CN105420280A - Method for biosynthesizing nano-selenium through bacillus licheniformis - Google Patents
Method for biosynthesizing nano-selenium through bacillus licheniformis Download PDFInfo
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Abstract
The invention discloses a method for biosynthesizing nano-selenium through bacillus licheniformis. The method comprises the steps of slope seed culture, first-level seed culture, second-level seed culture and fermentation production. The selected bacillus licheniformis can resist high-concentration selenite and can resist sodium selenite with the highest concentration of 2000 mg/L. The efficiency of converting the sodium selenite into red active nano-selenium reaches up to 80% or above. The bacillus licheniformis belongs to probiotics, is safe to use and can be directly used as a feed additive to be added into livestock and poultry feed, and selenium-enriched meat food can be produced for people by feeding livestock and poultry with the nano-selenium produced through the bacillus licheniform. According to the selected bacillus licheniformis, the requirements for culture media and culture conditions needed during culture are low, the culture cost is low, and the method for biosynthesizing the nano-selenium through the bacillus licheniformis has the advantages that operation is easy, preparation is convenient and the cost is low, and is applicable to industrial large-scale production.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method utilizing Bacillus licheniformis biosynthesizing nanometer selenium.
Background technology
Selenium is necessary trace element, has very large importance to the health of organism.Selenium is the important component part of seleno-protein, in its structure and enzyme, serve as important role, and the Triiodothyronine that enlivens generated as a kind of antioxidant and catalyzer is exactly best example.In human body, the amplification of the normal operation of selenium energy Promote immunity system, effectively inhibiting HIV, can also strengthen motility of sperm simultaneously, improves reproductive performance, reduce the probability and antitumous effect of miscarrying.Selenium deficiency easily makes health enter unhealthy emotion state usually, thus susceptible disease and infection virus.
The selenium of human body mainly comes from animals and plants food, and the soil of China 72% lacks selenium (≤0.6mg/kg), belong to the serious country lacking selenium, the selenium that people take in from diet is fewer, easily cause the diseases such as Keshan disease, therefore need extra supplement selenium to meet human health demand.
At occurring in nature, selenium mainly exists with three kinds of forms, inorganic selenium (as Sodium Selenite), organoselenium (Fructus Hordei Germinatus selenium, selenium yeast etc.) and nanometer selenium (particle diameter is at the zeroth order selenium of 5-200nm), but their biological activity and physiological-toxicity are also deposited, need when supplement selenium to consider factor that is active and toxicity.In acute toxicity (LD50) standard, inorganic selenium is 15mg/kg, and organoselenium is 30-40mg/kg, and nanometer selenium is then up to 113mg/kg.In subchronic toxicity, in feed, the content of inorganic selenium or organoselenium can cause rat body weight to decline and liver cirrhosis when 4-5mg/kg, and nanometer selenium content also there will not be above-mentioned phenomenon when 6mg/kg.In biological effect, the efficiency of the external removing hydroxyl radical free radical of nanometer selenium is 5 times of inorganic selenium, is 25 times of organoselenium.Therefore, nanometer selenium becomes the optimal selection of other form selemium nutrition preparations alternative or medicament.
Nanometer selenium has redness, black and brown three kinds of forms, only has red nano selenium just to have physiologically active.The production method of current nanometer selenium mainly contains chemical synthesis and biological synthesis process, biological nanometer selenium than chemosynthesis nanometer selenium evenly, form is more regular, and biosynthetic nanometer selenium is high temperature resistant, more stable, not easily changes into black or brown nanometer selenium.Biosynthesizing nanometer selenium mainly utilizes micro-reduction selenite to produce, the bacterial classification reported at present have the red bacterium of fixed nitrogen (
rhodoacterazotoformans), milk-acid bacteria (
lactobacillus), genus bacillus (
bacillus), intestinal bacteria (
escherichiacoli) etc.But due to most of microbe not easily fermentative production, the characteristic of not easily preserving, can not suitability for industrialized production be realized.Genus bacillus is owing to can produce the stronger gemma of resistance, the poor environment in ability process of manufacture and condition, and adding and be easy to preserve, is the good microbial strains of suitability for industrialized production nanometer selenium.But the genus bacillus mentioned in existing report is not high to selenite tolerance, is unfavorable for suitability for industrialized production yet.
Summary of the invention
The object of the invention is to the shortcoming overcoming prior art, provide a kind of production performance good, can resisting high-concentration selenite and Efficient Conversion selenite generating the genus bacillus of red active nano selenium and utilize the method for its biosynthesizing nanometer selenium.
Object of the present invention is achieved through the following technical solutions: a kind of method utilizing Bacillus licheniformis biosynthesizing nanometer selenium, and it comprises the following steps:
S1. inclined-plane seed culture: be inoculated in by Bacillus licheniformis on nutrient agar inclined-plane, cultivates 36 ~ 48h at 28 ~ 45 DEG C, obtains inclined-plane kind;
S2. first order seed is cultivated: be inoculated in liquid fermentation medium by inclined-plane kind, liquid fermentation medium 100ml is housed in 1L triangular flask, and 28 ~ 45 DEG C of vibration cultivation 12 ~ 18h, obtain level liquid kind;
S3. secondary seed is cultivated: level liquid kind be seeded in the filling of 300L liquid fermenting, described liquid fermenting is filled with 210L liquid fermentation medium, and cultivation 12 ~ 18h are cultivated in 28 ~ 45 DEG C of vibrations, obtain secondary liquid kind;
S4. fermentative production: secondary liquid kind be seeded in the filling of 2T liquid fermenting, described liquid fermenting is filled with 1.4T liquid fermentation medium, cultivation 18 ~ 36h are cultivated in 28 ~ 45 DEG C of vibrations, obtain nanometer selenium;
Wherein, described liquid fermentation medium is prepared from by the raw material of following concentration: fish meal: 1 ~ 10g/L, yeast powder: 1 ~ 5g/L, rice meal: 1 ~ 5g/L, bitter salt: 0.1 ~ 0.5g/L, potassium primary phosphate: 0.1 ~ 1g/L, sodium-chlor: 0.1 ~ 0.5g/L, Repone K: 0.1 ~ 0.5g/L, calcium carbonate: 0.1 ~ 1g/L, Sodium Selenite: 500mg/L, pH value is 7.2 ~ 7.4.
Further, described Bacillus licheniformis is purchased from China General Microbiological culture presevation administrative center, and deposit number is CGMCC1.6510.
Further, the nutrient agar described in step S1 is prepared from by the raw material of following concentration: extractum carnis: 2 ~ 5g/L, peptone: 8 ~ 15g/L, sodium-chlor: 3 ~ 7g/L, agar: 12 ~ 18g/L, and pH value is 7.0 ~ 7.2.
Further, the speed of described shaking culture is 180 ~ 220rpm.
The present invention has the following advantages:
1. the Bacillus licheniformis selected of the present invention, the selenite of energy resisting high-concentration, the general tolerable concentration of use bacterial classification before reported is about 170mg/L, and the suitableeest concentration of sodium selenite of Bacillus licheniformis that the present invention selects is 500mg/L, the Sodium Selenite of the highest ability 2000mg/L, and also conversion Sodium Selenite becomes the efficiency of red active nano selenium very high, can more than 80% be reached, therefore, the present invention can ensure disposablely to produce a large amount of nanometer selenium, reduces production cost;
2. the Bacillus licheniformis selected of the present invention, its bacterial classification belongs to probiotic bacterium, use safety, Ministry of Agriculture's " fodder additives kind catalogue (2010) " regulation can directly be added to inside animal and fowl fodder as fodder additives, and feeding by the nanometer selenium that this bacterial classification is produced, can to produce rich selenium meat product edible for the mankind for livestock and poultry;
3. the Bacillus licheniformis selected of the present invention, when it is cultivated, required substratum and culture condition require simple, toxigenic capacity is low, and utilizes the method for Bacillus licheniformis biosynthesizing nanometer selenium to have the advantages such as simple to operate, easy to prepare, is applicable to industrialization scale operation.
Accompanying drawing explanation
Fig. 1 is nanometer selenium aspect graph prepared by the inventive method, and magnification is 3000 times;
Fig. 2 is nanometer selenium aspect graph prepared by the inventive method, and magnification is 5000 times.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described, and protection scope of the present invention is not limited to the following stated.
embodiment 1:utilize a method for Bacillus licheniformis biosynthesizing nanometer selenium, it comprises the following steps:
S1. inclined-plane seed culture: be inoculated in by Bacillus licheniformis on nutrient agar inclined-plane, cultivates 36h at 28 DEG C, obtains inclined-plane kind; Nutrient agar is prepared from by the raw material of following concentration: extractum carnis: 2g/L, peptone: 8g/L, sodium-chlor: 3g/L, agar: 12g/L, and pH value is 7.0;
S2. first order seed is cultivated: be inoculated in liquid fermentation medium by inclined-plane kind, liquid fermentation medium 100ml is housed in 1L triangular flask, and 12h is cultivated in 28 DEG C of vibrations, and the speed of shaking culture is 180rpm, obtains level liquid kind;
S3. secondary seed is cultivated: level liquid kind be seeded in the filling of 300L liquid fermenting, described liquid fermenting is filled with 210L liquid fermentation medium, and 12h are cultivated in 28 DEG C of vibrations, and the speed of shaking culture is 180rpm, obtains secondary liquid kind;
S4. fermentative production: secondary liquid kind be seeded in the filling of 2T liquid fermenting, described liquid fermenting is filled with 1.4T liquid fermentation medium, 18h are cultivated in 28 DEG C of vibrations, and the speed of shaking culture is 180rpm, obtains nanometer selenium; Carry out living bacteria count counting to product, bacteria concentration can reach 12,000,000,000/mL.It is 85% that Sodium Selenite changes into nanometer selenium efficiency.
Wherein, described liquid fermentation medium is prepared from by the raw material of following concentration: fish meal: 1g/L, yeast powder: 1g/L, rice meal: 1g/L, bitter salt: 0.1g/L, potassium primary phosphate: 0.1g/L, sodium-chlor: 0.1g/L, Repone K: 0.1g/L, calcium carbonate: 0.1g/L, Sodium Selenite: 500mg/L, pH value is 7.2.
embodiment 2:utilize a method for Bacillus licheniformis biosynthesizing nanometer selenium, it comprises the following steps:
S1. inclined-plane seed culture: be inoculated in by Bacillus licheniformis on nutrient agar inclined-plane, cultivates 48h at 45 DEG C, obtains inclined-plane kind; Nutrient agar is prepared from by the raw material of following concentration: extractum carnis: 5g/L, peptone: 15g/L, sodium-chlor: 7g/L, agar: 18g/L, and pH value is 7.2;
S2. first order seed is cultivated: be inoculated in liquid fermentation medium by inclined-plane kind, liquid fermentation medium 100ml is housed in 1L triangular flask, and 18h is cultivated in 45 DEG C of vibrations, and the speed of shaking culture is 220rpm, obtains level liquid kind;
S3. secondary seed is cultivated: level liquid kind be seeded in the filling of 300L liquid fermenting, described liquid fermenting is filled with 210L liquid fermentation medium, and 18h are cultivated in 45 DEG C of vibrations, and the speed of shaking culture is 220rpm, obtains secondary liquid kind;
S4. fermentative production: secondary liquid kind be seeded in the filling of 2T liquid fermenting, described liquid fermenting is filled with 1.4T liquid fermentation medium, 36h are cultivated in 45 DEG C of vibrations, and the speed of shaking culture is 220rpm, obtains nanometer selenium; Carry out living bacteria count counting to product, bacteria concentration can reach 11,000,000,000/mL, and it is 83% that Sodium Selenite changes into nanometer selenium efficiency.
Wherein, described liquid fermentation medium is prepared from by the raw material of following concentration: fish meal: 10g/L, yeast powder: 5g/L, rice meal: 5g/L, bitter salt: 0.5g/L, potassium primary phosphate: 1g/L, sodium-chlor: 0.5g/L, Repone K: 0.5g/L, calcium carbonate: 1g/L, Sodium Selenite: 500mg/L, pH value is 7.4.
embodiment 3:utilize a method for Bacillus licheniformis biosynthesizing nanometer selenium, it comprises the following steps:
S1. inclined-plane seed culture: be inoculated in by Bacillus licheniformis on nutrient agar inclined-plane, cultivates 40h at 34 DEG C, obtains inclined-plane kind; Nutrient agar is prepared from by the raw material of following concentration: extractum carnis: 3g/L, peptone: 10g/L, sodium-chlor: 5g/L, agar: 14g/L, and pH value is 7.1;
S2. first order seed is cultivated: be inoculated in liquid fermentation medium by inclined-plane kind, liquid fermentation medium 100ml is housed in 1L triangular flask, and 14h is cultivated in 34 DEG C of vibrations, and the speed of shaking culture is 200rpm, obtains level liquid kind;
S3. secondary seed is cultivated: level liquid kind be seeded in the filling of 300L liquid fermenting, described liquid fermenting is filled with 210L liquid fermentation medium, and 14h are cultivated in 34 DEG C of vibrations, and the speed of shaking culture is 200rpm, obtains secondary liquid kind;
S4. fermentative production: secondary liquid kind be seeded in the filling of 2T liquid fermenting, described liquid fermenting is filled with 1.4T liquid fermentation medium, 25h are cultivated in 34 DEG C of vibrations, and the speed of shaking culture is 200rpm, obtains nanometer selenium; Carry out living bacteria count counting to product, bacteria concentration can reach 11,200,000,000/mL, and it is 88% that Sodium Selenite changes into nanometer selenium efficiency.
Wherein, described liquid fermentation medium is prepared from by the raw material of following concentration: fish meal: 4g/L, yeast powder: 2g/L, rice meal: 2g/L, bitter salt: 0.2g/L, potassium primary phosphate: 0.5g/L, sodium-chlor: 0.2g/L, Repone K: 0.2g/L, calcium carbonate: 0.4g/L, Sodium Selenite: 500mg/L, pH value is 7.3.
embodiment 4:utilize a method for Bacillus licheniformis biosynthesizing nanometer selenium, it comprises the following steps:
S1. inclined-plane seed culture: be inoculated in by Bacillus licheniformis on nutrient agar inclined-plane, cultivates 45h at 40 DEG C, obtains inclined-plane kind; Nutrient agar is prepared from by the raw material of following concentration: extractum carnis: 4g/L, peptone: 12g/L, sodium-chlor: 6g/L, agar: 16g/L, and pH value is 7.2;
S2. first order seed is cultivated: be inoculated in liquid fermentation medium by inclined-plane kind, liquid fermentation medium 100ml is housed in 1L triangular flask, and 16h is cultivated in 40 DEG C of vibrations, and the speed of shaking culture is 210rpm, obtains level liquid kind;
S3. secondary seed is cultivated: level liquid kind be seeded in the filling of 300L liquid fermenting, described liquid fermenting is filled with 210L liquid fermentation medium, and 16h are cultivated in 40 DEG C of vibrations, and the speed of shaking culture is 210rpm, obtains secondary liquid kind;
S4. fermentative production: secondary liquid kind be seeded in the filling of 2T liquid fermenting, described liquid fermenting is filled with 1.4T liquid fermentation medium, 30h are cultivated in 40 DEG C of vibrations, and the speed of shaking culture is 210rpm, obtains nanometer selenium; Carry out living bacteria count counting to product, bacteria concentration can reach 13,000,000,000/mL, and it is 81% that Sodium Selenite changes into nanometer selenium efficiency.
Wherein, described liquid fermentation medium is prepared from by the raw material of following concentration: fish meal: 8g/L, yeast powder: 4g/L, rice meal: 4g/L, bitter salt: 0.4g/L, potassium primary phosphate: 0.8g/L, sodium-chlor: 0.4g/L, Repone K: 0.4g/L, calcium carbonate: 0.8g/L, Sodium Selenite: 500mg/L, pH value is 7.2.
Claims (4)
1. utilize a method for Bacillus licheniformis biosynthesizing nanometer selenium, it is characterized in that, it comprises the following steps:
S1. inclined-plane seed culture: be inoculated in by Bacillus licheniformis on nutrient agar inclined-plane, cultivates 36 ~ 48h at 28 ~ 45 DEG C, obtains inclined-plane kind;
S2. first order seed is cultivated: be inoculated in liquid fermentation medium by inclined-plane kind, liquid fermentation medium 100ml is housed in 1L triangular flask, and 28 ~ 45 DEG C of vibration cultivation 12 ~ 18h, obtain level liquid kind;
S3. secondary seed is cultivated: level liquid kind be seeded in the filling of 300L liquid fermenting, described liquid fermenting is filled with 210L liquid fermentation medium, and cultivation 12 ~ 18h are cultivated in 28 ~ 45 DEG C of vibrations, obtain secondary liquid kind;
S4. fermentative production: secondary liquid kind be seeded in the filling of 2T liquid fermenting, described liquid fermenting is filled with 1.4T liquid fermentation medium, cultivation 18 ~ 36h are cultivated in 28 ~ 45 DEG C of vibrations, obtain nanometer selenium;
Wherein, described liquid fermentation medium is prepared from by the raw material of following concentration: fish meal: 1 ~ 10g/L, yeast powder: 1 ~ 5g/L, rice meal: 1 ~ 5g/L, bitter salt: 0.1 ~ 0.5g/L, potassium primary phosphate: 0.1 ~ 1g/L, sodium-chlor: 0.1 ~ 0.5g/L, Repone K: 0.1 ~ 0.5g/L, calcium carbonate: 0.1 ~ 1g/L, Sodium Selenite: 500mg/L, pH value is 7.2 ~ 7.4.
2. a kind of method utilizing Bacillus licheniformis biosynthesizing nanometer selenium as claimed in claim 1, it is characterized in that, described Bacillus licheniformis is purchased from China General Microbiological culture presevation administrative center, and deposit number is CGMCC1.6510.
3. a kind of method utilizing Bacillus licheniformis biosynthesizing nanometer selenium as claimed in claim 1, it is characterized in that, nutrient agar described in step S1 is prepared from by the raw material of following concentration: extractum carnis: 2 ~ 5g/L, peptone: 8 ~ 15g/L, sodium-chlor: 3 ~ 7g/L, agar: 12 ~ 18g/L, pH value is 7.0 ~ 7.2.
4. a kind of method utilizing Bacillus licheniformis biosynthesizing nanometer selenium as claimed in claim 1, it is characterized in that, the speed of described shaking culture is 180 ~ 220rpm.
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| 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 |
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| CN115838770A (en) * | 2022-11-29 | 2023-03-24 | 安徽科技学院 | Method for preparing biological nano-selenium by using bacillus sp HZ3 |
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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 |
| 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 |
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| CN107539963A (en) * | 2016-11-11 | 2018-01-05 | 王鲁宁 | Utilize the method for aerobic microbiological particle preparation nanometer selenium |
| CN107881127B (en) * | 2018-01-23 | 2022-03-22 | 陕西省微生物研究所 | Bacillus amyloliquefaciens Lxz-41 and method for controllably preparing nano-selenium by using same |
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| CN109402007B (en) * | 2018-11-12 | 2021-12-24 | 中国科学院合肥物质科学研究院 | Biological nano-selenium producing strain and method for preparing biological nano-selenium by using strain |
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| CN109593679A (en) * | 2018-12-26 | 2019-04-09 | 大连雪龙都市农业有限公司 | A kind of nanometer selenium microbial bacterial agent and the preparation method and application thereof |
| CN113308401A (en) * | 2021-05-24 | 2021-08-27 | 青岛农业大学 | Nano-selenium synthetic bacteria, screening method of nano-selenium synthetic bacteria and application of nano-selenium synthetic bacteria |
| CN114806936A (en) * | 2022-04-14 | 2022-07-29 | 上海市农业科学院 | Bacillus licheniformis with antibacterial and elemental selenium synthesis capacity and application thereof |
| CN115838770A (en) * | 2022-11-29 | 2023-03-24 | 安徽科技学院 | Method for preparing biological nano-selenium by using bacillus sp HZ3 |
| CN115838770B (en) * | 2022-11-29 | 2024-04-02 | 安徽科技学院 | Method for preparing biological nano-selenium by using bacillus sp HZ3 |
| WO2024113498A1 (en) * | 2022-11-29 | 2024-06-06 | 安徽科技学院 | Method for preparing biological nano-selenium using bacillus. sp hz3 |
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