CN102703513A - Method for preparing red elemental selenium by utilizing super-selenium resistance microorganism - Google Patents
Method for preparing red elemental selenium by utilizing super-selenium resistance microorganism Download PDFInfo
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- CN102703513A CN102703513A CN2012101676506A CN201210167650A CN102703513A CN 102703513 A CN102703513 A CN 102703513A CN 2012101676506 A CN2012101676506 A CN 2012101676506A CN 201210167650 A CN201210167650 A CN 201210167650A CN 102703513 A CN102703513 A CN 102703513A
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Abstract
The invention relates to a method for preparing red elemental selenium by utilizing a super-selenium resistance microorganism. The preparation method is characterized by comprising the following steps of: a, breeding a target bacterial strain; b, preparing a selenium-contained culture medium; c, inoculating and culturing; d, preparing the red elemental selenium; e, producing the red elemental selenium; and f, harvesting the product. According to the method for preparing the red elemental selenium by utilizing the super-selenium resistance microorganism, the super-selenium resistance microorganism capable of normally surviving in a high-selenium environment is adopted, wherein the super-selenium resistance microorganism has the characteristic of reducing inorganic selenium into the red elemental selenium, can be used for preparing low-toxicity nanometer organic selenium and is wide in application prospect.
Description
Technical field
The present invention relates to a kind ofly utilize ultra anti-selenium mikrobe to prepare the method for red elemental selenium.
Background technology
At present reported that the mikrobe that can tolerate Sodium Selenite mainly has: intestinal bacteria, the logical Salmonella CH34 in Rolls, Crimson rhodospirillum, the red bacterium of pod membrane, Pseudomonas fluorescens, the red bacterium of fixed nitrogen, yeast etc.But the concentration of tolerance selenium is not high, and the concentration that can tolerate Sodium Selenite like the logical Salmonella CH43 in Rolls is 6mmol/L, and the concentration of intestinal bacteria tolerance Sodium Selenite is up to 20mmol/L.Wang Dongliang etc. (2007) report one strain has height endurability to Sodium Selenite, and can Sodium Selenite be reduced to the photosynthetic bacterium S3 of red elemental selenium, and through being accredited as the red bacterium of fixed nitrogen, its anti-selenium ability reaches 125mM, is higher than this concentration and just can't have grown.Yeast has higher enrichment and the ability that transforms selenium, is generally about 300 μ g/g, through the anti-selenium domestication of gradient, DES chemomorphosis and ultraviolet mutagenesis, can bring up to 1026.96 μ g/g to the dosis tolerata of selenium.Plum superfine (2011) is when carrying out Rhodopseudomonas palustris to the research of selenite reduction detoxification, and the selenite addition is 25mg/L.
Grerbisu.et.al (1995) report bacterium is between the 0.6-5mM time in concentration of sodium selenite, and bacillus can be reduced to simple substance selenium with Sodium Selenite.Li Ruiping etc. (2005) report bacillus HBS4 has higher biological tolerance to Sodium Selenite, still can grow up to 11mM in concentration of sodium selenite, but along with the increasing of selenium concentration, and the restraining effect of bacterium is strengthened.
Certain micro-organisms has tolerance to selenium, and reason is the red elemental selenium that inorganic selenium is transformed into the colloidal state of nontoxic or low toxicity.1985, Nuttal just once proposed the hypothesis of colloidal state red elemental selenium biologically active under specific environment.Experiment confirm in recent years, red elemental selenium that biological reducing obtains or red elemental selenium be a kind of be nuclear with protein, red elemental selenium is the selenium-albumen composition of film, diameter is less than 200nm, toxicity is little, to thermally-stabilised, is considered to the mode of selenium biologic detoxication.Mikrobe through a series of redox reactions, is realized the conversion of the different valence state of selenium in the metabolism of selenium, play the detoxification to selenium, and reduces the murder by poisoning to bacterium itself.This red elemental selenium immunomodulatory, anti-oxidant, delaying senility and suppressing aspects such as tumour all has good biological activity; External removing hydroxyl radical free radical efficient is 5 times of inorganic selenium; 2.5 times of organoselenium; And toxicity has only 1/7 of Sodium Selenite, has higher research and using value in the biological activity of selenium aspect utilizing.
Zheng Qingshan etc. (2002) discover; Red elemental selenium can obviously suppress the tobacco-specific nitrosamines compounds and bring out the active oxygen radical that human bronchial epithelial cell produces; And can directly or indirectly remove O2 and the H2O2 that produces in the human embryo lung (HEL) epithelial cell canceration process, disclosing red elemental selenium has provide protection to the oxidative damage of cell due to the tobacco-specific nitrosamines compounds.
Summary of the invention
The purpose of this invention is to provide and a kind ofly utilize ultra anti-selenium mikrobe to prepare the method for red elemental selenium safely and effectively.
The present invention is a kind of to utilize ultra anti-selenium mikrobe to prepare the method for red elemental selenium, it is characterized in that comprising following steps:
A. breeding goal bacterial strain:
Restrain-5 grams with 2 and take from samples such as the selenium slag in selenium mining area, soil; Join and place 35 ℃ to increase bacterium cultivation 6 hours in 500 milliliters of broth cultures; Be inoculated on the nutrient agar with transfering loop picking meat soup bacterium liquid, put 35 ℃ and cultivated 24-48 hours, separate single bacterium colony and carry out gramstaining respectively; Choose Gram-positive bacillus at 1000 times of oily mirror microscopicallies; Have oval brood cell, the brood cell is positioned at thalline central authorities, and the Bacillus bacterium of not expanding or expanding slightly is an aimed strain;
B. preparation contains seleno culture medium:
In nutrient agar, add 1.0% glucose,, contain selenium 800 ug dosage interpolation Sodium Selenite by every milliliter of substratum and process solid medium to increase the supply of carbon;
C. inoculation and cultivation:
The aimed strain that step a is selected is inoculated into respectively with transfering loop and contains selenium 800 ug/ ml, contains on the nutrient agar of sugar 1%, puts in 35 ℃ of incubators and cultivates, and the bacterial strain that produced red elemental selenium in 48 hours is decided to be the work bacterial strain;
D. prepare red elemental selenium:
In red inoculation to 100 milliliter broth culture on the step c substratum, put 35 ℃ and cultivated 6 hours, increase bacterium again;
E. produce red elemental selenium:
Enrichment liquid is inoculated into selenium content 800ug/ml, contains on the nutrient agar of glucose 1.0%, put 35 ℃ and cultivated 24-72 hours, become big to bacterium colony, red elemental selenium is piled up, just can be gathered in the crops when whole substratum reddens;
F. harvest product:
Red lawn on the substratum scraped drying both can get product in 65 ℃ of baking ovens of underlying.
The bacterial strain that the present invention is separated to from the band sample of high selenium zone selenium mineral deposit generally has higher tolerance to Sodium Selenite; The ultra anti-selenium bacillus of 2 strains wherein; The tolerance of selenium and reducing power are surpassed 46500ug/ml and 33000ug/ml respectively, really belong to exception, explain to be grown in high selenium zone, particularly to be grown in mikrobe on the band of selenium mineral deposit; For adapting to high selenium living environment, selenium had natural superpower tolerance and reducing power.
A kind of method system that utilizes ultra anti-selenium mikrobe to prepare red elemental selenium of the present invention is employed in the ultra anti-selenium mikrobe of ability normal existence under the high selenium environment; Has the characteristic that the elemental selenium of inorganic states is reduced to red elemental selenium; Preparation low toxicity and nano level organoselenium have broad application prospects.
The present invention from high selenium zone selenium mineral deposit band gather sample, in broth culture, carry out enrichment, the cultivation of isolation and selection property obtains single bacterial strain.Carry out anti-selenium aptitude tests then, the Bacillus bacterial strain screening that can tolerate high density selenium and can the selenium in the substratum be reduced to red elemental selenium comes out, and is used to prepare the work bacterial strain of red elemental selenium.
Institute of the present invention strain separated finally is accredited as bacillus licheniformis and Bacillus subtillis, all is the bacterium that extensively distributes in the physical environment, and is useful and harmless to environment and people and animal.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail, but the working of an invention mode is not limited thereto.
Gather the selenium slag from the area, fishing dyke selenium mineral deposit of bestowing favour, take by weighing 3 grams, put into meat soup (commercially available finished product), put 35 ℃ and increase bacterium cultivation 6 hours.
Take by weighing 3.2 gram nutrient agar (commercially available finished product) adding distil water 100 ml in the 250ml triangular flask, heating for dissolving, add a cover the sponge plug, conventional autoclaving, 5 sterile petri dish of packing are processed solid medium.
Be seeded on the nutrient agar with strok method with transfering loop picking enrichment liquid, put 35 ℃ and cultivated 24 hours, separate single bacterium colony.
Carry out gramstaining, under 1000 power microscopes, observe, with the Gram-positive bacillus, the brood cell is arranged, the brood cell is positioned at thalline central authorities, and the bacterial strain that thalline does not expand or expands is slightly confirmed as aimed strain.
Take by weighing 3.2 gram nutrient agars in the 250ml triangular flask, adding distil water 100 ml, heating for dissolving; Add 0.18 gram Sodium Selenite (contain selenium 0.08 gram, promptly every ml substratum contains selenium 800 micrograms) again, add 1 gram glucose, mixing is jumped a queue, conventional autoclaving, and 5 sterile petri dish of packing are processed and are contained the selenium solid medium.
Choose the aimed strain on the nutrient agar with transfering loop, process bacteria suspension with SPSS, be seeded to respectively on the Se-containing nutritive nutrient agar with strok method, put 35 ℃ and cultivated 24-72 hours, red bacterium colony long on the substratum is decided to be the work bacterial strain.
The bacterial strain of will working increases bacterium again with broth culture (method is the same), will increase liquid and be seeded to (method for making is the same) on the Se-containing nutritive nutrient agar with strok method, puts 35 ℃ and cultivates 24-72 hours, and bacterial colony is bright red and is fused into the sheet lawn.
Scrape red lawn from substratum, put the dry dry product that gets in 65 ℃ of baking ovens.
The biological red elemental selenium detected result that the present invention produces is following: SH20120502003 number report of Enshi quality inspection: selenium content 19667.80ug/g.
Claims (1)
1. one kind is utilized ultra anti-selenium mikrobe to prepare the method for red elemental selenium, it is characterized in that comprising following steps:
A. breeding goal bacterial strain:
Restrain-5 grams with 2 and take from samples such as the selenium slag in selenium mining area, soil; Join and place 35 ℃ to increase bacterium cultivation 6 hours in 500 milliliters of broth cultures; Be inoculated on the nutrient agar with transfering loop picking meat soup bacterium liquid, put 35 ℃ and cultivated 24-48 hours, separate single bacterium colony and carry out gramstaining respectively; Choose Gram-positive bacillus at 1000 times of oily mirror microscopicallies; Have oval brood cell, the brood cell is positioned at thalline central authorities, and the Bacillus bacterium of not expanding or expanding slightly is an aimed strain;
B. preparation contains seleno culture medium:
In nutrient agar, add 1.0% glucose,, contain selenium 800 ug dosage interpolation Sodium Selenite by every milliliter of substratum and process solid medium to increase the supply of carbon;
C. inoculation and cultivation:
The aimed strain that step a is selected is inoculated into respectively with transfering loop and contains selenium 800 ug/ ml, contains on the nutrient agar of sugar 1%, puts in 35 ℃ of incubators and cultivates, and the bacterial strain that produced red elemental selenium in 48 hours is decided to be the work bacterial strain;
D. prepare red elemental selenium:
In red inoculation to 100 milliliter broth culture on the step c substratum, put 35 ℃ and cultivated 6 hours, increase bacterium again;
E. produce red elemental selenium:
Enrichment liquid is inoculated into selenium content 800ug/ml, contains on the nutrient agar of glucose 1.0%, put 35 ℃ and cultivated 24-72 hours, become big to bacterium colony, red elemental selenium is piled up, just can be gathered in the crops when whole substratum reddens;
F. harvest product:
Red lawn on the substratum scraped drying both can get product in 65 ℃ of baking ovens of underlying.
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361380A (en) * | 2013-07-18 | 2013-10-23 | 山西大学 | Method for preparation of red elemental selenium by photosynthetic bacteria |
| CN103952363A (en) * | 2014-05-16 | 2014-07-30 | 华中农业大学 | Bacillus ES2-45 capable of reducing selenite to produce nano-selenium and application thereof |
| CN104774875A (en) * | 2015-01-29 | 2015-07-15 | 中国农业大学 | Method for preparing biological nanoselenium by using Rahnella aquatilis |
| CN105441489A (en) * | 2015-11-11 | 2016-03-30 | 大连理工大学 | Method for synthesizing selenium and bismuth selenide nano material by using lysinibacillus sp. |
| CN106011021A (en) * | 2016-07-06 | 2016-10-12 | 安徽师范大学 | Method for separating, purifying and identifying bacteria with super selenium endurance |
| CN106148480A (en) * | 2016-07-06 | 2016-11-23 | 安徽师范大学 | A kind of super resistance to selenium antibacterial research method to the reduction of selenite |
| 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 |
| CN106479927A (en) * | 2016-11-02 | 2017-03-08 | 中国农业大学 | Method and its application using Bacillus licheniformis biosynthesiss nanometer selenium |
| CN106566788A (en) * | 2016-11-02 | 2017-04-19 | 中国农业大学 | Method for biological synthesis of nano-selenium by using silver-resistant Bacillus edaphicus and application thereof |
| CN106566790A (en) * | 2016-11-02 | 2017-04-19 | 中国农业大学 | Method for biosynthesis of nano-selenium by utilization of Agrobacterium tumefaciens and application thereof |
| CN106884000A (en) * | 2017-04-25 | 2017-06-23 | 安徽天邦生物技术有限公司 | A kind of preparation and application of spindle lysine bacillus selenium-rich microbial agent |
| CN109182128A (en) * | 2018-10-16 | 2019-01-11 | 广西壮族自治区农业科学院农业资源与环境研究所 | A kind of strain culturing method of Efficient Conversion Selenium in Soil |
| CN112831435A (en) * | 2021-01-15 | 2021-05-25 | 武汉华硒生物科技有限公司 | Separated nitrogen-producing pseudomonas and application thereof in reduction of selenium element |
| CN113430136A (en) * | 2021-07-09 | 2021-09-24 | 华中农业大学 | Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium |
| CN113912458A (en) * | 2021-10-24 | 2022-01-11 | 恩施硒谷科技股份有限公司 | Method for preparing micro-ecological slow-release organic selenium fertilizer by using super-selenium-resistant microorganisms |
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Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361380A (en) * | 2013-07-18 | 2013-10-23 | 山西大学 | Method for preparation of red elemental selenium by photosynthetic bacteria |
| CN103952363A (en) * | 2014-05-16 | 2014-07-30 | 华中农业大学 | Bacillus ES2-45 capable of reducing selenite to produce nano-selenium and application thereof |
| CN103952363B (en) * | 2014-05-16 | 2016-05-04 | 华中农业大学 | Reduction selenite produces bacillus ES2-45 and the application of nanometer selenium |
| CN104774875A (en) * | 2015-01-29 | 2015-07-15 | 中国农业大学 | Method for preparing biological nanoselenium by using Rahnella aquatilis |
| CN105441489A (en) * | 2015-11-11 | 2016-03-30 | 大连理工大学 | Method for synthesizing selenium and bismuth selenide nano material by using lysinibacillus sp. |
| CN105441489B (en) * | 2015-11-11 | 2018-11-09 | 大连理工大学 | A method of synthesizing selenium and bismuth selenide nano material using lysine bacillus |
| CN106011021A (en) * | 2016-07-06 | 2016-10-12 | 安徽师范大学 | Method for separating, purifying and identifying bacteria with super selenium endurance |
| CN106148480A (en) * | 2016-07-06 | 2016-11-23 | 安徽师范大学 | A kind of super resistance to selenium antibacterial research method to the reduction of selenite |
| 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 |
| CN106190905B (en) * | 2016-07-18 | 2018-07-10 | 中国农业大学 | Utilize the method and its application of bacillus subtilis biosynthesis nanometer selenium |
| CN106566790A (en) * | 2016-11-02 | 2017-04-19 | 中国农业大学 | Method for biosynthesis of nano-selenium by utilization of Agrobacterium tumefaciens and application thereof |
| CN106479927B (en) * | 2016-11-02 | 2018-06-26 | 中国农业大学 | Utilize the method and its application of bacillus licheniformis biosynthesis nanometer selenium |
| CN106566788A (en) * | 2016-11-02 | 2017-04-19 | 中国农业大学 | Method for biological synthesis of nano-selenium by using silver-resistant Bacillus edaphicus and application thereof |
| CN106479927A (en) * | 2016-11-02 | 2017-03-08 | 中国农业大学 | Method and its application using Bacillus licheniformis biosynthesiss nanometer selenium |
| CN106884000A (en) * | 2017-04-25 | 2017-06-23 | 安徽天邦生物技术有限公司 | A kind of preparation and application of spindle lysine bacillus selenium-rich microbial agent |
| CN109182128A (en) * | 2018-10-16 | 2019-01-11 | 广西壮族自治区农业科学院农业资源与环境研究所 | A kind of strain culturing method of Efficient Conversion Selenium in Soil |
| CN112831435A (en) * | 2021-01-15 | 2021-05-25 | 武汉华硒生物科技有限公司 | Separated nitrogen-producing pseudomonas and application thereof in reduction of selenium element |
| CN112831435B (en) * | 2021-01-15 | 2021-10-08 | 武汉华硒生物科技有限公司 | Separated nitrogen-producing pseudomonas and application thereof in reduction of selenium element |
| CN113430136A (en) * | 2021-07-09 | 2021-09-24 | 华中农业大学 | Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium |
| CN113912458A (en) * | 2021-10-24 | 2022-01-11 | 恩施硒谷科技股份有限公司 | Method for preparing micro-ecological slow-release organic selenium fertilizer by using super-selenium-resistant microorganisms |
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