CN114480224A - Method for biosynthesizing nano-selenium by utilizing bacillus subtilis and application thereof - Google Patents
Method for biosynthesizing nano-selenium by utilizing bacillus subtilis and application thereof Download PDFInfo
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- CN114480224A CN114480224A CN202210279516.9A CN202210279516A CN114480224A CN 114480224 A CN114480224 A CN 114480224A CN 202210279516 A CN202210279516 A CN 202210279516A CN 114480224 A CN114480224 A CN 114480224A
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Abstract
The invention relates to a method for biosynthesizing nano-selenium by utilizing bacillus subtilis and application thereof, which comprises the following steps: step 1, activating and culturing strains by using a culture medium; step 2, preparing the activated culture solution obtained in the step 1 into seed solution; step 3, fermenting the seed liquid obtained in the step 2; and 4, separating and purifying the nano selenium from the fermentation product in the step 3. The nano-selenium strain capable of realizing high yield by the method belongs to probiotics, and can be applied to related fields of medicine industry, food industry, feed industry, fertilizer industry, cosmetic industry and the like.
Description
Technical Field
The invention relates to the technical field of microbiology and biological nano-selenium preparation, in particular to a method for preparing an organic liquid fertilizer for biologically synthesizing nano-selenium by utilizing bacillus subtilis and application thereof.
Background
Selenium (Selenium) is a trace element essential to animals and humans, and is involved in a variety of metabolic pathways in the human body. Selenium deficiency in humans can cause various diseases, such as muscular atrophy, cardiovascular, skeletal or immune system diseases, and increase the risk of cancer, while excessive selenium can cause serious toxicity to the human body. The human body has a narrow adaptive range to selenium, the Chinese Nutrition society and the FAO/WHO/IAEA joint expert Committee determine that the suitable range of the human body intake is 60-250 mug/d, the safe dose is 400 mug/d, and the poisoning dose is 800 mug/d. Because a natural selenium-deficient band exists in the territory of China, the distribution of selenium is extremely unbalanced, the daily selenium intake of people in the selenium-deficient area is seriously insufficient, and safe and efficient selenium supplement is not slow for people in the selenium-deficient area of China.
Selenium in nature has various forms, and the negative divalent selenide includes hydrogen selenide (H)2Se), metal selenides, methyl selenides, selenoaminoacids, and the like; tetravalent selenium is mainly SeO2、H2SeO3And SeO3 2-Several forms; hexavalent selenium is mainly H2SeO4And SeO4 2-. In industrial production, grey elemental selenium can be prepared by oxidizing selenium in a negative divalent form and reducing selenium in a tetravalent or hexavalent form. In recent years, people prepare red nano-selenium by chemical and physical methods, and discover that various microorganisms (fungi, bacteria and actinomycetes) can convert hexavalent and tetravalent inorganic selenium salts into red nano-selenium.
It was found that the median lethal dose of sodium selenite orally administered to rats was about 7mg/kg, the median lethal dose of selenomethylcysteine was about 15mg/kg, the median lethal dose of selenomethionine was about 26mg/kg, and the median lethal dose of chemically synthesized nano-selenium was about 105 mg/kg. The biological activities of the nano-selenium and the selenomethionine in organs such as blood, liver, kidney and the like have no obvious difference, and the nano-selenium has obvious effect on inhibiting tumors and is superior to the selenomethionine, so the nano-selenium has the advantage of being safer and more effective in selenium supplement compared with inorganic and organic selenium.
The biological nano selenium is simple substance state nano selenium obtained by reducing inorganic selenium through microbial conversion and separating and purifying. The biological nano-selenium has the characteristics of no toxicity, high biological activity, large specific surface area, safety, high efficiency and the like, and is better than other selenium sources. Because the biological nano-selenium has obvious advantages in the aspect of supplementing selenium for human bodies and the characteristics of safety, high efficiency, environmental protection and the like in microbial transformation, a plurality of research reports about microbial transformation of nano-selenium exist. However, no research on post-industrial production and application has been reported.
Disclosure of Invention
The invention designs a method for biologically synthesizing nano selenium by utilizing bacillus subtilis and application thereof, and solves the technical problem that in the prior art, application of elemental nano selenium obtained by reducing inorganic selenium through microbial transformation and separating and purifying is not available.
In order to solve the technical problems, the invention adopts the following scheme:
a method for biosynthesizing nano-selenium by utilizing bacillus subtilis comprises the following steps:
step 1, activating and culturing strains by using a culture medium;
step 2, preparing the activated culture solution obtained in the step 1 into seed solution;
step 3, fermenting the seed liquid obtained in the step 2;
and 4, separating and purifying the nano selenium from the fermentation product in the step 3.
Preferably, in step 1, the strain BS8900 is inoculated on a slant of NSB medium, and the strain is activated and cultured for more than 16 hours at 37 ℃ by using ZFM medium.
Preferably, in step 1, the formulation of ZFM medium is: 5g/L glucose, 5g/L yeast extract and 10g/L KH peptone2PO40.9g/L，K2HPO40.9g/L, agar 15g/L, pH 7.0-7.2.
Preferably, in step 2, the activated BS8900 strain is formulated into 10 with sterile PBS7.0 buffer8The bacterial suspension of CFU/mL is inoculated in an FM liquid culture medium in an inoculation amount of 1%, and is subjected to shaking culture at 37 ℃ by a shaking table, the rotating speed is 225rpm, and the culture time is 12 h.
Preferably, in step 2, the formula of the FM liquid medium is: 5g/L glucose, 5g/L yeast extract and 10g/L KH peptone2PO40.9g/L，K2HPO40.9g/L，Na2SeO3 20mM，pH 7.0-7.2。
Preferably, in step 3, the volume of the HR fermentation medium is controlled to be 80% of the volume of the fermentation tank, the seed solution obtained in step 2 is inoculated into the fermentation tank according to the inoculum size of 3.3%, the fermentation temperature is controlled to be 37 ℃, the stirring speed is 120rpm, and the volume of the fermentation liquid: volume of ventilation per minute 1: 0.2, the tank pressure is 0.05MP, and the fermentation is carried out for 48 to 60 hours.
Preferably, in step 3, the formulation of the HR fermentation medium is: 15g/L glucose, 5g/L yeast extract and 10g/L KH peptone2PO40.9g/L，K2HPO40.9g/L，Na2SeO38mM，pH 7.0-7.2。
Preferably, in step 4, the collected red precipitate is suspended in PBS7.0 buffer solution in a fermentation liquid tank, and is subjected to ultrafiltration circulation for 2-3 times by a hollow fiber membrane with 100KD, and the precipitate is resuspended by water with the volume of 1/10 of fermentation liquid, and the concentration of concentrated nano selenium is 10 times of the fermentation concentration and reaches 50 mM; placing the obtained bacterial suspension back to a fermentation tank, heating the bacterial suspension to 90 ℃ by steam, stirring at the speed of 250rpm, and keeping the temperature for 1 hour; then rapidly cooling to 25 ℃, wherein the stirring speed is 250 rpm; to obtain thallus lysate and nano-selenium solution, and performing ultrafiltration by 7KD hollow fiber membrane to obtain nano-selenium solution.
A nano-selenium solution, characterized by: synthesized using the methods described above.
A selenium-rich organic liquid fertilizer is characterized in that: comprises nano selenium solution, wherein the content of biological nano selenium is 2000 mug/L.
The method for biologically synthesizing nano-selenium by utilizing the bacillus subtilis and the application thereof have the following beneficial effects:
1. the nano-selenium strain capable of realizing high yield by the method belongs to probiotics, and can be applied to related fields of medicine industry, food industry, feed industry, fertilizer industry, cosmetic industry and the like.
2 the yield of the nano-selenium biosynthesized by the strain in the method is higher than 800ug/L reported in the current literature.
Drawings
FIG. 1: the photo of the biological synthesis nano-selenium strain BS8900 used in the invention;
the strain belongs to bacillus subtilis;
FIG. 2: the electron microscope picture of the strain BS8900 used in the invention;
the shape of the thallus in an electron microscope is rod-shaped;
FIG. 3: the invention relates to a strain diagram under different concentrations of sodium selenite.
Detailed Description
The invention is further illustrated below with reference to fig. 1 to 2:
the invention relates to a method for biologically synthesizing nano selenium by separating and purifying nano selenium from a fermentation product, wherein the method comprises the steps of separating and obtaining a Bacillus subtilis BS8900 capable of tolerating high-concentration sodium selenite from the soil of a gynostemma pentaphylla selenium-rich tea planting base in Ankang city of Shaanxi province, and the concentration of hexavalent and/or tetravalent inorganic selenium salt in a fermentation culture medium of the Bacillus subtilis BS8900 is 0.001-70mM, preferably 1-20mM, in combination with an ultraviolet mutagenesis test. The fermentation product comprises a thallus concentrate obtained by ultrafiltration of fermentation liquor by a 100KD hollow fiber membrane, a thallus suspension and a thallus lysate obtained by ultrafiltration of a 7KD hollow fiber membrane.
As shown in FIG. 3, the sodium selenite (molar concentration mmol) is 50, the screening charts of UV mutagenesis, 40, 30, 20 and 5, and the strain charts obtained by UV mutagenesis are from left to right and from top to bottom.
The invention provides a method for biosynthesizing nano selenium by utilizing bacillus subtilis BS8900, which comprises the following steps:
s1, strain activation:
performing activated culture on the strain by using a ZFM culture medium, wherein the ZFM culture medium has the formula: 5g/L glucose, 5g/L yeast extract, 10g/L peptone and KH2PO40.9g/L，K2HPO40.9g/L, 15g/L agar, pH 7.0-7.2; the strain BS8900 was inoculated on NSB medium slant and cultured at 37 ℃ for 16 hours.
S2, preparation of seed liquid:
the FM liquid culture medium for seed culture comprises the following components in percentage by weight: 5g/L glucose, 5g/L yeast extract, 10g/L peptone and KH2PO40.9g/L，K2HPO40.9g/L，Na2SeO320mM, pH 7.0-7.2; the activated BS8900 strain was prepared into 10 with sterile PBS7.0 buffer8The bacterial suspension of CFU/mL is inoculated in an FM liquid culture medium in an inoculation amount of 1%, and is subjected to shaking culture at 37 ℃ by a shaking table, the rotating speed is 225rpm, and the culture time is 12 h.
S3, fermentation in a fermentation tank:
the fermentation culture adopts an HR fermentation culture medium, and the formula of the HR fermentation culture medium is as follows: 15g/L glucose, 5g/L yeast extract and 10g/L KH peptone2PO40.9g/L，K2HPO40.9g/L，Na2SeO38mM pH 7.0-7.2; controlling the volume of the culture medium to be 80% of the volume of the fermentation tank, inoculating the seed liquid into the fermentation tank according to the inoculation amount of 3.3%, controlling the fermentation temperature to be 37 ℃, the stirring speed to be 120rpm, and the ventilation volume to be 1: 0.2 (fermentation liquid volume: volume of ventilation volume per minute), 0.05MP of tank pressure, and 48-60 hours of fermentation. The yield of the nano selenium can reach 5 mM.
S4, separating and purifying nano selenium from the fermentation product:
the method for separating and purifying the biological nano selenium from the fermentation product comprises the following steps:
and (5) putting the fermentation liquor into a tank, and performing ultrafiltration by using a 100KD hollow fiber membrane to collect thalli and nano selenium. Suspending the collected red precipitate with PBS7.0 buffer solution, ultrafiltering with 100KD hollow fiber membrane for 2-3 times, suspending the precipitate with 1/10 volume of water (preferably purified water), and concentrating to obtain 50mM nano-selenium concentration 10 times of fermentation concentration. The resulting bacterial suspension was placed back in the fermentor and steam heated to 90 degrees with a stirring speed of 250rpm for 1 hour. Then rapidly cooled to 25 degrees with a stirring speed of 250 rpm. To obtain thallus lysate and nano-selenium solution, and performing ultrafiltration by 7KD hollow fiber membrane to obtain nano-selenium solution.
The invention provides a selenium-rich organic fertilizer prepared from the biological nano selenium solution, wherein the content of biological nano selenium is 2000 mug/L.
In one specific embodiment of the invention, the biological nano-selenium with the selenium content of 0.1g/L is prepared into 2000 mug/L of 5 tons of selenium-rich organic liquid fertilizer by 100L of solution,
table 1 shows that different yields of nano-selenium were obtained with corresponding amounts of sodium selenite.
Sodium selenite is used in the step 2 and the step 3, selenium in the sodium selenite belongs to inorganic selenium and is used as a substrate in a culture medium, and the inorganic selenium is biologically converted into nano selenium through the culture of a strain BS 8900. When the content of the selenium reaches 50mmol of sodium selenite, the BS8900 stops growing, the conversion capacity is 0, and the nano selenium cannot be obtained.
The invention is described above with reference to the accompanying drawings, it is obvious that the implementation of the invention is not limited in the above manner, and it is within the scope of the invention to adopt various modifications of the inventive method concept and solution, or to apply the inventive concept and solution directly to other applications without modification.
Claims (10)
1. A method for biosynthesizing nano-selenium by utilizing bacillus subtilis comprises the following steps:
step 1, activating and culturing strains by using a culture medium;
step 2, preparing the activated culture solution obtained in the step 1 into seed solution;
step 3, fermenting the seed liquid obtained in the step 2;
and 4, separating and purifying the nano selenium from the fermentation product in the step 3.
2. The method for biosynthesizing nano-selenium using Bacillus subtilis as claimed in claim 1, wherein: in step 1, the strain BS8900 is inoculated on the slant of NSB culture medium, and the strain is activated and cultured for more than 16 hours at 37 ℃ by using ZFM culture medium.
3. The method for biosynthesizing nano-selenium using bacillus subtilis as defined in claim 2, wherein: in the step 1, the formula of the ZFM culture medium is as follows: 5g/L glucose, 5g/L yeast extract and 10g/L KH peptone2PO40.9g/L，K2HPO40.9g/L, agar 15g/L, pH 7.0-7.2.
4. The method for biosynthesizing nano-selenium using bacillus subtilis as defined in claim 1, wherein: in step 2, the activated BS8900 strain is prepared into 10 by using sterile PBS7.0 buffer solution8The bacterial suspension of CFU/mL is inoculated in an FM liquid culture medium in an inoculation amount of 1%, and is subjected to shaking culture at 37 ℃ by a shaking table, the rotating speed is 225rpm, and the culture time is 12 h.
5. The method for biosynthesizing nano-selenium using bacillus subtilis as defined in claim 4, wherein: in the step 2, the formula of the FM liquid culture medium is as follows: 5g/L glucose, 5g/L yeast extract and 10g/L KH peptone2PO40.9g/L，K2HPO40.9g/L，Na2SeO3 20mM，pH 7.0-7.2。
6. The method for biosynthesizing nano-selenium using bacillus subtilis as defined in claim 1, wherein: in step 3, controlling the volume of the HR fermentation medium to be 80% of the volume of the fermentation tank, inoculating the seed solution obtained in step 2 into the fermentation tank according to the inoculation amount of 3.3%, controlling the fermentation temperature to be 37 ℃, the stirring speed to be 120rpm, and the volume of the fermentation liquid: volume of ventilation per minute 1: 0.2, the tank pressure is 0.05MP, and the fermentation is carried out for 48 to 60 hours.
7. The method for biosynthesizing nano-selenium using bacillus subtilis as defined in claim 6, wherein: in step 3, the HR fermentation medium formula is as follows: 15g/L glucose, 5g/L yeast extract and 10g/L KH peptone2PO40.9g/L，K2HPO40.9g/L，Na2SeO38mM，pH 7.0-7.2。
8. The method for biosynthesizing nano-selenium using bacillus subtilis as defined in claim 1, wherein: in the step 4, putting the fermentation liquor into a tank, suspending the collected red precipitate by PBS7.0 buffer solution, performing ultrafiltration circulation for 2-3 times by a hollow fiber membrane with 100KD, and suspending the precipitate by water with the volume of 1/10 fermentation liquor, wherein the concentration of concentrated nano selenium is 10 times of the fermentation concentration and reaches 50 mM; placing the obtained bacterial suspension back to a fermentation tank, heating the bacterial suspension to 90 ℃ by steam, stirring at the speed of 250rpm, and keeping the temperature for 1 hour; then rapidly cooling to 25 ℃, wherein the stirring speed is 250 rpm; to obtain thallus lysate and nano-selenium solution, and performing ultrafiltration by 7KD hollow fiber membrane to obtain nano-selenium solution.
9. A nano-selenium solution, characterized by: synthesized using the method of claims 1-9.
10. A selenium-rich organic liquid fertilizer is characterized in that: comprises the nano-selenium solution of claim 9, wherein the biological nano-selenium accounts for 2000 μ g/L.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115261257A (en) * | 2022-05-25 | 2022-11-01 | 武汉轻工大学 | Selenium-rich bacillus subtilis L11 derived from silage forage and application thereof |
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| CN106190905A (en) * | 2016-07-18 | 2016-12-07 | 中国农业大学 | Utilize method and the application thereof of bacillus subtilis biosynthesis nanometer selenium |
| US20200010857A1 (en) * | 2016-12-14 | 2020-01-09 | Uti Limited Partnership | Rhodococcus aetherivorans bcp1 as cell factory for the production of intracellular tellurium and/or selenium nanostructures (nanoparticles or nanorods) under aerobic conditions |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261257A (en) * | 2022-05-25 | 2022-11-01 | 武汉轻工大学 | Selenium-rich bacillus subtilis L11 derived from silage forage and application thereof |
| CN115261257B (en) * | 2022-05-25 | 2023-04-25 | 武汉轻工大学 | Selenium-enriched bacillus subtilis L11 from silage and application thereof |
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