduction method to synthesize stable nanometer selenium with high bioactivity. At present, many microorganisms have been found to convert highly toxic selenite into non-toxic nanometer selenium through detoxification mechanisms, and the synthesized nanometer selenium has the advantages of small particle size, stable properties and high bioactivity. However, the conversion rate of selenite to nanometer selenium by the currently reported and applied microorganisms is generally not high, and high-toxicity hydrogen selenide or organic selenium by-products are easily produced, which hinders the promotion of nanometer selenium biosynthesis technology in practical production and application. Therefore, obtaining efficient nanometer selenium synthesis microorganisms is of great significance to promote the development of nanometer selenium biosynthesis industry. SUMMARY

[0003] The purpose of the present application is to provide a nanometer selenium synthesis bacteria and a method and application thereof for synthesizing nanometer selenium.

[0004] In order to achieve the purpose of the present application, the technical solution adopted by the present application is as follows: A nanometer selenium synthesis bacteria, named soil sporosarcina (Sporosarcina siccitae) CH23, was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on September 23, 2025, and the address of the preservation center is No. 59 Building, 5th Floor, Institute of Guangzhou Martyrs Road 100, Postcode 510070, and the preservation number is GDMCC No: 67015. Sporosarcina soli

[0005] The second purpose of the present application is to provide the application of soil sporosarcina (Sporosarcina siccitae) CH23 in synthesizing nanometer selenium. Sporosarcina soli The third purpose of the present application is to provide a method for synthesizing nanometer selenium by microorganisms, comprising the following steps:

[0006] Synthesizing nanometer selenium by reducing selenium-containing salt using the above-mentioned soil sporosarcina (Sporosarcina siccitae) CH23. Sporosarcina soli Preferably, the specific steps are as follows:

[0007] ​​​(1) strain activation: inoculate soil-borne sphaerodes CH23 into solid activation medium and incubate at constant temperature until the colonies grow on the surface of the solid medium; (2) seed culture: pick single colony of soil-borne sphaerodes CH23 and inoculate into liquid medium for constant temperature shaking culture to obtain seed culture liquid; (3) fermentation culture: inoculate soil-borne sphaerodes CH23 seed culture liquid into fermentation medium containing sodium selenite, incubate at constant temperature to obtain fermentation product rich in nano-selenium.

[0008] Preferably, the fermentation product rich in nano-selenium synthesized by soil-borne sphaerodes CH23 is centrifuged for the first time, the supernatant is discarded, the precipitate is obtained, the precipitate is washed with 1.0-1.5% NaCl solution for three times, then the precipitate is resuspended in TE buffer, lysozyme is added to destroy the cell wall of the bacteria, then the bacteria are ultrasonically broken, centrifuged for the second time, the precipitate is discarded, the supernatant containing nano-selenium is collected, the supernatant is centrifuged for the third time, the precipitate is collected, and the precipitate is washed with deionized water for 2-4 times to obtain pure nano-selenium particles.

[0009] Further preferably, in step (1), soil-borne sphaerodes CH23 is inoculated into solid activation medium at a volume ratio of 1%-5%, and the solid medium contains 10-25 g / L of pancreas tryptone, 5-10 g / L of sodium chloride, 2-5 g / L of soybean papain hydrolysate, 1.5-4 g / L of potassium phosphate dibasic, 1.5-5 g / L of glucose, and 15-20 g / L of agar powder, with a pH range of 6.5-7.5, a culture temperature of 25-35°C, and a culture time of 24-48 h, until colonies with a diameter of 2-4 mm are formed.

[0010] Further preferably, in step (2), the activated soil-borne sphaerodes CH23 single colony is inoculated into liquid medium, and the liquid medium contains 10-25 g / L of pancreas tryptone, 5-10 g / L of sodium chloride, 2-5 g / L of soybean papain hydrolysate, 1.5-4 g / L of potassium phosphate dibasic, and 1.5-5 g / L of glucose, with a pH range of 6.5-7.5, a culture condition of 25-35°C and a rotation speed of 120-200 r / min, and a shaking culture time of 20-36 h.

[0011] Further preferably, in step (3), the seed culture liquid is inoculated into fermentation medium containing sodium selenite at a volume ratio of 1-5%, and the liquid fermentation medium contains 5-15 g / L of tryptone, 2-10 g / L of yeast extract, 5-10 g / L of sodium chloride, and 1-20 mM of sodium selenite, with a culture condition of 25-35°C and a rotation speed of 120-200 r / min, and a shaking culture time of 24-72 h.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] The beneficial effects of this invention are as follows: 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.

[0017] 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

[0018] 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); Figure 2Growth of S. sibiricum CH23 in liquid fermentation medium containing 5mM sodium selenite in Example 2 of the present application; Figure 3 Concentration of sodium selenite and nano-selenium in liquid fermentation medium containing 5mM sodium selenite with time for S. sibiricum CH23 in Example 2 of the present application; Figure 4 SEM image of S. sibiricum CH23 strain grown in control liquid medium without sodium selenite in Example 3 of the present application; Figure 5 SEM image of S. sibiricum CH23 strain grown in experimental liquid fermentation medium containing 5mM sodium selenite in Example 3 of the present application; Figure 6 TEM image of S. sibiricum CH23 strain grown in control liquid medium without sodium selenite in Example 3 of the present application; Figure 7 TEM image of S. sibiricum CH23 strain grown in experimental liquid fermentation medium containing 5mM sodium selenite in Example 3 of the present application; Figure 8 Growth of S. sibiricum CH23 strain in liquid fermentation medium containing different concentrations of sodium selenite in Example 4 of the present application. DETAILED DESCRIPTION

[0019] To further illustrate the features and effects of the present application, the present application will be described in more detail with reference to the embodiments and the accompanying drawings.

[0020] Example 1 Isolation and purification of nano-selenium synthesis bacteria, including the following steps: 1. Collect activated sludge from Guangzhou City, Guangdong Province, take 1g of activated sludge and add it to 50mL of 0.01M sterilized PBS, mix thoroughly, and prepare an activated sludge suspension; 2. Enrichment of nano-selenium biosynthesis strain: take 1mL of activated sludge suspension and add it to 50mL of liquid enrichment medium containing 5mM sodium selenite for culture, the composition of the enrichment medium is tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, sodium selenite 20mM, the solvent is water, and the preparation method is to mix the components uniformly and sterilize for use. The culture conditions are 30°C, 150r / min, constant temperature shaking culture for 72h, and the enrichment liquid of nano-selenium biosynthesis bacteria is obtained; 3. Take 1mL of enrichment liquid, use 0.01M sterilized PBS to dilute it by 10 1 -10 6The 0.1 mL of each dilution of the enrichment liquid was uniformly coated on a solid culture medium containing 5 mM sodium selenite, and the solid culture medium was prepared by mixing 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 18 g / L of agar powder, and 5 mM of sodium selenite with water as the solvent, and sterilizing the mixture for standby use. The mixture was placed in a 30°C constant temperature incubator for 48 hours of culture; 4. After red colonies grew on the plate, a red single colony was selected for streak isolation and purification on a new solid culture medium containing 5 mM sodium selenite, and the plate was cultured at 30°C for 48 hours. This step was repeated 2-3 times until a pure strain was obtained.

[0021] 5. The pure strain was inoculated into a liquid seed culture medium, and the liquid seed culture medium was prepared by mixing 17 g / L of tryptone, 5 g / L of sodium chloride, 3 g / L of soybean papain hydrolysate, 2.5 g / L of potassium phosphate dibasic, and 2.5 g / L of glucose with water as the solvent, and sterilizing the mixture for standby use. The culture conditions were 30°C, 150 r / min, and 24 hours of shaking culture. The strain suspension was stored by freezing with 15% sterilized glycerol; 6. The pure strain was sent to Qianke Biotechnology Co., Ltd. for identification (the sequence of its 16s rRNA is shown as SEQ ID NO. 1), and was named as Soil Sporosarcina (Sporosarcina Sporosarcina soli ) CH23. On September 23, 2025, it was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, China, with a postal code of 510070, and a preservation number of GDMCC No. 67015.

[0022] Example 2 Optimal culture time of the nanometer selenium synthesis bacteria Soil Sporosarcina (Sporosarcina Sporosarcina soli ) CH23 for biosynthesis of nanometer selenium, including the following steps: 1. Strain activation: Soil Sporosarcina (Sporosarcina Sporosarcina soli ) CH23 was inoculated on a solid activation culture medium containing 17 g / L of tryptone, 5 g / L of sodium chloride, 3 g / L of soybean papain hydrolysate, 2.5 g / L of potassium phosphate dibasic, and 2.5 g / L of glucose, with an agar powder of 17 g / L and a pH of 7.0, and the solvent was water. The mixture was uniformly mixed, sterilized for standby use, and cultured at 30°C for 24 hours until a colony with a diameter of about 3 mm was formed; 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 for isothermal shaking culture at 30°C and 150 rpm for 24 h to obtain the seed culture solution. 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. 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.

[0023] 5. After the culture is completed, the remaining selenite and the content of biosynthesized nano-selenium in the solution are tested; 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. Furthermore, 99% of the consumed selenite was converted into nano-selenium, and the resulting nano-selenium had high purity and contained almost no other selenium byproducts.

[0024] Example 3 Characteristics of the biosynthesis of nano-selenium by the soil-sporogenous spore-forming Micrococcus CH23 1. The seed culture solution of Example 2 was added to the experimental group liquid fermentation medium containing tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L and sodium selenite 5 mM at a volume ratio of 1% inoculation amount, the culture conditions were 30°C, 150 r / min, and the culture time was 36 h, and a group of liquid medium containing tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L was set as the control group, and the same culture conditions were used for culture; 2. Scanning electron microscope observation: After the sample after the end of the culture was treated by ethanol gradient dehydration, the Czech Tescan MIRA LMS scanning electron microscope was used for imaging and photographing, and the results of the blank control group are shown in Figure 4 , and the results of the experimental group are shown in Figure 5 ; 3. Transmission electron microscope observation: glutaraldehyde was added to 0.01M PBS to prepare a fixing solution containing 2.5% glutaraldehyde, and the sample after the end of the culture was fixed with 2.5% glutaraldehyde fixing solution at 4°C overnight. The treated sample was fixed on a copper grid, and the Hitachi H-7650 transmission electron microscope was used for imaging and photographing, and the results of the blank control group are shown in Figure 6 , and the results of the experimental group are shown in Figure 7 .

[0025] Example 4 The tolerance test of S. selenatarsenitrificans CH23 to selenite includes the following steps: A liquid culture medium containing tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L and sodium selenite 0.01-100 mM was prepared, and the seed culture solution of S. selenatarsenitrificans CH23 of Example 2 was added to the liquid culture medium with a final concentration of 0 mM, 0.1 mM, 1 mM, 5 mM, 20 mM and 100 mM sodium selenite respectively at a volume ratio of 1% inoculation amount, the culture temperature was 30°C, the rotation speed was 150 r / min, and the culture time was 48 h; The experimental results are shown in Figure 8 , S. selenatarsenitrificans CH23 can grow well in 1-20 mM sodium selenite medium and synthesize a large amount of red nanoselenium.

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, comprising the steps of: 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: (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: the seed culture of S. terrestris (CH23) was inoculated into liquid fermentation medium containing sodium selenite, and constant temperature oscillation culture was carried out to obtain fermentation product rich in nano-selenium. Sporosarcina soli )CH23 seed culture liquid was inoculated into liquid fermentation medium containing sodium selenite, and constant temperature oscillation culture was carried out to obtain fermentation product rich in nano-selenium.

5. The method of claim 4, wherein, Sporosarcina gregaria (ATCC 12777) Sporosarcina soli The first centrifugation was performed on the synthesized selenium-rich fermentation product of S. gregaria (ATCC 12777) CH23 GDMCC No. 67015, and the supernatant was discarded to obtain the precipitate. The precipitate was washed three times with 1.0-1.5% NaCl solution, and then resuspended in TE buffer. Lysozyme was added to break the cell wall, followed by ultrasonic disruption. The second centrifugation was performed, and the precipitate was discarded to collect the supernatant containing selenium nanoparticles. The third centrifugation was performed on the supernatant, and the precipitate was collected and washed 2-4 times with deionized water to obtain pure selenium nanoparticles.

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) inoculates the activated soil Sporosarcina into a liquid medium, and the liquid medium comprises 10-25 g / L of pancreas tryptone, 5-10 g / L of sodium chloride, 2-5 g / L of soybean papain hydrolysate, 1.5-4 g / L of dipotassium hydrogen phosphate, and 1.5-5 g / L of glucose, and has a pH range of 6.5-7.5, and is cultured at 25-35°C and a rotating speed of 120-200 r / min for 20-36 hours. Sporosarcina soli The step (2) inoculates the activated soil Sporosarcina into a liquid medium, and the liquid medium comprises 10-25 g / L of pancreas tryptone, 5-10 g / L of sodium chloride, 2-5 g / L of soybean papain hydrolysate, 1.5-4 g / L of dipotassium hydrogen phosphate, and 1.5-5 g / L of glucose, and has a pH range of 6.5-7.5, and is cultured at 25-35°C and a rotating speed of 120-200 r / min for 20-36 hours.

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

9. The method of claim 5, wherein, The centrifugation conditions are: 10,000-15,000*g for 10-20 min for the first centrifugation, 3,000-8,000*g for 5-15 min for the second centrifugation, and 10,000-20,000*g for 20-30 min for the third centrifugation.

10. The method of claim 5, wherein, The final concentration of lysozyme is 10-15 mg / mL, and the action condition is 37 °C for 1-1.5 h; and the ultrasonic crushing condition of the bacterial cells is: ultrasonic on ice, ultrasonic power is 100-180 W, ultrasonic operation for 5 s and pause for 5 s, and total operation time is 20-30 min.

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